Hydrogen sulfide activates Ca²⁺ sparks to induce cerebral arteriole dilatation

Abstract

Hydrogen sulfide (H₂S) is a gaseous vasodilator produced by endothelial cells. Mechanisms by which H₂S induces vasodilatation are unclear. We tested the hypothesis that H₂S dilates cerebral arterioles by modulating local and global intracellular Ca²⁺ signals in smooth muscle cells. High-speed confocal imaging revealed that Na₂S, an H₂S donor, increased Ca²⁺ spark frequency ∼1.43-fold and decreased global intracellular Ca²⁺ concentration ([Ca²⁺]_i) by ∼37 nm in smooth muscle cells of intact piglet cerebral arterioles. In contrast, H₂S did not alter Ca²⁺ wave frequency. In voltage-clamped (−40 mV) cells, H₂S increased the frequency of iberiotoxin-sensitive, Ca²⁺ spark-induced transient Ca²⁺-activated K⁺ (K_Ca) currents ∼1.83-fold, but did not alter the amplitude of these events. H₂S did not alter the activity of single K_Ca channels recorded in the absence of Ca²⁺ sparks in arteriole smooth muscle cells. H₂S increased SR Ca²⁺ load ([Ca²⁺]_SR), measured as caffeine (10 and 20 mm)-induced [Ca²⁺]_i transients, ∼1.5-fold. H₂S hyperpolarized (by ∼18 mV) and dilated pressurized (40 mmHg) cerebral arterioles. Iberiotoxin, a K_Ca channel blocker, reduced H₂S-induced hyperpolarization by ∼51%. Iberiotoxin and ryanodine, a ryanodine receptor channel inhibitor, reduced H₂S-induced vasodilatation by ∼38 and ∼37%, respectively. In summary, our data indicate that H₂S elevates [Ca²⁺]_SR, leading to Ca²⁺ spark activation in cerebral arteriole smooth muscle cells. The subsequent elevation in transient K_Ca current frequency leads to membrane hyperpolarization, a reduction in global [Ca²⁺]_i and vasodilatation.

Key points

• Hydrogen sulfide (H₂S), a gas produced by endothelial cells, relaxes smooth muscle cells within the vascular wall to increase organ blood flow and lower systemic blood pressure.

• Mechanisms by which H₂S produces vasodilatation in the cerebral circulation are unclear.

• We demonstrate that H₂S increases the quantity of calcium ions (Ca²⁺) contained with the sarcoplasmic reticulum (SR), the intracellular Ca²⁺ store, of cerebral arteriole smooth muscle cells.

• This elevation in SR Ca²⁺ stimulates the generation of local intracellular Ca²⁺ signals called Ca²⁺ sparks, which in turn activate Ca²⁺-sensitive potassium (K_Ca) channels on the cell membrane, leading to membrane hyperpolarization and vasodilatation.

• Elucidating this novel mechanism of H₂S-induced vasodilatation is important to better understand physiological control of blood flow within the brain.

Introduction

Hydrogen sulfide (H₂S), a physiological gasotransmitter, is generated in mammalian cells through the metabolism of l-cysteine by cystathionine β-synthase and cystathionine γ-lyase (Porter et al. 1974; Allsop & Watts, 1975; Wang, 2002). H₂S modulates blood flow, blood pressure, synaptic neurotransmission, immune response, hormone secretion and muscle relaxation (Fiorucci et al. 2005; Leffler et al. 2006; Austgen et al. 2011). H₂S induces vasodilatation in many different vascular beds, including rat mesenteric arteries, aorta, tibial arteries and piglet cerebral arterioles (Cheng et al. 2004; Liu & Bian, 2010; Schleifenbaum et al. 2010; Leffler et al. 2011; Liang et al. 2011). Several vascular ion channels have been reported to be involved in H₂S-induced vasodilatation, including ATP-sensitive K⁺ (K_ATP) channels, Ca²⁺-activated K⁺ (K_Ca) channels, KCNQ channels and L-type Ca²⁺ channels (Cheng et al. 2004; Dombkowski et al. 2004; Schleifenbaum et al. 2010; Leffler et al. 2011; Zuidema et al. 2010; Liang et al. 2011). However, smooth muscle cell ion channels that are specifically targeted by H₂S are unclear as are the mechanisms by which H₂S modulates these proteins.

In smooth muscle cells, ion channels generate and regulate local and global intracellular Ca²⁺ signals, which can control vascular contractility (Jaggar et al. 2000). Conversely, local and global intracellular Ca²⁺ signals can regulate the activity of plasma membrane ion channels, which feed back to modify local and global intracellular Ca²⁺ signalling (Jaggar et al. 2000). The regulation of local and global Ca²⁺ signals by H₂S in vascular smooth muscle cells is unclear. Establishing such regulation may reveal mechanisms by which this gaseous vasodilator controls vascular contractility.

