Article1 November 2001free access The vasorelaxant effect of H2S as a novel endogenous gaseous KATP channel opener Weimin Zhao Weimin Zhao Department of Physiology, University of Saskatchewan, Saskatoon, SK, Canada, S7N 5E5 Search for more papers by this author Jing Zhang Jing Zhang Department of Physiology, University of Saskatchewan, Saskatoon, SK, Canada, S7N 5E5 Search for more papers by this author Yanjie Lu Yanjie Lu Department of Physiology, University of Saskatchewan, Saskatoon, SK, Canada, S7N 5E5 Search for more papers by this author Rui Wang Corresponding Author Rui Wang Department of Physiology, University of Saskatchewan, Saskatoon, SK, Canada, S7N 5E5 Search for more papers by this author Weimin Zhao Weimin Zhao Department of Physiology, University of Saskatchewan, Saskatoon, SK, Canada, S7N 5E5 Search for more papers by this author Jing Zhang Jing Zhang Department of Physiology, University of Saskatchewan, Saskatoon, SK, Canada, S7N 5E5 Search for more papers by this author Yanjie Lu Yanjie Lu Department of Physiology, University of Saskatchewan, Saskatoon, SK, Canada, S7N 5E5 Search for more papers by this author Rui Wang Corresponding Author Rui Wang Department of Physiology, University of Saskatchewan, Saskatoon, SK, Canada, S7N 5E5 Search for more papers by this author Author Information Weimin Zhao1, Jing Zhang1, Yanjie Lu1 and Rui Wang 1 1Department of Physiology, University of Saskatchewan, Saskatoon, SK, Canada, S7N 5E5 *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:6008-6016https://doi.org/10.1093/emboj/20.21.6008 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Hydrogen sulfide (H2S) 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 H2S in the regulation of vascular contractility, the modulation of H2S production in vascular tissues, and the underlying mechanisms. Intravenous bolus injection of H2S transiently decreased blood pressure of rats by 12–30 mmHg, which was antagonized by prior blockade of KATP channels. H2S relaxed rat aortic tissues in vitro in a KATP channel-dependent manner. In isolated vascular smooth muscle cells (SMCs), H2S directly increased KATP channel currents and hyperpolarized membrane. The expression of H2S-generating enzyme was identified in vascular SMCs, but not in endothelium. The endogenous production of H2S from different vascular tissues was also directly measured with the abundant level in the order of tail artery, aorta and mesenteric artery. Most importantly, H2S production from vascular tissues was enhanced by nitric oxide. Our results demonstrate that H2S is an important endogenous vasoactive factor and the first identified gaseous opener of KATP channels in vascular SMCs. Introduction Hydrogen sulfide (H2S) has been best known for decades as the toxic gas dubbed ‘gas of rotten eggs’ (Winder and Winder, 1933; Smith and Gosselin, 1979). Less recognized, however, is the fact that H2S is also a biological gas endogenously generated from cysteine in a reaction catalysed by cystathionine β-synthase (CBS) and/or cystathionine γ-lyase (CSE) (Stipanuk and Beck, 1982; Hosoki et al., 1997). By analogy to other endogenous gaseous molecules, such as nitric oxide (NO) and carbon monoxide (CO) (Wang et al., 1997a,b), H2S was hypothesized to fulfil a physiological role in regulating cardiovascular functions, distinctive from its toxicological effect. In line with this idea was the study showing that H2S, at physiological concentrations, facilitated the induction of hippocampal long-term potentiation (Abe and Kimura, 1996). To date, the cardiovascular effects of both endogenous and exogenous H2S have not been fully understood. Hosoki et al. (1997) demonstrated that H2S relaxed rat aortic tissues in vitro. However, the cellular mechanisms for this vascular effect of H2S, as well as its physiological significance, remained to be examined. Does H2S affect the cardiovascular function in vivo of whole animal or in vitro at the cellular level? Do the cardiovascular effects of H2S have physiological significance? NO and CO mediate vasorelaxation by increasing the cellular cGMP activity and/or stimulating KCa channels in vascular smooth muscle cells (SMCs). Does H2S also act