Hydrogen sulfide dilates cerebral arterioles by activating smooth muscle cell plasma membrane K_ATP channels

Abstract

Hydrogen sulfide (H₂S) is a gaseous signaling molecule that appears to contribute to the regulation of vascular tone and blood pressure. Multiple potential mechanisms of vascular regulation by H₂S exist. Here, we tested the hypothesis that piglet cerebral arteriole smooth muscle cells generate ATP-sensitive K⁺ (K_ATP) currents and that H₂S induces vasodilation by activating K_ATP currents. Gas chromatography/mass spectrometry data demonstrated that after placing Na₂S, an H₂S donor, in solution, it rapidly (1 min) converts to H₂S. Patch-clamp electrophysiology indicated that pinacidil (a K_ATP channel activator), Na₂S, and NaHS (another H₂S donor) activated K⁺ currents at physiological steady-state voltage (−50 mV) in isolated cerebral arteriole smooth muscle cells. Glibenclamide, a selective K_ATP channel inhibitor, fully reversed pinacidil-induced K⁺ currents and partially reversed (∼58%) H₂S-induced K⁺ currents. Western blot analysis indicated that piglet arterioles expressed inwardly rectifying K⁺ 6.1 (K_ir6.1) channel and sulfonylurea receptor 2B (SUR2B) K_ATP channel subunits. Pinacidil dilated pressurized (40 mmHg) piglet arterioles, and glibenclamide fully reversed this effect. Na₂S also induced reversible and repeatable vasodilation with an EC₅₀ of ∼30 μM, and this effect was partially reversed (∼55%) by glibenclamide. Vasoregulation by H₂S was also studied in pressurized resistance-size cerebral arteries of mice with a genetic deletion in the gene encoding SUR2 (SUR2 null). Pinacidil- and H₂S-induced vasodilations were smaller in arterioles of SUR2 null mice than in wild-type controls. These data indicate that smooth muscle cell K_ATP currents control newborn cerebral arteriole contractility and that H₂S dilates cerebral arterioles by activating smooth muscle cell K_ATP channels containing SUR2 subunits.

hydrogen sulfide (H₂S) is endogenously synthesized from l-cysteine by cystathionine β-synthase or cystathionine γ-lyase (13, 44). It is becoming apparent that H₂S is a physiologically significant signaling molecule (25, 26, 29). Previous studies have described the cardiovascular effects of exogenous and endogenous H₂S in several species. H₂S dilates blood vessels, controls blood pressure, and protects the heart from ischemia-reperfusion injury (48). Most studies investigating vasodilation by H₂S have been performed in rodents and have examined large arteries, aortic rings, mesenteric arteries, and measured blood pressure (7, 32, 42). This study was designed to examine the regulation of cerebral arteriole contractility by H₂S in newborn pigs.

H₂S can affect a variety of ion channels and receptors (45). H₂S-induced vasorelaxation of systemic arteries and aorta has been demonstrated to occur because of endothelium-dependent dilators, Cl⁻/HCO₃⁻ exchanger, KCNQ channels, and ATP-sensitive K⁺ (K_ATP) channels (7, 24, 39, 45). K_ATP channels are hetero-octameric complexes containing four pore-forming, inwardly rectifying (K_ir6.1 or K_ir6.2) channel subunits, together with four sulfonylurea receptors (SURs), which are ATP-binding cassette family proteins (43, 45, 46). Molecular diversity exists between tissues regarding their K_ir and SUR isoform (SUR1, SUR2A, and SUR2B) expression and channel composition (4). A number of pharmacological studies have provided functional evidence that smooth muscle cell K_ATP channels regulate the tone of adult cerebral arteries in vitro and in vivo (20, 21, 33). We recently reported that exogenous and endogenous H₂S dilates piglet cerebral arterioles in vivo and that this vasodilation is inhibited by glibenclamide, suggesting the involvement of K_ATP channels (26). Newborn piglet cerebral arteriole smooth muscle cell K_ATP currents have not been described nor has diameter regulation by these channels been studied in vitro. This study aimed to determine whether newborn arterial smooth muscle cells generate K_ATP currents. Additional objectives of this study were to determine the effects of H₂S on K_ATP currents and to investigate the contribution of K_ATP channels to H₂S-induced dilation of arterioles isolated from other influences present in the intact brain.

