Role for PKCβ in enhanced endothelin-1-induced pulmonary vasoconstrictor reactivity following intermittent hypoxia

Intermittent hypoxia (IH) resulting from sleep apnea causes both systemic and pulmonary hypertension. Enhanced endothelin-1 (ET-1)-induced vasoconstrictor reactivity is thought to play a central role in the systemic hypertensive response to IH. However, whether IH similarly increases pulmonary vasoreactivity and the signaling mechanisms involved are unknown. The objective of the present study was to test the hypothesis that IH augments ET-1-induced pulmonary vasoconstrictor reactivity through a PKCβ-dependent signaling pathway. Responses to ET-1 were assessed in endothelium-disrupted, pressurized pulmonary arteries (～150 μm inner diameter) from eucapnic-IH [(E-IH) 3 min cycles, 5% O(2)-5% CO(2)/air flush, 7 h/day; 4 wk] and sham (air-cycled) rats. Arteries were loaded with fura-2 AM to monitor vascular smooth muscle (VSM) intracellular Ca(2+) concentration ([Ca(2+)](i)). E-IH increased vasoconstrictor reactivity without altering Ca(2+) responses, suggestive of myofilament Ca(2+) sensitization. Consistent with our hypothesis, inhibitors of both PKCα/β (myr-PKC) and PKCβ (LY-333-531) selectively decreased vasoconstriction to ET-1 in arteries from E-IH rats and normalized responses between groups, whereas Rho kinase (fasudil) and PKCδ (rottlerin) inhibition were without effect. Although E-IH did not alter arterial PKCα/β mRNA or protein expression, E-IH increased basal PKCβI/II membrane localization and caused ET-1-induced translocation of these isoforms away from the membrane fraction. We conclude that E-IH augments pulmonary vasoconstrictor reactivity to ET-1 through a novel PKCβ-dependent mechanism that is independent of altered PKC expression. These findings provide new insights into signaling mechanisms that contribute to vasoconstriction in the hypertensive pulmonary circulation.     

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.     

PKC activates BKCa channels in rat pulmonary arterial smooth muscle via cGMP-dependent protein kinase

Normally, signaling mechanisms that activate large-conductance, calcium- and voltage-activated potassium (BK(Ca)) channels in pulmonary vascular smooth muscle cause pulmonary vasodilatation. BK(Ca)-channel modulation is important in the regulation of pulmonary arterial pressure, and inhibition (decrease in the opening probability) of the BK(Ca) channel has been implicated in the development of pulmonary vasoconstriction. Protein kinase C (PKC) causes pulmonary vasoconstriction, but little is known about the effect of PKC on BK(Ca)-channel activity in pulmonary vascular smooth muscle. Accordingly, studies were done to determine the effect of PKC on BK(Ca)-channel activity using patch-clamp studies in pulmonary arterial smooth muscle cells (PASMCs) of the Sprague-Dawley rat. The PKC activators phorbol myristate acetate (PMA) and thymeleatoxin opened BK(Ca) channels in single Sprague-Dawley rat PASMC. The activator response to both PMA and thymeleatoxin on BK(Ca)-channel activity was blocked by G?-6983, which selectively blocks PKC-alpha, -delta, -gamma, and -zeta, and by rottlerin, which selectively inhibits PKC-delta. In addition, the specific cyclic GMP-dependent protein kinase antagonist KT-5823 blocked the responses to PMA and thymelatoxin, whereas the specific cyclic AMP-dependent protein kinase blocker KT-5720 had no effect. In isolated pulmonary arterial vessels, both PMA and forskolin caused vasodilatation, which was inhibited by KT-5823, G?-6983, or the BK(Ca)-channel blocker tetraethylammonium. The results of this study indicate that activation of specific PKC isozymes increases BK(Ca)-channel activity in Sprague-Dawley rat PASMC via cyclic GMP-dependent protein kinase, which suggests a unique signaling mechanism for vasodilatation.     

