Study On Actions Of Methane And Hydrogen Sulphide On Colonic Motility

Part I Effects and mechanisms of methane and hydrogen sulphide on colon motility of ratsObjective To investigate the effects and mechanisms of methane (CH4) and hydrogen sulphide (H2S) on colon motility of rats.Methods The longitudinal muscle (LM) and circular muscle (CM) strips of proximal colon were isolated from rats. A isometric force transducer and a biology signal collection system were applied to observe the effects of CH4and H2S on spontaneous contractile activity of muscle strips. The effects of CH4or H2S on muscle strips were observed when these strips were pretreated with tetrodotoxin (TTX), N-nitro-L-arginine methylester (L-NAME), tetraethylammonium (TEA) or glibencla mide (Gli). Single cell of colonic smooth muscle was isolated by collagenase, then whole-cell patch clamp technique was used to record voltage dependent potassium current (IKV) and large conductance Ca2+-activated K+current (IBKca) in the absence or presence of CH4.Results CH4significantly attenuated the contractile amplitude of the LM strips [from (1.12±0.27) g to (0.99±0.31) g, n=19, P=0.013], whereas there were no changes in some LM strips (n=6) in the presence of methane. The inhibitory effect of CH4still existed when the LM were pretreated with TTX or L-NAME. CH4had no effects on the contractile cycle time of LM and the contractile activity of CM.3%CH4solution significantly increased the density of IKV [from (13.26±1.02) pA/pF to (18.54±1.42) pA/pF, at+80mV, n=11, P=0.001], compared to the control group, whereas methane had no effect on IBKca. NaHS(0.01～1mM) concentration-dependently inhibited the spontaneous contractions of the LM and CM (n=10, P<0.001). The inhibitory effect of NaHS (0.3mM) still existed when the strips were pretreated with TTX or L-NAME. And the inhibitory effect of NaHS (0.3mM) was significantly reduced by TEA or Gli (n=10, P<0.05).Conclusion CH4can inhibit contractile activity of proximal colonic LM probablely by activing voltage dependent potassium channel and increasing IKV. CH4can inhibit contractile activity of proximal colonic LM and CM probablely by activing ATP-sensitive potassium. Part II Actions of hydrogen sulfide on colonic hypermotility in a rat model of chronic stressObjective To investigate the potential role of H2S in chronic stress-induced colonic hypermotility.Methods Male Wistar rats were submitted daily to1h of water avoidance stress (WAS) or sham WAS (SWAS) for10consecutive days. Organ bath recordings, H2S production, immunohistochemistry and western blotting were performed on rat colonic samples to investigate the role of endogenous H2S in repeated WAS-induced hyperm otility. Organ bath recordings and western blotting were used to detect the role of KATP channels in repeated WAS.Results Repeated WAS increased the number of fecal pellets per hour and the area under the curve of the spontaneous contractions of colonic strips, and the AUC of contractions induced by acetylcholine (Ach) and KCl (n=10, P<0.05). Repeated WAS decreased the endogenous production of H2S. and the expression of H2S-producing enzymes in the colon devoid of mucosa and submucosa (n=10, P<0.001). CSE was strongly expressed in the cytosols of the circular and longitudinal smooth muscle cells and the nucleus of the myenteric plexus neurons. CBS was primarily localized in the cytosols of myenteric plexus neurons and weakly localized in the epithelial cells. Inhibitors of H2S-producing enzymes increased the contractile activity of colonic strips in the SWAS rats (n=10, P<0.001). Repeated WAS treatment resulted in up-regulation of Kir6.1and SUR2B of KATP channels in the colon devoid of mucosa and submucosa (n=10,P<0.05). NaHS (0.01～1mM) concentration-dependently inhibited the spontaneous contractions of the strips and the NaHS IC50for the WAS rats was significantly lower than that for the SWAS rats (n=10, P<0.0001). The inhibitory effect of NaHS was significantly reduced by glibenclamide (n=10, P<0.0001).Conclusion The colonic hypermotility induced by repeated WAS may be associated with the decreased production of endogenous H2S. The increased expression of the subunits of KATP channels in colonic smooth muscle cells may be a defensive response to repeated WAS. H2S donor may have potential clinical utility in treating chronic stress-induced colonic hypermotility.