on these cellular targets? NO and CO can be released from both SMCs and endothelial cells. Is H2S synthesized in SMCs or endothelial cells or both? Once released, NO and CO act directly on SMCs independent of endothelium. Is the vascular effect of H2S mediated by endothelium? The endogenous stimuli for the synthesis and release of NO and CO have been identified. What are the endogenous regulators of H2S synthesis and release? Answers to these questions will not only help to establish the role of H2S as another endogenous gaseous vasoactive factor, but also provide novel mechanisms for the fine regulation of vascular tone. The purpose of the present study was to assess the physiological role of H2S in the regulation of cardiovascular functions, the modulation of H2S production in vascular tissues, and the underlying mechanisms. The vasoactive effects of H2S were investigated by measuring the blood pressure change of rats in vivo, vascular tension development in vitro and K+ channel currents in isolated vascular SMCs. Molecular biology and biochemistry techniques were used to detect the expression of H2S-generating enzymes and the endogenous levels of H2S. Our study indicates that H2S is an endogenous vasorelaxant factor that activates KATP channels and hyperpolarizes membrane potential of vascular SMCs. Results The cardiovascular effects of H2S in vivo An intravenous bolus injection of H2S at 2.8 and 14 μmol/kg body weight provoked a transient (29.5 ± 3.6 s) decrease in mean arterial blood pressure of anaesthetized rats by 12.5 ± 2.1 and 29.8 ± 7.6 mm Hg, respectively (n = 3 for each group, P < 0.05) (Figure 1A). Heart rate was not altered by H2S injection (Figure 1B). An 18 ± 3 mm Hg decrease of blood pressure was also observed after a bolus intravenous injection of a KATP channel opener pinacidil (2.8 μmol/kg) (n = 3, P < 0.01), mimicking the hypotensive effect of H2S. Although a bolus intravenous or intraperitoneal injection of glibenclamide (a KATP channel blocker) at 2.8 μmol/kg did not alter mean blood pressure (106 ± 2.3 mm Hg compared with 109 ± 8.2 mm Hg, P > 0.05, n = 3), pretreatment of animals for 20 min with glibenclamide significantly reduced by 83% the hypotensive effect of H2S (n = 3, P < 0.05). Prior injection of vehicle used for preparing glibenclamide did not alter the hypotensive effect of H2S (P < 0.05). Figure 1.The effect of H2S in vivo on mean arterial blood pressure (BP) and heart rate of rats. (A) Intravenous injection of H2S induced significant decrease in BP. This effect was mimicked by intravenous injection of pinacidil (2.8 μmol/kg) and antagonized by a prior intravenous injection of glibenclamide (2.8 μmol/kg). (B) Effect of H2S on rat heart rate. Heart rate was recorded 30 s after intravenous injection of PBS (control), H2S or pinacidil. *P < 0.05 compared with control. Download figure Download PowerPoint Characterization of the H2S-induced vasorelaxation Unless otherwise stated, each experiment shown in this section was composed of eight aortic rings. H2S induced a concentration-dependent relaxation of the phenylephrine (PHE)-precontracted rat aortic tissues (IC50, 125 ± 14 μM) (Figure 2A). For instance, at a concentration of 180 μM, H2S relaxed the tissue by 63 ± 2.2% (P < 0.05). A positive cooperativity for H2S in operating its acting sites on vascular smooth muscles was evidenced by the calculated Hill coefficient of 4.9 (Ruiz et al., 1999). Pretreatment of the endothelium-intact tissues with L-NAME (NG-nitro-L-arginine methyl ester, 100 μM) to block endogenous NO production from endothelium shifted the H2S concentration-dependent relaxation curve to the right with IC50 changed to 220 ± 12 μM (P < 0.05) (Figure 2B). Similar results were obtained by just removing endothelium from the aortic tissues (Figure 2C). Moreover, co-application of charybdotoxin (50 nM) and apamin (50 nM) to the endothelium-intact tissues reduced the H2S-induced vasorelaxation (Figure 2C). Figure 2.The H2S-induced relaxation of rat aortic rings and the underlying mechanisms. (A) Relaxation of the PHE-precontracted tissues by H2S in the form of either standard NaHS solution (square) or H2S gas-saturated solution (circle). (B) Inhibitory effect of L-NAME (100 μM, 20 min, circle) on the H2S-induced relaxation (control, square). (C) The effects of H2S (180 μM) on the endothelium-free or endothelium-intact aortic tissues pretreated with L-NAME or charybdotoxin (ChTX)/apamin. (D) The relaxant effect of H2S was not affected by pretreating the tissues with SQ22536, SOD or catalase, respectively. (E) The effect of ODQ treatment (10 μM for 10 min) on the relaxant effects of SNP (0.1 μM) or H2S (600 μM). n = 8 for each data point. *P < 0.05 compared with control. Download figure Download PowerPoint Further studies were carried out to identify the involvement of various signal transduction pathways in the vascular effect of H2S. Treatment of tissues with indomethacin (10 μM, not shown) (Rodriguez-Martinez et al., 1998) or staurosporine (30 nM, not shown) (Hattori et al., 1995; Huang, 1996) or SQ22536 (100 μM) (Talpain et al., 1995) did not change the effect of H2S (Figure 2D), disapproving the involvement of prostaglandin, protein kinase C or cAMP pathways, respectively. The generation of superoxide anion or hydrogen peroxide by H2S (Nicholls, 1961) was unlikely responsible for the H2S-induced vasorelaxation since the inclusion of superoxide dismutase (SOD, 160 U/ml) and catalase (1000 U/ml) (Rodriguez-Martinez et al., 1998) failed to alter the effect of H2S (Figure 2D). The vasorelaxation induced by sodium nitroprusside (SNP), a NO donor, was virtually abolished by the specific inhibitor of the soluble guanylyl cyclase, 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ). However, the vasorelaxant effect of H2S was not blocked by ODQ (Figure 2E). Clearly, the vasorelaxant effect of H2S was not mediated by the cGMP pathway. Involvement of K+ channel activities in the H2S-induced vasorelaxation Different relaxation potencies of H2S on vascular tissues precontracted by high or low concentration of KCl. To examine whether the H2S-induced vasorelaxation was mediated by the increased potassium conductance, aortic rings were precontracted with either 20 or 100 mM KCl and the vasorelaxant effect of H2S was then examined. The contraction forces induced by 20 and 100 mM KCl were 0.86 ± 0.08 and 1.58 ± 0.08 g, respectively. In general, H2S induced a greater relaxation of the vascular tissues precontracted by low concentration of KCl (20 mM) than by PHE. For instance, the threshold concentrations of H2S to initiate relaxation were 18 and 60 μM for 20 mM KCl- and PHE-precontracted tissues, respectively. The maximum vascular relaxation induced by H2S was 90 ± 8.2% or 19 ± 3.9% when the tissues were precontracted with 20 or 100 mM KCl, respectively (Figure 3A). This difference in the relaxation potency of H2S represents the portion of relaxation possibly mediated by potassium conductance. Figure 3.The K+ channel-mediated vascular effects of H2S. (A) The relaxant effect of H2S on the aortic tissues precontracted with 20 or 100 mM KCl. (B) Inhibitory effect of TEA on the H2S-induced vasorelaxation. The concentration-dependent vasorelaxant effects of H2S with or without TEA (10 mM) pretreatment of aortic rings were determined. (C) The H2S (600 μM)-induced vasorelaxation was not affected by pretreatment of aortic tissues with either 10 μM iberiotoxin (IbTX) or 2.5 mM 4-aminopyridine (4-AP). *P < 0.05 compared with control, n = 8. (D) The vasoactive effects of pinacidil (left) and H2S (right) on the precontracted aortic tissues. The vasorelaxant effect of H2S (600 μM) was examined in the presence of glibenclamide at different concentrations. *P < 0.05 compared with control, **P < 0.05 compared with the H2S group in the absence of glibenclamide, n = 8. Download figure Download PowerPoint Involvement of KCa channels in the vascular effects of H2S. In order to identify the role of specific types of K+ channels in the H2S-induced relaxation, aortic rings were incubated with either 10 mM tetraethylammonium (TEA), 100 nM charybdotoxin or 100 nM iberiotoxin (IbTX) (two specific KCa channel inhibitors) for 20 min prior to the application of H2S. In the presence of TEA, the H2S-induced vasorelaxation was completely inhibited (Figure 3B). At this high concentration, TEA is known