Excessive accumulation of ammonia in the brain is a causative factor of hepatic encephalopathy (HE), a neuropsychiatric syndrome associated with acute or chronic liver failure (Felipo and Butterworth 2002). Brain edema related to astrocytic swelling is a major cause of death in patients with acute HE. According to the current theory, ammonia causes astrocytic swelling by triggering a vicious cycle of oxidative/nitrosative stress (ONS) (Schliess et al. 2006; Häussinger and Görg 2010) and intramitochondrial accumulation of glutamine (Albrecht and Norenberg 2006). In cultured astrocytes, ammonia induces formation of reactive oxygen and nitrogen species (ROS/RNS) (Murthy et al. 2001; Schliess et al. 2002), in a process which also involves nitric oxide (NO) formation by overactivation of N-methyl-d-aspartate receptor (Schliess et al. 2002; Zielińska et al. 2003). The major toxic ROS species formed in ammonia-treated cultured astrocytes or brain slices is the superoxide anion (•O2−) stemming from activation of NADPH oxidase isoforms, the activation involving serine phosphorylation on the p47phox regulatory subunit of NADPH oxidase (Reinehr et al. 2007).
Molecular consequences of increased ROS/RNS production include protein and nucleic acids oxidation (Görg et al. 2008), protein tyrosine nitration and S-nitrosylation (Schliess et al. 2004), and modifications of lipid peroxidation (Swapna et al. 2006; Singh et al. 2008), all of which contribute to the pathogenesis of HE. Therefore, attenuation of free radicals production may be a desired target of HE therapy. We speculated that natriuretic peptides (NPs) are the compounds with a potential to counteract ONS produced by ammonia in the CNS. The hypothesis was essentially based on the data showing that NPs attenuate excessive ROS production in the cardiovascular system and liver cells (Woodard and Rosado 2008; De Vito et al. 2010). Of note, all three NPs: atrial natriuretic peptide (ANP), brain natriuretic peptide and C-type natriuretic peptide (CNP) are also present in the brain (Herman et al. 1996; Potter et al. 2006). NPs exert their actions by binding to natriuretic peptide receptors (NPRs): ANP binds preferentially to NPR-A, and brain natriuretic peptide and CNP to NPR-B, respectively (Lucas et al. 2000). All NPs bind with equal affinity to NPR-C (Lucas et al. 2000). NPR-A and NPR-B are particulate, cell membrane-bound guanylyl cyclase receptors which mediate the signal by increasing cGMP. NPR-C, earlier ascribed a role of a ‘clearance receptor’ removing the peptides to the periphery does not possess guanylyl cyclase activity (Potter et al. 2006; Rose and Giles 2008) and appears to have a complex signaling mechanism of its own. The best known immediate consequence of NPR-C activation is inhibition of adenylyl cyclase (cAMP) via guanine nucleotide inhibitory proteins (Gi proteins) (Potter et al. 2006). However, NPR-C is also coupled to endothelial nitric oxide synthase (eNOS)-dependent signal transduction, in which NO activates a soluble form of guanylate cyclase and in turn leads to production of cGMP (Murthy et al. 1998).
In peripheral tissues, NPs were found to attenuate ROS production by interacting with all three receptor classes. Thus, in hepatocytes and Kupffer cells, ANP reduced oxidative stress by activation of NPR-A/B (Pella 1991; Kiemer and Vollmar 1998; Carini et al. 2003). In turn, enhanced oxidative stress in vascular smooth muscle cells (VSMC) from hypertensive rats, was reduced by activation of NPR-C with its specific agonist, cANP(4–23) (Saha et al. 2008). In astrocytes, NPs have been reported to elicit physiologically meaningful downstream signals by interacting with NPR-A (Boran and Garcia 2007; Prado et al. 2010) or NPR-B (Zielińska et al. 2007), but neither the effects of NPs on ONS, nor functioning of NPR-C in general have been studied in these cells. Although the presence of NPR-C in astrocytes was identified on the basis of ligand displacement analysis (Sumners and Tang 1992), no detailed functional characteristics of the receptor was provided. Basing on the observation that cANP(4–23) reduced accumulation of ROS in VSMC (Saha et al. 2008), we envisaged the possibility that NPs may attenuate ammonia-induced ROS formation and that this may involve interaction with NPR-C. Here, we report that (i) ANP and CNP reduces ROS formation both in ammonia-treated and -non-treated astrocytes, (ii) the ROS-reducing effect also occurred upon incubation with the NPR-C-interacting ANP analog, cANP(4–23), and (iii) the superoxide anion was the major ROS species accumulating following ammonia treatment and its accumulation was reduced both by ANP and cANP(4–23). The study documented the presence of a functional NPR-C receptor on astrocytes the stimulation of which counteracts oxidative stress in these cells. Moreover, cANP(4–23) attenuated, by an as yet unknown mechanism, ammonia-induced accumulation of NO, and could in this way reduce nitrosative stress.
