T N and T Y contributed equally to this work.
The expression and function of histamine H3 receptors in pancreatic beta cells
Version of Record online: 10 DEC 2013
© 2013 The British Pharmacological Society
British Journal of Pharmacology
Volume 171, Issue 1, pages 171–185, January 2014
How to Cite
Nakamura, T., Yoshikawa, T., Noguchi, N., Sugawara, A., Kasajima, A., Sasano, H. and Yanai, K. (2014), The expression and function of histamine H3 receptors in pancreatic beta cells. British Journal of Pharmacology, 171: 171–185. doi: 10.1111/bph.12429
- Issue online: 10 DEC 2013
- Version of Record online: 10 DEC 2013
- Accepted manuscript online: 30 SEP 2013 09:24AM EST
- Manuscript Accepted: 16 SEP 2013
- Manuscript Revised: 6 SEP 2013
- Manuscript Received: 24 JAN 2013
- HIROMI Medical Research Foundation
- histamine H3 receptor;
- pancreatic beta cells;
- insulin secretion
- Top of page
- Discussion and conclusions
- Conflicts of interest
Background and Purpose
Histamine and its receptors in the CNS play important roles in energy homeostasis. Here, we have investigated the expression and role of histamine receptors in pancreatic beta cells, which secrete insulin.
The expression of histamine receptors in pancreatic beta cells was examined by RT-PCR, Western blotting and immunostaining. Insulin secretion assay, ATP measurement and calcium imaging studies were performed to determine the function and signalling pathway of histamine H3 receptors in glucose-induced insulin secretion (GIIS) from MIN6 cells, a mouse pancreatic beta cell line. The function and signalling pathway of H3 receptors in MIN6 cell proliferation were examined using pharmacological assay and Western blotting.
Histamine H3 receptors were expressed in pancreatic beta cells. A selective H3 receptor agonist, imetit, and a selective inverse H3 receptor agonist, JNJ-5207852, had inhibitory and facilitatory effects, respectively, on GIIS in MIN6 cells. Neither imetit nor JNJ-5207852 altered intracellular ATP concentration, or intracellular calcium concentration stimulated by glucose and KCl, indicating that GIIS signalling was affected by H3 receptor signalling downstream of the increase in intracellular calcium concentration. Moreover, imetit attenuated bromodeoxyuridine incorporation in MIN6 cells. The phosphorylation of cAMP response element-binding protein (CREB), which facilitated beta cell proliferation, was inhibited, though not significantly, by imetit, indicating that activated H3 receptors inhibited MIN6 cell proliferation, possibly by decreasing CREB phosphorylation.
Conclusions and Implications
Histamine H3 receptors were expressed in mouse beta cells and could play a role in insulin secretion and, possibly, beta cell proliferation.
- CaMK II
calcium/calmodulin-dependent PKs II
cAMP response element-binding protein
glucose-induced insulin secretion
histamine H3 receptor gene knockout
Hdc gene knockout
Krebs–Ringer bicarbonate buffer
vesicular monoamine transporter 2
- Top of page
- Discussion and conclusions
- Conflicts of interest
Histamine is involved in a wide variety of physiological events including allergic reactions, gastric acid secretion, smooth muscle contraction and the sleep–wake cycle (Haas et al., 2008). This amine exerts its diverse effects through interactions with four different histamine receptors: H1 (Fukui et al., 1994), H2 (Gantz et al., 1991), H3 (Lovenberg et al., 1999) and H4 (Liu et al., 2001), which belong to the GPCR superfamily (nomenclature of receptors follows Alexander et al., 2013).
The human histamine H3 receptor, first cloned by Lovenberg in 1999, is a Gi/o-coupled receptor composed of 445 amino acids. H3 receptors are expressed in brain and neuroendocrine tissues such as enterochromaffin-like cells and adrenal cortex (Lovenberg et al., 1999). When ligands bind to H3 receptor-activated Gi proteins (Clark and Hill, 1996), they inhibit adenylyl cyclase and lead to decreased cAMP concentrations, followed by inactivation of PKA (Lovenberg et al., 1999). In addition to the well-known cAMP–PKA pathway, H3 receptors are involved in various types of intracellular signalling, such as the MAPK pathway (Drutel et al., 2001), the PI3K pathway and changes in intracellular Ca2+ concentration (Leurs et al., 2005). Interestingly, H3 receptors display intrinsic, constitutive activity in the absence of ligands. The H3 receptor agonists activate these receptors, whereas an H3 receptor inverse agonist, which has the opposite effect of an agonist, reduces H3 receptor activity to below the normal level (Morisset et al., 2000). In fact, native brain H3 receptors have a high constitutive activity, so the corresponding inverse agonists thioperamide and FUB465 increase histamine release with Gi inactivation, although the H3 receptor agonist imetit reduces histamine release with Gi activation (Morisset et al., 2000).
Since the inhibitory effect of neuronal histamine on food intake was first reported in 1973 (Clineschmidt and Lotti, 1973), many studies have shown that histamine acts as a neurotransmitter in energy homeostasis, modulating hypothalamic neuronal activities (Jorgensen et al., 2007). A H3 receptor inverse agonist increased the hypothalamic concentration of histamine, leading to reduced food intake and body weight (Malmlof et al., 2005). Conversely, a H3 receptor agonist suppressed hypothalamic histamine release and elicited feeding behaviour in rats (Chiba et al., 2009). Betahistine, a partial inverse agonist at H3 receptors, induced significant weight loss with minimal adverse events in women under 50 years of age (Barak et al., 2008). Moreover, the centrally acting H3 receptor protean agonist proxyfan reduced blood glucose level and exhibited anti-diabetic properties in mice (Henry et al., 2011). Based on all these findings, neuronal H3 receptors have attracted substantial attention as a potential therapeutic target in obesity (Leurs et al., 2005).
Although peripheral metabolic organs, as well as the CNS, are involved in a rigorous control of energy homeostasis, the direct actions of histamine on peripheral metabolic organs, such as liver, muscles and pancreatic islets of Langerhans, remain to be elucidated. Previous reports showed that muscles (Lovenberg et al., 2000) and liver (Heron et al., 2001) did not express H3 receptors, but it remained unclear whether pancreatic beta cells, which have similar properties to neurons, did express this receptor. Pancreatic beta cells in the islets of Langerhans crucially regulate energy homeostasis through appropriate insulin secretion in response to blood glucose level. These beta cells express a variety of GPCRs, modulating beta cell functions (Ahren, 2009). This is evident in the success of glucagon-like peptide 1 analogues, which increase insulin secretion and promote beta cell growth by activating stimulatory GPCRs (Drucker, 2006). Other GPCRs that regulate beta cell functions include neurotransmitter receptors such as muscarinic M3 cholinoceptors (Gautam et al., 2006) and α2A-adrenoceptors (Devedjian et al., 2000; Rosengren et al., 2010).
