Our study shows that the H2S producing enzyme cystathionine β-synthase (CBS) is expressed by a subpopulation of primary sensory neurons (Figs. 1, 2 &3) and is upregulated in a rat model of IBS-like chronic visceral hyperalgesia (Fig. 4). CBS upregulation may contribute to chronic visceral hyperalgesia since a CBS inhibitor significantly attenuates the AWR scores in neonatal AA-treated rats (Fig. 5). In addition, H2S donor NaHS greatly enhanced the frequency of action potentials of DRG neurons in vitro (Fig. 6). These data strongly suggest that CBS-H2S signaling may play an important role in "functional" visceral pain i.e. pain occurring in the absence of overt structural or inflammatory processes.
There is considerable support for a role of H2S as a neuromodulator [24–26] or an endogenous gaseous transmitter . In physiological conditions, H2S has been found to regulate key neuronal functions, including induction of long-term potentiation and modulation of NMDA receptor currents in the hippocampus [24, 28]. H2S has been reported to produce inward or outward currents on dorsal raphe serotonergic neurons in vitro . H2S can also regulate the release of corticotrophin-releasing hormone from the hypothalamus . H2S is an important endogenous vasoactive factor and is an identified gaseous opener of KATP channels in vascular smooth muscle cells .
Endogenous H2S is also an important mediator of inflammation in a variety of models [6, 31]. Along with this, there is growing evidence of its involvement in nociception in both somatic [32, 33] and visceral [11, 14, 15] organs. However, this role is likely to be complex as suggested by the somewhat conflicting reports in the literature. Distrutti and colleagues have shown that systemic administration of H2S donors attenuates the response to CRD in both healthy and post-colitic rats; this effect is sensitive to glibenclamide, suggesting that it is mediated by KATPchannels [14, 15]. On the other hand, intracolonic H2S donor NaHS enhanced spontaneous visceral pain behavior as well as referred hyperalgesia and spinal ERK expression in mice, an effect that appears to be mediated by T-type calcium channels as it is blocked by mibefradil but not by verapamil (an L-type channel blocker) or glibenclamide . The reasons for these discrepant findings may include but not limit to H2S concentration, effect of inflammation on H2S action and H2S action sites. The concentration of H2S may not be an explanation for the different results since the same dose of H2S used by two different groups produced the different effects [11, 14, 15]. Tissue inflammation may have an influence on H2S actions. In this animal model, however, no histological signs of inflammation/injuries or significant changes in MPO activities were observed in the colons 8 weeks after neonatal AA treatment as reported previously . Thus, the different effects of H2S in AA-treated rats were not due to inflammation/injury. The site of action or/and the source of H2S may be most likely related to the different effect of H2S as has been suggested in somatic pain models . Thus, systemic administration of exogenous H2S may activate central antinociceptive mechanisms whereas peripheral H2S administration or endogenous sources may invoke pro-nociceptive effects. Further experiments on the mechanism of H2S signaling pathway are warranted.
CBS and systathionine γ-lyase (CSE) are two important enzymes for generation of endogenous H2S [1–5]. These two enzymes have been found in many types of mammalian cells in the central nervous system as well as peripheral tissues [10, 25, 27, 34, 35]. Both enzymes have also been shown to be expressed by rat colonic tissue . CSE and CBS have also been localized to colonic enteric neurons and CSE, but not CBS, to interstitial cells of Cajal in guinea pig colon . We have confirmed previous studies on the expression of CBS and CSE in the colon and further have shown that CBS, but not CSE, is expressed by colon-specific sensory neurons (Fig. 1), where it is localized to nociceptive neurons, indicating that CBS is a major enzyme responsible for the endogenous production of H2S in these cells. We have also shown that CBS expression in both sensory neurons and the colon is dynamic and is upregulated in a model of chronic non-inflammatory visceral hypersensitivity. Theoretically, enhanced H2S production from either a colonic or a neuronal source can affect the function of sensory neurons in our model and contribute to both enhanced pain as well as the secretomotor response that has previously been shown in guinea pigs . An additional source of H2S comes from sulfate-reducing bacteria in the GI tract [36–38]. Further research will indicate the relative importance of these various sources in health and disease. Our studies showed that CBS inhibitor attenuated the AWR scores in neonatal AA-treated rats (Fig. 5B) and no significant effect was seen in control rats (Fig. 5C), suggesting that this was not a non-specific analgesic effect. This also suggests that the role of CBS in signaling colonic distension may not be as important in health as in the sensitized state. Taken together, our results suggest but do not prove that CBS may be an important source of endogenous H2S and a credible therapeutic target for visceral pain syndromes.
Although the detailed mechanisms by which H2S induces visceral hyperalgesia have yet to be fully investigated, our data and that of others suggest that colonic nociceptors are a prime site of action. H2S has been shown to enhance the excitability of enteric neurons, possibly via TRPV1 receptors on extrinsic afferent terminals . Others have shown that stimulation with H2S enhances T-type calcium currents in small sensory neurons in vitro . In this study, we provide new evidence for the first time to show that H2S donor NaHS increased the number of action potentials evoked by electrical stimulation in colon specific DRG neurons (Fig. 6), indicating that H2S may increase the neuronal excitability. This effect may result from the previously reported activation of T-type calcium channels and/or involve potassium and/or sodium ion channels. In addition, we showed that CBS-ir was present abundantly in small and medium-size neurons of DRGs and they co-localized with TRPV1 and P2X3 receptors, suggesting a possible interaction between these molecules. Both of these receptors are upregulated in our model of IBS-like pain [16, 17] and further studies are needed to investigate whether and how H2S modulates their function and/or expression.
In conclusion, although there is a discrepancy of H2S effects in the literature, our data demonstrate that CBS-H2S signaling pathways may play a role in chronic visceral hyperalgesia, even in the absence of overt inflammation of the colon wall. Our results also identify CBS as a potential target for novel agents for the treatment of visceral pain in IBS and related disorders.