Intermittent hypoxia in rats reduces activation of Ca²⁺ sparks in mesenteric arteries

This study provides the first evidence that hydrogen sulfide (H₂S) increases Ca²⁺ spark activity through an endothelium-dependent effect to contribute to H₂S-induced dilation. Additionally, it appears that endothelial H₂S is required for depolarization-induced activation of Ca²⁺ sparks in intact arteries.

Abstract

Ca⁺ sparks are vascular smooth muscle cell (VSMC) Ca²⁺-release events that are mediated by ryanodine receptors (RyR) and promote vasodilation by activating large-conductance Ca²⁺-activated potassium channels and inhibiting myogenic tone. We have previously reported that exposing rats to intermittent hypoxia (IH) to simulate sleep apnea augments myogenic tone in mesenteric arteries through loss of hydrogen sulfide (H₂S)-induced dilation. Because we also observed that H₂S can increase Ca²⁺ spark activity, we hypothesized that loss of H₂S after IH exposure reduces Ca²⁺ spark activity and that blocking Ca²⁺ spark generation reduces H₂S-induced dilation. Ca²⁺ spark activity was lower in VSMC of arteries from IH compared with sham-exposed rats. Furthermore, depolarizing VSMC by increasing luminal pressure (from 20 to 100 mmHg) or by elevating extracellular [K⁺] increased spark activity in VSMC of arteries from sham rats but had no effect in arteries from IH rats. Inhibiting endogenous H₂S production in sham arteries prevented these increases. NaHS or phosphodiesterase inhibition increased spark activity to the same extent in sham and IH arteries. Depolarization-induced increases in Ca²⁺ spark activity were due to increased sparks per site, whereas H₂S increases in spark activity were due to increased spark sites per cell. Finally, inhibiting Ca²⁺ spark activity with ryanodine (10 μM) enhanced myogenic tone in arteries from sham but not IH rats and blocked dilation to exogenous H₂S in arteries from both sham and IH rats. Our results suggest that H₂S regulates RyR activation and that H₂S-induced dilation requires Ca²⁺ spark activation. IH exposure decreases endogenous H₂S-dependent Ca²⁺ spark activation to cause membrane depolarization and enhance myogenic tone in mesenteric arteries.

NEW & NOTEWORTHY

This study provides the first evidence that hydrogen sulfide (H₂S) increases Ca²⁺ spark activity through an endothelium-dependent effect to contribute to H₂S-induced dilation. Additionally, it appears that endothelial H₂S is required for depolarization-induced activation of Ca²⁺ sparks in intact arteries.

epidemiological studies have established that sleep apnea is an independent risk factor for cardiovascular disease, in particular hypertension (31). Potential mechanisms to explain this association include increased sympathetic activity (5), endothelial dysfunction (22), systemic inflammation (36), and increased endothelin-1-dependent vasoconstriction (10). We previously reported that arteries from rats made hypertensive by exposure to intermittent hypoxia (IH) to simulate the hypoxic episodes of sleep apnea have enhanced myogenic tone and depolarized vascular smooth muscle cell (VSMC) membrane potential (E_m) through an apparent loss of the vasodilator hydrogen sulfide (H₂S) (14, 19, 35, 37). Because previous studies have linked H₂S-mediated vasodilation with Ca²⁺ spark activation and VSMC hyperpolarization (12, 25), this study examined the effect of IH exposure on that pathway.

Ca²⁺ sparks are spatially and temporally limited Ca²⁺-release events from ryanodine-sensitive Ca²⁺ channels (RyR) in the sarcoplasmic reticulum (SR) of muscle cells. In cerebral artery VSMC, Ca²⁺ sparks activate large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels and cause E_m hyperpolarization, followed by decreased activity of L-type voltage-gated calcium channels (VGCC) and decreased cytosolic [Ca²⁺] (26). Ca²⁺ spark activity is increased in response to E_m depolarization downstream of increased intraluminal pressure and subsequent stretch of VSMC. This increase in Ca²⁺ spark activity has been hypothesized to act as an intrinsic negative feedback mechanism to regulate myogenic tone (11, 18). Ca²⁺ sparks are also regulated by cyclic nucleotides after activation of G protein-coupled receptors. Both cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) have been shown to increase spark activity (29). The gasotransmitter H₂S has recently been shown to be another activator of Ca²⁺ sparks in cerebral arteries (25) and in mesenteric arteries (12).

