Hydrogen sulfide (H2S) has been shown recently to function as an important gasotransmitter. The present study investigated the vascular effects of H2S, both exogenously applied and endogenously generated, on resistance mesenteric arteries of rats and the underlying mechanisms. Both H2S and NaHS evoked concentration-dependent relaxation of in vitro perfused rat mesenteric artery beds (MAB). The sensitivity of MAB to H2S (EC50, 25.2 +/- 3.6 microM) was about fivefold higher than that of rat aortic tissues. Removal of endothelium or coapplication of charybdotoxin and apamin to endothelium-intact MAB significantly reduced the vasorelaxation effects of H2S. The H2S-induced relaxation of MAB was partially mediated by ATP-sensitive K+ (KATP) channel activity in vascular smooth muscle cells. Pinacidil (EC50, 1.7 +/- 0.1 microM, n=6) mimicked, but glibenclamide (10 microM, n=6) suppressed, the vasorelaxant effect of H2S. KATP channel currents in isolated mesenteric artery smooth muscle cells were significantly augmented by H2S. L-cysteine, a substrate of cystathionine-gamma-lyase (CSE), at 1 mM increased endogenous H2S production by sixfold in rat mesenteric artery tissues and decreased contractility of MAB. DL-propargylglycine (a blocker of CSE) at 10 microM abolished L-cysteine-dependent increase in H2S production and relaxation of MAB. Our results demonstrated a tissue-specific relaxant response of resistance arteries to H2S. The stimulation of KATP channels in vascular smooth muscle cells and charybdotoxin/apamin-sensitive K+ channels in vascular endothelium by H2S represents important cellular mechanisms for H2S effect on MAB. Our study also demonstrated that endogenous CSE can generate sufficient H2S from exogenous L-cysteine to cause vasodilation. Future studies are merited to investigate direct contribution of endogenous H2S to regulation of vascular tone.

ATP-sensitive K+ (K(ATP)) channels in vascular smooth muscle cells (VSMC) are important targets for endogenous metabolic regulation and exogenous drug therapy. H2S, as a novel gasotransmitter, has been shown to relax rat aortic tissues via opening of K(ATP) channels. However, interaction of H2S, exogenous-applied or endogenous-produced, with K(ATP) channels in resistance artery VSMC has not been delineated. In the present study, using the whole-cell and single-channel patch-clamp technique, we demonstrated that exogenous H2S activated K(ATP) channels and hyperpolarized cell membrane in rat mesenteric artery VSMC. H2S enhanced the amplitude of whole-cell K(ATP) currents with an EC50 value of 116 +/- 8.3 microM and increased the open probability of single K(ATP) channels. H2S hyperpolarized membrane potentials by -12 mV in nystatin-perforated VSMC. Furthermore, inhibition of endogenous H2S production with D,L-propargylglycine (PPG) reduced whole-cell K(ATP) currents. PPG alone had no effect on unitary K(ATP) channel currents in cell-free membrane patches. In addition, effects of H2S on K(ATP) channels and membrane potentials were independent of cGMP-mediated phosphorylation. This study demonstrated modulation of K(ATP) channel activity by exogenous and endogenous H2S in resistance artery VSMC, thus helping elucidate cardiovascular functions of this endogenous gas.

1. Hydrogen sulfide (H(2)S) is a signalling gasotransmitter. It targets different ion channels and receptors, and fulfils its various roles in modulating the functions of different systems. However, the interaction of H(2)S with different types of ion channels and underlying molecular mechanisms has not been reviewed systematically. 2. H(2)S is the first identified endogenous gaseous opener of ATP-sensitive K(+) channels in vascular smooth muscle cells. Through the activation of ATP-sensitive K(+) channels, H(2)S lowers blood pressure, protects the heart from ischemia and reperfusion injury, inhibits insulin secretion in pancreatic beta cells, and exerts anti-inflammatory, anti-nociceptive and anti-apoptotic effects. 3. H(2)S inhibited L-type Ca(2+) channels in cardiomyocytes but stimulated the same channels in neurons, thus regulating intracellular Ca(2+) levels. H(2)S activated small and medium conductance K(Ca) channels but its effect on BK(Ca) channels has not been consistent. 4. H(2)S-induced hyperalgesia and pro-nociception seems to be related to the sensitization of both T-type Ca(2+) channels and TRPV(1) channels. The activation of TRPV(1) and TRPA(1) by H(2)S is believed to result in contraction of nonvascular smooth muscles and increased colonic mucosal Cl(-) secretion. 5. The activation of Cl(-) channel by H(2)S has been shown as a protective mechanism for neurons from oxytosis. H(2)S also potentiates N-methyl-d-aspartic acid receptor-mediated currents that are involved in regulating synaptic plasticity for learning and memory. 6. Given the important modulatory effects of H(2)S on different ion channels, many cellular functions and disease conditions related to homeostatic control of ion fluxes across cell membrane should be re-evaluated.

