Hydrogen sulfide regulates Na+/H+ exchanger activity via stimulation of phosphoinositide 3-kinase/Akt and protein kinase G pathways.

Intracellular pH (pH(i)) is an important endogenous modulator of cardiac function. Inhibition of Na(+)/H(+) exchanger-1 (NHE-1) protects the heart by preventing Ca(2+) overload during ischemia/reperfusion. Hydrogen sulfide (H(2)S) has been reported to produce cardioprotection. The present study was designed to investigate the pH regulatory effect of H(2)S in rat cardiac myocytes and evaluate its contribution to cardioprotection. It was found that sodium hydrosulfide (NaHS), at a concentration range of 10 to 1000 µM, produced sustained decreases in pH(i) in the rat myocytes in a concentration-dependent manner. NaHS also abolished the intracellular alkalinization caused by trans-(±)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide methane-sulfonate hydrate (U50,488H), which activates NHEs. Moreover, when measured with an NHCl(4) prepulse method, NaHS was found to significantly suppress NHE-1 activity. Both NaHS and cariporide or [5-(2-methyl-5-fluorophenyl)furan-2-ylcarbonyl]guanidine (KR-32568), two NHE inhibitors, protected the myocytes against ischemia/reperfusion injury. However, coadministration of NaHS with KR-32568 did not produce any synergistic effect. Functional study showed that perfusion with NaHS significantly improved postischemic contractile function in isolated rat hearts subjected to ischemia/reperfusion. Blockade of phosphoinositide 3-kinase (PI3K) with 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), Akt with Akt VIII, or protein kinase G (PKG) with (9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]]enzodiazocine-10-carboxylic acid, methyl ester (KT5823) significantly attenuated NaHS-suppressed NHE-1 activity and/or NaHS-induced cardioprotection. Although KT5823 failed to affect NaHS-induced Akt phosphorylation, Akt inhibitor did attenuate NaHS-stimulated PKG activity. In conclusion, this work demonstrated for the first time that H(2)S produced cardioprotection via the suppression of NHE-1 activity involving a PI3K/Akt/PKG-dependent mechanism.

Endogenous hydrogen sulphide mediates the cardioprotection induced by ischemic postconditioning.

The present study aimed to investigate the role of hydrogen sulphide (H2S) in the cardioprotection induced by ischemic postconditioning and to examine the underlying mechanisms. Cardiodynamics and myocardial infarction were measured in isolated rat hearts. Postconditioning with six episodes of 10-s ischemia (IPostC) significantly improved cardiodynamic function, which was attenuated by the blockade of endogenous H2S production with d-l-propargylglycine. Moreover, IPostC significantly stimulated H2S synthesis enzyme activity during the early period of reperfusion. However, d-l-propargylglycine only attenuated the IPostC-induced activation of PKC-alpha and PKC-epsilon but not that of PKC-delta, Akt, and endothelial nitric oxide synthase (eNOS). These data suggest that endogenous H2S contributes partially to the cardioprotection of IPostC via stimulating PKC-alpha and PKC-epsilon. Postconditioning with six episodes of a 10-s infusion of NaHS (SPostC) or 2 min continuous NaHS infusion (SPostC2) stimulated activities of Akt and PKC, improved the cardiodynamic performances, and reduced myocardial infarct size. The blockade of Akt with LY-294002 (15 microM) or PKC with chelerythrine (10 microM) abolished the cardioprotection induced by H2S postconditioning. SPostC2, but not SPostC, also additionally stimulated eNOS. We conclude that endogenous H2S contributes to IPostC-induced cardioprotection. H2S postconditioning confers the protective effects against ischemia-reperfusion injury through the activation of Akt, PKC, and eNOS pathways.

Characterization of a novel, water-soluble hydrogen sulfide-releasing molecule (GYY4137): new insights into the biology of hydrogen sulfide.

BACKGROUND: The potential biological significance of hydrogen sulfide (H(2)S) has attracted growing interest in recent years. The aim of this study was to characterize a novel, water-soluble, slow-releasing H(2)S compound [morpholin-4-ium 4 methoxyphenyl(morpholino) phosphinodithioate (GYY4137)] and evaluate its use as a tool to investigate the cardiovascular biology of this gas. METHODS AND RESULTS: The acute vasorelaxant effect of drugs was assessed in rat aortic rings and perfused rat kidney in vitro and in the anesthetized rat in vivo. The chronic effect of GYY4137 on blood pressure in normotensive and spontaneously hypertensive rats was determined by tail-cuff plethysmography. GYY4137 released H(2)S slowly both in aqueous solution in vitro and after intravenous or intraperitoneal administration in anesthetized rats in vivo. GYY4137 caused a slow relaxation of rat aortic rings and dilated the perfused rat renal vasculature by opening vascular smooth muscle K(ATP) channels. GYY4137 did not affect rat heart rate or force of contraction in vitro. GYY4137 exhibited antihypertensive activity as evidenced by ability to reduce N(G)-nitro-L-arginine methyl ester-evoked hypertension in the anesthetized rat and after chronic (14-day) administration in spontaneously hypertensive rats. CONCLUSIONS: These results identify GYY4137 as a slow-releasing H(2)S compound with vasodilator and antihypertensive activity. GYY4137 is likely to prove useful in the study of the many and varied biological effects of H(2)S. GYY4137 may also prove of therapeutic value in cardiovascular disease.

