ET-1-induced pulmonary vasoconstriction shifts from ET(A)- to ET(B)-receptor-mediated reaction after preconstriction.

Endothelin-1 (ET-1) has been reported to induce pulmonary vasoconstriction via either ET(A) or ET(B) receptors, and vasorelaxation after ET-1 injection has been observed. Our study investigated the effects of ET-1 in isolated rabbit lungs, which were studied at basal tone (part I) and after preconstriction (U-46619; part II). Pulmonary arterial pressure (PAP) and lung weight gain were monitored continuously. In part I, ET-1 (10(-8) M; n = 6; control) was injected after pretreatment with the ET(A)-receptor antagonist BQ-123 (10(-6) M; n = 6) or the ET(B)-receptor antagonist BQ-788 (10(-6) M; n = 6). The same protocol was carried out in part II after elevation of pulmonary vascular tone. ET-1 induced an immediate PAP increase (DeltaPAP 4.3 +/- 0.4 mmHg at 10 min) that was attenuated by pretreatment with BQ-123 (P < 0.05 at 10 min and P < 0.01 thereafter) and that was more pronounced after BQ-788 (P < 0.01 at 10 min and P < 0.001 thereafter). In part II, ET-1 induced an immediate rise in PAP with a maximum after 5 min (DeltaPAP 6.3 +/- 1.4 mmHg), leveling off at DeltaPAP 3.2 +/- 0.2 mmHg after 15 min. Pretreatment with BQ-123 failed to attenuate the increase. BQ-788 significantly reduced the peak pressure at 5 min (0.75 +/- 0.4 mmHg; P < 0.001) as well as the plateau pressure thereafter (P < 0.01). We conclude that ET-1 administration causes pulmonary vasoconstriction independent of basal vascular tone, and, at normal vascular tone, the vasoconstriction seems to be mediated via ET(A) receptors. BQ-788 treatment resulted in even more pronounced vasoconstriction. After pulmonary preconstriction, ET(A) antagonism exerted no effects on PAP, whereas ET(B) antagonism blocked the PAP increase. Therefore, ET-1-induced pulmonary vasoconstriction is shifted from an ET(A)-related to an ET(B)-mediated mechanism after pulmonary vascular preconstriction.     

Mechanism of hypoxic pulmonary vasoconstriction involves ET(A) receptor-mediated inhibition of K(ATP) channel

There is controversy on the role of endothelin (ET)-1 in the mechanism of hypoxic pulmonary vasoconstriction (HPV). Although HPV is inhibited by ET-1 subtype A (ET(A))-receptor antagonists in animals, it has been reported that ET(A)-receptor blockade does not affect HPV in isolated lungs. Thus we reassessed the role of ET-1 in HPV in both rats and isolated blood- and physiological salt solution (PSS)-perfused rat lungs. In rats, the ET(A)-receptor antagonist BQ-123 and the nonselective ET(A)- and ET(B)-receptor antagonist PD-145065, but not the ET(B)-receptor antagonist BQ-788, inhibited HPV. Similarly, BQ-123, but not BQ-788, attenuated HPV in blood-perfused lungs. In PSS-perfused lungs, either BQ-123, BQ-788, or the combination of both attenuated HPV equally. Inhibition of HPV by combined BQ-123 and BQ-788 in PSS-perfused lungs was prevented by costimulation with angiotensin II. The ATP-sensitive K(+) (K(ATP))-channel blocker glibenclamide also prevented inhibition of HPV by BQ-123 in both lungs and rats. These results suggest that ET-1 contributes to HPV in both isolated lungs and intact animals through ET(A) receptor-mediated suppression of K(ATP)-channel activity.     

