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Hydrogen sulfide ([H.sub.2]S), an endogenous gaseous mediator, produces both vasorelaxation and vasoconstriction at different concentrations. We found in the present study that NaHS, an [H.sub.2]S donor, produced stronger vasorelaxant and weaker vasoconstrictive effects in HEPES solution compared with those achieved in Krebs solution. We further screened the buffer components and found that bicarbonate (HC[O.sup.-.sub.3]) was the ion to influence the effect of [H.sub.2]S. After examining the vasorelaxant effects of acetylcholine, a vasodilator by releasing nitric oxide, and isoprenaline, a [beta]-adrenoceptor agonist, in HEPES and Krebs buffers, we found the HC[O.sup.-.sub.3]-dependent effect was specific to [H.sub.2]S. Blockade of anion exchanger-2 activity with 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) or with HC[O.sup.-.sub.3]-free solution abolished the vasoconstrictive effect of NaHS. Moreover, NaHS decreased nitric oxide level in the rat aorta in HC[O.sup.-.sub.3]-containing buffer, but this effect was abolished by HC[O.sup.-.sub.3]-free buffer or DIDS. In summary, we found for the first time that [H.sub.2]S stimulates anion exchanger to transport extracellular HC[O.sup.-.sub.3] in exchange for intracellular superoxide anions, which may further inactivate nitric oxide and induces vasoconstriction. anion exchanger; superoxide anions doi: 10.1152/ajpcell.00105.2010.

Systemic vascular resistance ([R.sub.sys]) of freshwater turtles increases substantially during anoxia, but the underlying mechanisms are not fully understood. We investigated whether hydrogen sulfide ([H.sub.2]S), an endogenously produced metabolite believed to be an [O.sub.2] sensor/ transducer of vasomotor tone, contributes to the increased [R.sub.sys] of anoxic red-eared slider turtles (Trachemys scripta). Vascular infusion of the [H.sub.2]S donor Naris in anesthetized turtles at 21[degrees]C and fully recovered normoxic turtles at 5[degrees]C and 21[degrees]C revealed [H.sub.2]S to be a potent vasoconstrictor of the systemic circulation. Likewise, wire myography of isolated turtle mesenteric and pulmonary arteries demonstrated [H.sub.2]S to mediate an anoxia-induced constriction. Intriguingly, however, Naris did not exert vasoconstrictory effects during anoxia (6 h at 21[degrees]C; 14 days at 5[degrees]C) when plasma [H.sub.2]S concentration, estimated from the colorimetric measurement of plasma acid-labile sulfide concentration, likely increased by ~3-and 4-fold during anoxia at 21[degrees]C, and 5[degrees]C, respectively. Yet, blockade of endogenous [H.sub.2]S production by DL-propargylglycine or hydroxylamine (0.44 mmol/kg) partially reversed the decreased systemic conductance ([G.sub.sys]) exhibited by 5[degrees]C anoxic turtles. These findings suggest that the signal transduction pathway of [H.sub.2]S-mediated vasoactivity is either maximally activated in the systemic circulation of anoxic turtles and/or that it is oxygen dependent. red-eared slider; anoxia; temperature; cardiovascular; systemic resistance; pulmonary resistance; blood pressure; heart doi: 10.1152/ajpregu.00521.2009.