Pharmacological analysis identified the H3 receptor as an autoreceptor that inhibiting histamine release from neurons (Arrang et al., 1983). Subsequently, extensive investigations have revealed that brain H3 receptors regulate the release of various neurotransmitters, such as ACh, glutamate and GABA, and are involved in mediating the effect of histamine in numerous physiological processes, including the sleep–wake cycle and cognition (Haas et al., 2008). In peripheral tissues, H3 receptors are expressed in neuroendocrine organs and regulate their functions. For example, the activation of H3 receptors inhibited the release of adrenocorticotropic hormone and prolactin from the pituitary gland (Knigge et al., 1999) and of histamine from enterochromaffin-like cells (Kidd et al., 1996).
These lines of evidence suggested that histamine receptors might be involved in the functions of pancreatic beta cells. Therefore, we investigated histamine receptor expression in pancreatic beta cells and the role of histamine receptors in insulin secretion and beta cell proliferation.
- Top of page
- Discussion and conclusions
- Conflicts of interest
All animal care and experimental procedures used in this study were in accordance with the Principles for Care and Use of Research Animals of Tohoku University, Sendai, Japan. All experiments involving animals are reported in accordance with the ARRIVE guidelines for reporting experiments involving animals (Kilkenny et al., 2010; McGrath et al., 2010). Male wild-type (WT) ICR mice were purchased from Japan SLC (Hamamatsu, Japan). Male H3 receptor gene knockout (H3KO) C57BL/6 mice (Okuda et al., 2009) and male histidine decarboxylase gene knockout (HdcKO) C57BL/6 mice (Ohtsu and Watanabe, 2003) were maintained in our laboratory. All mice were maintained on a 12 h light/dark cycle (0800/2000 h) in a humidity- and temperature-controlled room, and were allowed ad libitum access to water and food (Labo MR Breeder; Nosan, Yokohama, Japan). Twenty-five WT ICR mice, five H3KO mice and three HdcKO mice were used in this study.
MIN6 cells (Miyazaki et al., 1990), a cell line derived from mouse pancreatic islet beta cells (kindly donated by Professor Jun-ichi Miyazaki, Faculty of Medicine, Osaka University; passage 20–35), were maintained in DMEM (Sigma, St. Louis, MO, USA) containing 4500 mg·L−1 d-glucose, 10% fetal calf serum (FCS; Thermo, Waltham, MA, USA), 100 U·mL−1 penicillin G potassium (Wako, Osaka, Japan) and 50 mg·L−1 streptomycin sulphate (Wako) at 37°C in 5% CO2 in a humidified incubator.
Isolation of mouse pancreatic islets
WT ICR mice (12 weeks old) were anaesthetized with isoflurane (initial dose 3.0%, subsequent dose 2.5%, air flow 2 L·min−1) then laparotomized. The pancreata were perfused with Hank's solution (Nissui, Tokyo, Japan) through a 27 G needle placed into the common bile duct. The removed pancreata were then digested using collagenase (Wako) as described previously (Okamoto et al., 1975), and pancreatic islets were isolated by handpicking under a stereomicroscope. We used ICR mice for the preparation of islets because bile ducts of C57BL/6 mice were smaller and difficult to cannulate.
Reverse transcription (RT)-PCR
Total RNA was isolated from mouse pancreatic islets and MIN6 cells using NucleoSpin RNAII mini spin kits (Macherey-Nagel, Duren, Germany), and 500 ng of this RNA was then reverse-transcribed using PrimeScriptII (Takara-Bio, Otsu, Japan). Diluted reverse-transcribed samples (equivalent to 5 ng total RNA) were amplified by 35 cycles of PCR (10 s at 98°C, 30 s at 58°C and 90 s at 72°C) with specific oligonucleotide primers using an ABI 2710 thermal cycler (Applied Biosystems, Foster City, CA, USA). The primers used to detect histamine receptors are shown in Table 1. RT-PCR in the absence of reverse transcriptase was used as negative control. For positive controls, we used cDNA from mouse hypothalamus for H1 receptors and H3 receptors, and cDNA from mouse cardiac muscle and mouse mast cells was used for H2 and H4 receptors respectively.
|Sense primers||Antisense primers||Product size (bp)|
|Hdc||5′- TGTGACGTCCCAGTTTCCA-3′||5′- GTGGATCACGAAGACCCTGT-3′||777|
|Actb||5′- TGTTACCAACTGGGACGACA-3′||5′-GTACTTGCGCTCAGGAGGAG -3′||687|
Mouse H3 receptor antibody production
Rabbit anti-mouse H3 receptor antibody was produced for us by Operon Biotechnology (Tokyo, Japan). Rabbits (n = 2) were immunized with keyhole limpet haemocyanin-conjugated mouse H3 receptor peptides (amino acids: 142–155 and 428–442). An adequate increase in serum antibody titre was confirmed by elisa. Collected serum was purified by antigen-specific affinity chromatography, and the purified antibody was used to detect mouse H3 receptor protein.
H3 receptor positive control for Western blot
The mouse H3 receptor gene was cloned from WT ICR mouse islet cDNA and inserted into the pCI-neo vector (Promega, Fitchburg, WI, USA) using the In-Fusion PCR Cloning System (Clontech Bioinformatics, Mountain View, CA, USA). The plasmid construct was amplified in Escherichia coli and purified using a NucleoBond Xtra Maxi-prep kit (Macherey-Nagel). Purified plasmid was transfected into CHO cells using Lipofectamine LTX (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Transfected CHO cells were lysed with RIPA buffer (Thermo), and the cell lysate was used as the positive control in Western blots.
