Hydrogen sul®de (H 2 S) has been traditionally viewed as a toxic gas. It is also, however, endogenously generated from cysteine metabolism. We attempted to assess the physiological role of H 2 S in the regulation of vascular contractility, the modulation of H 2 S production in vascular tissues, and the underlying mechanisms. Intravenous bolus injection of H 2 S transiently decreased blood pressure of rats by 12± 30 mmHg, which was antagonized by prior blockade of K ATP channels. H 2 S relaxed rat aortic tissues in vitro in a K ATP channel-dependent manner. In isolated vascular smooth muscle cells (SMCs), H 2 S directly increased K ATP channel currents and hyperpolarized membrane. The expression of H 2 S-generating enzyme was identi®ed in vascular SMCs, but not in endothelium. The endogenous production of H 2 S from different vascular tissues was also directly measured with the abundant level in the order of tail artery, aorta and mesenteric artery. Most importantly, H 2 S production from vascular tissues was enhanced by nitric oxide. Our results demonstrate that H 2 S is an important endogenous vasoactive factor and the ®rst identi®ed gaseous opener of K ATP channels in vascular SMCs.

Zhao, Weimin, and Rui Wang. H2S-induced vasorelaxation and underlying cellular and molecular mechanisms. Am J Physiol Heart Circ Physiol 283: H474-H480, 2002. First published April 18, 2002 10.1152/ajpheart.00013. 2002 is endogenously generated in vascular smooth muscle cells. The signal transduction pathways involved in the vascular effects of H2S have been unclear and were investigated in the present study. H 2S induced a concentration-dependent relaxation of rat aortic tissues that was not affected by vascular denervation. The vasorelaxant potency of H2S was attenuated by the removal of the endothelium. Similarly, the blockade of nitric oxide synthase or the coapplication of the Ca 2ϩ -dependent K ϩ channel blockers apamin and charybdotoxin reduced the H2S-induced relaxation of the endothelium-intact aortic tissues. Sodium nitroprusside (SNP)-induced relaxation was completely abolished by either 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) or NS-2028, two soluble guanylate cyclase inhibitors. Instead of inhibition, ODQ and NS-2028 potentiated the H2S-induced vasorelaxation, which was suppressed by superoxide dismutase. The vasorelaxant effect of H2S was also significantly attenuated when Ca 2ϩ -free bath solution was used. Finally, pretreatment of aortic tissues with H2S reduced the relaxant response of vascular tissues to SNP. Our results demonstrate that the vascular effect of H2S is partially mediated by a functional endothelium and dependent on the extracellular calcium entry but independent of the activation of the cGMP pathway. aorta; guanosine 3Ј,5Ј-cyclic monophosphate; endothelium H 2 S HAS BEEN GENERALLY CONSIDERED as a toxic gas found in the contaminated environmental atmosphere. Its major toxic effects are the toxication of central nervous system and the inhibition of the respiratory system (2, 10, 28). H 2 S can also be produced endogenously from L-cysteine by cystathionine ␤-synthase (CBS) and/or cystathionine ␥-lyase (CSE) (23,24). The expression of these enzymes has been detected in various tissues (13). Our recent study (31) demonstrated that CSE was expressed in the rat aorta, tail artery, mesenteric artery, and pulmonary artery, whereas the expression of CBS was not detectable. Under physiological conditions, tissue content of H 2 S in the brain has been determined to be between 50 and 160 M (1). The endogenous production of H 2 S from rat vascular tissues has also been demonstrated in our previous study (31). With the use of a modified sulfide electrode method, the H 2 S concentration of rat serum was determined to be ϳ46 M (31). These observations speak for the potential physiological functions of H 2 S in the cardiovascular system.The relaxant effects of exogenously applied H 2 S on intestinal and vascular smooth muscles have been reported (13). In both the aorta and portal vein of rats, H 2 S induced a dose-dependent relaxation, but other thiol-containing endogenous substances such as cysteine and glutathione did not have any vasorelaxant effect (13). More importantly, we found ...

H2S is an important gasotransmitter with a vasorelaxant property. The modulation of endogenous H2S generation from different tissues and the functional consequence of this modulation are not clear. In the present study, the production of H2S from vascular tissues as well as the liver and ileum of rats was measured. The H2S production rate was significantly greater in rat liver than rat vascular tissues. H2S production in rat aortae, ileum, and liver tissues was upregulated by sodium nitroprusside in a cGMP-dependent fashion. Amino-oxyacetate (AOA) (1 mM) abolished H2S production in liver tissues and partially inhibited H2S production in the ileum, while D,L-propargylglycine (PPG) at a similar concentration only slightly inhibited H2S production in liver. Intraperitoneal injection PPG, but not AOA, significantly suppressed H2S production in liver, aorta, and ileum tissues. The systolic blood pressure of rats was significantly increased 2-3 weeks after i.p. injection of PPG. It is concluded that the endogenous production of H2S could be modulated by NO. AOA and PPG have different capacities in regulating the endogenous production of H2S in different types of tissues.

