Hydrogen Sulfide as Endothelium-Derived Hyperpolarizing Factor Sulfhydrates Potassium Channels
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Nitric oxide, the classic endothelium-derived relaxing factor (EDRF), acts through cyclic GMP and calcium without notably affecting membrane potential. A major component of EDRF activity derives from hyperpolarization and is termed endothelium-derived hyperpolarizing factor (EDHF). Hydrogen sulfide (H2S) is a prominent EDRF, since mice lacking its biosynthetic enzyme, cystathionine γ-lyase (CSE), display pronounced hypertension with deficient vasorelaxant responses to acetylcholine.
The purpose of this study was to determine if H2S is a major physiological EDHF.
Methods and Results:
We now show that H2S is a major EDHF because in blood vessels of CSE-deleted mice, hyperpolarization is virtually abolished. H2S acts by covalently modifying (sulfhydrating) the ATP-sensitive potassium channel, as mutating the site of sulfhydration prevents H2S-elicited hyperpolarization. The endothelial intermediate conductance (IKCa) and small conductance (SKCa) potassium channels mediate in part the effects of H2S, as selective IKCa and SKCa channel inhibitors, charybdotoxin and apamin, inhibit glibenclamide-insensitive, H2S-induced vasorelaxation.
H2S is a major EDHF that causes vascular endothelial and smooth muscle cell hyperpolarization and vasorelaxation by activating the ATP-sensitive, intermediate conductance and small conductance potassium channels through cysteine S-sulfhydration. Because EDHF activity is a principal determinant of vasorelaxation in numerous vascular beds, drugs influencing H2S biosynthesis offer therapeutic potential.
Multiple molecular mechanisms regulate blood vessel relaxation, with nitric oxide (NO) well established as a mediator of endothelium-dependent vasorelaxation (endothelium-derived relaxing factor, EDRF).1–3 Whereas NO acts by both stimulating cyclic GMP (cGMP) levels and in a cGMP-independent manner to influence calcium disposition and sensitivity,4 blood vessel relaxation and tone are also prominently mediated by endothelium-dependent hyperpolarization.5–7 Numerous substances have been advanced as putative endothelium-derived hyperpolarizing factors (EDHFs) including metabolites of arachidonic acid from cyclooxygenase, prostacyclin (PGI2), epoxyeicosatrienoic acids (EETs) derived from cytochrome P450, lipoxygenase [12-(s)-hydroxyeicosatetraenoic acid (12-S-HETE)], reactive oxygen species, hydrogen peroxide (H2O2), potassium ions (K+), vasoactive peptides, as well as NO itself.5–8 It has also been suggested that EDHF function may be mediated through direct coupling between endothelial and smooth muscle cells by myoendothelial gap junctions composed of connexins.5–7
Recently, hydrogen sulfide (H2S) has been shown to be a major EDRF, formed in vascular endothelial cells from cysteine by cystathionine γ-lyase (CSE), which is calcium-calmodulin dependent.9 Whereas CSE appears to play a significant role in the cardiovascular system, 2 other enzymes have also been shown to generate H2S in various tissues, namely cystathionine β-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST). In blood vessels however, CBS appears to play a negligible role in the production of H2S,10 whereas the precise role of 3-MST has yet to have been defined despite its presence in vascular endothelium.11 Acetylcholine-mediated blood vessel relaxation, however, is markedly reduced in CSE-deleted mice, which manifest increased blood pressure comparable to levels in mice lacking endothelial NO synthase (NOS).9,12 Using genetic deletion of CSE and other approaches, we now show that H2S is a major EDHF acting by chemically modifying sulfhydryl groups—sulfhydration—of potassium channels.