Our previous observation that IH exposure in rats enhances myogenic tone in mesenteric arteries through the loss of H₂S production led us to hypothesize that IH reduces H₂S-dependent Ca²⁺ spark activity, leading to depolarization and enhanced myogenic tone in mesenteric arteries. Studies were designed to determine how H₂S regulates Ca²⁺ sparks in VSMC.

METHODS

Animals.

Male Sprague-Dawley rats (275–325 g) were used for all studies and were exposed to IH as described previously (20). Briefly, animals were housed in Plexiglas boxes with free access to food and water and exposed to either IH (nadir 5% O₂:5% CO₂ to peak 21% O₂:0% CO₂) or air only cycling at a rate of 20 cycles/h for 7 h/day for 14 days (20). This IH protocol reduces Po₂ to ∼35 mmHg, maintains Pco₂ at ∼30 mmHg (32), and increases arterial blood pressure about 15 mmHg (2, 8, 35). On the day of the experiment, animals were anesthetized with pentobarbital sodium (200 mg/kg, ip) and mesenteric arteries were collected for Ca²⁺ imaging, dilation studies, E_m recordings, and myogenic tone studies. All animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the University of New Mexico School of Medicine and conform to National Institutes of Health guidelines for animal use.

Isolated vessel preparation.

The intestinal arcade was removed and placed in a Silastic-coated Petri dish containing chilled physiological saline solution (PSS; in mmol/l: 129.80 NaCl, 5.40 KCl, 0.83 MgSO₄, 0.43 NaH₂PO₄, 19.00 NaHCO₃, 1.80 CaCl₂, and 5.50 glucose). Fifth- to seventh-order artery segments (80–120-μm inner diameter) were dissected from the mesenteric vascular arcade and placed in fresh PSS. Arteries were transferred to a vessel chamber (Living Systems Instrumentation), cannulated onto glass micropipettes, and secured with silk ligatures. The arteries were pressurized to 60 mmHg with PSS using a servo-controlled peristaltic pump (Living Systems Instrumentation) and superfused with warmed, oxygenated PSS at a rate of 5 ml/min.

Vessel reactivity studies.

Luminal pressure was increased in 40-mmHg steps from 20 to 180 mmHg using a servo-controlled peristaltic pump. Myogenic tone was allowed to develop for a minimum of 5 min at each pressure step. Pressure was reduced, and arteries were subsequently incubated with Ca²⁺-free PSS for 60 min. The pressure curve was repeated to determine passive diameter at each pressure. Myogenic tone was calculated as [(Ca²⁺-free diameter) − (Ca²⁺-containing diameter)/(Ca²⁺-free diameter)]*100.

In separate arteries, tone was induced to 50% resting diameter using phenylephrine, and dilation responses to the H₂S donor, NaHS (10 μM), were recorded. Dilation was calculated as [(NaHS diameter − PE diameter)/(Ca²⁺-free diameter)] − (PE diameter*100) and represents reversal of active tone.

Ca²⁺ spark imaging.

Arteries used for Ca²⁺ spark imaging studies were incubated in a fluo-4 AM (10 μmol/l, Invitrogen) solution containing 0.25% pluronic acid in HEPES buffer (in mmol/l: 134.000 NaCl, 6.000 KCl, 1.000 MgCl, 2.000 CaCl₂, 10.000 HEPES, 0.026 EDTA, 10.000 glucose, pH 7.4) for 60 min at 28°C before cannulation. After being loaded with fluo-4, arteries were transferred to a vessel chamber, cannulated as described above, and pressurized to 60 mmHg. After 5-min equilibration in oxygenated PSS at 32°C, fluo-4-loaded arteries were excited at 488 nm by a solid-state laser, and emitted light > 500 nm was collected using an Olympus IX71 microscope with a ×60 water-immersion lens and a spinning-disk confocal scanning unit (Andor Technology). A 75 × 50 μm area was imaged at 50–60 Hz using a laser power of 15%.

Spark analysis.