Three primary Ca²⁺ signals occur in arterial smooth muscle cells, termed Ca²⁺ sparks, Ca²⁺ waves and global [Ca²⁺]_i (Jaggar et al. 2000). Ca²⁺ sparks occur due to the concerted opening of multiple sarcoplasmic reticulum (SR) ryanodine receptor (RyR) channels (Nelson et al. 1995; Jaggar et al. 2000). Ca²⁺ sparks activate nearby plasma membrane K_Ca channels, leading to transient K_Ca currents that hyperpolarize the membrane potential. Membrane hyperpolarization reduces voltage-dependent Ca²⁺ channel activity, leading to a reduction in global intracellular Ca²⁺ concentration and vasodilatation (Nelson et al. 1995). Ca²⁺ waves are propagating SR Ca²⁺ release events that occur due to the activation of SR inositol trisphosphate-gated Ca²⁺ release channels and RyR channels (Jaggar, 2007). Global [Ca²⁺]_i is the spatially homogeneous [Ca²⁺]_i to which plasma membrane Ca²⁺ influx and SR Ca²⁺ release can contribute (Jaggar et al. 2000). Voltage-dependent L-type Ca²⁺ (Ca_V1.2) channels are a major contributor to global [Ca²⁺]_i (Jaggar et al. 2000). Local Ca²⁺ gradients, termed Ca²⁺ sparklets, are generated by the opening of voltage-dependent Ca²⁺ channels and contribute to global [Ca²⁺]_i (Santana & Navedo, 2009).

Here, we investigated the regulation of local and global [Ca²⁺]_i signals by H₂S in cerebral arteriole smooth muscle cells, the underlying mechanisms, and the involvement in H₂S-induced cerebral arteriole dilatation. Data indicate that H₂S elevates [Ca²⁺]_SR, which stimulates Ca²⁺ sparks, leading to an increase in transient K_Ca current frequency, a reduction in global [Ca²⁺]_i and vasodilatation. In contrast, H₂S did not alter the activity of Ca²⁺ waves or directly regulate single K_Ca channels. These data define a novel mechanism of action of H₂S and indicate that an elevation in SR Ca²⁺ load ([Ca²⁺]_SR)is a specific mechanism of vasodilatation induced by this gasotransmitter.

Methods

Tissue preparation

All procedures used were approved by the University of Tennessee Health Science Center Animal Care and Use Committee. The authors have read, and the experiments comply with the policies and regulations of The Journal of Physiology given by Drummond (2009). Newborn pigs (1–3 days old, 1–2.5 kg body weight) were anaesthetized with ketamine hydrochloride (33 mg kg⁻¹i.m.) and acepromazine (3.3 mg kg⁻¹i.m.). The brain was removed and maintained in ice-cold (4°C) Hepes-buffered physiological saline solution (PSS) containing (in mm): 134 NaCl, 6 KCl, 2 CaCl₂, 1 MgCl₂, 10 Hepes, and 10 glucose, with pH adjusted to 7.4 with NaOH. Isolated arterioles (50–200 μm) were dissected from the brain and cleaned to remove basolateral connective tissue. Individual smooth muscle cells were dissociated from cerebral arterioles using a procedure similar to that previously described (Jaggar, 2001). Arterioles were endothelium denuded by intraluminal injection of air for 1 min, as previously described (Adebiyi et al. 2008). Unless stated otherwise, all procedures were performed at room temperature (22–25°C).

Confocal Ca²⁺ imaging

Piglet cerebral arterioles (∼2 mm in length) were placed in Hepes-buffered PSS containing 10 μm fluo-4 AM and 0.05% Pluronic F-127 for 20 min at room temperature followed by a 30 min wash with Hepes-buffered PSS to allow indicator deesterification. Bath solution that contained (in mm): 110 NaCl, 30 KCl, 10 Hepes, 2 CaCl₂, 1 MgCl₂, and 10 glucose (adjusted to pH 7.4 with NaOH) was used to depolarize arteries to approximately −40 mV, as previously described (Jaggar, 2001). Fluorescence images were collected using a Noran OZ laser-scanning confocal microscope (Noran Instruments, Middleton, WI, USA) and a ×60 water immersion objective (1.2 NA) attached to a TE300 microscope (Nikon), as described previously (Jaggar, 2001). Fluo-4 AM was illuminated at 488 nm using a krypton–argon laser, and emitted light >500 nm was captured. Images (56.3 × 52.8 μm) were recorded every 16.67 ms (i.e. 60 images per second). Custom analysis software (kindly provided by Dr M. T. Nelson, University of Vermont) was used to detect Ca²⁺ signals in smooth muscle cells. For detection of Ca²⁺ sparks, an area 1.54 × 1.54 μm (7 × 7 pixels, i.e. 2.37 μm²) in each image (F) was divided by a baseline (F₀) that was determined by averaging 10 images without Ca²⁺ spark activity. The entire image area was analysed to detect Ca²⁺ sparks. A Ca²⁺ spark was identified as a local increase in F/F₀ that was >1.2. Arterial Ca²⁺ spark frequency (measured in Hz) was calculated by averaging values from at least two different areas of the same arteriole. Ca²⁺ waves were defined as an elevation in F/F₀ >1.2 that propagated for at least 20 μm. Ca²⁺ waves were detected by placing 2.2 × 2.2 μm (10 × 10 pixels, i.e. 4.84 μm²) in each smooth muscle cell and by using a method similar to that for Ca²⁺ spark detection. Changes in local or global [Ca²⁺]_i were calculated using the pseudoratio method: [Ca²⁺] = KR/(K/([Ca²⁺]_rest+ 1 –R)). K is the apparent affinity of fluo-4 AM for Ca²⁺ (770 nm; Woodruff et al. 2002), R is the fractional fluorescence increase (F/F₀), and [Ca²⁺]_rest is [Ca²⁺]_i at F₀. [Ca²⁺]_rest was 224 nm, as previously determined by ratiometric imaging of fura-2 in newborn cerebral arterioles (Li et al. 2006). Global [Ca²⁺]_i fluorescence was calculated from the same images used for Ca²⁺ spark and wave analysis and was the mean pixel value of 100 different images acquired during a 10 s period (Cheranov & Jaggar, 2004; Xi et al. 2005; Cheranov & Jaggar, 2006).

