The Role Of Hydrogen Sulfide In Regulation Of Gastric Fundus Smooth Muscle Tension And Its Mechanism In Mice

Hydrogen sulfide(H2S) is a colorless, flammable acid gas with a smell of rotten eggs at low concentrations while with no odor at high concentrations for it can paralyze the olfactory nerve. Hydrogen sulfide is an acute toxic gas. Careless inhaling high concentrations of hydrogen sulfide can be fatal in a short time. Eye, respiratory and central nervous system can be affected by low concentrations of hydrogen sulfide. Our focus is only confined to its toxicity over the years. However, recent studies have found that hydrogen sulfide exists widely in a range of mammals, and it is an important protective member in cells. Its main synthase, cystathionine-β- synthase(cystathionine-β-synthase, CBS) and cystathionine-γ- lyase(cysta-thionine-γ-lyase, CSE) are widely expressed in tissues of mammals, with difference in the distribution. For instance, CBS is predominant in nerve cells while CSE is predominant in the cardiovascular system, colon and liver. Endogenous H2 S extensively is involved in many physiological and pathological processes of the diseases. It is considered to be the third gaseous signaling molecule, following the nitric oxide(NO) and carbon monoxide(CO) in recent years. H2 S was found to regulate mitochondrial ATP generation. ATP-sensitive potassium channels play significant roles in insulin secretion process, and it is reported that H2 S participates in the process of type 2 diabetes. It is discovered that H2 S can affect ATP-sensitive potassium channel of cardiovascular system and respiratory system, including the tension lower, breathing enhancement and so on. It is valued that H2 S fights against hypoxic-ischemic myocardial injury for H2 S can activate protein kinase C and ATP-sensitive potassium channel in rat. On the other hand, H2 S can promote myocardial cell proliferation and vascular smooth muscle cells proliferation and migration.Food digestion depends on the mechanical movement of the gastrointestinal tract, which is crucial for the smooth muscle self-control. Self-regulation exists in the gastric smooth muscle apart from neuroregulation and humoral regulation. H2 S is probably one of the important members of the self-regulating mechanisms. It has been confirmed that endogenous H2 S can be produced in gastrointestinal tissue; Bacterial decomposition in the digestive tract, especially in the colon will produce H2 S. Therefore, it is crucial to clarify the effect of H2 S on the regulation of gastrointestinal motility.In our study, electrical physiology and molecular biology were used to study the mechanism of H2 S in mouse gastric smooth muscle. First, we studied the effect of H2 S on the classical calcium-dependent contraction pathway. We used immunohistochemical methods to detect the expression of H2 S synthase in gastric smooth muscle cells in mice; We evaluated the effect of NaHS on the tension of strips in mice smooth muscle; We made use of the intracellular recording method to detect the influence of H2 S on membrane potential; Whole-cell patch-clamp method was used to detect the impact of H2 S on the current of the voltage-dependent potassium channels and L- type calcium channels in single smooth muscle cells; Calcium imaging was used to detect the impact of H2 S on intracellular calcium concentration. Secondly, we explore the effect of H2 S on NO production and its mechanism. We used immunohistochemical methods to detect the expressions of H2 S synthase and NO synthase in gastric smooth muscle cells in mice; Electrochemical method was used to directly detect the generation of H2 S and NO; We evaluated the effect of NaHS and SNP on the tension of strips in mice; Western blot was used to detect the effe of H2 S on NO synthase regulation; We utilized siRNA interference to detect the effect of endogenous H2 S on NO synthase. Results as follows:The role of hydrogen sulfide in regulation of gastric fundus smooth muscle tension and ionic mechanism1. Western blot results show that the H2 S synthase protein CBS and CSE are expressed in cultured gastric smooth muscle cells in mice;2. NaHS(H2S donor) can increase gastric smooth muscle tone; NO is considered as a negative control; CBS blocker AOAA, reduce the tension, while NO synthase inhibitor, L-NAME, increase the tension; AOAA can significantly reduce the tension, but CSE blocker, PAG, almost does not affect the tension; NaHS can reverse the tension reduced by AOAA to nearly normal control levels. The pretreatment with selective specific voltage-dependent potassium channel blocker, 4-AP, blocke the excitatory effect of NaHS on smooth muscle; The pretreatment with L- type calcium channel blocker, nicardipine, blockethe excitatory effect of NaHS on smooth muscle.3. The intracellular recording results show that NaHS depolarize the membrane potential of smooth muscle cells.4. The whole-cell patch-clamp recording show that NaHS can reduce current of voltage-dependent potassium channel and NaHS can increase the current of L- type calcium channel.5. The calcium fluorescent calcium imaging shows that NaHS can increase the concentration of intracellular calcium, which can be blocked by nicardipine.CONCLUSIONS: we show that the H2S-producing enzymes CBS and CSE are expressed in the gastric fundus in mice. H2 S at physiological concentrations may excite the fundus smooth muscles and induce tonic contraction. Endogenous H2 S depolarizes the membrane potential via the inhibition of voltage-dependent potassium channels. NaHS-induced depolarization activates L-type calcium channels, raises the intracellular calcium level and finally increases the tension of gastric fundus.The role of hydrogen Sulfide on nitric oxide synthesis and its mechanism1. Western blot results show that the H2 S synthase protein CBS and CSE are expressed in cultured gastric smooth muscle cells in mice; The NO synthase protein eNOS and nNOS are expressed in cultured gastric smooth muscle cells in mice.2. Na HS can increase the tension of gastric fundus; SNP decreasethe tension of gastric fundus; NaHS can reverse the effect of SNP. NO synthase inhibitor, L-NAME, can reduce the effect of NaHS-increased tension. HNO decrease the tension of gastric fundus; The negative control, KOH, did not change the tension significantly.3. The electrochemical method detected the generation of H2 S and NO in the smooth muscle tissue. It’s found that the NO generation measured directly by the electrochemical method can be significantly increased by AOAA.4. Western blot method shows that NaHS can reduce the phosphorylation of eNOS synthase at serine 1177 site, the phosphorylation of Akt at serine 308 site and the phosphorylation of at threonine 473 site; PI3 K inhibitor, LY294002, can block NaHS-induced decrease of phosphorylation of eNOS synthase at serine 1177 site; AOAA, can increase the phosphorylation of eNOS synthase at serine 1177 site, the phosphorylation of Akt at serine 308 site and the phosphorylation of at threonine 473 site; Given CBSsiRNA interference for 48 hours, the results show that CBSsiRNA can increase the phosphorylation of eNOS synthase at serine 1177 site, the phosphorylation of at threonine 473 site and the phosphorylation of Akt at serine 308 site; CBSsiRNA interference increases the total eNOS protein expression, but it does not change the total amount of Akt kinase significantly. CONCLUSIONS:(1) H2 S and NO synthase are expressed in the stomach fundus smooth muscle, and the cells can generate H2 S and NO stablely;(2) H2 S can increase gastric tension and NO can reduce gastric tension;(3) H2 S decreases the eNOS enzyme activity via PI3 K / Akt pathway thereby increased muscle tension.SUMMARYThe above results can be summarized as follows:1. H2 S can be produced by CBS and CSE in the mouse gastricfundus smooth muscle tissue; NO can be produced by eNOS andnNOS in the mouse gastric fundus smooth muscle tissue.2. H2 S can increase the smooth muscle tension via calciumdependent pathway.3. H2 S can increase the smooth muscle tension via PI3K/Akt/eNOSpathway.