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Regulation of Contractile Activity in Longitudinal Muscle of Rat Ileum By Hydrogen Sulfide
Munenori Nagao*1, Judith a. Duenes1, David R. Linden3, Gianrico Farrugia2,3, Michael G. Sarr1
1Dept. of Surgery and Gastroenterology Research Unit, Mayo Clinic, Rochester, MN; 2Dept. of Internal Medicine, Mayo Clinic, Rochester, MN; 3Dept. of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN
BACKGROUND: Endogenous hydrogen sulfide (H2S) is a newly discovered modulator of GI motility; however, the mechanisms of action of H2S are not well understood. AIM: To determine effects and mechanisms of action of H2S on contractile activity in longitudinal muscle of rat ileum. METHODS: Ileal longitudinal muscle strips from 12 Lewis rats were prepared to measure contractile activity in a temperature controlled tissue chamber. Sodium hydrosulfide (NaHS; 10-5 to 10-3M) was used as an exogenous donor of H2S yielding bath solution levels of H2S of approximately 1 to 180 μM. Physiologic endogenous levels of H2S are thought to be 50-200 µM. Effects of NaHS were evaluated on spontaneous contractile activity and after pre-contraction with bethanechol (3x10-6M). L-cysteine (10-4, 10-3, and 10-2M), the substrate for production of H2S, was used as an endogenous donor of H2S. We evaluated involvement of A) Extrinsic nerves with atropine (10-7M), phentolamine (10-5M), and propranolol (5x10-6M); B) Enteric nervous system with tetrodotoxin (10-6M); C) Nitric oxide with L-NG-nitro arginine (L-NNA; 10-4 and 10-3M); D) Visceral primary afferent nerve fibers with capsaicin (10-5 and 10-4M); E) K+ATP channels with glibenclamide (10-4 and 10-3M); and F) K+Ca channel activity with apamin (10-6 and 5x10-6M). Area under the contractile curve was used to compare the effects. RESULTS: NaHS exhibited a prominent dose-dependent inhibitory effect on spontaneous and bethanechol-stimulated contractile activity (p<0.01) with effects starting at 5x10-4M and also decreased baseline tone. L-cysteine had no inhibitory effect. Establishing non-adrenergic and non-cholinergic conditions, blockade of all neural activity by tetrodotoxin, blocking NO production by L-NNA, blocking visceral primary afferents by capsaicin, blocking K+ATP channels by glibenclamide, or blocking K+Ca channels by apamin, had no effect on the inhibitory effect of 10-3M NaHS on contractile activity. CONCLUSION: H2S at physiologic levels inhibits contractile activity of ileal smooth muscle. The inhibitory effect of H2S in rat ileum does not appear to be mediated by intrinsic or extrinsic neural pathways, nitric oxide, or activity of visceral afferent nerve fibers, K+ATP channels, or K+Ca channels in ileal longitudinal muscle suggesting that the mechanisms of action of H2S proposed in other tissues do not appear to be operative in rat ileal longitudinal muscle.
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