Patch-clamp electrophysiology

Isolated smooth muscle cells were allowed to adhere to a glass coverslip in the bottom of a chamber for 10 min before experimentation. K⁺ currents were measured using the amphotericin B perforated-patch configuration with an Axopatch 200B amplifier (Axon Instruments, Union City, CA, USA). The bath solution was Hepes-buffered PSS, and the pipette solution contained (in mm): 110 potassium aspartate, 30 KCl, 10 NaCl, 1 MgCl₂, 10 Hepes, and 0.05 EGTA, with pH adjusted to 7.2 using KOH. Membrane currents were filtered at 1 kHz and digitized at 4 kHz. In each patch under each condition, transient K_Ca current frequency and amplitude or K_Ca channel activity was calculated from at least 5 min of continuous gap-free data.

Sarcoplasmic reticulum Ca²⁺ load measurements

[Ca²⁺]_SR was estimated by measuring the amplitude of [Ca²⁺]_i transients induced by caffeine (10 or 20 mm), a RyR channel activator (Cheranov & Jaggar, 2002, 2004; Xi et al. 2005). Endothelium-denuded arterioles were incubated in Hepes-buffered PSS containing 5 μm fura-2 AM and 0.1% Pluronic F-127 for 30 min at room temperature. After wash, arteries were allowed to de-esterify the indicator for 15 min. Fura-2 AM was alternately excited with 340 or 380 nm light using a xenon arc lamp and a personal computer-driven hyperswitch (Ionoptix, Milton, MA, USA). Background corrected ratios were collected every 1 s at 510 nm using a Dage MTI integrating CCD camera (IonOptix).

Pressurized artery diameter measurements

A cerebral arteriole segment ∼2 mm in length was cannulated at each end in a temperature-controlled perfusion chamber (Living Systems Instrumentation, Burlington, VT, USA). The chamber was continuously perfused with PSS of the following composition (mm): 112 NaCl, 4.8 KCl, 24 NaHCO₃, 1.8 CaCl₂, 1.2 MgSO₄, 1.2 KH₂PO₄ and 10 glucose, equilibrated with a mixture of 21% O₂, 5% CO₂ and 74% N₂, and maintained at 35°C. 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. Arterioles were observed with a charge-coupled device camera attached to an inverted microscope (Nikon TS 100). Arteriole diameter was measured by using the automatic edge-detection function of IonWizard software (Ionoptix, Milton, MA, USA) and digitized at 1 Hz using a personal computer. Tested compounds were applied via chamber perfusion to maintain constant concentration during experiments.

Pressurized artery membrane potential measurements

Arterioles were maintained at 40 mmHg for 2 h to ensure steady-state myogenic tone had occurred prior to obtaining membrane potential recordings. Membrane potential was measured by impaling arterioles with glass microelectrodes filled with 3 m KCl (50–90 mΩ) from the adventitial side. Membrane potential was recorded using a WPI FD223 amplifier, pCLAMP 9.2 software (Molecular Devices) and a personal computer. Successful intracellular impalements required a rapid negative potential change upon insertion; a stable voltage for at least 25 s; a fast positive voltage deflection upon removal, and a <10% change in tip resistance after impalement.

Statistical analysis

Values are reported as means ± SEM; n refers to the number of events analysed, unless otherwise specified. Student's t test was used for comparison of paired data, except for statistical analysis of Ca²⁺ spark frequency and amplitude and Ca²⁺ wave frequency, where a non-parametric Wilcoxon's matched pairs test was used. ANOVA with the Student–Newman–Keuls post hoc test was used for multiple group comparison. P < 0.05 was considered significant.

Chemicals

Fluo-4 AM, fura-2 AM and Pluronic F-127 were purchased from Molecular Probes (Eugene, OR, USA). Iberiotoxin was purchased from California Peptide Research Inc. (Napa, CA, USA). Unless otherwise stated, all other chemicals were obtained from Sigma Chemical (St Louis, MO, USA).

