Intermittent Hypoxia in Rats Increases Myogenic Tone Through Loss of Hydrogen Sulfide Activation of Large-Conductance Ca²⁺-Activated Potassium Channels

Abstract

Rationale

Myogenic tone, an important regulator of vascular resistance, is dependent on vascular smooth muscle (VSM) depolarization, can be modulated by endothelial factors, and is increased in several models of hypertension. Intermittent hypoxia (IH) elevates blood pressure and causes endothelial dysfunction. Hydrogen sulfide (H₂S), a recently described endothelium-derived vasodilator, is produced by the enzyme cystathionine γ-lyase (CSE) and acts by hyperpolarizing VSM.

Objective

Determine whether IH decreases endothelial H₂S production to increase myogenic tone in small mesenteric arteries.

Methods and Results

Myogenic tone was greater in mesenteric arteries from IH than Shamfrom sham rat arteries, and VSM membrane potential was depolarized in IH in comparison with Shamsham arteries. Endothelium inactivation or scavenging of H₂S enhanced myogenic tone in Shamsham arteries to the level of IH. Inhibiting CSE also enhanced myogenic tone and depolarized VSM in Shamsham but not IH arteries. Similar results were seen in cerebral arteries. Exogenous H₂S dilated and hyperpolarized Shamsham and IH arteries, and this dilation was blocked by iberiotoxin, paxilline, and KCl preconstriction but not glibenclamide or 3-isobutyl-1-methylxanthine. Iberiotoxin enhanced myogenic tone in both groups but more in Shamsham than IH. CSE immunofluorescence was less in the endothelium of IH than in Shamsham mesenteric arteries. Endogenouse H₂S dilation was reduced in IH arteries.

Conclusions

IH appears to decrease endothelial CSE expression to reduce H₂S production, depolarize VSM, and enhance myogenic tone. H₂S dilatation and hyperpolarization of VSM in small mesenteric arteries requires BK_Ca channels.

In epidemiological studies, obstructive sleep apnea (OSA) is an independent risk factor for hypertension and other cardiovascular diseases.¹ Previously, we reported that exposing rats to eucapnic intermittent hypoxia (IH), a model of sleep apnea, elevates systemic blood pressure and arterial constrictor sensitivity to ET-1² with an associated increase in vascular reactive oxygen species (ROS). Furthermore, the antioxidant tempol prevents IH-induced hypertension.³ These results suggest that IH might also augment nonagonist-induced vasoconstriction.

Myogenic, or spontaneously developed tone, can augment agonist-induced increases in blood pressure⁴ through increased vascular resistance. Furthermore, myogenic tone is increased in some experimental models of hypertension.⁵ Myogenic tone appears to be initiated by activation of mechanosensitive cation channels, leading to membrane depolarization and opening of L-type voltage-gated Ca²⁺ channels (L-type VGCC).⁶ Modulation of this pathway, leading to elevated resting myogenic tone, could therefore contribute to systemic hypertension.

H₂S, a recently described vasodilator, is produced endogenously from L-cysteine by 3 enzymes: cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3MST).⁷ CSE has been reported to be the primary source of H₂S in the vasculature, although 3MST may also contribute in some vascular beds. H₂S is a reducing compound that can react with superoxide anion (O₂⁻) to form sulfite⁸ or with nitric oxide (NO) to form a nitrosothiol, potentially limiting the bioavailability of both gasotransmitters.⁹ Most studies attribute H₂S vasodilation to activation of vascular smooth muscle (VSM) ATP-sensitive potassium channels (K_ATP),¹⁰ but other mechanisms have also been reported.¹¹ Genetic deletion of CSE in mice elevates blood pressure.¹² In this study, CSE expression was primarily in the endothelium of mesenteric arteries, and large mesenteric arteries from CSE^−/− mice exhibited endothelial dysfunction.

We hypothesized that IH decreases endothelium-dependent H₂S generation to enhance myogenic tone in small mesenteric arteries. We observed that small mesenteric arteries from IH-exposed rats have increased myogenic tone and depolarized VSM membrane potential (E_m). Increased myogenic tone in IH arteries was mimicked in sham arteries by disrupting the endothelium, inhibiting CSE, or scavenging H₂S. Inhibiting CSE depolarized VSM in sham but not IH arteries. Exogenous H₂S dilated and hyperpolarized sham and IH arteries, and both effects were prevented by large-conductance Ca²⁺-activated potassium channel (BK_Ca) blockade. BK_Ca blockade also augmented myogenic tone more in sham than in IH arteries. These results suggest that H₂S is an endogenous endothelium-dependent regulator of myogenic tone in small mesenteric arteries that acts through the activation of BK_Ca channels and that IH exposure impairs this pathway.