Reactive oxygen and nitrogen species (ROS and RNS, respectively) generate nitrotyrosine and activate latent resident myocardial matrix metalloproteinases (MMPs). Although in chronic heart failure (CHF) there is robust increase in ROS, RNS, and MMP activation, recent data suggest that hydrogen sulfide ([H.sub.2]S, a strong antioxidant gas) is cardioprotective. However, the role of [H.sub.2]S in mitigating oxidative and proteolytic stresses in cardiac remodeling/apoptosis in CHF was unclear. To test the hypothesis that [H.sub.2]S ameliorated cardiac apoptosis and fibrosis by decreasing oxidative and proteolytic stresses, arteriovenous fistula (AVF) was created in wild-type (C57BL/6J) mice. The hearts were analyzed at 0, 2, and 6 wk after AVF. To reverse the remodeling, AVF mice were treated with NariS (an H2S donor, 30 [micro]mol/l in drinking water) at 8 and 10 wk. The levels of MMPs were measured by gelatin-gel zymography. The levels of nitrotyrosine, tissue inhibitors of metalloproteinase (TIMPs), [[beta].sub.1]-integrin, and a disintegrin and metalloproteinase-12 (ADAM-12) were analyzed by Western blots. The levels of pericapillary and interstitial fibrosis were identified by Masson trichrome stains. The levels of apoptosis were measured by identifying the TdT-mediated dUTP nick end labeling (TUNEL)positive cells and caspase-3 levels. The results suggested robust nitrotyrosine and MMP activation at 2 and 6 wk of AVF. The treatment with [H.sub.2]S donor mitigated nitrotyrosine generation and MMP activation (i.e., oxidative and proteolytic stresses). The levels of TIMP-1 and TIMP-3 were increased and TIMP-4 decreased in AVF hearts. The treatment with [H.sub.2]S donor reversed this change in TIMPs levels. The levels of ADAM-12, apoptosis, and fibrosis were robust and integrin were decreased in AVF hearts. The treatment with [H.sub.2]S donor attenuated the fibrosis, apoptosis, and decrease in integrin. fibrosis; extracellular matrix; ADAM; integrin; matrix metalloproteinase; tissue inhibitor of metalloproteinase; left ventricle hypertrophy; collagen; TUNEL; apoptosis; homocysteine doi: 10.1152/ajpheart.00682.2009

Am J Physiol Regul Integr Comp Physiol 298: R51-R60, 2010. First published November 4, 2009; doi: 10.1152/ajpregu.00576.2009.--Hypoxic pulmonary vasoconstriction (HVC), an intrinsic and assumed ubiquitous response of mammalian pulmonary blood vessels, matches regional ventilation to perfusion via an unknown O2-sensing mechanism. Global pulmonary hypoxia experienced by individuals suffering from chronic obstructive pulmonary disease or numerous hypoventilation syndromes, including sleep apnea, often produces maladaptive pulmonary hypertension, but pulmonary hypertension is not observed in diving mammals, where profound hypoxia is routine. Here we examined the response of cow and sea lion pulmonary arteries (PA) to hypoxia and observed the expected HVC in the former and a unique hypoxic vasodilation in resistance vessels in the latter. We then used this disparate response to examine the [O.sub.2]-sensing mechanism. In both animals, exogenous [H.sub.2]S mimicked the vasoactive effects of hypoxia in isolated PA. [H.sub.2]S-synthesizing enzymes, cystathionine [beta]-synthase, cystathionine [gamma]-lyase, and 3-mercaptopyruvate sulfur transferase, were identified in lung tissue from both animals by one-dimensional Western blot analysis and immunohistochemistry. The relationship between [H.sub.2]S production/consumption and [O.sub.2] was examined in real time by use of amperometric [H.sub.2]S and [O.sub.2] sensors. H2S was produced by sea lion and cow lung homogenate in the absence of [O.sub.2], but it was rapidly consumed when [O.sub.2] was present. Furthermore, consumption of exogenous [H.sub.2]S by cow lung homogenate, PA smooth muscle cells, and heart mitochondria was [O.sub.2] dependent and exhibited maximal sensitivity at physiologically relevant [Po.sub.2] levels. These studies show that HVC is not an intrinsic property of PA and provide further evidence for [O.sub.2]-dependent [H.sub.2]S metabolism in [O.sub.2] sensing. hypoxic vasoconstriction; pinniped; pulmonary hypertension