We show that both pinacidil, a K_ATP channel activator, and H₂S activate cerebral arteriole smooth muscle cell K_ATP currents, leading to vasodilation. Glibenclamide, a plasma membrane and mitochondrial K_ATP channel inhibitor, reversed pinacidil and H₂S-induced K⁺ current activation and vasodilation. H₂S-induced vasodilation was attenuated in cerebral arteries of SUR2 null mice, indicating that plasma-membrane K_ATP channels mediate this response. These data indicate that exogenous H₂S activates arteriole smooth muscle cell plasma-membrane K_ATP channels containing SUR2, leading to vasodilation.

MATERIALS AND METHODS

Tissue preparation and arteriole smooth muscle cell isolation.

All procedures involving animals were approved by the University of Tennessee Health Science Center Animal Care and Use Committee. Newborn pigs (1–3 days old, 1.5–2.5 kg; Nichols Hog Farm; Olive Branch, MS) were anesthetized with ketamine hydrochloride (33 mg/kg im) and acepromazine (3.3 mg/kg im). Mice with a targeted deletion of exons 14–18 in the Abcc9 gene encoding SUR2 (SUR2 null mice) were generated as previously described (8, 9). Male and female mice (∼20 g) were euthanized with an overdose of pentobarbital sodium (130 mg/kg ip). Brains were removed and placed into oxygenated ice-cold (4°C) physiological saline solution (PSS) of the following composition: (in mM) 112 NaCl, 4.8 KCl, 24 NaHCO₃, 1.8 CaCl₂, 1.2 MgSO₄, 1.2 KH₂PO4, and 10 glucose, which was gassed with 21% O₂-5% CO₂-74% N₂ to pH 7.4. Piglet pial arterioles (100–250 μm in diameter) were dissected from the cerebral cortical surface. Murine posterior cerebral and cerebellar arteries (∼200 μm in diameter) were collected. Arterioles and arteries were cleaned of connective tissue. Individual smooth muscle cells were enzymatically dissociated from cerebral arterioles using a procedure previously described (17). Cells were maintained in ice-cold (4°C) isolation solution and used for experiments within 6 h.

H₂S measurements using gas chromatography/mass spectrometry.

Detection and quantification of H₂S released from Na₂S and NaHS was made by comparison to standards with known concentrations of H₂S. These standards were made by dilution from water saturated with H₂S. Saturated H₂S solution (10⁻¹ M at 25°C) was made by placing water in a vial (50% of the volume) with the headspace filled with 100% H₂S gas. The vial was vortexed well and the headspace gas replaced with 100% H₂S three times at 5-min intervals with vortexing in between. The vial was then left for at least 30 min with intermittent vortexing to ensure equilibration between the gas and liquid. Aliquots of the H₂S-saturated water were used to make standard curves. Because the total H₂S in the vial and the volume of liquid are known, the mass spectral peak of the headspace gas would be the same as that of a 300-μl sample containing the same molarity as the standard. H₂S in 20 μl of the headspace gas was identified and quantified by gas chromatography/mass spectrometry (GC/MS; 5975C series; Agilent Technologies, Santa Clara, CA). Gas chromatography was run on a 0.32 mm × 30 m fused silica HP-PLOT-U column (Agilent Technologies). The gas chromatography was programmed with an initial oven temperature of 45°C, a ramp to 75°C over 30 s, hold for 1 min, ramp to 85°C over 6 s, and hold for 4 min, before returning to 45°C. The carrier gas was H₂ at 3.0 ml/min. H₂S area counts against molarity (0.37, 0.74, 1.9, 3.7, 5.6, 9.2, and 18.5 μM in 300 μl buffer) were linear and passed through zero/zero (r² = 0.998). H₂S concentrations in Na₂S samples were calculated based on the counts from known H₂S samples freshly diluted from saturated stock for each experiment.

Different concentrations of Na₂S (1, 10, and 100 μM) were made by dissolving Na₂S in buffer containing (in mM) 134 NaCl, 6 KCl, 2 CaCl₂, 1 MgCl₂, 10 HEPES, and 10 glucose (pH 7.4) at room temperature (25°C). Three hundred microliters of each Na₂S sample concentration was placed immediately into a separate 2-ml air-tight vial. To obtain a time course of H₂S production, each Na₂S sample was vortexed well and kept upside down at 25°C for 1, 10, 20, or 40 min to allow H₂S release from Na₂S and equilibration of H₂S between liquid and headspace gas.

Patch-clamp electrophysiology.