Results

H₂S activates Ca²⁺ sparks and reduces global [Ca²⁺]_i in smooth muscle cells of intact cerebral arterioles

The regulation of local and global Ca²⁺ signals by H₂S was measured in smooth muscle cells of piglet cerebral arteriole segments. For this study, we used Na₂S as an H₂S donor. Na₂S (10 μm) increased mean Ca²⁺ spark frequency from ∼2.8 Hz to 4.0 Hz, or 1.43-fold (Fig. 1A and B). In contrast, H₂S did not change mean Ca²⁺ spark amplitude or Ca²⁺ wave frequency (Fig. 1C and D). H₂S decreased mean global F/F₀ by ∼16%, which translates to a reduction in global intracellular Ca²⁺ concentration [Ca²⁺]_i from 224 ± 29 nm (Li et al. 2006) to 187 ± 12 nm (Fig. 1E). These data indicate that H₂S elevates Ca²⁺ spark frequency and reduces global [Ca²⁺]_i, but does not alter Ca²⁺ waves in cerebral arteriole smooth muscle cells.

Figure 1. Hydrogen sulfide (H₂S) regulates local and global Ca²⁺ signals in cerebral arteriole smooth muscle cells.

A, confocal images in upper panels illustrate average fluo-4 fluorescence in smooth muscle cells in the same arteriole in control and Na₂S (10 μm). White boxes illustrate locations where sparks occurred during 10 s of imaging. Coloured boxes illustrate locations from where corresponding normalized fluorescence (F/F₀) over time traces from each cell shown in the lower panels were determined. Middle panels show two representative Ca²⁺ sparks that occurred at locations labelled as a and b in each condition. Lower panels indicate intracellular Ca²⁺ changes that occurred in corresponding coloured boxes over time with Ca²⁺ waves evident in a proportion of cells. H₂S increased Ca²⁺ spark frequency (B), but did not change Ca²⁺ spark amplitude (C) (n = 8 for each condition). D, H₂S did not alter Ca²⁺ wave frequency (n = 8 for each condition). E, H₂S decreased global [Ca²⁺]_i from 224 ± 29 nm to 187 ± 12 nm (n = 8). *P < 0.05 vs. control.

H₂S activates transient K_Ca currents in isolated arteriole smooth muscle cells

Ca²⁺ sparks activate transient K_Ca currents in cerebral artery and arteriole smooth muscle cells (Jaggar et al. 2000, 2002). H₂S regulation of Ca²⁺ spark-induced transient K_Ca currents was measured in isolated cerebral arteriole smooth muscle cells using the perforated patch-clamp configuration. At a membrane potential of −40 mV, Na₂S (10 μm) increased mean transient K_Ca current frequency from ∼0.23 to 0.42 Hz, or 1.83-fold (Fig. 2A and B). In contrast, Na₂S did not alter mean transient K_Ca current amplitude (Fig. 2A and C). To further investigate Na₂S regulation of transient K_Ca current amplitude, events were divided into small (<25 pA), medium (25–50 pA) and large (>50 pA) amplitude groups, as we have done previously (Li et al. 2008). Na₂S did not significantly alter the amplitude distribution of these groups (Fig. 2A and D). When applied in the presence of Na₂S, iberiotoxin, a selective K_Ca channel blocker, essentially abolished transient K_Ca currents, reducing mean frequency and amplitude to ∼0.5 and 10.2% of that in Na₂S (Fig. 2B and C). These data indicate that H₂S-induced Ca²⁺ spark activation leads to an elevation in transient K_Ca current frequency, but does not alter transient K_Ca current amplitude, in cerebral arteriole smooth muscle cells.

Figure 2. H₂S increases transient K_Ca current frequency in cerebral arteriole smooth muscle cells.

A, representative recording illustrating Na₂S (10 μm) induced an elevation in transient K_Ca current frequency, but not amplitude. B, mean transient K_Ca current frequency in the same cells in control (n = 11 cells) and Na₂S (10 μm, n = 11 cells), and in Na₂S+iberiotoxin (IBTX, 100 nm, n = 5 cells). C, transient K_Ca current mean amplitude was not altered by Na₂S (n = 11 cells), but was reduced by iberiotoxin (IBTX, n = 5 cells). D, Na₂S did not change the proportion of transient K_Ca currents in each transient K_Ca current amplitude category (n = 11 cells for control and Na₂S). All currents were recorded at a steady holding potential of −40 mV. *P < 0.05 vs. control. #P < 0.05 vs. Na₂S.

H₂S does not directly activate K_Ca channels in cerebral arteriole smooth muscle cells

In piglet cerebral arteriole smooth muscle cells, a proportion (∼40%) of Ca²⁺ sparks do not activate a transient K_Ca current (Jaggar et al. 2002). Carbon monoxide, a gasotransmitter that elevates K_Ca channel Ca²⁺ sensitivity induces coupling of K_Ca channels to these smaller amplitude Ca²⁺ sparks (Jaggar et al. 2002; Xi et al. 2004; Li et al. 2006). To investigate the possibility that H₂S stimulates transient K_Ca currents through an effect on K_Ca channels, the regulation of single K_Ca channels by H₂S was measured in isolated cerebral arteriole smooth muscle cells. Thapsigargin (100 nm), a SR Ca²⁺-ATPase (SERCA) inhibitor, was used to deplete [Ca²⁺]_SR and abolish transient K_Ca currents. From a control activity (NP_o) of 0.35 ± 0.1, H₂S did not alter K_Ca channel activity (95 ± 17% of control, P > 0.05; Fig. 3). These data indicate that H₂S does not activate K_Ca channels, further supporting other data here that H₂S activates K_Ca channels solely by elevating Ca²⁺ spark frequency.