Spark movies were analyzed using SparkAn software, developed by A. D. Bonev and M. T. Nelson (University of Vermont). Ten images without spark activity were averaged to determine background fluorescence levels (F₀). Regions of interest of 10 pixels² (4 μm²) were used to detect sparks with a minimum F/F₀ of 1.2. The average F/F₀ was 1.27 ± 0.20 with a duration of 0.057 ± 0.003 s, and neither parameter varied between conditions. Each image contained 10–20 cells, and spark frequency was averaged for all cells visible. The number of active sites was counted for each field of view and divided by number of cells to obtain spark sites/cell. To determine the effect of inhibitors and activators on spark activity, a baseline level of sparks was recorded, and then arteries were equilibrated for 15 min before a second recording was made. Where possible, the order of conditions was randomized. For irreversible inhibitors, vehicle time controls were performed. In some arteries, the endothelium was disrupted by gentle abrasion of the lumen with a strand of moose mane as previously described (14). To evaluate the effect of modest depolarization on Ca²⁺ spark activity, extracellular [KCl] was increased from 5 to 15 mM, the minimum concentration to cause VSMC depolarization (Table 2) without a significant vasoconstriction.

VSMC E_m measurement.

Small mesenteric arteries (80–100-μm inner diameter) were cannulated and pressurized to 20 mmHg. After 60-min equilibration, VSMC were pierced from the exterior using sharp electrodes (filling solution 3M KCl in ddH₂O, R = 40–60 MΩ). E_m was recorded for at least 1 min from 3–8 sites and averaged to obtain n = 1 for each artery. Then intraluminal pressure was increased to 100 mmHg, and E_m was recorded from another 3–8 sites and averaged. In another set of arteries pressurized to 60 mmHg, E_m was recorded in normal PSS (5 mM K⁺) and high K-PSS (15 mM K⁺ with KCl substituted equimolar for NaCl).

Statistical analysis.

Myogenic tone, dilation, and spark frequency were analyzed using two-way ANOVA with Student-Newman-Keuls post hoc analysis for differences between groups. E_m differences were evaluated using a paired t-test. For all studies, P < 0.05 was considered statistically significant.

RESULTS

IH reduces RyR-mediated regulation of myogenic tone.

To determine whether Ca²⁺ spark activation regulates myogenic tone in mesenteric arteries, tone was assessed with and without blockade of RyR using ryanodine (10 μM). Vehicle-treated arteries from IH rats developed significant tone, whereas arteries from sham rats did not (Fig. 1), consistent with previous work (14). Furthermore, ryanodine treatment enhanced myogenic tone in arteries from sham rats but not in arteries from IH rats (Fig. 1). These results are consistent with Ca²⁺ sparks acting to inhibit arterial myogenic tone under normal conditions. This inhibition is impaired following IH exposure, consistent with loss of H₂S-dependent activation of RyR through loss of Ca²⁺ spark activity.

Fig. 1.

Effect of ryanodine (10 μM) on myogenic tone in mesenteric arteries from sham (A) and intermittent hypoxia (IH) (B) rats; n = 6–7 animals/group. *P < 0.05 vs. vehicle.

IH impairs basal Ca²⁺ spark activity.

Ca²⁺ spark activity was measured in VSMC in sham and IH arteries pressurized to 60 mmHg using software identification of Ca²⁺ sparks with F/F₀ ≥ 1.2 of fluorescence (Fig. 2A). Spark activity was significantly lower in endothelium-intact arteries from IH rats compared with arteries from sham rats (Fig. 2B).

Fig. 2.

Effect of IH treatment on basal Ca²⁺ spark activity. A: example trace of sparks in a region of interest in arteries from a sham (red) or IH (blue) rat. B: basal spark activity in sham and IH vascular smooth muscle cell (VSMC) in an artery pressurized to 60 mmHg; n = 16 sham, 21 IH. *P < 0.05 vs. sham.

IH impairs activation of Ca²⁺ spark activity.