Isolated arteriole smooth muscle cells were placed into an experimental chamber containing Ca²⁺-free HEPES buffer and allowed to settle for 10 min. Potassium currents were measured using the whole cell patch-clamp configuration (Axopatch 200B and Clampex 9.2; Molecular Devices, Downingtown, PA). To minimize voltage-dependent K⁺ currents, K_ATP currents were recorded at a steady membrane potential of −50 mV using a continuous gap-free acquisition protocol. The pipette solution contained (in mM) 10 HEPES, 102 KCl, 38 KOH, 10 EGTA, 1 MgCl₂, 1 CaCl₂, 0.1 NaATP, 0.1 NaADP, 0.2 NaGTP, and 10 glucose (pH 7.2 with HCl). The calculated free Ca²⁺ concentration in this solution is 13 nM (WEBMAXC, Stanford University, Stanford, CA). The bath solution contained (in mM) 10 HEPES, 60 KCl, 80 NaCl, 1 MgCl₂, 0.1 CaCl₂, 1 tetraethylammonium, and 10 glucose (pH 7.4 with KOH) (Fig. 2, C and D) or (in mM) 10 HEPES, 140 KCl, 1 MgCl₂, 0.1 CaCl₂, 1 tetraethylammonium, and 10 glucose (pH 7.4 with KOH) (Fig. 2A, B and D). Data analysis was performed off-line using Clampfit 9.2 (Molecular Devices). The bath solution was continuously perfused to maintain constant H₂S concentrations in the chamber.

Fig. 2.

Piglet cerebral arteriole smooth muscle cells generate ATP-sensitive K⁺ (K_ATP) currents that are activated by H₂S. Pinacidil (10 μM, A), Na₂S (10 μM, B), and NaHS (20 μM, C) activate glibenclamide (Glib)-sensitive K⁺ currents. D: mean data: Glib, n = 4; pinacidil and pinacidil + Glib (10 μM), n = 6 for each; Na₂S and Na₂S + Glib (10 μM), n = 6 for each; and NaHS and NaHS + Glib (20 μM), n = 6 for each. All currents were recorded at a steady holding potential of −50 mV. *P < 0.05 compared with pinacidil, Na₂S, or NaHS, respectively.

Pressurized artery diameter measurements.

An arteriole segment ∼2 mm in length was cannulated at each end in a temperature-controlled perfusion chamber (Living Systems Instrumentation, Burlington, VT). The chamber was continuously perfused with PSS, equilibrated with a mixture of 21% O₂-5% CO₂-74% N₂, and maintained at 35°C. Arterioles were observed with a charge-coupled device camera attached to an inverted microscope (Nikon TE 200). Arteriole diameter was measured by using the automatic edge-detection function of IonWizard software (Ionoptix; Milton, MA) and digitized at 1 Hz using a personal computer. Steady-state changes in intravascular pressure were achieved by elevating and lowering an attached reservoir and monitored using a pressure transducer. Intraluminal PSS was static during experiments. Tested compounds were applied via chamber perfusion. The presence of intact endothelium was confirmed by observing bradykinin (10 nM)-induced vasodilation.

Western blot analysis.

To determine total protein, arteries were homogenized in a lysis buffer of the following composition: 50 mM Tris·HCl, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, and 0.1% SDS. Cellular debris was removed by centrifugation at 8,000 rpm for 10 min. Samples were denatured by adding 5× Laemmli buffer containing 2-mercaptoethanol (2%) and boiled for 3 min. Total protein was loaded onto 7.5% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes. Membranes were cut so that the same lysate could be probed for both K_ir6.1 and SUR2B proteins. Membranes were incubated either with goat polyclonal anti-K_ir6.1 (1:100, Santa Cruz Biotechnology, CA) or rabbit polyclonal anti-SUR2B (BNJ-40, 1:500, a kind gift from Dr. Jonathan Makielski, University of Wisconsin, Ref. 37) primary antibodies overnight at 4°C in Tris-buffered solution (TBS) containing 0.1% Tween 20 (TBS-T) and 5% nonfat dry milk. After being washed with TBS-T, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 2 h, followed by washing with TBS-T. The membranes were then developed using Supersignal West Pico chemiluminescent substrate (Thermo Scientific), and digital images were obtained using a Kodak FX Pro imaging system.

Chemicals.

Glibenclamide and pinacidil were purchased from Tocris (Ellisville, MO). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO).