Effect of changes in pH on wall tension in isolated rat pulmonary artery: role of the RhoA/Rho-kinase pathway

Pulmonary arteries (PA) are resistant to the vasodilator effects of extracellular acidosis in systemic vessels; the mechanism underlying this difference between systemic and pulmonary circulations has not been elucidated. We hypothesized that RhoA/Rho-kinase-mediated Ca2+ sensitization pathway played a greater role in tension development in pulmonary than in systemic vascular smooth muscle and that this pathway was insensitive to acidosis. In arterial rings contracted with the alpha1-agonist phenylephrine (PE), the Rho-kinase inhibitor Y-27632 (< or =3 microM) induced greater relaxation in precontracted PA rings than in aortic rings. In PA rings stimulated by PE, the activation of RhoA was greater than in aorta. Normocapnic acidosis (NA) induced a smaller relaxation in precontracted PA than in aorta. However, in the presence of nifedipine and thapsigargin, when PE-induced contraction was predominantly mediated by Rho-kinase, the relaxant effect of NA was reduced and similar in both vessel types. Furthermore, in the presence of Y-27632, NA induced a greater relaxation in both PA and aorta, which was similar in both vessels. Finally, in alpha-toxin-permeabilized smooth muscle, PE-induced contraction at constant Ca2+ activity was inhibited by Y-27632 and unaffected by acidosis. These results indicate that Ca2+ sensitization induced by the RhoA/Rho-kinase pathway played a greater role in agonist-induced vascular smooth muscle contraction in PA than in aorta and that tension mediated by this pathway was insensitive to acidosis. The predominant role of the RhoA/Rho-kinase pathway in the pulmonary vasculature may account for the resistance of this circulation to the vasodilator effect of acidosis observed in the systemic circulation.     

Analysis of responses to endothelins in the rabbit pulmonary and systemic vascular beds

The effects of two isoforms of human endothelin (ET) on the pulmonary and systemic vascular beds were compared in the anesthetized intact-chest rabbit under conditions of constant pulmonary blood flow and left atrial pressure. Intralobar bolus injections of ET-1 (0.1-1 micrograms) and ET-3 (1-3 micrograms) produced modest vasoconstriction in the pulmonary vascular bed, whereas both peptides decreased systemic arterial pressure. The pulmonary vasoconstrictor response to ET-1 and ET-3 was inhibited by intralobar infusion of nitrendipine but was not altered by indomethacin. In contrast to the small effects of ET-1 and ET-3 on intact pulmonary resistance vessels, both peptides markedly contracted isolated pulmonary conductance vessels, with greater activity on venous than on arterial segments. Intravenous bolus injection of ET-1 (0.1-0.3 micrograms) or ET-3 (0.3-1 microgram) decreased systemic arterial pressure, increased cardiac output, and markedly decreased systemic vascular resistance. Higher doses of ET-1 produce a biphasic systemic vascular response with a prominent secondary pressor component. The present data suggest that the pulmonary vasoconstrictor activity of ET-1 is greater than that of ET-3 and their pressor activity depends on an extracellular source of calcium. The pulmonary and systemic hemodynamic effects of ET-1 and ET-3 in the rabbit do not depend on cyclooxygenase products. The systemic vasodilator response to ET-1 is not altered by first-pass lung transit. Furthermore the systemic vasodilator response to both peptides occurs independent of activation of muscarinic, beta 2-adrenergic, and platelet-activating factor receptors. Although ET-1 and ET-3 were initially reported as vasoconstrictor peptides, the present data suggest that, by having unique and potent systemic vasodilator activity, ET-1 and ET-3 act differently in the systemic and pulmonary vascular beds under resting conditions in the rabbit.     

Comparison of vascular and respiratory effects of endothelin-1 in the pig

The haemodynamic and respiratory responses caused by i.v. administration of endothelin-1 (ET-1) (20-100 pmol/kg) were studied in anaesthetized spontaneously breathing pigs. Intravenous bolus administration of synthetic ET-1 (40-100 pmol/kg) caused a transient decrease followed by a long-lasting increase in mean pulmonary arterial pressure and dose dependent vasoconstriction both in the systemic and pulmonary circulations. The effect on pulmonary arterial pressure was biphasic, with an initial transient fall followed by a long-lasting dose dependent increase. A biphasic response of the systemic mean arterial pressure was demonstrated only at a high dose of ET-1 (100 pmol/kg). ET-1 administration did not significantly change breathing pattern or phasic vagal input, but caused a significant decrease in passive compliance. Passive resistances or active compliance and resistances of the respiratory system were not modified. These results suggest that in the pig ET-1 is a more potent constrictor of vascular than of bronchial smooth muscle. The vasoconstrictor activity was greater in the pulmonary than the systemic circulations.     