to block many different types of K+ channels (Nelson and Quayle, 1995). The H2S-induced vasorelaxation was not affected by iberiotoxin (Figure 3C) or charybdotoxin (not shown), suggesting that big-conductance KCa channels might not be responsible for the H2S-induced vasorelaxation. Involvement of voltage-dependent K + (Kv) channels in the vascular effects of H2S. A previous study has shown that 4-AP specifically inhibited the Kv channel with an IC50 of 1.4 mM (Remillard and Leblanc, 1996). In the present study, 2.5 mM 4-AP was used to treat vascular tissues 20 min before the application of H2S. The relaxant effect of H2S was not affected by 4-AP, eliminating the involvement of Kv channels (Figure 3C). Involvement of KATP channels in the vascular effects of H2S. To elucidate whether an ATP-sensitive K+ channel (KATP) was the target of H2S, the interaction of H2S with known KATP channel modulators was examined. The H2S-induced vasorelaxation was inhibited in a concentration-dependent manner by glibenclamide (IC50, 140 μM) (Figure 3D). The H2S effect was also mimicked by pinacidil that relaxed the PHE-precontracted vascular tissues in a concentration-dependent manner (IC50, 2.4 μM) (Figure 3D). The direct effect of H2S on KATP channel currents and membrane potential in single vascular SMCs After exposure to 300 μM H2S, KATP channel currents in rat aortic SMCs were significantly increased in amplitude (Figure 4A). This excitatory effect of H2S was fully manifested 3 min after the application and nullified immediately after washing out H2S from the bath solution. Pinacidil (5 μM) also increased KATP channel currents, similar to the effect of H2S (Figure 4B). The effects of H2S and pinacidil on KATP channel currents were observed in a wide test potential range (−40 to +60 mV). The reversal potential of KATP channels in these cells was not altered by either H2S or pinacidil (Figure 4A and B, right panels). Figure 4.The effect of H2S and pinacidil on KATP channel currents in rat aortic SMCs. (A) The effect of H2S on KATP channel currents. The original records from one cell (left) and the mean current–voltage relationship of six cells (right) in the absence and then presence of H2S (300 μM) are shown. (B) The effect of pinacidil (5 μM) on KATP channel currents. The original records from one cell (left) and the mean current–voltage relationship of five cells (right) in the absence and then presence of pinacidil are shown. Dashed line indicates zero current level. (C) The antagonistic effect of glibenclamide (Gli, 5 μM) on the effect of H2S or pinacidil (Pin) on KATP channel currents (holding potential, −60 mV; test potential, +40 mV). (D) The membrane hyperpolarization induced by H2S (300 μM) or pinacidil (5 μM), respectively. *P < 0.05 compared with control; n = 3–7 for each group. Download figure Download PowerPoint On average, H2S (300 μM) and pinacidil (5 μM) increased KATP channel currents from 85.8 ± 12.4 to 149.0 ± 21.4 pA and from 99.1 ± 5.1 to 191.8 ± 28.1 pA (test potential, +40 mV), respectively (Figure 4C). Increased KATP channel currents by H2S would lead to membrane hyperpolarization, resulting in smooth muscle relaxation. This hypothesis was tested by directly measuring membrane potential change using the conventional whole-cell patch–clamp technique and the results are shown in Figure 4D. After exposing SMCs to H2S (300 μM) or pinacidil (5 μM), the cell membrane was hyperpolarized from −35.7 ± 2.0 to −53.3 ± 2.5 mV or from −36.6 ± 4.5 to −56.7 ± 5.0 mV, respectively. The hyperpolarization developed within 3 min of the application of H2S or pinacidil. Glibenclamide (5 μM) alone had no effect on resting membrane potential (n = 6). The hyperpolarization induced by H2S or pinacidil was reversed to the control level 3–5 min after the subsequently applied glibenclamide to the same cells (n = 5). Using the perforated whole-cell recording technique, we also found that H2S (300 μM) significantly hyperpolarized the resting membrane potential from −51.2 ± 6.5 to −69.2 ± 7.6 mV (n = 6, P < 0.05). It may be argued that the increase in KATP channel currents in the presence of H2S resulted from the interfered ATP metabolism by