Data analysis.

Values are given as means ± SE. Student's t-test was used to compare two groups of paired or unpaired data, as appropriate. P < 0.05 was considered significant. Concentration-response curves were fit with a Boltzmann equation to obtain the half-maximal effective concentration (EC₅₀) value.

RESULTS

H₂S production from Na₂S and reaction time course.

GC/MS was used to measure the amount and rate of H₂S generation by Na₂S, a H₂S donor. With a pH of 7.4 and at a temperature of 25°C, Na₂S concentrations of 1, 10, and 100 μM produced ∼0.47, 4.84, and 49.83 μM of gaseous H₂S, respectively (Fig. 1A). The regression of H₂S concentration versus Na₂S concentration was linear (Fig. 1A). After Na₂S was dissolved in HEPES-buffered PSS, H₂S concentrations in the gas phase reached a maximum level by the first time point (1 min), and there was no significant difference when compared with longer reaction times (10, 20, and 40 min) (Fig. 1B). These data indicate that H₂S production from Na₂S is very fast with H₂S reaching stable levels within 1 min of Na₂S addition. Assuming a complete dissociation of Na₂S, these data suggest that at pH 7.4 with 1.7 ml gas and 300 μl liquid, gaseous H₂S constitutes almost 50% of the total sulfide.

Fig. 1.

Hydrogen sulfide (H₂S) production by Na₂S determined by gas chromatography/mass spectrometry of headspace gas. A: H₂S production by Na₂S (1–100 μM) fit with a regression function (n = 9, 12, 9 for each data point). Errors bars are provided for each data point but are small and occluded by the symbols. Mean ± SE values are 0.47 ± 0.04, 4.84 ± 0.43, and 49.83 ± 4.1 μM for 1, 10, 100 μM of Na₂S, respectively. Experimental data points were fit with a linear regression function (Y = A + B × X), where X and Y are Na₂S and H₂S concentrations (in μM), respectively (R² = 1), and A is the intercept and B the slope, giving A = −0.0843 ± 0.06327 and B = 0.4991 ± 0.0011. B: time course of H₂S production from 1 or 10 μM Na₂S (n = 3 to 4 for each data point).

H₂S activates K_ATP currents in cerebral arteriole smooth muscle cells.

To test the hypothesis that K_ATP channels are a target of H₂S, K⁺ currents were measured in isolated newborn piglet arteriole smooth muscle cells using the conventional whole cell patch-clamp configuration. Currents were measured at a physiological steady-state voltage of −50 mV using solutions designed to limit large-conductance Ca²⁺-activated K⁺ channel activity. With 140 mM [K⁺] in the bath solution, pinacidil (10 μM), a K_ATP channel activator, increased mean inward current by ∼75 pA (Fig. 2, A and D). Glibenclamide (10 μM), a K_ATP channel inhibitor, did not alter K⁺ currents when applied alone but abolished pinacidil-activated K⁺ currents (Fig. 2, A and D). Na₂S (10 μM) increased mean inward current by ∼48 pA (Fig. 2, B and D). Subsequent application of glibenclamide reduced mean Na₂S-induced K⁺ currents by ∼28 pA or by ∼58% (Fig. 2, B and D). Similar data were obtained when using NaHS, another H₂S-releasing molecule, which activated whole cell K⁺ currents that were partially inhibited by glibenclamide (20 μM) (Fig. 2, C and D). These data indicate that two different H₂S donors activate K_ATP currents in cerebral arteriole smooth muscle cells.

Piglet cerebral arterioles express K_ir6.1 and SUR2B subunits and are regulated by K_ATP channel modulators.

Expression of K_ATP channel subunits in piglet cerebral arterioles was unclear. Western blot analysis of piglet cerebral arteriole protein lysate revealed expression of both SUR2B (∼135 kDa mol mass) and K_ir6.1 (∼51 kDa mol mass) subunits (Fig. 3A). We next studied the myogenic response of piglet cerebral arterioles. At 40 mmHg, arterioles constricted from a mean passive diameter of 239 ± 11 μm to a myogenic diameter of 138 ± 6 μm or by 42% (n = 21). To establish the functional significance of smooth muscle cell K_ATP channels containing SUR2B and K_ir6.1 subunits, diameter responses to pinacidil were measured in myogenic arterioles. Pinacidil (10 μM) increased mean arteriole diameter by ∼33 μm (Fig. 3, B and C). Glibenclamide (10 μM) did not alter arteriole diameter when applied alone but fully reversed pinacidil-induced vasodilation (Fig. 3, B and C). These data indicate that smooth muscle cell K_ATP channel subunits are expressed and that K_ATP channels control diameter in piglet cerebral arterioles.