Contribution of the K(Ca) channel to membrane potential and O2 sensitivity is decreased in an ovine PPHN model

Ca2+-sensitive K+ (K(Ca)) channels play an important role in mediating perinatal pulmonary vasodilation. We hypothesized that lung K(Ca) channel function may be decreased in persistent pulmonary hypertension of the newborn (PPHN). To test this hypothesis, pulmonary artery smooth muscle cells (PASMC) were isolated from fetal lambs with severe pulmonary hypertension induced by ligation of the ductus arteriosus in fetal lambs at 125-128 days gestation. Fetal lambs were killed after pulmonary hypertension had been maintained for at least 7 days. Age-matched, sham-operated animals were used as controls. PASMC K+ currents and membrane potentials were recorded using amphotericin B-perforated patch-clamp techniques. The increase in whole cell current normally seen in response to normoxia was decreased (333.9 +/- 63.6% in control vs. 133.1 +/- 16.0% in hypertensive fetuses). The contribution of the K(Ca) channel to the whole cell current was diminished in hypertensive, compared with control, fetal PASMC. In PASMC from hypertensive fetuses, a change from hypoxia to normoxia caused no change in membrane potential compared with a -14.6 +/- 2.8 mV decrease in membrane potential in PASMC from control animals. In PASMC from animals with pulmonary hypertension, 4-aminopyridine (4-AP) caused a larger depolarization than iberiotoxin, whereas in PASMC from control animals, iberiotoxin caused a larger depolarization than 4-AP. These data confirm the hypothesis that the contribution of the K(Ca) channel to membrane potential and O2 sensitivity is decreased in an ovine model of PPHN, and this may contribute to the abnormal perinatal pulmonary vasoreactivity associated with PPHN.     

Inhaled nitric oxide-induced rebound pulmonary hypertension: role for endothelin-1

Clinically significant increases in pulmonary vascular resistance have been noted on acute withdrawal of inhaled nitric oxide (NO). Endothelin (ET)-1 is a vasoactive peptide produced by the vascular endothelium that may participate in the pathophysiology of pulmonary hypertension. The objectives of this study were to determine the effects of inhaled NO on endogenous ET-1 production in vivo in the intact lamb and to determine the potential role of ET-1 in the rebound pulmonary hypertension associated with the withdrawal of inhaled NO. Seven 1-mo-old vehicle-treated control lambs and six PD-156707 (an ET(A) receptor antagonist)-treated lambs were mechanically ventilated. Inhaled NO (40 parts per million) was administered for 24 h and then acutely withdrawn. After 24 h of inhaled NO, plasma ET-1 levels increased by 119.5 +/- 42.2% (P < 0.05). Western blot analysis revealed that protein levels of preproET-1, endothelin-converting enzyme-1alpha, and ET(A) and ET(B) receptors were unchanged. On acute withdrawal of NO, pulmonary vascular resistance (PVR) increased by 77.8% (P < 0.05) in control lambs but was unchanged (-5.5%) in PD-156707-treated lambs. Inhaled NO increased plasma ET-1 concentrations but not gene expression in the intact lamb, and ET(A) receptor blockade prevented the increase in PVR after NO withdrawal. These data suggest a role for ET-1 in the rebound pulmonary hypertension noted on acute withdrawal of inhaled NO.     

Nitric oxide-endothelin-1 interactions after acute ductal constriction in fetal lambs

Acute partial compression of the fetal ductus arteriosus (DA) results in an initial increase in pulmonary blood flow (PBF) that is followed by acute vasoconstriction. The objective of the present study was to determine the role of nitric oxide (NO)-endothelin-1 (ET-1) interactions in the acute changes in pulmonary vascular tone after in utero partial constriction of the DA. Twelve late-gestation fetal lambs (132-140 days) were instrumented to measure vascular pressures and left PBF. After a 24-h recovery period, acute constriction of the DA was performed by partially inflating a vascular occluder, and the hemodynamic variables were observed for 4 h. In control lambs (n = 7), acute ductal constriction initially increased PBF by 627% (P < 0.05). However, this was followed by active vasoconstriction, such that PBF was restored to preconstriction values by 4 h. This was associated with a 43% decrease in total NO synthase (NOS) activity (P < 0.05) and a 106% increase in plasma ET-1 levels (P < 0.05). Western blot analysis demonstrated no changes in lung tissue endothelial NOS, preproET-1, endothelin-converting enzyme-1, or ET(B) receptor protein levels. The infusion of PD-156707 (an ET(A) receptor antagonist, n = 5) completely blocked the vasoconstriction and preserved NOS activity. These data suggest that the fetal pulmonary vasoconstriction after acute constriction of the DA is mediated by NO-ET-1 interactions. These include an increase in ET(A) receptor-mediated vasoconstriction and an ET(A) receptor-mediated decrease in NOS activity. The mechanisms of these NO-ET-1 interactions, and their role in mediating acute changes in PBF, warrant further studies.     

