Orexin receptor antagonism represents a novel approach for the treatment of insomnia that directly targets sleep/wake regulation. Several such compounds have entered into clinical development, including the dual orexin receptor antagonists, suvorexant and almorexant. In this study we have used equilibrium and kinetic binding studies with the orexin-2 (OX2) selective antagonist radioligand, [3H]-EMPA, to profile several orexin receptor antagonists. Furthermore, selected compounds were studied in cell-based assays of inositol phosphate accumulation and extracellular-regulated kinase (ERK)-1/2 phosphorylation in CHO cells stably expressing the OX2 receptor that employ different agonist incubation times (30 and 5 minutes, respectively). EMPA, suvorexant, almorexant and TCS-OX-29 all bind to the OX2 receptor with moderate to high affinity (pKI values ≥ 7.5), whereas the primarily OX1 selective antagonists SB-334867 and SB-408124 displayed low affinity (pKI values ca. 6). Competition kinetic analysis showed that the compounds displayed a range of dissociation rates from very fast (TCS-OX2-29, koff = 0.22 min-1) to very slow (almorexant, koff = 0.005 min-1). Notably, there was a clear correlation between association rate and affinity. In the cell-based assays, fast-offset antagonists EMPA and TCS-OX2-29 displayed surmountable antagonism of orexin-A agonist activity. However, both suvorexant and particularly almorexant cause concentration-dependent depression in the maximal orexin-A response, a profile that is more evident with a shorter agonist incubation time. Analysis according to a hemi-equilibrium model suggests that antagonist dissociation is slower in a cellular system than in membrane binding; under these conditions almorexant effectively acts as a pseudo-irreversible antagonist.
The DNA damage response (DDR), consisting of an orchestrated network of proteins effecting repair and signalling to cell cycle arrest, to allow time to repair, is essential for cell viability and to prevent DNA damage being passed on to daughter cells. The DDR is dysregulated in cancer with some pathways upregulated and others downregulated or lost. Up-regulated pathways can confer resistance to anticancer DNA damaging agents. Therefore, inhibitors of key components of these pathways have the potential to prevent this therapeutic resistance. Conversely, defects in a particular DDR pathway may lead to dependence on a complementary pathway. Inhibition of this complementary pathway may result in tumour-specific cell killing. Thus inhibitors of the DDR have the potential to increase the efficacy of DNA damaging chemotherapy and radiotherapy and have single agent activity against tumours with a specific DDR defect. This review describes the compounds that have been designed to inhibit specific DDR targets and summarises the pre-clinical and clinical evaluation of these inhibitors of DNA damage signalling and repair.
Bcl-2 is a central regulator of cell survival that is overexpressed in the majority of small cell lung cancers (SCLC) and contributes to both malignant transformation and therapeutic resistance. The purpose of this work was to study the key factors that determine the sensitivity of SCLC cells to BH3 mimetic S1 and the mechanism underlying the resistance of BH3 mimetics.
Western blot was used to evaluate the contribution of Bcl-2 family members to the cellular response of SCLC cell lines to S1. Acquired resistant cells were derived from initially sensitive H1688 cells. Quantitative PCR and gene silencing were performed to investigate Bcl-2 up-regulation.
A progressive increase in the relative levels of Bcl-2 and phosphorylated Bcl-2 (pBcl-2) characterized the increased de novo and acquired resistance of SCLC cell lines. Furthermore, acute treatment of S1 induced Bcl-2 expression and phosphorylation. We showed that BH3 mimetics including S1 and ABT-737 induced ER stress and then activated MEK/ERK pathway. The dual function of MEK/ERK pathway in defining BH3 mimetics was illustrated: ERK1/2 activation leaded to Bcl-2 transcriptional up-regulation and sustained phosphorylation in naïve and acquired resistant SCLC cells. pBcl-2 played a key role in creating resistance of S1 and ABT-737 not only by sequestrating pro-apoptotic proteins, but also a positive feedback to promote ERK1/2 activation.
These results provide significant novel insights into the molecular mechanisms for crosstalk between ER stress and endogenously apoptotic pathways in SCLC following BH3 mimetics treatment.
Two-state and operational models of both agonism and allosterism are compared to identify and characterize common pharmacologic parameters. To account for receptor-dependent basal response constitutive receptor activity is considered in the operational models. By arranging two-state models as the fraction of active receptors and operational models as the fractional response relative to the maximum effect of the system a one-by-one correspondence between parameters is found. The comparative analysis allows a better understanding of complex allosteric interactions. Particularly, inclusion of constitutive receptor activity in the operational model of allosterism allows the description of modulators able to lower the basal response of the system; that is, allosteric modulators with negative intrinsic efficacy. Theoretical simulations and overall goodness of fit of the models to simulated data suggest that models are feasible for their application to experimental data and constitute one step forward in receptor theory formalism.
Understanding the neurochemical basis for cognitive function is one of the major goals of neuroscience, with a potential impact on the diagnosis, prevention and treatment of a range of psychiatric and neurological disorders. In this review, the focus will be on a biochemical pathway that remains under-recognised in its implications for brain function, even though it can be responsible for moderating the activity of two neurotransmitters fundamentally involved in cognition – glutamate and acetylcholine. Since this pathway – the kynurenine pathway of tryptophan metabolism - is induced by immunological activation and stress it also stands in an unique position to mediate the effects of environmental factors on cognition and behaviour. Targetting the pathway for new drug development could, therefore, be of value not only for the treatment of existing psychiatric conditions, but also for preventing the development of cognitive disorders in response to environmental pressures.
Peroxisome proliferator-activated receptor (PPAR)γ has been reported to be a protective regulator in ischemia/reperfusion (I/R) injury. The receptor for advanced glycation end-products (RAGE) plays a major role in the innate immune response and its expression is associated with PPARγ activation. Several angiotensin receptor blockers possess partial agonist activities toward PPARγ. Therefore, this study investigated the mechanism of losartan, particularly on PPARγ activation and RAGE signaling pathways during hepatic I/R.
Mice were subjected to 60 min of ischemia followed by 6 h of reperfusion. Losartan (0.3, 1, 3, and 10 mg·kg-1) was administered 1 h prior to ischemia and immediately before reperfusion. GW9662, a PPARγ antagonist, was administered 30 min prior to first pretreatment of losartan.
Losartan enhanced the DNA binding activity of PPARγ in I/R. Losartan attenuated the increased serum alanine aminotransferase activity, tumor necrosis factor-α, and interleukin-6 levels and nuclear concentrations of nuclear factor kappa B in I/R. GW9662 reversed these beneficial effects. Losartan caused a decrease in apoptosis, as assessed by TUNEL assay, release of cytochrome c, and cleavage of caspase-3; and these effects were abolished by GW9662 administration. Losartan attenuated not only I/R-induced RAGE overexpression, but also its downstream early growth response-1-dependent macrophage inflammatory protein-2 protein level and phosphorylation of p38, extracellular signal-regulated kinase, c-Jun N-terminal kinase, and subsequent c-Jun phosphorylation. GW9662 reversed these effects of losartan administration.
Our findings suggest that losartan ameliorates I/R-induced liver damage through PPARγ activation and down-regulation of the RAGE signaling pathway.
After its acute administration, the electrophysiological effects of ORM-10103, a new sodium/calcium exchanger (NCX) inhibitor, on the NCX and L-type Ca2+ currents and on the formation of early and delayed afterdepolarizations were studied.
Ion currents were recorded by using a voltage clamp technique in canine single ventricular cells, and action potentials were obtained from canine and guinea-pig ventricular preparations with the use of microelectrodes.
ORM-10103 significantly reduced both the inward and outward NCX currents with estimated EC50 values of 780 nM and 960 nM, respectively. Even at a high concentration (10 μM), ORM-10103 did not significantly change the L-type Ca2+ current or the maximum rate of depolarization (dV/dtmax), indicative of the fast inward Na+ current. 10 μM ORM-10103 did not affect the amplitude or the dV/dtmax of the slow response action potentials recorded from guinea-pig papillary muscles, which suggested the lack of its effect on the L-type Ca2+ current. ORM-10103 did not influence Na+/K+ pump and the main K+ currents of canine ventricular myocytes except the rapid delayed rectifier K+ current, which is slightly diminished by the drug at 3 μM concentration. The amplitude of pharmacologically induced early and delayed afterdepolarizations were significantly decreased by ORM-10103 (3 and 10 μM) in a concentration-dependent manner.
It is concluded that specific inhibition of the NCX current by ORM-10103 can abolish triggered arrhythmias and may result in antiarrhythmic electrophysiological effects.
We previously reported that adenosine, acting at adenosine A2A receptors (A2AR), inhibits osteoclast (OC) differentiation in vitro in vitro (A2AR activation OC formation reduces by half) and in vivo. For a better understanding how adenosine A2AR stimulation regulates OC differentiation we dissected the signaling pathways involved in A2AR signaling.
OC differentiation was studied as TRAP+ multinucleated cells following M-CSF/RANKL stimulation of either primary murine bone marrow cells or the murine macrophage line, RAW264.7, in presence/absence of the A2AR agonist CGS21680, the A2AR antagonist ZM241385, PKA activators (8-Cl-cAMP 100nM, 6-Bnz-cAMP) and the PKA inhibitor (PKI). cAMP was quantitated by EIA and PKA activity assays were carried out. Signaling events were studied in PKA knockdown (lentiviral shRNA for PKA) RAW264.7 cells (scrambled shRNA as control). OC marker expression was studied by RT-PCR.
A2AR stimulation increased cAMP and PKA activity which and were reversed by addition of ZM241385. The direct PKA stimuli 8-Cl-cAMP and 6-Bnz-cAMP inhibited OC maturation whereas PKI increased OC differentiation. A2AR stimulation inhibited p50/p105 NFκB nuclear translocation in control but not in PKA KO cells. A2AR stimulation activated ERK1/2 by a PKA-dependent mechanism, an effect reversed by ZM241385, but not p38 and JNK activation. A2AR stimulation inhibited OC expression of differentiation markers by a PKA-mechanism.
A2AR activation inhibits OC differentiation and regulates bone turnover via PKA-dependent inhibition of NFκB nuclear translocation, suggesting a mechanism by which adenosine could target bone destruction in inflammatory diseases like Rheumatoid Arthritis.
Thioredoxin-interacting protein (TXNIP), a regulator of cellular oxidative stress, is related to the NOD-like receptor 3 (NLRP3) inflammasome activation, inflammation and lipid metabolism, suggesting its role in the pathogenesis of nonalcoholic fatty liver disease (NAFLD) in diabetes. This study investigated whether TXNIP was involved in type 1 diabetes-associated NAFLD, and anti-oxidants quercetin and allopurinol targeting TXNIP improved these disorders.
Diabetes was induced in male SD rats by a single intraperitoneal injection of 55 mg/kg streptozotocin (STZ). Quercetin and allopurinol were orally given to diabetic rats for 7 weeks. Hepatic function, oxidative stress, inflammation and lipid level were determined. Rat BRL-3A and human HepG2 cells were exposed to high glucose (30 mM) in the presence or absence of anti-oxidants, or TXNIP siRNA transfection or caspase-1 inhibitor Ac-YVAD-CMK.
Quercetin and allopurinol significantly inhibited TXNIP over-expression and NLRP3 inflammasome activation, up-regulated peroxisome proliferator-activated receptor alpha (PPARα) and down-regulated sterol regulatory element binding protein 1c (SREBP-1c), SREBP-2, fatty acid synthase (FAS) and liver X receptor α (LXR α), accompanying with reactive oxygen species (ROS) and interleukin-1β reduction in diabetic rat liver. These effects were confirmed in hepatocyte cell models, showing that TXNIP down-regulation contributed to suppression of the NLRP3 inflammasome activation, inflammation, PPARα/SREBPs dysexpression.
These results demonstrate that hepatic TXNIP inhibition by quercetin and allopurinol contributes to the improvement of liver inflammation and lipid accumulation under hyperglycemia condition. Therefore, quercetin and allopurinol targeting hepatic TXNIP may have therapeutic application to prevent type 1 diabetes-associated NAFLD.
Normal pregnancy is associated with decreased vascular resistance and increased release of vasodilators. Endothelin-1 (ET-1) causes vasoconstriction via endothelin receptor type A (ETAR), but could activate ETBR in the endothelium and release vasodilator substances. However, the role of ETBR in the regulation of vascular function during pregnancy and the vascular mediators involved are unclear.
Pressurized mesenteric microvessels from pregnant and virgin Sprague-Dawley rats were loaded with fura-2/AM for simultaneous measurement of diameter and [Ca2+]i.
High KCl (51 mM) and phenylephrine caused increases in vasoconstriction and [Ca2+]i that were similar in pregnant and virgin rats. ET-1 caused vasoconstriction that was less in pregnant than virgin rats, with small increases in [Ca2+]i. Pretreatment with the ETBR antagonist BQ-788 caused greater enhancement of ET-1-induced vasoconstriction in pregnant rats. Acetylcholine (ACh) caused endothelium-dependent relaxation and decreased [Ca2+]i, and was more potent in pregnant than virgin rats. ET-1+ETAR antagonist BQ-123, and the ETBR agonists sarafotoxin 6c (S6c) and IRL-1620 caused greater vasodilation in pregnant than virgin rats with no changes in [Ca2+]i, suggesting upregulated ETBR-mediated relaxation pathways. ACh, S6c and IRL-1620-induced relaxation was reduced by the NO synthase inhibitor L-NAME and abolished by tetraethylammonium or endothelium removal. Western blots revealed greater amount of ETBR in intact microvessels of pregnant than virgin rats, but reduced levels in endothelium-denuded microvessels, supporting a role of endothelial ETBR.
The enhanced ETBR-mediated microvascular relaxation may contribute to the decreased vasoconstriction and vascular resistance during pregnancy.
Chronic ethanol abuse and haemorrhagic shock are major causes of global mortality and both induce profound hepato- and immunotoxic effects via activation of NF-κB. Here, we examined the role of chronic ethanol intake upon the pathophysiological derangements after haemorrhagic shock with subsequent resuscitation (H/R) with special respect to the contribution of NF-κB.
Especially bred mice whose enhanced green fluorescent protein (EGFP) expression is under the transcriptional control of NF-κB cis-elements were fed an ethanol containing Lieber-DeCarli or an isocaloric control-diet over 4 weeks, and were then pairwise subjected to H/R. Liver tissues and peripheral blood were harvested at 2 and 24 h after resuscitation.
Chronic ethanol feeding prior to H/R increased mortality at 24 h and elevated liver injury. This was associated with an upregulation of NF-κB dependent genes, and with an enhanced IL-6 release. Ethanol increased the production of NF-κB driven ICAM-1 and EGFP content in liver tissue. Interestingly, 2 h after H/R and prior to chronic ethanol intake we observed the highest proportion of NF-κB activated non parenchymal cells, and a NF-κB dependent increase in polymorphonuclear leukocyte CD11b expression.
Chronic ethanol intake exacerbates liver injury after H/R, which is accompanied by an overwhelming hepatic and systemic immune response. Our findings contribute to an increasing body of evidence implicating NF-κB transcription factor as a key player in the orchestration of the immune response in haemorrhagic shock patients with a history of chronic ethanol abuse.
Previously, we demonstrated that glucosamine (GlcN) exerts a suppressive effect on lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) through the inhibition of nuclear factor-kappaB (NF-κB) activation in BV2 mouse microglial cells. The purpose of the present study was to examine the mechanisms by which GlcN inhibits NF-κB activation.
BV2 cells were stimulated with LPS and/or GlcN. NF-κB/c-Rel activities were studied by EMSA, nuclear translocation, reporter assay or chromatin immunoprecipitation. Wheat germ agglutinin precipitation or galactosyltransferase assay were used to measure O-linked N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) of c-Rel. Protein-protein interaction was examined by co-immunoprecipitation method.
In BV2 microglial cells, the anti-inflammatory effect of GlcN appears to be mediated by prevention of prolonged NF-κB/c-Rel activation. LPS-induced activation of c-Rel was accompanied by an increase in the extent of O-GlcNAcylation of the protein and GlcN suppressed LPS effect. O-GlcNAcylation of c-Rel was not dependent on activation-induced nuclear protein translocation. LPS promoted a c-Rel-O-GlcNAc transferase (OGT) interaction and c-Rel-p50/p105 interaction, whereas GlcN suppressed these interactions. Whereas knockdown of OGT reduced the c-Rel O-GlcNAcylation and c-Rel-p50 interaction in response to LPS, it did affect neither the binding of c-Rel to the iNOS promoter nor the transcriptional activity of c-Rel.
Study the inhibition of NF-κB/c-Rel activation by GlcN will facilitate the development of pharmacological strategies or may provide a good therapeutic option seeking to prevent excessive NO formation when targeting inflammatory diseases of the periphery or central nervous system.
In order to describe drug action at a G protein-coupled receptor (GPCR), a full understanding of the pharmacological terms affinity, efficacy and potency is necessary. This is true whether comparing the ability of different agonists to produce a measurable response in a cell or tissue, or whether determining the relative ability of an agonist to activate a single receptor subtype and produce multiple responses. There is a great deal of interest in the μ opioid receptor (MOPr) and the ligands that act at this GPCR, not only because of the clinically important analgesic effects produced by MOPr agonists, but also because of their liability to induce adverse effects such as respiratory depression and dependence. Our understanding of the mechanisms underlying these effects, as well as the ability to develop new, more effective MOPr drugs depends upon the accurate determination of the efficacy with which these ligands induce coupling of MOPr to downstream signalling events. In this review, which is written with the minimum of mathematical content, the basic meaning of terms including efficacy, intrinsic activity and intrinsic efficacy is discussed, along with their relevance to the field of MOPr pharmacology, and in particular in relation to biased agonism at this important GPCR.
Histamine H3 receptors are best known as presynaptic receptors inhibiting the release of histamine, as well as other neurotransmitters including acetylcholine and dopamine. However, in the dorsal and ventral striatum, the vast majority of H3 receptors are actually located postsynaptically on medium sized spiny output neurons. These cells also contain large numbers of dopamine (D1 and D2) receptors and it has been shown that H3 receptors form heterodimers with both dopamine receptors. Thus the anatomical localization of H3 receptors suggests a complex interaction that could both enhance and inhibit the dopaminergic neurotransmission. Dopamine especially within the striatal complex plays a crucial role in the development of addiction, both in the initial reinforcing effects of drugs of abuse, as well as in maintenance, relapse and reinstatement of drug taking behaviour. It is therefore conceivable that H3 receptors can moderate the development and maintenance of drug addiction. In the present paper, we will review the current literature on the involvement of H3 receptors in drug addiction and will try to explain these data in a theoretical framework, as well as provide suggestions for further research.
Oncogenic mutations in RAS or BRAF can drive the inappropriate activation of the extracellular signal-regulated kinases, ERK1/2. In many cases tumour cells adapt to become addicted to this deregulated ERK1/2 signalling for their proliferation, providing a therapeutic window for tumour-selective growth inhibition. As a result, inhibition of ERK1/2 signalling by BRAF or MEK1/2 inhibitors is an attractive therapeutic strategy. Indeed, the first BRAF inhibitor, vemurafenib, has now been approved for clinical use, whilst clinical evaluation of MEK1/2 inhibitors is at an advanced stage. Despite this progress it is apparent that tumour cells adapt quickly to these new, targeted agents so that tumours with acquired resistance can emerge within 6-9 months of primary treatment. One of the major reasons for this is that tumour cells typically respond to BRAF or MEK1/2 inhibitors by undergoing a G1 cell cycle arrest rather than dying. Indeed, although inhibition of ERK1/2 invariably increases the expression of pro-apoptotic BCL2 family proteins, tumour cells undergo minimal apoptosis. This cytostatic response may simply provide the cell with the opportunity to adapt and acquire resistance. Here we discuss recent studies that demonstrate that combination of BRAF or MEK1/2 inhibitors with inhibitors of pro-survival BCL2 proteins is synthetic lethal for ERK1/2-addicted tumour cells. This combination effectively transforms the cytostatic response of BRAF and MEK1/2 inhibitors into a striking apoptotic cell death response. This not only augments the primary efficacy of BRAF and MEK1/2 inhibitors but delays the onset of acquired resistance to these agents, validating their combination in the clinic.
The Kaposi Sarcoma-associated herpesvirus G protein-coupled receptor (vGPCR) is a key molecule in the pathogenesis of Kaposi Sarcoma, where it increases Nuclear Factor kappa B (NFκB) gene expression and activates the NFκB pathway. We investigated whether the less calcemic vitamin D analog TX 527 inhibited the proliferation of endothelial cells transformed by vGPCR by modulation of the NFκB pathway.
Endothelial cells transformed by vGPCR (SVEC-vGPCR) were treated with TX 527. Proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) and cell cycle by flow cytometry. mRNA and protein levels were measured by qRT-PCR and immunoblot analysis, respectively.
TX 527, similarly to bortezomib (0. 5 nM), a proteasome inhibitor that inhibits the activation of NFκB, reduced proliferation and induced G0/G1 cell cycle arrest in SVEC-vGPCR. TX 527 like 1α,25(OH)2D3,biological active form of vitamin D, decreased the activity of NFκB comparable with the effect of bortezomib. Time-response studies showed that TX 527 significantly decreased NFκB and increased IκBα mRNA and protein levels. The increase of IκBα was accompanied by a reduction in p65/NFκB translocation to the nucleus. These responses were abolished when vitamin D receptor (VDR) expression was suppressed by stable transfection of shRNA against VDR. In parallel with NFκB inhibition, there was a down-regulation of inflammatory genes such as IL-6, CCL2/MCP and CCL20/MIP3α.