Protein kinase C inhibits BKCa channel activity in pulmonary arterial smooth muscle

Signaling mechanisms that elevate cyclic AMP (cAMP) activate large-conductance, calcium- and voltage-activated potassium (BKCa) channels in pulmonary vascular smooth muscle and cause pulmonary vasodilatation. BKCa channel modulation is important in the regulation of pulmonary arterial pressure, and inhibition (closing) of the BKCa channel has been implicated in the development of pulmonary vasoconstriction. Protein kinase C (PKC) causes pulmonary vasoconstriction, but little is known about the effect of PKC on BKCa channel activity. Accordingly, studies were done to determine the effect of PKC activation on cAMP-induced BKCa channel activity using patch-clamp studies in pulmonary arterial smooth muscle cells (PASMC) of the fawn-hooded rat (FHR), a recognized animal model of pulmonary hypertension. Forskolin (10 microM), a stimulator of adenylate cyclase and an activator of cAMP, opened BKCa channels in single FHR PASMC, which were blocked by the PKC activators phorbol 12-myristate 13-acetate (100 nM) and thymeleatoxin (100 nM). The inhibitory response by thymeleatoxin on forskolin-induced BKCa channel activity was blocked by G?-6983, which selectively blocks the alpha, beta, delta, gamma, and zeta PKC isozymes, and G?-6976, which selectively inhibits PKC-alpha, PKC-beta, and PKC-mu, but not by rottlerin, which selectively inhibits PKC-delta. Collectively, these results indicate that activation of specific PKC isozymes inhibits cAMP-induced activation of the BKCa channel in pulmonary arterial smooth muscle, which suggests a unique signaling pathway to modulate BKCa channels and subsequently cAMP-induced pulmonary vasodilatation.     

ET-1 activates Ca2+ sparks in PASMC: local Ca2+ signaling between inositol trisphosphate and ryanodine receptors

Ca+ sparks originating from ryanodine receptors (RyRs) are known to cause membrane hyperpolarization and vasorelaxation in systemic arterial myocytes. By contrast, we have found that Ca2+ sparks of pulmonary arterial smooth muscle cells (PASMCs) are associated with membrane depolarization and activated by endothelin-1 (ET-1), a potent vasoconstrictor that mediates/modulates acute and chronic hypoxic pulmonary vasoconstriction. In this study, we characterized the effects of ET-1 on the physical properties of Ca2+ sparks and probed the signal transduction mechanism for spark activation in rat intralobar PASMCs. Application of ET-1 at 0.1-10 nM caused concentration-dependent increases in frequency, duration, and amplitude of Ca2+ sparks. The ET-1-induced increase in spark frequency was inhibited by BQ-123, an ETA-receptor antagonist; by U-73122, a PLC inhibitor; and by xestospongin C and 2-aminoethyl diphenylborate, antagonists of inositol trisphosphate (IP3) receptors (IP3Rs). However, it was unrelated to sarcoplasmic reticulum Ca2+ content, activation of L-type Ca2+ channels, PKC, or cADP ribose. Photorelease of caged-IP3 indicated that Ca2+ release from IP3R could cross-activate RyRs to generate Ca2+ sparks. Immunocytochemistry showed that the distributions of IP3Rs and RyRs were similar in PASMCs. Moreover, inhibition of Ca2+ sparks with ryanodine caused a significant rightward shift in the ET-1 concentration-tension relationship in pulmonary arteries. These results suggest that ET-1 activation of Ca2+ sparks is mediated via the ETA receptor-PLC-IP3 pathway and local Ca2+ cross-signaling between IP3Rs and RyRs; in addition, this novel signaling mechanism contributes significantly to the ET-1-induced vasoconstriction in pulmonary arteries.     