Methods

An expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org.

Animals

Male Sprague–Dawley rats were exposed 7 hours per day to either IH or sham cycling as described previously.¹³ 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

Fourth- or fifth-order mesenteric artery segments (i.d. <100 μm) or middle cerebral arteries were cannulated, pressurized to 60 mm Hg, loaded with fura-2 am to record intracellular [Ca²⁺] and superfused with warmed, oxygenated Kreb’s buffer. Diameter changes and vessel wall calcium concentration ([Ca²⁺]) were recorded using a microscopy system with edge-detection software.

Pressure–Response Curves

Luminal pressure was increased from 20 to 180 mm Hg in 20-mm Hg steps and diameter changes recorded. After incubation in Ca²⁺-free buffer for 60 minutes, the pressure curve was repeated to determine passive diameter, and myogenic tone was calculated as ([(Ca²⁺-free diameter)−(Ca²⁺-containing diameter)]/(Ca²⁺-free diameter))*100. Vessel wall [Ca²⁺] was recorded simultaneously.

Membrane Potential Recordings

Pressurized arteries at 37°C were impaled through the adventitia with glass intracellular microelectrodes (tip resistance 40 to 120 mol/LΩ). Criteria for acceptance of E_m recordings were (1) an abrupt negative deflection of E_m as the microelectrode was advanced into a cell; (2) stable E_m for at least 1 minute; and (3) an abrupt change in E_m to ≈0 mV after the electrode was retracted from the cell.

Results

Myogenic Tone in Intact and Denuded Arteries

Myogenic tone was greater in mesenteric arteries from IH rats in comparison with arteries from sham rats (Figure 1A, left). There was also a greater increase in VSM [Ca²⁺] in the IH arteries (Figure 1A, right). Endothelium disruption slightly elevated myogenic tone in IH arteries but greatly augmented tone in sham arteries so that myogenic tone was not different between sham and IH endothelium-disrupted arteries (Figure 1B, left). Endothelial disruption also elevated VSM [Ca²⁺] in both groups, and the Ca²⁺ response to pressure was not different between groups (Figure 1B, right). The enhanced myogenic tone in IH arteries was not due to vessel wall hypertrophy (Online Figure I).

Figure 1. Myogenic tone and vascular smooth muscle (VSM) [Ca²⁺] in mesenteric arteries is enhanced by intermittent hypoxia (IH) treatment.

A, myogenic tone and VSM [Ca²⁺] in sham and IH arteries. B, myogenic tone and VSM [Ca²⁺] in sham and IH arteries±endothelium. Values are means±SE; n=5 to 7 per group. *P<0.05 IH versus sham. #P<0.05 sham versus sham treated. %P<0.05 IH versus IH treated.

Effect of Inhibiting Endothelial Vasodilator Production on Myogenic Tone

Myogenic tone was evaluated in arteries from sham rats treated with the following inhibitors: 100 μmol/L N^G-nitro-l-arginine (L-NNA, NOS inhibitor), 10 μmol/L indomethacin (cyclooxygenase blocker), 10 μmol/L SKF 525A (cytochrome p450 inhibitor), 250 U/mL PEG-catalase (H₂O₂ scavenger), 100 nmol/L apamin and 1 μmol/L TRAM-34 (SK and IK potassium channel blockers), and 10 μmol/L Cr(III) mesoporphyrin IX (heme oxygenase inhibitor) (Figure 2). None had a significant effect.

Figure 2. Inhibitors of previously described endothelium-produced vasodilators do not affect myogenic tone in sham arteries.

Myogenic tone was assessed at 100 mm Hg in untreated sham arteries (vehicle) or after pharmacological inhibition or endothelium disruption (EC disrupted). Values are means±SE; n=3 to 6 per group. *P<0.05 versus sham intact.