Hydrogen sulfide (H(2)S) is an endogenous opener of K(ATP) channels in many different types of cells. However, the molecular mechanism for an interaction between H(2)S and K(ATP) channel proteins remains unclear. The whole-cell patch-clamp technique and mutagenesis approach were used to examine the effects of H(2)S on different K(ATP) channel subunits, rvKir6.1 and rvSUR1, heterologously expressed in HEK-293 cells. H(2)S stimulated coexpressed rvKir6.1/rvSUR1 K(ATP) channels, but had no effect on K(ATP) currents generated by rvKir6.1 expression alone. Intracellularly applied sulfhydryl alkylating agent (N-ethylmaleimide, NEM), oxidizing agent (chloramine T, CLT), and a disulfide bond-oxidizing enzyme (protein disulfide isomerase) did not alter H(2)S effects on this recombinant channels. CLT, but not NEM, inhibited basal rvKir6.1/rvSUR1 currents, and both abolished the stimulatory effects of H(2)S on K(ATP) currents, when applied extracellularly. After selective cysteine residues (C6S and C26S but not C1051S and C1057S) in the extracellular loop of rvSUR1 subunits were point-mutated, H(2)S lost its stimulatory effects on rvKir6.1/rvSUR1 currents. Our results demonstrate that H(2)S interacts with Cys6 and Cys26 residues of the extracellular N terminal of rvSUR1 subunit of K(ATP) channel complex. Direct chemical modification of rvSUR1 subunit protein constitutes a molecular mechanism for the activation of K(ATP) channels by H(2)S.

Taken together, H₂S is an EDHF. The identification of H2S as an EDHF will not only solve one of the long-lasting perplexing puzzles for the mechanisms underlying endothelium-dependent vasorelaxation, but also shed light on potential therapeutic effects of H₂S on pathological abnormalities in peripheral resistance arteries.

Molecular basis of native voltage-dependent K(+) (Kv) channels in smooth muscle cells (SMCs) from rat mesenteric arteries was investigated. The whole cell patch-clamp study revealed that a 4-aminopyridine-sensitive delayed rectifier K(+) current (I(K)) was the predominant K(+) conductance in these cells. A systematic screening of the expression of 18 Kv channel genes using RT-PCR technique showed that six I(K)-encoding genes (Kv1.2, Kv1.3, Kv1.5, Kv2.1, Kv2.2, and Kv3.2) were expressed in mesenteric artery. Although no transient outward Kv currents (I(A)) were recorded in the studied SMCs, transcripts of multiple I(A)-encoding genes, including Kv1.4, Kv3.3, Kv3.4, Kv4.1, Kv4.2, and Kv4.3 as well as I(A)-facilitating Kv beta-subunits (Kvbeta1, Kvbeta2, and Kvbeta3), were detected in mesenteric arteries. Western blot analysis demonstrated that four I(K)-related Kv channel proteins (Kv1.2, Kv1. 3, Kv1.5, and Kv2.1) were detected in mesenteric artery tissues. The presence of Kv1.2, Kv1.3, Kv1.5, and Kv2.1 channel proteins in isolated SMCs was further confirmed by immunocytochemistry study. Our results suggest that the native I(K) in rat mesenteric artery SMCs might be generated by heteromultimerization of Kv genes.

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Reduced β-cell mass and increased activities of ATP-sensitive K(+) channels in pancreatic β cells are associated with the pathogenesis of diabetes. Cystathionine γ-lyase (CSE) is a major hydrogen sulfide (H(2)S)-producing enzyme in pancreatic β cells. Herein, we examine the effects of genetic and pharmacologic ablation of CSE on β-cell functions and their correlation with streptozotocin (STZ)-induced diabetes. Compared with wild-type mice, CSE knockout (CSE KO) mice that received STZ injections exhibited a delayed onset of diabetic status. The application of dl-propargylglycine (PPG) to inhibit CSE activity protected wild-type mice from STZ-induced hyperglycemia and hypoinsulinemia. STZ significantly increased pancreatic H(2)S production in wild-type mice but not in CSE KO mice. STZ induced more apoptotic β-cell death in wild-type mice than in CSE KO mice. STZ exposure decreased the viability of cultured INS-1E cells, which was partly reversed by PPG co-treatment. STZ also significantly stimulated H(2)S production in cultured INS-1E cells. In addition, STZ stimulated ATP-sensitive K(+) currents in pancreatic β cells from wild-type mice but not in the presence of PPG or in β cells from CSE KO mice. Sodium hydrosulfide injection instantly increased blood glucose, decreased plasma insulin, and deteriorated glucose tolerance in mice. Take together, these results provide evidence that the CSE/H(2)S system plays a critical role in regulating β-cell functions.