H2S. However, the reversibility of H2S effect was not in favour of this argument. Moreover, in the aforementioned experiments, the cells were dialysed with the pipette solution that contained a pre-determined ATP concentration, i.e. 0.5 mM. In another set of experiments, the concentrations of ATP in the pipette solution were intentionally altered (Figure 5A). Similar KATP current densities were observed in the cells with 0.2 or 1 mM ATP in the pipette solution. The excitatory effect of H2S on KATP currents was not altered by changing intracellular ATP concentrations within this range (P > 0.05). In the presence of 3 mM ATP in the pipette solution, however, the KATP current density was significantly smaller (5.1 ± 0.7 pA/pF) than that with 0.2–1 mM ATP (P < 0.05). The increase in KATP channel currents induced by H2S was also significantly decreased in the presence of 3 mM ATP (49.7 ± 2.7% increase) as compared with the effect of H2S in the presence of 0.2 mM ATP (87.3 ± 12.2% increase) (P < 0.05) (Figure 5A). Furthermore, pinacidil (5 μM) increased the KATP currents in aortic SMCs by 72.4 ± 10.5% (n = 5, P < 0.05 compared with control) or 107.6 ± 32.2% (n = 5, P < 0.05 compared with control) in the presence of 0.2 or 1 mM ATP in the pipette solution, respectively (P > 0.05 between the effects of pinacidil at the two ATP concentrations). In the presence of 3 mM ATP in the pipette solution, pinacidil (5 μM) increased KATP currents by 44.7 ± 4.9% (n = 4, P < 0.05), which was significantly smaller than the effect of pinacidil at 0.2 or 1 mM ATP (P < 0.05). These results demonstrated a low ATP sensitivity of the KATP channels in the examined vascular SMCs and revealed that the effect of KATP channel openers was reduced in the presence of high intracellular ATP concentrations (Quayle et al., 1995). Figure 5.The modulation of KATP channel currents by H2S with different intracellular ATP concentrations and by glibenclamide (Gli) in rat aortic SMCs. (A) The ATP-dependence of the H2S (300 μM)-induced increases in KATP channel currents. Holding potential, −60 mV; test potential, +40 mV. *P < 0.05 compared with control. (B) The time course of the inhibition of KATP currents by glibenclamide. Aortic SMCs were dialysed with 0.2 mM ATP and 1 mM GDP. The voltage pulses of +30 mV were applied from a holding potential of −60 mV. Inset shows the representative of original current traces before and after the application of 10 μM glibenclamide. Download figure Download PowerPoint To further characterize the KATP channels in these cells, the effect of glibenclamide was studied. The H2S-stimulated or pinacidil-stimulated KATP channel currents were significantly reduced by glibenclamide (5 μM, n = 6 for each group) to the control level (Figure 4C). Glibenclamide per se did not change the basal KATP channel current under our recording conditions (5.4 mM KCl in the bath solution and 0.5 mM ATP in the pipette solution) (n = 5, P > 0.05). In the presence of 1 mM GDP in the pipette solution containing 0.2 mM ATP, the basal KATP current density was significantly increased to 22.3 ± 4.9 pA/pF (n = 8) in comparison with the KATP current density of 13.0 ± 1.4 pA/pF recorded in the absence of GDP and presence of 0.2 mM ATP in the pipette solution (n = 4) (test potential, +40 mV; P < 0.05). Under this modified condition, glibenclamide reduced the basal KATP current density by 47.3 ± 9.2% (n = 8, test potential at +40 mV, P < 0.01) (Figure 5B). The identification, differential expression and cloning of the H2S-generating enzymes in vascular tissues To corroborate the physiological importance of the vasorelaxant effect of H2S, the endogenous sources of H2S were determined in vascular tissues. Using RT–PCR, a PCR-amplified 234 bp fragment of CSE, but not CBS (not shown), was detected in endothelium-free rat pulmonary artery, mesenteric artery, tail artery and aorta as well as rat liver (Figure 6A). To ascertain that the amplified PCR product was free of genomic DNA contamination, every RNA sample was simultaneously amplified by PCR without reverse transcriptase treatment. Under these conditions, no PCR product was detected. The gel-purified PCR products of CSE from rat liver