Figure 3. H₂S does not alter single K_Ca channel activity in intact arteriole smooth muscle cells.

A, representative trace illustrating that H₂S did not change single K_Ca channel activity in an arteriole smooth muscle cell. B, mean data (n = 6 cells). All currents were recorded at a steady holding potential of 0 mV. Cells were pre-treated with thapsigargin (100 nm) to inhibit Ca²⁺ spark-induced transient K_Ca currents.

Na₂S elevates sarcoplasmic reticulum Ca²⁺ load

[Ca²⁺]_SR regulates Ca²⁺ spark frequency (Wellman et al. 2001; Cheranov & Jaggar, 2002). Therefore, the regulation of [Ca²⁺]_SR by H₂S was studied by measuring the amplitude of caffeine-induced [Ca²⁺]_i transients in endothelium-denuded piglet cerebral arterioles. Caffeine (10 mm)-stimulated [Ca²⁺]_i transients under control conditions (Fig. 4A and B). Na₂S increased caffeine (10 mm)-induced [Ca²⁺]_i transients to 161 ± 13% (1st application) and 153 ± 11% (2nd application) of control (n = 5 arterioles, P < 0.05; Fig. 4). Conceivably, Na₂S may elevate caffeine-induced [Ca²⁺]_i transients by increasing RyR caffeine sensitivity. To investigate this possibility, experiments were repeated using a higher caffeine concentration (20 mm). Na₂S similarly increased caffeine (20 mm)-induced [Ca²⁺]_i transients to 149 ± 14% (1st application) and 167 ± 12% (2nd application) of control (n = 5 arterioles, P < 0.05). Data were not significantly different when comparing the first and second caffeine applications at 10 and 20 mm (P > 0.05 for each). These data indicate that H₂S elevates [Ca²⁺]_SR.

Figure 4. H₂S elevates [Ca²⁺]_SR.

A, original trace illustrating that Na₂S (10 μm) increased caffeine (Caff, 10 mm)-induced [Ca²⁺]_i transients in an isolated piglet cerebral arteriole. 340/380, 340 to 380 nm ratio. B, mean change in fura-2 AM ratio in two sequential control conditions and Na₂S (n = 5 arterioles). *P < 0.05 vs. 1st caffeine-induced [Ca²⁺]_i transient.

H₂S induces iberiotoxin-sensitive hyperpolarization in pressurized cerebral arterioles

To investigate the functional significance of H₂S-induced transient K_Ca current activation, the membrane potential of pressurized arterioles was measured using glass microelectrodes. At an intravascular pressure of 40 mmHg, mean arteriole membrane potential was ∼−30.3 mV (Fig. 5A and B). Na₂S (10 μm) hyperpolarized pressurized arterioles to ∼−47.9 mV, or by 17.6 mV (Fig. 5A and B). Application of iberiotoxin, a selective K_Ca channel blocker, in the presence of Na₂S returned mean membrane potential to ∼−39.0 mV, inhibiting the Na₂S-induced hyperpolarization by ∼51% (Fig. 5A and B).

Figure 5. H₂S induces iberiotoxin-sensitive hyperpolarization in pressurized arterioles.

A, original recordings of membrane potential upon microelectrode impalement in pressurized (40 mmHg) arterioles in control, Na₂S (10 μm) and Na₂S (10 μm) + iberiotoxin (100 nm). Dotted line indicates 0 mV. Scale bars are 20 s. B, mean data (control, n = 6; Na₂S, n = 5; Na₂S + iberiotoxin, n = 5). *P < 0.05 when compared with control and # when compared with Na₂S.

H₂S dilates cerebral arterioles by activating RyR and K_Ca channels

Edge-detection myography was performed to study contractility regulation by H₂S and involvement of Ca²⁺ spark and K_Ca channel activation in pressurized arterioles. At 40 mmHg, arterioles constricted from a mean passive diameter of 231 ± 10 μm to a myogenic diameter of 132 ± 8 μm, or by 43% (n = 11). Na₂S (10 μm) increased mean arteriole diameter by ∼33 μm (Fig. 6A and C). Iberiotoxin (100 nm) or ryanodine, a RyR channel blocker (10 μm), did not alter the diameter of pressurized arterioles when applied alone (Fig. 6B and D). In contrast, iberiotoxin partially reversed Na₂S-induced vasodilatation from ∼32 to 20 μm, or by 38% (Fig. 6A and B). Ryanodine also partially reversed Na₂S-induced vasodilatation from ∼35 to 22 μm, or by 37% (Fig. 6C and D). These data indicate that H₂S-induced cerebral arteriole vasodilatation occurs in part via Ca²⁺ spark-induced K_Ca channel activation in piglet cerebral arteriole smooth muscle cells.

