Leslie C. Fuchs

Angiotensin II Relaxes Microvessels Via the AT2 Receptor and Ca2+-Activated K+ (BKCa) Channels

Angiotensin II (Ang II) is one of the most potent vasoconstrictor substances, yet paradoxically, Ang II may dilate certain vascular beds via an undefined mechanism. Ang II–induced vasoconstriction is mediated by the AT1 receptor, whereas the relative expression and functional importance of the AT2 receptor in regulating vascular resistance and blood pressure are unknown. We now report that Ang II induces relaxation of mesenteric microvessels and that this vasodilatory response was unaffected by losartan, an AT1 receptor antagonist, but was inhibited by PD123,319, a selective antagonist of AT2 receptors. In addition, reverse transcriptase–polymerase chain reaction studies revealed high amounts of AT2 receptor mRNA in smooth muscle from these same microvessels. Ang II–induced relaxation was inhibited by either tetraethylammonium or iberiotoxin, suggesting involvement of the large-conductance, calcium- and voltage-activated potassium (BKCa) channel. Subsequent whole-cell and single-channel patch-clamp studies on single myocytes demonstrated that Ang II increases the activity of BKCa channels. As in our tissue studies, the effect of Ang II on BKCa channels was inhibited by PD123,319, but not by losartan. In light of these consistent findings from tissue physiology, molecular studies, and cellular/molecular physiology, we conclude that Ang II relaxes microvessels via stimulation of the AT2 receptor with subsequent opening of BKCa channels, leading to membrane repolarization and vasodilation. These findings provide evidence for a novel endothelium-independent vasodilatory effect of Ang II.

Vascular and hemodynamic effects of behavioral stress in borderline hypertensive and Wistar-Kyoto rats

In borderline hypertensive rats (BHR), behavioral stress produces hypertension, which has been attributed to increases in sympathetic nervous system activity and peripheral changes in vascular structure. However, the mechanisms mediating development of stress-induced hypertension have not been well defined. Experiments were designed to determine hemodynamic effects and changes in small mesenteric artery (≈300 μm) vascular reactivity in response to 10 days of air-jet stress (2 h/day) in BHR and in Wistar-Kyoto (WKY) rats. The acute stress-induced increase in mean arterial pressure (AP) was impaired in WKY rats compared with BHR on day 1, and habituation developed to the increase in AP in BHR, but not WKY rats. Conversely, WKY rats adapted to the stress-induced tachycardia to a larger extent than BHR. The mechanisms mediating endothelium-dependent relaxation to acetylcholine (ACh) were altered in small mesenteric arteries isolated from WKY rats and BHR after 10 days of air-jet stress. Inhibition of nitric oxide synthase activity had a significantly larger inhibitory effect on ACh-induced relaxation in vessels from stressed compared with control BHR. Also, cyclooxygenase products contributed to ACh-induced relaxation of small mesenteric arteries from stressed WKY rats, but not control WKY rats. Endothelium-independent relaxation to nitroprusside was impaired in vessels from stressed WKY rats, but not stressed BHR. Finally, contraction to phenylephrine was impaired in vessels from stressed BHR, but not WKY rats. In conclusion, changes in vascular reactivity induced by air-jet stress appear to correlate with, and may contribute to, the differential hemodynamic adaptations to stress observed in WKY rats and BHR.In borderline hypertensive rats (BHR), behavioral stress produces hypertension, which has been attributed to increases in sympathetic nervous system activity and peripheral changes in vascular structure. However, the mechanisms mediating development of stress-induced hypertension have not been well defined. Experiments were designed to determine hemodynamic effects and changes in small mesenteric artery (approximately 300 microns) vascular reactivity in response to 10 days of air-jet stress (2 h/day) in BHR and in Wistar-Kyoto (WKY) rats. The acute stress-induced increase in mean arterial pressure (AP) was impaired in WKY rats compared with BHR on day 1, and habituation developed to the increase in AP in BHR, but not WKY rats. Conversely, WKY rats adapted to the stress-induced tachycardia to a larger extent than BHR. The mechanisms mediating endothelium-dependent relaxation to acetylcholine (ACh) were altered in small mesenteric arteries isolated from WKY rats and BHR after 10 days of air-jet stress. Inhibition of nitric oxide synthase activity had a significantly larger inhibitory effect on ACh-induced relaxation in vessels from stressed compared with control BHR. Also, cyclooxygenase products contributed to ACh-induced relaxation of small mesenteric arteries from stressed WKY rats, but not control WKY rats. Endothelium-independent relaxation to nitroprusside was impaired in vessels from stressed WKY rats, but not stressed BHR. Finally, contraction to phenylephrine was impaired in vessels from stressed BHR, but not WKY rats. In conclusion, changes in vascular reactivity induced by air-jet stress appear to correlate with, and may contribute to, the differential hemodynamic adaptations to stress observed in WKY rats and BHR.