These results suggest that the antiproliferative effects of the vitamin D analog TX 527 in SVEC-vGPCR occur by modulation of the NFκB pathway and are VDR- dependent.
Degenerative dementia is mainly caused by Alzheimer's disease and/or cerebrovascular abnormalities. Disturbance of the intracellular calcium homeostasis is central to the pathophysiology of neurodegeneration. In Alzheimer's disease, enhanced calcium load may be brought about by extracellular accumulation of amyloid-β. Recent studies suggest that soluble forms facilitate influx through calcium-conducting ion channels in the plasma membrane, leading to excitotoxic neurodegeneration. Calcium channel blockade attenuates amyloid-β-induced neuronal decline in vitro and is neuroprotective in animal models. Vascular dementia, on the other hand, is caused by cerebral hypoperfusion and may benefit from calcium channel blockade due to relaxation of the cerebral vasculature. Several calcium channel blockers have been tested in clinical trials of dementia and the outcome is heterogenous. Nimodipine as well as nilvadipine prevent cognitive decline in some trials, whereas other calcium channel blockers failed. In trials with a positive outcome, blood pressure reduction did not seem to play a role in preventing dementia, indicating a direct protecting effect on neurons. An optimisation of calcium channel blockers for the treatment of dementia may involve an increase of selectivity for presynaptic calcium channels and an improvement of the affinity to the inactivated state. Novel low molecular weight compounds suitable for proof-of-concept studies are now available.
Histamine and prostaglandin E2 (PGE2), directly and via their effects on other cells, regulate the behaviour of vascular smooth muscle (VSM), but their effects on human VSM are incompletely resolved.
The effects of PGE2 on histamine-evoked changes in intracellular free Ca2+ concentration ([Ca2+]i) and adenylyl cyclase (AC) activity were measured in populations of cultured human aortic smooth muscle cells (ASMC). Selective ligands of histamine and EP receptors were used to identify the receptors that mediate the responses.
Histamine, via H1 receptors, stimulates an increase in [Ca2+]i that is entirely mediated by activation of inositol 1,4,5-trisphosphate receptors. Selective stimulation of EP2 or EP4 receptors attenuates histamine-evoked Ca2+ signals, but the effects of PGE2 on both Ca2+ signals and AC activity are largely mediated by EP2 receptors.
Two important inflammatory mediators, histamine via H1 receptors, and PGE2 acting largely via EP2 receptors, exert opposing effects on [Ca2+]i in human ASMC.
Anti-tumour therapies based on the use PARAs (pro-apoptotic receptor agonists), including TRAIL (TNF-Related Apoptosis inducing Ligand) or monoclonal antibodies targeting TRAIL-R1 or TRAIL-R2, have been disappointing so far, despite clear evidence of clinical activity and lack of adverse events for the vast majority of these compounds, whether combined or not with conventional or targeted anti-cancer therapies. This brief review aims at discussing the possible reasons for the lack of apparent success of these therapeutic approaches and at providing hints in order to rationally design optimal protocols based on our current understanding of TRAIL signalling regulation or resistance for future clinical trials.
Since the identification of the histamine H4 receptor subtype (H4R) several ligands activating this receptor have been described and more compounds are in the development process. These ligands are well characterized in pharmacological assays, including radioligand competition binding studies, GTPγS and GTPase assays. In most cases, these experiments are performed in transfected cell lines, expressing unnaturally high levels of target receptors and G-protein signaling components. In this study we investigated the specific properties of H4R ligands in native cells.
Histamine and five different H4R agonists – 4-Methylhistamine, UR-PI376, Clobenpropit, VUF8430 and ST-1006 – were characterized in native human monocytes. The ligands (concentrations of 10μM to 10nM) were tested for inhibition of IL-12p70 secretion from human monocytes and the effect of the H2R antagonist ranitidine and the H4R antagonist JNJ7777120 on their action was investigated.
Histamine and all the tested agonists reduced IL-12p70 secretion into monocyte supernatants by 40% to 70%. The potencies varied with pEC50 values ranging from 5.7 to 6.9, depending on the agonist used. All potencies were lower than the ones determined in the original investigations of the compounds. Pre-treatment of monocytes with H2R or H4R antagonists showed that some H4R ligands also had low activity at the H2R.
Our study demonstrates discrepancies between the potencies obtained from pharmacological assays and from assays with native human cells, indicating the importance of H4R ligand evaluation in native cells.
We previously showed that apolipoprotein A-I (ApoA-I) mimetic peptide infusions induce regression of aortic valve stenosis (AVS) in rabbits. This study aimed at determining the effects of ApoA-I mimetic therapy in mice with calcific or fibrotic AVS.
57 apolipoprotein E-deficient (ApoE-/-) mice and 19 mice with Werner progeria gene deletion (WrnΔhel/Δhel) received high-fat diets during 20 weeks. Following AVS establishment, mice were randomized to receive saline (placebo group, n=29 ApoE-/-; n=9 Wrn) or ApoA-I mimetic peptide infusions (ApoA-I treated group, 100 mg/kg, n=28 ApoE-/-; 50 mg/kg, n=10 Wrn), 3 times/week for 4 weeks. We evaluated the treatment effects on AVS using serial echocardiograms and valve histology.
Aortic valve area (AVA) increased in both ApoE-/- and Wrn mice treated with the ApoA-I mimetic compared to placebo (ApoE-/-: 0.624 [0.606-0.641] vs 0.587 [0.569-0.605] mm2, P=0.0039; Wrn: 0.664 [0.642-0.686] vs 0.597 [0.574-0.621] mm2, P=0.0002). Maximal sinus wall thickness was lower in ApoA-I treated ApoE-/- mice (61.5±7.3 vs 69.1±11.8 μm for placebo; P=0.016). The type I/ III collagen ratio was lower in the sinus wall of ApoA-I treated ApoE-/- mice compared to placebo (1.16 (0.86;9.50) vs. 1.80 (0.72;4.16), P=0.020). Total collagen content was reduced in aortic valves of ApoA-I treated Wrn mice (80.4±10.8 vs 90.6±4.2% for placebo; P=0.023). Our 3D computer model and numerical simulations confirmed that reduced aortic root wall thickness resulted in improved AVA.
ApoA-I mimetic treatment reduces AVS by decreasing remodeling and fibrosis of the aortic root and valve in mice.
Excitatory amino acid transporters (EAATs) in the central nervous system (CNS) contribute to the clearance of glutamate released during neurotransmission. The aim of this study was to explore the role of EAATs in the regulation of locus coeruleus (LC) neurons by glutamate.
We measured the effect of different EAAT subtype inhibitors/enhancers on glutamate- and KCl-induced activation of LC neurons in rat slices. EAAT2-3 expression in the LC was also characterized by immunohistochemistry.
The EAAT2-5 inhibitor DL-TBOA (100 μM), but not the EAAT2,4,5 inhibitor t-PDC (100 μM) or the EAAT2 inhibitor DHK (100 μM), enhanced the glutamate- and KCl-induced activation of the firing rate of LC neurons. These effects were blocked by ionotropic but not metabotrobic glutamate receptor antagonists. DHK (100 μM) was the only EAAT inhibitor that increased the spontaneous firing rate of LC cells, an effect that was due to inhibition of EAAT2 and subsequent AMPA receptor activation. Chronic treatment with ceftriaxone (200 mg kg-1 i.p., once daily, 7 days), an EAAT2 expression enhancer, increased the actions of glutamate and DHK, suggesting a functional impact of EAAT2 up-regulation on the glutamatergic system. Immuhistochemical data revealed the presence of EAAT2 and EAAT3 surrounding noradrenergic neurons and EAAT2 on glial cells in the LC.
These results remark the importance of EAAT2 and EAAT3 in the regulation of rat LC by glutamate. Neuronal EAAT3 would be responsible for terminating the action of synaptically released glutamate, whereas glial EAAT2 would regulate tonic glutamate concentrations in this nucleus.
Presynaptic nicotinic acetylcholine receptors (nAChRs) and adenosine A2A receptors (A2ARs) are involved in the control of dopamine release and are putative therapeutic targets in Parkinson's disease and addiction. Since A2ARs have been reported to interact with nAChRs, here we aimed at mapping the possible functional interaction between A2ARs and nAChRs in rat striatal dopaminergic terminals.
We pharmacologically characterized the release of dopamine and defined the localization of nAChR subunits in rat striatal nerve terminals in vitro, and carried out locomotor behavioral sensitization in rats in vivo.
In striatal nerve terminals, the selective A2AR agonist CGS21680 (30 nM) inhibited, while the A2AR antagonist ZM241385 potentiated the nicotine-stimulated [3H]dopamine release. Upon blockade of the α6 subunit-containing nAChRs, the remaining nicotine-stimulated [3H]dopamine release was no longer modulated by A2AR ligands. In the locomotor sensitization experiments, nicotine enhanced the locomotor activity on day 7 of repeated nicotine injection, an effect which no longer persisted after one week of drug withdrawal. Notably, ZM241385-injected rats developed locomotor sensitization to nicotine already on day 2, which remained persistent upon nicotine withdrawal.
These results provide the first evidence for a functional interaction between nicotinic and adenosine A2A receptors in striatal dopaminergic terminals, with likely therapeutic consequences for smoking, Parkinson's disease and other dopaminergic disorders.
Our previous study demonstrated that 6-(pyrrolidin-1-yl)-2-(3-methoxyphenyl)quinazolin-4-one (HMJ38) was the most potent antitubulin agent. Here, HMJ38 was used as a lead compound to develop more potent anticancer agents and examine the anticancer mechanisms.
According to computer-aided drug design, 2-aryl-6-substituted quinazolinones (MJ compounds) were designed and synthesized by extending HMJ38's structure through introducing substituents at C-2 and C-6 positions. The cytotoxicity of MJ compounds toward human cancer cells was examined by trypan blue exclusion assay. Microtubule distribution was visualized using TubulinTrackerTM Green reagent. The protein expression of cell cycle regulators and c-jun N-terminal kinase (JNK) were detected by Western blot analysis.
MJ65–70 exhibited strong anti-proliferative effect toward melanoma M21, lung squamous carcinoma CH27, lung non-small carcinoma H460, hepatoma Hep3B and oral cancer HSC-3 cells. 6-(Pyrrolidin-1-yl)-2-(naphthalen-1-yl)quinazolin-4-one (MJ66) displayed excellent activity against M21 cells with IC50 about 0.033 μM. Treatment of CH27 or HSC-3 cells with MJ65–70 resulted in significant mitotic arrest accompanied by increasing multiple asters of microtubules. JNK protein expression was involved in the MJ65–70-induced CH27 and M21 cell death. In accordance with the MJ65–70-induced cell cycle arrest at G2/M phase, remarkable increases in cyclin B1 and Bcl-2 phosphorylation were also observed.
MJ65–70 are dual-targeted, tubulin- and JNK-binding, anticancer agents and induce cancer cells death through upregulation of JNK and interfering dynamics of tubulin. Our work provides a new strategy and mechanism for developing dual-targeted anticancer drug, and may contribute to clinical anticancer drug discovery and application.
Glucocorticoids are highly effective therapies for a range of inflammatory diseases. Advances in the understanding of the diverse molecular mechanisms underpinning glucocorticoid action suggest that anti-inflammatory molecules with reduced side-effect liabilities can be discovered. Here we set out to explore whether modification of the 17α position of the steroid nucleus could generate molecules with a unique pharmacological profile, and to determine whether such molecules would retain anti-inflammatory activity.
The pharmacological properties of GW870086 were compared to fluticasone propionate (FP) using a range of cellular and in vivo model systems, including extensive gene expression profiling.
GW870086 repressed inflammatory cytokine release from lung epithelial cells in a similar manner to FP, but antagonised the effect of dexamethasone on MMTV driven reporter gene transactivation. GW870086 had a strong effect on the expression of some glucocorticoid regulated genes (such as COX-2), while having minimal impact on the expression of other known target genes (such as SGK). GW870086 retained the ability to strengthen tight junctions in epithelial cell culture, but unlike FP was unable to protect the culture from elastase mediated damage. In murine models of irritant-induced contact dermatitis and ovalbumin-induced allergic inflammation models, GW870086 showed comparable anti-inflammatory efficacy to FP.
GW870086 is a potent anti-inflammatory compound with a unique ability to regulate only a subset of those genes that are normally affected by classical glucocorticoids. It has the potential to become a new topical steroid with a different safety profile to existing therapies.
The blood-brain barrier (BBB) restricts drug penetration to the brain preventing effective treatment of patients suffering from brain tumors. Intraarterial injection of short-chain alkylglycerols (AGs) opens the BBB and increases delivery of molecules to rodent brain parenchyma in vivo. The mechanism underlying AG-mediated modification of BBB permeability is still unknown. The aim of the present work was to test the effects of AGs on barrier properties of cultured brain microvascular endothelial cells.
The effects of 1-O-pentylglycerol and 2-O-hexyldiglycerol were examined using an in vitro BBB model consisting of primary rat brain endothelial cells co-cultured with rat cerebral glial cells. Integrity of the paracellular tight junction (TJ)-based permeation route was analyzed by functional assays, immunostaining for junctional proteins, freeze fracture electron microscopy, and analysis of claudin-claudin trans-interaction.
AG treatment (5 min) led to a significant (P<0.05), reversible reduction of transendothelial electrical resistance and an increase in blood-brain barrier permeability for fluorescein accompanied by changes in cell morphology and immunostaining for claudin-5 and β-catenin. These short-term changes were not mirrored in alterations of interendothelial TJ strand complexity or the trans-interaction of claudin-5.
AG-mediated increase in brain endothelial paracellular permeability is short, reversible and does not affect TJ strand complexity. Redistribution of junctional proteins and alterations in the cell shape indicate the involvement of cytoskeleton in the action of AGs. These data confirm the results from in vivo studies on rats and mice characterizing alkylglycerols as adjuvants to transiently open the BBB.
Biological nitrogen oxide signaling and stress is an area of extreme clinical, pharmacological, toxicological, biochemical and chemical research interest. The utility of nitric oxide and derived species as signaling agents is due to their novel and vast chemical interactions with a variety of biological targets. Herein, the chemistry associated with the interaction of the biologically relevant nitrogen oxide species with fundamental biochemical targets is discussed. Specifically, the chemical interactions of nitrogen oxides with nucleophiles (e.g. thiols), metals (e.g. hemeproteins) and paramagnetic species (e.g. dioxygen and superoxide) are addressed. Importantly, the terms associated with the mechanisms by which NO (and derived species) react with their respective biological targets have been defined by numerous past chemical studies. Thus, in order to assist researchers in referring to chemical processes associated with nitrogen oxide biology, the vernacular associated with these chemical interactions is addressed.
Targeted chemotherapy using small-molecule inhibitors of angiogenesis and proliferation is a promising strategy for cancer therapy.
N-methyl-4-(4-(3-(trifluoromethyl)benzamido)phenoxy)picolinamide4- methylbenzenesulfonate (YL529) was developed via computer-aided drug design, de novo synthesis and high-throughput screening. The biochemical, pharmacodynamical, and toxicological profiles of YL529 were investigated using kinase and cell viability assay, zebrafish and mice tumor xenograft models.
In vitro, YL529 selectively inhibited the activities of vascular endothelial growth factor receptors VEGFR2/VEGFR3 and serine/threonine kinase RAF kinase. YL529 inhibited VEGF165-induced phosphorylation of VEGFR2, as well as proliferation, migration, invasion and tube formation of human umbilical vascular endothelial cells (HUVECs). It also significantly blocked vascular formation and angiogenesis in zebrafish model. Moreover, YL529 strongly attenuated the proliferation of A549 cell line through disrupting RAF/MEK/MAPK (mitogen-activated protein kinase) pathway. Oral administration of YL529 (37.5-150 mg-1·kg-1·day-1) to nude mice bearing established tumor xenografts significantly prevented the growth (60-80%) of A549, SPC-A1, A375, OS-RC-2 and HCT116 tumors without detectable toxicity. Tumors from animals inoculated with the lung cancer cell lines SPC-A1 and A549 and the colon carcinoma cell line HCT116 revealed that YL529 treatment markedly reduced microvessel density and increased tumor cell apoptosis.
YL529, an orally active multikinase inhibitor, shows the therapeutic potential for solid tumors, and warrants further investigation as a candidate anticancer agent.
The intermediate-conductance Ca2+-activated K+ channel KCa3.1 modulates the Ca2+ response through the control of the membrane potential in the immune system. We investigated the role of KCa3.1 on the pathogenesis of delayed-type hypersensitivity (DTH) in auricular lymph node (ALN) CD4+ T-lymphocytes of oxazolone (Ox)-induced DTH model mice.
The expression patterns of KCa3.1 and its possible transcriptional regulators were compared among ALN T-lymphocytes of three groups [non-sensitized (Ox-/-), Ox-sensitized but non-challenged (Ox+/-), and Ox-sensitized and -challenged (Ox+/+)] using real-time PCR, Western blotting and flow cytometry. KCa3.1 activity was measured by whole-cell patch clamp and the voltage-sensitive dye imaging. The effects of KCa3.1 blockade were examined by the administration of selective KCa3.1 blockers.
Significant up-regulation of KCa3.1a was observed in CD4+ T-lymphocytes of Ox+/- and Ox+/+, without any evident changes in the expression of the dominant-negative form, KCa3.1b. Negatively correlated with this, the repressor element-1 silencing transcription factor, REST was significantly down-regulated. Pharmacological blockade of KCa3.1 resulted in an accumulation of the CD4+ T-lymphocytes of Ox+/+ at the G0/G1 phase of the cell cycle, and also significantly recovered not only the pathogenesis of DTH, but also the changes in the KCa3.1 expression and activity in the CD4+ T-lymphocytes of Ox+/- and Ox+/+.
The up-regulation of KCa3.1a in conjunction with the down-regulation of REST may be involved in CD4+ T-lymphocyte proliferation in the ALNs of DTH model mice, and KCa3.1 may be an important target for therapeutic intervention in allergy diseases such as DTH.
Albuminuria is an important biomarker of renal dysfunction and is a major mediator of renal damage and fibrosis during kidney disease. The mechanisms underlying albumin-induced renal fibrosis remain unclear. There has been significant interest in γ-secretase activity in tubular epithelial cells in recent times; however, its potential role in albumin-induced fibrosis has not been investigated.
The primary aim of this study was to examine the role of γ−secretase in albumin-induced fibrotic effects in proximal tubular cells. The effects of increasing albumin concentrations on fibrosis indicators and mediators in the human HK-2 cell line were examined in the presence and absence of a γ-secretase inhibitor, Compound E.
Treatment with albumin resulted in a number of pro-fibrotic effects including upregulation of fibronectin, TGF-β1 and the EGF-R. Interestingly, similar effects were observed in response to treatment with the γ-secretase inhibitor, Compound E. Co-treatment of cells with albumin and an EGF-R inhibitor AG-1478 resulted in significant inhibition of the observed pro-fibrotic effects suggesting a major role for the EGF-R in albumin-induced fibrotic events. Albumin-induced effects on the EGF-R appeared to be mediated through inhibition of γ-secretase activity and were dependent on ERK mitogen activated protein kinase signalling.
These results provide novel insights into mechanisms of albumin-induced fibrotic effects in tubular epithelial cells suggesting important roles for the γ-secretase and the EGF-R. These results suggest that the proposed use of γ-secretase inhibitors as anti-fibrotic agents requires further investigation.
The 5-HT3 receptor is a ligand-gated ion channel that is modulated allosterically by various compounds including colchicine, alcohols and volatile anaesthetics. However the positive allosteric modulators (PAMs) identified to date have low affinity, which hinders investigation due to non-selective effects at pharmacologically active concentrations. The present study identifies 5-chloroindole (Cl-indole) as a potent PAM of the 5-HT3 receptor.
5-HT3 receptor function was assessed by the increase in intracellular calcium and single cell electrophysiological recordings in HEK293 cells stably expressing the h5-HT3A receptor and also the mouse native 5-HT3 receptor that increases neuronal contraction of bladder smooth muscle.
Cl-indole (1-100 μM) potentiated agonist (5-HT) and particularly partial agonist ((S)-zacopride, DDP733, RR210, quipazine, dopamine, 2-methyl-5-HT, SR57227A, mCPBG)) induced h5-HT3A receptor mediated responses. This effect of Cl-indole was also apparent at the mouse native 5-HT3 receptor. Radioligand binding studies identified that Cl-indole induced a small (∼2-fold) increase in the apparent affinity of 5-HT for the h5-HT3A receptor, whereas there was no effect upon the affinity of the antagonist, tropisetron. Cl-indole was able to re-activate desensitised 5-HT3 receptors. In contrast to its effect on the 5-HT3 receptor, Cl-indole did not alter human nicotinic α7 receptor responses.
The present study identifies Cl-indole as a relatively potent and selective PAM of the 5-HT3 receptor; such compounds will aid investigation of the molecular basis for allosteric modulation of the 5-HT3 receptor and may assist the discovery of novel therapeutic drugs targeting this receptor.
The influence of the neurotransmitter histamine on spontaneous and stimulation-evoked release of glutamate, aspartate, GABA, and acetylcholine (ACh) in the nucleus accumbens (NAc) was investigated in vivo.
Using the push-pull-superfusion technique, histaminergic compounds were applied to the NAc and neurotransmitter release was assessed. In some experiments the fornix/fimbria of the hippocampus was electrically stimulated by a microelectrode and evoked potentials were monitored in the NAc.