Increased hydrogen peroxide downregulates soluble guanylate cyclase in the lungs of lambs with persistent pulmonary hypertension of the newborn

Similar to infants born with persistent pulmonary hypertension of the newborn (PPHN), there is an increase in circulating endothelin-1 (ET-1) and decreased cGMP-mediated vasodilation in an ovine model of PPHN. These abnormalities lead to vasoconstriction and vascular remodeling. Our previous studies have demonstrated that reactive oxygen species (ROS) levels are increased in pulmonary arterial smooth muscle cells (PASMC) exposed to ET-1. Thus the initial objective of this study was to determine whether the development of pulmonary hypertension in utero is associated with elevated production of the ROS hydrogen peroxide (H(2)O(2)) and if this is associated with alterations in antioxidant capacity. Second we wished to determine whether chronic exposure of PASMC isolated from fetal lambs to H(2)O(2) would mimic the decrease in soluble guanylate cyclase expression observed in the ovine model of PPHN. Our results indicate that H(2)O(2) levels are significantly elevated in pulmonary arteries isolated from 136-day-old fetal PPHN lambs (P 0.05). In addition, we determined that catalase and glutathione peroxidase expression and activities remain unchanged. Also, we found that the overnight exposure of fetal PASMC to a H(2)O(2)-generating system resulted in significant decreases in soluble guanylate cyclase expression and nitric oxide (NO)-dependent cGMP generation (P 0.05). Finally, we demonstrated that the addition of the ROS scavenger catalase to isolated pulmonary arteries normalized the vasodilator responses to exogenous NO. As these scavengers had no effect on the vasodilator responses in pulmonary arteries isolated from age-matched control lambs this enhancement appears to be unique to PPHN. Overall our data suggest a role for H(2)O(2) in the abnormal vasodilation associated with the pulmonary arteries of PPHN lambs.     

Developmental changes in endothelin expression and activity in the ovine fetal lung

Mechanisms that regulate endothelin (ET) in the perinatal lung are complex and poorly understood, especially with regard to the role of ET before and after birth. We hypothesized that the ET system is developmentally regulated and that the balance of ET(A) and ET(B) receptor activity favors vasoconstriction. To test this hypothesis, we performed a series of molecular and physiological studies in the fetal lamb, newborn lamb, and adult sheep. Lung preproET-1 mRNA levels, tissue ET peptide levels, and cellular localization of ET-1 expression were determined by Northern blot analysis, peptide assay, and immunohistochemistry in distal lung tissue from fetal lambs between 70 and 140 days (term = 145 days), newborn lambs, and ewes. Lung mRNA expression for the ET(A) and ET(B) receptors was also measured at these ages. We found that preproET-1 mRNA expression increased from 113 to 130 days gestation. Whole lung ET protein content was highest at 130 days gestation but decreased before birth in the fetal lamb lung. Immunolocalization of ET-1 protein showed expression of ET-1 in the vasculature and bronchial epithelium at all gestational ages. ET(A) receptor mRNA expression and ET(B) receptor mRNA increased from 90 to 125 and 135 days gestation. To determine changes in activity of the ET(A) and ET(B) receptors, we studied the effect of selective antagonists to the ET(A) or ET(B) receptors at 120, 130, and 140 days of fetal gestation. ET(A) receptor-mediated vasoconstriction increased from 120 to 140 days, whereas blockade of the ET(B) receptor did not change basal fetal pulmonary vascular tone at any age examined. We conclude that the ET system is developmentally regulated and that the increase in ET(A) receptor gene expression correlates with the onset of the vasodilator response to ET(A) receptor blockade. Although ET(B) receptor gene expression increases during late gestation, the balance of ET receptor activity favors vasoconstriction under basal conditions. We speculate that changes in ET receptor activity play important roles in regulation of pulmonary vascular tone in the ovine fetus.     