NaHS (10 μM) administration increased Ca²⁺ spark activity in VSMC in arteries from sham rats (Fig. 3A). Conversely, inhibiting endogenous synthesis of H₂S via cystathionine γ-lyase (CSE) with the CSE inhibitor β-cyanoalanine (BCA; 100 μM) decreased sparks in arteries from sham rats. However, CSE inhibition did not affect spark activity in arteries from IH rats. Similarly, depolarizing VSMC with elevated [KCl] (15 mM) or by increasing luminal pressure from 20 to 100 mmHg also increased Ca²⁺ spark activity in arteries from sham but not IH rats, suggesting that IH exposure impairs VSMC Ca²⁺ spark activation by certain stimuli. To determine whether cyclic nucleotide activation of Ca²⁺ sparks was also impaired in arteries from IH rats, phosphodiesterase was inhibited with 3-isobutyl-1-methylxanthine (IBMX, 40 μM) to elevate cyclic nucleotide activation of RyR (29). IBMX increased spark activity in arteries from both groups, showing that RyR are present and can generate Ca²⁺ sparks (Fig. 3A). Raw traces of Ca²⁺ sparks in sham arteries show increased activity at basally active sites as well as recruitment of previously inactive sites (Fig. 3B).

Fig. 3.

A: summary data of changes in Ca²⁺ spark activity from baseline (Δ spark activity) in endothelium-intact arteries from sham and IH rats in response to increased H₂S (NaHS, 10 μM), inhibition of H₂S production (β-cyanoalanine, BCA, 100 μM), depolarization (KCl, 15 mM), stretch-induced depolarization (increased luminal pressure from 20 to 100 mmHg) or phosphodiesterase inhibition (3-isobutyl-1-methylxanthine, IBMX, 40 μM); n = 5–6 animals/group. *P < 0.05 vs. sham within treatment. B: representative images and traces of Ca²⁺ spark activity in regions of interest (boxes on image) in an artery from a sham rat in normal physiological saline solution (PSS) (5.4 mM KCl) or high KCl (15 mM) (top). This concentration of KCl (15 mM) increased Ca²⁺ sparks in sham arteries but did not cause vasoconstriction so that paired images could be analyzed. Bottom: comparison of arteries from sham and IH rats in normal PSS.

Endothelium is required for H₂S- and depolarization-induced Ca²⁺ spark activation.

The endothelium was disrupted in arteries from sham rats to determine whether the site of action for H₂S regulation of Ca²⁺ spark activity is the endothelium. Endothelium removal suppressed changes in spark activity to all perturbations except for phosphodiesterase inhibition (Fig. 4). Thus H₂S- and depolarization-dependent spark activation both require intact endothelium, but activation of sparks by cyclic nucleotide elevation is endothelium independent.

Fig. 4.

Changes in Ca²⁺ spark activity from baseline in arteries from sham rats with endothelium intact (endothelial cell, EC intact) or disrupted (EC disrupted) in response to increased H₂S (NaHS, 10 μM), inhibition of H₂S production (BCA, 100 μM), depolarization (KCl, 15 mM), stretch-induced depolarization (increased luminal pressure from 20 to 100 mmHg), or phosphodiesterase inhibition (IBMX, 40 μM); n = 5–6 animals/group. *P < 0.05 vs. EC intact within treatment.

Depolarization-induced increase in Ca²⁺ spark activity is H₂S dependent.

VSMC E_m was recorded to determine whether the diminished spark response to KCl and increased luminal pressure in arteries from the IH rats was caused by a smaller change in VSMC E_m. We observed that either increased luminal pressure or increased [KCl] caused similar depolarization in arteries from sham and IH rats (Fig. 5, insets). In arteries from sham rats, inhibiting CSE to decrease H₂S production (BCA, 100 μM) decreased basal Ca²⁺ spark activity and prevented increases in spark activity after exposure to KCl (Fig. 5A). BCA treatment also prevented increased Ca²⁺ spark activity after a pressure step (Fig. 5B). Therefore, Ca²⁺ spark activation in response to small depolarizations appears to require endogenous H₂S. In contrast with VSMC in arteries from sham rats, VSMC in arteries from IH rats have fewer spark sites/cell (Fig. 6A) but a similar activity at individual sites (Fig. 6B). KCl depolarization increased the total number of sparks/site per second (Fig. 6B) without increasing the number of spark sites/cell.

Fig. 5.