Endothelin-1-induced contraction of mesenteric small arteries is mediated by ryanodine receptor Ca2+ channels and cyclic ADP-ribose.

Contraction of vascular smooth muscle by endothelin-1 is dependent on extracellular and intracellular Ca2+. However, the role of ryanodine-sensitive Ca2+ stores in endothelin-1-induced contraction is unknown. Vascular contraction was measured in mesenteric small arteries (200-300 microm intraluminal diameter) isolated from Sprague-Dawley rats and maintained at a constant intraluminal pressure of 40 mm Hg. The presence of functional ryanodine receptor Ca2+ release channels (RyRC) was demonstrated by the finding that ryanodine (10 microM), which locks the RyRC in a subconductance state, produced significant contraction of small arteries in the presence of 15 mM KCl. This effect was inhibited by dantrolene (10 microM), a RyRC inhibitor. Dantrolene significantly reduced the ET(A) receptor-mediated contraction to endothelin-1 (10(-11)-10(-9) M). The ability of dantrolene to reverse contraction induced by endothelin-1 was also determined. Dantrolene (1-10 microM) produced concentration-dependent relaxation of vessels precontracted to 38+/-3% of resting diameter with endothelin-1 but had no effect in vessels precontracted to a similar degree with phenylephrine or KCl. Because activation of RyRC may be dependent on production of cyclic ADP-ribose, the effect of nicotinamide (2 mM), an inhibitor of ADP-ribosyl cyclase, on contraction to endothelin-1 was determined. Nicotinamide had an inhibitory effect similar to that produced by dantrolene. A combination of nicotinamide and dantrolene had no greater effect than either agent alone, suggesting a common pathway for cyclic ADP-ribose and RyRC. In summary, endothelin-1 induces contraction of small mesenteric arteries through ET(A) receptor-mediated production of cyclic ADP-ribose and activation of RyRC.

Effects of antisense oligonucleotide to iNOS on hemodynamic and vascular changes induced by LPS

Lipopolysaccharide (LPS) causes impaired vascular contractility proposed to be mediated by induction of nitric oxide synthase (iNOS). Antisense (AS) oligonucleotide inhibits the translation of target mRNA into functional proteins. We hypothesize that in vivo pretreatment with AS oligonucleotide targeted to iNOS mRNA can prevent LPS-induced hyporeactivity to norepinephrine (NE). Three groups of conscious male Wistar rats received one of the following: saline, AS, or mismatch (MM) oligonucleotide at 0.4 mg/kg iv at 12 and 24 h before LPS (5 mg/kg iv). The fourth group received saline only. Mean arterial pressure (MAP) and heart rate (HR) were continuously recorded before and 6 h after LPS or saline administration. Aorta, lung lavage, and lung tissue were collected for determination of iNOS protein expression and NOS activity. Small mesenteric arteries ( approximately 250 micron) were isolated, denuded of endothelium, and maintained at a constant intraluminal pressure of 40 mmHg for study in vitro. LPS produced significant tachycardia that was not altered by AS or MM oligonucleotide. AS, but not MM oligonucleotide, reduced the accumulation of cGMP, the increase in conversion of L-[3H]arginine to L-[3H]citrulline, and iNOS protein expression in tissue from LPS-treated rats. Small mesenteric arterial contraction to NE was significantly impaired in vessels from LPS-treated rats and was restored by AS, but not MM, oligonucleotide. In a rat model of septic shock, AS oligonucleotide to iNOS mRNA inhibits NOS activity and iNOS protein expression and prevents the vascular hyporeactivity to NE, which may contribute to hypotension in shock.

Anesthesia with sodium pentobarbital enhances lipopolysaccharide-induced cardiovascular dysfunction in rats.