Chronic O2 exposure in the newborn rat results in decreased pulmonary arterial nitric oxide release and altered smooth muscle response to isoprostane.

Chronic oxygen exposure in the newborn rat results in lung isoprostane formation, which may contribute to the pulmonary hypertension evident in this animal model. The purpose of this study was to investigate the pulmonary arterial smooth muscle responses to 8-iso-prostaglandin F(2alpha) (8-iso-PGF(2a)) in newborn rats exposed to 60% O2 for 14 days. Because, in the adult rat, 8-iso-PGF(2alpha) may have a relaxant effect, mediated by nitric oxide (NO), we also sought to evaluate the pulmonary arterial NO synthase (NOS) protein content and NO release in the newborn exposed to chronic hyperoxia. Compared with air-exposed control animals, 8-iso-PGF(2a) induced a significantly greater force (P < 0.01) and reduced (P < 0.01) relaxation of precontracted pulmonary arteries in the 60% O2-treated animals. These changes were reproduced in control pulmonary arteries by NOS blockade by using NG-nitro-L-arginine methyl ester. Pulmonary arterial endothelial NOS was unaltered, but the inducible NOS protein content was significantly decreased (P < 0.01) in the experimental group. Pulmonary (P < 0.05) and aortic (P < 0.01) tissue ex vivo NO accumulation was significantly reduced in the 60% O2-treated animals. We speculate that impaired pulmonary vascular tissue NO metabolism after chronic O2 exposure potentiates 8-iso-PGF(2alpha)-induced vasoconstriction in the newborn rat, thus contributing to pulmonary hypertension.     

Nitric oxide-endothelin-1 interactions after surgically induced acute increases in pulmonary blood flow in intact lambs

Several congenital heart defects require surgery that acutely increases pulmonary blood flow (PBF). This can lead to dynamic alterations in postoperative pulmonary vascular resistance (PVR) and can contribute to morbidity and mortality. Thus the objective of this study was to determine the role of nitric oxide (NO), endothelin (ET)-1, and their interactions in the alterations of PVR after surgically induced increases in PBF. Twenty lambs underwent placement of an aortopulmonary vascular graft. Lambs were instrumented to measure vascular pressures and PBF and studied for 4 h. Before and after shunt opening, lambs received an infusion of saline (n = 9), tezosentan, an ETA- and ETB -receptor antagonist (n = 6), or Nomega-nitro-L-arginine (L-NNA), a NO synthase (NOS) inhibitor (n = 5). In control lambs, shunt opening increased PBF by 117.8% and decreased PVR by 40.7% (P < 0.05) by 15 min, without further changes thereafter. Plasma ET-1 levels increased 17.6% (P < 0.05), and total NOS activity decreased 61.1% (P < 0.05) at 4 h. ET-receptor blockade (tezosentan) prevented the plateau of PBF and PVR, such that PBF was increased and PVR was decreased compared with controls at 3 and 4 h (P < 0.05). These changes were associated with an increase in total NOS activity (+61.4%; P < 0.05) at 4 h. NOS inhibition (L-NNA) after shunt placement prevented the sustained decrease in PVR seen in control lambs. In these lambs, PVR decreased by 15 min (P < 0.05) but returned to baseline by 2 h. Together, these data suggest that surgically induced increases in PBF are limited by vasoconstriction, at least in part by an ET-receptor-mediated decrease in lung NOS activity. Thus NO appears to be important in maintaining a reduction in PVR after acutely increased PBF.     

Rho kinase activation maintains high pulmonary vascular resistance in the ovine fetal lung