Isolated pancreatic islets from 12-week-old mice (n = 5) and MIN6 cells were lysed with RIPA buffer. We used the lysate of H3 receptor-overexpressing CHO cells as positive control. The lysate of CHO cells transfected with plasmid DNA containing no insert was used as negative control. Samples were denatured at 75°C for 5 min. Aliquots containing 10 μg of protein were applied to 5–20% gradient polyacrylamide gels (Bio-Rad, Hercules, CA, USA) and separated by SDS-PAGE at constant voltage of 200 V for 30 min. After SDS-PAGE, the samples were transferred to a PVDF membrane by blotting at a constant current of 2.5 A for 3 min. The PVDF membrane was blocked by agitating in 5% non-fat dry milk solution for 1 h, then immunoblotted with rabbit anti-mouse H3 receptor antibody (0.4 μg·mL−1 in 5% BSA solution) or rabbit anti-β-actin antibody (Cell Signaling Technology, Danvers, MA, USA; 1:2000 dilution), and incubated at room temperature for 1 h. At the end of the incubation period, the membrane was washed then incubated with a HRP-labelled donkey anti-rabbit antibody (Thermo) as the secondary antibody (1:25 000 dilution in non-fat dry milk solution) at room temperature for 1 h. Immunoreactive bands were detected using an ECL Western Blotting Analysis System (PerkinElmer, Waltham, MA, USA), and bands were recorded using the Ez-capture MG chemiluminescence imaging system (Atto, Tokyo, Japan).
Pancreata were quickly removed from 12-week-old WT (n = 5), H3KO (n = 3) and HdcKO (n = 3) mice. Isolated pancreata were fixed in 10% paraformaldehyde and embedded in paraffin. The sections were then deparaffinized and blocked with 10% goat serum at room temperature for 15 min. To detect insulin, the sections were incubated with mouse anti-insulin antibody (Sigma; 1:2000 dilution) for 2 h at room temperature, and then treated with the secondary antibody: Alexa Fluor (Invitrogen) 568-conjugated goat anti-mouse antibody (1:100 dilution). To detect mouse H3 receptors, Hdc and histamine in mouse specimens, samples were incubated with rabbit anti-mouse H3 receptor antibody (4 μg·mL−1), rabbit anti-Hdc antibody (Progen Biotechnik, Heidelberg, Germany; 1:200 dilution) and rabbit antihistamine antibody (Sigma; 1:50 dilution), respectively, overnight at 4°C, and then treated with the secondary antibody: Alexa Fluor 488-conjugated goat anti-rabbit antibody (Invitrogen). For double staining of insulin and H3 receptors, Hdc or histamine, samples were incubated with anti-insulin antibody for 2 h at room temperature, then washed and labelled with antibodies to H3 receptors, Hdc or histamine and incubated overnight at 4°C. Specimens from H3KO and HdcKO mice were used as negative controls for H3 receptors and Hdc respectively. As a negative control for histamine, the antihistamine antibody was incubated in 1 mM histamine buffer at room temperature for 30 min before it was added to the specimens.
Anonymized paraffinized human specimens (n = 2) were obtained from Tohoku University pathological library. The use of human pancreatic samples was approved by the ethical committee of Tohoku University Graduate School of Medicine. Because the human samples exhibited high autofluorescence, we were unable to use the same staining method as we used for the mouse samples and instead we used non-fluorescent methods to stain human sections. Deparaffinized sections were incubated with mouse anti-insulin antibody (Sigma; 1:2000 dilution) then treated with chromogenic Vector Red (Vector, Burlingame, CA, USA). After washing with PBS, rabbit anti-human H3 receptor antibody (Acris, Herford, Germany; 1:100 dilution) was added to the incubation buffer, followed by treatment with chromogenic HistoGreen (Cosmobio, Tokyo, Japan). Parallel sections were incubated with non-immune rabbit Ig fraction (DAKO, Glostrup, Denmark) instead of human H3 receptor antibody as a negative control. To detect Hdc and histamine in human specimens, deparaffinized sections were incubated with rabbit antihistamine antibody (Sigma) and rabbit anti-Hdc antibody (Progen Biotechnik), respectively, overnight at 4°C, and then treated with goat anti-rabbit antibody conjugated with HRP (Nissui, Tokyo, Japan). Colour was developed by diaminobenzidine, followed by haematoxylin contrast staining.
MIN6 cells, seeded into a 24 well plate at a density of 2.0 × 105 cells per well, were incubated in 1 mL of DMEM containing 5% horse serum (Gibco, Carlsbad, CA, USA) ±1 mM histidine (Wako) for 1, 2, 4, 8 and 24 h at 37°C in 5% CO2. In this assay, we used horse serum as substitute for FCS because FCS contains much higher concentration of histaminase, a histamine-degrading enzyme (Dy et al., 1982). After harvesting the medium from each well, the histamine concentration in the medium was measured by HPLC (Yamatodani et al., 1985). Histamine contents in the medium without MIN6 cells were used as blank controls.
MIN6 cells, seeded into a 24 well plate at a density of 2.0 × 105 cells per well, were pre-incubated in 500 μL of Krebs–Ringer bicarbonate buffer (KRB) containing 2.8 mM glucose for 30 min at 37°C in 5% CO2. Following pre-incubation, MIN6 cells were stimulated in 500 μL of KRB containing 2.8 or 16.7 mM glucose or 20 mM KCl ±1 μM imetit (Sigma), a selective H3 receptor agonist (Garbarg et al., 1992), or 1 μM JNJ-5207852 (kindly donated by Dr. Nicholas Carruthers, Johnson & Johnson Pharmaceutical Research and Development, USA), a potent and selective H3 receptor inverse agonist, for 1 h at 37°C in 5% CO2 (Barbier et al., 2004; Jia et al., 2006; Bonaventure et al., 2007). Imetit and JNJ-5207852 were both dissolved in distilled water. The insulin concentration in KRB was determined using an insulin elisa kit (Morinaga Institute of Biological Science, Yokohama, Japan). Another insulin secretion experiment was designed to examine the effects of imetit efflux after blockade of regulatory G proteins using Pertussis toxin (PTX; Sigma; Clark et al., 1993). This was performed using the same insulin secretion experimental protocol described above, with the addition of KRB buffer containing PTX (10 ng·mL−1).
Measurement of intracellular ATP concentration
We measured intracellular ATP concentration in static cells, which is a method commonly used in pancreatic beta cell research (Uchizono et al., 2004). MIN6 cells, seeded into a 96 well plate at a density of 2.0 × 104 cells per well, were incubated under the same conditions used for insulin secretion. After the buffer was removed from the plate, intracellular ATP concentration was quantitated using Cell Titer-glo (Promega) using a luminometer (Berthold Technologies, Bad Wildbad, Germany; Lin and Anseth, 2011).