A: changes in Ca²⁺ spark activity in response to increased KCl in arteries with intact endothelium from sham rats treated with vehicle or BCA (100 μM). Inset: membrane potential (E_m) in VSMC in pressurized arteries (60 mmHg) from sham or IH rats in PSS with normal (5.4 mM) or elevated (15.0 mM) KCl. *Different from vehicle for P < 0.05; #different from 5 mM KCl for P < 0.05; $different from sham for P < 0.05. B: changes in Ca²⁺ spark activity in response to increased luminal pressure (from 20 to 100 mmHg) in arteries from sham rats with endothelium intact treated with vehicle or BCA (100 μM). Membrane potential in VSMC in arteries from sham or IH rats pressurized to low (20 mmHg) or high (100 mmHg) pressures. *Different from vehicle; #different from 20 mmHg; or $different from Sham for P < 0.05; n = 5–9 animals/group.

Fig. 6.

Ca²⁺ spark activity in arteries from sham or IH rats. The average number of sites/cell (A) or average number of sparks/site per second (B) observed in arteries from sham (Open bars) and IH (shaded bars) rats exposed to different KCl concentrations after treatment with vehicle (open bars) or BCA (100 μM, hatched bars). *Different from 5.4 mM KCl; #different from vehicle; or $different from sham for P < 0.05; n = 7 (sham) or 6 (IH) animals.

RyR are required for NaHS-induced dilation.

Previous work from our laboratory demonstrated that endogenous H₂S production limits generation of myogenic tone in arteries from sham rats, but arteries from both sham and IH rats dilate in response to an exogenous H₂S donor, NaHS. Because NaHS-induced Ca²⁺ spark activity is similar in VSMC from sham and IH rats (Fig. 3A), dilation to NaHS was assessed in small mesenteric arteries from sham and IH rats in the absence and presence of ryanodine (10 μM). In arteries pressurized to 100 mmHg and constricted to ∼50% resting diameter with phenylephrine, arteries from both sham and IH rats demonstrated a robust dilation to NaHS, which was almost fully blocked by pretreatment with ryanodine (Fig. 7). Therefore, Ca²⁺ sparks appear to contribute to NaHS-induced dilation in arteries from both sham and IH rats.

Fig. 7.

Dilation to NaHS in mesenteric arteries pressurized to 100 mmHg in the presence of ryanodine (10 μM) or vehicle (ddH₂O). Arteries were contracted to 50% resting diameter with phenylephrine, and dilation was expressed as a percentage reversal of contraction so that 100% would be complete dilation. *P < 0.05 vs. vehicle; n = 6 animals/group.

DISCUSSION

This study establishes that ryanodine channel activity inhibits myogenic tone, but this effect is lost after IH exposure (Fig. 1). The loss of a response to ryanodine is accompanied by decreased spark activity in arteries from IH rats (Fig. 2). Exogenous H₂S increases Ca²⁺ spark activity above baseline, whereas inhibiting H₂S synthesis decreases Ca²⁺ spark activity in arteries from sham but not IH rats, consistent with endogenous H₂S regulating Ca²⁺ spark activity (Fig. 3) and validating that H₂S activates Ca²⁺ sparks in these arteries. In arteries from sham rats, depolarizing VSMC increases Ca²⁺ spark activity as expected, but this is impaired in arteries from IH rats and is prevented by inhibiting H₂S synthesis (Fig. 3). Thus endogenous H₂S appears to be required for depolarization-induced increases in Ca²⁺ spark activity. Disrupting the endothelium eliminated exogenous H₂S regulation of Ca²⁺ spark activity and abrogated the effect of CSE inhibition (Fig. 4). Inhibiting CSE in arteries with intact endothelium also prevented KCl and pressure-induced increases in Ca²⁺ spark activity (Fig. 5). Together, these data show that H₂S facilitates basal and depolarization-induced Ca²⁺ spark activity and suggest that this effect is mediated through actions on the endothelium. Furthermore, NaHS-induced dilation was prevented by ryanodine blockade of spark activity (Fig. 7), suggesting that Ca²⁺ spark activity contributes to H₂S-induced dilation. Finally, CSE inhibition and endothelium disruption did not affect Ca²⁺ spark activity in arteries from IH rats, consistent with our previous observation that IH leads to decreased endothelial expression of CSE (10). These data support the conclusion that H₂S regulates Ca²⁺ spark-mediated dilation as illustrated in the diagram in Fig. 8. Loss of this pathway after exposure to IH for 14 days may contribute to increased vasoconstriction and elevated blood pressure in this model of sleep apnea-induced hypertension (6, 9, 20).