Elevated level of homocysteine (Hcy), known as hyperhomocysteinemia (HHcy), is associated with end-stage renal diseases. Hcy metabolizes in the body to produce hydrogen sulfide ([H.sub.2]S), and studies have demonstrated a protective role of HRS in end-stage organ failure. However, the role of [H.sub.2]S in HHcy-associated renal diseases is unclear. The present study was aimed to determine the role of [H.sub.2]S in HHcy-associated renal damage. Cystathionine-[beta]-synthase heterozygous (CBS+/-) and wild-type (WT, C57BL/6J) mice with two kidney (2-K) were used in this study and supplemented with or without NariS (30 [micro]mol/l, [H.sub.2]S donor) in the drinking water. To expedite the HHcy-associated glomerular damage, uninephrectomized (1-K) CBS(+/-) and 1-K WT mice were also used with or without NariS supplementation. Plasma Hcy levels were elevated in CBS(+/-) 2-K and 1-K and WT 1-K mice along with increased proteinuria, whereas, plasma levels of [H.sub.2]S were attenuated in these groups compared with WT 2-K mice. Interestingly, [H.sub.2]S supplementation increased plasma [H.sub.2]S level and normalized the urinary protein secretion in the similar groups of animals as above. Increased activity of matrix metalloproteinase (MMP)-2 and -9 and apoptotic cells were observed in the renal cortical tissues of CBS(+/-) 2-K and 1-K and WT 1-K mice; however, [H.sub.2]S prevented apoptotic cell death and normalized increased MMP activities. Increased expression of desmin and downregulation of nephrin in the cortical tissue of CBS(+/-) 2-K and 1-K and WT 1-K mice were ameliorated with [H.sub.2]S supplementation. Additionally, in the kidney tissues of CBS(+/-) 2-K and 1-K and WT 1-K mice, increased superoxide ([[O.sub.2].sup..-]) production and reduced glutathione (GSH)-to-oxidized glutathione (GSSG) ratio were normalized with exogenous [H.sub.2]S supplementation. These results demonstrate that HHcy-associated renal damage is related to decreased endogenous [H.sub.2]S generation in the body. Additionally, here we demonstrate with evidence that [H.sub.2]S supplementation prevents HHcy-associated renal damage, in part, through its antioxidant properties. cystathionine-[beta] synthase; cystathionine-[gamma] lyase; homocysteine; matrix metalloproteinase

Hydrogen sulfide ([H.sub.2]S) is one of three endogenous gases, along with carbon monoxide (CO) and nitric oxide (NO), that exert a variety of important vascular actions in vivo. Although it has been demonstrated that CO or NO can trigger the development of a preconditioned phenotype in postischemic tissues, it is unclear whether [H.sub.2]S may also induce protection in organs subsequently exposed to ischemia-reperfusion (I/R). In light of these observations, we postulated that preconditioning with the exogenous [H.sub.2]S donor sodium hydrosulfide (NaHS-PC) would inhibit leukocyte rolling (LR) and adhesion (LA) induced by I/R. We used intravital microscopic techniques to demonstrate that NaHS-PC 24 h, but not 1 h, before I/R causes postcapillary venules to shift to an anti-inflammatory phenotype in wild-type (WT) mice such that these vessels fail to support LR and LA during reperfusion. The protective effect of NaHS-PC on LR was largely abolished by coincident pharmacological inhibition of NO synthase (NOS) in WT animals and was absent in endothelial NOS-deficient ([eNOS.sup.-/-]) mice. A similar pattern of response was noted in WT mice treated concomitantly with NaHS plus p38 mitogen-activated protein kinase (MAPK) inhibitors (SB 203580 or SK-86002). Whereas the reduction in LA induced by antecedent NaHS was attenuated by pharmacological inhibition of NOS or p38 MAPK in WT mice, the antiadhesive effect of NaHS was still evident in [eNOS.sup.-/-] mice. Thus NaHS-PC prevents LR and LA by triggering the activation of an eNOS- and p38 MAPK-dependent mechanism. However, the role of eNOS in the antiadhesive effect of NaHS-PC was less prominent than its effect to reduce LR. ischemia; reperfusion; sodium hydrosulfide; endothelial nitric oxide synthase-deficient mice; leukocyte rolling; leukocyte adhesion