Myograph Measurements of Vascular Tension
The segments (1–1.5 mm in length) of mesenteric arteries or aortas from 8- to 12-week-old male animals were used for myograph measurements of vascular tension as described before.13 Briefly, the mice were heparinized 1 hour before euthanasia. Once euthanized, the arteries were carefully excised and cleaned from the surrounding fat and placed in a Petri dish containing ice-cold Krebs-Ringer bicarbonate solution at pH 7.4 (concentrations in mM: 118.3 NaCl, 4.7 KCl, 1.6 CaCl2, 1.2 KH2PO4, 25 NaHCO3, 1.2 MgSO4, and 11.1 dextrose). The vessels were then carefully placed in the multiwire myograph system DMT 610 mol/L bubbling with continuous of oxygen gas (95% O2 and 5% CO2) at 37°C and incrementally stretched for optimized contractility. Phenylephrine was then applied to preconstrict the vessels, after which changes in vascular tension were recorded with application of different pharmacological agents. In some experiments, endothelium removal was performed as described before.14
Vessel Diameter Measurements
Vessels were prepared as described above, cannulated at both ends with glass micropipettes (80–100 μm), secured with nylon monofilament suture, and placed in a microvascular chamber (Living Systems, Burlington, VT). Vessels were studied in the absence of flow and maintained at a constant transmural pressure of 70 mm Hg as described before.15,16 The chamber was superfused with Krebs-Ringer bicarbonate solution, maintained at 37°C, pH 7.4, and gassed with 95% O2 and 5% CO2. The chamber was then placed on the stage of an inverted microscope (Nikon TMS-F) connected to a video camera (Panasonic CCTV camera). The vessel image was projected on a video monitor, and the internal diameter continuously determined by a video dimension analyzer (Living Systems Instrumentation) with BIOPAC data acquisition system (Santa Barbara, CA). Changes in vessel diameter were measured with application of different pharmacological agents.
Membrane Potential Measurements
Membrane potentials were measured as described before17–19 but with modifications. Briefly, vessels were prepared as above, fixed by pinning one end and cut open in the longitudinal plane. Each corner was pulled out enough, and pinned (0.125 mm diameter tungsten pins) such that the cellular layers remained intact. The vessels were maintained at 37°C in Krebs-Ringer bicarbonate solution at pH 7.4, loaded with 100 nmol/L DiBAC4(3) dye (Molecular Probes, Carlsbad, CA) or fluorometric imaging plate reader red dye (Molecular Devices, Sunnyvale, CA) and maintained in the dark for 30 minutes. The majority of the experiments were conducted using the DiBAC dye unless otherwise indicated. Each tissue was then mounted under a fluorescent microscope (Nikon Eclipse 80i Microscope with Roper Scientific Camera) and the system set at an exposure time of 100 ms with a sampling rate of 3 images per second. FITC filter (fluorescein isothiocyanate) was used because the dye has an excitation of 488 nm and a peak emission of 518 nm. Changes in fluorescence intensities were then recorded with addition of various drugs in small volumes without disturbing the focus. A similar process was used for cultured cells, but the FlexStation-3 fluorescence microplate reader system (Molecular Devices, Sunnyvale, CA) was used instead.
S-Sulfhydration (Modified Biotin Switch) Assay
The assay was carried out as described previously,20 but with modifications. Briefly, arteries or cells treated with appropriate stimulants such as NaHS or acetylcholine were homogenized in HEN buffer (250 mmol/L HEPES-NaOH, pH 7.7, 1 mmol/L EDTA, 0.1 mmol/L Neocuproine) supplemented with 100 μmol/L deferoxamine (DFO) and centrifuged at 13 000g for 30 minutes at 4°C. Lysates (240 μg) were added to blocking buffer (HEN buffer plus 25% SDS and 20 mmol/L methyl methanethiosulfonate [MMTS]) at 50°C for 20 minutes with frequent vortexing. The MMTS was then removed by acetone and the proteins precipitated at −20°C for 20 minutes. After acetone removal, the proteins were resuspended in HENS (HEN+SDS) buffer. To the suspension was added 4 mmol/L biotin-N-[6-(biotinamido)hexyl]-3′-(2′-pyridyldithio)propionamide (HPDP) in DMSO without ascorbic acid. After incubation for 4 hours at 25°C, biotinylated proteins were precipitated by streptavidin-agarose beads, which were then washed with HENS buffer. The biotinylated proteins were eluted by SDS-PAGE sample buffer and subjected to Western blot analysis.
CSE Activity Assays
CSE protein was purified and its activity assayed using the tissue homogenate method as described previously,21 with the exception of a preincubation step with 100 nmol/L S-nitroso-glutathione (GSNO) at 37°C for 2 hours.