Fig. 8.

Summary illustrating our hypothesis that H₂S generated by cystathionine γ-lyase (CSE) in ECs initiates a signal within EC that sensitizes VSMC ryanodine receptor (RyR) activation by depolarization-induced Ca²⁺ influx through voltage-gated Ca²⁺ channels (VGCC). The pathway inhibits contraction of VSMC and is impaired after 2 wk of IH exposure.

Myogenic tone regulation of appropriate organ perfusion is gaining recognition as a potential therapeutic target for preventing end-organ damage in diabetes, hypertension, and aging (30). Thus the H₂S pathway is a potential therapeutic target to modulate myogenic tone and alleviate vascular disease in patients with sleep apnea and other populations. These findings provide a theoretical basis for exploring the upregulation of endogenous H₂S signaling as a prophylactic and therapeutic target in peripheral vascular disease.

Ca²⁺ sparks have been shown to regulate myogenic tone and vascular function through activation of VSMC BK_Ca channels (26), hyperpolarizing VSMC E_m (7). The activation of a cluster of BK_Ca channels by RyR-dependent Ca²⁺ sparks causes a spontaneous transient outward current (STOC) (28). These STOCs sum to cause a steady-state E_m hyperpolarization of 10 mV or greater (23) so that depolarization-induced increases in Ca²⁺ spark activity act as a feedback regulator of membrane potential and vascular tone (17). Our data suggest that this feedback regulation is impaired after acute inhibition of H₂S generation in arteries from healthy rats or after loss of H₂S synthesis in arteries from rats chronically exposed to IH.

Hypoxia has been previously shown to inhibit Ca²⁺ spark-dependent dilation. Acute hypoxia uncouples Ca²⁺ sparks from BK_Ca channel activation, reducing their hyperpolarizing effect (40). This effect is mediated by reduced Ca²⁺ sensitivity of BK_Ca channels, with no effect on Ca²⁺ spark activity. In contrast, our data show that 2 wk of IH decreases both basal and depolarization-induced Ca²⁺ spark activity over the same time course that myogenic (14) and agonist-induced vasoconstriction increase (1). Furthermore, the IH-induced decrease in Ca²⁺ spark activity is persistent under the normoxic conditions in which experiments were conducted, whereas hypoxic uncoupling of Ca²⁺ sparks and BK_Ca channels is seen only during acute hypoxic exposure. Thus the effects of acute, sustained hypoxia vs. chronic intermittent exposure to hypoxia appear to be distinct. Our previous observations that IH exposure decreases endothelial expression of CSE (14) and the current data showing that IH abolishes the effect of CSE inhibition to decrease Ca²⁺ spark activity point to H₂S as a primary regulator of Ca²⁺ spark activity and basal vasoreactivity.

In arteries from sham rats, either inhibiting CSE or abrading the endothelium similarly decreased basal Ca²⁺ spark activity and prevented depolarization-induced increases in Ca²⁺ spark activity (Figs. 3 and 4). Because CSE is primarily expressed in the endothelium in small mesenteric arteries (12), endogenous endothelium-derived H₂S appears requisite for depolarization-induced Ca²⁺ spark activation. Potential mechanisms for H₂S facilitation of Ca²⁺ spark generation include direct effects on VSMC RyR or VGCC, the source and trigger, respectively, of Ca²⁺ sparks (17). However, exogenous H₂S does not increase Ca²⁺ spark activity in endothelium-denuded arteries, suggesting that neither RyR nor VGCC are direct targets of H₂S. Rather, H₂S appears to release an endothelial factor that increases coupling of VGCC to RyR. Future studies to identify this factor should help to elucidate this novel pathway for vasodilation.