H₂S Regulation of Myogenic Tone

Arteries were treated with bismuth (III) subsalicylate (BSS,10 μmol/L) to scavenge H₂S, and myogenic tone was evaluated. BSS enhanced myogenic tone in sham but not IH arteries, eliminating differences in pressure-induced constriction between groups (Figure 3A, left). BSS also reduced VSM [Ca²⁺] in IH arteries but had no effect in sham arteries (Figure 3A, right). Because BSS nonselectively scavenges H₂S and several H₂S-producing enzymes are present in the vascular wall, the effect of CSE inhibitors was evaluated. The CSE inhibitor β-cyano-l-alanine (BCA, 100 μmol/L) enhanced myogenic tone similarly to BSS in sham arteries with no effect in IH arteries (Figure 3B, left). BCA also enhanced VSM [Ca²⁺] in sham arteries but had no effect in IH arteries, normalizing the response between groups (Figure 3B, right). A second inhibitor of CSE, DL propargylglycine (PAG, 100 μmol/L), also enhanced myogenic tone in sham arteries but reduced myogenic tone in IH arteries (Online Figure II, left). PAG also enhanced VSM [Ca²⁺] in sham arteries and reduced VSM [Ca²⁺] in IH arteries (Online Figure II, right). Myogenic curves in middle cerebral arteries from sham and IH rats demonstrated that BCA also elevates myogenic tone in sham but not IH cerebral arteries (Online Figure III).

Figure 3. Myogenic tone in sham mesenteric arteries is enhanced by treatment with H₂S antagonists bismuth (III) subsalicylate (BSS) (A) and β-cyano-l-alanine (BCA) (B).

Values are means±SE; n=5 to 7 per group. #P<0.05 sham versus sham treated. *P<0.05 intermittent hypoxia (IH) versus sham within treatment. %P<0.05 IH versus IH treated.

Cysteine-Induced Vasodilation

The endogenous substrate cysteine increases CSE synthesis of H₂S.¹⁴ Arteries preconstricted with PE were dilated with cysteine (1 μmol/L). Cysteine caused a greater dilation in sham than in IH arteries (Figure 4). Addition of BCA significantly reduced cysteine dilation in sham but not IH arteries, although sham arteries still dilated to a greater extent in the presence of BCA. Endothelium disruption likewise significantly reduced cysteine dilation in sham but not IH arteries, normalizing the response between groups (Figure 4).

Figure 4. Endothelial CSE activity as measured by cysteine-induced dilation is reduced in intermittent hypoxia (IH) arteries. –EC=endothelial disruption.

BCA=β-cyano-l-alanine; n=5 per group. Values are means±SE. *P<0.05 versus sham untreated. #P<0.05 sham versus IH within treatment.

Mechanism of H₂S-Mediated Vasodilation

To determine whether IH arteries have decreased sensitivity to H₂S, we examined dilation to the H₂S donor NaHS in endothelium-intact arteries constricted with PE to 50% resting diameter. NaHS dilated both sham and IH arteries, with slightly greater dilation in IH than in sham arteries (Figure 5A). The K_ATP blocker glibenclamide (10 μmol/L) was used to determine whether K_ATP channels mediate this dilation as reported in larger arteries and in the perfused mesenteric bed.¹⁰ Glibenclamide had little effect in sham or IH arteries (Figure 5B and 5C), although it blocked pinacidil-induced dilation (Online Figure IV). In contrast, the BK_Ca channel inhibitors iberiotoxin (IbTx, 100 nmol/L) and paxilline (1 μmol/L) significantly reduced NaHS dilation in both groups (Figure 5B and 5C and Online Figure V). The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) did not reduce NaHS induced dilation in either group (Online Figure VI). Arteries constricted to 50% resting diameter with KCl did not dilate to NaHS (Online Figure VII).

Figure 5. Blockade of BK_Ca channels inhibits NaHS-induced vasodilation and enhances myogenic tone in small mesenteric arteries.

NaHS dilates both sham and intermittent hypoxia (IH) arteries (A). NaHS dilation in sham arteries is inhibited by iberiotoxin (IbTx) but not glibenclamide (Glib) (B). NaHS dilation in IH arteries is inhibited by IbTx but not Glib (C). D, Myogenic tone in sham and IH arteries±IbTx. E, VSM [Ca²⁺] in sham and IH arteries±IbTx. Values are means±SE. *P<0.05 sham versus IH untreated. #P<0.05 untreated versus IbTx within sham. %P<0.05 untreated versus Glib within group. †P<0.05 untreated versus IbTx within IH. IbTx=iberiotoxin, 100 nmol/L; Glib=glibenclamide, 10 μmol/L.

