Glia and NO nicotine: a perfect harmony for secretory control

Abstract

In a recent issue of The Journal of Physiology, MacEachern et al. (2011) describe an intriguing new regulatory mechanism for nicotinic cholinergic-induced fluid secretion that may have important pathophysiological implications in the digestive tract. Secretion of fluid and electrolytes by channels and transporters on intestinal epithelial cells is required for normal tissue homeostasis, and this process is tightly regulated by local cholinergic neurons. Inflammatory diarrheal disease is often associated with altered cholinergic innervation, and elevated nitric oxide (NO) production has widely been implicated in modulating this fluid secretion (Martinez-Augustin et al. 2009). Interestingly, nicotine has been shown to confer significant health benefits, with a reduced incidence of diarrhoea in patients with active colitis (Sandborn, 1999). However, because intestinal epithelial cells lack the nicotinic receptor and, thus, are unresponsive to nicotinic agonists, this begs the question as to how nicotine modulates fluid secretion in intact intestinal tissues. MacEachern et al. identify enteric glia as novel cellular regulators of epithelial chloride secretion in the mouse colon, by virtue of their reactive capacity to generate NO in response to nicotinic signals (vide infra). Enteric glia are emerging as important new homeostatic regulators of intestinal mucosal function, having recently been shown to orchestrate epithelial proliferation, restitution and barrier functions (Savidge et al. 2007). These astrocyte-like cells are highly reactive to stress and tissue damage, and are prolific producers of NO generated by a nitric oxide synthase isoform that is normally inducible (iNOS). Enteric glia also regulate nitrergic neuronal function by providing a rate-limiting l-arginine substrate shuttle to neuronal nitric oxide synthase (nNOS) (Nagahama et al. 2001). MacEachern et al show rapid nicotinic stimulation of NO production in both enteric neurons and glia via activation of their respective NOS isoforms. This finding is supported by immunofluorescence detection of the nicotinic receptor α3 subunit on both cell types. Importantly, these nicotinic-NO signals have drastically different outcomes on chloride secretion, and appear to be neuronal plexus specific. In colonic mucosa containing the submucosal (but not the myenteric) plexus, chloride secretion is highly dependent on neuronally derived NO, as ion transport is blocked by the neurotoxin tetrodotoxin (TTX) and by the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO). By contrast, in intact tissues containing both neuronal plexi, an NO-dependent inhibitory response is also recorded, leading the authors to propose that iNOS-derived NO from enteric glia attenuates nicotinic-induced fluid secretion. Control experiments included hexamethonium inhibition demonstrating specificity of the nicotinic response, and an equipotent chloride secretory response to forskolin ruling out diffusion artifacts in mucosal versus intact tissue preparations. Thus, the authors postulate that the myenteric plexus provides an important nicotinic-glia-NO mechanism that may represent a commonly overlooked anti-secretory signal in Ussing chamber studies (Fig. 1). Figure 1 Hypothetical illustration of dimethylphenylpiperazinium (DMPP)-induced nicotinic-NO signaling on epithelial ion transport in mouse colon Because NO is already known to mediate cholinergic signals, and it co-localizes with cholinergic neurons in the myenteric plexus, it is suggested that this molecule may act as an intermediate in the signalling cascade. Previous reports have shown NO to directly or indirectly modulate epithelial ion transport (Martinez-Augustin et al. 2009), further strengthening this notion (supported in this study by exogenous NO dose-dependently recapitulating both stimulatory and inhibitory chloride secretion in intact tissues). However, exogenous NO failed to alter chloride secretion in stripped mucosa, demonstrating a dependency on the myenteric plexus to mobilize stimulatory and inhibitory signals in response to NO. Mucosa-derived NO may also potentiate nicotinic signals by sensitizing the receptor-cascade itself. These important new mechanistic insights do raise many intriguing questions. Firstly, are the therapeutic benefits of nicotine restricted to modulating fluid secretion, or do nicotinic-glia-NO signals also regulate motor function and correlate with dysmotility? Secondly, is this signalling mechanism a compensatory absorptive response specific to the distal colon, since the caecum was not similarly affected in an earlier study by the same group (Green et al. 2004)? Thirdly, does this inhibitory mechanism promote bicarbonate ion secretion as a compensatory response, to stabilize mucin and defensin protein folding for innate immunity (Garcia et al. 2009)? Finally, what is the nature of the NO-intermediate signals? These greatly potentiate nicotinic receptor-mediated chloride secretion in the mucosa, yet in intact tissues they mediate nicotinic-independent, TTX-sensitive signals that modulate ion transport (albeit at pharmacological doses of NO donor). It is increasingly appreciated that many diverse signalling cascades associated with NO production are attributed to stable S-nitrosothiol (SNO) intermediates that act via covalent modification of cysteine residues in target molecules (S-nitrosylation), and that aberrant S-nitrosylation may play a role in disease aetiology (Savidge, 2011). Because cysteine residues are often key regulators of protein function, S-nitrosylation represents a physiologically important posttranslational mechanism analogous to O-phosphorylation, which is known to regulate nicotinic receptor signalling. Because S-nitrosylation is known to directly modulate G-protein coupled receptor activation and ion channel activity, it will be interesting to test whether S-nitrosothiols represent intermediates of NO signalling in this instance.