Shear Stress Experiments
Human aortic endothelial cells (HAEC) were grown to ≈80% confluence and subjected to a laminar shear stress of 20 dyne/cm2 for 24 hours, using a cone-and-plate viscometer as described earlier.22,23 The cells were then scrapped for CSE activity assay.
Additional Methods can be found as an Online Supplement available at http://circres.ahajournls.org.
Cholinergic Vasorelaxation and Hyperpolarization are Significantly Reduced in CSE−/− and Glibenclamide-Treated Vessels
We confirm the importance of H2S in mediating muscarinic cholinergic-dependent vasorelaxation of the smaller mesenteric artery (diameter of 80–200 μm in mice) and larger aorta (diameter of 350–450 μm in mice), using force-tension myography and vessel diameter measurements (Figure 1A and Online Figures I, A, and IV, A). We eliminated influences of NOS and cyclooxygenase (COX) products by treatment with appropriate inhibitors (L-NAME 100 μmol/L and indomethacin 10 μmol/L, respectively). L-NAME nearly abolishes NO generation in both wild-type and CSE knockout vessels (Online Figure II), which display similar basal NO productions (Online Figure II). In the mesenteric arteries, the overall cholinergic relaxation, of which about 75–80% is NOS/COX independent, is reduced by ≈60% in CSE deleted animals (Figure 1A). Conversely, in the aorta, cholinergic relaxation appears to be primarily NOS/COX dependent and is reduced by less than 25% in CSE knockout vessels treated with NOS/COX inhibitors (Online Figure IV, A). To study the effects of H2S on cellular membrane potential, we used 2 potentiometric fluorescent probes (Online Figure III): (1) DiBAC, a probe with slow response time, and (2) fluorometric imaging plate reader, a newer dye with increased sensitivity and rapid response time. Although the authors acknowledge that electrophysiological techniques such as whole-cell patch clamping are the gold standard for investigating channel function, the use of fluorescent voltage-sensitive dyes to interrogate channels in a rapid, high throughput and economical manner is rapidly emerging. Indeed, studies have shown dye responses to ligand-evoked activation of potassium channels to be comparable with whole-cell patch clamp measurements.17–19 Using the dyes, we find that cholinergic relaxation is associated with pronounced hyperpolarization of about 13–16 mV in mesenteric arteries and about 6–8 mV in the aorta (Figure 1B and Online Figure IV, B). For the NOS/COX independent system, CSE deletion virtually abolishes hyperpolarization. The importance of potassium channels for cholinergic vasorelaxation is evident in that vasorelaxation is markedly reduced in the presence of 30 mmol/L KCl, which fully blocks all potassium channels (Online Figure I, B). Several potassium channels have been implicated in vasorelaxation, with the ATP-sensitive channels closely linked to H2S and EDHF.24,25 The channel inhibitor glibenclamide reduces hyperpolarization about ≈65% (Figure 1B and Online Figure IV, B). Thus, cholinergic vasorelaxation primarily reflects H2S hyperpolarizing cells through the ATP-sensitive potassium channels. Cholinergic vasorelaxation in mouse mesenteric artery (Figure 1C) as well as vasorelaxation and hyperpolarization in rat mesenteric artery (Figure 1D and Online Figure V) are also largely independent of NOS and COX and prevented by glibenclamide. The same is true for hyperpolarization in rat aorta, even though overall vasorelaxation is dependent on the NOS/COX system (Online Figure IV, C and D). In addition, the CSE inhibitor propargylglycine significantly reduces cholinergic hyperpolarization in mesenteric arteries (Figure 1D). Since elevated reactive oxygen species (ROS) might contribute to endothelium dysfunction, we measured differences in ROS levels in the vessels of wild-type and CSE knockout mice. We did not however observe any significant difference in basal ROS production between wild-type and knockout arteries (Online Figure VI).