BK_Ca Channel Regulation of Myogenic Tone

Because the NaHS dilation experiments implicated BK_Ca channels as a target for H₂S, the effect of IbTx on myogenic tone was evaluated. IbTx elevated myogenic tone at all pressures in both sham and IH arteries, and normalized myogenic tone between groups (Figure 5D). Likewise, IbTx elevated VSM [Ca²⁺] in sham and IH arteries, eliminating differences between groups (Figure 5E). NS1619 (BK_Ca channel opener) dilation in arteries constricted with PE and incubated with BCA was similar in both groups (Online Figure VIII).

Membrane Potential Recordings

Sharp electrodes were used to record E_m in sham and IH arteries at 60 and 140 mm Hg luminal pressure. Although the increase in pressure caused a significant depolarization in both groups, IH arteries had a more depolarized E_m at both 60 and 140 mm Hg than did sham (Figure 6A and 6B). Treatment with BCA depolarized only sham arteries, normalizing E_m between groups but increased luminal pressure depolarized E_m in both groups in the presence of the inhibitor (Figure 6A and 6B). The H₂S donor, NaHS (10⁻⁵ mol/L), hyperpolarized E_m in both sham and IH arteries constricted with 10⁻⁶ mol/L PE, and this hyperpolarization was prevented by IbTx (Figure 6C and 6D).

Figure 6. H₂S hyperpolarizes vascular smooth muscle cells through activation of BK_Ca channels and loss of endogenous H₂S depolarizes intermittent hypoxia (IH) vascular smooth muscle cell E_m.

A, Representative traces of sharp electrode E_m measurements±BCA. B, Summary data from experiments in A. C, Representative traces of sharp electrode E_m measurements before and after±NaHS addition in sham arteries. D, Summary data from experiments in C. Values are means±SE; n=5 to 6 per group. #P<0.05 within group. *P<0.05 versus sham vehicle.

ROS and NO Regulation of Myogenic Tone

Because H₂S reacts with superoxide to form sulfite and can also react with NO, pressure-induced constriction was evaluated in the presence of the superoxide dismutase mimetic tiron or L-NNA. Tiron slightly reduced myogenic tone in IH arteries but did not affect tone in sham arteries (Figure 7A, left). However, myogenic tone was still greater in IH than sham arteries at 140 mm Hg. Tiron did not affect VSM [Ca²⁺] in either group, and IH arteries had a significantly higher VSM [Ca²⁺] than sham arteries with this treatment (Figure 7A, right). L-NNA tended to reduce myogenic tone in IH arteries at 100 mm Hg but had no effect on sham arteries so that tone was greater in IH arteries at 100 mm Hg but not at 140 mm Hg (Figure 7B, left). L-NNA had no significant effect on VSM [Ca²⁺] in either group (Figure 7B, right).

Figure 7. Tiron and L-NNA partially reduced myogenic tone in intermittent hypoxia (IH) arteries to sham levels.

A, Tiron effect on sham and IH myogenic tone. B, L-NNA effect on sham and IH myogenic tone. Values are means±SE. *P<0.05 sham treated versus IH treated. % P<0.05 IH versus IH treated.

CSE Immunofluorescence, Western Blot, and qPCR

Immunofluorescence imaging in small mesenteric arteries assessed expression of CSE. With the morphology of costained nuclei to assess cell type, CSE expression was apparent in endothelial, VSM, and adventitia layers of small mesenteric arteries (Figure 8A). CSE expression was highest in the adventitial layer of sham arteries. CSE expression was decreased in the endothelial layer of IH arteries versus sham (Figure 8B). Western blots for CSE using tissue homogenates of 1st- through 5th-order mesenteric arteries showed that CSE expression was actually greater in arteries from IH rats (Figure 8C and 8D). To resolve this discrepancy between immunofluorescence and Western blot data, we evaluated immunofluorescence in 1st-order mesenteric arteries. In these larger arteries, CSE expression was greatest in the adventitia in both groups and significantly greater in IH than in sham arteries (Online Figure IX). Quantitative real-time PCR of CSE mRNA using whole mesenteric artery homogenates was performed, which showed no differences between sham and IH (Online Figure X).