Mechanisms that maintain high pulmonary vascular resistance (PVR) in the fetal lung are poorly understood. Activation of the Rho kinase signal transduction pathway, which promotes actin-myosin interaction in vascular smooth muscle cells, is increased in the pulmonary circulation of adult animals with experimental pulmonary hypertension. However, the role of Rho kinase has not been studied in the fetal lung. We hypothesized that activation of Rho kinase contributes to elevated PVR in the fetus. To address this hypothesis, we studied the pulmonary hemodynamic effects of brief (10 min) intrapulmonary infusions of two specific Rho kinase inhibitors, Y-27632 (15-500 microg) and HA-1077 (500 microg), in chronically prepared late-gestation fetal lambs (n = 9). Y-27632 caused potent, dose-dependent pulmonary vasodilation, lowering PVR from 0.67 +/- 0.18 to 0.16 +/- 0.02 mmHg x ml(-1) x min(-1) (P < 0.01) at the highest dose tested without lowering systemic arterial pressure. Despite brief infusions, Y-27632-induced pulmonary vasodilation was sustained for 50 min. HA-1077 caused a similar fall in PVR, from 0.39 +/- 0.03 to 0.19 +/- 0.03 (P < 0.05). To study nitric oxide (NO)-Rho kinase interactions in the fetal lung, we tested the effect of Rho kinase inhibition on pulmonary vasoconstriction caused by inhibition of endogenous NO production with nitro-L-arginine (L-NA; 15-30 mg), a selective NO synthase antagonist. L-NA increased PVR by 127 +/- 73% above baseline under control conditions, but this vasoconstrictor response was completely prevented by treatment with Y-27632 (P < 0.05). We conclude that the Rho kinase signal transduction pathway maintains high PVR in the normal fetal lung and that activation of the Rho kinase pathway mediates pulmonary vasoconstriction after NO synthase inhibition. We speculate that Rho kinase plays an essential role in the normal fetal pulmonary circulation and that Rho kinase inhibitors may provide novel therapy for neonatal pulmonary hypertension.     

ET－1，NO和缺氧对肺动脉平滑肌细胞钙内流及胶原合成的影响

肺血管平滑肌细胞是肺血管收缩反应的主要执行者,也是肺血管结构重建的重要参与者。本研究观察了内皮素１（ＥＴ１）,一氧化氮（ＮＯ）和缺氧对培养的新生小牛肺动肺平滑肌细胞（ＰＡＳＭＣ）钙内流以及胶原合成的影响。结果表明ＥＴ１和缺氧可促进ＰＡＳＭＣ的钙内流,ＮＯ供应剂硝普钠（ＳＭＰ）可抑制ＥＴＩ诱导的钙内流,其作用呈剂量依赖性,ＳＮＰ还可以剂量依赖地抑制ＰＡＳＭＣ的胶原合成,而缺氧可促进ＰＡＳＭＣ的胶原合成。     

Cellular localization of the endothelin receptor subtypes ET(A) and ET(B) in the rat heart and their differential expression in coronary arteries,veins, and capillaries

In the heart, the endothelin (ET)/endothelin-receptor system is markedly involved in pathophysiological mechanisms underlying various cardiac diseases. Based upon pharmacological studies both ET-receptor subtypes take part in the regulation of coronary vascular tone, however, their detailed cellular distribution in the coronary vascular bed based upon direct mRNA and protein detection is unknown. This issue was addressed in the rat heart by means of non-radioactive in situ hybridization, RT-PCR, and immunohistochemistry. Expression of vascular ET(A)-receptors was detected in arterial smooth muscle and capillary endothelium while ET(B)-receptors were present in arterial, venous, and capillary endothelium, and in arterial and venous smooth muscle cells. This differential distribution of the ET-receptor subtypes supports the concept that ET(A)- as well as ET(B)-receptors mediate arterial vasoconstriction, while postcapillary vascular resistance is exclusively regulated by ET(B)-receptors. The observed capillary endothelial expression of the ET(A)-receptor correlates with the known ability of ET(A)-receptor antagonists to attenuate increases in cardiac microvascular permeability during endotoxin shock and ischemia/reperfusion injury.     

Chronic intrauterine pulmonary hypertension decreases calcium-sensitive potassium channel mRNA expression