Measurement of intracellular Ca2+ concentration
Because intracellular Ca2+ concentration changes much faster than ATP, we could not use static measurement methods as for ATP but instead, it was necessary to measure changes dynamically by perfusion experiments. MIN6 cells, seeded into 35 mm diameter glass-bottomed dishes at a density of 6.0 × 105 cells per dish, were pre-incubated in KRB containing 2.8 mM glucose and 2 μM fura-2/AM (Invitrogen) for 30 min at 37°C (Nakazaki et al., 1998). Following pre-incubation, MIN6 cells were continuously perfused with preheated buffer at 2 mL·min−1 on the stage of an inverted microscope (Olympus, Tokyo, Japan). Cells were excited alternately at 340 and 380 nm. Light emitted through a 510 nm filter was detected using a cooled CCD camera (Hamamatsu Photonics, Hamamatsu, Japan) and analysed with MetaMorph software (Molecular Devices, Sunnyvale, CA, USA).
Bromodeoxyuridine (BrdU) assay
MIN6 cells, seeded at equal numbers of 2.0 × 104 cells per well into a 96 well plate, were incubated in DMEM containing 2.8 mM glucose and 100 pM insulin (Sigma) at 37°C for 1, 2, 4, 8 or 24 h. Fifteen minutes after the start of the incubation, 1 μM BrdU was added for the remainder of the incubation period. BrdU incorporation was measured using a BrdU Labelling and Detection Kit3 (Roche, Basel, Switzerland), and absorbance at 405 nm was measured with a spectrophotometer (Molecular Devices).
Measurement of intracellular cAMP concentration
MIN6 cells seeded into a 24 well plate were stimulated using the same protocol as for insulin secretion. At the end of the incubation period, the cells were lysed in 0.1 M HCl. The intracellular cAMP concentration was measured using a cAMP EIA kit (Cayman, Ann Arbor, MI, USA). The protein concentration of the cell lysate was also measured using the Bradford protein assay.
cAMP response element-binding protein (CREB) and Akt phosphorylation assay
MIN6 cells, seeded into a 6 well plate at a density of 1.0 × 106 cells per well, were pre-incubated in KRB containing 2.8 mM glucose for 2 h then incubated in 16.7 mM glucose/KRB ±1 μM imetit for 15 min. After incubation, MIN6 cells were lysed with RIPA buffer (Thermo) containing protease inhibitor (Roche). SDS-PAGE was performed following the same protocol as for detection of H3 receptor protein. Samples were then transferred to PVDF membranes and immunoblotted with rabbit anti-CREB antibody, anti-phosphorylated CREB antibody, anti-Akt antibody, anti-phosphorylated Akt antibody (all at 1:1000 dilution) and anti-β-actin antibody (1:2000 dilution; all from Cell Signaling Technology). Immunoreactive bands were detected using HRP-labelled anti-rabbit antibody (Thermo; 1:25 000 dilution) as a secondary antibody and visualized with an ECL Western Blotting Analysis System (PerkinElmer).
Data are expressed as the mean ± SE. Statistical analysis was performed using Student's t-test. Differences were considered significant at P-values less than 0.05. IC50, Imax, EC50 and Emax parameters were calculated using Prism 5 software (GraphPad Software, La Jolla, CA, USA) with non-linear regression analysis. All experiments were performed more than once. No significant differences were found between repeated experiments.
- Top of page
- Discussion and conclusions
- Conflicts of interest
H3 receptors are expressed in pancreatic beta cells
RT-PCR was used to identify the histamine receptor subtypes expressed in primary mouse islets. H3 receptor mRNA was highly expressed in mouse islets (Figure 1A), whereas mRNA for none of the other histamine receptors were detectable. Expression of H3 receptor protein in mouse pancreatic islets was confirmed by Western blotting (Figure 1B). Pancreatic islets consist mainly of α-cells and beta cells, and immunohistochemical analysis of pancreatic islets showed that insulin-positive beta cells (stained red) also expressed H3 receptorprotein (stained green), indicating that H3 receptors were expressed in insulin-positive mouse beta cells (Figure 1C). Moreover, immunohistochemical analysis showed that human pancreatic beta cells also express H3 receptors (Figure 1D). These results together suggest that pancreatic beta cells, in general, express H3 receptors.
Hdc and histamine are expressed in pancreatic beta cells
We next investigated the expression of histamine and of Hdc (Ohtsu, 2010), the enzyme essential for histamine synthesis, by RT-PCR and immunostaining pancreatic sections. MIN6 cells and mouse pancreatic islets both expressed Hdc mRNA (Figure 1A). The exocrine pancreas did not express histamine or Hdc; in contrast, mouse and human pancreatic islets expressed both (Figure 2A–C), indicating that histamine synthesized by Hdc in pancreatic islets could stimulate H3 receptors in an autocrine or paracrine manner.
Histamine is secreted by MIN6 cells
To evaluate whether MIN6 cells generate and secrete histamine, we measured the amount of histamine in the medium after histidine loading. Histamine secretion from MIN6 cells increased in a time-dependent manner in the presence of histidine (Figure 2D), suggesting that mouse pancreatic beta cells synthesized and secreted histamine.
Activation of H3 receptors inhibits glucose-induced insulin secretion (GIIS) in MIN6 cells
We used MIN6 cells to assay expression of functional H3 receptors in pancreatic beta cells because pancreatic islets contain 10–30% of other endocrine cells, such as glucagon-releasing α-cells and somatostatin-releasing δ-cells. The MIN6 cell line, derived from mouse pancreatic beta cells, is widely used in research on pancreatic beta cell function because MIN6 cells are sensitive to the presence of glucose and secrete insulin in response to it. We confirmed that MIN6 cells expressed both H3 receptor mRNA (Figure 1A) and protein (Figure 1B).