Figure 8. Intermittent hypoxia (IH) reduces endothelial cell CSE in small mesenteric arteries.

A, Representative images. B, Immunofluorescence summary data; n=4 per group. C, Western blot of CSE in 1st- to 5th-order mesenteric arteries from sham and IH rats. Tissue for Western blot contained adventitial connective tissue (ADV), but adipose tissue was removed prior to homogenization. D, Summary data from blot in C. CSE expression normalized to β-actin. ADV = adventitial connective tissue; EC = endothelial cells. + Positive control brain lysate; n=5 per group. *P<0.05 sham versus IH. #P<0.05 sham endothelium versus sham adventitia. %P<0.05 sham vascular smooth muscle (VSM) versus sham endothelium.

H₂S Assay

To verify that BCA reduces H₂S production, we evaluated enzymatic production of H₂S in kidney homogenates. Sham and IH kidney homogenates generated similar quantities of H₂S, and this production was significantly reduced by either 100 μmol/L or 1 mmol/L BCA (Online Figure XI). Similar to the effect of 1 mmol/L BCA, 100 μmol/L BCA reduced CSE H₂S production by ≈20%. Plasma levels of H₂S were also not different between sham and IH (Online Figure XII).

There are 3 major new findings from these studies. First, IH in rats enhances myogenic tone in small mesenteric arteries by decreasing H₂S-dependent dilation. Second, H₂S limits myogenic tone in mesenteric arteries in an endothelium-dependent manner. Third, H₂S dilation in mesenteric arteries is mediated by BK_Ca activation, a previously unreported mechanism of H₂S-induced vasodilation. Both functional dilation studies and recordings of VSM membrane potential support these conclusions. This is in contrast to previous reports of dilation by higher concentrations of H₂S in aorta and in larger mesenteric arteries, suggesting that H₂S may have dose-dependent mechanisms of action that vary in different size arteries.

Both pressure-induced increases in myogenic tone and VSM [Ca²⁺] are greater in mesenteric arteries after IH, consistent with the widely accepted theory of myogenic tone in which constriction is dependent on depolarization-induced Ca²⁺ influx.⁶ Although myogenic tone can be increased by vessel wall hypertrophy through the “structural amplifier” mechanism,¹⁵ there was no increase in wall thickness in IH arteries, suggesting functional rather than structural mechanisms account for this increased tone.

Endothelial dysfunction is a commonly reported consequence of sleep apnea,¹⁶ and the current studies suggest that reduced endothelial dilation causes the enhanced myogenic tone in IH arteries. That is, endothelium inactivation increased both myogenic tone and VSM [Ca²⁺] more in sham than in IH arteries, suggesting that endothelial factors are reduced or do not reach the smooth muscle in IH arteries. Of interest, endothelial disruption greatly enhances VSM [Ca²⁺] in sham arteries with only a moderate increase in tone over that in the IH endothelium-intact group, whereas a relatively small enhancement of tone but a large increase in VSM [Ca²⁺] is seen in IH arteries. Thus the enhanced myogenic tone in IH arteries appears to be dependent on greater VSM [Ca²⁺], but an element of Ca²⁺ sensitization may also contribute. In contrast to our findings, Phillips et al observed no effect of IH on myogenic tone in gracilis arterioles.¹⁷ In this study, rats were exposed to 1 minute of 10% O₂ every 4 minutes without supplemental CO₂ to prevent hypocapnia and no effects on arterial pressure were observed. In our study, rats were exposed to ≈15 seconds of O₂ <10% every 3 minutes with supplemental CO₂ and experienced a sustained increase in arterial pressure, suggesting different exposure paradigms may differentially affect myogenic reactivity. However, Phillips also found that their IH exposure impairs endothelium-dependent dilation in skeletal muscle arteries.¹⁸ Thus endothelial function may have a greater impact on myogenic tone in mesenteric arteries than in the more myogenically active skeletal muscle arteries.

Multiple pharmacological inhibitors were used in an attempt to identify the product or effect of the endothelium limiting myogenic tone in sham but not in IH mesenteric arteries. Inhibiting NOS, cyclooxygenase, cytochrome P450 enzymes, heme oxygenase, scavenging H₂O₂ or blocking endothelium-derived hyperpolarization with SK and IK blockers did not mimic endothelial disruption, in contrast to studies in other vascular beds in which NO limits myogenic tone.¹⁹ These studies suggested that a novel endothelial product minimizes myogenic tone in small mesenteric arteries.