KCl and Glibenclamide Markedly Diminish H2S Vasorelaxation and Hyperpolarization in Intact and Endothelium-Denuded Mesenteric Arteries
The vasorelaxing and hyperpolarizing actions of applied H2S involve potassium channels, since they are blocked by 30 mmol/L KCl, which fails to alter NO responses (Figure 2A and 2B and Online Figure VIII, A and B). The H2S-mediated vasorelaxation is not affected by changes in the buffer oxygen concentration as relaxation is comparable in buffer bubbled with 95% oxygen and HEPES buffer containing the ambient 21% oxygen (Online Figure VII). H2S acts primarily through ATP-sensitive potassium channels, as glibenclamide (5 μmol/L) markedly reduces the H2S precursor sodium hydrogen sulfide (NaHS)-elicited vasorelaxation and hyperpolarization (Figure 2A and 2B and Online Figure VIII, A and B). In contrast, glibenclamide fails to influence relaxation in response to the NO donor sodium nitroprusside (1 μmol/L). NO but not H2S mediated vasorelaxation is prevented by the cGMP pathway inhibitors ODQ (sGC inhibitor) and KT5823 (PKG inhibitor) (Online Figure IX). Since charybdotoxin and apamin also inhibit a component of the H2S induced vasorelaxation (Figure 2A), IKCa and SKCa channels may in part mediate the effects of H2S, consistent with the findings of Wang et al.24 The combination of glibenclamide and charybdotoxin/apamin abolishes all H2S-mediated vasorelaxation (Figure 2A).
H2S is generated by CSE in the endothelium of blood vessels, and, like NO, diffuses to the adjacent smooth muscle to elicit vasorelaxation.9 We confirm that the actions of H2S-induced vasorelaxation through the ATP-sensitive potassium channels reflect direct influences on the vascular smooth muscle, as in endothelium-denuded mesenteric artery, NaHS relaxation is abolished by glibenclamide, which fails to alter effects of NO (Figure 2C). H2S can also hyperpolarize endothelial cells, as primary cultures of wild-type but not CSE knockout mouse aortic endothelial cells are hyperpolarized on acetylcholine stimulation (Figure 2D). This effect is mediated not by ATP-sensitive potassium channels, but by the combination of IKCa/SKCa channels, as hyperpolarization is completely blocked by charybdotoxin/apamin (Figure 2D). In addition, in cultured human endothelial cells (HAECs), H2S-mediated hyperpolarization is unaffected by either glibenclamide or the BKca channel blocker iberiotoxin, but is significantly diminished by the IKca channel blocker TRAM-34 (Figure 2E). We have previously demonstrated that chemical stimulation of endothelial cells with ACh or the Ca2+ ionophore A23187 increases CSE activity.9 We now observe an increase in CSE activity in cultured HAECs after shear stress suggesting that H2S, and hence EDHF activity, can be induced not only by cholinergic means, but also by a physiological mechanical stimulus (Online Figure X).
Physiological Sulfhydration of Kir 6.1-C43 Activates the Channel Causing Hyperpolarization
Because sulfhydration appears to be a principal means whereby H2S signals,21 we wondered whether vasorelaxation reflects sulfhydration of its target potassium channels. Both the Kir 6.1 subunit of ATP-sensitive potassium channels overexpressed in HEK293 cells and IKca channels from human aortic endothelial cells are sulfhydrated by NaHS in a DTT-sensitive fashion (Figure 3A and Online Figure XI). Kir 6.1 is basally sulfhydrated in cells overexpressing wild-type CSE but not in cells lacking CSE or containing catalytically-inactive CSE (Figure 3B). Cholinergic stimulation of mouse aorta enhances sulfhydration of Kir 6.1 in wild-type but not CSE mutant mice (Figure 3C).
To link sulfhydration to channel function, we overexpressed Kir 6.1 in HEK293 cells in which NaHS-elicited hyperpolarization is blocked by glibenclamide, just as in blood vessels (Figure 3D). To identify the sulfhydrated cysteine residue, we modeled Kir 6.1 on the basis of the established structure of the highly homologous Kir 3.1 (Figure 3E).26 Kir 6.1 has 9 cysteines with cysteine-43 (C43), which lies close to the surface, responding selectively to oxidative insults.27 C43 is the principal target of sulfhydration in Kir 6.1, as sulfhydration of the channel is abolished with C43S mutation (Figure 3F, inset). NaHS-elicited hyperpolarization is significantly reduced in Kir 6.1-C43S mutants, but responses to the channel opener cromakalim remain preserved (Figure 3F and Online Figure XII, A and B). Thus, H2S vasorelaxation reflects hyperpolarization mediated by the opening of Kir 6.1 channels through their sulfhydration at C43. The channel openers pinacidil and cromakalim elicit hyperpolarization comparable to NaHS in HEK293 cells (Online Figure XII, C).
