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BACKGROUND: Endogenous hydrogen sulfide (H₂S) appears to be a new gasotransmitter that modulates small intestinal motility, but mechanisms of action of H₂S are unknown in gut smooth muscle. AIM: To determine effects and mechanisms of action of H₂S on contractile activity in circular muscle of rat jejunum. METHODS: Jejunal circular muscle strips from Lewis rats were mounted in temperature-controlled tissue chambers to measure contractile activity. Sodium hydrosulfide (NaHS) was used as an exogenous donor of H₂S yielding physiologic levels of H₂S of 10^-5, 10^-4, 2x10^-4, and 5x10^-4 M, resp. We evaluated effects of NaHS on spontaneous contractile activity and after precontraction with bethanechol (10^-4 M). L-cysteine (10^-4, 10^-3 and 10^-2 M), the substrate for endogenous production of H₂S via H₂S-producing enzymes, was also evaluated. We used atropine (10^-7 M), phentolamine (10^-5 M), and propranolol (5x10^-6 M) together to establish non-adrenergic/non-cholinergic (NANC) conditions, and in separate experiments tetrodotoxin (10^-6 M), capsaicin (10^-5 and 10^-4 M), L-N^G-nitro arginine (L-NNA; 10^-4 and 10^-3 M), glibenclamide (10^-5 and 10^-4 M), apamin (10^-6 and 5x10^-6 M) , and calyculin A (10^-6 M) to study involvement of the NANC and enteric nervous system, primary visceral afferent nerve fibers, nitric oxide, K⁺_ATP channel, K⁺_Ca channel, and myosin light chain phosphatase activity, respectively, in mediating the inhibitory action of H₂S. Motility was quantitated as area under the contractile curve and used to compare the effects. RESULTS: NaHS exhibited a marked, dose-dependent inhibitory effect (Fig 1) on spontaneous contractile activity (p<0.05); threshold dose 10^-4 M, ID₅₀ (50% inhibitory dose) = 3x10^-4 M. However, surprisingly, NaHS did not inhibit bethanechol-stimulated contractile activity. L-cysteine also exerted a dose-dependent inhibitory effect on contractile activity (Fig 2) (p<0.05). Establishment of NANC conditions, blockade of all neural activity by tetrodotoxin, exposure to capsaicin (block visceral afferents), L-NNA (block NO production), glibenclamide (block K⁺_ATP channels), or apamin (block K⁺_Ca channels) had no effect on (i.e. did not block) the inhibitory response to 5x10^-4 M NaHS. After exposure to calyculin A (block myosin light chain phosphatase), the inhibitory effect of NaHS was prevented. CONCLUSIONS: H₂S at physiologic concentrations and L-cysteine inhibit contractile activity of rat jejunal circular muscle. The inhibitory effect of H₂S on jejunal circular muscle does not appear to be mediated via K⁺_ATP channels as in the vascular tree or by the more classic pathways via the enteric nervous system, a nitric oxide pathway, activity of visceral afferent nerve fibers, or K⁺_Ca channels but rather via myosin light chain phosphatase. (Support RO1-DK39337 [MGS])

Fig 1

Fig 2