Fig. 3.

K_ATP current activation induces vasodilation in pressurized (40 mmHg) piglet cerebral arterioles. A: Western blot of piglet cerebral arteriole lysate. The top blot indicates sulfonylurea receptor 2B (SUR2B) at ∼135 kDa, whereas the bottom blot illustrates inwardly rectifying K⁺ 6.1 (K_ir6.1) channel at ∼51 kDa. B: representative trace illustrating Glib (10 μM) reversal of pinacidil (10 μM)-induced vasodilation in a pressurized (40 mmHg) piglet cerebral artery. C: mean data: Glib (10 μM), pinacidil (10 μM), and pinacidil + Glib (10 μM) (n = 6 for each). *P < 0.05 compared with pinacidil.

Glibenclamide attenuates H₂S-induced vasodilation in piglet cerebral arterioles.

Physiological functions of H₂S were studied in pressurized (40 mmHg) piglet cerebral arterioles. Na₂S induced a mean vasodilation of ∼20 μm that was reversible upon washout (Fig. 4, A and C). Na₂S-induced vasodilation was also repeatable (a second Na₂S-induced vasodilation was 107 ± 20% of the vasodilation induced by a prior Na₂S application, n = 5, P > 0.05, Fig. 4A). Performing a concentration-response curve to Na₂S (0.1–1,000 μM) indicated an EC₅₀ of 30.4 ± 5.3 μM (Fig. 4B). Na₂S-induced vasodilation was partially (by ∼55%) reversed by glibenclamide (10 μM) (Fig. 4, C and D). In contrast, glibenclamide did not alter vasodilation induced by isoproterenol, an adrenergic receptor agonist and smooth muscle-specific vasodilator of piglet cerebral arterioles [Δdiameter: isoproterenol (1 μM), 74 ± 10 μm; isoproterenol + glibenclamide (10 μM), 78 ± 9 μm; n = 4, P > 0.05] (22, 28). These data indicate that H₂S induces vasodilation via K_ATP channel-dependent and -independent mechanisms in isolated newborn cerebral arterioles. In contrast, isoproterenol causes vasodilation via K_ATP channel-independent mechanisms.

Fig. 4.

H₂S dilates pressurized (40 mmHg) piglet cerebral arterioles by activating K_ATP currents. A: representative traces illustrating diameter responses to a first and second application of Na₂S (10 μM) in the same pressurized piglet cerebral arteriole. B: concentration-dependent vasodilation induced by Na₂S in pressurized piglet cerebral arteries (n = 2, 9, 3, 8, 7, 2, and 2 for each). Mean data are fit with a Boltzmann function. C: exemplar trace illustrating that Na₂S (1 μM)-induced vasodilation is partially reversed by Glib (10 μM). D: mean data for Na₂S (1 μM) and Na₂S + Glib (10 μM) (n = 6 for each). *P < 0.05 when compared with Na₂S.

Na₂S- and pinacidil-induced vasodilation is attenuated in cerebral arteries of SUR2 null mice.

To investigate molecular targets of H₂S-induced vasodilation, we used cerebral arteries of SUR2 null mice. Pressurized (60 mmHg) arteries from wild-type and SUR2 null mice developed similar levels of myogenic tone (wild-type, 17 ± 1%, n = 19; and SUR2 null mice, 15 ± 4%, n = 8, P > 0.05). Wild-type and SUR2 null mouse arteries also had similar mean passive diameters of 200 ± 5 (n = 19) and 196 ± 10 μm, respectively, as determined by removal of bath Ca²⁺ at 60 mmHg (n = 8, P > 0.05). Pinacidil and Na₂S dilated pressurized wild-type cerebral arteries by ∼11 and 10 μm, respectively (Fig. 5). In contrast, pinacidil and Na₂S dilated pressurized SUR2 null arteries by ∼2 and 5 μm, respectively (Fig. 5). Therefore, mean pinacidil- and Na₂S-induced vasodilation was ∼18 and ∼50% of that in wild-type controls. These data indicate that pinacidil- and H₂S-induced cerebral artery dilation occurs through plasma membrane K_ATP channels containing SUR2.