Superfusion of the NAc with the H1 receptor antagonist triprolidine (50 μM) decreased spontaneous outflow of glutamate, aspartate, and ACh, while release of GABA remained unaffected. Superfusion with histamine elevated release of ACh, without influencing that of the amino acids. Electrical stimulation of the fornix/fimbria enhanced the output of amino acids and ACh within the NAc. The evoked outflow of glutamate and ACh was diminished on superfusion with triprolidine, while release of aspartate and GABA was not affected. Superfusion of the NAc with histamine intensified the stimulation-evoked release of glutamate and Ach. Histamine also elevated the stimulation-induced release of aspartate, without influencing that of GABA. Presuperfusion with triprolidine abolished the reinforced effect of histamine on stimulation-evoked transmitter release within the NAc.
Neuronal histamine activates H1 receptors and increases spontaneous release of glutamate, aspartate, and ACh within the NAc. Stimulation of the hippocampal fornix/fimbria tract also enhances release of glutamate and ACh within the NAc and this effect is intensified by H1 receptor stimulation within the NAc. The latter effects, which are mediated by hippocampal afferences, might play an important role in mnemonic performance and in emotional processes such as anxiety and stress disorders.
Oestrogens can interact directly with membrane receptors and channels and can activate vascular BKCa channels. We hypothesised that novel oestrogen derivatives could relax smooth muscle by an extracllular effect on the α and β1 subunits of the BKCa channel, rather than at an intracellular site.
We studied the effects of novel oestrogens on the tension of pre-contracted isolated rat aortic rings, and on the electrophysiological properties of HEK 293 cells expressing the hSloα or hSloα+β1 subunits. Two of the derivatives incorporated a quaternary ammonium side-chain making them membrane impermeable.
Oestrone, oestrone oxime and Quat DME-oestradiol relaxed pre-contracted rat aorta, but only Quat DME-oestradiol induced relaxation was iberiotoxin-sensitive. However, only potassium currents recorded in HEK 293 cells over-expressing both hSloα and hSloβ1 were activated by oestrone, oestrone oxime and Quat DME-oestradiol.
The novel oestrogens were able to relax smooth muscle, but through different mechanisms. In particular, oestrone oxime required the presence of the endothelium to exert much of its effect, whilst Quat DME-oestradiol depended both on nitric oxide and BKCa channel activation. The activation of BKCa currents in HEK 293 cells expressing hSloα+β1 by Quat DME-oestradiol is consistent with an extracellular binding site between the two subunits. The binding site resides between the extracellular N terminal of the α subunit and the extracellular loop between TM1 and 2 of the β1 subunit. Membrane- impermeant Quat DME-oestradiol, lacks an exchangeable hydrogen on the A ring obviating antioxidant activity.
The aim of the study was to determine whether KCNQ channels are functionally expressed in bladder smooth muscle cells and to investigate their physiological significance in bladder contractility.
KCNQ channels were examined at the genetic, protein, cellular and tissue level in guinea-pig bladder smooth muscle using RT-PCR, immunofluorescence, patch-clamp electrophysiology, calcium imaging, detrusor strip myography and a panel of KCNQ activators and inhibitors.
KCNQ subtypes 1-5 are expressed in bladder detrusor smooth muscle. Detrusor strips typically displayed TTX-insensitive myogenic spontaneous contractions which were increased in amplitude by KCNQ channel inhibitors XE991, linopirdine or chromanol 293B. Contractility was inhibited by the KCNQ channel activators flupirtine or meclofenamic acid (MFA). The frequency of Ca2+-oscillations in SMC contained within bladder tissue sheets was increased by XE991. Outward currents in dispersed bladder smooth muscle cells, recorded under conditions where BK and KATP currents were minimal, were significantly reduced by XE991, linopirdine or chromanol and enhanced by flupirtine or MFA. XE991 depolarized the cell membrane and could evoke transient depolarizations in quiescent cells. Flupirtine (20μM) hyperpolarized the cell membrane with a simultaneous cessation of any spontaneous electrical activity.
These novel findings reveal the role of KCNQ currents in the regulation of the resting membrane potential of detrusor smooth muscle cells and their important physiological function in the control of spontaneous contractility in the guinea-pig bladder.
Eicosapentaenoic acid (EPA), has been shown to suppress immune cell responses such as cytokine production and downstream prostaglandin production in vitro. Studies in vivo, however, have used EPA as a minor constituent of fish oil with variable results. We investigated the effects of EPA on systemic inflammatory responses as pure EPA has not been evaluated on immune/ inflammatory responses in vivo.
Rabbits were administered polyinosinic: polycytidylic acid (poly I:C) i.v. before and after oral treatment with EPA for 42 days (given daily). The responses to interleukin-1β (IL-1β) and tumour necrosis factor-α (TNF-α) were also studied. Immediately following administration of poly I:C, body temperature was continuously monitored and blood samples were taken. Plasma levels of IL-1β, prostaglandin E2 (PGE2), and 15-deoxy-Δ12,14-PGJ2 (15-dPGJ2) were measured by enzyme immunoassay.
Following EPA treatment the fever response to poly I:C was markedly suppressed compared to pretreatment responses. This was accompanied by a parallel reduction in the polyI:C-stimulated elevation in plasma levels of IL-1β and PGE2. Paradoxically, the levels of 15-dPGJ2were higher following EPA treatment. EPA treatment did not significantly alter the fever response or plasma levels of PGE2 in response to either IL-1β or TNF-α.
Oral treatment with EPA can suppress immune/ inflammatory responses in vivo via a suppression of upstream cytokine production resulting in a decreased fever response and indirectly reducing circulating levels of PGE2. EPA also enhances the production of the cytoprotective prostanoid 15-dPGJ2 indicating the therapeutic benefit of EPA.
Drug interference with normal hERG protein trafficking substantially reduces the channel density in the plasma membrane and thereby poses an arrhythmic threat. The chemical substructures important for hERG trafficking inhibition were investigated using pentamidine as a model drug. Furthermore, the relationship between acute ion channel block and correction of trafficking by dofetilide was studied.
hERG and Kir2.1 trafficking in HEK293 cells was evaluated by western blot and immunofluorescence microscopy after treatment with pentamidine and 6 pentamidine analogues, and correction with dofetilide and 4 dofetilide analogues that either display in- or decreased levels of acute IKr inhibition. Molecular dynamics simulations were used to address mode, number, and type of interactions between hERG and dofetilide analogues.
Structural modifications of pentamidine differentially affected plasma membrane levels of hERG and Kir2.1. Modification of the phenyl ring or substituents directly attached to it had the largest impact, emphasising the importance of these chemical residues in ion channel binding. PA-4 had the mildest effects on both ion channels (hERG: control 1.0±0.0 vs PA-4 0.65±0.06; Kir2.1: control 1.0±0.0 vs PA-4 0.80±0.12). Dofetilide corrected pentamidine-induced hERG, but not Kir2.1 trafficking defects. Dofetilide analogues that display high channel affinity, mediated by pi-pi stacks and hydrophobic interactions, were also able to correct hERG protein levels, while rescue by analogues that have low hERG affinity was ineffective.
Our results show that drug-induced trafficking defects can be minimized if certain chemical features are avoided or ‘synthesised out’, which could be of interest in future drug designs and development. Further analysis of such features in hERG trafficking correctors may facilitate the design of a non-blocking corrector for trafficking defective hERG proteins in both congenital and acquired LQTS.
The sphingosine 1-phosphate receptor subtype 1 (S1P1R) is modulated by phosphorylated FTY720 (pFTY720), which causes S1P1R internalisation preventing lymphocyte migration thus limiting autoimmune response. Studies indicate that internalised S1P1Rs continue to signal, maintaining an inhibition of cAMP, thus raising question whether the effects of pFTY720 are due to transient initial agonism, functional antagonism, and/or continued signalling. To further investigate this, the current study first determined if continued S1P1R activation is pathway specific.
Using human and rat astrocyte cultures, the effects of S1P1R activation on cAMP, pERK and Ca2+ signalling was investigated. In addition, to examine the role of S1P1R redistribution on these events, a novel biologic (MNP301) that prevented pFTY720-mediated S1P1R redistribution was engineered.
The data showed that pFTY720 induced long lasting S1P1R redistribution and continued cAMP signalling in rat astrocytes. In contrast, pFTY720 induced a transient increase of Ca2+ in astrocytes and subsequent antagonism of Ca2+ signalling. Notably, while leaving pFTY720-induced cAMP signalling intact, the novel MNP301 peptide attenuated S1P1R-mediated Ca2+ and pERK signalling in cultured rat astrocytes.
These findings suggested that pFTY720 causes continued cAMP signalling that is not dependent on S1P1R redistribution and induces functional antagonism of Ca2+ signalling after transient stimulation. To our knowledge, this is the first report demonstrating that pFTY720 causes continued signalling in one pathway (cAMP) versus functional antagonism of another pathway (Ca2+) and which also suggests that redistributed S1P1Rs may have differing signalling properties from those expressed at the surface.
Nebulized saline solutions are used in the treatment of multiple pulmonary diseases including cystic fibrosis (CF), asthma and COPD. The benefits of these therapies include improved lung function, phlegm clearance and fewer lung infections. The thiocyanate anion (SCN) is a normal component of the airway epithelial lining fluid (ELF) secreted by pulmonary epithelia with antioxidant and host defense functions. We sought to test if SCN could be nebulized to combat lung infection by bolstering innate immune defense and antioxidant capacity.
We established an effective antioxidant concentration of SCN in vitro using a bronchiolar epithelial cell line. We then developed a nebulization method of SCN in mice that increased ELF SCN above this concentration up to 12 hours and used this method in a prolonged Pseudomonas aeruginosa infection model to test if increasing SCN improved host defense and infection outcomes.
SCN protected against cytotoxicity in vitro from acute and sustained exposure to inflammation-associated oxidative stress. Nebulized SCN effectively reduced bacterial load, infection-mediated morbidity and airway inflammation in mice infected with P. aeruginosa. SCN also sustained adaptive increases in reduced glutathione (GSH) in infected mice.
SCN is a dually protective molecule able to both enhance host defense and decrease tissue injury and inflammation as an antioxidant. Nebulized SCN could be developed to combat lung infections and inflammatory lung disease.
Dietary indole derivatives, indole-3-carbinol (I3C), and diindolylmethane (DIM), possess anti-cancer properties and exhibit the characteristics of aryl hydrocarbon receptor (AhR) ligands. Because AhR activation has recently been shown to regulate T cell differentiation, we tested the hypothesis that I3C and DIM may mediate anti-inflammatory properties by promoting regulatory T cell (T-regs) differentiation while inhibiting Th17 cells.
We investigated the therapeutic efficacy of I3C and DIM against experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS). The efficacy was evaluated based on clinical scores of paralysis, histopathology, serum cytokines, and infiltration of T cells in the CNS. We next studied the mechanism of induction of T cells against myelin oligodendrocyte glycoprotein (MOG35-55) peptide, both in vivo and in vitro, specifically investigating the differentiation of T-regs and Th17 cells, and determined if indoles were acting through AhR.
Pre-treatment of EAE mice with I3C or DIM completely prevented the clinical symptoms and cellular infiltration into the CNS. Also, post-treatment of EAE with I3C or DIM also proved highly effective in curtailing the overall severity of the disease. In addition, I3C or DIM promoted the generation of T-regs, while downregulating the induction of MOG-specific Th17 cells. The regulation of FoxP3 induction and suppression of Th17 cells by indoles in vivo and in vitro were found to be AhR-dependent.
Together, our studies demonstrate for the first time that I3C and DIM may serve as novel therapeutics to suppress neuroinflammation seen during MS through activation of AhR.
The 5-HT3 receptor antagonist palonosetron is an important pharmacotherapy for the treatment of emesis and nausea during cancer therapy. Its clinical efficacy is thought to result from its unique binding and clearance characteristics and receptor down-regulation mechanisms. We investigated the mechanisms by which palonosetron exerts its long-term inhibition of 5-HT3 receptors in order to better understand its clinical efficacy.
Cell surface receptors (recombinantly expressed 5HT3A or 5HT3A/B in COS-7 cells) were monitored using [3H]granisetron binding and ELISA after exposure to palonosetron. Receptor endocytosis was investigated using immunofluorescence microscopy.
Chronic exposure to palonosetron reduces the number of available cell surface [3H]granisetron binding sites. This downregulation is not sensitive to either low temperature or pharmacological inhibitors of endocytosis (dynasore or nystatin) suggesting that internalisation does not play a role. This was corroborated by our observation that there is no change in cell surface 5-HT3 receptor levels or increase in endocytic rate. We find that palonosetron exhibits slow dissociation from the receptor over many hours, with a significant proportion of binding sites being occupied for at least 4 days. Furthermore, our observations suggest that chronic receptor downregulation involves interactions with an allosteric binding site.
Our results indicate that palonosetron acts as a pseudo-irreversible antagonist causing prolonged inhibition of 5-HT3 receptors due to its very slow dissociation. In addition, an irreversible binding mode persists at least four days. Allosteric receptor interactions appear to play a role in this phenomenon.
Retigabine is a recently approved anti-epileptic agent which activates Kv7.2-7.5 potassium channels. An important role for these channels in vascular regulation is emerging, but the vascular effects of retigabine in the conscious state are unknown. In the present study the regional haemodynamic responses to retigabine were assessed in conscious rats.
Male Sprague–Dawley rats were chronically instrumented with pulsed Doppler flow probes to measure regional haemodynamic responses to retigabine under control conditions and during acute hypertension induced by infusion of angiotensin II (AII) and arginine vasopressin (AVP). Further experiments were performed to elucidate the roles of β-adrenoceptor activation in the responses to retigabine in vivo and in vitro.
Under normotensive conditions, retigabine caused dose-dependent hypotension and hindquarters vasodilatation, with small, transient renal and mesenteric vasodilatations. In the acutely hypertensive state, the renal and mesenteric, but not hindquarters, vasodilatations were enhanced. The hindquarters response to retigabine was mediated, in part, by β2-adrenoceptors. However, in vitro experiments confirmed that retigabine did not act as a β-adrenoceptor agonist.
These results, showing differential vasodilator effects of retigabine, broadly support other studies identifying Kv7 channels as mediators of vascular tone, highlighting the different responses under normotensive and hypertensive conditions, and a role for β-adrenoceptor activation.
Background and purpose: Doxorubicin is effective against breast cancer but its major side-effect is cardiotoxicity. The aim of this study was to determine whether the efficacy of doxorubicin on cancer cells could be increased in combination with PPARγ agonists or chrono-optimisation by exploiting the diurnal cycle. Experimental approach: We determined cell toxicity using MCF-7 cancer cells, neonatal rat cardiac myocytes and fibroblasts in this study. Key results: Doxorubicin damages the contractile filaments of cardiac myocytes and affects cardiac fibroblasts by significantly inhibiting collagen production and proliferation at the level of the cell cycle. Cyclin D1 protein levels decreased significantly following doxorubicin treatment indicative of a G1/S arrest. PPARγ agonists with doxorubicin increased the toxicity to MCF-7 cancer cells without affecting cardiac cells. Rosiglitazone and ciglitazone both enhanced anticancer activity when combined with doxorubicin (e.g. 50% cell death for doxorubicin at 0.1 μM compared to 80% cell death when combined with rosiglitazone). Thus, the therapeutic dose of doxorubicin could be reduced 20-fold through combination with the PPARγ agonists thereby reducing adverse effects on the heart. The presence of melatonin also significantly increased doxorubicin toxicity, in cardiac fibroblasts (1μM melatonin) but not in MCF-7 cells. Conclusions and implications. Our data demonstrate, for the first time, that circadian rhythms play an important role in doxorubicin toxicity in the myocardium; doxorubicin should be administered mid-morning, when circulating levels of melatonin are low, and in combination with rosiglitazone to increase therapeutic efficacy in cancer cells while reducing the toxic effects on the heart.
The current lack of disease-modifying therapeutics to manage neurological and neurodegenerative conditions justifies the development of more efficacious agents. One distinct pathway leading to neuronal death in these conditions and which represents a very promising and attractive therapeutic target is parthanatos, involving overactivation of poly (ADP-ribose) polymerase-1 (PARP-1). We therefore sought to identify small molecules that could be neuroprotective by targeting the pathway.
Using HeLa cells, we developed and optimised an assay for high-throughput screening of about 5,120 small molecules. Structure-activity relationship study was carried out in HeLa and SH-SY5Y cells for molecules related to the initial active compound. The neuroprotective ability of each active compound was tested in cortical neuronal cultures.
4′-methoxyflavone (4MF) showed activity by preventing the decrease in cell viability of HeLa and SH-SY5Y cells caused by the DNA-alkylating agent, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), which induces parthanatos. A similar compound from the SAR study, 3′,4′-dimethoxyflavone (DMF), also showed significant activity. Both compounds reduced the synthesis and accumulation of poly (ADP-ribose) polymer and protected cortical neurones against cell death induced by N-methyl-D-aspartate (NMDA).
Our data reveal additional neuroprotective members of the flavone class of flavonoids and show that methoxylation of the parent flavone structure at position 4′ confers parthanatos-inhibiting activity while additional methoxylation at position 3′, reported by others to improve metabolic stability, does not destroy the activity. These molecules may therefore serve as leads for the development of novel neurotherapeutics for the management of neurological and neurodegenerative conditions.
Neurodegenerative diseases present progressive neurological disorder induced by cell death or apoptosis. Catalpol, an iridoid glucoside separated from the root of Rehmannia glutinosa Libosch, exists broadly in many plant families. Despite the evidence shows that Catalpol performs effective anti-apoptosis active role on LPS (lipopolysaccharides) -induced neurodegeneration, but the mechanism is not very clear so far. In view of the potency effects of Catalpol in anti-neurodegeneration, the following project was designed to elucidate whether Catalpol was capable of preventing apoptosis induced by LPS in an experimental model of neurodegeneration in vitro.
Apoptosis was induced by adding LPS (80ng/ml) in PC12 cells which were pre-treated with Catalpol for 12h.Then we analyzed the intracellular ROS, apoptosis and intracellular calcium concentration ([Ca2+]i) by flow cytometer or laser confocal scanning microscope. Simultaneously, we analyzed the protein expression of Bcl-2, Bax and CAMKII-depended ASK-1/JNK/p38 signaling cascade in PC12 cells by western blot.
Our data showed that Catalpol could stimulate the expression of Bcl-2 and constrain the expression of Bax. Catalpol could attenuate the increase in Ca2+ concentration induced by LPS in PC12 cells as well as down-regulation of CAMK phosphorylation. The result indicated that Catalpol could prevent CAMKII-depended ASK-1/JNK/p38 signaling cascade remarkably, which resulted in effective anti-apoptosis.
The data presented here provide new mechanistic insights into the connections between the CAMKII-depended ASK-1/JNK/p38 signaling cassette and the protective effect of Catalpol on apoptosis induced by LPS in PC12 cells.
Recent human studies suggest that recreational cannabis strains that are relatively high in cannabidiol (CBD) content produce less cognitive impairment than do strains with negligible CBD and similar Δ9tetrahydrocannabinol (THC) content. Self-selection in such studies means it is impossible to rule out additional variables which may determine both cannabis strain selection and basal cognitive performance level. Controlled laboratory studies can better determine a direct relationship.
In this study, adult male rhesus monkeys were assessed on visuo-spatial Paired-Associates Learning and self-ordered spatial search memory tasks, as well as additional tests of motivation and manual dexterity. Subjects were challenged with THC (0.2, 0.5 mg/kg, i.m.) in randomized order and evaluated in the presence or absence of 0.5 mg/kg CBD.
CBD attenuated the effects of THC on paired-associates learning and a bimanual motor task without affecting the detrimental effects of THC on a self-ordered spatial search task of working memory. CBD did not significantly reverse THC-induced impairment of a progressive ratio or a rotating turntable task.
This study provides direct evidence that CBD can oppose the cognitive impairing effects of THC, it does so in a task-selective manner, when administered simultaneously and at a 1:1 ratio with THC. The addition of CBD to THC-containing therapeutic products may therefore help to ameliorate unwanted cognitive side-effects.
Oxytocin (OT) plays a major role in the control of male sexual responses. Notably, blockade of OT receptors has been reported to inhibit ejaculation in animals. The study aimed to investigate the action of a highly selective, non-peptide OT antagonist GSK557296 in a model of pharmacologically-induced ejaculation in anaesthetised rats. The site of action was assessed by investigating different delivery routes for this compound.
Urethane-anaesthetised Wistar rats were implanted with a cerebral ventricle cannula for intracerebroventricular (i.c.v.) injections or with a subdural catheter for intrathecal (i.t.) GSK557296 injections. Occurrence of ejaculation was assessed following i.v. 7-OH-DPAT, a dopamine D3 receptor agonist. In addition, seminal vesicle pressures (SVP) and bulbospongiosus muscle (BS) electromyogram (EMG) were recorded as physiological markers of emission and expulsion phases of ejaculation respectively.
Highest i.v. GSK557296 dose reduced occurrence of ejaculation and increases in SVP but had no effect on BS-EMG. I.c.v. GSK557296 dose-dependently inhibited ejaculation, increases in SVP and BS contractions. At spinal thoracic level, GSK557296 dose-dependently inhibited ejaculation and increases in SVP but BS-EMG was impaired only with the highest dose. When delivered at lumbar level, GSK557296 dose-dependently inhibited ejaculation, increases in SVP and BS contractions.
In the 7-OH-DPAT–induced ejaculation model, GSK557296 acts peripherally and centrally to inhibit ejaculation with different modalities. Blockade of brain OT receptors seems to be the most effective mechanism of action. Targeting central OT receptors with highly selective antagonist seems a promising approach for the treatment of premature ejaculation.