GIIS is one of the most important functions of pancreatic beta cells, enabling precise maintenance of glucose homeostasis. We investigated the role of H3 receptors expressed in MIN6 cells by stimulating MIN6 cells with glucose-containing KRB buffer in the absence or presence of the H3 receptor agonist imetit. Insulin secretion increased more than twofold over basal levels in response to high concentrations of glucose (16.7 mM). This insulin secretion associated with high glucose levels was inhibited by imetit (P < 0.05), whereas basal secretion at 2.8 mM glucose with imetit was maintained at close to control levels (Figure 3A). Subsequent experiments were therefore performed with high glucose (16.7mM). We examined the effects of various concentrations of imetit (10 nM to 100 μM) on insulin secretion. As shown in Figure 3B, imetit inhibited GIIS in a dose-dependent manner, with an IC50 of 1.95 μM and an Imax value of 68%. To confirm whether imetit reduced GIIS via the Gi activation of H3 receptors, we treated MIN6 cells with PTX, an inhibitor of Gi protein activity (Clark et al., 1993). The reduction in insulin secretion seen with 100 μM imetit was completely blocked by exposure to PTX (Figure 3E), confirming that imetit inhibited GIIS by activating the Gi protein associated with the H3 receptors. These results indicate that H3 receptors negatively regulate insulin secretion in pancreatic beta cells, via Gi proteins.
The H3 receptor inverse agonist, JNJ-5207852, increases GIIS in a dose-dependent manner
Next, we investigated the effects of an H3 receptor inverse agonist, JNJ-5207852, on insulin secretion. We assumed that an H3 receptor inverse agonist would increase insulin secretion from MIN6 cells because of the high constitutive activity of H3 receptors in neurons (Morisset et al., 2000). As expected, the level of insulin secretion stimulated by 16.7 mM glucose was increased by 25% in the presence of 1 μM JNJ-5207852 (P < 0.05; Figure 3C), whereas basal insulin secretion was not affected by the inverse agonist. JNJ-5207852 increased GIIS in a dose-dependent manner, with an EC50 of 13.8 μM and an Emax of 103% (Figure 3D). These results indicate that H3 receptors expressed in pancreatic beta cells are constitutively active and that H3 receptor inverse agonists could be used as insulin secretagogues.
Inhibition of GIIS mediated by H3 receptors is not accompanied by changes in ATP or intracellular Ca2+ concentration
We next investigated which part of the GIIS pathway was affected by intracellular H3 receptor signalling. The GIIS signalling pathway can be divided into four steps, as follows (Leibiger et al., 2008): (i) glucose imported into beta cells is metabolized to ATP; (ii) increased ATP induces closure of ATP-sensitive potassium channels followed by plasma membrane depolarization; (iii) depolarization opens voltage-dependent calcium channels, leading to Ca2+ influx; and (iv) increased intracellular Ca2+ evokes insulin granule exocytosis.
Because an increase in ATP concentration due to glucose uptake and metabolism is necessary for insulin secretion, we first examined the effects of imetit on ATP concentrations. The intracellular ATP concentration was increased by stimulation with 16.7 mM glucose, but imetit did not reduce the intracellular ATP concentration at either a low or a high glucose concentration (Figure 4A), suggesting that intracellular signalling from the H3 receptor affected the process of GIIS signalling after the increase in intracellular ATP concentration. Next, we investigated the effect of imetit on insulin secretion induced by 20 mM KCl, which directly depolarizes the plasma membrane and leads to a raised intracellular Ca2+ concentration, followed by insulin secretion. Insulin secretion in response to 20 mM KCl was reduced by imetit (Figure 4C). The extent of inhibition of KCl-induced insulin secretion by imetit was quite similar to that of GIIS (22 vs. 24% reduction), suggesting that intracellular signalling from the H3 receptor affects the steps downstream of membrane depolarization, increasing intracellular Ca2+ concentration and/or insulin granule exocytosis.
We next examined the effects of imetit on the increase in intracellular Ca2+ concentration in response to 20 mM KCl or 16.7 mM glucose. Imetit did not affect the increase in intracellular Ca2+ concentration in response to 20 mM KCl (transient changes in Ca2+; 1.74 ± 0.21 vs. 1.75 ± 0.09; n = 4; Figure 4E) and 16.7 mM glucose (transient changes in Ca2+; 0.87 ± 0.08 vs. 0.81 ± 0.10; n = 4; Figure 4G). These results suggest that intracellular signalling from the H3 receptor inhibits GIIS downstream of Ca2+ influx.
We also investigated the effect of JNJ-5207852 and found that it did not change the ATP concentration at either low or high glucose concentrations (Figure 4B). The extent of the stimulation of KCl-induced insulin secretion by JNJ-5207852 was similar to that of GIIS (38 vs. 39%; Figures 3C and 4D). Moreover, the intracellular Ca2+ concentration induced by 20 mM KCl (transient changes in Ca2+; 1.88 ± 0.20 vs. 1.83 ± 0.20; n = 4; Figure 4F) or 16.7 mM glucose (transient changes in Ca2+; 1.35 ± 0.22 vs. 1.32 ± 0.25; n = 4; Figure 4H) was not affected by JNJ-5207852.
To summarize all the results of the GIIS signalling experiments, H3 receptor signalling did not alter either the intracellular ATP or Ca2+ concentration.
Imetit inhibits the proliferation of MIN6 cells
We investigated the effect of H3 receptors on the proliferation of MIN6 cells because a variety of GPCRs expressed in pancreatic beta cells are known to regulate beta cell proliferation (Ahren, 2009). BrdU incorporation increased more than threefold in response to insulin and glucose, compared with basal BrdU incorporation (Figure 5A). This BrdU incorporation in the presence of insulin and glucose was significantly inhibited by imetit after 24 h incubation (Figure 5A). Imetit inhibited BrdU incorporation in a time-dependent manner (Figure 5A). Insulin stimulates beta cell proliferation through the phosphorylation of Akt (Saltiel and Kahn, 2001), but Akt phosphorylation was not affected by imetit (Figure 5B). However, glucose stimulates beta cell proliferation through the cAMP–CREB pathway (Jhala et al., 2003; Hussain et al., 2006). Imetit tended to reduce the intracellular concentration of cAMP stimulated by 16.7 mM glucose, although this reduction was not significant (Figure 5C), and also led to a small reduction in the level of phosphorylated CREB (Figure 5D). These results indicate that agonists at the H3 receptor inhibit MIN6 cell proliferation, possibly through the cAMP–CREB pathway.
Discussion and conclusions
- Top of page
- Discussion and conclusions
- Conflicts of interest
We demonstrate for the first time that H3 receptors were expressed in mouse and human pancreatic beta cells. Some H3 receptor-positive cells at the margin of mouse pancreatic islets did not correspond to insulin-positive cells, as shown in Figure 1C. WT mouse pancreatic islets typically exhibit a core and mantle structure, comprising a core of beta cells surrounded by a mantle of α-, δ- and PP-cells (Boyer et al., 2006). H3 receptors could be expressed not only in beta cells but also in these other cell types. H3 receptor protein was detected in the cytoplasm in addition to the plasma membrane (Figure 1C,D), suggesting that some of the H3 receptors are internalized by ligand binding in pancreatic beta cells (Shi et al., 2012).
