Benjimen R. Walker

Role of Endothelin in Intermittent Hypoxia-Induced Hypertension

Clinical studies suggest that sleep apnea causes systemic hypertension. In addition, patients with sleep apnea have elevated plasma levels of endothelin-1 (ET-1). We hypothesized that the intermittent hypoxia/hypercapnia (IH) associated with sleep apnea causes hypertension by increasing ET-1 production. To test this hypothesis, rats with arterial and venous catheters were placed in Plexiglas chambers. IH rat chambers were flushed with an N2-CO2 mixture for 90 seconds to achieve hypoxia/hypercapnia (5% O2–5% CO2) followed by 90 seconds of compressed air to achieve normoxia (21% O2–0% CO2). Control rat chambers were flushed with 90 seconds of air-air cycles. Cycles for both groups were repeated 8 hours per day for 11 days. Resting mean arterial pressure (MAP) and heart rate were recorded daily before the start of exposure. After 11 days, MAP was significantly elevated in IH rats compared with initial MAP (109±5 mm Hg initial, 139±11 mm Hg day 11) and compared with air-air rats (110±4 mm Hg). On day 11, cumulative doses of PD145065 (a nonselective ET-receptor antagonist) were administered intravenously to the rats breathing room air. PD145065 caused a dose-dependent decrease in MAP in IH rats but did not alter MAP in air-air rats. Plasma ET-1 measured by radioimmunoassay was significantly increased on days 5 and 11 in the IH rats compared with day 1 and compared with air-air rats. There was no significant change in plasma ET-1 over time in air-air rats. We conclude that IH exposure increases both MAP and plasma ET-1 and that the increased ET-1 may contribute to the hypertension.

Reactive oxygen species mediate RhoA/Rho kinase-induced Ca2+ sensitization in pulmonary vascular smooth muscle following chronic hypoxia

Recent evidence supports a prominent role for Rho kinase (ROK)-mediated pulmonary vasoconstriction in the development and maintenance of chronic hypoxia (CH)-induced pulmonary hypertension. Endothelin (ET)-1 contributes to the pulmonary hypertensive response to CH, and recent studies by our laboratory and others indicate that pulmonary vascular reactivity following CH is largely independent of changes in vascular smooth muscle (VSM) intracellular free calcium concentration ([Ca(2+)](i)). In addition, CH increases generation of reactive oxygen species (ROS) in pulmonary arteries, which may underlie the shift toward ROK-dependent Ca(2+) sensitization. Therefore, we hypothesized that ROS-dependent RhoA/ROK signaling mediates ET-1-induced Ca(2+) sensitization in pulmonary VSM following CH. To test this hypothesis, we determined the effect of pharmacological inhibitors of ROK, myosin light chain kinase (MLCK), tyrosine kinase (TK), and PKC on ET-1-induced vasoconstriction in endothelium-denuded, Ca(2+)-permeabilized small pulmonary arteries from control and CH (4 wk at 0.5 atm) rats. Further experiments examined ET-1-mediated, ROK-dependent phosphorylation of the regulatory subunit of myosin light chain phosphatase (MLCP), MYPT1. Finally, we measured ET-1-induced ROS generation in dihydroethidium-loaded small pulmonary arteries and investigated the role of ROS in mediating ET-1-induced, RhoA/ROK-dependent Ca(2+) sensitization using the superoxide anion scavenger, tiron. We found that CH increases ET-1-induced Ca(2+) sensitization that is sensitive to inhibition of ROK and MLCK, but not PKC or TK, and correlates with ROK-dependent MYPT1(Thr696) phosphorylation. Furthermore, tiron inhibited basal and ET-1-stimulated ROS generation, RhoA activation, and VSM Ca(2+) sensitization following CH. We conclude that CH augments ET-1-induced Ca(2+) sensitization through ROS-dependent activation of RhoA/ROK signaling in pulmonary VSM.