Calcium-sensitive potassium (K(Ca)) channels play a critical role in mediating perinatal pulmonary vasodilation. Because infants with persistent pulmonary hypertension of the newborn (PPHN) have blunted vasodilator responses to birth-related stimuli, we hypothesized that lung K(Ca) channel gene expression is decreased in PPHN. To test this hypothesis, we measured K(Ca) channel gene expression in distal lung homogenates from both fetal lambs with severe pulmonary hypertension caused by prolonged compression of the ductus arteriosus and age-matched, sham-operated animals (controls). After at least 9 days of compression of the ductus arteriosus, fetal lambs were killed. To determine lung K(Ca) channel mRNA levels, primers were designed against the known sequence of the K(Ca) channel and used in semiquantitative RT-PCR, with lung 18S rRNA content as an internal control. Compared to that in control lambs, lung K(Ca) channel mRNA content in the PPHN group was reduced by 26 +/- 6% (P < 0.02), whereas lung voltage-gated K(+) 2.1 mRNA content was unchanged. We conclude that lung K(Ca) channel mRNA expression is decreased in an ovine model of PPHN. Decreased K(Ca) channel gene expression may contribute to the abnormal pulmonary vascular reactivity associated with PPHN.     

Alterations in ET-1, not nitric oxide,in 1-week-old lambs with increased pulmonary blood flow

Altered pulmonary vascular reactivity is a source of morbidity and mortality for children with congenital heart disease and increased pulmonary blood flow. Nitric oxide (NO) and endothelin (ET)-1 are important mediators of pulmonary vascular reactivity. We hypothesize that early alterations in endothelial function contribute to the altered vascular reactivity associated with congenital heart disease. The objective of this study was to characterize endothelial function in our lamb model of increased pulmonary blood flow at 1 wk of life. Eleven fetal lambs underwent in utero placement of an aortopulmonary vascular graft (shunt) and were studied 7 days after delivery. The pulmonary vasodilator response to both intravenous ACh (endothelium dependent) and inhaled NO (endothelium independent) was similar in shunted and control lambs. In addition, tissue NO(x), NO synthase (NOS) activity, and endothelial NOS protein levels were similar. Conversely, the vasodilator response to both ET-1 and 4Ala-ET-1 (an ET(B) receptor agonist) were attenuated in shunted lambs, and tissue ET-1 concentrations were increased (P < 0.05). Associated with these changes were an increase in ET-converting enzyme-1 protein and a decrease in ET(B) receptor protein levels (P < 0.05). These data demonstrate that increased pulmonary blood flow induces alterations in ET-1 signaling before NO signaling and suggest an early role for ET-1 in the altered vascular reactivity associated with increased pulmonary blood flow.     

Superoxide dismutase restores eNOS expression and function in resistance pulmonary arteries from neonatal lambs with persistent pulmonary hypertension

Endothelial nitric oxide (NO) synthase (eNOS) expression and activity are decreased in fetal lambs with persistent pulmonary hypertension (PPHN). We sought to determine the impact of mechanical ventilation with O(2) with or without inhaled NO (iNO) or recombinant human SOD (rhSOD) on eNOS in the ductal ligation model of PPHN. PPHN lambs and age-matched controls were ventilated with 100% O(2) for 24 h alone or combined with 20 ppm iNO continuously or a single dose of rhSOD (5 mg/kg) given intratracheally at delivery. In 1-day spontaneously breathing lambs, eNOS expression in resistance pulmonary arteries increased relative to fetal levels. eNOS expression increased in control lambs ventilated with 100% O(2), but not in PPHN lambs. Addition of iNO or rhSOD increased eNOS expression and decreased generation of reactive oxygen species (ROS) in PPHN lambs relative to those ventilated with 100% O(2) alone. However, only rhSOD restored eNOS function, increased tetrahydrobiopterin (BH(4)), a critical cofactor for eNOS function, and restored GTP cyclohydrolase I expression in isolated vessels and lungs from PPHN lambs. These data suggest that ventilation of PPHN lambs with 100% O(2) increases ROS production, blunts postnatal increases in eNOS expression, and decreases available BH(4) in PPHN lambs. Although the addition of iNO or rhSOD diminished ROS production and increased eNOS expression, only rhSOD improved eNOS function and levels of available BH(4). Thus therapies designed to decrease oxidative stress and restore eNOS coupling, such as rhSOD, may prove useful in the treatment of PPHN in newborn infants.     