We also show for the first time that the H3 receptor plays an important role in insulin secretion and in beta cell proliferation. Interestingly, an H3 receptor inverse agonist, JNJ-5207852, increased GIIS, indicating that H3 receptor inverse agonists could be novel insulin secretagogues. Although conventional insulin secretagogues, such as the sulfonylureas, have strong effects on insulin secretion from pancreatic beta cells, they induce weight gain because of the elevated glucose utilization of metabolic organs (UK Prospective Diabetes Study Group, 1998). As a result, obese patients with diabetes taking sulfonylureas need to diet to lose weight, but it is sometimes difficult to alter their dietary habits (Halford et al., 2010). In contrast, H3 receptor inverse agonists have been shown to suppress food intake and reduce body weight by modulating histaminergic neurons (Malmlof et al., 2005; Barak et al., 2008; Chiba et al., 2009).
Pancreatic islets and/or autonomic nerves adjacent to the islets contain neurotransmitters such as ACh and noradrenaline and also express the enzymes that synthesize them (Gautam et al., 2006; Rosengren et al., 2010). These neurotransmitters bind to their GPCRs in beta cells and modulate beta cell functions. The physiological serum concentration of histamine is about 5 nM (Maintz and Novak, 2007), which is insufficient to stimulate H3 receptors. In fact, pancreatic beta cells have been shown to express vesicular monoamine transporter 2 (VMAT2), which transports histamine to secretory vesicles (Saisho et al., 2008). A VMAT2-specific antagonist, tetrabenazine, reportedly plays an important role in histamine transport into secretory vesicles (Erickson et al., 1995). Therefore, it is possible that histamine synthesized by Hdc is transported to secretory vesicles via VMAT2 and that histamine released from beta cells regulates beta cell functions in an autocrine and/or paracrine manner.
The potencies of the H3 receptor ligands in regulating GIIS by MIN6 cells were somewhat lower (Fig. 3B,D) than expected from their high potencies in other systems. The IC50 of imetit at the H3 receptors affecting insulin secretion by MIN6 cells was 100 times higher than its IC50 for histamine release by neurons (Garbarg et al., 1992). Because the level of receptor expression influences drug potency (Wilson et al., 1996), the high potency of H3 receptor ligands in neurons could be attributed to the high neuronal expression of H3 receptors. In fact, neurons express the highest levels of H3 receptors and thus agonists have a much lower IC50 value, compared with those in other cells and tissues. The IC50 value of imetit at the H3 receptors in MIN6 cells (about 2 μM) is not extremely high, compared with those in other non-neuronal tissues. For example, in rat mesenteric resistance arteries, another potent H3 receptor agonist, α-methyl histamine, had a IC50 value of about 10 μM (Sun et al., 2010). Moreover, the expression of H3 receptors in pancreatic beta cells was higher than that in MIN6 cells (Figure 1B). In addition, histamine and Hdc are both expressed in both mouse and human pancreatic islets (Figure 2), so the local histamine concentration might be high enough to stimulate the H3 receptors in pancreatic beta cells.
To elucidate the regulatory mechanisms underlying the effects of activated H3 receptors, we also investigated the effects of imetit and JNJ-5207852 on the insulin secretion signalling pathway. Neither imetit nor JNJ-5207852 increased intracellular ATP or Ca2+ concentrations, even though both ligands significantly affected insulin secretion. These results suggest that H3 receptor-mediated signalling is involved in insulin granular exocytosis, secondary to the increases in ATP and Ca2+. Renstrom et al. (1996) reported that adrenaline reduced insulin secretion by interfering with exocytosis downstream of the elevation in intracellular Ca2+ concentration through the activation of calcineurin. Recently, Rosengren et al. (2010) confirmed that activation of α2A-adrenoceptors, Gi/o-type GPCRs, inhibited insulin granule exocytosis through calcineurin activation, indicating that H3 receptors might inhibit insulin granule exocytosis through the same activation of calcineurin. GIIS is also amplified by calcium/calmodulin-dependent PK II (CaMK II). CaMK II phosphorylates synapsin I which, in turn, increases insulin exocytosis (Yamamoto et al., 2003). Activation of H3 receptors down-regulated CaMK II activity (Torrent et al., 2005), suggesting that H3 receptors might inhibit insulin granule exocytosis by reducing CaMK II activity coupled to the reduction of synapsin I phosphorylation. However, the precise mechanism(s) that are utilised following H3 receptor activation need to be confirmed in further studies.
Although proliferation of human beta cells in vivo is still controversial (Cnop et al., 2009), pancreatic beta cells need to proliferate to maintain their numbers and the number of pancreatic beta cells is dramatically reduced in patients with diabetes (Butler et al., 2003), so the molecular mechanism underlying impaired beta cell proliferation is interesting in terms of therapeutic potential. Various studies have shown that H3 receptors are involved in cell proliferation, but their role was different among different tissues. Activation of H3 receptors decreased the proliferation of biliary cells (Francis et al., 2007) and hepatoma cells (Davenas et al., 2008) through the inhibition of a cAMP-dependent pathway. In contrast, H3 receptor activation protected against neurotoxicity by stimulating the PI3K/Akt pathway (Bongers et al., 2007). The effects of activating H3 receptors on proliferation probably depend on cell-specific, intracellular signalling. We showed here that H3 receptor activation by imetit inhibited MIN6 cell proliferation (Figure 5A). Imetit also showed a trend towards decreased cAMP concentration and inhibition of the phosphorylation of CREB, a downstream target of cAMP. This pathway plays an important role in mediating intracellular signalling from the factors that promote beta cell proliferation, such as incretin (Jansson et al., 2008) and calcineurin (Soleimanpour et al., 2010). Therefore, H3 receptor activation in pancreatic beta cells might transduce signals to the cAMP–CREB pathway, but not the PI3K/Akt pathway, resulting in inhibition of beta cell proliferation. H3 receptor antagonists/inverse agonists may have proliferative effects on pancreatic beta cells by enhancing the phosphorylation of CREB, and this is supported by the fact that JNJ-5207852 was shown to increase intracellular cAMP concentration in a human neuroblastoma cell line (Barbier et al., 2004). However, further details of the effects of H3 receptor activation on beta cell proliferation need to be elucidated.