Impaired endothelin-induced vasoconstriction in coronary arteries of Zucker obese rats is associated with uncoupling of [Ca2+]i signaling

Although insulin resistance (IR) is a major risk factor for coronary artery disease, little is known about the regulation of coronary vascular tone in IR by endothelin-1 (ET-1). We examined ET-1 and PGF(2alpha)-induced vasoconstriction in isolated small coronary arteries (SCAs; approximately 250 microM) of Zucker obese (ZO) rats and control Zucker lean (ZL) rats. ET-1 response was assessed in the absence and presence of endothelin type A (ET(A); BQ-123), type B (ET(B); BQ-788), or both receptor inhibitors. ZO arteries displayed reduced contraction to ET-1 compared with ZL arteries. In contrast, PGF(2alpha) elicited similar vasoconstriction in both groups. ET(A) inhibition diminished the ET-1 response in both groups. ET(B) inhibition alone or in combination with ET(A) blockade, however, restored the ET-1 response in ZO arteries to the level of ZL arteries. Similarly, inhibition of endothelial nitric oxide (NO) synthase with N(omega)-nitro-l-arginine methyl ester (l-NAME) enhanced the contraction to ET-1 and abolished the difference between ZO and ZL arteries. In vascular smooth muscle cells from ZO, ET-1-induced elevation of myoplasmic intracellular free calcium concentration ([Ca2+]i) (measured by fluo-4 AM fluorescence), and maximal contractions were diminished compared with ZL, both in the presence and absence of l-NAME. However, increases in [Ca2+]i elicited similar contractions of the vascular smooth muscle cells in both groups. Analysis of protein and total RNA from SCA of ZO and ZL revealed equal expression of ET-1 and the ET(A) and ET(B) receptors. Thus coronary arteries from ZO rats exhibit reduced ET-1-induced vasoconstriction resulting from increased ET(B)-mediated generation of NO and diminished elevation of myoplasmic [Ca2+]i.     

Endothelin in the splanchnic vascular bed of DOCA-salt hypertensive rats

Vascular capacitance is reduced by endothelin-1 (ET-1) in deoxycorticosterone (DOCA)-salt hypertensive rats. This may contribute to hypertension development. Because the splanchnic blood vessels (especially veins) are important in determining vascular capacitance, we tested the hypothesis that ET-1 levels in the splanchnic vasculature are elevated in hypertensive DOCA-salt compared with normotensive rats. Tissue ET-1 content was measured by ELISA in aorta, vena cava, superior mesenteric artery and vein, and small mesenteric arteries and veins from normotensive sham-operated (sham) and 4-wk DOCA-salt rats. We also determined ET-1 concentration in aortic and portal venous blood (draining the nonhepatic splanchnic organs) in anesthetized and conscious sham and DOCA-salt rats before and after acute blockade of ETB receptor-mediated plasma clearance of ET-1. Results showed a higher ET-1 content in veins than in arteries of similar size. However, ET-1 content was similar in vessels from sham and DOCA-salt rats, except in aorta and superior mesenteric artery, where ET-1 content was greater in DOCA-salt rats. ET-1 concentration was significantly higher in portal venous than in aortic blood, indicating net nonhepatic splanchnic release (nNHSR) of ET-1. However, nNHSR of ET-1 was similar in sham and DOCA-salt rats. Although nNHSR of ET-1 increased significantly after ETB receptor blockade in sham rats, it was completely unchanged in DOCA-salt rats. These data suggest that, despite the absence of ETB receptor-mediated plasma clearance of ET-1, neither the venous peptide content nor the net release of ET-1 is increased in the splanchnic vasculature of DOCA-salt rats. These results argue against the hypothesis that increased venomotor tone in DOCA-salt hypertension is caused by increased ET-1 concentration around splanchnic venous smooth muscle cells.     