Augmented Endothelin Vasoconstriction in Intermittent Hypoxia-Induced Hypertension

We reported previously that simulating sleep apnea in rats by exposing them 7 hours per day to intermittent hypoxia/hypercapnia (IH) elevates plasma endothelin-1 and causes hypertension, which is reversed by an endothelin-1 antagonist. We hypothesized that in this model of sleep apnea–induced hypertension, vascular sensitivity to endothelin-1 is increased in combination with the elevated plasma endothelin-1 to cause the endothelin-1–dependent hypertension. In small mesenteric arteries with endothelial function disabled by passing air through the lumen, diameter and vessel wall [Ca2+] were recorded simultaneously. IH arteries demonstrated increased constrictor sensitivity to endothelin-1 (percentage max constriction 100±0% IH versus 80±10% Sham; P<0.05). This was accompanied by increased calcium sensitivity of IH arteries. In contrast, constrictor sensitivity and increases in vessel wall [Ca2+] to KCl and phenylephrine were not different between IH and Sham arteries. We have shown previously that endothelin-1 constriction in mesenteric arteries is mediated by endothelin A receptors. In the current study, the selective increase in endothelin-1 constriction in IH resistance arteries was accompanied by increased expression of endothelin A receptor expression (densitometry units 271±23 IH versus 158±25 Sham; P<0.05). Thus, IH hypertension appears to cause alterations in signaling components unique to endothelin-1 at the receptor level and in postreceptor signaling that increases calcium sensitivity during endothelin A activation. Future studies will determine the specific changes in vascular smooth muscle signaling in IH hypertension causing this augmented contractile phenotype.

Maintained upregulation of pulmonary eNOS gene and protein expression during recovery from chronic hypoxia

We previously demonstrated augmented endothelium-derived nitric oxide (EDNO)-dependent pulmonary arterial dilation and increased arterial endothelial nitric oxide synthase (eNOS) levels in chronic hypoxic (CH) and monocrotaline (nonhypoxic) models of pulmonary arterial hypertension. Therefore, we hypothesized that the long-term elevation of arterial eNOS levels associated with CH is related to pulmonary hypertension or some factor(s) associated with hypertension and not directly to hypoxia. To test this hypothesis, we examined responses to the EDNO-dependent dilator ionomycin in U-46619-constricted, isolated, saline-perfused lungs from control rats, CH (4 wk at 380 mmHg) rats, and rats previously exposed to CH but returned to normoxia for 4 days or 2 wk. Microvascular pressure was assessed by double-occlusion technique, allowing calculation of segmental resistances. In addition, vascular eNOS immunoreactivity was assessed by quantitative immunohistochemistry, and eNOS mRNA abundance was determined by RT-PCR assays. Our findings indicate that 4-day and 2-wk posthypoxic rats exhibit persistent pulmonary hypertension, likely due to maintained arterial remodeling and polycythemia associated with prior exposure to CH. Furthermore, arterial dilation to ionomycin was augmented in lungs from each experimental group compared with controls. Finally, arterial eNOS immunoreactivity and whole lung eNOS mRNA levels remained elevated in posthypoxic animals. These findings suggest that altered vascular mechanical forces or vascular remodeling contributes to enhanced EDNO-dependent arterial dilation and upregulation of arterial eNOS in various models of established pulmonary hypertension.

Chronic hypoxia induces Rho kinase-dependent myogenic tone in small pulmonary arteries