One of the limitations of this study is that all the functional analyses of H3 receptors were performed in MIN6 cells, a cell line derived from a mouse pancreatic tumour (Miyazaki et al., 1990). Although MIN6 cells secrete amounts of insulin equivalent to cultured normal islet cells, the MIN6 cell line may not retain all the physiological properties of pancreatic beta cells. Moreover, the local histamine concentration in pancreatic islets in vivo has not been determined, so further studies are needed to elucidate the in vivo function of H3 receptors expressed in pancreatic beta cells.
In the present study, we investigated the role of H3 receptors in pancreatic beta cells using imetit and JNJ-5207852. The insulin secretion study clearly demonstrates that activation of H3 receptors inhibits insulin secretion, possibly by regulating exocytosis. In addition, H3 receptors could be involved in the proliferation of pancreatic beta cells, possibly through the phosphorylation of CREB. These lines of evidence might indicate that the H3 receptor could play an important role in beta cell functions.
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- Discussion and conclusions
- Conflicts of interest
This study was supported by a grant from the HIROMI Medical Research Foundation. We thank Professors T. Watanabe and H. Okamoto at Tohoku University for encouraging us to perform this study. We also express our gratitude to Professor Jun-ichi Miyazaki at Osaka University, Dr. Nicholas Carruthers at Johnson & Johnson Pharmaceutical Research and Development and Dr. Jun Sukegawa at Tohoku University for providing experimental materials. Finally, we appreciate the technical assistance provided by F. Naganuma, R. Harada, A. Mohsen, K. Shibuya and Tohoku University Graduate School of Medicine Pathological Platform in the experiments.
Conflicts of interest
- Top of page
- Discussion and conclusions
- Conflicts of interest
The authors declare no conflict of interest.
- Top of page
- Discussion and conclusions
- Conflicts of interest
- 2009). Islet G protein-coupled receptors as potential targets for treatment of type 2 diabetes. Nat Rev Drug Discov 8: 369–385. (
- 2013). The Concise Guide to PHARMACOLOGY 2013/14: Overview. Br J Pharmacol 170: 1449–1867. et al. (
- 1983). Auto-inhibition of brain histamine release mediated by a novel class (H3) of histamine receptor. Nature 302: 832–837. , , (
- 2008). Effect of histaminergic manipulation on weight in obese adults: a randomized placebo controlled trial. Int J Obes (Lond) 32: 1559–1565. , , , , (
- 2004). Acute wake-promoting actions of JNJ-5207852, a novel, diamine-based H3 antagonist. Br J Pharmacol 143: 649–661. , , , , , et al. (
- 2007). Histamine H3 receptor antagonists: from target identification to drug leads. Biochem Pharmacol 73: 1084–1096. , , , , , et al. (
- 2007). The Akt/GSK-3beta axis as a new signaling pathway of the histamine H(3) receptor. J Neurochem 103: 248–258. , , , , , et al. (
- 2006). Complementation rescue of Pdx1 null phenotype demonstrates distinct roles of proximal and distal cis-regulatory sequences in pancreatic and duodenal expression. Dev Biol 298: 616–631. , , , , , (
- 2003). Beta-cell deficit and increased beta-cell apoptosis in humans with type 2 diabetes. Diabetes 52: 102–110. , , , , , (
- 2009). Acute central administration of immepip, a histamine H3 receptor agonist, suppresses hypothalamic histamine release and elicits feeding behavior in rats. Brain Res Bull 79: 37–40. , , , (
- 1996). Sensitivity of histamine H3 receptor agonist-stimulated [35S]GTP gamma[S] binding to pertussis toxin. Eur J Pharmacol 296: 223–225. , (
- 1993). Guanine nucleotides and pertussis toxin reduce the affinity of histamine H3 receptors on AtT-20 cells. Agents Actions 40: 129–134. , , (
- 1973). Histamine: intraventricular injection suppresses ingestive behavior of the cat. Arch Int Pharmacodyn Ther 206: 288–298. , (
- 2009). The long lifespan and low turnover of human islet beta cells estimated by mathematical modelling of lipofuscin accumulation. Diabetologia 53: 321–330. , , , , , et al. (
- 2008). Autoregulation of McA-RH7777 hepatoma cell proliferation by histamine H3 receptors. J Pharmacol Exp Ther 326: 406–413. , , , (
- 2000). Transgenic mice overexpressing alpha2A-adrenoceptors in pancreatic beta-cells show altered regulation of glucose homeostasis. Diabetologia 43: 899–906. , , , , , et al. (
- 2006). The biology of incretin hormones. Cell Metab 3: 153–165. (
- 2001). Identification of rat H3 receptor isoforms with different brain expression and signaling properties. Mol Pharmacol 59: 1–8. , , , , , et al. (
- 1982). Presence of histaminase in fetal calf sera. J Immunol Methods 48: 261–265. , , (
- 1995). Reserpine- and tetrabenazine-sensitive transport of 3H-histamine by the neuronal isoform of the vesicular monoamine transporter. J Mol Neurosci 6: 277–287. , , , (
- 2007). H3 histamine receptor agonist inhibits biliary growth of BDL rats by downregulation of the cAMP-dependent PKA/ERK1/2/ELK-1 pathway. Lab Invest 87: 473–487. , , , , , et al. (
- 1994). Molecular cloning of the human histamine H1 receptor gene. Biochem Biophys Res Commun 201: 894–901. , , , , , et al. (
- 1991). Molecular cloning of the human histamine H2 receptor. Biochem Biophys Res Commun 178: 1386–1392. , , , , , et al. (
- 1992). S-[2-(4-imidazolyl)ethyl]isothiourea, a highly specific and potent histamine H3 receptor agonist. J Pharmacol Exp Ther 263: 304–310. , , , , , et al. (
- 2006). A critical role for beta cell M3 muscarinic acetylcholine receptors in regulating insulin release and blood glucose homeostasis in vivo. Cell Metab 3: 449–461. , , , , , et al. (
- UK Prospective Diabetes Study Group (1998). Intensive blood-glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with type 2 diabetes (UKPDS 33). UK Prospective Diabetes Study (UKPDS) Group. Lancet 352: 837–853.