Fig. 5.

Pinacidil- and Na₂S-induced vasodilation is attenuated in pressurized (60 mmHg) cerebral arteries of SUR2 null mice. A and B: representative traces illustrating pinacidil (5 μM)- and Na₂S (5 μM)-induced vasodilation in cerebral arteries of wild-type (WT) and SUR2 null mice. C: mean data for pinacidil (5 μM; n = 15 for WT, n = 6 for SUR2 null) and Na₂S (5 μM; n = 14 for WT, n = 4 for SUR2 null). *P < 0.05 compared with WT mice.

DISCUSSION

Novel findings of this study are as follows: 1) upon dissolution in aqueous medium, Na₂S rapidly converts to gaseous H₂S; 2) pinacidil- and glibenclamide-sensitive K_ATP currents are present in newborn piglet cerebral arteriole smooth muscle cells and regulate arteriole contractility; 3) piglet cerebral arterioles express K_ir6.1 and SUR2B subunits; 4) exogenous H₂S derived from Na₂S or NaHS activates K_ATP currents in isolated piglet cerebral arteriole smooth muscle cells; 5) H₂S dilates pressurized piglet cerebral arterioles by activating K_ATP channels; and 6) pinacidil- and H₂S-induced vasodilations are attenuated in cerebral arteries from SUR2 null mice. These data indicate that H₂S dilates cerebral arteries and arterioles in part by activating smooth muscle cell K_ATP channels containing SUR2 subunits.

In the majority of studies published to date, NaHS has been used as an exogenous H₂S donor, with Na₂S used to a lesser extent (5). Recently, phase 1 clinical trials in humans have used pharmaceutical grade Na₂S for therapeutic treatment of myocardial infarction (31). Here, we used both Na₂S and NaHS as H₂S donors. These molecules are effective donors that contribute only micromolar Na⁺ as an additional atom and dilate similarly to H₂S gas in vivo (27). For consideration of H₂S generated, we used GC/MS of the headspace gas in a sealed vial. With inclusion of a gas phase, the amount dissolved in the liquid will be reduced. In the liquid phase there is dissolved H₂S gas, HS⁻ and S²⁻ (H₂S↔HS⁻↔S²⁻). The quantity of total sulfide in the vial will remain the same, but the reaction will be shifted to the left as H₂S diffuses to the gas phase at equilibrium (equal partial pressures of H₂S in gas and liquid). The amount in gas and liquid will be determined by the quantity of the gas space relative to the liquid quantity, the pH (lower pH moves reaction left), and temperature (assuming barometric pressure is constant). We used GC/MS to measure H₂S in headspace gas to determine both rate of production and levels of H₂S generated by Na₂S. Our quantitative analysis results indicate that Na₂S produces H₂S quickly, reaching a maximum level in headspace gas within 1 min of being dissolved in pH 7.4 buffer. Based on these data, in the present study cells or pressurized arteries were continuously perfused with fresh donors during experiments to maintain constant H₂S concentrations in experimental chambers. With the assumption that all Na₂S was ionized, ∼50% was gaseous H₂S at all Na₂S concentrations tested between 1 and 100 μM. These measurements are somewhat higher than previous reports for dissolved H₂S concentrations at neutral pH (33%) and pH 7.4 (40%) in physiological solutions at 25°C (14, 47). The difference could be explained by the experimental conditions. In these experiments the gas volume was 1,700 μl, the liquid volume was 300 μl, the temperature was constant, and the pH was 7.4. Under these conditions the total sulfide was distributed ∼50% between the liquid and gas.

Earlier studies and reviews have measured a variety of H₂S concentrations in blood from different species: ∼10 μM H₂S in Wistar rat blood, ∼50 μM H₂S in Sprague-Dawley rat blood, and 10–100 μM H₂S in human blood (11, 30, 51). Millimolar concentrations of H₂S or H₂S donors have typically been used to study cardiac and pulmonary protective effects of H₂S (6, 23). Our data demonstrate that low micromolar H₂S concentrations activate K_ATP currents in cerebral arteriole smooth muscle cells and induce vasodilation in cerebral arterioles and arteries. We found that 300 μM and 1 mM Na₂S induce an alkaline shift in pH from 7.4 to 7.54 and 7.75, respectively, whereas lower concentrations of H₂S donors did not alter pH. Therefore, physiological functions of H₂S described here cannot be attributed to pH changes. However, such alterations in pH may explain some results in studies where high H₂S concentrations have been used.