A requirement for the endothelium in depolarization-induced Ca²⁺ spark activation contrasts with previous reports of depolarization regulation of Ca²⁺ sparks and STOCs in isolated VSMCs. For example, short depolarizing pulses increased Ca²⁺ spark activity in isolated vein myocytes (3), and depolarization from −50 to −10 mV increased STOCs in VSMC from Sprague Dawley rat cerebral arteries in a nifedipine-dependent manner (15). Thus large depolarizations in isolated VSMC increase Ca²⁺ influx through VGCC to elicit Ca²⁺ sparks. However, no previous studies have addressed the ability of depolarization to elicit Ca²⁺ sparks in endothelium-denuded arteries or evaluated the effect of a modest depolarization on Ca²⁺ spark activity in isolated VSMC. Indeed, some reports indicate a dissociation between Ca²⁺ spark activity and VSMC E_m. Koide et al. (24) reported in 2011 that cerebral artery VSMC from rats with subarachnoid hemorrhage had depolarized E_m and decreased Ca²⁺ spark activity, illustrating that depolarization does not always increase Ca²⁺ sparks and STOCs. Yang et al. reported (38) that VSMC from cerebral but not cremaster arterioles exhibited Ca²⁺ sparks even though both had similar E_m and that spark activity could be increased by caffeine but not by changes in resting E_m. Thus several laboratories have observed dissociations between E_m and spark activation in isolated myocytes. In studies showing depolarization-mediated activation of Ca²⁺ spark activity, the degree of change in E_m is larger than that used in the present study (16). In the present study, the low concentration of KCl used might cause a modest hyperpolarization of the endothelial cell (EC) attributable to activation of K_IR. We did not directly measure this because it is not possible to cut open these small arteries to expose the EC. If indeed there is hyperpolarization of the EC, this should increase Ca²⁺, which could be a stimulus for H₂S release. With the stretch, there are also reports of increases in EC [Ca²⁺] so that this might be a common pathway to implicate Ca²⁺ in the final VSMC response. Thus VSMC depolarization increases spark activity, especially in cerebral artery myocytes, but this does not exclude an additional modulatory role of endothelial H₂S on Ca²⁺ spark activation in response to a modest depolarization.

The mechanism of this modulation is not clear, but H₂S appears to stimulate the release of an endothelial-derived factor (12) rather than exerting a direct effect on the VSMC. Because inhibition of CSE appears to decrease the number of active sites/cell, it appears that the H₂S pathway increases the sensitivity of RyR to increased Ca²⁺ influx. In contrast, we observed that KCl-induced depolarization of the VSMC E_m increases the number of sparks/site, consistent with increase activator Ca²⁺ delivery through VGCC. This suggests that, at any given E_m, H₂S enhances the coupling between RyR and VGCC Ca²⁺ influx, consistent with our observation that increasing KCl from 5.4 to 15.0 mM similarly depolarized VSMC from sham and IH rats, but this depolarization only increased Ca²⁺ spark activity in arteries with a functional H₂S system. Future investigations are needed to directly test this hypothesis.

In summary, this study provides the first evidence that H₂S increases Ca²⁺ spark activity through an endothelium-dependent effect to contribute to H₂S-induced dilation. Additionally, it appears that endothelial H₂S is required for depolarization-induced activation of Ca²⁺ sparks in intact arteries. Finally, both H₂S activity and Ca²⁺ spark activity are impaired by IH exposure, leading to enhanced myogenic tone, and may contribute to therapy resistant hypertension common in the sleep apnea population (27).

GRANTS

This work was supported by American Heart Association grants HL7736 and 10PRE4050028 to O. Jackson-Weaver, American Heart Association grant 12POST8690008 to J. Osmond, grant HL088151 to L. Gonzalez Bosc, grant HL95640 to B. Walker, and grants HL82799 and HL123301 to N. Kanagy.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

Author contributions: O.J.-W., J.M.O., L.V.G.B., B.R.W., and N.L.K. conception and design of research; O.J.-W., J.M.O., J.S.N., and N.L.K. performed experiments; O.J.-W., J.M.O., J.S.N., L.V.G.B., and N.L.K. analyzed data; O.J.-W., J.M.O., J.S.N., L.V.G.B., B.R.W., and N.L.K. interpreted results of experiments; O.J.-W., J.M.O., J.S.N., and N.L.K. prepared figures; O.J.-W. and J.M.O. drafted manuscript; O.J.-W., J.M.O., J.S.N., L.V.G.B., B.R.W., and N.L.K. edited and revised manuscript; O.J.-W., J.M.O., J.S.N., L.V.G.B., B.R.W., and N.L.K. approved final version of manuscript.