H₂S is a recently described endothelial product that mediates vasodilation and regulates blood pressure.⁷ In contrast to the effects of the other inhibitors, the H₂S scavenger BSS greatly increased myogenic tone in sham but not IH arteries. The similarity of H₂S scavenging to endothelial disruption suggests that endothelial H₂S minimizes myogenic tone but is impaired by IH treatment. Although BSS tended to increase VSM [Ca²⁺] in sham arteries, the increase was not significant and BSS decreased VSM [Ca²⁺] in IH arteries, suggesting that the scavenger may have nonspecific effects such as dissociation into salicylate,²⁰ a cyclooxygenase inhibitor.

BSS scavenges H₂S nonselectively, so pharmacological inhibitors were used to determine whether H₂S production in the sham arteries is from CSE, the reported primary vascular source.⁷ CSE inhibition with either BCA or PAG also elevated myogenic tone in sham arteries but not in IH arteries, similar to BSS scavenging of H₂S. Likewise, BCA elevated VSM [Ca²⁺] in sham but not IH arteries so that tone and [Ca²⁺] were not different between groups in the presence of the inhibitor. In cerebral arteries, BCA also enhanced myogenic tone in sham but not IH arteries. The concentration of BCA used (100 μmol/L) was verified to be as effective at reducing H₂S production in kidney homogenates as was a higher concentration (1 mmol/L) used in some studies. These results suggest that endothelial CSE produces H₂S to minimize myogenic tone and IH impairs this pathway in multiple vascular beds. Unexpectedly, PAG, but not BCA or BSS, reduced myogenic tone and [Ca²⁺] in IH arteries and increased VSM [Ca²⁺] in sham arteries. Because PAG inhibits the entire cystathionine synthaselike family of proteins and is thus not specific to CSE, it is possible that one of this large family of proteins that includes several ion channels and transporters²¹ is required for myogenic tone after IH treatment, although what enzyme it is or what function it fulfills is unknown.

Dilation to excess CSE substrate cysteine was also evaluated. Cysteine dilation was greater in sham than in IH arteries and significantly reduced by CSE inhibition in sham but not IH arteries, suggesting that mesenteric arteries from IH rats have reduced CSE function. Consistent with H₂S being endothelium derived, endothelial disruption reduced cysteine dilation in sham but not in IH arteries.

CSE expression is reduced in the endothelium of IH arteries, providing a likely cause of decreased H₂S dilation following IH treatment. In contrast to studies in mice,¹² CSE was readily apparent in all 3 layers of the vasculature with the greatest expression in the adventitial layer. Indeed, Western blot analysis of CSE protein in homogenates of the entire mesenteric vascular tree revealed a significant increase in total CSE in IH arteries in comparison with sham arteries, similar to the increased immunofluoresence observed in the adventitial layer of IH 1st-order mesenteric arteries. It is interesting that although CSE is expressed in all vessel wall layers, disrupting the endothelium prevents CSE’s vasoregulatory function. One possibility is that CSE in smooth muscle and adventitial cells produces little vasoactive H₂S or is associated with enzymes that rapidly degrade H₂S. Alternatively, the endothelium may be the target rather than the source of the H₂S. Indeed, recent studies by Schleifenbaum et al²² demonstrated vascular effects of periad-ventitial H₂S production in rat aorta. However, in the mesenteric arteries, endothelial CSE appears to promote vasodilation and IH appears to impair this pathway.

The H₂S donor NaHS dilated PE-constricted arteries from both groups with a slightly greater dilation in IH arteries, demonstrating that IH arteries have an intact response to H₂S and that loss of endogenous H₂S may even cause compensatory upregulation of H₂S targets. Similarly, midsized mesenteric arteries from CSE^–/– mice exhibit greater dilation to exogenous H₂S than do arteries from wild-type littermates.¹² Thus enhanced myogenic tone in IH arteries is likely caused by loss of production or increased scavenging of H₂S. We observed NaHS-dependent dilation at much lower concentrations than has been previously reported with a consistent dilation in response to H₂S in the nanomole-per-liter range. This low concentration, more comparable to tissue H₂S levels measured by gas chromatography as 17 nmol/L,²³ suggests that small arteries may be more sensitive to H₂S than are larger arteries. We found that basal and cysteine-induced H₂S production from arterial homogenates was below the detection limit of a colorometric assay capable of detecting 100 nmol/L levels, although this same assay detected BCA-sensitive H₂S production in kidney homogenates. Thus exposure to high concentrations of exogenous H₂S likely elicits pharmacological rather than physiological effects of this molecule.