1 Carbon monoxide (CO) induced a concentration-dependent relaxation of isolated rat tail artery tissues which were precontracted with phenylephrine or U-46619. This vasorelaxing eect of CO was independent of the presence of the intact endothelium. 2 The CO-induced vasorelaxation was partially inhibited by the blockade of either the cyclicGMP pathway or big-conductance calcium-activated K (K Ca ) channels. When both the cyclicGMP pathway and K Ca channels were blocked, the CO-induced vasorelaxation was completely abolished. 3 Incubation of vascular tissues with hemin, in order to enhance the endogenous production of CO, suppressed the phenylephrine-induced vasocontraction in a time-and concentration-dependent manner. The hemin-induced suppression of the vascular contractile response to phenylephrine was abolished after the vascular tissues were co-incubated with either oxyhaemoglobin or zinc protoporphyrin-IX, suggesting an induced endogenous generation of CO from vascular tissues. 4 The eect of hemin incubation on vascular contractility did not involve the endogenous generation of nitric oxide. 5 Our results suggest that CO may activate both a cyclicGMP signalling pathway and K Ca channels in the same vascular tissues, and that the endogenously generated CO may signi®cantly aect the vascular contractile responses.

Abstract-Heme oxygenase (HO) and carbon monoxide (CO) participate in the homeostatic control of cardiovascular functions, including the regulation of blood pressure (BP). Upregulation of the HO/CO system has been shown to lower BP in young (8 weeks) but not in adult (20 weeks) spontaneously hypertensive rats (SHR). The underlying mechanism for this selective effect, however, has been unknown and was investigated in the present study. Key Words: cyclic GMP Ⅲ hypertension, experimental Ⅲ rats, spontaneously hypertensive Ⅲ mesenteric arteries Ⅲ heme oxygenase T he homeostatic control of biological functions is maintained by a wide array of metabolic pathways capable of triggering the necessary corrective mechanisms to maintain cellular functions within physiological limits. Among these metabolic pathways is the catalytic breakdown of heme by heme oxygenase (HO) to carbon monoxide (CO), bilirubin, and iron. HO is a microsomal enzyme with three distinct isoforms, namely, inducible (HO-1) and constitutive forms (HO-2 and HO-3). 1 HO-1 is a 32-kDa protein that is not constitutively present in cells but expressed after exposure of cells to different stimuli. 2 This enzyme is believed to play a predominant role for CO generation during pathophysiological episodes. HO-2 is a 36-kDa protein that is normally expressed in many organs under physiological conditions. 3 For example, HO-2 expressed in endothelial and smooth muscle layers of blood vessels generates CO that intrinsically modulates vascular tone. 4 HO-3 (33 kDa) shares Ϸ90% homology with HO-2. 1 HO-3 is devoid of catalytic activity and considered important in the regulation of heme proteins. 1 CO has been shown to induce relaxation of vascular smooth muscle cells (VSMC) by stimulating soluble gyanylyl cyclase (sGC), opening calcium-activated K ϩ channels, and acting on cytochrome P450. 5 sGC is a heme-containing heterodimer composed of ␣ and ␤ subunits. ␣ 1 and ␤ 1 of the catalytic domain have molecular weights of 82 kDa and 70 kDa, respectively, whereas the regulatory domain is made up of ␣ 2 and ␤ 2 subunits. The activation of sGC converts GTP to cGMP. Intracellular cGMP regulates biological functions by activating cGMP-dependent protein kinases, directly gating ion channels, 5,6 or regulating phosphodiesterase hydrolysis. The activation of the sGC/cGMP pathway is one of major mechanisms for CO-induced vasorelaxation. The synchronized activities of the HO/CO-sGCc/GMP system may constitute an important metabolic pathway in the modulation of blood pressure (BP).The HO/CO metabolic pathway is believed to be involved in the regulation of basal tone of resistance blood vessels, 7 growth of VSMC, and vascular remodeling. 8 Many recent reports have shown that VSMC constitute a major site for HO-1 induction. 9 Examples include hemin-induced dilation of small arteries of the peritoneum 10 and HO-1 induction in VSMC of human atherosclerotic lesions. 11 Also, CO produced from heme metabolism in blood vessels 12 was reported to elicit relaxation 13 through the elevation of c...