Figure 6. H₂S dilates pressurized (40 mmHg) piglet cerebral arterioles via RyR and K_Ca channel activation.

A, representative trace illustrating that iberiotoxin (IBTX, 100 nm), partially reverses vasodilatation induced by Na₂S (10 μm). B, mean data (IBTX, n = 5; Na₂S and Na₂S + IBTX, n = 9 for each). C, representative trace illustrating that ryanodine (Rya, 10 μm) partially reverses vasodilatation induced by Na₂S (10 μm). D, mean data (Rya, Na₂S, and Na₂S + Rya, n = 6 for each). *P < 0.05 vs. Na₂S.

Discussion

Here, we investigated the regulation of local and global intracellular Ca²⁺ signals by H₂S in smooth muscle cells of piglet cerebral arterioles. Major findings of this study are that (1) H₂S activates Ca²⁺ sparks but not Ca²⁺ waves, and reduces global intracellular Ca²⁺ concentration ([Ca²⁺]_i), (2) H₂S activation of Ca²⁺ sparks leads to an increase in transient K_Ca current frequency, (3) H₂S increases [Ca²⁺]_SR, (4) H₂S does not directly activate K_Ca channels, (5) H₂S hyperpolarizes pressurized cerebral arterioles via K_Ca channel activation, and (6) H₂S dilates pressurized cerebral arterioles and this dilatation is partially reversed by RyR and K_Ca channel blockers. Collectively, these data indicate that H₂S elevates [Ca²⁺]_SR, which stimulates Ca²⁺ sparks that increase transient K_Ca currents, leading to membrane hyperpolarization, a reduction in global [Ca²⁺]_i and vasodilatation.

H₂S is generated endogenously in a wide variety of mammalian tissues, including brain, liver, heart, aorta and kidney (Ubuka, 2002). Physiological concentrations of H₂S in rat and human blood are between 5 and 50 μm (Zhao et al. 2001; Ubuka, 2002; Li et al. 2005; Elsey et al. 2010). In piglet cerebrospinal fluid, we measured a mean H₂S concentration during stimulation with hypercapnia of approximately 4 μm (Leffler et al. 2011). Here, we used Na₂S, a commonly used H₂S donor, to elevate extracellular H₂S. Recent phase 1 human clinical trials have also used Na₂S as a potential therapeutic treatment for myocardial infarction (Li et al. 2009). Na₂S at 10 μm generates ∼5 μm H₂S gas in physiological saline solution at room temperature within 1 min (Liang et al. 2011). Therefore, the Na₂S concentration studied here produces H₂S concentrations within the physiological range. In donating H₂S, Na₂S contributes only micromolar Na⁺ as an additional atom. Bath solutions used for our experiments contain 140 mm Na⁺. A 10 μm Na⁺ elevation will have insignificant effects on Ca²⁺ sparks, transient K_Ca currents and arteriole contractility. We have previously shown that Na₂S and NaHS, another H₂S donor, produce quantitatively similar K_ATP current activation in cerebral arteriole smooth muscle cells, indicating that these donors act through H₂S donation (Liang et al. 2011).

In arterial smooth muscle cells, a single Ca²⁺ spark activates multiple K_Ca channels, leading to a transient K_Ca current (Nelson et al. 1995). Asynchronous transient K_Ca currents hyperpolarize arterial membrane potential, thereby reducing voltage-dependent Ca²⁺ channel activity and [Ca²⁺]_i, leading to vasodilatation (Jaggar et al. 2000). Several vasodilators, including those that activate adenylyl cyclase or soluble guanylyl cyclase, activate Ca²⁺ sparks (Porter et al. 1998; Cheranov & Jaggar, 2006; Mandala et al. 2007; Li et al. 2008). Such Ca²⁺ spark stimulation elevates transient K_Ca current frequency, leading to membrane hyperpolarization, a reduction in voltage-dependent Ca²⁺ channel activity, a decrease in [Ca²⁺]_i, and vasodilatation (Jaggar & Nelson, 2000; Cheng & Lederer, 2008). Here, we provide the first direct evidence that H₂S activates Ca²⁺ sparks in arteriole smooth muscle cells. H₂S increased Ca²⁺ spark frequency, but did not alter Ca²⁺ spark amplitude. Consistent with this result, H₂S increased mean transient K_Ca current frequency, but did not alter mean amplitude. We have previously demonstrated that transient K_Ca current amplitude is not normally distributed in piglet cerebral arteriole smooth muscle cells (Li et al. 2008). Therefore, H₂S-induced changes in transient K_Ca current amplitude may not be revealed by comparing mean data. Previous studies from our laboratory indicated that although carbon monoxide (CO) elevates the effective coupling of K_Ca channels to Ca²⁺ sparks, astrocyte-derived CO did not alter transient K_Ca current mean amplitude (Jaggar et al. 2002; Xi et al. 2004; Li et al. 2008). Categorizing transient K_Ca currents into three amplitude groups indicated that CO increased the frequency of smaller amplitude transient K_Ca currents more than larger events, thereby depressing the mean amplitude (Li et al. 2008). The emergence of a new population of transient K_Ca currents occurs due to CO-induced coupling of previously uncoupled smaller amplitude Ca²⁺ sparks to K_Ca channels (Jaggar et al. 2002). Similar to our previous study, data here indicate that the largest proportion of transient K_Ca currents are smaller events (<25 pA) in cerebral arteriole smooth muscle cells (Li et al. 2008). In contrast to CO, which elevates K_Ca channel sensitivity to Ca²⁺ sparks (Jaggar et al. 2002; Li et al. 2008), H₂S did not alter transient K_Ca current amplitude distribution. These data indicate that H₂S does not elevate effective coupling of K_Ca channels to Ca²⁺ sparks. Consistent with a lack of effect of H₂S on transient K_Ca current mean amplitude or amplitude distribution, H₂S also did not alter single K_Ca channel activity in intact arteriole smooth muscle cells. These data provide further support for our finding that H₂S activates transient K_Ca currents specifically by elevating Ca²⁺ spark frequency. In contrast to our findings, NaHS, another H₂S donor, at 0.1–1 mm, had no effect on transient K_Ca currents, but inhibited transient K_Ca currents at a very high concentration (4 mm) in guinea-pig gastric myocytes (Zhao et al. 2009).