Although H₂S vasodilation can be mediated by activation of K_ATP channels,¹⁰ the K_ATP channel blocker glibenclamide had little effect on NaHS-induced dilation. However, H₂S dilation was prevented by constricting arteries with depolarizing concentrations of KCl, confirming that H₂S activates potassium channels to cause vasodilation. BK_Ca channels can also limit myogenic tone,²⁴ and H₂S activates BK_Ca channels in rat pituitary tumor cells.²⁵ The selective BK_Ca blockers IbTx and paxilline blocked NaHS-induced dilation in both groups across the lower portion of the curve, suggesting that H₂S dilates small mesenteric arteries by activating BK_Ca channels. Vascular BK_Ca channels contain a redox-sensitiveCa²⁺ domain in the cytoplasmic C-terminal, providing a potential mechanism for H₂S sensitivity.²⁶ However, there have been conflicting reports of H₂S effects on BK_Ca channels. In pituitary tumor cells, H₂S activates BK_Ca channels but in carotid body glomus cells H₂S decreased BK_Ca-mediated current.²⁷ A recent study in vascular endothelial cells also demonstrated that BK_Ca channels are activated by H₂S.²⁸ Thus BK_Ca channel– dependent vasodilation in small mesenteric arteries may be mediated by similar effects on the endothelium, or perhaps there are regional differences in channel expression between vascular beds. Endothelial BK_Ca-mediated hyperpolarization could enhance production of vasoactive agents such as NO, and thus we would expect this production to be reduced in IH arteries. This loss could explain the apparent Ca²⁺ sensitization seen in the IH artery myogenic curves, due to the effect of NO to reduce Ca²⁺ sensitization.²⁹ The depolarized state of IH arteries could also activate Rho kinase-Ca²⁺ sensitization as seen in pulmonary arteries.³⁰

Another reported vascular target of H₂S is the voltage-dependent potassium channel, KCNQ.^22,31 Several studies suggest that H₂S produced in adventitial adipocytes activates this channel to cause vasodilation. Our immunofluorescence studies revealed high expression of CSE in the adventitial layer. However, myogenic tone was augmented by endothelium disruption, and cysteine-induced dilation was almost eliminated by disrupting the endothelium, suggesting that adventitial CSE does not play a major role in vasodilation of small mesenteric arteries.

In addition to blocking NaHS dilation, IbTx enhanced myogenic tone and VSM [Ca²⁺] more in sham than IH arteries, consistent with lower BK_Ca channel activity in IH arteries as the cause of greater pressure-induced Ca²⁺ influx and constriction. Furthermore, resting E_m was depolarized in small mesenteric arteries from IH rats in comparison with sham arteries at both 60 and 140 mm Hg and inhibiting CSE-caused depolarization only in sham arteries. Thus endogenous H₂S appears to contribute to resting E_m, and loss of H₂S leads to depolarization in IH arteries. In parallel to the dilation studies, NaHS caused a BK_Ca-dependent hyperpolarization in PE treated sham and IH arteries, returning E_m to nearly the pre-PE potentials. Because the BK_Ca activator, NS1619, dilated both sham and IH arteries similarly, reduced H₂S activation of BK_Ca channels is apparently not due to a lower ability of the channels to be activated.

Recent studies have demonstrated that in some arteries, ECs also express BK_Ca channels, and these can contribute to VSMC hyperpolarization, at least in disease states such as hypoxia.³² The data presented here do not establish the cellular location of H₂S-activated BK_Ca channels, and the recent observation that H₂S activates BK_Ca channels in endothelial cells²⁸ suggests the endothelium may be the target for the IbTx-sensitive vasodilation by H₂S reported here, suggesting future studies on the site of action of H₂S. An additional potential mechanism for H₂S-induced dilation is inhibition of phosphodiesterases to elevate vascular levels of cGMP and cAMP.¹¹ However, the phosphodiesterase inhibitor IBMX did not affect NaHS-induced dilation, suggesting that inhibition of phosphodiesterase does not contribute to H₂S dilation in this vascular bed.