Smooth muscle F-actin disassembly and RhoA/Rho-kinase signaling during endotoxin-induced alterations in pulmonary arterial compliance

Endotoxemia is associated with changed pulmonary vascular function with respect to vasoreactivity, endothelial permeability, and activation of inducible nitric oxide synthase II (NOSII). However, whether altered passive arterial wall mechanics contribute to this endotoxin-induced pulmonary vascular dysfunction is still unknown. Therefore, we investigated whether endotoxin affects the passive arterial mechanics and compliance of isolated rat pulmonary arteries. Pulmonary arteries of pentobarbital-anesthetized Wistar rats (n = 55) were isolated and exposed to Escherichia coli endotoxin (50 microg/ml) for 20 h. Endotoxin increased pulmonary artery diameter and compliance (transmural pressure = 13 mmHg) in an endothelium-, Ca2+-, or NOSII-induced NO release-independent manner. Interestingly, the endotoxin-induced alterations in the passive arterial mechanics were accompanied by disassembly of the smooth muscle cell (SMC) F-actin cytoskeleton. Disassembly of F-actin by incubation of control arteries with the cytoskeleton-disrupting agent cytochalasin B or the Rho-kinase inhibitor Y-27632 induced a similar increase in passive arterial diameter and compliance. In contrast, RhoA activation by lysophosphatidic acid prevented the endotoxin-induced alterations in the pulmonary SMC F-actin cytoskeleton and passive mechanics. In conclusion, these findings indicate that disassembly of the SMC F-actin cytoskeleton and RhoA/Rho-kinase signaling act as mediators of endotoxin-induced changes in the pulmonary arterial mechanics. They imply the involvement of F-actin rearrangement and RhoA/Rho-kinase signaling in endotoxemia-induced vascular lung injury.     

Endothelin-1 Augments Na+/H+ Exchange Activity in Murine Pulmonary Arterial Smooth Muscle Cells via Rho Kinase

Excessive production of endothelin-1 (ET-1), a potent vasoconstrictor, occurs with several forms of pulmonary hypertension. In addition to modulating vasomotor tone, ET-1 can potentiate pulmonary arterial smooth muscle cell (PASMC) growth and migration, both of which contribute to the vascular remodeling that occurs during the development of pulmonary hypertension. It is well established that changes in cell proliferation and migration in PASMCs are associated with alkalinization of intracellular pH (pH_i), typically due to activation of Na⁺/H⁺ exchange (NHE). In the systemic vasculature, ET-1 increases pH_i, Na⁺/H⁺ exchange activity and stimulates cell growth via a mechanism dependent on protein kinase C (PKC). These results, coupled with data describing elevated levels of ET-1 in hypertensive animals/humans, suggest that ET-1 may play an important role in modulating pH_i and smooth muscle growth in the lung; however, the effect of ET-1 on basal pH_i and NHE activity has yet to be examined in PASMCs. Thus, we used fluorescent microscopy in transiently (3–5 days) cultured rat PASMCs and the pH-sensitive dye, BCECF-AM, to measure changes in basal pH_i and NHE activity induced by increasing concentrations of ET-1 (10⁻¹⁰ to 10⁻⁸ M). We found that application of exogenous ET-1 increased pH_i and NHE activity in PASMCs and that the ET-1-induced augmentation of NHE was prevented in PASMCs pretreated with an inhibitor of Rho kinase, but not inhibitors of PKC. Moreover, direct activation of PKC had no effect on pH_i or NHE activity in PASMCs. Our results indicate that ET-1 can modulate pH homeostasis in PASMCs via a signaling pathway that includes Rho kinase and that, in contrast to systemic vascular smooth muscle, activation of PKC does not appear to be an important regulator of PASMC pH_i.     