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Oxidative/nitrosative stress resulting from excessive intracellular accumulation of ROS and RNS is one of the key mechanisms by which ammonia accumulating in brain during HE impairs astrocytic metabolism and function, leading to cell swelling and cerebral edema (Blei 2008; Häussinger and Görg 2010). Earlier studies with cultured astrocytes have demonstrated that induction of ROS/RNS and its pathophysiological manifestations in astrocytes in culture and/or in the brain in situ, can be prevented or attenuated by pharmacological manipulation with antioxidants (Murthy et al. 2001; Jayakumar et al. 2006), inhibitors of NO synthesis (Schliess et al. 2002; Zielińska et al. 2003), or by inhibiting the synthesis or mitochondrial transport of glutamine, the mediator of the oxidative stress induced by ammonia at the mitochondrial level (Pichili et al. 2007; Rama Rao et al. 2010; and references therein). In cardiovascular tissue and liver cells, NPs have proven effective in countering oxidative stress caused by a variety of causes (De Vito et al. 2010; for other references see introductory paragraphs). In this study, we assessed the potential of NPs to modulate production of ROS/RNS in ammonia-treated astrocytes.
In the present study, both ANP and CNP were found to significantly reduce ROS accumulation. Most interestingly, ROS accumulation was also attenuated by an ANP analog, cANP(4–23), which is a specific agonist of NPR-C. Experiments with peripheral and neuroendocrine tissues revealed that NPR-C is coupled to inhibitory G proteins (Gi), the downstream signal depending upon the protein involved; coupling to Giα inhibits adenylyl cyclase, whereas interaction with Giβ,γ stimulates phospholipase C (reviewed by Rose and Giles 2008). The cANP(4–23)-induced decrease of cAMP level demonstrated in the present study documents the presence of a functional NPR-C receptor in astrocytes capable of signaling downstream the Giα–adenylyl cyclase route. However, cANP(4–23) failed to counter the accumulation of cAMP evoked by ammonia treatment. This intriguing observation appears compatible with earlier evidence that ammonia increases cAMP synthesis in glial cells by multiple mechanisms which may not be affected by activation of Giα (Faff et al. 1996). Down-regulation of Giα protein expression in astrocytes by exposure to cANP(4–23) resembled the effect described by Saha et al. (2008) in VSMC and is likewise to be considered as evidence for the presence of a functional receptor in these cells. In VSMC from SHR rats, NPR-C activation decreased NADPH oxidase activity and subsequent accumulation of •O2−, in parallel with decreasing Giα protein expression. In the present study, cANP(4–23) likewise decreased excess •O2− production by ammonia, as did native ANP. The reduced ability of cANP(4–23) to attenuate ammonia-induced accumulation of •O2− when Giα protein was blocked with an antibody (Fig. 6) bespeaks direct involvement of the NADPH oxidase–Giα protein pathway. However, modulation of NADPH oxidase activity by Giα must have occurred independently of altered Giα expression because: (i) reduction of Giα expression in control rats by cANP(4–23) was not associated with reduced accumulation of •O2−, and (ii) in contrast to VSMC from SHR rats, no elevation of Giα protein expression was observed in ammonia-treated astrocytes. Evidently, although the amount of Giα was reduced by ammonia, it turned out to be still enough to effectively respond both to cANP(4–23) and ANP. From the theoretical perspective, ammonia could increase •O2− generation in astrocytes by mechanisms other than the NADPH oxidase reaction, such as enzymatic oxidation of xanthine or non-enzymatic generation of redox-reactive compounds such as semi-ubiquinone (Dröge 2002).To our knowledge, no study examining the effects of ammonia on the other routes of •O2− generation has been reported as yet.
As ROS present in control cells are engaged in physiological control of cell metabolism and function (for a review see Dröge 2002), their reduction by cANP(4–23) in astrocytes not treated with ammonia may be a regulatory phenomenon. The relatively non-toxic classes of ROS the decrease of which was recorded in control cells with the use of carboxy-H2DCFDA probe may have included singlet oxygen, hydrogen peroxide, hydroxyl radical, and peroxyl radical (Dröge 2002).