Many organs suffer from ischemic injuries that reduce the ability to generate sufficient energy, which is required for functional maintenance and repair. Erythropoietin (EPO) has the potential to ameliorate ischemic tissue by pleiotropic effects. The aim of this study was to investigate a small molecule EH-201 (2,3,5,4′-tetrahydroxystilbene-2-o-beta-d-glucoside, THSG) with a potent EPO induction effect in nonhaematopoietic cells and examine the compound's therapeutic potential in ischemic disorders.
Mice kidney slices, primary hepatocytes, primary cardiomyocytes, and C2C12 myoblasts were exposed to the EH-201. The effects of treatment on EPO, haemoglobin expression, and mitochondrial biogenesis were analysed. In vivo, doxorubicin-induced cardiomyopathic mice were treated with EH-201. The mice were subjected to the endurance test, electrocardiography and echocardiography, and a histological examination of the isolated hearts was performed. EH-201 was also administered to cisplatin-induced nephropathic mice.
We demonstrated that EH-201 is a potent EPO-inducer and activates EPO-mediated mitochondrial function and haemoglobin expression in nonhaematopoietic cells. Using EPO and EPO receptor neutralising antibodies in mechanistic studies, we confirmed that EH-201 enhances EPO-EPOR autocrine activity. EH-201 robustly increased the endurance performance activity of healthy and cardiomyopathic mice during hypoxic stress, with induction of myocardial mitochondrial biogenesis and haemoglobin expression, as well as improved cardiac function. EH-201 also ameliorated anaemia and renal dysfunction in nephropathic mice.
The enhancement and recovery of cellular functions through the stimulation of mitochondrial activity and haemoglobin production in nonhaematopoietic cells by an inducer of endogenous EPO might serve as a potential therapeutic strategy for ischemic diseases.
Interleukin (IL)-22, a member of the IL-10 cytokine family, has recently gained significant attention as protective principle in murine models of diseases driven by epithelial injury. Alike its biochemical and functional sibling IL-10, IL-22 elicits cellular activation primarily by engaging the signal transducer and activator of transcription (STAT)-3 track of cellular activation. Exclusively produced by leukocytes but targeting foremost cells of epithelial origin, IL-22 is supposed to be a specialized cytokine messenger acting between leukocytic and non-leukocytic cell compartments. Unresponsiveness of leukocytes to IL-22 mirrors tightly controlled IL-22 receptor expression and likely relates to apparent lack of instant adverse effects after systemic IL-22 administration to mice. Anti-apoptotic, pro-proliferative, and pro-regenerative characteristics are outstanding among biological properties of this cytokine. Specifically, application of IL-22 associates with tissue protection and/or regeneration in murine models of infection/microbe-driven inflammation at host/environment interfaces, ventilator-induced lung injury, pancreatitis, and liver damage. On the whole, pre-clinical studies suggest therapeutic administration of seemingly well-tolerated recombinant IL-22 for treatment of an array of acute diseases that manifest at epithelial tissues. However, the feasibility of prolonged administration of this cytokine is expected to be restrained by a tumourigenic potential of the IL-22/STAT3 axis. IL-22, moreover, apparently displays an inherent context-specific capacity to amplify distinct aspects of autoimmune inflammation. Herein, prospects, expectations, and restrictions of IL-22 administration in tissue protective therapy are being discussed.
The long-standing debate that polyphenol secondary metabolites from dietary plants are important nutritional components continues due to compelling evidence for their abilities to ameliorate degenerative conditions including, cancer, neurological disorders, and cardiovascular disease. The clinical use of polyphenols is not, however, main-stream as issues regarding poor selectivity, dosage, toxicity and delivery methods are unresolved. The paper by Rieder et al. (2012) suggests that the lack of selectivity, at least for the stilbene, resveratrol, may not be a major limiting factor. The present commentary is a critique of this significant finding that is focused on deciding how the use of resveratrol as clinical medicine could be advanced, and how this new information integrates with current knowledge of polyphenol physiological effects. This commentary suggests that the multi-target nature of polyphenols may be translated into reliable therapy using the current systems/network pharmacology approach concerned with developing viable therapeutic agents that achieve specific effects through interactions with a wide array of targets.
Pulmonary hypertension is characterized by enhanced pulmonary vascular resistance, right ventricular hypertrophy, and increased right ventricular systolic pressure. Here, we investigated the effects of a N-acylhydrazone derivative, 3,4-dimethoxyphenyl-N-methyl-benzoilidrazide (LASSBio-1359), on the therapeutic treatment of monocrotaline induced pulmonary hypertension in rats.
Male Wistar rats were divided into saline (control), monocrotaline, monocrotaline + vehicle and monocrotaline + LASSBio-1359 groups. Pulmonary hypertension was induced by a single intraperitoneal injection of monocrotaline (60 mg/kg). Starting at 2 weeks after monocrotaline injection, oral LASSBio-1359 (50 mg/kg) or vehicle was administrated once daily for 14 days, after which the rats were sacrificed.
Monocrotaline administration increased the right ventricular systolic pressure, right ventricular to left ventricle plus septum weight ratio, and pulmonary arteriolar thickening. Monocrotaline also promoted endothelial dysfunction of the pulmonary artery rings. These alterations were improved by treatment with LASSBio-1359. The right ventricular systolic pressure was reduced from 49.59 ± 5.08 (monocrotaline group) to 30.37 ± 2.44 mmHg (monocrotaline + LASSBio-1359 group; P < 0.05). Treatment with LASSBio-1359 decreased the right ventricular area, wall thickness and the collagen volume fraction of pulmonary arterioles. LASSBio-1359 also restored the left ventricular cardiac output and pulmonary artery acceleration time of monocrotaline-injected rats.
The results indicate that LASSBio-1359 reversed right ventricular hypertrophy, endothelial dysfunction, and pulmonary vascular remodeling in rats with pulmonary hypertension, probably through the activation of adenosine A2A receptors.
Synaptic vesicle protein 2A (SV2A) is the specific binding site of the anti-epileptic drug levetiracetam (LEV) and its higher affinity analogue UCB30889. Moreover, the protein has been well validated as a target for anticonvulsant therapy. Here, we report the identification of UCB1244283 acting as a SV2A positive allosteric modulator of UCB30889.
UCB1244283 was characterized in vitro using radioligand binding assays with [3H]UCB30889 on recombinant SV2A expressed in HEK cells and on rat cortex. In vivo, the compound was tested in sound-sensitive mice.
Saturation binding experiments in the presence of UCB1244283 demonstrated a five-fold increase in the affinity of [3H]UCB30889 for human recombinant SV2A, combined with a two fold increase of the total number of binding sites. Similar results were obtained on rat cortex. In competition binding experiments, UCB1244283 potentiated the affinity of UCB30889 while the affinity of LEV remained unchanged. UCB1244283 significantly slowed down both the association and dissociation kinetics of [3H]UCB30889. Following i.c.v. administration in sound-sensitive mice, UCB1244283 showed a clear protective effect against both tonic and clonic convulsions.
These results indicate that UCB1244283 can modulate the conformation of SV2A, thereby inducing a higher affinity state for UCB30889. Our results also suggest that the conformation of SV2A per se might be an important determinant of its functioning, especially during epileptic seizures. Therefore, agents that act on the conformation of SV2A might hold great potential in the search for new SV2A-based anticonvulsant therapies.
Quercetin is a natural polyphenolic flavonoid that displays antidiabetic properties in vivo. Its mechanism of action on insulin secreting β-cells is poorly documented. In this work, we have analyzed in β cells the effect of quercetin both on insulin secretion and on the intracellular calcium concentration ([Ca2+]i) in the absence of any co-stimulating factor.
Experiments were performed on both INS-1 cell line and rat isolated pancreatic islets. Insulin release was quantified by the homogeneous time resolved fluorescence (HTRF) method. Variations in [Ca2+]i were measured using the ratiometric fluorescent Ca2+ indicator Fura-2. Ca2+ channel currents were recorded with the whole-cell patch-clamp technique.
Quercetin induced concentration-dependent insulin secretion and rise in [Ca2+]i. These effects were not modified by the SERCA inhibitor thapsigargin (1 μmol.L-1), but were nearly abolished by the L-type Ca2+ channel antagonist nifedipine (1 μmol.L-1). Like the agonist Bay K 8644, quercetin enhanced the L-type Ca2+ current by shifting its voltage-dependent activation towards negative potentials, leading to the rise of [Ca2+]i and insulin secretion. The effects of quercetin were unchanged in the presence of a maximally active concentration of Bay K 8644 (1 μmol.L-1), with the two drugs having cumulative effects on [Ca2+]i.
Taken together, our results show that quercetin stimulates insulin secretion by increasing Ca2+ influx through an interaction with L-type Ca2+ channels at a site different from that of Bay K 8644. These data contribute to a better understanding of quercetin mechanism of action on insulin secretion.
Although cannabinoid CB2 receptor ligands have been widely characterised in recombinant systems in vitro, little pharmacological characterisation has been performed in tissues natively expressing CB2 receptors. The aim of this study was to compare the pharmacology of CB2 receptor ligands in tissue natively expressing CB2 receptors (human, rat and mouse spleen) and hCB2-transfected CHO cells.
We tested the ability of well-known cannabinoid CB2 receptor ligands to stimulate or inhibit [35S]GTPγS binding to mouse, rat and human spleen membranes and to hCB2-transfected CHO cell membranes. cAMP assays were also performed in hCB2-CHO cells.
The data presented demonstrate that: (1) CP 55,940, WIN 55,212-2 and JWH 133 behave as CB2 receptor full agonists both in spleen and hCB2-CHO cells, in both [35S]GTPγS and cAMP assays; (2) JWH 015 behaves as a low efficacy agonist in spleen as well as in hCB2-CHO cells when tested in the [35S]GTPγS assay, whilst it displays full agonism when tested in the cAMP assay using hCB2-CHO cells; (3) (R)-AM 1241 and GW 405833 behave as agonists in the [35S]GTPγS assay using spleen; instead it behaves as a low efficacy inverse agonist in hCB2-CHO cells; (4) SR 144528, AM 630 and JTE 907 behave as CB2 receptor inverse agonists in all the tissues.
Our results demonstrate that CB2 receptor ligands can display differential pharmacology when assays are conducted in tissues that natively express CB2 receptors and imply that conclusions from recombinant CB2 receptors should be treated with caution.
The homeostatic control of arterial blood pressure is well understood with changes in blood pressure (BP) resulting from changes in cardiac output (CO) and/or total peripheral resistance (TPR). Drug effects on this interrelationship have not been analyzed in a mechanism-based and quantitative manner. This is important since it may constitute a basis for the prediction of drug effects on BP. This investigation aimed to describe, in a mechanism-based and quantitative manner, the effects of drugs with different mechanisms of action (MoA) on the interrelationship between BP, CO and TPR.
The cardiovascular effects of 6 drugs with diverse MoA's, (amlodipine, fasudil, enalapril, propranolol, hydrochlorothiazide and prazosin) were characterized in spontaneously hypertensive rats. The rats were chronically instrumented with ascending aortic flow probes and/or aortic catheters/radiotransmitters for continuous recording of CO and/or BP. Data were analyzed in conjunction with independent information on the time course of drug concentration using a mechanism-based PKPD modeling approach.
By simultaneous analysis of the effects of 6 different compounds, the dynamics of the interrelationship between BP, CO and TPR, were quantified. System-specific parameters could be distinguished from drug-specific parameters indicating that the developed model is drug-independent.
A system-specific model characterizing the interrelationship between BP, CO and TPR has been obtained, which can be used to quantify and predict cardiovascular drug effects and to elucidate the MoA for novel compounds. Ultimately, the proposed PKPD model may allow prediction of BP effects in humans based on preclinical data.
3'5'-cyclic nucleotide phosphodiesterase (PDE) 4 is expressed in several inflammatory and immune cells, and PDE4 catalyses the hydrolysis of cAMP to 5'AMP down-regulating cAMP signalling in cells. Mitogen-activated protein kinase phosphatase-1 (MKP-1) is an endogenous p38 mitogen-activated protein kinase signalling suppressor and limits inflammatory gene expression and inflammation. In the present study, we investigated the effect of a PDE4 inhibitor rolipram on MKP-1 expression and whether MKP-1 is involved in the anti-inflammatory effects of rolipram.
The effect of rolipram on TNF production was investigated in J774 mouse macrophage cell line and in primary mouse peritoneal macrophages (PM) from wild-type (WT) and MKP-1(–/–) mice. We also investigated the effect of rolipram on carrageenan-induced paw inflammation in WT and MKP-1(–/–) mice.
MKP-1 expression was enhanced by rolipram, by a non-selective PDE inhibitor IBMX and by a cAMP analog 8-Br-cAMP in J774 cells and in PM. Enhanced MKP-1 mRNA expression by rolipram was reversed by a PKA inhibitor. Rolipram, IBMX and 8-Br-cAMP also inhibited TNF production. Rolipram inhibited TNF production in PMs from WT mice but, interestingly, not in PMs from MKP-1(–/–) mice. Furthermore, rolipram attenuated carrageenan-induced paw inflammation in WT but not in MKP-1(–/–) mice.
PDE4 inhibitor rolipram enhanced the expression of MKP-1, and MKP-1 mediated, at least partly, the anti-inflammatory effects of PDE4 inhibition. The results suggest that compounds that enhance MKP-1 expression and/or MKP-1 activity hold potential as novel anti-inflammatory drugs.
8-Bromoadenosine 3′,5′-cyclic monophosphate (8-Br-cAMP); cAMP responsive element (CRE); cAMP responsive element binding protein (CREB); extracellularly-regulated protein kinase (ERK); foetal bovine serum (FBS); glyceraldehyde-3-phosphate dehydrogenase (GAPDH); dimethyl sulfoxide (DMSO); interleukin (IL); Jun N-terminal kinase (JNK); lipopolysaccharide (LPS); mitogen-activated protein kinase (MAPK); mitogen-activated protein kinase-activated protein kinase 2 (MK2); MAP kinase phosphatase-1 (MKP-1); 3'5'-cyclic nucleotide phosphodiesterase (PDE); protein kinase A (PKA); protein kinase A inhibitor 6-22 amide (PKAi); peritoneal macrophages (PM); tumour necrosis factor (TNF)
Chronic elevation in intracellular Ca2+ concentration participates in death of skeletal muscle from mdx mice, a model for Duchenne muscular dystrophy (DMD). Candidate pathways mediating this Ca2+ overload involve store-operated channels (SOC) and stretch-activated channels (SAC), which are modulated by the Ca2+-independent form of phospholipase A2 (iPLA2). We investigated the effect of doxorubicin (Dox), a chemotherapeutic agent reported to inhibit iPLA2 in other systems, on the activity of this enzyme and on the consequences on Ca2+ handling and muscle function in mdx mice.
Effects of Dox on PLA2 activity, ROS production, and on Ca2+ influx were investigated in C2C12 and mdx myotubes. The mechanism of Dox-mediated iPLA2 inhibition was evaluated using purified 6x His-tagged enzyme. Aequorin technology was used to assess Ca2+ concentrations underneath the plasma membrane. Isolated muscles were exposed to fatigue protocols and eccentric contractions to evaluate the effects of Dox on muscle function.
Dox at 1-30 μM inhibited iPLA2 activity in cells and of the purified enzyme. Dox also inhibited SAC- but not SOC-mediated Ca2+ influx in myotubes. Stimulated elevations of Ca2+ concentrations below the plasmalemma were also blocked. Exposure of excised muscle to Dox was not deleterious to force production and promoted recovery from eccentric contractions.
Dox showed efficacy against targets known to play a role in the pathology of DMD, namely iPLA2 and SAC. The potent SAC inhibitory effect of Dox is a novel finding that can explain partly the cardiomyopathy seen in chronic anthracycline treatment.
Corticosteroid insensitivity is a major therapeutic problem for some inflammatory diseases including chronic obstructive pulmonary disease (COPD), and it is known to be induced by reduced histone deacetylase (HDAC)-2 activities via activation of the phosphoinositide 3-kinase (PI3K) pathway. The aim of this study is to evaluate effects of a novel macrolide/fluoroketolide, solithromycin (SOL, CEM-101) on corticosteroid sensitivity induced by oxidative stress.
Corticosteroid sensitivity was determined by IC50/EC50 of dexamethasone (Dex) on TNFα-induced CXCL8 production in U937 monocytic cell line and peripheral blood mononuclear cells (PBMC) from COPD patients. Activities of HDAC and protein phosphatase 2A (PP2A) were measured by fluorescence-based assay in cells exposed to hydrogen peroxide (H2O2). We also investigated steroid insensitive airway neutrophilia in cigarette smoke exposed mice in vivo.
SOL (10 μM) restored Dex sensitivity in PBMC from COPD patients, H2O2-treated U937 cells and PMA-differentiated U937 cells. In addition, SOL restored HDAC activity with concomitant inhibition of Akt phosphorylation as surrogate marker of PI3K activation. The inhibition of Akt phosphorylation by SOL was due to increased PP2A phosphatase activity, which was reduced in COPD and oxidative stress model. Other known macrolides, such as eryhthromycin, clarithromycin and azithromycin were significantly less effective in these responses. In cigarette smoke-exposed mice, SOL (100 mg kg-1, po) showed significant but weak inhibition of neutrophilia, whereas Dex (10mg kg-1, po) showed no such effect. However, a combination of SOL and Dex inhibited neutrophilia by over 50%.
SOL has potential as novel therapy for corticosteroid-insensitive diseases such as COPD.
Mitochondria are a drug target in mitochondrial dysfunction diseases and in antiparasitic chemotherapy. While zebrafish is increasingly used as a biomedical model, its potential for mitochondrial research remains relatively unexplored. Here we perform the first systematic analysis of how mitochondrial respiratory chain inhibitors affect zebrafish development and cardiovascular function, and assess multiple quinones, inc. ubiquinone-mimetics idebenone and decylubiquinone, and the antimalarial atovaquone.
Zebrafish (Danio rerio) embryos were chronically and acutely exposed to mitochondrial inhibitors and quinone analogues. Concentration-response curves, developmental and cardiovascular phenotyping were performed together with sequence analysis of inhibitor-binding mitochondrial subunits in zebrafish vs. mouse, human and parasites. Phenotype rescuing was assessed in co-exposure assays.
Complex I and II inhibitors induced developmental abnormalities, but their sub-maximal toxicity was not additive, suggesting active alternative pathways for complex III feeding. Complex III inhibitors evoked a direct normal-to-dead transition. ATP synthase inhibition arrested gastrulation. Menadione induced hypochromic anemia when transiently present following primitive erythropoiesis. Atovaquone was over 1,000-fold less lethal in zebrafish than reported for Plasmodium falciparum, and its toxicity partly rescued by the ubiquinone precursor 4-hydroxibenzoate. Idebenone and decylubiquinone delayed rotenone- but not myxothiazol- or antimycin-evoked cardiac dysfunction.
This study characterizes pharmacologically-induced mitochondrial dysfunction phenotypes in zebrafish, laying the foundation for comparison with future studies addressing mitochondrial dysfunction in this model organism. It has relevant implications for interpreting zebrafish disease models linked to complex I/II inhibition. Further, it evidences zebrafish's potential for in vivo efficacy or toxicity screening of ubiquinone analogues or antiparasitic mitochondria-targeted drugs.
Inhibition of apoptosis may attenuate irreversible injury associated with reperfusion. In the current study, we focused on the cardioprotective effects and the underlying mechanism of sodium tanshinone IIA silate (STS) against oxygen-glucose deprivation/reperfusion (OGD/R) damage in H9c2 cardiomyocytes and the underlying mechanisms.
We investigated the cardioprotective effects of STS in a cellular oxygen glucose deprivation/recovery (OGD/R) model of ischemia/reperfusion. Apoptosis of cells was observed using Hoechst 33342-based fluorescence microscopy, and Annexin V-FITC based flow cytometry. Caspase-3 and caspase-8 activities and mitochondrial membrane potential (MMP) were assessed using commercial kits. TNF-α in the supernatant fractions were measured with sandwich ELISA, and protein levels assayed using Western blot.
STS inhibited OGD/R-induced apoptosis significantly by suppressing JNK mediated NF-κB activation, TNF-α expression, caspase-3 and caspase-8 activation; and the Bax/Bcl-2 ratio. Additionally, positive feedback between NF-κB and TNF-α and amplification of TNF-α were inhibited, suggesting that STS plays a protective role against apoptosis in cardiomyocytes, even upon activation of pro-inflammatory cytokines. Interestingly, the cardioprotective effects of STS on OGD/R-induced apoptosis and promotion of cell survival were attenuated upon inhibition of PI3K.
The inhibitory effects of STS on TNF-α and positive feedback signaling of the NF-κB/TNF-α pathways may play important roles in myocardial protection against ischemia/reperfusion. These protective effects of STS are mediated by suppressing JNK activity through activation of the PI3K-Akt pathway.
Schizophrenia is a highly debilitating disorder characterised by hallucinations and delusions, but also impaired cognition such as memory. While hallucinations and delusions are the main target for pharmacological treatment, cognitive impairments are hardly treated. Evidence exists for a role of histamine in cognitive deficits in schizophrenia providing the possibility for a histamine based treatment to be developed. Histamine H3-antagonists have shown to improve memory performance in experimental animals, but these effects have hardly been investigated in humans within the context of impaired cognition in schizophrenia and using sensitive measures of brain activity. In the present study the effects of betahistine (H3-antagonist/H1-agonist) on learning and memory, and associated brain activity were assessed.