K_ATP channels regulate the contractility of a wide variety of smooth muscle cells, including those from coronary, pulmonary, mesenteric, and cerebral arteries, as well as bladder and airway (3, 23, 38). To date, no electrophysiological studies have identified K_ATP currents in newborn cerebral arteriole smooth muscle cells. Here, we demonstrate that glibenclamide alone did not alter baseline K⁺ currents or arteriole diameter, indicating that smooth muscle cell K_ATP channels do not contribute to baseline K⁺ currents or contractility in pressurized newborn cerebral arterioles. However, pinacidil activated K⁺ currents and dilated pressurized piglet cerebral arterioles and these effects were abolished by glibenclamide. Therefore, we provide the first direct evidence that K_ATP channels can generate K⁺ currents in newborn cerebral arteriole smooth muscle cells and that activation of these currents induces vasodilation.

H₂S stimulates K_ATP currents in rat mesenteric artery and aortic smooth muscle cells and pancreatic β-cells (45). Our data are the first to demonstrate that H₂S activates K_ATP currents in cerebral arteriole smooth muscle cells and dilates cerebral arterioles and arteries. Na₂S dilated pressurized cerebral arterioles with an EC₅₀ of ∼30 μM. In contrast, glibenclamide did not alter isoproterenol-induced vasodilation, indicating that K_ATP channels do not contribute to effects of this smooth muscle-specific vasodilator. Recently, using GC/MS, we measured basal H₂S concentrations of ∼600 nM in piglet cortical surface cerebrospinal fluid (26). Dilator concentrations measured here in vitro are near the range we have also measured in cortical surface cerebrospinal fluid during hypercapnia (∼4 μM) (26). Similarly, in vivo dilation of pial arterioles to topical H₂S had a threshold of <10 μM (26). Therefore, concentrations of H₂S that induced dilation here are consistent with those in vivo.

H₂S-induced K_ATP current activation and vasodilation were only partially reversed by glibenclamide at a concentration that fully blocked pinacidil-induced K⁺ currents and pinacidil-induced vasodilation. These data suggest that H₂S can activate K⁺ channels other than K_ATP, an effect that would also contribute to vasodilation. Consistent with this conclusion, 4-aminopyridine, a voltage-gated K⁺ channel blocker, attenuated relaxation induced by millimolar NaHS in agonist-contracted coronary artery rings (6). XE991, a KCNQ channel blocker, also attenuated NaHS-induced relaxation in aortic rings (39). However, in vivo glibenclamide completely blocked pial arteriole dilation to topical H₂S (26). The reasons for greater effectiveness of glibenclamide in blocking dilation to H₂S in vivo than in vitro are not known. One explanation for the slightly different glibenclamide sensitivity may be the size of the arterioles studied in vivo (50 μm) and in vitro (200 μm). In addition, the relative contribution of K_ATP currents to vasodilation may depend on the H₂S concentration used. Additional experiments will be required to test these hypotheses.