Myogenic tone in the pulmonary vasculature of normoxic adult animals is minimal or nonexistent. Whereas chronic hypoxia (CH) increases basal tone in pulmonary arteries, it is unclear if a portion of this elevated tone is due to development of myogenicity. Since basal arterial RhoA activity and Rho kinase (ROK) expression are augmented by CH, we hypothesized that CH elicits myogenic reactivity in pulmonary arteries through ROK-dependent vascular smooth muscle (VSM) Ca(2+) sensitization. To test this hypothesis, we assessed the contribution of ROK to basal tone and pressure-induced vasoconstriction in endothelium-disrupted pulmonary arteries [50-300 microm inner diameter (ID)] from control and CH [4 wk at 0.5 atmosphere (atm)] rats. Arteries were loaded with fura-2 AM to continuously monitor VSM intracellular Ca(2+) concentration ([Ca(2+)](i)). Basal VSM [Ca(2+)](i) was not different between groups. The ROK inhibitor, HA-1077 (100 nM to 30 microM), caused a concentration-dependent reduction of basal tone in CH arteries but had no effect in control vessels. In contrast, PKC inhibition with GF109203X (1 microM) did not alter basal tone. Furthermore, significant vasoconstriction in response to stepwise increases in intraluminal pressure (5-45 mmHg) was observed at 12, 15, 25, and 35 mmHg in arteries (50-200 microm ID) from CH rats. This myogenic reactivity was abolished by HA-1077 (10 microM) but not by GF109203X. VSM [Ca(2+)](i) was unaltered by HA-1077, GF109203X, or increases in pressure in either group. Myogenicity was not observed in larger vessels (200-300 microm ID). We conclude that CH induces myogenic tone in small pulmonary arteries through ROK-dependent myofilament Ca(2+) sensitization.

Intermittent Hypoxia in Rats Increases Myogenic Tone Through Loss of Hydrogen Sulfide Activation of Large-Conductance Ca 2+ -Activated Potassium Channels

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 &ggr;-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 sham control rat arteries, and VSM membrane potential was depolarized in IH in comparison with sham arteries. Endothelium inactivation or scavenging of H2S enhanced myogenic tone in sham arteries to the level of IH. Inhibiting CSE also enhanced myogenic tone and depolarized VSM in sham but not IH arteries. Similar results were seen in cerebral arteries. Exogenous H2S dilated and hyperpolarized sham 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 sham than IH. CSE immunofluorescence was less in the endothelium of IH than in sham 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 BKCa channels.

Role of endothelial carbon monoxide in attenuated vasoreactivity following chronic hypoxia.

Chronic hypoxic exposure has been previously demonstrated to attenuate systemic vasoconstrictor activity to a variety of agents. This attenuated responsiveness is observed not only in conscious animals but in isolated vascular preparations as well. Because hypoxia has been documented to increase heme oxygenase (HO) levels and the subsequent production of the vasodilator CO in vitro, we hypothesized that the blunted reactivity observed with chronic hypoxia (CH) may be in part due to increased HO activity. In thoracic aortic rings from CH rats, cumulative dose-response curves to phenylephrine (PE) in the presence of the nitric oxide (NO) synthase inhibitor N ω-nitro-l-arginine (l-NNA) and the HO inhibitor zinc protoporphyrin 9 (ZnPPIX) elicited increased contractility compared with CH rings treated with onlyl-NNA. Similar results were observed in rings incubated overnight with the HO-inducing agent sodium m-arsenite. In contrast, contractile responses in rings from control rats were unaffected by the HO inhibitor. Furthermore, endothelium-denuded rings from either control or CH rats did not exhibit an increase in reactivity to PE following ZnPPIX incubation. ZnPPIX had no effect on relaxant responses to the NO donor S-nitroso- N-penicillamine, suggesting that its actions were specific to HO inhibition. Finally, aortic rings exhibited dose-dependent relaxant responses to exogenous CO that were endothelium independent and blocked by an inhibitor of soluble guanylyl cyclase. The other products of HO enzyme activity, iron and biliverdin, were without effect on vasoreactivity. Thus we conclude that the attenuated vasoreactivity to PE following CH is likely to involve the induction of endothelial HO and the subsequent enhanced production of CO.Chronic hypoxic exposure has been previously demonstrated to attenuate systemic vasoconstrictor activity to a variety of agents. This attenuated responsiveness is observed not only in conscious animals but in isolated vascular preparations as well. Because hypoxia has been documented to increase heme oxygenase (HO) levels and the subsequent production of the vasodilator CO in vitro, we hypothesized that the blunted reactivity observed with chronic hypoxia (CH) may be in part due to increased HO activity. In thoracic aortic rings from CH rats, cumulative dose-response curves to phenylephrine (PE) in the presence of the nitric oxide (NO) synthase inhibitor Nomega-nitro-L-arginine (L-NNA) and the HO inhibitor zinc protoporphyrin 9 (ZnPPIX) elicited increased contractility compared with CH rings treated with only L-NNA. Similar results were observed in rings incubated overnight with the HO-inducing agent sodium m-arsenite. In contrast, contractile responses in rings from control rats were unaffected by the HO inhibitor. Furthermore, endothelium-denuded rings from either control or CH rats did not exhibit an increase in reactivity to PE following ZnPPIX incubation. ZnPPIX had no effect on relaxant responses to the NO donor S-nitroso-N-penicillamine, suggesting that its actions were specific to HO inhibition. Finally, aortic rings exhibited dose-dependent relaxant responses to exogenous CO that were endothelium independent and blocked by an inhibitor of soluble guanylyl cyclase. The other products of HO enzyme activity, iron and biliverdin, were without effect on vasoreactivity. Thus we conclude that the attenuated vasoreactivity to PE following CH is likely to involve the induction of endothelial HO and the subsequent enhanced production of CO.

