Background
Hydrogen sulfide (H
2S), a gas synthesized by sulfate reducing colonic bacteria and the endogenous enzymes cystathionine-
β-synthetase (CBS) and cystathionine-
γ-lyase (CSE) [
1‐
5], is increasingly recognized as a biologically important signaling molecule in various tissues and processes including pain and inflammation [
6,
7]. Its putative role as a neurotransmitter is supported by recent reports on its effects on hippocampal neurons as well as capsaicin-sensitive peripheral sensory neurons [
8‐
10]. With respect to the latter, there is evidence that intraplantar injection of NaHS (a commonly used H
2S donor) in rat hindpaws produces mechanical hyperalgesia through activation of T-type Ca
2+ channels [
11], supporting a pro-nociceptive role for H
2S. Further, H
2S generation is also enhanced in the formalin [
12] and carrageenan [
13] model of persistent inflammatory pain. Systemic injections of H
2S donors in rats suppress responses to colorectal distention (CRD) by activating K
ATP channels [
14,
15], suggesting a possible anti-nociceptive effect. On the other hand, colonic administration of H
2S enhances pain behaviors in response to CRD in mice [
11]. However, the role of H
2S in non-inflammatory visceral hypersensitivity is not known. Our aim was therefore to study the potential role of H
2S in the pathogenesis of chronic visceral hyperalgesia (CVH) in a well characterized rat model of irritable bowel syndrome (IBS), developed in our laboratory [
16,
17]. Our results show that CVH in this model is associated with an upregulation of CBS expression in both thoracolumbar (TL) and lumbarsacral (LS) DRG and that the use of a CBS inhibitor attenuates colonic hypersensitivity. H
2S enhances the excitability of colon specific sensory neurons
in vitro and together these findings indicate an important role for H
2S signaling in IBS-like visceral hyperalgesia. Parts of this work have been published previously in an abstract form [
18].
Discussion
Our study shows that the H
2S 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, H
2S donor NaHS greatly enhanced the frequency of action potentials of DRG neurons
in vitro (Fig.
6). These data strongly suggest that CBS-H
2S 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 H
2S as a neuromodulator [
24‐
26] or an endogenous gaseous transmitter [
27]. In physiological conditions, H
2S 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]. H
2S has been reported to produce inward or outward currents on dorsal raphe serotonergic neurons
in vitro [
29]. H
2S can also regulate the release of corticotrophin-releasing hormone from the hypothalamus [
30]. H
2S is an important endogenous vasoactive factor and is an identified gaseous opener of K
ATP channels in vascular smooth muscle cells [
27].
Endogenous H
2S 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 H
2S 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 K
ATPchannels [
14,
15]. On the other hand, intracolonic H
2S 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 [
11]. The reasons for these discrepant findings may include but not limit to H
2S concentration, effect of inflammation on H
2S action and H
2S action sites. The concentration of H
2S may not be an explanation for the different results since the same dose of H
2S used by two different groups produced the different effects [
11,
14,
15]. Tissue inflammation may have an influence on H
2S 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 [
16]. Thus, the different effects of H
2S in AA-treated rats were not due to inflammation/injury. The site of action or/and the source of H
2S may be most likely related to the different effect of H
2S as has been suggested in somatic pain models [
33]. Thus, systemic administration of exogenous H
2S may activate central antinociceptive mechanisms whereas peripheral H
2S administration or endogenous sources may invoke pro-nociceptive effects. Further experiments on the mechanism of H
2S signaling pathway are warranted.
CBS and systathionine γ-lyase (CSE) are two important enzymes for generation of endogenous H
2S [
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 [
15]. 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 [
10]. 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 H
2S 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 H
2S 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 [
10]. An additional source of H
2S 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 H
2S and a credible therapeutic target for visceral pain syndromes.
Although the detailed mechanisms by which H
2S 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. H
2S has been shown to enhance the excitability of enteric neurons, possibly via TRPV1 receptors on extrinsic afferent terminals [
10]. Others have shown that stimulation with H
2S enhances T-type calcium currents in small sensory neurons
in vitro [
11]. In this study, we provide new evidence for the first time to show that H
2S donor NaHS increased the number of action potentials evoked by electrical stimulation in colon specific DRG neurons (Fig.
6), indicating that H
2S 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 H
2S 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.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
All authors have read and approved the final manuscript. GYX designed, performed and supervised the experiments, analyzed the data, prepared the figures and wrote the manuscript; MS and SFZ performed the experiments; JHW, JDZC and PJP coordinated the project, helped to interpret the data, and edited the manuscript.