H₂S has been reported to both activate and inhibit K_Ca channels. Previous studies indicated that H₂S activated K_Ca channels in different tissues (Sitdikova et al. 2010; Zuidema et al. 2010; Jackson-Weaver et al. 2011). Iberiotoxin blocked H₂S-induced vasodilatation in rat mesenteric arteries, decreased H₂S-induced K_Ca channel activation in rat pituitary tumour cell outside-out patches, and reversed an H₂S-induced elevation in whole-cell K_Ca current in cultured human microvascular endothelial cells (Sitdikova et al. 2010; Zuidema et al. 2010; Jackson-Weaver et al. 2011). In contrast, H₂S inhibited carotid body glomus cell K_Ca channels and recombinant K_Ca channels expressed in human embryonic kidney cells (Telezhkin et al. 2009; Li et al. 2010). Explanations for these different observations may include channel subunit composition, including whether and which β subunit isoforms are present and the α:β ratio, cell type and species studied, H₂S concentrations used, and whether experiments were performed in intact cells or inside-out membrane patches, which may modify pH and oxidation state, and thus the proportion of H₂S to HS⁻. It is also important to note that we have shown 300 μm and 1 mm Na₂S induce an alkaline shift in pH from 7.4 to 7.54 and 7.75, respectively, in physiological saline solution (Liang et al. 2011). In contrast, lower concentrations of H₂S donors, similar to those used here, do not alter pH. Therefore, pH alterations may underlie results in studies where high concentrations of H₂S donor or H₂S gas have been used. Of note, alkaline pH shifts Ca²⁺ sparks to waves in arterial smooth muscle cells and has been demonstrated to induce vasoconstriction (Austin & Wray, 1993; Heppner et al. 2002). Intracellular acidification also inhibits K_Ca channels by reducing Ca²⁺ sensitivity and open time in rabbit tracheal smooth muscle cells (Kume et al. 1990).

An elevation in [Ca²⁺]_SR increases Ca²⁺ spark frequency in cardiac myocytes and smooth muscle cells (Satoh et al. 1997; Cheranov & Jaggar, 2002). To examine mechanisms by which H₂S increased Ca²⁺ spark frequency, we measured H₂S regulation of [Ca²⁺]_SR. Na₂S increased the amplitude of [Ca²⁺]_i transients produced by two different millimolar caffeine concentrations similarly. These data indicate that Na₂S elevates [Ca²⁺]_SR. In contrast, Na₂S did not alter global [Ca²⁺]_i in these experiments. This is expected, because for these experiments the bath solution contained 6 mm K⁺, in contrast to the experiments shown in Fig. 1 where the bath solution contained 30 mm K⁺ to depolarize smooth muscle cells to physiological voltage. In 6 mm K⁺, unpressurized arterial potential is ∼−60 mV, and global [Ca²⁺]_i, Ca²⁺ spark frequency, and K_Ca channel activity is low. Under this condition, an Na₂S-induced elevation in Ca²⁺ sparks and thus, K_Ca channel activity would not be expected to further reduce global [Ca²⁺]_i. These data indicate that an Na₂S-induced elevation in [Ca²⁺]_SR stimulates an increase in Ca²⁺ spark frequency, and thus transient K_Ca current frequency. Mechanisms by which H₂S elevates [Ca²⁺]_SR are likely to involve SERCA activation. Phospholamban is an endogenous inhibitor of SERCA (Hutter et al. 2002; Toyoshima et al. 2003). Protein kinase C, cAMP/cGMP-dependent protein kinases and Ca²⁺–calmodulin-dependent protein kinase II phosphorylate phospholamban, leading to an increase in SERCA activity (Wegener et al. 1986; Simmerman et al. 1986; Karczewski et al. 1997). H₂S can also S-sulfhydrate cysteine residues, including in K_ATP channels, which elevates channel activity (Mustafa et al. 2011). Conceivably, H₂S may activate SERCA through direct sulfhyrdration or via regulation of an upstream SERCA regulator, including phospholamban. It is possible that H₂S also activates RyR channels and thus, Ca²⁺ sparks, by a SR Ca²⁺ load-independent mechanism. Future studies will investigate further the mechanisms by which H₂S activates RyR channels to elevate Ca²⁺ spark frequency.

