Nancy L. Kanagy

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.

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.

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.

Chronic hypertension produced by infusion of endothelin in rats.

Endothelin, a potent vasoconstrictor peptide synthesized by the vascular smooth muscle endothelium, was chronically infused into male Sprague-Dawley rats to determine whether a long-term increase in circulating endothelin levels would cause a sustained elevation in mean arterial pressure. Rats were catheterized, housed in metabolic cages, and maintained on a fixed 6 meq/day sodium intake throughout the experiment with daily measurements including mean arterial pressure, heart rate, water intake, urine output, urinary sodium excretion, urinary potassium excretion, cardiac output, total peripheral resistance, and stroke volume. Infusion of endothelin-1 (ET-1) at rates of 3, 5, or 7.5 pmol/kg/min for 7 days was associated with significant, sustained, and dose-dependent increases in mean arterial pressure and smaller less consistent elevations in total peripheral resistance. Other parameters were unaffected. Similar results were observed in rats receiving endothelin-3 (ET-3), except that a higher dose of ET-3 was required. These results indicate that elevated blood levels of endothelin could produce a maintained hypertension without sodium or water retention and that the hemodynamic basis for the increased mean arterial pressure is similar to that seen in most other forms of experimental and clinical hypertension.

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.

Impairment of coronary endothelial cell ETB receptor function after short-term inhalation exposure to whole diesel emissions

Air pollutant levels positively correlate with increases in both acute and chronic cardiovascular disease. The pollutant diesel exhaust (DE) increases endothelin (ET) levels, suggesting that this peptide may contribute to DE-induced cardiovascular disease. We hypothesized that acute exposure to DE also enhances ET-1-mediated coronary artery constrictor sensitivity. Constrictor responses to KCl, U-46619, and ET-1 were recorded by videomicroscopy in pressurized intraseptal coronary arteries from rats exposed for 5 h to DE (300 microg/m(3)) or filtered air (Air). ET-1 constriction was augmented in arteries from DE-exposed rats. Nitric oxide synthase (NOS) inhibition [N(omega)-nitro-L-arginine (L-NNA), 100 microM] and endothelium inactivation augmented ET-1 responses in arteries from Air but not DE rats so that after either treatment responses were not different between groups. DE exposure did not affect KCl and U-46619 constrictor responses, while NOS inhibition augmented KCl constriction equally in both groups. Thus basal NOS activity does not appear to be affected by DE exposure. The endothelin type B (ET(B)) receptor antagonist BQ-788 (10 microM) inhibited ET-1 constriction in DE but not Air arteries, and constriction in the presence of the antagonist was not different between groups. Cytokine levels were not different in plasma from DE and AIR rats, suggesting that acute exposure to DE does not cause an immediate inflammatory response. In summary, a 5-h DE exposure selectively increases constrictor sensitivity to ET-1. This augmentation is endothelium-, NOS-, and ET(B) receptor dependent. These data suggest that DE exposure diminishes ET(B) receptor activation of endothelial NOS and augments ET(B)-dependent vasoconstriction. This augmented coronary vasoreactivity to ET-1 after DE, coupled with previous reports that DE induces production of ET-1, suggests that ET-1 may contribute to the increased incidence of cardiac events during acute increases in air pollution levels.

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.

Mechanisms of diesel-induced endothelial nitric oxide synthase dysfunction in coronary arterioles.

Background and objective Increased air pollutants correlate with increased incidence of cardiovascular disease potentially due to vascular dysfunction. We have reported that acute diesel engine exhaust (DE) exposure enhances vasoconstriction and diminishes acetylcholine (ACh)-induced dilation in coronary arteries in a nitric oxide synthase (NOS)-dependent manner. We hypothesize that acute DE inhalation leads to endothelial dysfunction by uncoupling NOS. Methods Rats inhaled fresh DE (300 μg particulate matter/m3) or filtered air for 5 hr. After off-gassing, intraseptal coronary arteries were isolated and dilation to ACh recorded using videomicroscopy. Results Arteries from DE-exposed animals dilated less to ACh than arteries from air-exposed animals. NOS inhibition did not affect ACh dilation in control arteries but increased dilation in the DE group, suggesting NOS does not normally contribute to ACh-induced dilation in coronary arteries but does contribute to endothelial dysfunction after DE inhalation. Cyclooxygenase (COX) inhibition did not affect ACh dilation in the DE group, but combined inhibition of NOS and COX diminished dilation in both groups and eliminated intergroup differences, suggesting that the two pathways interact. Superoxide scavenging increased ACh dilation in DE arteries, eliminating differences between groups. Tetrahydrobiopterin (BH4) supplementation with sepiapterin restored ACh-mediated dilation in the DE group in a NOS-dependent manner. Superoxide generation (dihydroethidium staining) was greater in DE arteries, and superoxide scavenging, BH4 supplementation, or NOS inhibition reduced the signal in DE but not air arteries. Conclusion Acute DE exposure appears to uncouple NOS, increasing reactive oxygen species generation and causing endothelial dysfunction, potentially because of depletion of BH4 limiting its bioavailability.

Chronic infusion of angiotensin receptor antagonists in the hypothalamic paraventricular nucleus prevents hypertension in a rat model of sleep apnea

Sleep apnea is characterized by increased sympathetic activity and is associated with systemic hypertension. Angiotensin (Ang) peptides have previously been shown to participate in the regulation of sympathetic tone and arterial pressure in the hypothalamic paraventricular nucleus (PVN) neurons. We investigated the role of endogenous Ang peptides within the PVN to control blood pressure in a rat model of sleep apnea-induced hypertension. Male Sprague-Dawley rats (250 g), instrumented with bilateral guide cannulae targeting the PVN, received chronic infusion of Ang antagonists (A-779, Ang-(1-7) antagonist; losartan and ZD7155, AT(1) antagonists; PD123319, AT(2) receptor antagonist, or saline vehicle). A separate group received an infusion of the GABA(A) receptor agonist (muscimol) to inhibit PVN neuronal activity independent of angiotensin receptors. After cannula placement, rats were exposed during their sleep period to eucapnic intermittent hypoxia (IH; nadir 5% O(2); 5% CO(2) to peak 21% O(2); 0% CO(2)) 20 cycles/h, 7 h/day, for 14 days while mean arterial pressure (MAP) was measured by telemetry. In rats receiving saline, IH exposure significantly increased MAP (+12±2 mm Hg vs. Sham -2±1 mm Hg P<0.01). Inhibition of PVN neurons with muscimol reversed the increase in MAP in IH rats (MUS: -9±4 mm Hg vs. vehicle +12±2 mm Hg; P<0.01). Infusion of any of the Ang antagonists also prevented the rise in MAP induced by IH (A-779: -5±1 mm Hg, losartan: -9±4 mm Hg, ZD7155: -11±4 mm Hg and PD123319: -4±3 mm Hg; P<0.01). Our results suggest that endogenous Ang peptides acting in the PVN contribute to IH-induced increases in MAP observed in this rat model of sleep apnea-induced hypertension.

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.