Hydrogen sulfide is gaining acceptance as an endogenously produced modulator of tissue function. The present paradigm of [H.sub.2]S (diprotonated, gaseous form of hydrogen sulfide) as a tissue messenger consists of [H.sub.2]S being released from the desulfhydration of L-cysteine at a rate sufficient to maintain whole tissue hydrogen sulfide concentrations of 30 [micro]M to >100 [micro]M, and these tissue concentrations serve a messenger function. Utilizing physiological concentrations of L-cysteine and aerobic conditions, we found that catabolism of hydrogen sulfide by mouse liver and brain homogenates exceeded the rate of enzymatic release of this compound such that measureable hydrogen sulfide release was less with tissue-containing vs. tissue-free buffers. Analyses of the gas space over rapidly homogenized mouse brain and liver indicated that in situ tissue hydrogen sulfide concentrations were only about 15 nM. Human alveolar air measurements indicated negligible free [H.sub.2]S concentrations in blood. We conclude rapid tissue catabolism of hydrogen sulfide maintains whole tissue brain and liver concentrations of free hydrogen sulfide that are three orders of magnitude less than conventionally accepted values and only 1/5,000 of the hydrogen sulfide concentration (100 [micro]M) required to alter cellular function in vitro. For hydrogen sulfide to serve as an endogenously produced messenger, tissue production and catabolism must result in intracellular microenvironments with a sufficiently high hydrogen sulfide concentration to activate a local signaling mechanism, while whole tissue concentrations remain very low. cell signaler; nitric oxide; cytochrome c oxidase

The present study aimed to investigate the role of hydrogen sulphide ([H.sub.2]S) 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 [H.sub.2]S production with D-L-propargylglycine. Moreover, IPostC significantly stimulated [H.sub.2]S 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 [H.sub.2]S 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 [micro]M) or PKC with chelerythrine (10 [micro]M) abolished the cardioprotection induced by [H.sub.2]S postconditioning. SPostC2, but not SPostC, also additionally stimulated eNOS. We conclude that endogenous [H.sub.2]S contributes to IPostC-induced cardioprotection. [H.sub.2]S postconditioning confers the protective effects against ischemia-reperfusion injury through the activation of Akt, PKC, and eNOS pathways. Akt; protein kinase C; hydrogen sulphide; endothelial nitric oxide synthase

[O.sub.2] chemoreceptors elicit cardiorespiratory reflexes in all vertebrates, but consensus on [O.sub.2]-sensing signal transduction mechanism(s) is lacking. We recently proposed that hydrogen sulfide ([H.sub.2]S) metabolism is involved in [O.sub.2] sensing in vascular smooth muscle. Here, we examined the possibility that [H.sub.2]S is an [O.sub.2] sensor in trout chemoreceptors where the first pair of gills is a primary site of aquatic [O.sub.2] sensing and the homolog of the mammalian carotid body. Intrabuccal injection of [H.sub.2]S in unanesthetized trout produced a dose-dependent bradycardia and increased ventilatory frequency and amplitude similar to the hypoxic response. Removal of the first, but not second, pair of gills significantly inhibited [H.sub.2]S-mediated bradycardia, consistent with the loss of aquatic chemoreceptors, mRNA for [H.sub.2]S-synthesizing enzymes, cystathionine [beta]-synthase and cystathionine [gamma]-lyase, was present in branchial tissue. Homogenized gills produced [H.sub.2]S enzymatically, and [H.sub.2]S production was inhibited by [O.sub.2], whereas mitochondrial [H.sub.2]S consumption was [O.sub.2] dependent. Ambient hypoxia did not affect plasma [H.sub.2]S in unanesthetized trout, but produced a [Po.sub.2]-dependent increase in a sulfide moiety suggestive of increased [H.sub.2]S production. In isolated zebrafish neuroepithelial cells, the putative chemoreceptive cells of fish, both hypoxia and [H.sub.2]S, produced a similar ~10-mV depolarization. These studies are consistent with [H.sub.2]S involvement in [O.sub.2] sensing/signal transduction pathway(s) in chemoreceptive cells, as previously demonstrated in vascular smooth muscle. This novel mechanism, whereby [H.sub.2]S concentration ([[H.sub.2]S]) is governed by the balance between constitutive production and oxidation, tightly couples tissue [[H.sub.2]S] to [Po.sub.2] and may provide an exquisitely sensitive, yet simple, [O.sub.2] sensor in a variety of tissues. hydrogen sulfide concentration