Sixteen healthy volunteers (8 female) aged between 18 and 50 years received two oral doses of betahistine 48 mg separated by 30 minutes, and placebo on separate days according to a two way, double blind, cross-over design. Volunteers performed an N-back working memory task and a spatial paired associates learning task while being scanned using a magnetic resonance imaging scanner.
Task related activity changes in well-defined networks and performance were observed. No betahistine induced changes in brain activity were found in these networks. Alternatively, liberal whole brain analyses showed activity changes in areas outside task networks, like the lateral geniculate nucleus.
Clear effects of betahistine on working memory could not be established. Future studies may use higher doses and explore the role of histamine in visual information processing.
Chronic Myeloid Leukaemia (CML) is a clonal myeloproliferative disorder characterised by the presence of a fusion oncogene BCR-ABL, which encodes a protein with constitutive tyrosine kinase activity. The implementation of tyrosine kinase inhibitors (TKI) marked a major advance in CML therapy, however there are problems with current treatment. For example, relapse occurs when these drugs are discontinued in the majority of patients who have achieved a complete molecular response (CMR) on TKI and these agents are less effective in patients with mutations in the BCR-ABL kinase domain. Importantly, TKI can effectively target proliferating mature cells, but do not eradicate quiescent leukaemic stem cells (LSC), therefore allowing disease persistence despite treatment. It is essential that alternative strategies are used to target the LSC population. BCR-ABL activation is responsible for the modulation of different signalling pathways, which allows the LSC fraction to evade cell death. Several pathways have been shown to be modulated by BCR-ABL, including PI3K/AKT/mTOR, JAK-STAT and autophagy signalling pathways. Targeting components of these survival pathways, alone or in combination with TKI, therefore represents an attractive potential therapeutic approach for targeting the LSC. However, many pathways are also active in normal stem cells. Therefore potential targets must be validated to effectively eradicate CML stem cells while sparing normal counterparts. This review summarises the main pathways modulated in CML stem cells, recent developments and the use of novel drugs to target components in these pathways which may be used to target the LSC population.
Arylamine N-acetyltransferase (NAT) plays an important role in metabolism and detoxification of many amine-containing compounds including drugs and environmental carcinogens through chemical modification of the substrates with an acetyl group. Recent studies have suggested that NATs are also involved in cancer cell growth and inhibition of the enzymes may be a potential target for cancer chemotherapy. Three-dimensional (3D) structures are available for NATs from both prokaryotes and eukaryotes. These structures provide valuable insights into the acetylation mechanism and active site features. Moreover, the structural determinants that govern substrate/inhibitor-binding specificity are unraveled. This allows a more intuitive understanding of the structure-activity relationships for NAT substrates and inhibitors. Further the structural elucidation on NATs has led to powerful tools in designing small molecule inhibitors that are anticipated to alleviate cancer due to the important role of the enzyme in cancer biology.
N-arachidonoyl glycine is a member of lipoamino acids with vasorelaxant properties. We aimed to explore the mechanisms of the NAGly action on unstimulated and agonist-stimulated endothelial cells.
The effects of NAGly on endothelial electrical signaling were studied in combination with vascular reactivity.
In EA.hy926 cells, the sustained hyperpolarization to histamine was sensitive to bepridil and KB-R7943, a non-selective Na+/Ca2+ exchanger (NCX) inhibitor and an inhibitor of reversed mode NCX, respectively. In cells dialyzed with Cs+-based Na+-containing solution the outwardly rectifying current with typical characteristics of NCX was augmented following histamine exposure, further increased upon external Na+ withdrawal and inhibited by bepridil. In a URB597- and GDPβS-insensitive manner, NAGly (0.3-30 μM) mode-independently suppressed NCX currents, [Ca2+]i elevation evoked by Na+ removal and the hyperpolarization to histamine. In rat aorta, NAGly opposed endothelial hyperpolarization and relaxation to acetylcholine. In unstimulated EA.hy926 cells, NAGly potentiated whole-cell current attributable to large conductance Ca2+-dependent K+ (BKCa) channels in a GDPβS-insensitive paxilline-sensitive manner and produced a sustained hyperpolarization. In cell-free inside-out patches NAGly stimulated single BKCa channel activity.
Our data identify NCX as a Ca2+ entry pathway in endothelial cells and NAGly as a potent G-protein-independent modulator of endothelial electrical signaling. Our data reveal dual effect of NAGly on endothelial electrical responses. In agonist-prestimulated cells, NAGly opposes hyperpolarization and relaxation via inhibition of NCX-mediated Ca2+ entry, while in unstimulated cells the compound promotes hyperpolarization via receptor-independent activation of BKCa channels.
High-Altitude Pulmonary Edema (HAPE) experienced under high altitude conditions is attributed to mitochondrial redox distress. Hence, hypobaric hypoxia (HH)-induced alteration in expression of mitochondrial biogenesis and dynamics genes was determined in rat lung. Further such alteration was correlated with expression of mitochondrial DNA (mtDNA)-encoded oxidative phosphorylation (mtOXPHOS) genes. The prophylactic effect of dexamethasone in counteracting the HH-induced mitochondrial distress was used as control to understand adaptation to high-altitude exposure.
Rats pre-treated with dexamethasone were exposed to normobaric normoxia (NN) or HH. HH-induced injury was assessed as an increase in lung: water content, tissue damage and oxidant generation. Mitochondrial number, mtDNA content and mtOXPHOS activities were measured to determine mitochondrial function. The expression of mitochondrial biogenesis, dynamics and mtOXPHOS genes was studied.
HH-induced lung injury was associated with decreased: mitochondrial number, mtDNA content and mtOXPHOS activities. HH exposure decreased the nuclear gene ERRα which interacts with PGC-1α in controlling mitochondrial metabolism. Consequently, mtOXPHOS transcripts are repressed under HH. Further, HH modulated mitochondrial dynamics by decreasing Mfn2 and augmenting Fis1 and Drp1 expression. Nevertheless, dexamethasone treatment under NN (i.e. adaptation to HH) did not affect mitochondrial biogenesis and dynamics but increased mtOXPHOS transcripts. Further, mtOXPHOS activities increased together with reduced oxidant generation. Also, dexamethasone pre-treatment normalized ERRα along with mitochondrial dynamics genes and increased mtOXPHOS transcripts to elicit the mitochondrial function under HH.
HH stress (HAPE)-mediated mitochondrial dysfunction is due to repressed ERRα and mtOXPHOS transcripts. Thus, ERRα-mediated protection of mitochondrial-bioenergetics might be the likely candidate required for lung adaptation to HH.
The purpose of this study was to assess cerebral perfusion changes following systemic administration of the recreational drug 3,4 methylendioxymethamphetamine (MDMA “Ecstasy”) to rats.
Cerebral perfusion was quantified using bolus-tracking arterial spin labelling (btASL) magnetic resonance imaging (MRI). Rats received MDMA (20 mg/kg; i.p.) and were assessed 1, 3 or 24 hr later. Rats received MDMA (5 or 20 mg/kg; i.p.) and were assessed 3 hr later. In addition, rats received MDMA (5 or 10 mg/kg; i.p.) or saline four times daily over 2 consecutive days and were assessed 8 weeks later. Perfusion-weighted images were generated in a 7T MRI scanner and experimental data was fitted to a quantitative model of cerebral perfusion to generate mean transit time (MTT), capillary transit time (CTT) and signal amplitude.
MDMA reduces MTT and CTT and increases amplitude in somatosensory and motor cortex 1 and 3 hr following administration, indicative of an increase in perfusion. Prior exposure to MDMA provoked a long term reduction in cortical 5-HT concentration but did not produce a sustained effect on cerebral cortical perfusion. The response to acute MDMA challenge (20mg/kg; i.p.) was attenuated in these animals indicating adaptation in response to prior MDMA exposure.
MDMA provokes changes in cortical perfusion which are quantifiable by btASL MRI, a neuroimaging tool with translational potential. Future studies are directed towards elucidation of the mechanisms involved and correlating changes in cerebrovascular function with potential behavioural deficits associated with drug use.
Emerging evidence indicates that the balance between pro-inflammatory cytokines (PICs) and anti-inflammatory cytokines (AICs) within the brain is an important determinant in the outcome of hypertension. However, the mechanism by which this dysregulation occurs is not known. We aimed to investigate whether AngII induces imbalance between PIC and AIC by modulating downstream transcription factors, nuclear factor kappaB (NFκB) and cyclic AMP response-element binding protein (CREB), and whether AngII-induced effects are mediated by Glycogen synthase kinase-3β (GSK-3β).
CATH.a neurons were exposed to AngII (10 nM-1μM) over a preset time course. In another set of experiments, GSK-3β was knock-down by using lentivirus containing short hairpin RNA targeting GSK-3β (L-sh-GSK3β) before AngII exposure. Cell extracts were subjected to RT-PCR, immunoblot, and immunoprecipitation
AngII caused time-dependent increase in PICs (TNF-α and IL-1β) and reduction in AIC (IL-10). AngII exposure caused reduced phosphorylated CREB(Ser-133) and increased p-NFκB(Ser-276) levels leading to reduced CREB-CBP and increased NFκB-CBP binding. These results were accompanied by increased activation of GSK-3β as indicated by increased p-GSK3(Tyr-216) to p-GSK3(Ser-9) ratio. In a subsequent study, pretreatment with L-sh-GSK3β attenuated AngII-induced alterations in PICs and IL-10 by augmenting CREB-CBP and attenuating NFκB-CBP binding.
Collectively, these findings are the first to provide direct evidence that AngII-induced dysregulation in cytokines is mediated by GSK-3β-mediated alterations in downstream transcription factors in neuronal cells. Our data also reveals that AngII-induced effects could be alleviated by GSK-3β inhibition suggesting GSK-3β as an important therapeutic target for hypertension that is characterized by increased PICs and NFκB activation.
Long-term high-fat diet (HFD) consumption has been shown to cause insulin resistance, which is characterized by hyperinsulinemia with metabolic inflexibility. Insulin resistance is associated with cardiac sympathovagal imbalance, cardiac dysfunction, and cardiac mitochondrial dysfunction. Dipeptidyl peptidase-4 (DPP-4) inhibitors, vildagliptin and sitagliptin, are oral anti-diabetic drugs often prescribed in patients with cardiovascular disease. Therefore, in this study, we sought to determine the effects of vildagliptin and sitagliptin in a murine model of insulin resistance.
Male Wistar rats weighing 180-200 g, were fed either a normal diet (20 % energy from fat) or a HFD (59% energy from fat) for 12 weeks. These rats were then divided into 3 subgroups to receive vildagliptin (3 mg/kg/day), sitagliptin (30 mg/kg/day), or vehicle for another 21 days. Metabolic parameters, oxidative stress, heart rate variability (HRV), cardiac function, and cardiac mitochondrial function were determined.
Rats that received HFD developed insulin resistance characterized by increased body weight, plasma insulin, total cholesterol, and oxidative stress levels along with a decreased high-density lipoprotein (HDL) level. Moreover, cardiac dysfunction, depressed HRV, cardiac mitochondrial dysfunction, and cardiac mitochondrial morphology changes were observed in HFD rats. Both vildagliptin and sitagliptin decreased plasma insulin, total cholesterol, and oxidative stress as well as increased HDL level. Furthermore, vildagliptin and sitagliptin attenuated cardiac dysfunction, prevented cardiac mitochondrial dysfunction, and completely restored HRV.
Both vildagliptin and sitagliptin share similar efficacy in cardioprotection in obese insulin resistant rats.
We investigated the effects of aging on the contributions of nitric oxide (NO) and endothelium-dependent hyperpolarization (EDH) to endothelium-dependent relaxation in saphenous arteries of male and female C57BL/6J mice aged 12, 34, and 64 weeks.
Vasomotor responses of saphenous arteries were analyzed by wire-myography in the absence and presence of stimuli of the endothelium, inhibitors of NO-synthase, and inhibitors and stimulants of small (KCa2.3) and intermediate (KCa3.1) conductance calcium-activated potassium channels.
Arterial relaxing responses to sodium-nitroprusside and to acetylcholine (ACh) in the absence of pharmacological inhibitors (indomethacin and L-NAME), were similar in all age groups and sexes but those mediated by endothelium-derived NO were slightly but significantly increased in 64-week-old male mice. In the presence of inhibitors, 12-week-old animals showed pronounced ACh-induced relaxation, which was significantly reduced in 34- and 64-week-old mice of both sexes. The EDH-related component of ACh-induced relaxations was abolished by TRAM-34 (KCa3.1 blocker) or UCL 1684 (KCa2.3 blocker). Although the maximal relaxation induced by NS309 (KCa activator) was not affected by aging, the sensitivity for NS309 significantly decreased with aging. The presence of SKA-31 (KCa modulator) potentiated relaxations induced by ACh in arteries of 12-week-old but not older mice.
In a small muscular artery of mice of either sex, total endothelium-dependent relaxation is not affected by age. However, possibly due to changes in KCa channel function, the contribution of EDH to endothelium-dependent relaxations decreased with age. The contribution of endothelium-derived NO increases in old male mice.
Brain vascular endothelial cells express histamine H1 and H2 receptors, which regulate brain capillary permeability. We tested the hypothesis that also H3 and H4 receptor are expressed in these cells and may thus play roles in permeability regulation.
An immortalized rat brain endothelial cell line RBE4 was used to assess the presence of H3R and H4R. RT-PCR and sequencing were used to identify the receptor mRNAs. Receptor stimulation was done with histamine and immepip, and specific inverse agonists/antagonists ciproxifan and JNJ 7777120 were used to block H3R and H4R, respectively.
RT-PCR on mRNA extracted from cultured immortalized RBE4 cells revealed two Hrh4 transcripts, one full length H4 receptor (CDS 1173bp), and one with a 164bp deletion. Also two Hrh3 receptor isoform mRNAs were expressed in RBE4 cells, and sequencing showed them to be the full-length H3R and the 144 bp deletion form. Both histamine and immepip (H3R and H4R agonist) activated the Erk1/2 MAPK pathway in cultured brain microvessel endothelial cells and in vivo in brain blood vessels through histamine H4 receptor activation. In RBE4 cells, the H4R specific inverse agonists / antagonist JNJ 7777120 but not ciproxifan, the H3 receptor antagonist, dose dependently blocked the effect.
The results show that both Hrh3 and Hrh4 receptor are expressed in rat brain endothelial cells, and activation of H4R activates the Erk1/2 cascade. H3R and H4R in endothelial cells are potentially important for regulation of BBB permeability, including trafficking of immunecompetent cells.
Small molecule glucokinase activators (GKAs) have been associated with potent antidiabetic efficacy and hepatic steatosis in rodents. This study reports the discovery of S 50131 and S 51434, two novel GKAs with an original scaffold and an atypical pharmacological profile.
In vitro compound activity was assessed by measuring the ability to activate recombinant glucokinase, stimulate glycogen synthesis in rat hepatocytes, and increase insulin secretion from rat pancreatic islets of Langerhans. In vivo efficacy and safety were evaluated after oral administration in db/db mice by measuring glycaemia, HbA1c, and dyslipidemia-associated events.
S 50131 and S 51434 activated the GK enzyme and were able to stimulate glycogen synthesis in hepatocytes and insulin secretion from pancreatic islets. Unexpectedly, whilst both compounds effectively lowered glycaemia after acute oral administration, they did not decrease HbA1c after a 4-week treatment in db/db mice. This lack of antidiabetic efficacy was associated with increased plasma free fatty acids (FFAs), contrasting with the effect of GKA50 and N00236460, two GKAs with sustained HbA1c lowering activity but neutral regarding plasma FFAs. S 50131, but not S 51434, also induced hepatic steatosis, as did GKA50 and N00236460. However, a shorter, 4-day treatment resulted in increased hepatic triglycerides without changing the plasma FFA levels, demonstrating dynamic alterations in the lipid profile over time.
In addition to corroborating the occurrence of dyslipidemia with GKAs, these findings provide new insights into understanding how such compounds may sustain or lose efficacy over time.
Hydrogen sulfide (H2S) is a signaling molecule that belongs to the gasotransmitter family. Two major sources for endogenous enzymatic production of H2S are cystathionine beta synthase (CBS) and cystathionine gamma lyase (CSE). In the present study we examined the selectivity of commonly used pharmacological inhibitors of H2S biosynthesis towards CSE and CBS.
To address this question human CSE or CBS enzymes were expressed and purified from E. coli as fusion proteins with glutathione-S-transferase. After purification the activity of the recombinant enzymes was tested using the methylene blue method.
β-cyanoalanine (BCA), a CSE inhibitor, was more potent in inhibiting CSE than propargylglycine (PAG) (IC50 14+0.2 μM vs 40+8 μM, respectively). Similarly to PAG, L-aminoethoxyvinylglycine only inhibited CSE, but did so at much lower concentrations. On the other hand, aminooxyacetic acid (AOAA), a frequently used CBS inhibitor, was more potent in inhibiting CSE compared to BCA and PAG (IC50 1.1+0.1μM); the IC50 for AOAA for inhibiting CBS was 8.5+0.7 μM. In line with our biochemical observations, relaxation to L-cysteine was blocked by AOAA in aortic rings that lacked CBS expression. Trifluoroalanine and hydroxylamine, two compounds that have also been used to block H2S biosynthesis, blocked the activity of CBS and CSE. Trifluoroalanine had a four-fold lower IC50 for CBS vs CSE, while hydroxylamine was 60-fold more selective against CSE.
In conclusion, although PAG, AVG and BCA exhibit selectivity in inhibiting CSE vs. CBS, no selective pharmacological CBS inhibitor is currently available.
Histamine and especially H3R regulate drug dependence related behaviors. Conflicting data has been published on whether histamine is inhibitory to the rewarding effects of abused drugs. The purpose of this study was to clarify the role of histamine in the addictive effects of alcohol.
Alcohol-CPP was used to measure alcohol reward. Alcohol-induced locomotor stimulation, alcohol consumption and kinetics were also assessed. mRNA levels were quantified using radioactive in situ hybridization.
Low doses of H3R antagonists (JNJ-10181457 and JNJ-39220675) inhibited alcohol reward in WT mice. However, H3R antagonists did not inhibit alcohol reward in HDC KO mice and lack of histamine did not alter alcohol consumption. H3R antagonists thus inhibit alcohol reward in a histamine dependent manner. Furthermore, WT and HDC KO mice were similarly stimulated by alcohol. Dopamine D1 and D2 receptors, STEP61 and DARPP-32 mRNA expression levels in striatal subregions were unaltered in HDC KO mice. No differences were seen in alcohol kinetics in HDC KO versus WT control animals. In addition, JNJ-39220675 had no effect on alcohol kinetics in WT mice.
These data suggest that histamine is required in the H3R-mediated inhibition of alcohol-CPP and support the hypothesis that the brain histaminergic system has an inhibitory role in alcohol reward. Increasing neuronal histamine release via H3R blockade could therefore be a novel means to treat alcohol dependence.
Down's syndrome (DS) is a common genetic cause of intellectual disability yet there are no drug therapies. Mechanistic studies in a model of DS (Ts65Dn mice) demonstrated that impaired cognitive function is due to excessive neuronal inhibitory tone. These deficits can be normalized by low doses of GABAA receptor (GABAAR) antagonists in adult animals. In this study, we explore the therapeutic potential of pentylenetetrazole (PTZ), a GABAAR antagonist which has a history of safe use in man.
Long-term memory was assessed by the Novel Object Recognition (NOR) test in different cohorts of Ts65Dn mice after a delay following a short-term chronic treatment with PTZ. Seizure susceptibility, as an index of treatment safety, was studied by means of EEG, behavior and hippocampus morphology. EEG spectral analysis was used as a biometric of the treatment.
PTZ has a broad therapeutic window (0.03-3mg.kg-1) that is >10-1000 fold below the seizure threshold for this drug, and chronic PTZ treatment does not lower the seizure threshold. Remarkably, short-term, low, chronic dose regimens of PTZ elicit long-lasting (>1week) normalization of cognitive function in young and aged mice. PTZ effectiveness is time of day dependent: cognitive performance improves when PTZ is delivered during the light (inactive) phase, but not during the dark (active) phase. Chronic PTZ treatment results in EEG power normalization.
PTZ at very low dosage can be administered safely, produces long lasting cognitive improvements and has the potential of fulfilling an unmet therapeutic need in DS.
Thermostabilisation by mutagenesis is one method which has facilitated the determination of high resolution structures of the adenosine A2A receptor (A2AR). Sets of mutations were identified which both thermostabilised the receptor and resulted in preferential agonist (Rag23 mutant) or antagonist (Rant5 and Rant21) binding forms as assessed by radioligand binding analysis. While the ligand binding profiles of these mutants are known the effects these mutations have on receptor activation and downstream signaling are less well characterised.
Here we have investigated the effects of the thermostabilising mutations on receptor activation using a yeast cell growth assay. The assay employs an engineered Saccharomyces cerevisiae, MMY24, which couples receptor activation to cell growth.
Analysis of the receptor activation profile revealed that the WT A2AR had considerable constitutive activity. In contrast the Rag23, Rant5 and Rant21 thermostabilised mutants all exhibited no constitutive activity. While the preferentially antagonist binding mutants Rant5 and Rant21 showed a complete lack of agonist-induced activity, the Rag23 mutant showed high levels of agonist-induced receptor activity. Further analysis using a mutant intermediate between Rag23 and WT indicated that the loss of constitutive activity observed in the agonist responsive mutants was not due to reduced G-protein coupling.
The loss of constitutive activity may be an important feature of these thermostabilised GPCRs. In addition, the constitutively active and agonist-induced active conformations of the A2AR are distinct.
Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a polyglutamine expansion in the huntingtin protein. We have previously demonstrated that the cell signaling of the metabotropic glutamate receptor 5 (mGluR5) is altered in a mouse model of HD. Although mGluR5-dependent protective pathways are more activated in HD neurons, intracellular Ca2+ release is also more pronounced, which could contribute to excitotoxicity. In the present study we aim to investigate whether mGluR5 positive allosteric modulators (PAMs) could activate protective pathways without triggering high levels of Ca2+ release and be neuroprotective in HD.
We performed a neuronal cell death assay to determine which drugs are neuroprotective, western blot and Ca2+ release experiments to investigate the molecular mechanisms involved in this neuroprotection, and object recognition task to determine whether the tested drugs could ameliorate HD memory deficit.
We find that mGluR5 PAMs can protect striatal neurons from the excitotoxic neuronal cell death promoted by elevated concentrations of glutamate and NMDA. mGluR5 PAMs are capable of activating AKT without triggering increased intracellular Ca2+ concentration and AKT blockage leads to loss of PAMs-mediated neuroprotection. Importantly, PAMs’ potential as drugs to treat neurodegenerative diseases is highlighted by the neuroprotection exerted by mGluR5 PAMs on striatal neurons from a mouse model of HD, BACHD. Moreover, mGluR5 PAMs can activate neuroprotective pathways more robustly in BACHD mice and ameliorate HD memory deficit.
mGluR5 PAMs are potential drugs to treat neurodegenerative diseases, especially HD.
It is well recognized that vasopressin modulates the neurogenic control of the circulation. Here we report the central mechanisms by which vasopressin modulates cardiovascular response to stress induced by immobilization.
Experiments were performed in conscious male Wistar rats equipped with radiotelemetric device for continuous measurement of hemodynamic parameters: systolic and diastolic blood pressure (BP) and heart rate (HR). The functioning of the spontaneous BRR was evaluated using the sequence method and the following parameters were evaluated: BRR sensitivity (BRS) and BRR effectiveness index (BEI).
Under baseline physiological conditions intracerebroventricular injection of 100ng and 500ng of selective non-peptide V1a- or V1b- or V2-receptor antagonist did not modify BP, HR and BRR. Rats exposed to 15-minutes-long stress by immobilization exhibited increase of BP, HR, reduction of BRS and no change in BEI. Pre-treatment of rats with V1a-receptor antagonist did not modulate the BP, HR, BRS and BEI response to stress. Pre-treatment of rats with V1b-receptor and V2-receptor antagonist, at both doses, prevented BRR desensitization and tachycardia but failed to modulate stress induced hypertension.
Vasopressin by the stimulation of central V1b- and V2-like receptors mediates stress-induced tachycardia and BRR desensitization. If these mechanisms are involved BRR desensitization in heart failure and hypertension associated with poor outcome, they could be considered as novel targets for cardiovascular drug development.
In small arteries, SKCa and IKCa channels restricted to the vascular endothelium generate hyperpolarization that underpins the NO- and PGI2-independent, EDHF response that is the predominate endothelial mechanism for vasodilatation. As neuronal IKCa channels can be negatively regulated by PKA, we investigated whether β-adrenoceptor stimulation, which signals through cAMP/PKA, might influence endothelial cell hyperpolarization and as a result modify the associated vasodilatation.
Rat isolated small mesenteric arteries were pressurized to measure vasodilatation and endothelial cell [Ca2+]i, mounted in a wire myograph to measure smooth muscle membrane potential or dispersed into endothelial cell sheets for membrane potential recording.
Intraluminal perfusion of β-adrenoceptor agonists inhibited endothelium-dependent dilatation to ACh (1 nM – 10 μM) without modifying the associated changes in endothelial cell [Ca2+]i. The inhibitory effect of β-adrenoceptor agonists was mimicked by direct activation of adenylyl cyclase with forskolin, blocked by the β-adrenoceptor antagonists propranolol (non-selective), atenolol (β1) or the PKA inhibitor KT-5720; but remained unaffected by ICI 118,551 (β2) or glibenclamide (KATP channel blocker). Endothelium-dependent hyperpolarization to ACh was also inhibited by β-adrenoceptor stimulation in both intact arteries and in endothelial cells sheets. Blocking IKCa (with 1 μM TRAM-34) but not SKCa (50 nM apamin) channels prevented β-adrenoceptor agonists from suppressing either hyperpolarization or vasodilatation to ACh.
In resistance arteries, endothelial cell β1-adrenoceptors link to inhibit endothelium-dependent hyperpolarization and the resulting vasodilatation to ACh. This effect appears to reflect inhibition of endothelial IKCa-channels and may be one consequence of raised circulating catecholamines.
The 5-HT3B subunit was first cloned in 1999, and co-expression with the 5-HT3A subunit results in heteromeric 5-HT3AB receptors that are functionally distinct from homomeric 5-HT3A receptors. The affinities of competitive ligands at the two receptor subtypes are usually similar, but those of non-competitive antagonists that bind in the pore often differ. A competitive ligand and allosteric modulator that distinguishes 5-HT3A from 5-HT3AB receptors has recently been described, and the number of non-competitive antagonists identified with this ability has increased in recent years. In this review we discuss the differences between 5-HT3A and 5-HT3AB receptors and describe the possible sites of action of compounds that can distinguish between them.
Perivascular adipose tissue (PVAT) releases adipose tissue-derived hyperpolarizing factors (ADHFs) which may partly act by opening myocyte K+ channels. The present study in rat and mouse mesenteric arteries aimed to identify the myocyte K+ channel activated by PVAT and to determine whether adiponectin contributed to the hyperpolarizing effects of PVAT.
Myocyte membrane potential was recorded from de-endothelialised, non-contracted rat and mouse mesenteric arteries in the presence and absence of PVAT.
The β3 adrenoceptor agonist, CL-316,243 (10μM), generated PVAT-dependent, iberiotoxin-sensitive myocyte hyperpolarizations resulting from BKCa channel opening and which were partially blocked by L-NMMA (100μM). Adiponectin (5μg ml-1) also produced iberiotoxin-sensitive hyperpolarizations in PVAT-denuded arterioles. Activation of myocyte AMP-activated protein kinase (AMPK) using 5μM A-769662 also induced BKCa-mediated hyperpolarizations. Dorsomorphin abolished hyperpolarizations to CL-316,243, adiponectin and A769662. In vessels from Adipo-/- mice, hyperpolarizations to CL-316,243 were absent whereas those to A-769662 and adiponectin were normal. In rat vessels, adipocyte-dependent hyperpolarizations were blocked by glibenclamide and clotrimazole but those to NS1619 (33 μM), were unaltered.
Under basal, non-contracted conditions, β3 -adrenoceptor stimulation of PVAT releases an ADHF which is probably adiponectin. This activates AMPK to open myocyte BKCa channels indirectly and additionally liberates nitric oxide which also contributes to the observed PVAT-dependent myocyte hyperpolarizations. Clotrimazole and glibenclamide each reversed hyperpolarizations to adiponectin and A-769662, suggesting the involvement of myocyte TRPM4 channels in the ADHF-induced myocyte electrical changes mediated via the opening of BKCa channels.
Molecular and non-invasive imaging are rapidly emerging fields in preclinical cancer drug discovery. This is driven by the need to develop more efficacious and safer treatments, the advent of molecular targeted therapeutics, and the requirements to reduce and refine current preclinical in vivo models. Such bioimaging strategies include magnetic resonance imaging (MRI), positron emission tomography (PET), single positron emission computed tomography (SPECT), ultrasound, and optical approaches such as bioluminescence and fluorescence imaging. These molecular imaging modalities have several advantages over traditional screening methods, not least the ability to quantitatively monitor pharmacodynamic changes at the cellular and molecular level in living animals non-invasively in real-time. This review aims to provide an overview of non-invasive molecular imaging techniques, highlighting the strengths, limitations and versatility of these approaches in preclinical cancer drug discovery and development.
GPR18 is a recently deorphaned lipid receptor that is activated by the endogenous lipid N-arachidonoyl glycine (NAGly) as well the behaviorally–inactive atypical cannabinoid, abnormal cannabidiol (Abn-CBD). The presence and/or function of any GPR18-based ocular signaling system remains essentially unstudied. The objectives of this research are: 1) to determine the disposition of GPR18 receptors and ligands in anterior murine eye, 2) examine the effect of GPR18 activation on intraocular pressure (IOP) in a murine model, including knockout mice for CB1, CB2 and GPR55.
IOP was measured in mice following topical application of Abn-CBD, NAGly, or the GPR55/GPR18 agonist O-1602, alone or with injection of the GPR18 antagonist, O-1918. GPR18 protein localization was assessed with immunohistochemistry. Endocannabinoids were measured using LC/MS-MS.
GPR18 protein was expressed most prominently in the ciliary epithelium and the corneal epithelium and, interestingly, in the trabecular meshwork. The GPR18 ligand, NAGly, was also detected in mouse eye at a level comparable to that seen in the brain. Abn-CBD and NAGly, but not O-1602, significantly reduced IOP in all mice tested. The antagonist, O-1918, blocked the effects of Abn-CBD and NAGly.
We present evidence for a functional GPR18-based signaling system in the murine anterior eye, including receptors and ligands. GPR18 agonists, Abn-CBD and NAGly, reduce IOP independently of CB1, CB2, or GPR55. These findings suggest that GPR18 may serve as a desirable target for the development of novel ocular hypotensive medications.
Mast cell stabilising drugs work by inhibiting the release of allergic mediators from mast cells and are used clinically to prevent allergic reactions to common allergens. Despite the relative success of the most commonly prescribed mast cell stabiliser, disodium cromoglycate, in use for the preventative treatment of bronchial asthma, allergic conjunctivitis and vernal keratoconjunctivitis there, there still remains an urgent need to design new substances that are less expensive and require less frequent dosing schedules. In this regard, recent developments towards the discovery of the next generation of mast cell stabilising drugs has included studies on substances isolated from natural sources, biological, newly synthesised compounds and drugs licensed for other indications. The diversity of natural products evaluated range from simple phenols, alkaloids, terpenes to simple amino acids. While in some cases their precise mode of action remains unknown it has nevertheless sparked interest in the development of synthetic derivatives with improved pharmacological properties. Within the purely synthetic class of inhibitors, particular attention has been devoted to the inhibition of important signalling molecules including spleen tyrosine kinase and Janus kinase. The statin class of cholesterol-lowering drugs as well as nilotinib, a tyrosine kinase inhibitor, are just some examples of clinically used drugs that have been evaluated for their anti-allergic properties. Here, we examine each approach under investigation, summarise the test data generated and offer suggestions for further pre-clinical evaluation before their therapeutic potential can be realised.
The presence of the histamine H4 receptor (H4R) was previously reported in benign and malignant lesions and cell lines derived from the human mammary gland. The aim of this work was to evaluate the effects of H4R ligands on the survival, tumour growth rate and metastatic capacity of breast cancer in an experimental model.
Xenograft tumours of the highly invasive human breast cancer cell line MDA-MB-231 were established in immune deficient nude mice. The following H4R agonists were employed: histamine (5 mg·kg-1), clozapine (1 mg·kg-1), and the experimental compound JNJ28610244 (10 mg·kg-1).
Data indicate that developed tumours were highly undifferentiated, expressed H4R and exhibited high levels of histamine content and proliferation marker (PCNA) while displaying low apoptosis. Mice of the untreated group displayed a median survival of 60 days, and a tumour doubling time of 7.4±0.6 days. A significant decrease in tumour growth evidenced by an augment of the tumour doubling time was observed in the H4R agonist groups (13.1±1.2, P<0.01 in histamine group; 15.1±1.1, P<0.001 in clozapine group; 10.8±0.7, P<0.01 in JNJ28610244 group). This effect was associated with a decrease in the PCNA expression levels, and also reduced intratumoural vessels in histamine and clozapine treated mice. Histamine significantly increased median survival (78 days; Log-rank Mantel-Cox Test, P=0.0025; Gehan-Breslow-Wilcoxon Test, P=0.0158) and tumoural apoptosis.
Histamine through the H4R exhibits a crucial role in tumour progression. Therefore, H4R ligands offer a novel therapeutic potential as adjuvants for breast cancer treatment.
Because tumor necrosis factors (TNFs) are major mediators of inflammation and inflammation-related diseases, the United States Food and Drug Administration (FDA) has approved blockers of the cytokine, TNF-α, which include chimeric TNF antibody (Infliximab), humanized TNF-α antibody (Humira), and soluble TNF receptor-II (Enbrel). TNF blockers are now being used for the treatment of osteoarthritis, inflammatory bowel disease, psoriasis, and ankylosis at a total cumulative market value of more than $20 billion/year. Besides being expensive ($15,000-20,000/person/year), these drugs must be injected and have enough adverse effects to be given a black label warning by the FDA. In the current report, we describe an alternative, curcumin (diferuloylmethane), a component of turmeric (Curcuma longa) that is very inexpensive, orally bioavailable, and highly safe in humans, yet can block TNF-α action and production in in vitro models, in animal models, and in humans. In addition, we provide evidence for curcumin's activities against all of the diseases for which TNF blockers are being used. Mechanisms by which curcumin inhibits the production and the cell signaling pathways activated by this cytokine are also discussed. With health care costs and safety being major issues today, this golden spice may help provide the solution.
Homocysteine is an intermediate product formed during the metabolism of methionine, and increases in cells with folate deficiency. Patients with hyperhomocysteinemia tend to be susceptible to developing cardiovascular diseases (CVD). In this study, we examined the molecular mechanisms underlying the antiproliferative and antimigratory effects of folate on homocysteine-challenged rat aortic smooth muscle cells (RASMC).
We evaluated the protective mechanisms of folate against homocysteine challenge using the gain-of-function and loss-of-function of p21/p27, AKT, and RhoA in homocysteine-treated RASMC.
Folate upregulated p21/p27 through a Src/ERK-dependent mechanism that accounted for its antiproliferative effects on RASMC. Folate protected RASMC from the effects of homocysteine by reducing AKT1, FAK, paxillin, and p190RhoGAP activation/phosphorylation as well as the cytosolic levels of p21 and p27, and increasing RhoA activation. The gain-of-function of AKT1, but not of AKT2, induced p21/p27 phosphorylation and increased cytosolic p21/p27 levels in a manner similar to that of homocysteine treatment. By contrast, and similar to folate treatment, DNAKT1 counteracted these effects. Additionally, we showed AKT to be an upstream target of FAK activation, and verified the involvement of RhoA activation in the antimigratory effects of folate in RASMC by overexpressing CARhoA. The addition of Y27632 (a RhoA inhibitor) and DNRhoA counteracted the antimigratory effects, confirming RhoA involvement.
This study was the first to show the antiproliferative and antimigratory effects of folate on homocysteine-challenged RASMC, and to delineate the mechanisms underlying folate-mediated protection against the proatherosclerotic effects of homocysteine.
The neuropeptide calcitonin gene-related peptide (CGRP) is reported to play an important role in migraine. It is expressed throughout the trigeminovascular system. Antagonists targeting the CGRP receptor have been developed and have shown efficacy in clinical trials for migraine. However, no CGRP antagonist is yet approved for treating this condition. The molecular composition of the CGRP receptor is unusual because it comprises two subunits. One is a G protein-coupled receptor (GPCR), the calcitonin receptor-like receptor (CLR). This associates with receptor activity-modifying protein (RAMP) 1 to yield a functional receptor for CGRP. However, RAMP1 also associates with the calcitonin receptor, creating a receptor for the related peptide amylin but this also has high affinity for CGRP. Other combinations of CLR or the calcitonin receptor with RAMPs can also generate receptors that are responsive to CGRP. CGRP potentially modulates an array of signal transduction pathways downstream of activation of these receptors, in a cell type-dependent manner. The physiological significance of these signalling processes remains unclear but may be a potential avenue for refining drug design. This complexity has prompted us to review the signalling and expression of CGRP and related receptors in the trigeminovascular system. This reveals that more than one CGRP responsive receptor may be expressed in key parts of this system and that further work is required to determine their contribution to CGRP physiology and pathophysiology.
The histamine H4 receptor, originally thought to signal merely through Gαi-proteins, has recently been shown to also recruit and signal via β-arrestin2. Following the discovery that the reference antagonist indolecarboxamide JNJ 7777120 appears to be a partial agonist in β-arrestin2 recruitment, we have identified additional biased hH4R ligands that preferentially couple to Gαi or β-arrestin2 proteins. In this study we explored ligand and receptor regions that are important for biased hH4R signalling.
We evaluated a series of 48 indolecarboxamides with subtle structural differences for their ability to induce hH4R-mediated Gαi-protein signalling or β-arrestin2 recruitment. Subsequently, a FLAP 3D-QSAR analysis correlated intrinsic activity values to structural ligand requirements. Moreover, a hH4R homology model was used to identify receptor regions important for biased hH4R signalling.
One indolecarboxamide (75) with a nitro substituent on position R7 of the aromatic ring displayed an equal preference for the Gαi and β-arrestin2 pathway and was classified as unbiased hH4R ligand. The other 47 indolecarboxamides were β-arrestin2 biased agonists. Intrinsic activities of the unbiased as well as β-arrestin2-biased indolecarboxamides to induce β-arrestin2 recruitment could be correlated to different ligand features and hH4R regions.
Small structural modifications result in diverse intrinsic activities for unbiased (75) and β-arrestin2-biased indolecarboxamides. Analysis of ligand and receptor features revealed efficacy hotspots responsible for biased-β-arrestin2 recruitment. This knowledge is useful for the design of hH4R ligands with biased intrinsic activities and aids our understanding of the mechanism of H4R activation.
The recently proposed binding mode of 2-aminopyrimidines to the human histamine H4 receptor (hH4R) suggests an interaction between the 2-amino group of these ligands with a glutamic acid residue E1825.46 in transmembrane (TM) helix 5 of hH4R. Interestingly, substituents at this 2-position of the pyrimidine are also in close proximity to cysteine residue C983.36 in TM3. We hypothesized that an ethenyl group at this position can form a covalent bond with C983.36 by functioning as Michael acceptor. A covalent pyrimidine analogue will not only prove the proposed binding mode, it is also a valuable tool in H4R research, amongst others enabling additional studies with respect to ligand residence time and firmly stabilizing a specific receptor conformation.
We designed and synthesized 4-(4-methylpiperazin-1-yl)-6-phenyl-2-vinylpyrimidine (VUF14480), and pharmacologically characterized this compound in hH4R radioligand binding, G protein activation, and β-arrestin2 recruitment experiments. The reactivity of VUF14480 as covalent binder was both chemically and pharmacologically assessed.
VUF14480 is a partial agonist in hH4R-mediated G protein signalling and β-arrestin2 recruitment. VUF14480 bound covalently to the hH4R with submicromolar affinity. Serine-substitution of C983.36 prevented this covalent interaction.
The possible covalent interaction of VUF14480 with C983.36 confirmed our previously proposed 2-aminopyrimidine binding pose. VUF14480 will be an interesting tool compound to stabilize hH4R in a specific conformation.
Endocannabinoids such as anandamide (AEA) are important lipid ligands regulating cell proliferation, differentiation, and apoptosis. Their levels are regulated by hydrolase enzymes, the fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MGL). Here, we investigated whether FAAH or AEA are involved in nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/antioxidant responsive element (ARE) pathway.
To analyze the effects of AEA or FAAH inhibition by the URB597 inhibitor or FAAH/siRNA on the activation of Nrf2-ARE signaling pathway and heme oxygenase-1 (HO-1) induction and transcription.
Endogenous AEA was detected in the immortalized human mammary epithelial MCF-10A cells (0.034ng/106 cells) but not in MCF-7 or MDA-MB-231 breast cancer cells. Since breast tumor cells express FAAH abundantly, we examined the effects of FAAH in Nrf2/antioxidant pathway. We found that inhibition of FAAH by the URB597 inhibitor induced antioxidant HO-1 in breast cancer cells and MCF-10A cells. RNAi-mediated knockdown of FAAH or treatment with AEA-activated ARE-containing reporter induced HO-1 mRNA and protein expression, independent from the cannabinoid receptors, CB1, CB2, or TRPV1. Furthermore, URB597, AEA and siRNA-FAAH treatments induced the nuclear translocation of Nrf2, while siRNA-Nrf2 treatment and Keap1 expression blocked AEA, URB597 and si-FAAH from activation of ARE reporter and HO-1 induction. siRNA-HO-1 treatment decreased the viability of breast cancer cells and MCF-10A cells.
These data uncovered a novel mechanism by which inhibition of FAAH or exposure to AEA induced HO-1 transcripts and implicating AEA and FAAH as direct modifiers in signaling mediated activation of Nrf2-HO-1 pathway, independent of cannabinoid receptors.
Histamine, serotonin and dopamine are biogenic amines devoted to intercellular communication with multiple effects on human pathophysiology. They are all products of two highly homologous enzymes, histidine decarboxylase and L-aromatic amino acid decarboxylase, and transmit their signals through different receptors and signal transduction mechanisms. Polyamines derived from ornithine (putrescine, spermidine and spermine) are mainly involved in intracellular effects related to cell proliferation and death mechanisms. This review summarizes structural and functional evidence on connections among elements of all these amine metabolic/signalling networks (decarboxylases, transporters, oxidases, receptors, etc) in cellular/tissue contexts, apart from nervous and neuroendocrine systems, where the crosstalk among these amine-related elements can also have important pathophysiological consequences. The discussion highlights points that could be helpful to predict and/or discuss the effects of intervention strategies.