H₂S can combine with O₂^– and NO so that greater synthesis of either could inactivate H₂S after IH exposure.^8,9 Scavenging O₂^– with tiron or inhibiting NO with L-NNA slightly inhibited myogenic tone in IH but not sham arteries. However, myogenic tone was still elevated in IH in comparison with sham arteries. Thus increased scavenging of H₂S by endogenous reactive species may account for a small portion of the loss of H₂S inhibition of myogenic tone in IH arteries.

In light of the emerging role of H₂S as an oxygen sensor stabilized during acute hypoxia,^33–35 it is remarkable that vascular H₂S production is apparently decreased by IH. Therefore chronic IH may have a very different effect on H₂S than would a single acute exposure to hypoxia. Thus 1 hypoxic episode may elevate H₂S but days or weeks of IH exposure appear to downregulate CSE expression, at least in the endothelium, suggesting that additional research is needed to evaluate hypoxia regulation of CSE.

In conclusion, our results suggest that in small mesenteric arteries, H₂S production by endothelial CSE maintains low myogenic tone through E_m hyperpolarization. Two weeks of IH treatment reduces H₂S modulation of both VSM E_m and myogenic tone through decreased endothelial CSE expression and through a modest increase in scavenging by reactive oxygen and nitrogen species. H₂S dilates small endothelium-intact mesenteric arteries through activation of BK_Ca channels, a novel mechanism of vasorelaxation for this gaseous messenger. These studies implicate a unique mechanism of endothelial dysfunction in IH, and suggest that therapies targeting the H₂S signaling could be useful in combating vascular dysfunction and hypertension in sleep apnea patients.

Novelty and Significance.

What Is Known?

• Intermittent hypoxia (IH) is a model for sleep apnea-induced hypertension, and is associated with vascular dysfunction and elevated blood pressure in rats.

• Myogenic tone is a pressure-induced constriction of blood vessels that can be an important regulator of arterial resistance.

• Hydrogen sulfide (H₂S) is a recently described endothelium-derived vasodilator that is responsive to hypoxia.

What New Information Does This Article Contribute?

• H₂S causes vasodilation in small mesenteric arteries through the activation of large-conductance Ca²⁺-activated K⁺ channels (BK_Ca).

• H₂S dilation normally inhibits myogenic tone in small mesenteric arteries, but loss of H₂S production after IH exposure leads to increased myogenic tone.

• IH vascular smooth muscle cells (VSMC) are depolarized in relation to control cells because of this loss of H₂S.

IH enhances vascular contractility to endothelin-1, but its effect on myogenic tone is unclear. Here we report that myogenic tone in small mesenteric arteries was enhanced by IH, and that this effect was through loss of an endothelium-dependent effect. We found that H₂S reduced myogenic tone in control arteries but that this function was lost in IH arteries. The role of H₂Sin regulating myogenic tone is a novel finding and adds to the growing list of the physiological functions of this molecule. Endothelial expression of cystathionine γ-lyase, an H₂S generating enzyme, was reduced in IH arteries, suggesting a mechanism for this loss of H₂S. We also established that H₂S causes vasodilation in these arteries through hyperpolarization of VSMC, and that the BK_Ca channel mediates this effect. These results establish a novel mechanism of sleep apnea–induced hypertension, but may also have implications in a broad range of cardiovascular diseases that are affected by H₂S.

Non-standard Abbreviations and Acronyms

3MST

3-mercaptopyruvate sulfurtransferase

AOA

amino-oxyacetate

BCA

β-cyano-l-alanine

BK_Ca

large-conductance Ca²⁺-activated potassium channel

BSS

Bi (III) subsalicylate

CSE

cystathionine γ-lyase

IBMX

3-isobutyl-1-methylxanthine

IbTx

iberiotoxin

i.d.

inner diameter

IH

intermittent hypoxia

IK

intermediate-conductance Ca²⁺-activated potassium channel

KATP

ATP sensitive potassium channel

L-NNA

N^G-nitro-L-arginine

NOS

Nitric oxide synthase

PAG

DL propargylglycine

PE

phenylephrine

PSS

physiological saline solution

ROS

reactive oxygen species

SK

small-conductance Ca²⁺-activated potassium channel

VGCC

voltage gated Ca²⁺ channel

VSM

vascular smooth muscle