Oxidant stress from uncoupled nitric oxide synthase impairs vasodilation in fetal lambs with persistent pulmonary hypertension

Persistent pulmonary hypertension of newborn (PPHN) is associated with decreased NO release and impaired pulmonary vasodilation. We investigated the hypothesis that increased superoxide (O(2)(*-)) release by an uncoupled endothelial nitric oxide synthase (eNOS) contributes to impaired pulmonary vasodilation in PPHN. We investigated the response of isolated pulmonary arteries to the NOS agonist ATP and the NO donor S-nitroso-N-acetylpenicillamine (SNAP) in fetal lambs with PPHN induced by prenatal ligation of ductus arteriosus and in sham-ligated controls in the presence or absence of the NOS antagonist nitro-L-arginine methyl ester (L-NAME) or the O(2)(*-) scavenger 4,5-dihydroxy-1,3-benzenedisulfonate (Tiron). ATP caused dose-dependent relaxation of pulmonary artery rings in control lambs but induced constriction of the rings in PPHN lambs. L-NAME, the NO precursor L-arginine, and Tiron restored the relaxation response of pulmonary artery rings to ATP in PPHN. Relaxation to NO was attenuated in arteries from PPHN lambs, and the response was improved by L-NAME and by Tiron. We also investigated the alteration in heat shock protein (HSP)90-eNOS interactions and release of NO and O(2)(*-) in response to ATP in the pulmonary artery endothelial cells (PAEC) from these lambs. Cultured PAEC and endothelium of freshly isolated pulmonary arteries from PPHN lambs released O(2)(*-) in response to ATP, and this was attenuated by the NOS antagonist L-NAME and superoxide dismutase (SOD). ATP stimulated HSP90-eNOS interactions in PAEC from control but not PPHN lambs. HSP90 immunoprecipitated from PPHN pulmonary arteries had increased nitrotyrosine signal. Oxidant stress from uncoupled eNOS contributes to impaired pulmonary vasodilation in PPHN induced by ductal ligation in fetal lambs.     

Pulmonary vasoregulation by endothelin in conscious dogs after left lung transplantation.

We tested the hypothesis that regulation of the pulmonary circulation by endogenous endothelin (ET) during normoxia and hypoxia was altered in conscious dogs 1 mo after left lung autotransplantation (LLA). Sham-operated control and post-LLA dogs were chronically instrumented to measure the left pulmonary vascular pressure-flow (LP-Q) relationship. LP-Q plots were generated on separate days during normoxia and hypoxia (arterial PO(2) approximately 50 Torr) in the intact condition, after selective ET(A)-receptor inhibition (BQ-485), and after combined ET(A+B)-receptor inhibition (bosentan). Although LLA resulted in a chronic increase in pulmonary vascular resistance, the ET-receptor antagonists had no effect on the LP-Q relationship during normoxia in either group. The magnitude of hypoxic pulmonary vasoconstriction (HPV) was flow dependent in both groups, and the HPV response was potentiated post-LLA compared with control. ET(A)-receptor inhibition attenuated the HPV response to the same extent in both groups. ET(A+B)-receptor inhibition attenuated the HPV response to a greater extent than did ET(A)-receptor inhibition alone, and this effect was greater post-LLA compared with control. Plasma ET-1 concentration only increased during hypoxia in the LLA group. These results indicate that ET does not regulate the baseline LP-Q relationship in either group. Both ET(A)- and ET(B)-receptor activation mediate a component of HPV in conscious dogs, and the vasoconstrictor influence of ET(B)-receptor activation is enhanced post-LLA.     

Decreased association of HSP90 impairs endothelial nitric oxide synthase in fetal lambs with persistent pulmonary hypertension

Persistent pulmonary hypertension of newborn (PPHN) is associated with decreased nitric oxide (NO) release and impaired pulmonary vasodilation. We investigated the hypothesis that decreased association of heat shock protein 90 (HSP90) with endothelial NO synthase (eNOS) impairs NO release and vasodilation in PPHN. The responses to the NOS agonist ATP were investigated in fetal lambs with PPHN induced by prenatal ligation of ductus arteriosus, and in sham ligation controls. ATP caused dose-dependent vasodilation in control pulmonary resistance arteries, and this response was attenuated in PPHN vessels. The response of control pulmonary arteries to ATP was attenuated by NG-nitro-l-arginine methyl ester (l-NAME), a NOS antagonist, and geldanamycin, an inhibitor of HSP90-eNOS interaction. The attenuated response to ATP observed in PPHN was improved by pretreatment of vessels with l-NAME or 4,5-dihydroxy-1,3-benzene-disulfonate, a superoxide scavenger. Pulmonary arteries from PPHN lambs had decreased basal levels of HSP90 in association with eNOS. Association of HSP90 with eNOS and NO release increased in response to ATP in control pulmonary artery endothelial cells, but not in cells from PPHN lambs. Decreased HSP90-eNOS interactions may contribute to the impaired NO release and vasodilation observed in the ductal ligation model of PPHN.     