Histamine exerts the capacity to influence the activity of immune cells including neutrophils and plays a pivotal role in inflammatory processes which are a complex network of cellular and humoral events. Since oxidative burst linked with the production of reactive oxygen species is a principal functional manifestation of activated neutrophils, the effects of histamine receptor agonists and antagonists on the oxidative burst of neutrophils is reviewed. A role of well characterized histamine H1 and H2 receptors is discussed and compared to that of recently discovered H4 receptor.
The gastrointestinal hormone motilin has been known for >40 years but after identification of its receptor and subsequent development of new tools and methods, a reappraisal of its actions is required. Firstly, it is important to note that motilin and ghrelin are family members (high receptor identity; similar genomic organisation, gastrointestinal distribution and abilities to stimulate gastrointestinal motility) yet each fails to recognise the ligand of the other and whereas ghrelin and ghrelin receptors are widespread outside the gastrointestinal tract, motilin and its receptors are largely restricted to the gastrointestinal tract. Secondly, although some studies suggest motilin has activity in rodents, most do not and receptor pseudogenes exist in rodents. Thirdly, motilin preferentially operates by facilitating enteric cholinergic activity rather than directly contracting the muscle, contrasting with the relatively high expression of receptor immunoreactivity on muscle. This activity is ligand-dependent, with short-lasting actions of motilin contrasting with longer-lasting actions of the non-selective and selective motilin receptor agonist's erythromycin and GSK962040. Finally, the use of erythromycin (also an antibiotic drug) to treat patients requiring acceleration of gastric emptying has led to concerns over safety and potential exacerbation of antibiotic resistance. Replacement motilin receptor agonists derived from erythromycin (motilides) have been unsuccessful. New, non-motilide, small molecule receptor agonists designed to minimise self-desensitisation, are now entering clinical trials for mechanistically-relevant patients undergoing enteral feeding or with diabetic gastroparesis. Thus for the translational pharmacologist, the study of motilin illustrates the need to avoid over-reliance on artificial systems, on structural information and on animal studies.
Calcitonin gene-related peptide (CGRP) is a member of the calcitonin family of peptides. It is a widely distributed neuropeptide implicated in conditions such as neurogenic inflammation. With other members of the calcitonin family, it shares an N-terminal disulphide-bonded ring which is essential for biological activity, an area of potential alpha helix, and a C-terminal amide. CGRP binds to the calcitonin receptor-like receptor (CLR) in complex with receptor activity modifying protein 1 (RAMP1), a member of the family B (or Secretin-like) G protein-coupled receptors (GPCRs). It can also activate other CLR or calcitonin-receptor/RAMP complexes. This 37 amino acid peptide comprises the N-terminal ring that is required for receptor activation (residues 1-7); an alpha helix (residues 8-18), a region incorporating a beta bend (residues 19-26) and the C-terminal portion (residues 27-37), that is characterised by bends between residues 28-30 and 33-34. A few residues have been identified that seem to make major contributions to receptor binding and activation, with a larger number contributing either to minor interactions (which collectively may be significant) or, to maintaining the conformation of the bound peptide. It is not clear if CGRP follows the pattern of other family B GPCRs in binding largely as an alpha helix.
Sensory neurons play important roles in many disorders, including inflammatory diseases such as sepsis. Sepsis is a potentially lethal systemic inflammatory reaction to a local bacterial infection, affecting thousands of patients annually. Although associated with a high mortality rate, sepsis outcome depends on the severity of systemic inflammation, which can be directly influenced by several factors including the immune response of the patient. Currently, there are a lack of effective drugs to treat sepsis, and thus there is an need to develop new drugs to improve sepsis outcome. Several mediators involved in the formation of sepsis have now been identified, but the mechanisms underlying the pathology remain poorly understood. The transient receptor potential vanilloid 1 (TRPV1) receptor and the neuropeptide substance P (SP) have recently been demonstrated as important targets for sepsis and are located on sensory neurones and non-neuronal cells. Herein, we highlight and review the importance of sensory neurones for the modulation of sepsis, with specific focus on recent findings relating to TRPV1 and SP, with their distinct abilities to alter the transition from local to systemic inflammation and also modify the overall sepsis outcome. We also emphasise the protective role of TRPV1 in this context.
Obesity is a major worldwide public health issue. The physiological systems that regulate body weight are thus of great interest as targets for anti-obesity agents. Peptidergic systems are critical to the regulation of energy homeostasis by key regions in the hypothalamus and brain stem. A number of neuropeptide systems have therefore been investigated as potential treatments for obesity. Blocking orexigenic peptide signals such as neuropeptide Y, melanin-concentrating hormone, orexins, relaxin-3 and galanin-like peptide or stimulating anorectic signalling pathways used by peptides such as the melanocortins, ciliary neurotrophic factor and brain derived neurotrophic factor, are approaches that have shown some promise, but which have also highlighted possible concerns. Manipulation of central peptidergic systems poses a number of therapeutic problems, including brain access and side effects. Given that the homeostatic defence of body weight may limit the effectiveness of any single target therapy developed, a combination therapy approach may offer the best hope for the effective prevention and treatment of obesity.
N-3 long chain polyunsaturated fatty acids (n-3 LC PUFAs), in particular α-linolenic acid (18:3n-3), eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3) are receiving much attention because of their presumed beneficial health effects. To explain these, a variety of mechanisms have been proposed, but their interactions with the endocannabinoid system have received relatively little attention so far. However, it has already been shown some time ago that consumption of n-3 LC PUFAs not only affects the synthesis of prototypic endocannabinoids like anandamide, but also stimulates the formation of specific n-3 LC PUFA-derived conjugates with ethanolamine, dopamine, serotonin or other amines. Some of these fatty amides show overlapping biological activities with those of typical endocannabinoids, whereas others possess distinct and sometimes largely unknown receptor affinities and other properties. The ethanolamine and dopamine conjugates of DHA have been the most investigated thus far. These mediators may provide promising new leads to in the field of inflammatory and neurological disorders and for other pharmacological applications, including their use as carrier molecules for neurotransmitters to target the brain. Furthermore, combinations of n-3 LC PUFA-derived fatty acid amides, their precursors and FAAH inhibitors offer possibilities to optimise their effects in health and disease.
The glycoprotein IIb/IIIa receptor is the final common pathway of platelet aggregation, regardless of the agonist, and thus represents an ideal therapeutic target for blocking coronary thrombosis. In this paper the anti-platelet and antithrombotic actions of Z4A5, a new glycoprotein IIb/IIIa receptor inhibitor, were evaluated in the canine model of acute unstable angina.
Z4A5 was given intravenously as a bolus followed by 60 min of continuous infusion at dose of 30 μg/kg + 1 μg/kg/min, 30 μg/kg + 5 μg/kg/min, or 300 μg/kg + 5 μg/kg/min. In the model of platelet dependent cyclic flow reduction (CFR) in injured, stenosed left circumflex coronary artery, the antithrombotic effect was evaluated in CFR frequency. Platelet aggregation and coagulation parameters were determined by platelet rich plasma and platelet poor plasma, respectively.
In a coronary thrombosis model, dose-dependent reduction of CFR frequency was observed during the Z4A5 infusion and returned to baseline levels 60 min after the termination of the infusion in low dose. Tongue bleeding time increased marginally with an effective dose (30 μg/kg + 5 μg/kg/min), without dramatic changes in haemodynamic or coagulation parameters. Furthermore, inhibited ADP-induced platelet aggregation and prolonged bleeding time observed during Z4A5 infusion reverted to baseline levels 60 min after the termination of the infusion.
These results indicate that Z4A5 is an effective antithrombotic agent for coronary artery thrombosis with a rapid-on and rapid-off pharmacological profile, which could be used as an alternative treatment of coronary artery ischemic syndromes.
Binge eating disorder (BED) is characterized by excessive food intake during short periods of time. Recent evidence suggest that alterations in the endocannabinoid signalling could be involved in the pathophysiology of BED. In this study we investigated whether the pharmacological manipulation of the endocannabinoid transmission may be effective in modulating the aberrant eating behaviour present in a validated rat model of BED.
Binge-type eating was induced in female rats by providing limited access to an optional source of fat dietary (margarine). Rats were divided into three groups, all with ad libitum access to chow and water: Control (C), with no access to margarine; Low-restriction (LR), with 2h margarine access 7 days/week; High-restriction (HR), with 2h margarine access 3 days/week.
As compared to LR group, HR group displayed higher consumption of margarine accompanied by an increasing in body weight. The cannabinoid CB1/CB2 receptor (CB1R/CBR2) agonist Δ9-tetrahydrocannabinol (THC) significantly increased margarine intake selectively in LR rats, while the fatty acid amide hydrolase inhibitor URB597 showed no effect. The CB1R inverse agonist/antagonist rimonabant dose-dependently reduced margarine intake in HR rats. Notably, in HR rats, chronic treatment with a low dose of rimonabant induced a selective long-lasting effect on margarine intake that did not develop tolerance, and produced a significant and persistence reduction of body weight.
Chronic pharmacological blockade of CB1Rs reduces binge eating behaviour in female rats and may prove effective in treating BED, with an associated significant reduction in the body weight.
The development of potent and selective inhibitors of the biosynthesis of the endocannabinoid 2-AG via diacylglycerol lipases (DAGL) α and β is just starting to be considered as a novel and promising source of pharmaceuticals for the treatment of disorders that might benefit from a reduction of endocannabinoid tone, such as hyperphagia in obese subjects.
Three new fluorophosphonate compounds: 1-((fluoro(methyl)phosphoryl)oxy)-3-(penthyloxy)propan-2-yl oleate (O-7458); 1-ethoxy-3-((fluoro(methyl)phosphoryl)oxy)propan-2-yl oleate (O-7459); and 1-((fluoro(methyl)phosphoryl)oxy)-3-isopropoxypropan-2-yl oleate (O-7460) were synthesized and characterized in various enzymatic assays. O-7460 was tested on high fat diet intake in mice.
Of the new compounds, O-7460 exhibited the highest potency (IC50=690 nM) against the human recombinant DAGLα, and selectivity (IC50>10 μM) towards COS-7 cell and human monoacylglycerol lipase (MAGL), and rat brain fatty acid amide hydrolase (FAAH). Competitive activity-based protein profiling confirmed that O-7460 inhibits mouse brain MAGL only at concentrations > 10 μM, and showed that this compound has only one major “off-target”, i.e. the serine hydrolase KIAA1363. O-7460 did not exhibit measurable affinity for human recombinant CB1 or CB2 cannabinoid receptors (Ki>10 μM). In mouse neuroblastoma N18TG2 cells stimulated with ionomycin, O-7460 (10 μM) reduced 2-AG levels. When administered to mice, O-7460 dose-dependently (0-12 mg/kg, i.p.) inhibited the intake of a high fat diet over a 14 h observation period, and, subsequently, slightly but significantly reduced body weight.
O-7460 might be considered a useful pharmacological tool to investigate further the role played by 2-AG both in vitro and in vivo under physiological as well as pathological conditions.
This study determined the effect of JZL184, a selective inhibitor of monoacylglycerol lipase (MAGL), the enzyme which preferentially catabolises the endocannabinoid 2-arachidonoyl glycerol (2-AG), on inflammatory cytokines in the brain and plasma following an acute immune challenge. The receptor and molecular mechanisms involved were also investigated.
JZL184 and/or AM251 (CB1 antagonist) or AM630 (CB2 antagonist) were administered to rats 30 min prior to the administration of lipopolysaccharide (LPS), 2hrs following which cytokine expression/levels, MAGL activity, 2-AG, arachidonic acid and prostaglandin levels were measured in the frontal cortex, plasma and spleen.
JZL184 attenuated LPS-induced increases in IL-1β, IL-6, TNF-α and IL-10, but not IĸBα expression in the rat frontal cortex. AM251 attenuated the JZL184-induced decrease in frontal cortical IL-1β expression. Although arachidonic acid levels in the frontal cortex were reduced in JZL184-treated rats, MAGL activity, 2-AG, PGE2 and PGD2 levels remained unchanged. In comparison, MAGL activity was inhibited and 2-AG levels enhanced in the spleen following JZL184 administration. In the plasma, LPS-induced increases in TNF-α and IL-10 levels were attenuated by JZL184, an effect partially blocked by AM251. In addition, AM630 blocked the LPS-induced increases in plasma IL-1β in the presence, but not absence, of JZL184.
Inhibition of peripheral MAGL in the rat by JZL184 suppresses LPS-induced circulating cytokines which in turn may modulate central cytokine expression. The data provide further evidence for the therapeutic potential of targeting the endocannabinoid system for the treatment of central and peripheral inflammatory disorders.
Anandamide and 2-arachidonoylglycerol are neuromodulatory lipids interacting with cannabinoid receptors, whose availability is regulated by the balance between “on demand” generation and enzymatic degradation (by FAAH/MAGL). Given the reported effects of anandamide on dopamine transmission, we investigated the influence of endocannabinoids and URB597, a well-known FAAH inhibitor, on the expression of tyrosine hydroxylase (TH), the the rate-limiting enzyme in dopamine synthesis.
We investigated TH expression in N1E115 neuroblastoma using a reporter gene assay, as well as mRNA and protein quantifications. FAAH inhibition was confirmed by measuring radiolabeled substrates hydrolysis and endogenous endocannabinoids.
Anandamide decreased TH promoter activity in N1E115 cells through CB1 receptor activation. Unexpectedly, URB597 reduced TH expression (pEC50=8.7±0.2) through FAAH-independent mechanisms. Indeed, four structurally unrelated inhibitors of FAAH had no influence on TH expression, although all the inhibitors increased endocannabinoid levels. At variance with the endocannabinoid responses, the use of selective antagonists indicated that the URB597-mediated decrease in TH expression was not directed by the CB1 receptor, but rather by abnormal-cannabidiol-sensitive receptors and PPARs. Further supporting the physiological relevance of these in vitro data, URB597 administration resulted in reduced TH mRNA levels in mice brain.
While confirming the implication of endocannabinoids on the modulation of TH, we provide strong evidence for additional physiologically relevant off-target effects of URB597. In light of the numerous preclinical studies involving URB597, particularly in anxiety and depression, the existence of non-CB1 and non-FAAH mediated influences of URB597 on key enzymes of the catecholaminergic transmission system should be taken into account when interpreting the data.
Recent advances have improved our understanding of the renin-angiotensin system (RAS). These have included the recognition that angiotensin (Ang)-(1-7) is a biologically active product of the RAS cascade. The identification of the ACE homologue ACE2, which forms Ang-(1-7) from Ang II, and the GPCR Mas as an Ang-(1-7) receptor have provided the necessary biochemical and molecular background and tools to study the biological significance of Ang-(1-7). Most available evidence supports a counter-regulatory role for Ang-(1-7) by opposing many actions of Ang II on AT1 receptors, especially vasoconstriction and proliferation. Many studies have now shown that Ang-(1-7) by acting via Mas receptor exerts inhibitory effects on inflammation and on vascular and cellular growth mechanisms. Ang-(1-7) has also been shown to reduce key signalling pathways and molecules thought to be relevant for fibrogenesis. Here, we review recent findings related to the function of the ACE2/Ang-(1-7)/Mas axis and focus on the role of this axis in modifying processes associated with acute and chronic inflammation, including leukocyte influx, fibrogenesis and proliferation of certain cell types. More attention will be given to the involvement of the ACE2/Ang-(1-7)/Mas axis in the context of renal disease because of the known relevance of the RAS for the function of this organ and for the regulation of kidney inflammation and fibrosis. Taken together, this knowledge may help in paving the way for the development of novel treatments for chronic inflammatory and renal diseases.
]]>High-density lipoproteins (HDLs) represent a family of particles characterized by the presence of apolipoprotein A-I (apoA-I) and by their ability to transport cholesterol from peripheral tissues back to the liver. In addition to this function, HDLs display pleiotropic effects including antioxidant, anti-apoptotic, anti-inflammatory, anti-thrombotic or anti-proteolytic properties that account for their protective action on endothelial cells. Vasodilatation via production of nitric oxide is also a hallmark of HDL action on endothelial cells. Endothelial cells express receptors for apoA-I and HDLs that mediate intracellular signalling and potentially participate in the internalization of these particles. In this review, we will detail the different effects of HDLs on the endothelium in normal and pathological conditions with a particular focus on the potential use of HDL therapy to restore endothelial function and integrity.
]]>Inflammatory processes play both regenerative and destructive roles in multiple sclerosis, stroke, CNS trauma, amyotrophic lateral sclerosis and aging-related neurodegenerative diseases such as Alzheimer's, Parkinson's and Huntington's. Endogenous defence mechanisms against these pathologies include those that are directly neuroprotective, and those that modulate the expression of inflammatory mediators in microglia, astrocytes, and invading inflammatory cells. While a number of mechanisms and molecules have been identified that can directly promote neuronal survival, less is known about how the brain protects itself from harmful inflammation, and further, how it co-opts the healing function of the immune system to promote CNS repair. The two closely related neuroprotective peptides, vasoactive intestinal peptide (VIP) and pituitary adenylyl cyclase-activating peptide (PACAP), which are up-regulated in neurons and immune cells after injury and/or inflammation, are known to protect neurons, but also exert powerful in vivo immunomodulatory actions, which are primarily anti-inflammatory. These peptide actions are mediated by high-affinity receptors expressed not only on neurons, but also astrocytes, microglia and peripheral inflammatory cells. Well-established immunomodulatory actions of these peptides are to inhibit macrophage and microglia production and release of inflammatory mediators such as TNF-α and IFN-γ, and polarization of T-cell responses away from Th1 and Th17, and towards a Th2 phenotype. More recent studies have revealed that these peptides can also promote the production of both natural and inducible subsets of regulatory T-cells. The neuroprotective and immunomodulatory actions of VIP and PACAP suggest that receptors for these peptides may be therapeutic targets for neurodegenerative and neuroinflammatory diseases and other forms of CNS injury.
]]>PDEs restrict the positive inotropic effects of β-adrenoceptor stimulation by degrading cAMP. Hence, PDE inhibitors sensitize the heart to catecholamines and are therefore used as positive inotropes. On the downside, this is accompanied by exaggerated energy expenditure, cell death and arrhythmias. For many years, PDE3 was considered to be the major isoform responsible for the control of cardiac force and rhythm. However, recent work in gene-targeted mice and rodent cells has indicated that PDE4 is also involved. Furthermore, selective PDE4 inhibitors augment catecholamine-stimulated cAMP levels and induce arrhythmias in human atrial preparations, which suggests that PDE4 has a more prominent role in the human heart than anticipated, and that PDE4 inhibitors such as roflumilast may carry an arrhythmogenic risk. In this issue of the journal, a team of researchers from three laboratories report on the effect of PDE3 and PDE4 inhibitors on ventricular trabeculae from explanted human hearts. The key result is that the PDE4 inhibitor rolipram does not affect the positive inotropic effects of β1- or β2-adrenoceptor stimulation. Given that the ventricle rather than the atria is the critical region in terms of arrhythmogenic consequences, this is an important and reassuring finding.
This article is a commentary on the research paper by Molenaar et al., pp. 528–538 of this issue. To view this paper visit http://dx.doi.org/10.1111/bph.12167
PDE3 and/or PDE4 control ventricular effects of catecholamines in several species but their relative effects in failing human ventricle are unknown. We investigated whether the PDE3-selective inhibitor cilostamide (0.3–1 μM) or PDE4 inhibitor rolipram (1–10 μM) modified the positive inotropic and lusitropic effects of catecholamines in human failing myocardium.
Right and left ventricular trabeculae from freshly explanted hearts of 5 non-β-blocker-treated and 15 metoprolol-treated patients with terminal heart failure were paced to contract at 1 Hz. The effects of (-)-noradrenaline, mediated through β1 adrenoceptors (β2 adrenoceptors blocked with ICI118551), and (-)-adrenaline, mediated through β2 adrenoceptors (β1 adrenoceptors blocked with CGP20712A), were assessed in the absence and presence of PDE inhibitors. Catecholamine potencies were estimated from –logEC50s.
Cilostamide did not significantly potentiate the inotropic effects of the catecholamines in non-β-blocker-treated patients. Cilostamide caused greater potentiation (P = 0.037) of the positive inotropic effects of (-)-adrenaline (0.78 ± 0.12 log units) than (-)-noradrenaline (0.47 ± 0.12 log units) in metoprolol-treated patients. Lusitropic effects of the catecholamines were also potentiated by cilostamide. Rolipram did not affect the inotropic and lusitropic potencies of (-)-noradrenaline or (-)-adrenaline on right and left ventricular trabeculae from metoprolol-treated patients.
Metoprolol induces a control by PDE3 of ventricular effects mediated through both β1 and β2 adrenoceptors, thereby further reducing sympathetic cardiostimulation in patients with terminal heart failure. Concurrent therapy with a PDE3 blocker and metoprolol could conceivably facilitate cardiostimulation evoked by adrenaline through β2 adrenoceptors. PDE4 does not appear to reduce inotropic and lusitropic effects of catecholamines in failing human ventricle.
This article is commented on by Eschenhagen, pp 524–527 of this issue. To view this commentary visit http://dx.doi.org/10.1111/bph.12168
Gender differences in inflammation are well described, with females often showing more robust, oestrogen-associated responses. Here, we investigated the influence of gender, oestrogen and the anti-inflammatory protein annexin A1 (AnxA1) on lipopolysaccharide (LPS)-induced leukocyte–endothelial cell interactions in murine cerebral and mesenteric microvascular beds.
Intravital microscopy was used to visualize and quantify the effects of LPS (10 μg·per mouse i.p.) on leukocyte–endothelial interactions in male and female wild-type (WT) mice. The effects of ovariectomy ± oestrogen replacement were examined in WT and AnxA1-null (AnxA1−/−) female mice.