and vascular tissues were ligated with a synthesized deoxyooligonucleotide (Amersham Pharmacia Biotech) that contains the phage T7 promoter sequence and then sequenced manually using T7 primer. The sequences of our PCR-amplified CSE fragments from rat arteries matched the corresponding CSE sequence cloned from rat liver (DDBJ/EMBL/GenBank accession No. X53460), demonstrating that the same isoform of CSE gene was expressed in rat vascular tissues and liver. RNase protection assay revealed the abundant levels of CSE mRNA in different vascular tissues with an intensity rank of pulmonary artery, aorta, tail artery and mesenteric artery (Figure 6B). Due to the lack of commercially available antibodies against CSE, the protein levels of CSE in vascular tissues were not examined. Figure 6.Differential expression of CSE in rat vascular tissues. (A) RT–PCR analysis of the expression of CSE (234 bp) in rat liver, mesenteric artery, tail artery, pulmonary artery and aorta. (B) Quantitative comparison of CSE mRNA levels in rat tail artery, mesenteric artery, pulmonary artery and aorta with RPA. This is representative of three experiments. *P < 0.05 compared with mesenteric artery; **P < 0.01 compared with tail artery, mesenteric artery and aorta. A, artery. (C) The transcriptional expressions of CSE and β-actin in cultured SMCs and EC (endothelial cell) detected by RT–PCR. (D) In situ hybridization showing the location of CSE mRNA in rat aorta wall by antisense probe on the left and sense probe as control on the right. Download figure Download PowerPoint The differential expression of CSE between vascular SMCs and endothelial cells was studied further. Using RT–PCR, a 1123 bp fragment of CSE was detected in cultured rat aortic SMCs, but not in cultured vascular endothelial cells (Figure 6C). In situ hybridization was applied to further locate CSE mRNA in the aortic wall. The expression of CSE mRNA was clearly identified in the smooth muscle layer of the artery wall, but not in the endothelial layer (Figure 6D, left panel). To exclude the non-specific staining, the sense probe for CSE was used to hybridize rat aortic tissues under the same condition as for the antisense probe. Figure 6D (right panel) shows that no positive staining could be spotted using the sense probe for CSE. Finally, a PCR-based cloning technique was used with a pair of primers covering the translation initiation and termination codons of CSE to obtain a cDNA clone encoding the whole open reading frame (ORF) region of CSE. The PCR-amplified product of ∼1300 bp was detected in rat aorta, tail artery and mesenteric artery. We have cloned and sequenced two isoforms of CSE from rat mesenteric arteries, which contained an ORF of 1197 bp, encoding a 398 amino acid peptide. We further cloned and sequenced CSE from rat liver and found no differences among all the clones from artery and liver. The sequence data of our cloned rat vascular CSE and liver CSE have been submitted to GenBank (AB052882 and AY032875). The endogenous level of H2S and its regulation The physiological role of H2S could not be established before the endogenous level of this gas in vascular tissues or in circulation had been determined. Thus, the endogenous production of H2S in vascular tissues and in circulation was assayed. Figure 7A showed that various vascular tissues produced different levels of H2S. When the specific inhibitor of CSE, DL-propargylglycine, was added to the reaction medium at the final concentration of 20 mM (Hosoki et al., 1997), H2S production was completely abolished in all tested arteries (n = 3), indicating that the generation of H2S from vascular tissues was due to the specific catalytic activity of CSE. Using a modified sulfide electrode method, the H2S concentration of rat serum was determined to be 45.6 ± 14.2 μM (n = 4). Figure 7.Regulation of the endogenous H2S production in different rat tissues. (A) Accumulated endogenous H2S levels in rat tail artery (TA), mesenteric artery (MA), aorta and ileum. (B) H2S production rate of aorta tissues was stimulated by SNP in a concentration-dependent manner. *P < 0.05 compared with control, n = 3. (C) The SNAP-induced concentration-dependent