Anthopleura elegantissima,

v

v

v

Xenopus laevis

ASICs are sodium-gated ion channels member of the epithelial/degenerin Nachannel family, acting as “chemoelectrical transducers” by sensing the decrease of pH in the extracellular environment [ 233 ]. Six ASIC subunits have been characterized to date, which are encoded by four distinct genes (ASIC1-4), since ASIC1 and ASIC2 have been demonstrated to produce two functional splice variants (ASIC1a, ASIC1b, and ASIC2a, ASIC2b, respectively) [ 233 ]. Each ASIC subunit consists of short intracellular N- and C-terminals, two transmembrane domains (TM1 and TM2), and a large extracellular domain [ 234 ]. ASIC subunits can assemble as heteromeric or homomeric trimers to produce functional channels [ 235 ]. ASICs are directly gated by protons during tissue acidosis occurring as a consequence of ischemia, trauma, tumor, and surgery, in particular when the tissue pH value is below 7, and represent the major sensor of extracellular pH changes in nociceptive pathways [ 236 ]. ASIC activation by acidic pH induces membrane depolarization and action potentials firing in neuronal pain pathways involved in inflammatory pain and in different chronic pain conditions [ 8 235 ]. Functionally active ASIC1–ASIC4 channels have been localized in neurons from rat DRGs, and several findings show that an acidic environment is a potent stimulus for primary afferent neurons, inducing fast currents in DRG neurons [ 237 ]. In addition, immunohistochemistry studies revealed colocalization of ASICs with CGRP and substance P in small capsaicin-sensitive nociceptive neurons [ 238 ]. In the gastrointestinal tract, ASICs have been detected in extrinsic primary afferent neurons originating from the DRGs and NG [ 239 ]. Different functional ASICs are present as homomultimers, mainly represented by ASIC1a and ASIC3, or in heteromultimer combinations of different subunits, in glossopharyngeal, vagal, and spinal afferent neurons innervating the gut and other visceral organs (for a more detailed description of ASIC distribution in the gastrointestinal tract see the review by Holzer [ 239 ]. In particular, ASIC3, which represents the most important protonsensitive channel involved in modulation of moderate- to high-intensity pain sensation, has been almost exclusively localized to primary afferent neurons [ 240 242 ]. Interestingly, retrograde tracing studies have shown that the majority of the NG and DRG neurons projecting to the rat stomach express ASIC3-like immunoreactivity [ 243 ]. Accordingly, the soma of a high percentage of thoracolumbar mouse DRGs projecting to the mouse colon stained for ASIC3 and, to a minor extent, for ASIC2 and ASIC1 [ 244 ]. Proton activation of ASICs has been implicated in gastritis, peptic ulceration, and other gastrointestinal disorders associated with altered pain perception, suggesting that modulation of these channels may have potential implications for the relief of visceral gut pain [ 239 245 ]. In the upper gastrointestinal tract, ASIC3 has been suggested to play a major role in the development of inflammatory hypersensitivity to gastric acid, occurring during gastritis and peptic ulcer disease, both associated with painful symptoms [ 246 ]. In the lower part of the gut, ASIC activation correlates with development of hypersensitivity to colorectal distension, indicating that, analogously to TRP, ASIC may be affected by different stimuli, including mechanical stimuli [ 247 249 ]. In particular, in a mouse model of zymosan-induced sensitization of colonic mechanoreceptors, both ASIC3 and TRPV1 participated in development of chronic hypersensitivity to colorectal distension in the absence of inflammation, suggesting that both ASIC3 and TRPV1 may contribute to non-inflammatory visceral hypersensitivity, typical of IBS [ 249 ]. Intracolonic administration of butyrate in rats induced hyperresponsiveness to colorectal distension, associated with an upregulation of ASIC1a in colonic DRG neurons and in the spinal cord [ 250 251 ]. In these experimental conditions, among the different mediators of visceral hypersensitivity, NGF played a key role in crosslinking painful stimuli with spinal upregulation of ASICs and colonic hypersensitivity [ 252 253 ]. Interestingly, butyrate-induced upregulation of both ASIC1a and colonic hypersensitivity was prevented by NGF neutralization [ 251 ]. These studies suggest that molecules acting at ASICs may represent possible targets for the discovery of drugs active on visceral pain associated with functional gastrointestinal disorders, such as IBS. The sea anemone toxin APETx2 has been shown to modulate pain perception by interacting with ASICs. APETx2 is a 42 amino acid toxin with a defensin-like fold, derived from sea anemone,known to affect ASIC3 channels homomers as well as several heteromers of ASIC3 in combination with ASIC1a, ASIC1b, and ASIC2b [ 254 255 ]. APETx2 blocks ionic currents in the sensory neurons, shifting downward the affinity of ASIC3 for protons, through a not yet clear combined activity on these channels [ 255 ]. In rats, subcutaneous administration of APETx2 exerted a potent analgesic effect on inflammation-induced hyperalgesia by peripherally blocking ASIC3 on cutaneous nociceptors [ 241 ]. Several other studies resorting to rodent models have demonstrated the efficacy of APETx2 in reducing postoperative, osteoarthritic, muscular, and inflammatory pain [ 241 259 ]. In rats, intraperitoneal injection of APETx2 before experimentally inducing acute gastric mucosal lesions reduced gastric acidity, mucosal injury, and ASIC3 expression in thoracic DRG projecting to the stomach, suggesting a role for ASIC3 in the development of gastritis symptoms [ 63 ]. More recently, APETx2 displayed a bell-shaped dose–response curve in reducing gut visceral pain evaluated by acetic-acid-induced abdominal contractile responses in mice [ 64 ]. In this study, the toxin exerted an analgesic effect only at lower doses, while its efficacy diminished at higher doses, possibly owing to the nonspecific action of the compound. An important drawback concerning the analgesic efficacy of APETx2 is represented by its off-target effects on Na1.8, Na1.6, Na1.2, and cardiac hERG channels, which may influence its therapeutic potential [ 260 261 ]. Another important issue is represented by the ability of the toxin to partially modulate ASIC3 activation-mediated currents. The ability of APETx2 to reduce only a transient, but not a sustained, component of acid-induced ASIC3 current in whole cell patch clamp measurements inoocytes was considered a possible mechanism underlying its partial efficacy in modulating visceral pain. Indeed, another sea anemone toxin, Ugr9-1, which displayed a higher potency in reducing acidosis-associated pain conditions, was able to completely block ASIC3 currents in vitro [ 64 65 ]. The discovery of more selective and efficacious ASIC inhibitors represents an important challenge in order to target physiologically relevant and abnormally active ASICs involved in pathophysiological pain perception [ 262 ]. From this perspective, selective blockade of ASICs, in particular ASIC3, may provide interesting therapeutic tools to treat visceral hypersensitivity and pain associated with functional and inflammatory chronic gut diseases, such as IBS and IBD [ 239 ].