Hydrogen sulfide dilates rat mesenteric arteries by activating endothelial large-conductance Ca2+-activated K+ channels and smooth muscle Ca2+ sparks

We have previously shown that hydrogen sulfide (H₂S) reduces myogenic tone and causes relaxation of phenylephrine (PE)-constricted mesenteric arteries. This effect of H₂S to cause vasodilation and vascular smooth muscle cell (VSMC) hyperpolarization was mediated by large-conductance Ca(2+)-activated potassium channels (BKCa). Ca(2+) sparks are ryanodine receptor (RyR)-mediated Ca(2+)-release events that activate BKCa channels in VSMCs to cause membrane hyperpolarization and vasodilation. We hypothesized that H₂S activates Ca(2+) sparks in small mesenteric arteries. Ca(2+) sparks were measured using confocal microscopy in rat mesenteric arteries loaded with the Ca(2+) indicator fluo-4. VSMC membrane potential (Em) was measured in isolated arteries using sharp microelectrodes. In PE-constricted arteries, the H₂S donor NaHS caused vasodilation that was inhibited by ryanodine (RyR blocker), abluminal or luminal iberiotoxin (IbTx, BKCa blocker), endothelial cell (EC) disruption, and sulfaphenazole [cytochrome P-450 2C (Cyp2C) inhibitor]. The H₂S donor NaHS (10 μmol/l) increased Ca(2+) sparks but only in the presence of intact EC and this was blocked by sulfaphenazole or luminal IbTx. Inhibiting cystathionine γ-lyase (CSE)-derived H2S with β-cyano-l-alanine (BCA) also reduced VSMC Ca(2+) spark frequency in mesenteric arteries, as did EC disruption. However, excess CSE substrate homocysteine did not affect spark activity. NaHS hyperpolarized VSMC Em in PE-depolarized mesenteric arteries with intact EC and also hyperpolarized EC Em in arteries cut open to expose the lumen. This hyperpolarization was prevented by ryanodine, sulfaphenazole, and abluminal or luminal IbTx. BCA reduced IbTx-sensitive K(+) currents in freshly dispersed mesenteric ECs. These results suggest that H₂S increases Ca(2+) spark activity in mesenteric artery VSMC through activation of endothelial BKCa channels and Cyp2C, a novel vasodilatory pathway for this emerging signaling molecule.