Sulfhydration Augments ATP-Sensitive Potassium Channel Activity by Reducing Kir 6.1-ATP Binding and Enhancing Kir 6.1-PIP2 Binding
Physiological activation of the ATP-sensitive potassium channels is elicited by binding of its Kir subunits to the phospholipid phosphatidylinositol (4,5)-bisphosphate (PIP2)28 with concomitant reductions in binding to the inhibitor ATP.29 We wondered whether the regulation by H2S of Kir 6.1 stems from influences on its binding to ATP and PIP2, since cysteine-43 appears to be located within the ATP binding region and adjacent to the PIP2 binding region of Kir channels (Figure 4A and 4B and Online Figure XIII, A).29–32 In HEK293 cells, NaHS reduces ATP-Kir 6.1 binding (Figure 4C). Confirming that ATP-Kir 6.1 binding involves the sulfhydrated C43, we observed significantly more ATP binding to Kir 6.1-C43S mutants on treatment with NaHS compared with the wild-type Kir 6.1 (Figure 4D). Unlike its influences on ATP-Kir 6.1 interactions, NaHS markedly augments PIP2-Kir 6.1 binding (Figure 4E). In cells overexpressing wild-type active CSE, PIP2 binds Kir 6.1 with minimal binding in cells lacking CSE or containing the catalytically inactive enzyme (Online Figure XIV). Finally, we observed substantial reductions of PIP2 binding to Kir 6.1-C43S mutants (Figure 4F).
In summary, our findings establish H2S as a principal mediator of EDHF activity, as it satisfies all the major requirements of an EDHF candidate (Online Table I). EDHF activity is virtually abolished in two major vascular beds of CSE deleted mice. EDHF, like H2S, is produced by vascular endothelial cells on cholinergic stimulation in a calcium-calmodulin–dependent manner and both directly activate endothelial potassium channels, hyperpolarizing the cells while diffusing to adjacent smooth muscle cells where they function in a similar capacity.5–7 EDHF appears to function by covalently modifying cysteine residues of its targets, as reducing agents such as DTT reverse its effects.33 H2S also functions by sulfhydrating cysteine residues of key potassium channels in a DTT-sensitive manner. Hyperpolarization of endothelial and smooth muscle cells by H2S and EDHF leads to vasorelaxation that is independent of the NO-cGMP pathway.34 Unlike NO, which signals primarily in larger conductance vessels, EDHF activity is notably predominant in smaller vascular beds, the resistance blood vessels that determine blood pressure.5–7,34,35 This fits with our observations of a greater role for H2S in the mesenteric artery, a resistance vessel, than in the aorta, which displays more prominent NO-mediated relaxation. Recently, H2S has been shown to be an important endogenous vasorelaxant in smaller cerebral arteries.36 NO can inhibit the synthesis and release of EDHF,37 which might explain the prominence of EDHF in mesenteric arteries whose levels of eNOS, and therefore NO production, are less compared with the aorta.38 We find that NO can directly inhibit CSE activity in vitro with an IC50 of approximately 100 nmol/L (Online Figure XV).
It is important to note, however, that mediators beyond EDHF and EDRF do play significant vasoregulatory roles in different arteries. For example, studies have shown that CO plays an important role in renal vasoregulation, although the molecular mechanism of which has not entirely been worked out.39 Endothelium-dependent potassium channel activity does not appear to be involved in guinea pig uterine artery relaxation.40 H2O2 dilates coronary vasculature through a redox mechanism involving thiol oxidation through p38 map kinase.41 Although the variation in histology and physiology of vessels among different species appears to preclude the existence of a universal set of vasoregulatory molecules, EDHF or EDRF have nonetheless been repeatedly demonstrated to be the principal mechanism by which vascular tone is regulated.
Although some studies indicate that circulating H2S levels in the vasculature are less than 1 μmol/L42 there are numerous studies that show much larger concentrations of H2S ranging from 30–300 μmol/L in blood vessels as well as in numerous other tissues including the heart, lung, brain, liver, and kidney.10,43–50 Presumably, this generation of H2S by different tissues (particularly the liver) contributes to circulating plasma levels in the 30–300 μmol/L range. This may result in perfusion of the entire body with significant H2S concentrations. Our utilization of 100 μmol/L NaHS is in keeping with physiological concentration of the gas to which blood vessels might well be exposed.