Hydrogen sulfide ([H.sub.2]S) is an endogenously produced gaseous signaling molecule with diverse physiological activity. The potential protective effects of [H.sub.2]S have not been evaluated in the liver. The purpose of the current study was to investigate if [H.sub.2]S could afford hepatoprotection in a murine model of hepatic ischemia-reperfusion (I/R) injury. Hepatic injury was achieved by subjecting mice to 60 min of ischemia followed by 5 h of reperfusion. [H.sub.2]S donor (IK1001) or vehicle were administered 5 min before reperfusion. [H.sub.2]S attenuated the elevation in serum alanine aminotransferase (ALT) by 68.6% and aspartate aminotransferase (AST) by 70.8% compared with vehicle group. [H.sub.2]S-mediated cytoprotection was associated with an improved balance between reduced glutathione (GSH) vs. oxidized glutathione (GSSG), an attenuated formation of lipid hydroperoxides, and an increased expression of thioredoxin-1 (Trx-1). Furthermore, [H.sub.2]S inhibited the progression of apoptosis after I/R injury by increasing the protein expression of heat shock protein (HSP-90) and Bcl-2. These results indicate that [H.sub.2]S protects the murine liver against I/R injury through an upregulation of intracellular antioxidant and antiapoptotic signaling pathways. apoptosis; liver; glutathione; thioredoxin; heat shock protein 90

Hydrogen sulfide ([H.sub.2]S) is rapidly emerging as a biologically significant signaling molecule. Studies published before 2000 report low or undetectable [H.sub.2]S (usually as total sulfide) levels in blood or plasma, whereas recent work has reported sulfide concentrations between 10 and 300 [micro]M, suggesting it acts as a circulating signal. In the first series of experiments, we used a recently developed polarographic sensor to measure the baseline level of endogenous [H.sub.2]S gas and turnover of exogenous [H.sub.2]S gas in real time in blood from numerous animals, including lamprey, trout, mouse, rat, pig, and cow. We found that, contrary to recent reports, [H.sub.2]S gas was essentially undetectable (< 100 nM total sulfide) in all animals. Furthermore, exogenous sulfide was rapidly removed from blood, plasma, or 5% bovine serum albumin in vitro and from intact trout in vivo. To determine if blood [H.sub.2]S could transiently increase, we measured oxygen-dependent [H.sub.2]S production by trout hearts in vitro and in vivo. [H.sub.2]S has been shown to mediate ischemic preconditioning (IPC) in mammals. [PC is present in trout and, unlike mammals, the trout myocardium obtains its oxygen from relatively hypoxic systemic venous blood. In vitro, myocardial [H.sub.2]S production was inversely related to P[O.sub.2], whereas we failed to detect [H.sub.2]S in ventral aortic blood from either normoxic or hypoxic fish in vivo. These results provide an autocrine or paracrine mechanism for myocardial coupling of hypoxia to [H.sub.2]S in [PC, i.e., oxygen sensing, but they fail to provide any evidence that [H.sub.2]S signaling is mediated by the circulation. gasotransmitter; hydrogen sulfide metabolism; vascular signaling