- 2008). Histamine in the nervous system. Physiol Rev 88: 1183–1241. , , (
- 2010). Pharmacological management of appetite expression in obesity. Nat Rev Endocrinol 6: 255–269. , , , , (
- 2011). Antidiabetic properties of the histamine H3 receptor protean agonist proxyfan. Endocrinology 152: 828–835. , , , , , et al. (
- 2001). Expression analysis of the histamine H(3) receptor in developing rat tissues. Mech Dev 105: 167–173. , , , , , (
- 2006). Increased pancreatic beta-cell proliferation mediated by CREB binding protein gene activation. Mol Cell Biol 26: 7747–7759. , , , , , et al. (
- 2008). Glucose controls CREB activity in islet cells via regulated phosphorylation of TORC2. Proc Natl Acad Sci U S A 105: 10161–10166. , , , , , (
- 2003). cAMP promotes pancreatic beta-cell survival via CREB-mediated induction of IRS2. Genes Dev 17: 1575–1580. , , , , , et al. (
- 2006). Effects of histamine H3 antagonists and donepezil on learning and mnemonic deficits induced by pentylenetetrazol kindling in weanling mice. Neuropharmacology 50: 404–411. , , , , , et al. (
- 2007). Histamine and the regulation of body weight. Neuroendocrinology 86: 210–214. , , , (
- 1996). Autoregulation of enterochromaffin-like cell histamine secretion via the histamine 3 receptor subtype. Yale J Biol Med 69: 9–19. , , , , , (
- 2010). Animal research: reporting in vivo experiments: the ARRIVE guidelines. Br J Pharmacol 160: 1577–1579. , , , , (
- 1999). Stress-induced release of anterior pituitary hormones: effect of H3 receptor-mediated inhibition of histaminergic activity or posterior hypothalamic lesion. Neuroendocrinology 69: 44–53. , , , , , (
- 2008). Insulin signaling in the pancreatic beta-cell. Annu Rev Nutr 28: 233–251. , , (
- 2005). The histamine H3 receptor: from gene cloning to H3 receptor drugs. Nat Rev Drug Discov 4: 107–120. , , , (
- 2011). Cell-cell communication mimicry with poly(ethylene glycol) hydrogels for enhancing beta-cell function. Proc Natl Acad Sci U S A 108: 6380–6385. , (
- 2001). Cloning and pharmacological characterization of a fourth histamine receptor (H4) expressed in bone marrow. Mol Pharmacol 59: 420–426. , , , , , et al. (
- 1999). Cloning and functional expression of the human histamine H3 receptor. Mol Pharmacol 55: 1101–1107. , , , , , et al. (
- 2000). Cloning of rat histamine H3 receptor reveals distinct species pharmacological profiles. J Pharmacol Exp Ther 293: 771–778. , , , , (
- 2007). Histamine and histamine intolerance. Am J Clin Nutr 85: 1185–1196. , (
- 2010). Guidelines for reporting experiments involving animals: the ARRIVE guidelines. Br J Pharmacol 160: 1573–1576. , , , , (
- 2005). Influence of a selective histamine H3 receptor antagonist on hypothalamic neural activity, food intake and body weight. Int J Obes (Lond) 29: 1402–1412. , , , , , et al. (
- 1990). Establishment of a pancreatic beta cell line that retains glucose-inducible insulin secretion: special reference to expression of glucose transporter isoforms. Endocrinology 127: 126–132. , , , , , et al. (
- 2000). High constitutive activity of native H3 receptors regulates histamine neurons in brain. Nature 408: 860–864. , , , , , et al. (
- 1998). Repetitive mitochondrial Ca2+ signals synchronize with cytosolic Ca2+ oscillations in the pancreatic beta-cell line, MIN6. Diabetologia 41: 279–286. , , , , , et al. (
- 2010). Histamine synthesis and lessons learned from histidine decarboxylase deficient mice. Adv Exp Med Biol 709: 21–31. (
- 2003). New functions of histamine found in histidine decarboxylase gene knockout mice. Biochem Biophys Res Commun 305: 443–447. , (
- 1975). Inhibition by somatostatin of insulin release from isolated pancreatic islets. FEBS Lett 54: 103–105. , , , , (
- 2009). Methamphetamine- and 3,4-methylenedioxymethamphetamine-induced behavioral changes in histamine H3-receptor knockout mice. J Pharmacol Sci 111: 167–174. , , , , , et al. (
- 1996). Neurotransmitter-induced inhibition of exocytosis in insulin-secreting beta cells by activation of calcineurin. Neuron 17: 513–522. , , , (
- 2010). Overexpression of alpha2A-adrenergic receptors contributes to type 2 diabetes. Science 327: 217–220. , , , , , et al. (
- 2008). Relationship between pancreatic vesicular monoamine transporter 2 (VMAT2) and insulin expression in human pancreas. J Mol Histol 39: 543–551. , , , , , et al. (
- 2001). Insulin signalling and the regulation of glucose and lipid metabolism. Nature 414: 799–806. , (
- 2012). Identification and characterization of ZEL-H16 as a novel agonist of the histamine H3 receptor. PLoS ONE 7: e42185. , , , , , et al. (
- 2010). Calcineurin signaling regulates human islet β-cell survival. J Biol Chem 285: 40050–40059. , , , , , et al. (
- 2010). R-(-)-alpha-methylhistamine, a histamine H3 receptor agonist, induces endothelium-dependent vasodilation in rat mesenteric resistance arteries. Biol Pharm Bull 33: 58–63. , , , , , (
- 2005). H3 autoreceptors modulate histamine synthesis through calcium/calmodulin- and cAMP-dependent protein kinase pathways. Mol Pharmacol 67: 195–203. , , , , , et al. (
- 2004). Tacrolimus impairment of insulin secretion in isolated rat islets occurs at multiple distal sites in stimulus–secretion coupling. Endocrinology 145: 2264–2272. , , , , , (
- 1996). Agonist potency at the cloned human beta-3 adrenoceptor depends on receptor expression level and nature of assay. J Pharmacol Exp Ther 279: 214–221. , , , , , et al. (
- 2003). New aspects of neurotransmitter release and exocytosis: involvement of Ca2+/calmodulin-dependent phosphorylation of synapsin I in insulin exocytosis. J Pharmacol Sci 93: 30–34. , , , (
- 1985). High-performance liquid chromatographic determination of plasma and brain histamine without previous purification of biological samples: cation-exchange chromatography coupled with post-column derivatization fluorometry. J Chromatogr 344: 115–123. , , , , (