K_ATP channels are comprised of a K_ir6.1/6.2 channel and a SUR1, 2A, or 2B subunit (15). H₂S directly activates recombinant K_ATP channels composed of K_ir6.1 and SUR1 subunits by interacting with NH₂-terminal cysteine residues (18). However, arterial smooth muscle cell plasma membrane K_ATP channels are comprised of K_ir6.1 and SUR2B subunits, as supported by data here and in previous studies (1, 40). In addition, smooth muscle cells likely employ signaling pathways that are distinct from those present in immortalized cells used to express recombinant proteins. Thus H₂S-induced K_ATP channel activation in arterial smooth muscle cells may occur because of direct or indirect mechanisms (18, 45). K_ATP channels are located both on the plasma membrane and on mitochondria (12, 16). Glibenclamide inhibits both plasma membrane and mitochondrial K_ATP channels (49). Therefore, H₂S may have activated arteriole smooth muscle cell K⁺ currents and induced vasodilation by activating plasma membrane and/or mitochondrial K_ATP channels, particularly since the effects of H₂S on both K_ATP channels and mitochondrial function have been described (10, 35, 41). SUR2B does not appear to be a functional component of arterial smooth muscle cell mitochondrial K_ATP channels (1). Therefore, to study K_ATP channels activated by H₂S, we measured the functional responses in cerebral arteries of SUR2 null mice. Vascular smooth muscle cell K_ATP currents are abolished in SUR2 null mice (19). We have previously studied vasoregulation by intravascular pressure and by different K_ATP channel openers in mesenteric arteries of SUR2 null mice (1), but to our knowledge this study is the first to examine functional responses in SUR2 null cerebral arteries. Data indicate that SUR2 null mice cerebral and mesenteric arteries develop similar levels of myogenic tone to wild-type controls, indicating that K_ATP channels do not contribute to the myogenic response (1). Here, we show that wild-type and SUR2 null mouse cerebral arteries dilate similarly to removal of bath Ca²⁺ and have similar passive diameters, consistent with data in mesenteric arteries. These data indicate that SUR2 null arteries do not exhibit generalized alterations in vasodilation. Pinacidil-induced vasodilation was significantly smaller, but not abolished, in SUR2 null cerebral arteries, consistent with data in mesenteric arteries where vasodilation to this K_ATP channel opener was 10% of that in wild-type controls (1). The mechanism for the residual dilation is unclear but suggests a SUR2-independent target for pinacidil in arterial smooth muscle cells. K_ATP channel-independent mechanisms proposed to mediate pinacidil-induced vasodilation include stimulation of plasma membrane Ca²⁺ extrusion, a reduction in contractile apparatus Ca²⁺ sensitivity, and inhibition of GTP-binding protein-coupled phosphatidylinositol turnover (2, 34). Data with SUR2 null arteries indicate that approximately half of the H₂S-induced vasodilation occurs because of activation of arterial smooth muscle cell SUR2-containing K_ATP channels, results that support our evidence using pharmacological modulators. The H₂S-induced vasodilation present in SUR2 null arteries is consistent with our data indicating that although glibenclamide fully blocked pinacidil-induced K⁺ current activation and vasodilation, H₂S-induced K⁺ current activation and vasodilation were only partially reversed by glibenclamide. These findings indicate that H₂S also activates K⁺ channels other than K_ATP, leading to vasodilation. Future studies are needed to identify additional K⁺ channel(s) activated by Na₂S and whether Na₂S also causes vasodilation through K⁺ channel-independent mechanisms.

Murine cardiac myocytes express one full-length (130 kDa) and three short (28, 55, and 68 kDa) SUR2 forms (37, 50). Full-length SUR2 contains two nucleotide-binding domains and together with K_ir6.1 or -6.2 generate glibenclamide-sensitive K_ATP channels. All three SUR2 short forms lack nucleotide-binding domain 1. The 55 kDa form together with K_ir6.2 generates glibenclamide-insensitive K_ATP currents in cardiac myocytes. SUR2 null mouse cardiac myocytes lack full-length SUR2 but express the 55 kDa form in addition to the other two short forms (37). It is unclear whether wild-type and SUR2 null mouse cerebral artery smooth muscle cells express SUR2 short forms. However, glibenclamide partially reversed H₂S-induced vasodilation in wild-type arteries and H₂S-induced vasodilation was attenuated in SUR2 null arteries. Therefore, our data indicate that H₂S-induced vasodilation occurs because of the activation of K_ATP channels containing full-length SUR2B and K_ir6.1. Whether SUR2 short forms contribute to the glibenclamide-insensitive component of H₂S-induced vasodilation and the H₂S-induced vasodilation present in SUR2 null cerebral arteries remains to be determined.

At pH 7.4, H₂S is permeable to plasma membranes because its solubility in lipophilic solvents is about fivefold greater than in water (47). H₂S dissociates in aqueous solution to make the anionic species HS⁻. Both H₂S and HS⁻ may react with many intracellular targets, including peroxynitrite, peroxide, superoxide, metalloproteins, and cytochrome c (36). Therefore, both H₂S and HS⁻ may participate in cerebral arteriole dilation.

In conclusion, our data indicate that functional plasma membrane K_ATP channels are expressed in newborn piglet cerebral arteriole smooth muscle cells and that H₂S activates cerebral arteriole smooth muscle cell K_ir6.1- and SUR2B-containing K_ATP channels, leading to vasodilation. Our data also suggest that K⁺ channels other than K_ATP also contribute to H₂S-induced vasodilation.

GRANTS

This research was supported by National Heart, Lung, and Blood Institute Grants HL-67061 and HL-94378 (to J. H. Jaggar), HL-34059 and HL42851 (to C. W. Leffler), and HL-0789326 (to E. M. McNally).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).