Of the numerous substances that have been explored as potential mediators of EDHF, including potassium ions, lipoxygenase products, hydrogen peroxide, CNP (C-type natriuretic peptide), cytochrome P450-derived EETs, and even NO itself,6 there are few studies utilizing mutant mice indicating a physiological role for them as EDHF mediators. eNOS/COX-1 double knockout mice display reduced endothelium-dependent vasodilation but no significant attenuation of membrane potential change.51 Epoxide hydrolase knockouts manifest elevated EETs and hypotension, but no available membrane potential data support these molecules as EDHF.52 In contrast, the profoundly diminished vasorelaxation and hyperpolarization of CSE knockouts establishes H2S as a major EDHF. It is nonetheless possible that these other EDHF candidates may play important roles in modulating the formation or actions of H2S. As our studies have been confined to rodents, we do not know if they apply fully to human vasculature. However, vascular regulation is generally similar in humans and rodents.5,7
Sulfhydration is a physiological modification of cysteines in H2S target proteins analogous to S-nitrosylation by NO.21,53S-nitrosylation most often inhibits the function of its targets, while sulfhydration predominantly enhances activity.21,53 The importance of sulfhydration is indicated by the large proportion of proteins that are sulfhydrated and the considerable extent of sulfhydration, 10–25% for some major liver proteins including actin, β-tubulin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).21 The process of sulfhydration reflects the formation of a persulfide bond, which is an oxidative reaction. H2S is sometimes referred to as a reducing agent. Like many other substances, however, the redox potential of H2S enables it to act both as a reducing as well as an oxidizing agent. Some well-known examples of dual role substances are cysteine and glutathione, which despite being recognized as reducing agents, mediate the oxidizing processes of cysteinylation and glutathionylation of proteins, respectively.54 These modifications essentially appear to follow a similar chemistry as sulfhydration. This contrasts with substances such as DTT, which are very strong reducing agents and not likely to have oxidizing functions.55
Evidence that Kir 6.1 is physiologically sulfhydrated includes the demonstration of its sulfhydration basally as well as elicited by cholinergic simulation and H2S donors with sulfhydration abolished in CSE deleted tissues. Moreover, we established that sulfhydration involves a single cysteine, cysteine-43, whose mutation abolishes sulfhydration and the subsequent H2S-mediated hyperpolarization. Previously, we demonstrated sulfhydration of several dozen proteins, with the modification confirmed in vivo by mass spectrometry.21 The very low abundance of Kir 6.1 in vascular tissue, however, renders a mass spectrometric analysis not feasible.
Activation of Kir 6.1 is known to reflect its dissociation with ATP29 and binding to PIP2,28 which we also observe after sulfhydration at cysteine-43. As sulfhydration renders cysteines more electronegative, the modification at cysteine-43, which lies within the electropositive ATP binding region, might electrostatically hinder channel binding to ATP in addition to causing steric hindrance (Online Figure XIII, B). Since the PIP2 binding region lies adjacent to the ATP binding region, preclusion of ATP binding may provide PIP2 greater access to its binding site on the channel leading to enhanced channel activity.
Several studies suggest that myoendothelial gap junctions composed of connexins transduce endothelial to vascular smooth muscle hyperpolarization.56,57 Connexin 40–deleted mice, which lack the myoendothelial gap junctions, are hypertensive.56 Furthermore, inhibitors of gap junction attenuate smooth muscle hyperpolarization in rat mesenteric artery but have no effect on endothelial hyperpolarization.57 H2S stimulates endothelial IKca/SKca as well as smooth muscle ATP-sensitive potassium channels leading to hyperpolarization and vasorelaxation (Online Figure XVI). Given the clear implications of gap junctions regulating smooth muscle hyperpolarization, it is likely that H2S diffuses from endothelial to smooth muscle cells through gap junctions to sulfhydrate the cytosolic cysteine-43 of smooth muscle ATP-sensitive potassium channels. These potential mechanisms, however, remain to be explored.