Endothelin type A receptor antagonist normalizes blood pressure in rats exposed to eucapnic intermittent hypoxia

We have reported that eucapnic intermittent hypoxia (E-IH) causes systemic hypertension, elevates plasma endothelin 1 (ET-1) levels, and augments vascular reactivity to ET-1 and that a nonspecific ET-1 receptor antagonist acutely lowers blood pressure in E-IH-exposed rats. However, the effect of chronic ET-1 receptor inhibition has not been evaluated, and the ET receptor subtype mediating the vascular effects has not been established. We hypothesized that E-IH causes systemic hypertension through the increased ET-1 activation of vascular ET type A (ET(A)) receptors. We found that mean arterial pressure (MAP) increased after 14 days of 7 h/day E-IH exposure (109 +/- 2 to 137 +/- 4 mmHg; P < 0.005) but did not change in sham-exposed rats. The ET(A) receptor antagonist BQ-123 (10 to 1,000 nmol/kg iv) acutely decreased MAP dose dependently in conscious E-IH but not sham rats, and continuous infusion of BQ-123 (100 nmol.kg(-1).day(-1) sc for 14 days) prevented E-IH-induced increases in MAP. ET-1-induced constriction was augmented in small mesenteric arteries from rats exposed 14 days to E-IH compared with those from sham rats. Constriction was blocked by the ET(A) receptor antagonist BQ-123 (10 microM) but not by the ET type B (ET(B)) receptor antagonist BQ-788 (100 microM). ET(A) receptor mRNA content was greater in renal medulla and coronary arteries from E-IH rats. ET(B) receptor mRNA was not different in any tissues examined, whereas ET-1 mRNA was increased in the heart and in the renal medulla. Thus augmented ET-1-dependent vasoconstriction via vascular ET(A) receptors appears to elevate blood pressure in E-IH-exposed rats.

Chronic hypoxia augments depolarization-induced Ca2+ sensitization in pulmonary vascular smooth muscle through superoxide-dependent stimulation of RhoA

Rho kinase (ROCK)-dependent vasoconstriction has been implicated as a major factor in chronic hypoxia (CH)-induced pulmonary hypertension. This component of pulmonary hypertension is associated with arterial myogenicity and increased vasoreactivity to receptor-mediated agonists and depolarizing stimuli resulting from ROCK-dependent myofilament Ca(2+) sensitization. On the basis of separate lines of evidence that CH increases pulmonary arterial superoxide (O(2)(-)) generation and that O(2)(-) stimulates RhoA/ROCK signaling in vascular smooth muscle (VSM), we hypothesized that depolarization-induced O(2)(-) generation mediates enhanced RhoA-dependent Ca(2+) sensitization in pulmonary VSM following CH. To test this hypothesis, we determined effects of the ROCK inhibitor HA-1077 and the O(2)(-)-specific spin trap tiron on vasoconstrictor reactivity to depolarizing concentrations of KCl in isolated lungs and Ca(2+)-permeabilized, pressurized small pulmonary arteries from control and CH (4 wk at 0.5 atm) rats. Using the same vessel preparation, we examined effects of CH on KCl-dependent VSM membrane depolarization and O(2)(-) generation using sharp electrodes and the fluorescent indicator dihydroethidium, respectively. Finally, using a RhoA-GTP pull-down assay, we investigated the contribution of O(2)(-) to depolarization-induced RhoA activation. We found that CH augmented KCl-dependent vasoconstriction through a Ca(2+) sensitization mechanism that was inhibited by HA-1077 and tiron. Furthermore, CH caused VSM membrane depolarization that persisted with increasing concentrations of KCl, enhanced KCl-induced O(2)(-) generation, and augmented depolarization-dependent RhoA activation in a O(2)(-)-dependent manner. These findings reveal a novel mechanistic link between VSM membrane depolarization, O(2)(-) generation, and RhoA activation that mediates enhanced myofilament Ca(2+) sensitization and pulmonary vasoconstriction following CH.