Role of Rho-kinase signaling and endothelial dysfunction in modulating blood flow distribution in pulmonary hypertension

Rho-kinase-mediated vasoconstriction and endothelial dysfunction are considered two primary instigators of pulmonary arterial hypertension (PAH). However, their contribution to the adverse changes in pulmonary blood flow distribution associated with PAH has not been addressed. This study utilizes synchrotron radiation microangiography to assess the specific role, and contribution of, Rho-kinase-mediated vasoconstriction and endothelial dysfunction in PAH. Male adult Sprague-Dawley rats were injected with saline (Cont-rats) or monocrotaline (MCT-rats) 3 wk before microangiography was performed on the left lung. We assessed dynamic changes in vessel internal diameter (ID) in response to 1) the Rho-kinase inhibitor fasudil (10 mg/kg iv); or 2) ACh (3 μg · kg?1 · min?1), sodium nitroprusside (SNP, 5 μg · kg?1 · min?1), and N(ω)-nitro-l-arginine methyl ester (l-NAME, 50 mg/kg iv). We observed that MCT-rats had fewer vessels of the microcirculation compared with Cont-rats. The fundamental result of this study is that fasudil improved pulmonary blood flow distribution and reduced pulmonary pressure in PAH rats, not only by dilating already-perfused vessels (ID > 100 μm), but also by restoring blood flow to vessels that had previously been constricted closed (ID < 100 μm). Endothelium-dependent vasodilation was impaired in MCT-rats primarily in vessels with an ID < 200 μm. Moreover the vasoconstrictor response to l-NAME was accentuated in MCT-rats, but only in the 200- to 300-μm vessels. These results highlight the importance of Rho-kinase-mediated control and endothelial control of pulmonary vascular tone in PAH. Indeed, an effective therapeutic strategy for treating PAH should target both the smooth muscle Rho-kinase and endothelial pathways.     

Hypoxic constriction of porcine distal pulmonary arteries: endothelium and endothelin dependence

To determine the role of endothelium in hypoxic pulmonary vasoconstriction (HPV), we measured vasomotor responses to hypoxia in isolated seventh-generation porcine pulmonary arteries < 300 microm in diameter with (E+) and without endothelium. In E+ pulmonary arteries, hypoxia decreased the vascular intraluminal diameter measured at a constant transmural pressure. These constrictions were complete in 30-40 min; maximum at PO(2) of 2 mm Hg; half-maximal at PO(2) of 40 mm Hg; blocked by exposure to Ca(2+)-free conditions, nifedipine, or ryanodine; and absent in E+ bronchial arteries of similar size. Hypoxic constrictions were unaltered by indomethacin, enhanced by indomethacin plus N(G)-nitro-L-arginine methyl ester, abolished by BQ-123 or endothelial denudation, and restored in endothelium-denuded pulmonary arteries pretreated with 10(-10) M endothelin-1 (ET-1). Given previous demonstrations that hypoxia caused contractions in isolated pulmonary arterial myocytes and that ET-1 receptor antagonists inhibited HPV in intact animals, our results suggest that full in vivo expression of HPV requires basal release of ET-1 from the endothelium to facilitate mechanisms of hypoxic reactivity in pulmonary arterial smooth muscle.     

Role of ET-1 receptor binding and [Ca(2+)](i) in contraction of coronary arteries from DOCA-salt hypertensive rats

Hypertension is associated with an increase in coronary artery disease, but little is known about the regulation of coronary vascular tone by endothelin-1 (ET-1) in hypertension. The present study evaluated the mechanisms mediating altered contraction to ET-1 in coronary small arteries from deoxycorticosterone acetate (DOCA)-salt hypertensive rats. DOCA-salt rats exhibited an increase in systolic blood pressure and plasma ET-1 levels compared with placebo rats. Contraction to ET-1 (1 x 10(-11) to 3 x 10(-8) M), measured in isolated coronary small arteries maintained at a constant intraluminal pressure of 40 mmHg, was largely reduced in vessels from DOCA-salt rats compared with placebo rats. To determine the role of endothelin receptor binding in the impaired contraction to ET-1, (125)I-labeled ET-1 receptor binding was measured in membranes isolated from coronary small arteries. Maximum binding (fmol/mg protein) and binding affinity were similar in coronary membranes from DOCA-salt rats compared with placebo rats. Changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) were measured in freshly dissociated coronary small artery smooth muscle cells loaded with fura 2. ET-1 (10(-9) M) produced a 30 +/- 9% increase in [Ca(2+)](i) in smooth muscle cells from placebo rats, but had no effect on cells from DOCA-salt rats (2 +/- 2%). In summary, the ET-1-induced coronary artery contraction and increase in [Ca(2+)](i) are impaired in DOCA-salt hypertensive rats, whereas endothelin receptor binding is not altered. These results suggest endothelin receptor uncoupling from signaling mechanisms and indicate that impaired [Ca(2+)](i) signaling contributes to the decrease in ET-1-induced contraction of coronary small arteries in DOCA-salt hypertensive rats.     