Activation of a calcium-sensing receptor (Ca-SR) leads to increased intracellular calcium concentration and altered cellular activities. The expression of Ca-SR has been identified in both nonexcitable and excitable cells, including neurons and smooth muscle cells. Whether Ca-SR was expressed and functioning in cardiac myocytes remained unclear. In the present study, the transcripts of Ca-SR were identified in rat heart tissues using RT-PCR that was further confirmed by sequence analysis. Ca-SR proteins were detected in rat ventricular and atrial tissues as well as in isolated cardiac myocytes. Anti-(Ca-SR) Ig did not detect any specific bands after preadsorption with standard Ca-SR antigens. An immunohistochemistry study revealed the presence of Ca-SR in rat cardiac as well as other tissues.

The hollow fiber test has been developed for the preliminary in vivo assessment of cancer chemotherapeutic efficacy of selected natural products. Using this model, we have established growth conditions for HL-60, HUVEC, Ishikawa, KB, KB-V1, LNCaP, Lu1, MCF-7, Mel2, P-388, and SW626 cells implanted at the intraperitoneal (i.p.) and subcutaneous (s.c.) compartments of athymic mice. Five cytotoxic natural product isolates (2-6) were tested in this model, along with paclitaxel (taxol) (1). Among the compounds tested, dioscin (2) and 13-methoxy-15-oxozoapatlin (3) were found to be active, indicating their potential to function as cancer chemotherapeutic agents. On the other hand, ochraceolide A (4), alpha-lapachone (5), and 2-(1-hydroxyethyl)naphtha[2,3-b]furan-4,9-quinone (6), all of which were significantly cytotoxic to cultured mammalian cells, did not mediate significant responses with the hollow fiber model. In further xenograft studies using KB cells implanted at the subcutaneous site, compound 3 mediated a statistically significant response which was consistent with the response observed at the subcutaneous compartment in the hollow fiber tests. In sum, these studies illustrate the usefulness of the hollow fiber model in natural product drug discovery programs. Preliminary indications of potential therapeutic efficacy can be provided quickly at relatively low expense. Agents capable of mediating a response at the subcutaneous site would appear to warrant greatest attention.

Heme oxygenase (HO) and carbon monoxide (CO) have been implicated in the modulation of various cardiovascular functions including blood pressure (BP) regulation. Up-regulating the HO/CO system lowers BP in young (8-week-old) but not in adult (20-week-old) spontaneously hypertensive rats (SHRs). The mechanisms for this selective effect are largely unknown. We investigated the effects of HO-1 inducer, hemin, on the HO/COsoluble gyanylyl cyclase (sGC)/cGMP system in the aorta of prehypertensive (4-week-old) young and adult SHRs as well as age-matched Wistar-Kyoto rats (WKYs). Reduced expressions of HO-1, HO-2, and sGC proteins associated with depressed HO activity and cGMP levels were detected in young SHRs. These deficiencies were significantly reversed by hemin treatment. Macrophage infiltration of vascular tissues was more significant in adult SHRs than adult WKYs, but invisible in young SHRs and WKYs. Hemin treatment did not alter macrophage infiltration of vascular tissues in young SHRs. The same hemin administration resulted in a significant decrease in BP (from 148.6 ؎ 3.2 to 125.8 ؎ 2.6 mmHg, P < .01) in young SHRs, but not in prehypertensive or adult SHRs or WKYs of all ages. The HO inhibitor zinc protoporphyrin abrogated the hemin effect in young SHRs. Aortic tissues became desensitized to YC-1, an activator sGC, in adult SHRs. Thus, in young SHRs the expression and function of the HO/CO-sGC/cGMP system were suppressed, constituting a pathogenic mechanism for the development of hypertension. In adult SHRs, the HO/COsGC/cGMP system appeared normal, but desensitization of the sGC/cGMP pathway caused hypertension to prevail. IntroductionOne of the biggest health challenges in industrialized nations today is the high incidence and rapidly growing problem of hypertension, especially among the elderly (those older than 60 years) with approximately 50% morbidity. 1 Hypertension is a multifactorial pathology and considered the prevalent risk factor for many cardiovascular diseases including myocardial infarction, cardiac failure, and coronary and peripheral artery diseases. Among the known etiologic causes for hypertension is the disproportionate level of vasoconstrictors such as 20-hydroxyeicosatetraenoic acid (20-HETE) by cytochrome P450 monooxygenase 2 compared with that of vasodilators such as epoxyeicosatrienoic acids 3 and gasotransmitters. 4 All known gasotransmitters, including nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H 2 S), relax vascular tissues through various mechanisms. CO induces relaxation of vascular smooth muscle cells (SMCs) by several pathways comprising the stimulation of soluble gyanylyl cyclase (sGC), opening of calcium-activated K ϩ channel, or inhibition of cytochrome P450-dependent mono-oxygenase. 5 sGC with catalytic ␣ 1 (82 kDa) and ␤ 1 (70 kDa) subunits participates in a variety of physiologic processes responsible for converting guanosine triphosphatases to cGMP. Cyclic GMP regulates physiologic functions through gating of ion channels, 6,7 activating cGMP-depe...