491 Down-Regulation of MicroRNA-133a Due to Oxidative Stress Mediates Up-Regulation of RHOA Expression and Increase in Rho Kinase Activity and Gastric Muscle Contraction in Diabetes

Hydrogen sulfide(H 2 S), mainly produced endogenously by two enzymes, cystathionine γlyase(CSE) and cystathionine β-synthase(CBS), is a messenger molecule in the central nervous system, in the GI tract and in sympathetic prevertebral ganglia.Our previous study found, in mouse superior mesenteric ganglion(SMG), that exogenous H 2 S acts selectively on splanchnic nerve terminals to potentiate fast excitatory post synaptic potentials (F-EPSPs).In this study, we investigated the distribution of CSE and CBS and the role of endogenously produced H 2 S on central sympathetic input.Methods: The SMG, splanchnic nerve trunks and colonic nerve trunk were dissected from adult SJL/J mice.Immunostaining was performed with antibodies for CSE and CBS and for vesicular acetylcholine transporter(VAChT) to identify cholinergic nerve terminals.Microelectrodes were used for intracellular recordings.Results: CSE immunoreactivity(IR) was found in neurons and glia cells and did not co-localize with VAChT-IR.CBS-IR was found only in glia cells.To determine whether the F-EPSPs could be potentiated by endogenously released H 2 S, we tested the effect of inhibiting H 2 S breakdown with stigmatellin, a specific sulfide quinone reductase inhibitor, on F-EPSPs.Stigmatellin(1μM) significantly(P<0.05)increased the amplitude and the area of F-EPSPs evoked by splanchnic nerve stimulation(21.0±4.8mV and 441±107ms•mV with stigmatellin vs. 18.4±5.3mVand 338±108ms•mV before stigmatellin, n=4) but had no significant effect on F-EPSPs evoked by colonic nerve stimulation.In contrast, the CSE inhibitor, dl-propargylglycine(PAG, 1mM), significantly(P<0.05)decreased the amplitude and the area of F-EPSPs evoked by splanchnic nerve stimulation(23.7±3.9mV and 824±288ms•mV with PAG vs. 27.0±4.1mVand 1230±414ms•mV before PAG, n=6), but had no effect on F-EPSPs evoked by colonic nerve stimulation.Surprisingly, the CBS inhibitor, aminooxyacetic acid(AOAA, 0.5mM), increased the amplitude and the area of F-EPSPs evoked by splanchnic nerve stimulation(23.6±12.1mVand 651±455ms•mV with AOAA vs. 20.2±10.9mVand 438±318ms•mV before AOAA, n=4, P<0.05) and increased the amplitude and the area of F-EPSPs evoked by colonic nerve stimulation(36.2±8.2mV and 1609±468ms•mV with AOAA vs. 28.9±8.1mVand 1084±402ms•mV before AOAA, n=6, P<0.05).Bicuculline(20μM), a specific GABA-A receptor inhibitor, blocked the potentiating effect of AOAA suggesting that the AOAA induced potentiation of F-EPSPs was due to the inhibition of GABA transaminase-induced accumulation of GABA.Conclusion: We conclude that endogenously produced H 2 S in the mouse SMG modulates fast cholinergic synaptic input in a pathwayspecific manner.H 2 S modulates fast cholinergic synaptic input from central splanchnic nerve terminals but has no affect on input from peripheral cholinergic input.