Lipopolysaccharide (LPS)-induced hypotension and impaired aortic contraction to norepinephrine (NE) are thought to be consequent to induction of nitric oxide synthase (iNOS). Anesthesia is often employed in studies of the mechanisms mediating LPS-induced cardiovascular dysfunction in rats. Since sympathetic nervous system activity and compensatory mechanisms can be altered by anesthesia, this study was designed to determine a) if the cardiovascular dysfunction associated with LPS (5 mg/kg, i.v.)-induced endotoxin shock is enhanced in anesthetized compared with conscious male Wistar rats, and b) the potential role of iNOS in these responses to LPS. Arterial pressure and heart rate were continuously measured via a femoral arterial cannula. Six hours after LPS, conscious rats had a stable mean arterial pressure (MAP) and were tachycardic, while anesthetized rats showed a significant decrease in MAP without tachycardia. Small mesenteric arterioles (200–300 μm) were isolated, and the endothelium was removed six h after LPS. Intraluminal diameter was continuously recorded while vessels were maintained at a constant intraluminal pressure of 40 mmHg. Norepinephrine-induced contraction and oscillations/min were impaired to a greater extent in arterioles from LPS-treated anesthetized rats than in those from conscious rats. Calcium-dependent and -independent nitric oxide formation, reflected as cGMP accumulation, were also determined in aortic rings treated with a chelator of Ca2+, EGTA, or the inhibitor of nitric oxide synthase activity, L-NAME. In rings from saline-treated conscious and anesthetized rats, cGMP accumulation was significantly reduced by EGTA and L-NAME, indicating calcium-dependent constitutive (cNOS) activity. However, in aortic rings from LPS-treated conscious and anesthetized rats, cGMP accumulation was not affected by EGTA and was significantly greater in rings from anesthetized vs. conscious rats. These results suggest that cardiovascular dysfunction is more prominent in LPS-treated anesthetized vs. conscious rats. This effect may be related to increased induction of iNOS in the presence of anesthesia.

Behavioral stress alters coronary vascular reactivity in borderline hypertensive rats.

Background Behavioral stress has been proposed to contribute to the occurrence of myocardial ischemia. Objective To investigate the effect of chronic exposure to behavioral stress on the function and structure of the coronary artery of borderline hypertensive rats (BHR). Design BHR were either exposed to an air-jet stress for 2 h/day for 10 days or kept in their cage for 10 days. Methods After 10 days, hemodynamic measurements in conscious animals were recorded, and their hearts were removed for isolation of a left ventricular coronary artery for functional studies or for fixation by retrograde perfusion for study with scanning electron microscopy. Vascular reactivity was measured in isolated coronary arteries (≈250 μm) maintained at an intraluminal diameter of 40 mmHg while the intraluminal diameter was recorded continuously. Results The resting mean arterial pressure and heart rate in conscious, unrestrained BHR were not altered significantly by exposure to 10 days of 2 h/day air-jet stress. Coronary artery relaxation in response to the endothelium-dependent vasodilator acetylcholine was impaired in rats exposed to the air-jet stress compared with that in controls. An attenuated response to exogenous nitric oxide in coronary arteries from stressed BHR was confirmed by the finding of a reduced sensitivity to nitroprusside, which releases nitric oxide independently from the endothelium. However, relaxation of coronary arteries in response to isoproterenol, which acts independently from nitric oxide, was not altered. Coronary artery contraction in response to endothelin-1 and phenylephrine was not altered in vessels taken from BHR exposed to behavioral stress compared with that in vessels from control rats. Scanning electron microscopy of the endothelial surface of the septal coronary artery showed no difference between vessels from control and stressed BHR. Conclusion These results indicate that behavioral stress impairs endothelium-dependent and nitric oxide-mediated coronary relaxation, but does not alter α1-adrenoceptor or endothelin-1-mediated contraction. By impairing coronary artery vascular relaxation, chronic exposure to behavioral stress may contribute to myocardial ischemia.