Mechanics of caudal artery relaxation in control and hypertensive rats

Alterations of smooth muscle function can just as easily stem from mechanical alterations in its ability to relax as from alteration in contraction. Since a failure of arterial smooth muscle to relax may contribute to the development of hypertension, we felt it necessary to study the relaxation process in greater depth. The effect of load on the time course of relaxation of rat caudal artery smooth muscle was analyzed either by comparing afterloaded contractions against various loads or by imposing abrupt alterations in load. Unlike mammalian striated muscles in which relaxation was reported sensitive to loading conditions, relaxation in the smooth muscle of the rat caudal artery (n = 17) was found to be largely independent of loading conditions. This type of relaxation has been termed "inactivation-dependent" relaxation; it is typical of muscle tissue in which the calcium sequestering apparatus is poorly developed. Our results suggest that calcium resequestration, or some biochemical process downstream to it, is the rate-limiting step during relaxation in arterial smooth muscle and that this is not qualitatively different for hypertensive arterial smooth muscle. These analytic techniques were used in the study of relaxation of hypertensive vessels. Quantitative analysis of the relaxation curves showed that both isometric and isotonic relaxation time was prolonged in hypertensive arterial smooth muscle. Prolonged isotonic relaxation indicates that hypertensive arteries remain narrowed for prolonged periods compared with normotensive vessels. Such narrowed vessels may be a factor in the increased total peripheral resistance seen in genetic hypertension.     

Prolonged endothelin B receptor blockade causes pulmonary hypertension in the ovine fetus

Endothelin (ET)-1 contributes to regulation of pulmonary vascular tone and structure in the normal ovine fetus and in models of perinatal pulmonary hypertension. The hemodynamic effects of ET-1 are due to activation of its receptors. The ET(A) receptor mediates vasoconstriction and smooth muscle cell proliferation, whereas the ET(B) receptor mediates vasodilation. In a lamb model of chronic intrauterine pulmonary hypertension, ET(B) receptor activity and gene expression are decreased. To determine whether prolonged ET(B) receptor blockade causes pulmonary hypertension, we studied the hemodynamic effects of selective ET(B) receptor blockade with BQ-788. Animals were treated with an infusion of either BQ-788 or vehicle for 7 days. Prolonged BQ-788 treatment increased pulmonary arterial pressure and pulmonary vascular resistance (P < 0.05). The pulmonary vasodilator response to sarafotoxin 6c, a selective ET(B) receptor agonist, was attenuated after 7 days of BQ-788 treatment, demonstrating pharmacological blockade of the ET(B) receptor. Animals treated with BQ-788 had greater right ventricular hypertrophy and muscularization of small pulmonary arteries (P < 0. 05). Lung ET-1 levels were threefold higher in the animals treated with BQ-788 (P < 0.05). We conclude that prolonged selective ET(B) receptor blockade causes severe pulmonary hypertension and pulmonary vascular remodeling in the late-gestation ovine fetus.     

Differential prejunctional effect of captopril and saralasin on neurogenic vasoconstriction in pithed normotensive rats