What are the physiological and pathophysiologic consequences of these observations? It is clear that deletion of these potassium channels,58 as well as application of potent and selective channel inhibitors such as glibenclamide,59,60 causes hypertension similar to our earlier observations with CSE deleted animals.9 Recently, Ishii et al have shown that deletion of CSE does not significantly alter blood pressure in mice.61 It is important to note, however, that in this instance blood pressure was measured using the tail-cuff method which is not only less precise compared with the more invasive catheter measurements conducted by our laboratories,9 but also leads to highly variable measurements hindering proper analysis of the data. On the other hand, in addition to the data presented in this study on the CSE inhibitor propargylglycine, there is now clear evidence that selective CSE inhibitors, as well as pathological conditions such as intermittent hypoxia in which H2S is diminished, significantly increase vascular myogenic tone and therefore raise blood pressure.62
Thus, the finding that H2S is a major EDHF of resistance blood vessels that regulate blood pressure, as well as its novel mechanism of action may have important therapeutic implications. Drugs altering CSE activity or H2S-mediated channel sulfhydration may be beneficial in treating diverse vascular disorders including hypertension.
We thank Drs Yoshi Kurachi and Hiroshi Hibino (Osaka University, Japan) for providing the SUR2B cDNA construct and Drs Victor Miriel, David Yue, and Manu Ben Johny for advice on the membrane potential experiments.
Sources of Funding
This study was supported by a
Non-standard Abbreviations and Acronyms
bis-(1,3-dibutylbarbituric acid)trimethine oxonol
endothelium-derived hyperpolarizing factor
endothelium-derived relaxing factor
intermediate conductance potassium channel
L-NG-nitroarginine methyl ester
sodium hydrogen sulfide
nitric oxide synthase
small conductance potassium channel
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Novelty and Significance
What Is Known?
Hydrogen sulfide (H2S) is a gaseous signaling molecule. It is synthesized by cystathionine γ-lyase (CSE), which is confined predominantly to the vascular endothelium.
Mice lacking H2S are hypertensive and demonstrate impaired endothelium-dependent vasorelaxation. Thus, H2S acts as an endothelium-derived relaxing factor that mediates vascular relaxation and lowers blood pressure.
The effects of H2S, unlike those of nitric oxide, are mediated, in part, by the activation of the ATP-sensitive potassium channels (KATP) but are independent of cyclic GMP.
What New Information Does This Article Contribute?
H2S causes a redox-sensitive posttranslational modification, sulfhydration, of a single cysteine, C43, in the Kir 6.1 subunit of the KATP channel.
H2S-mediated sulfhydration enhances Kir 6.1 activity by reducing Kir 6.1-ATP binding and increasing Kir 6.1-PIP2 binding.
Hence, cholinergic, endothelium-dependent vasorelaxation and hyperpolarization are significantly reduced in vessels in which CSE is inhibited, in vessels from CSE-/- mice, or in which the KATP channel has been inhibited.
Sulfhydration of the calcium-dependent intermediate conductance potassium channel (IKca) contributes to H2S-dependent hyperpolarization of endothelial cells.
Emerging evidence suggests that H2S is an important gaseous signaling molecule in the vascular system, where it is produced by the endothelial enzyme CSE. It mediates vasorelaxation in part by activating vascular smooth muscle KATP channels. We found that cholinergic vasorelaxation and hyperpolarization are markedly reduced in CSE-/- and glibenclamide-treated vessels, indicating that H2S is a major endothelium-derived hyperpolarizing factor that causes vascular endothelial and smooth muscle cell hyperpolarization and vasorelaxation, since H2S mediates its effect by a novel redox-sensitive thiol-dependent, posttranslational modification of proteins by sulfhydration. Indeed, the Kir 6.1 KATP subunit C43S mutant expressed in HEK293 cells abolishes sulfhydration and significantly reduces H2S mediated hyperpolarization. Sulfhydration of C43 in the Kir 6.1 subunit of the KATP channel reduces ATP binding and enhances PIP2 binding, a process that leads to channel activation. Finally, H2S also leads to sulfhydration and hyperpolarization of endothelial cells through the IKca and SKca channels. These findings suggest that H2S is an important endothelium-derived hyperpolarizing factor; therefore, dysregulation of this pathway may be critical step in the development of vascular diseases such as hypertension.