LPS increased leukocyte adherence in the cerebral and mesenteric beds of both male and female WT mice; females showed exacerbated responses in the brain versus males, but not the mesentery. Ovariectomy further enhanced LPS-induced adhesion in the brain but not the mesentery; its effects were reversed by oestrogen treatment. OVX AnxA1−/− mice also showed exaggerated adhesive responses to LPS in the brain. However, these were unresponsive to ovariectomy and, paradoxically, responded to oestrogen with a pronounced increase in basal and LPS-induced leukocyte adhesion in the cerebrovasculature.
Our data confirm the fundamental role of AnxA1 in limiting the inflammatory response in the central and peripheral microvasculature. They also (i) show that oestrogen acts via an AnxA1-dependent mechanism to protect the cerebral, but not the mesenteric, vasculature from the damaging effects of LPS and (ii) reveal a paradoxical and potentially toxic effect of the steroid in potentiating the central response to LPS in the absence of AnxA1.
NAD(P)H oxidase and COX-1 participate in vascular damage induced by angiotensin II. We investigated the effect of rosuvastatin on endothelial dysfunction, vascular remodelling, changes in extracellular matrix components and mechanical properties of small mesenteric arteries from angiotensin II-infused rats.
Male rats received angiotensin II (120 ng·kg−1·min−1, subcutaneously) for 14 days with or without rosuvastatin (10 mg·kg−1·day−1, oral gavage) or vehicle. Vascular functions and morphological parameters were assessed by pressurized myography.
In angiotensin II-infused rats, ACh-induced relaxation was attenuated compared with controls, less sensitive to L-NAME, enhanced by SC-560 (COX-1 inhibitor) or SQ-29548 (prostanoid TP receptor antagonist), and normalized by the antioxidant ascorbic acid or NAD(P)H oxidase inhibitors. After rosuvastatin, relaxations to ACh were normalized, fully sensitive to L-NAME, and no longer affected by SC-560, SQ-29548 or NAD(P)H oxidase inhibitors. Angiotensin II enhanced intravascular superoxide generation, eutrophic remodelling, collagen and fibronectin depositions, and decreased elastin content, resulting in increased vessel stiffness. All these changes were prevented by rosuvastatin. Angiotensin II increased phosphorylation of NAD(P)H oxidase subunit p47phox and its binding to subunit p67phox, effects inhibited by rosuvastatin. Rosuvastatin down-regulated vascular Nox4/NAD(P)H isoform and COX-1 expression, attenuated the vascular release of 6-keto-PGF1α, and enhanced copper/zinc-superoxide dismutase expression.
Rosuvastatin prevents angiotensin II-induced alterations in resistance arteries in terms of function, structure, mechanics and composition. These effects depend on restoration of NO availability, prevention of NAD(P)H oxidase-derived oxidant excess, reversal of COX-1 induction and its prostanoid production, and stimulation of endogenous vascular antioxidant defences.
The α7 nicotinic ACh receptor subtype is abundantly expressed in the CNS and in the periphery. Recent evidence suggests that α7 nicotinic ACh receptor (nAChR) subtypes, which can be activated by an endogenous cholinergic tone comprising ACh and the α7 agonist choline, play an important role in chronic pain and inflammation. In this study, we evaluated whether type II α7 positive allosteric modulator PNU-120596 induces antinociception on its own and in combination with choline in the formalin pain model.
We assessed the effects of PNU-120596 and choline and the nature of their interactions in the formalin test using an isobolographic analysis. In addition, we evaluated the interaction of PNU-120596 with PHA-54613, an exogenous selective α7 nAChR agonist, in the formalin test. Finally, we assessed the interaction between PNU-120596 and nicotine using acute thermal pain, locomotor activity, body temperature and convulsing activity tests in mice.
We found that PNU-120596 dose-dependently attenuated nociceptive behaviour in the formalin test after systemic administration in mice. In addition, mixtures of PNU-120596 and choline synergistically reduced formalin-induced pain. PNU-120596 enhanced the effects of nicotine and α7 agonist PHA-543613 in the same test. In contrast, PNU-120596 failed to enhance nicotine-induced convulsions, hypomotility and antinociception in acute pain models. Surprisingly, it enhanced nicotine-induced hypothermia via activation of α7 nAChRs.
Our results demonstrate that type II α7 positive allosteric modulators produce antinociceptive effects in the formalin test through a synergistic interaction with the endogenous α7 agonist choline.
Nasal sensory nerves play an important role in symptoms associated with rhinitis triggered by environmental stimuli. Here, we propose that TRPV1 is pivotal in nasal sensory nerve activation and assess the potential of SB-705498 as an intranasal therapy for rhinitis.
The inhibitory effect of SB-705498 on capsaicin-induced currents in guinea pig trigeminal ganglion cells innervating nasal mucosa was investigated using patch clamp electrophysiology. A guinea pig model of rhinitis was developed using intranasal challenge of capsaicin and hypertonic saline to elicit nasal secretory parasympathetic reflex responses, quantified using MRI. The inhibitory effect of SB-705498, duration of action and potency comparing oral versus intranasal route of administration were examined.
SB-705498 concentration-dependently inhibited capsaicin-induced currents in isolated trigeminal ganglion cells (pIC50 7.2). In vivo, capsaicin ipsilateral nasal challenge (0.03–1 mM) elicited concentration-dependent increases in contralateral intranasal fluid secretion. Ten per cent hypertonic saline initiated a similar response. Atropine inhibited responses to either challenge.
SB-705498 inhibited capsaicin-induced responses by ∼50% at 10 mg·kg−1 (oral), non-micronized 10 mg·mL−1 or 1 mg·mL−1 micronized SB-705498 (intranasal) suspension.
Ten milligram per millilitre intranasal SB-705498, dosed 24 h prior to capsaicin challenge produced a 52% reduction in secretory response. SB-705498 (10 mg·mL−1, intranasal) inhibited 10% hypertonic saline responses by 70%.
The paper reports the development of a guinea pig model of rhinitis. SB-705498 inhibits capsaicin-induced trigeminal currents and capsaicin-induced contralateral nasal secretions via oral and intranasal routes; efficacy was optimized using particle-reduced SB-705498. We propose that TRPV1 is pivotal in initiating symptoms of rhinitis.
Chronic lung diseases such as asthma, COPD and pulmonary fibrosis are characterized by abnormal extracellular matrix (ECM) turnover. TGF-β is a key mediator stimulating ECM production by recruiting and activating lung fibroblasts and initiating their differentiation process into more active myofibroblasts. Glycogen synthase kinase-3 (GSK-3) regulates various intracellular signalling pathways; its role in TGF-β1-induced myofibroblast differentiation is currently largely unknown.
To determine the contribution of GSK-3 signalling in TGF-β1-induced myofibroblast differentiation.
We used MRC5 human lung fibroblasts and primary pulmonary fibroblasts of individuals with and without COPD. Protein and mRNA expression were determined by immunoblotting and RT-PCR analysis respectively.
Stimulation of MRC5 and primary human lung fibroblasts with TGF-β1 resulted in time- and dose-dependent increases of α-sm-actin and fibronectin expression, indicative of myofibroblast differentiation. Pharmacological inhibition of GSK-3 by SB216763 dose-dependently attenuated TGF-β1-induced expression of these myofibroblasts markers. Moreover, silencing of GSK-3 by siRNA or pharmacological inhibition by CT/CHIR99021 fully inhibited the TGF-β1-induced expression of α-sm-actin and fibronectin. The effect of GSK-3 inhibition on α-sm-actin expression was similar in fibroblasts from individuals with and without COPD. Neither smad, NF-κB nor ERK1/2 were involved in the inhibitory actions of GSK-3 inhibition by SB126763 on myofibroblast differentiation. Rather, SB216763 increased the phosphorylation of CREB, which in its phosphorylated form acts as a functional antagonist of TGF-β/smad signalling.
We demonstrate that GSK-3 signalling regulates TGF-β1-induced myofibroblast differentiation by regulating CREB phosphorylation. GSK-3 may constitute a useful target for treatment of chronic lung diseases.
Prostaglandin E2 (PGE2) has been implicated in the regulation of adhesion molecules, leukocyte adhesion and infiltration into inflamed site. However, the underlying mechanism therein involved remains ill-defined. In this study, we explored its cellular mechanism of action in the regulation of the intercellular adhesion molecule-1 (ICAM-1) expression in the brain endothelial cells.
bEnd.3 cells, the murine cerebrovascular endothelial cell line and primary mouse brain endothelial cells were treated with PGE2 with or without agonists/antagonists of PGE2 receptors and associated signalling molecules. ICAM-1 expression, Akt phosphorylation and activity of NF-κB were determined by reverse transcription polymerase chain reaction (RT-PCR), immunoblot analysis, luciferase assay and immunocytochemistry.
PGE2 significantly up-regulated the expression of ICAM-1, which was blocked by EP4 antagonist (ONO-AE2-227) and knock-down of EP4. PGE2 effects were mimicked by forskolin, dibutyryl cAMP (dbcAMP) and an exchange protein directly activated by cAMP (Epac) activator (8-Cpt-cAMP) but not a protein kinase A activator (N6-Bnz-cAMP). PGE2-induced ICAM-1 expression was reduced by knock-down of Epac1. A PI3K specific inhibitor (LY294002), Akt inhibitor VIII (Akti) and NF-κB inhibitors (Bay-11–7082 and MG-132) attenuated the induction of ICAM-1 by PGE2. PGE2, dbcAMP and 8-Cpt-cAMP induced the phosphorylation of Akt, IκB kinase and IκBα and the translocation of p65 to the nucleus and increased NF-κB dependent reporter gene activity, which was diminished by Akti.
Our findings suggest that PGE2 induces ICAM-1 expression via EP4 receptor and Epac/Akt/NF-κB signalling pathway in bEnd.3 brain endothelial cells, supporting its pathophysiological role in brain inflammation.
Retinopathy, as a common complication of diabetes, is a leading cause of reduced visual acuity and acquired blindness in the adult population. The aim of present study was to investigate the therapeutic effect of hydrogen sulfide on streptozotocin (STZ)-induced diabetic retinopathy in rats.
Rats were injected with a single i.p. injection of STZ (60 mg·kg−1) to induce diabetic retinopathy. Two weeks later, the rats were treated with NaHS (i.p. injection of 0.1 mL·kg−1·d−1 of 0.28 mol·L−1 NaHS, a donor of H2S) for 14 weeks.
Treatment with H2S had no significant effect on blood glucose in STZ-induced diabetic rats. Treatment with exogenous H2S enhanced H2S levels in both plasma and retinas of STZ-induced diabetic rats. Treatment with H2S in STZ-treated rats improved the retinal neuronal dysfunction marked by enhanced amplitudes of b-waves and oscillatory potentials and expression of synaptophysin and brain-derived neurotrophic factor, alleviated retinal vascular abnormalities marked by reduced retinal vascular permeability and acellular capillary formation, decreased vitreous VEGF content, down-regulated expressions of HIF-1α and VEGFR2, and enhanced occludin expression, and attenuated retinal thickening and suppressed expression of extracellular matrix molecules including laminin β1 and collagen IVα3 expression in retinas of STZ-induced diabetic rats. Treatment with H2S in retinas of STZ-induced diabetic rats abated oxidative stress, alleviated mitochondrial dysfunction, suppressed NF-κB activation and attenuated inflammation.
Treatment with H2S alleviates STZ-induced diabetic retinopathy in rats possibly through abating oxidative stress and suppressing inflammation.
Pitolisant, a histamine H3 receptor inverse agonist/antagonist is currently under Phase III clinical trials for treatment of excessive daytime sleepiness namely in narcoleptic patients. Its drug abuse potential was investigated using in vivo models in rodents and monkeys and compared with those of Modafinil, a psychostimulant currently used in the same indications.
Effects of Pitolisant on dopamine release in the nucleus accumbens, on spontaneous and cocaine-induced locomotion, locomotor sensitization were monitored. It was also tested in three standard drug abuse tests i.e. conditioned place preference in rats, self-administration in monkeys and cocaine discrimination in mice as well as in a physical dependence model.
Pitolisant did not elicit any significant changes in dopaminergic indices in rat nucleus accumbens whereas Modafinil increased dopamine release. In rodents, Pitolisant was without any effect on locomotion and reduced the cocaine-induced hyperlocomotion. In addition, no locomotor sensitization and no conditioned hyperlocomotion were evidenced with this compound in rats whereas significant effects were elicited by Modafinil. Finally, Pitolisant was devoid of any significant effects in the three standard drug abuse tests (including self-administration in monkeys) and in the physical dependence model.
No potential drug abuse liability for Pitolisant was evidenced in various in vivo rodent and primate models, whereas the same does not seem so clear in the case of Modafinil.
Blood-stage Plasmodium parasites cause morbidity and mortality from malaria. Parasite resistance to drugs makes development of new chemotherapies an urgency. Aminoacyl-tRNA synthetases have been validated as antimalarial drug targets. We explored long-term effects of borrelidin and mupirocin in lethal P. yoelii murine malaria.
Long-term (up to 340 days) immunological responses to borrelidin or mupirocin were measured after an initial 4 day suppressive test. Prophylaxis and cure were evaluated and the inhibitory effect on the parasites analysed.
Borrelidin protected against lethal malaria at 0.25 mg·kg−1·day−1. Antimalarial activity of borrelidin correlated with accumulation of trophozoites in peripheral blood. All infected mice treated with borrelidin survived and subsequently developed immunity protecting them from re-infection on further challenges, 75 and 340 days after the initial infection. This long-term immunity in borrelidin-treated mice resulted in negligible parasitaemia after re-infections and marked increases in total serum levels of antiparasite IgGs with augmented avidity. Long-term memory IgGs mainly reacted against high and low molecular weight parasite antigens. Immunofluorescence microscopy showed that circulating IgGs bound predominantly to late intracellular stage parasites, mainly schizonts.
Low borrelidin doses protected mice from lethal malaria infections and induced protective immune responses after treatment. Development of combination therapies with borrelidin and selective modifications of the borrelidin molecule to specifically inhibit plasmodial threonyl tRNA synthetase should improve therapeutic strategies for malaria.
Nitrite (NO2−) has recently been shown to represent a potential source of NO, in particular under hypoxic conditions. The aim of the current study was to compare the haemodynamic effects of NO2− in healthy volunteers and patients with stable congestive heart failure (CHF).
The acute haemodynamic effects of brachial artery infusion of NO2− (0.31 to 7.8 μmol·min−1) was assessed in normal subjects (n = 20) and CHF patients (n = 21).
NO2− infusion was well tolerated in all subjects. Forearm blood flow (FBF) increased markedly in CHF patients at NO2− infusion rates which induced no changes in normal subjects (anova: F = 5.5; P = 0.02). Unstressed venous volume (UVV) increased even with the lowest NO2− infusion rate in all subjects (indicating venodilation), with CHF patients being relatively hyporesponsive compared with normal subjects (anova: F = 6.2; P = 0.01). There were no differences in venous blood pH or oxygen concentration between groups or during NO2− infusion. Venous plasma NO2− concentrations were lower in CHF patients at baseline, and rose substantially less with NO2− infusion, without incremental oxidative generation of nitrate, consistent with accelerated clearance in these patients. Plasma protein-bound NO concentrations were lower in CHF patients than normal subjects at baseline. This difference was attenuated during NO2− infusion. Prolonged NO2− exposure in vivo did not induce oxidative stress, nor did it induce tolerance in vitro.
The findings of arterial hyper-responsiveness to infused NO2− in CHF patients, with evidence of accelerated transvascular NO2− clearance (presumably with concomitant NO release) suggests that NO2− effects may be accentuated in such patients. These findings provide a stimulus for the clinical exploration of NO2− as a therapeutic modality in CHF.
Nutrient sensing in the gut is believed to be accomplished through activation of GPCRs expressed on enteroendocrine cells. In particular, L-cells located predominantly in distal regions of the gut secrete glucagon-like peptide 1 (GLP-1) and peptide tyrosine-tyrosine (PYY) upon stimulation by nutrients and bile acids (BA). The study was designed to address the mechanism of hormone secretion in L-cells stimulated by the BA receptor G protein-coupled bile acid receptor 1 (GPBAR1).
A novel, selective, orally bioavailable, and potent GPBAR1 agonist, RO5527239, was synthesized in order to investigate L-cell secretion in vitro and in vivo in mice and monkey. In analogy to BA, RO5527239 was conjugated with taurine to reduce p.o. bioavailability yet retaining its potency. Using RO5527239 and tauro-RO5527239, the acute secretion effects on L-cells were addressed via different routes of administration.
GPBAR1 signalling triggers the co-secretion of PYY and GLP-1, and leads to improved glucose tolerance. The strong correlation of plasma drug exposure and plasma PYY levels suggests activation of GPBAR1 from systemically accessible compartments. In contrast to the orally bioavailable agonist RO5527239, we show that tauro-RO5527239 triggers PYY release only when applied intravenously. Compared to mice, a slower and more sustained PYY secretion was observed in monkeys.
Selective GPBAR1 activation elicits a strong secretagogue effect on L-cells, which primarily requires systemic exposure. We suggest that GPBAR1 is a key player in the intestinal proximal-distal loop that mediates the early phase of nutrient-evoked L-cell secretion effects.
To determine the minimally effective dose of cannabidiolic acid (CBDA) that effectively reduces lithium chloride (LiCl)-induced conditioned gaping reactions (nausea-induced behaviour) in rats and to determine if these low systemic doses of CBDA (5–0.1 μg·kg−1) relative to those of CBD could potentiate the anti-nausea effects of the classic 5-hydroxytryptamine 3 (5-HT3) receptor antagonist, ondansetron (OND).
We investigated the efficacy of low doses of CBDA to suppress acute nausea, assessed by the establishment of conditioned gaping to a LiCl-paired flavour in rats. The potential of threshold and subthreshold doses of CBDA to enhance the reduction of nausea-induced conditioned gaping by OND were then determined.
CBDA (at doses as low as 0.5 μg·kg−1) suppressed nausea-induced conditioned gaping to a flavour. A low dose of OND (1.0 μg·kg−1) alone reduced nausea-induced conditioned gaping, but when it was combined with a subthreshold dose of CBDA (0.1 μg·kg−1) there was an enhancement in the suppression of LiCl-induced conditioned gaping.
CBDA potently reduced conditioned gaping in rats, even at low doses and enhanced the anti-nausea effect of a low dose of OND. These findings suggest that combining low doses of CBDA and OND will more effectively treat acute nausea in chemotherapy patients.
We have previously shown that arginase inhibition alleviates hypertension associated with in a diabetic animal model. Here, we investigated the protective effect of arginase inhibition on hypertension in metabolic syndrome.
Metabolic syndrome was induced in rats by administration of fructose (10% in drinking water) for 12 weeks to induce vascular dysfunction. Three arginase inhibitors (citrulline, norvaline and ornithine) were administered daily in the last 6 weeks of study before and tail BP was recorded in conscious animals. Concentration response curves for phenylephrine (PE), KCl and ACh in addition to ACh-induced NO generation were obtained in thoracic aorta rings. Serum glucose, insulin, uric acid and lipid profile were determined as well as reactive oxygen species (ROS) and arginase activity.
Arginase activity was elevated in metabolic syndrome while significantly inhibited by citrulline, norvaline or ornithine treatment. Metabolic syndrome was associated with elevations in systolic and diastolic BP, while arginase inhibition significantly reduced elevations in diastolic and systolic BP. Metabolic syndrome increased vasoconstriction responses of aorta to PE and KCl and decreased vasorelaxation to ACh, while arginase inhibition completely prevented impaired responses to ACh. In addition, arginase inhibition prevented impaired NO generation and exaggerated ROS formation in metabolic syndrome. Furthermore, arginase inhibition significantly reduced hyperinsulinaemia and hypertriglyceridaemia without affecting hyperuricaemia or hypercholesterolaemia associated with metabolic syndrome.
Arginase inhibition alleviates hypertension in metabolic syndrome directly through endothelial-dependent relaxation/NO signalling protection and indirectly through inhibition of insulin resistance and hypertriglyceridaemia.
Ranolazine is an antianginal drug currently approved for treatment of angina pectoris in the United States. Recent studies have focused on its effects on neuronal channels and its possible therapeutic uses in the nervous system. We characterized how ranolazine affects the brain sodium channel, NaV1.2, and how its actions are modulated by low pH. In this way, we further explore ranolazine's potential as an anticonvulsant and its efficacy in conditions like those during an ischaemic stroke.
We performed whole-cell patch-clamp experiments on the voltage-gated sodium channel, NaV1.2. Experiments were performed with extracellular solution titrated to either pH 7.4 or pH 6.0 before and after ranolazine perfusion.
Ranolazine accelerates onset and slows recovery of fast and slow inactivation. Ranolazine increases the maximum probability of use-dependent inactivation and reduces macroscopic and ramp sodium currents at pH 7.4. pH 6.0 reduced the slowing of fast inactivation recovery and inhibited use-dependent block by ranolazine. In the presence of ranolazine, the time constants of slow inactivation recovery and onset were significantly increased at pH 6.0 relative to pH 7.4 with 100 μM ranolazine.
Our work provides novel insights into the modulation of brain sodium channel, NaV1.2, by ranolazine. We demonstrate that ranolazine binds NaV1.2 in a state-dependent manner, and that the effects of ranolazine are slowed but not abolished by protons. Our results suggest that further research performed on channels with epilepsy-causing mutations may prove ranolazine to be an efficacious therapy.