The present study describes a differential inhibitory effect of captopril and [Sar1 Ala8]angiotensin II (saralasin) on the neurogenic vasoconstriction in pithed normotensive rats. In pithed normotensive rats with intact kidneys captopril more profoundly inhibited the vasopressor response to spinal stimulation than observed for saralasin. Bilateral nephrectomy also diminished the hypertensive response to spinal stimulation. After bilateral nephrectomy, 1 h previously, captopril but not saralasin diminished the hypertensive response to spinal stimulation. After bilateral nephrectomy, 18-24 h previously, captopril did not produce an additional reduction of the vasopressor response to spinal stimulation. In contrast, saralasin significantly potentiated the neurogenic vasoconstriction. The results suggest that both captopril and saralasin diminish the hypertensive response to spinal stimulation by producing dilatation of vascular smooth muscle in pithed normotensive rats. Apart from this common mechanism, a differential effect of captopril and saralasin on the neurogenic vasoconstriction can be observed. In contrast to saralasin, captopril may depress the neurogenic vasoconstriction in pithed normotensive rats by blocking the sympathofacilitatory action induced by subpressor levels of angiotensin II (AII). In pithed normotensive rats, saralasin may mimic the sympathofacilitatory action of subpressor AII.     

Force-velocity relationships in hypertensive arterial smooth muscle

Increased total peripheral resistance is the cardinal haemodynamic disorder in essential hypertension. This could be secondary to alterations in the mechanical properties of vascular smooth muscle. Adequate study has not been made of the force-velocity (F-V) relationship in hypertensive arterial smooth muscle. Increased shortening in arterial smooth muscle would result in greater narrowing of arteries. The objectives of this investigation were to see if there is (i) increased shortening or increased maximum change in muscle length (delta Lmax where L stands for muscle length), (ii) an increased maximum velocity of shortening (Vmax) measured in l omicron per second where l omicron is the optimal muscle length for tension development, and (iii) a difference in maximum isometric tension (P omicron) developed in spontaneously hypertensive rat (SHR; N = 6) compared with normotensive Wistar Kyoto rat (WKY;N = 5) caudal artery strips. An electromagnetic muscle lever was employed in recording force-velocity data. Analysis of these data revealed the following: (a) the SHR mean P omicron of 6.21 +/- 1.01 N/cm2 was not different from the mean WKY P omicron of 6.97 +/- 1.64 N/cm2 (p greater than 0.05); (b) the SHR preparations showed greater shortening for all loads imposed; (c) the SHR Vmax of 0.016 l omicron/s was greater than the WKY Vmax of 0.013 l omicron/s (p less than 0.05). This study provides evidence that while hypertensive arterial smooth muscle is not able to produce more force than normotensive arterial smooth muscle, it is capable of faster and greater shortening. The latter could result in increased narrowing of hypertensive arteries and increased blood pressure.     

Attenuation of acute hypoxic pulmonary vasoconstriction and hypoxic pulmonary hypertension in mice by inhibition of Rho-kinase

RhoA GTPase mediates a variety of cellular responses, including activation of the contractile apparatus, growth, and gene expression. Acute hypoxia activates RhoA and, in turn, its downstream effector, Rho-kinase, and previous studies in rats have suggested a role for Rho/Rho-kinase signaling in both acute and chronically hypoxic pulmonary vasoconstriction. We therefore hypothesized that activation of Rho/Rho-kinase in the pulmonary circulation of mice contributes to acute hypoxic pulmonary vasoconstriction and chronic hypoxia-induced pulmonary hypertension and vascular remodeling. In isolated, salt solution-perfused mouse lungs, acute administration of the Rho-kinase inhibitor Y-27632 (1 x 10(-5) M) attenuated hypoxic vasoconstriction as well as that due to angiotensin II and KCl. Chronic treatment with Y-27632 (30 mg x kg(-1) x day(-1)) via subcutaneous osmotic pump decreased right ventricular systolic pressure, right ventricular hypertrophy, and neomuscularization of the distal pulmonary vasculature in mice exposed to hypobaric hypoxia for 14 days. Analysis of a small number of proximal pulmonary arteries suggested that Y-27632 treatment reduced the level of phospho-CPI-17, a Rho-kinase target, in hypoxic lungs. We also found that endothelial nitric oxide synthase protein in hypoxic lungs was augmented by Y-27632, suggesting that enhanced nitric oxide production might have played a role in the Y-27632-induced attenuation of chronically hypoxic pulmonary hypertension. In conclusion, Rho/Rho-kinase activation is important in the effects of both acute and chronic hypoxia on the pulmonary circulation of mice, possibly by contributing to both vasoconstriction and vascular remodeling.