Effect of in vivo fetal infusion of dexamethasone at 0.75 GA on fetal ovine resistance artery responses to ET-1

At 110-111 days gestation, instrumented fetal sheep were administered saline or dexamethasone (2.2 microgram. kg(-1). h(-1) iv) for 48 h. Measurement of fetal blood pressure showed a greater increase in dexamethasone-treated (n = 6) compared with control (n = 5) fetuses (7.3 +/- 2.3 vs. 0.6 +/- 2.3 mmHg, P < 0.05). Fetuses were delivered by cesarean section, and the femoral muscle and brain were obtained under halothane anesthesia. Femoral and middle cerebral arteries (approximately 320-micrometer internal diameter) were evaluated using wire myography. Sensitivity to KCl (2.5-125 mM) and the magnitude of the maximal vasoconstriction to 125 mM K(+) were similar in femoral and middle cerebral arteries from dexamethasone-treated vs. control fetuses. Acetylcholine-induced vasorelaxation was similar in femoral arteries from control and dexamethasone-treated fetuses. Middle cerebral arteries did not relax to acetylcholine. Sensitivity to endothelin-1 (ET-1; 0.1 pM-0.1 microM) and magnitude of the ET-1-induced vasoconstriction were greater in femoral arteries from dexamethasone-treated vs. control fetuses (P < 0.05). Autoradiographical studies with receptor-specific ligands demonstrated increased ET(A)-receptor binding, the principal receptor subtype, in femoral muscle vessels (P < 0.001) but decreased ET(A)-receptor binding in middle cerebral arteries (P < 0.01) from dexamethasone-treated compared with control fetuses. Relatively little ET(B)-receptor binding was evident in all tissues examined. We conclude that hyperreactivity to ET-1, due to increased ET(A)-receptor binding, may be involved in the dexamethasone-induced increase in peripheral vascular resistance in fetal sheep in vivo.     

Modulation of pulmonary endothelial endothelin B receptor expression and signaling: implications for experimental hepatopulmonary syndrome

The hepatopulmonary syndrome (HPS) results from intrapulmonary vasodilation in the setting of cirrhosis and portal hypertension. In experimental HPS, pulmonary endothelial endothelin B (ET(B)) receptor overexpression and increased circulating endothelin-1 (ET-1) contribute to vasodilation through enhanced endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) production. In both experimental cirrhosis and prehepatic portal hypertension, ET(B) receptor overexpression correlates with increased vascular shear stress, a known modulator of ET(B) receptor expression. We investigated the mechanisms of pulmonary endothelial ET(B) receptor-mediated eNOS activation by ET-1 in vitro and in vivo. The effect of shear stress on ET(B) receptor expression was assessed in rat pulmonary microvascular endothelial cells (RPMVECs). The consequences of ET(B) receptor overexpression on ET-1-dependent ET(B) receptor-mediated eNOS activation were evaluated in RPMVECs and in prehepatic portal hypertensive animals exposed to exogenous ET-1. Laminar shear stress increased ET(B) receptor expression in RPMVECs without altering mRNA stability. Both shear-mediated and targeted overexpression of the ET(B) receptor enhanced ET-1-mediated ET(B) receptor-dependent eNOS activation in RPMVECs through Ca(2+)-mediated signaling pathways and independent of Akt activation. In prehepatic portal hypertensive animals relative to control, ET-1 administration also activated eNOS independent of Akt activation and triggered HPS. These findings support that increased pulmonary microvascular endothelial ET(B) receptor expression modulates ET-1-mediated eNOS activation, independent of Akt, and contributes to the development of HPS.