Cyclooxygenase-2 mediates the delayed cardioprotection induced by hydrogen sulfide preconditioning in isolated rat cardiomyocytes.

We previously reported that hydrogen sulfide (H(2)S) preconditioning (SP) produces cardioprotection in isolated rat cardiomyocytes. The present study was designed to determine the involvement of cyclooxygenase-2 (COX-2) in the SP-induced delayed cardioprotection. Isolated cardiac myocytes were treated with NaHS (100 microM, a H(2)S donor) for 30 min and then cultured for 20 h followed by ischemia/reperfusion insults. SP significantly increased cell viability, percentage of rod-shaped cells, and myocyte contractility after 10 min of reperfusion. Given 30 min before and during lethal ischemia, two selective COX-2 inhibitors, NS-398 and celebrex, abrogated SP-induced cardioprotective effects. Moreover, SP upregulated the expression of COX-2 and increased PGE(2) production in the cardiac myocytes. These effects were significantly attenuated by glibenclamide, an ATP-sensitive K(+) channel (K(ATP)) blocker, and chelerythrine, a selective protein kinase C (PKC) inhibitor, suggesting that activation of both K(ATP) and PKC is required for the stimulation of COX-2. Additionally, NG-nitro-L: -arginine methyl ester, a nitric oxide synthase inhibitor, failed to regulate COX-2 protein expression but inhibited SP-enhanced COX-2 activity and PGE(2) production. In conclusion, we provided the first evidence that SP may produce delayed cardioprotection via K(ATP)/PKC dependent induction of COX-2 expression and via nitric oxide-induced COX-2 activation.

Cardioprotection induced by hydrogen sulfide preconditioning involves activation of ERK and PI3K/Akt pathways.

We previously reported that hydrogen sulfide (H(2)S) preconditioning (SP) produces cardioprotective effects against ischemia in rat cardiac myocytes. The present study aims to elucidate the signaling mechanisms involved in SP-induced cardioprotection by investigating the role of extracellular signal regulated kinase (ERK1/2) and phosphatidylinositol 3-kinase (PI3K)/Akt. We found that preconditioning with NaHS (a H(2)S donor) for three cycles significantly decreased myocardial infarct size and improved heart contractile function in the isolated rat hearts. NaHS (1-100 microM) concentration-dependently increased cell viability and percentage of rod-shaped cardiac myocytes. Blockade of ERK1/2 with PD 98059 or PI3K/Akt with LY-294002 or Akt inhibitor III during either preconditioning or ischemia periods significantly attenuated the cardioprotection of SP, suggesting that both ERK1/2 and PI3K/Akt triggered and mediated the cardioprotection of SP. Moreover, SP induced ERK1/2 and Akt phosphorylation in isolated hearts. The phosphorylation of ERK1/2 induced by SP was attenuated by either glibenclamide, an ATP-sensitive K(+) channel (K(ATP)) blocker, or chelerythrine, a specific protein kinase C (PKC) blocker. In addition, ischemic-preconditioning-induced ERK1/2 activation was reversed by inhibiting endogenous H(2)S production, suggesting that ERK1/2 activation induced by ischemic preconditioning was, at least partly, mediated by endogenous H(2)S. In conclusion, K(ATP)/PKC/ERK1/2 and PI3K/Akt pathways contributed to SP-induced cardioprotection.

H2S preconditioning-induced PKC activation regulates intracellular calcium handling in rat cardiomyocytes.

The present study was aimed to investigate the regulatory effect of protein kinase C (PKC) on intracellular Ca(2+) handling in hydrogen sulfide (H(2)S)-preconditioned cardiomyocytes and its consequent effects on ischemia challenge. Immunoblot analysis was used to assess PKC isoform translocation in the rat cardiomyocytes 20 h after NaHS (an H(2)S donor, 10(-4) M) preconditioning (SP, 30 min). Intracellular Ca(2+) was measured with a spectrofluorometric method using fura-2 ratio as an indicator. Cell length was compared before and after ischemia-reperfusion insults to indicate the extent of hypercontracture. SP motivated translocation of PKCalpha, PKCepsilon, and PKCdelta to membrane fraction but only translocation of PKCepsilon and PKCdelta was abolished by an ATP-sensitive potassium channel blocker glibenclamide. It was also found that SP significantly accelerated the decay of both electrically and caffeine-induced intracellular [Ca(2+)] transients, which were reversed by a selective PKC inhibitor chelerythrine. These data suggest that SP facilitated Ca(2+) removal via both accelerating uptake of Ca(2+) into sarcoplasmic reticulum and enhancing Ca(2+) extrusion through Na(+)/Ca(2+) exchanger in a PKC-dependent manner. Furthermore, blockade of PKC also attenuated the protective effects of SP against Ca(2+) overload during ischemia and against myocyte hypercontracture at the onset of reperfusion. We demonstrate for the first time that SP activates PKCalpha, PKCepsilon, and PKCdelta in cardiomyocytes via different signaling mechanisms. Such PKC activation, in turn, protects the heart against ischemia-reperfusion insults at least partly by ameliorating intracellular Ca(2+) handling.