upregulation of CSE transcriptional expression in cultured aortic SMCs, determined using northern blotting. The 28S ribosome RNA was assayed as the housekeeping control. n = 3, *P < 0.05 compared with control. Download figure Download PowerPoint The effect of NO on the endogenous production of H2S was examined by incubating homogenized rat vascular tissues with different concentrations of SNP, a NO donor, for 90 min. An accumulated H2S production was upregulated by SNP in a concentration-dependent manner (1–100 μM) (Figure 7B). Nitric oxide has been shown to regulate protein expression and synthesis, including growth factors, leukocyte adhesive proteins and extracellular matrix proteins (Kourembanas et al., 1993; Kolpakov et al., 1995; Zeiher et al., 1995). In our study, incubating the cultured vascular SMCs with SNAP (0.1 or 1 mM), another NO donor, for 6 h significantly increased the transcriptional level of CSE (Figure 7C). Discussion In the present study, the cardiovascular effects of H2S were demonstrated in vivo and in vitro. Intravenous injection of H2S provoked a transient but significant decrease in mean arterial blood pressure. Similar to our in vitro vascular contractility assay and patch–clamp studies, the H2S-induced decrease in blood pressure was antagonized by glibenclamide and mimicked by pinacidil. Glibenclamide and pinacidil are specific KATP channel blocker and opener, respectively (Beech et al., 1993; Quayle et al., 1994). Thus, these in vivo results indicated that the hypotensive effect of H2S was likely provoked by the relaxation of resistance blood vessels through the opening of KATP channels. The short duration of the hypo tensive effect of H2S could be attributed to the scavenging of H2S by metalloproteins, disulfide-containing proteins, thio-S-methyl-transferase and haem compounds. The administration of H2S as a bolus injection also partially explains the transient effect. Similarly, the hypotensive effect of pinacidil was also transient. Furthermore, our data showed that the in vivo hypotensive effect of H2S was due to a specific action on vascular smooth muscles, since heart rate was not significantly affected. Our in vitro study showed that H2S relaxed the isolated aortic tissues at concentrations as low as 18 and 60 μM for the aortic tissues precontracted with either 20 mM KCl or PHE, respectively. It has been reported that the normal blood level of H2S in Wistar rats was ∼10 μM (Mason et al., 1978). Our study demonstrated that the plasma level of H2S in SD rats was ∼50 μM. The tissue level of H2S is known to be higher than the circulating level. For instance, the physiological concentration of H2S in brain tissue has been reported to be ∼50–160 μM (Hosoki et al., 1997). Taken together, H2S is believed to induce vasorelaxation within a physiologically relevant concentration range. These findings strongly suggest that, similar to NO and CO, H2S is another intrinsic vasoactive gas factor. The physiological role of H2S in the cardiovascular system was further substantiated by its endogenous sources and regulated production process. Using RT–PCR, RPA and northern blotting, we detected the transcriptional expression of the H2S-generating enzyme CSE in all rat arteries tested. The whole sequence of the ORF of CSE had been cloned in this study from rat vascular tissues, which had not been done in any vascular tissues from any species until the present study. CSE has the capability to cleave L-cysteine to produce H2S, ammonium and pyruvate. This enzyme has a unique tissue distribution and was not detectable in brain and lungs (Smith and Gosselin, 1979; Abe and Kimura, 1996). Akin to the release of NO from endothelium by acetylcholine, the regulation of H2S production by other endogenous substances is an essential piece of evidence for establishing the physiological role of H2S. We showed for the first time that NO regulates the endogenous levels of H2S in vascular tissues via two mechanisms. First, NO increases CSE activity in vascular tissues. Incubating aortic tissue homogenate with a NO donor for 90 min significantly increased H2S generation in a concentration-dependent manner (Figure 7B). NO ma