Consistent with H₂S-induced Ca²⁺ spark and transient K_Ca current activation, our data indicate that H₂S hyperpolarizes pressurized arterioles and reduces global [Ca²⁺]_i in arteriole smooth muscle cells. These data are consistent with the established concept that transient K_Ca current activation leads to membrane hyperpolarization, which reduces voltage-dependent Ca²⁺ channel activity, and therefore, global [Ca²⁺]_i (Jaggar & Nelson, 2000; Cheng & Lederer, 2008). Recent evidence indicated that NaHS, an H₂S donor, produced iberiotoxin-sensitive membrane hyperpolarization in mesenteric arteries (Jackson-Weaver et al. 2011). In contrast, NaHS hyperpolarized coronary arteries via a 4-aminopyridine-sensitive mechanism (Cheang et al. 2010). Here, H₂S activated Ca²⁺ sparks and reduced global [Ca²⁺]_i, but did not alter Ca²⁺ waves. RyR channels can contribute to Ca²⁺ waves in arterial smooth muscle cells (Jaggar & Nelson, 2000). Conceivably, effects of H₂S on Ca²⁺ waves may be concentration dependent, with higher non-physiological H₂S concentrations, which can induce an alkaline shift in pH, stimulating RyR channels sufficiently to increase Ca²⁺ wave frequency (Heppner et al. 2002).

In pressurized piglet cerebral arterioles, iberiotoxin or ryanodine alone did not alter arterial diameter. This finding is consistent with previous measurements performed in vitro and in vivo indicating that baseline Ca²⁺ spark and K_Ca channel activity are too low in piglet cerebral arteriole smooth muscle cells to oppose pressure-induced vasoconstriction (Jaggar et al. 2002; Ahmed et al. 2004; Kanu & Leffler, 2007). We show that H₂S produces hyperpolarization and vasodilatation in pressurized arterioles. Iberiotoxin partially reversed H₂S-induced hyperpolarization and iberiotoxin and ryanodine both attenuated H₂S-induced vasodilatation. These data indicate that H₂S stimulates Ca²⁺ sparks that activate K_Ca channels, leading to hyperpolarization and vasodilatation. We have previously demonstrated that H₂S-induced vasodilatation in pressurized piglet cerebral arterioles is also partially inhibited by glibenclamide, a K_ATP channel-specific inhibitor (Liang et al. 2011). Therefore, our data indicate that H₂S dilates by activating both K_Ca and K_ATP channels in smooth muscle cells. Our previous in vivo study showed that glibenclamide can completely block H₂S (10 μm)-induced piglet pial arteriole dilatation in cranial window experiments (Leffler et al. 2011). The greater effect of glibenclamide in vivo could be explained by different experiment conditions and potential impact of other cells. The cranial windows are surgically implanted and closed. Under cranial window, cerebral arterioles are in contact with meninges, neurons and particularly astrocytes. Topically applied H₂S may affect not only pial arterioles but also astrocytes or neurons. Those cells also could affect pial arterial dilatation. Oxygen partial pressure is ∼40 mmHg in cranial window experiments, but ∼150 mmHg in the pressurized arterial experiments. Finally, the pressurized arterioles in the present study had mean diameters of ∼150 μm, while the arterioles measured in vivo were much smaller at ∼50 μm. Any or all of these differences may allow detection of a role for K_Ca channels in dilatation to H₂S that was not detected in the previous study in vivo. Collectively, these data indicate that H₂S can dilate piglet cerebral arterioles via the activation of both K_Ca and K_ATP channels, the former mediated through Ca²⁺ spark activation. Recent evidence indicates that K_ATP channels are the primary vasodilatory target of H₂S in non-pressurized mouse and rat aorta and mesenteric arteries (Cheng et al. 2004; Mustafa et al. 2011). Therefore, vascular origin, size, age and species may underlie different vasodilatory mechanisms for H₂S and specific ion channels targeted.

In summary, we demonstrate that H₂S elevates [Ca²⁺]_SR, which stimulates Ca²⁺ sparks, leading to an increase in transient K_Ca current frequency in cerebral arteriole smooth muscle cells. The H₂S-induced elevation in K_Ca channel activity produces membrane hyperpolarization that reduces global [Ca²⁺]_i, leading to vasodilatation.

Glossary

Abbreviations

RyR

ryanodine receptor

SERCA

SR Ca²⁺-ATPase

SR

sarcoplasmic reticulum

Author contributions

G.H.L. performed and analysed experiments, contributed to study design and wrote the manuscript; Q.X. performed experiments; J.H.J. and C.W.L. designed the study and wrote the manuscript. All authors approved the final version.