Decreased endothelin binding and [Ca2+]i signaling in microvessels of DOCA-salt hypertensive rats

Objectives and design The deoxycorticosterone acetate (DOCA)-salt model of hypertension is characterized by elevated vascular endothelin-1 (ET-1) and by reduced contraction to ET-1 in isolated mesenteric small arteries. The decreased contraction to ET-1 may be a compensatory mechanism caused by elevations in ET-1 and arterial pressure. The present study was designed to determine whether down-regulation of endothelin receptors or altered Ca2+ signaling contribute to the decreased contraction to ET-1. Methods and results Contraction to ET-1 (10−11 to 10−8 mol/l) was significantly reduced in isolated mesenteric small arteries (87–286 μm intraluminal diameter) from DOCA-salt rats compared with placebo rats. Membrane protein was obtained for measurement of [125I]ET-1 receptor binding and ETA receptor expression. Maximum binding was significantly reduced in vascular membranes from DOCA-salt rats (670 ± 71 fmol/mg protein) compared with placebo rats (1165 ± 75 fmol/mg protein), but binding affinity was unchanged. Conversely, ETA receptor protein was increased in DOCA-salt rat vessels. To assess Ca2+ signaling, freshly dissociated mesenteric small artery smooth muscle cells were loaded with fura-2 for measurement of the average myoplasmic free Ca2+ concentration ([Ca2+]i). The ET-1 (10−9 mol/l) induced increase in [Ca2+]i was significantly less in cells from DOCA-salt rats compared with from placebo rats. This effect was not due to a loss of L-type Ca2+ channels since expression was increased in membrane protein from DOCA-salt rats compared with placebo rats, as measured by Western blot analysis. Conclusions These findings indicate that decreases in receptor binding and Ca2+ signaling contribute to the impaired contraction to ET-1 in DOCA-salt hypertensive rats. However, these changes are not due to reduced expression of ETA receptors or L-type Ca2+ channels.

Effect of behavioral stress on coronary artery relaxation altered with aging in BHR.

Behavioral stress and aging are associated with an increase in vascular disease. This study determined the mechanisms contributing to changes in endothelium-dependent relaxation of isolated coronary arteries (300-350 μm) induced by exposure to 10 days of air-jet stress (2 h/day) in young (3 mo) and old (18 mo) male borderline hypertensive rats (BHR). Aging, alone, did not alter endothelium-dependent relaxation to acetylcholine (ACh) quantitatively but did alter the mechanisms contributing to relaxation to ACh, which was largely dependent on nitric oxide synthase (NOS) in vessels from old, but not young, BHR. Behavioral stress resulted in an enhanced relaxation to ACh that was dependent on NOS in vessels from young stressed compared with young control BHR. Conversely, relaxation to ACh was reduced in coronary arteries from old stressed compared with old control BHR. In vessels from old control BHR, there was an NOS-independent component of relaxation mediated by opening of K+ channels that was absent in vessels from old stressed BHR. The superoxide anion scavenger, tiron, partially restored relaxation, and inhibition of cyclooxygenase largely restored relaxation to ACh in vessels from old stressed BHR. In summary, the effect of behavioral stress was age dependent. ACh-induced relaxation of coronary arteries was enhanced in an NOS-dependent manner in young BHR and was impaired in old BHR due to superoxide anions, vasoconstrictor cyclooxygenase products, and a loss of K+ channel-mediated relaxation.Behavioral stress and aging are associated with an increase in vascular disease. This study determined the mechanisms contributing to changes in endothelium-dependent relaxation of isolated coronary arteries (300-350 micrometers) induced by exposure to 10 days of air-jet stress (2 h/day) in young (3 mo) and old (18 mo) male borderline hypertensive rats (BHR). Aging, alone, did not alter endothelium-dependent relaxation to acetylcholine (ACh) quantitatively but did alter the mechanisms contributing to relaxation to ACh, which was largely dependent on nitric oxide synthase (NOS) in vessels from old, but not young, BHR. Behavioral stress resulted in an enhanced relaxation to ACh that was dependent on NOS in vessels from young stressed compared with young control BHR. Conversely, relaxation to ACh was reduced in coronary arteries from old stressed compared with old control BHR. In vessels from old control BHR, there was an NOS-independent component of relaxation mediated by opening of K+ channels that was absent in vessels from old stressed BHR. The superoxide anion scavenger, tiron, partially restored relaxation, and inhibition of cyclooxygenase largely restored relaxation to ACh in vessels from old stressed BHR. In summary, the effect of behavioral stress was age dependent. ACh-induced relaxation of coronary arteries was enhanced in an NOS-dependent manner in young BHR and was impaired in old BHR due to superoxide anions, vasoconstrictor cyclooxygenase products, and a loss of K+ channel-mediated relaxation.