The involvement of the different NOS isoforms in the ammonia-induced increase of NO synthesis has been a matter of controversy in the literature. Schliess et al. (2002) found increased iNOS expression in ammonia-treated astrocytes. By contrast, studies in in vivo models of hyperammonemia or HE have invariably pointed to increased expression of eNOS mRNA (Hernandez et al. 2004; Sawara et al. 2009) and protein (Blei 2005) in different brain regions of symptomatic animals. Of note, eNOS mRNA expression was found normalized when HE symptoms retreated (Sawara et al. 2009). The present study tends to support the data obtained in the in vivo models; increased NOx accumulation in astrocytes treated with ammonia for 24 h was: (i) associated with increased eNOS mRNA expression (Fig. 8b) and (ii) not recorded when the constitutive forms of NOS (eNOS + nNOS) were preferentially blocked by l-NG-nitroarginine (Fig. 8a). The fact that ammonia increased the specific activity of NOS (Fig. S1) shows it also modulates the activity at the post-translational level. The specific activity of NOS was also increased by cANP(4–23). The observation that NPR-C stimulation activated NOS activity without modifying eNOS expression is analogous to what has been observed in the cardiovascular tissue stimulated with ANP (Elesgaray et al. 2008; William et al. 2008; Costa et al. 2010). The exact mechanism by which activation of NPR-C in astrocytes regulates eNOS activity, but also the question if enzyme isoforms other than eNOS are involved in the stimulation remain to be investigated. Of note, basing on the mechanism underlying the protection of hepatocytes against oxidative stress (Pella 1991; Kiemer and Vollmar 1998; Carini et al. 2003) contribution of NPR-A to the effect observed with ANP cannot be excluded and deserves more detailed analysis.
Interestingly, the increase of NOx induced by ammonia or cANP(4–23) was not reflected by an increase of cGMP accumulation. With regard to ammonia, the lack of increase could be due to down-regulation of soluble guanylate cyclase in ammonia-treated astrocytes (Konopacka et al. 2006). One other possibility which may apply to both agents is an increased activity of a feedback pathway in which cGMP synthesized in the NO-cGMP pathway activates cGMP-degrading phosphodiesterases; this pathway was found altered by ammonia (Monfort et al. 2004). In the case of ANP, cGMP accumulation was a straightforward reflection of the activation of NPR-A, the particulate form of guanylate cyclase which is present in the astrocytic membrane (Sumners and Tang 1992; Konopacka et al. 2006).
A decrease of ammonia-dependent NO production by cANP(4–23) indicates that selective activation of NPR-C may be effective in counteracting nitrosative stress evoked in astrocytes by ammonia. However, the mechanism underlying this effect is unknown. In macrophages, NPs destabilize iNOS mRNA (Kiemer and Vollmar 1998, 2001) and/or decrease the lipopolysaccharide-induced expression of CAT-2B, an arginine-transporting protein (Kiemer and Vollmar 2001). These mechanisms are unlikely to apply to ammonia-treated astrocytes because: (i) in contrast to the report of Schliess et al. (2002), the present study does not implicate iNOS in the ammonia-induced increase of NOx, and (ii) the effects described in macrophages are cGMP-dependent, implicating NPR-A rather than NPR-C. Possibility (ii) is thus difficult to reconcile with the intriguing absence of the NO-reducing effect of ANP in ammonia-treated astrocytes, because ANP also interacts with NPR-A and generates cGMP. Clearly, the relative roles of the different NOS isoforms in the ammonia-induced generation of NOx, and their involvement in the mechanism by which activation of NPR-C reduces the response require a more detailed investigation.
Activation of NPR-C also signals via a pathway involving phospholipase C, which streams down to protein kinase C δ and results in phosphorylation of mitogen-activated protein kinase p38 (Rose and Giles 2008). This pathway was not investigated in the present study and its presence in astrocytes needs to be confirmed. However, even if present, it is not very likely to be engaged in diminishing oxidative stress induced by ammonia or other ROS-generating agents in astrocytes; activation of MAPK actually mediates the deleterious effects of ROS in cultured astrocytes treated with ammonia (Jayakumar et al. 2006) or menadione (Zhu et al. 2009).
In conclusion, NPR-C receptor activation attenuates ammonia-induced formation of ROS and NO in astrocytes. As NO and ROS contribute jointly to ONS, the key mechanism underlying ammonia neurotoxicity, NPR-C-preferring analogs of ANP may in the future become a useful therapeutic modality in overt stages of HE at which blood and brain ammonia concentrations are significantly raised. To achieve this goal, future experimental studies should account for the ability of these analogs to counter ammonia neurotoxicity in vivo.
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Figure S1. Effect of pretreatment with cANP (4-23) on NOS activity in primary astrocyte cultures not treated (Control) and treated with 5 mmol/L ammonium chloride (Ammonia) for 24h. Results are mean ± SD (n=3). * p < 0.05, ** p< 0.01 vs Control; †p< 0.05 vs Ammonia.
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