The aim of the present study was to investigate the effect of hydrogen sulfide ([H.sub.2]S) signaling by nitric oxide (NO) in isolated rat aortas and cultured human umbilical vein endothelial cells (HUVECs). Both administration of [H.sub.2]S and NariS, as well as endogenous [H.sub.2]S, reduced NO formation, endothelial nitric oxide synthase (eNOS) activity, eNOS transcript abundance, and L-arginine (L-Arg) transport (all P < 0.01). The kinetics analysis of eNOS activity and L-Arg transport showed that [H.sub.2]S reduced [V.sub.max] values (all P < 0.01) without modifying [K.sub.m] parameters. Use of selective NOS inhibitors verified that eNOS [vs. inducible NOS (iNOS) and neuronal NOS (nNOS)] was the specific target of [H.sub.2]S regulation. [H.sub.2]S treatment (100 [micro]mol/l) reduced Akt phosphorylation and decreased eNOS phosphorylation at Ser1177. [H.sub.2]S reduced L-Arg uptake by inhibition of a system y+ transporter and decreased the CAT-1 transcript. [H.sub.2]S treatment reduced protein expression of eNOS but not of nNOS and iNOS. Pinacidil ([K.sub.ATP] channel opener) exhibited the similar inhibitory effects on the L-Arg/NOS/NO pathway. Glibenclamide ([K.sub.ATP] channel inhibitor) partly blocked the inhibitory effect of [H.sub.2]S and pinacidil. An in vivo experiment revealed that [H.sub.2]S downregulated the vascular L-Arg/eNOS/NO pathway after intraperitoneal injection of NariS (14 [micro]mol/kg) in rats. Taken together, our findings suggest that [H.sub.2]S downregulates the vascular L-Arg/NOS/NO pathway in vitro and in vivo, and the [K.sub.ATP] channel could be involved in the regulatory mechanism of [H.sub.2]S. nitric oxide; nitric oxide synthase; L-arginine transport

Hydrogen sulfide ([H.sub.2]S) is a gasotransmitter that regulates cardiovascular functions. The present study aimed to examine the hypothesis that chronic treatment with sodium hydrosulfide (NariS, an [H.sub.2]S donor) is able to prevent left-ventricular remodeling in spontaneously hypertensive rats (SHR). Four-week-old SHR were treated with NailS (10, 30, and 90 [micro]mol x [kg.sup.-1] x [day.sup.-1]), a combination of Naris (30 [micro]mol x [kg.sup.-1] x [day.sup.-1]) and glibenclamide (5 mg x [kg.sup.-1] x [day.sup.-1]), glibenclamide alone (5 mg x [kg.sup.-1] x [day.sup.-1]), hydralazine alone (10 mg x [kg.sup.-1] x [day.sup.-1]), and placebo for 3 too. At the end of the treatment period, variables such as cardiac geometry and function, intramyocardial arterioles ranging in diameter from 25 to 100 [micro]m, perivascular and interstitial collagen content, reactive oxygen species (ROS), thiol groups, conjugated dienes, and DNA base modification were examined. The novel finding of the present study is that chronic NariS treatment prevented the hypertrophy of intramyocardial arterioles and ventricular fibrosis, as well as decreased myocardial ROS and conjugated diene levels. The cardioprotective effects were blunted by coadministration of glibenclamide, suggesting a role of ATP-sensitive potassium channels in mediating the action of NariS. Hydralazine caused a comparable reduction of blood pressure compared with NariS treatment; however, it exerted no effect on the remodeling process or on ROS and conjugated diene levels. Moreover, NariS treatment caused an increase in myocardial thiol group levels, whereas DNA base modification was not altered by NariS treatment. In conclusion, the superior cardioprotective effects of NariS treatment are worthy to be further explored to develop novel therapeutic approaches for the treatment of cardiac remodeling in hypertension. left-ventricular remodeling; gasotransmitter; hypertension; hydrogen sulfide