Coronary vascular reactivity is improved by endothelin A receptor blockade in DOCA-salt hypertensive rats

Endothelin-1 (ET-1) is thought to play an important role in the development of deoxycorticosterone acetate (DOCA)-salt hypertension. Because hypertension is associated with an increased incidence of coronary artery disease, this study was designed to determine if coronary vascular contraction to ET-1 is altered in DOCA-salt hypertensive rats and to determine the effect of chronic treatment of DOCA-salt rats with the selective ETA receptor antagonist A-127722. Male Sprague-Dawley rats were divided into four groups: DOCA, Placebo, DOCA + A-127722, and Placebo + A-127722. A-127722 was administered in drinking water at a concentration of 8 mg/100 ml. After 3 wk, mean arterial pressure (MAP) was significantly enhanced in DOCA-salt compared with Placebo rats. A-127722 significantly inhibited the increase in MAP. Contraction to ET-1 (10-11 to 3 × 10-8 M) was measured in isolated coronary and mesenteric small arteries (200-300 μm, intraluminal diameter) maintained at a constant intraluminal pressure of 40 mmHg and was significantly impaired in vessels from DOCA-salt compared with Placebo rats. Dose-dependent contractions to KCl were also inhibited in coronary, but only minimally impaired in mesenteric, arteries of DOCA-salt rats. Inhibition of nitric oxide synthase activity did not restore contraction to ET-1 in coronary small arteries. However contractions to ET-1 were enhanced in mesenteric small arteries. Chronic treatment with A-127722 significantly restored contraction to ET-1 in coronary, but not in mesenteric, arteries of DOCA-salt rats. Because ETAreceptor blockade impairs the development of hypertension and improves coronary vascular reactivity, these data indicate that ET-1 plays an important role in coronary vascular dysfunction associated with DOCA-salt hypertension.Endothelin-1 (ET-1) is thought to play an important role in the development of deoxycorticosterone acetate (DOCA)-salt hypertension. Because hypertension is associated with an increased incidence of coronary artery disease, this study was designed to determine if coronary vascular contraction to ET-1 is altered in DOCA-salt hypertensive rats and to determine the effect of chronic treatment of DOCA-salt rats with the selective ETA receptor antagonist A-127722. Male Sprague-Dawley rats were divided into four groups: DOCA, Placebo, DOCA + A-127722, and Placebo + A-127722. A-127722 was administered in drinking water at a concentration of 8 mg/100 ml. After 3 wk, mean arterial pressure (MAP) was significantly enhanced in DOCA-salt compared with Placebo rats. A-127722 significantly inhibited the increase in MAP. Contraction to ET-1 (10(-11) to 3 x 10(-8) M) was measured in isolated coronary and mesenteric small arteries (200-300 micron, intraluminal diameter) maintained at a constant intraluminal pressure of 40 mmHg and was significantly impaired in vessels from DOCA-salt compared with Placebo rats. Dose-dependent contractions to KCl were also inhibited in coronary, but only minimally impaired in mesenteric, arteries of DOCA-salt rats. Inhibition of nitric oxide synthase activity did not restore contraction to ET-1 in coronary small arteries. However contractions to ET-1 were enhanced in mesenteric small arteries. Chronic treatment with A-127722 significantly restored contraction to ET-1 in coronary, but not in mesenteric, arteries of DOCA-salt rats. Because ETA receptor blockade impairs the development of hypertension and improves coronary vascular reactivity, these data indicate that ET-1 plays an important role in coronary vascular dysfunction associated with DOCA-salt hypertension.

Superoxide Anions Contribute to Impaired Endothelium-Dependent Relaxation in Coronary Arteries of Young Cardiomyopathic Hamsters

A genetic model of cardiomyopathy, the Syrian cardiomyopathic hamster, is characterized by myocardial necrosis and dysfunction which may be initiated by inadequate coronary blood flow. The mechanisms mediating coronary vasospasm observed during the development of cardiomyopathy in this model are unknown. The present study utilized isolated coronary arteries (150-250 pm diameter) from cardiomyopathic (M) and Golden Syrian control (C) hamsters to determine if endothelial dysfunction was present during the necrotic stage of cardiomyopathy (60-90 days of age). Intraluminal diameter was continuously recorded in coronary arteries maintained at an intraluminal pressure of 40 mxnhg. In pre-constricted vessels, relaxation to the endothelium-dependent vasodilator, acetylcholine (ACh), was impaired in vessels from M compared to C hamsters. Inhibition of nitric oxide synthase activity with N-nitro-L-arginine (LNA) significantly reduced relaxation to ACh in coronary arteries from C hamsters. However, LNA had little ef...