Exaggerated hypoxic pulmonary hypertension in endothelin B receptor-deficient rats

Mechanisms by which endothelin (ET)-1 mediates chronic pulmonary hypertension remain incompletely understood. Although activation of the ET type A (ET(A)) receptor causes vasoconstriction, stimulation of ET type B (ET(B)) receptors can elicit vasodilation or vasoconstriction. We hypothesized that the ET(B) receptor attenuates the development of hypoxic pulmonary hypertension and studied a genetic rat model of ET(B) receptor deficiency (transgenic sl/sl). After 3 wk of severe hypoxia, the transgenic sl/sl pulmonary vasculature lacked expression of mRNA for the ET(B) receptor and developed exaggerated pulmonary hypertension that was characterized by elevated pulmonary arterial pressure, diminished cardiac output, and increased total pulmonary resistance. Plasma ET-1 was fivefold higher in transgenic sl/sl rats than in transgenic controls. Although mRNA for prepro-ET-1 was not different, mRNA for ET-converting enzyme-1 was higher in transgenic sl/sl than in transgenic control lungs. Hypertensive lungs of sl/sl rats also produced less nitric oxide metabolites and 6-ketoprostaglandin F(1alpha), a metabolite of prostacyclin, than transgenic controls. These findings suggest that the ET(B) receptor plays a protective role in the pulmonary hypertensive response to chronic hypoxia.     

Altered role of smooth muscle endothelin receptors in coronary endothelin-1 and alpha1-adrenoceptor-mediated vasoconstriction in Type 2 diabetes

Regulation of vascular tone and blood flow involves interactions between numerous local and systemic vascular control signals, many of which are altered by Type 2 diabetes (T2D). Vascular responses to endothelin-1 (ET-1) are mediated by endothelin type A (ET(A)) and type B (ET(B)) receptors that have been implicated in cross talk with alpha(1)-adrenoceptors (alpha(1)-AR). ET(A) and ET(B) receptor expression and plasma ET-1 levels are elevated in T2D; however, whether this influences coronary alpha(1)-AR function has not been examined. Therefore, we examined the effect of ET(A) and ET(B) receptor inhibition on coronary vasoconstriction to ET-1 and alpha(1)-AR activation in a mouse model of T2D. Coronary vascular responses were examined in isolated mouse hearts from control and diet-induced T2D C57BL/6J mice. Responses to ET-1 and the selective alpha(1)-AR agonist phenylephrine (PE) were examined alone and in the presence of the nitric oxide synthase inhibitor N(omega)-nitro-l-arginine methyl ester (l-NAME) alone or in combination with selective ET(A) or ET(B) receptor inhibitors BQ-123 and BQ-788, respectively. Vasoconstriction to ET-1 was enhanced, whereas ET(B), but not ET(A), receptor blockade reduced basal coronary tone in T2D hearts. In the presence of l-NAME, ET(A) receptor inhibition attenuated ET-1 vasoconstriction in both groups, whereas ET(B) inhibition abolished this response only in control hearts. In addition, ET(A) inhibition enhanced alpha(1)-AR-mediated vasoconstriction in T2D, but not control, hearts following l-NAME treatment. Therefore, in this model, enhanced coronary ET-1 responsiveness is mediated primarily through smooth muscle ET(B) receptors, whereas the interaction with alpha(1)-ARs is mediated solely through the ET(A) receptor subtype.     

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.     

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.     

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.     

Effect of chronic and selective endothelin receptor antagonism on microvascular function in type 2 diabetes

Vascular dysfunction, which presents either as an increased response to vasoconstrictors or an impaired relaxation to dilator agents, results in worsened cardiovascular outcomes in diabetes. We have established that the mesenteric circulation in Type 2 diabetes is hyperreactive to the potent vasoconstrictor endothelin-1 (ET-1) and displays increased nitric oxide-dependent vasodilation. The current study examined the individual and/or the relative roles of the ET receptors governing vascular function in the Goto-Kakizaki rat, a mildly hyperglycemic, normotensive, and nonobese model of Type 2 diabetes. Diabetic and control rats received an antagonist to either the ET type A (ETA; atrasentan; 5 mg x kg(-1) x day(-1)) or type B (ET(B); A-192621; 15 or 30 mg x kg(-1) x day(-1)) receptors for 4 wk. Third-order mesenteric arteries were isolated, and vascular function was assessed with a wire myograph. Maximum response to ET-1 was increased in diabetes and attenuated by ETA antagonism. ETB blockade with 15 mg/kg A-192621 augmented vasoconstriction in controls, whereas it had no further effect on ET-1 hyperreactivity in diabetes. The higher dose of A-192621 showed an ETA-like effect and decreased vasoconstriction in diabetes. Maximum relaxation to acetylcholine (ACh) was similar across groups and treatments. ETB antagonism at either dose had no effect on vasorelaxation in control rats, whereas in diabetes the dose-response curve to ACh was shifted to the right, indicating a decreased relaxation at 15 mg/kg A-192621. These results suggest that ETA receptor blockade attenuates vascular dysfunction and that ETB receptor antagonism exhibits differential effects depending on the dose of the antagonists and the disease state.     

EDHF-mediated vasodilation involves different mechanisms in normotensive and hypertensive rat lungs

The role of endothelium-derived hyperpolarizing factor (EDHF) in regulating the pulmonary circulation and the participation of cytochrome P-450 (CYP450) activity and gap junction intercellular communication in EDHF-mediated pulmonary vasodilation are unclear. We tested whether tonic EDHF activity regulated pulmonary vascular tone and examined the mechanism of EDHF-mediated pulmonary vasodilation induced by thapsigargin in salt solution-perfused normotensive and hypoxia-induced hypertensive rat lungs. After blockade of both cyclooxygenase and nitric oxide synthase, inhibition of EDHF with charybdotoxin plus apamin did not affect either normotensive or hypertensive vascular tone or acute hypoxic vasoconstriction but abolished thapsigargin vasodilation in both groups of lungs. The CYP450 inhibitors 7-ethoxyresorufin and sulfaphenazole and the gap junction inhibitor palmitoleic acid, but not 18alpha-glycyrrhetinic acid, inhibited thapsigargin vasodilation in normotensive lungs. None of these agents inhibited the vasodilation in hypertensive lungs. Thus tonic EDHF activity does not regulate either normotensive or hypertensive pulmonary vascular tone or acute hypoxic vasoconstriction. Whereas thapsigargin-induced EDHF-mediated vasodilation in normotensive rat lungs involves CYP450 activity and might act through gap junctions, the mechanism of vasodilation is apparently different in hypertensive lungs.     

IL-1beta alters hemodynamics in newborn intestine: role of endothelin

Studies were carried out to determine the effects of IL-1beta on newborn intestinal hemodynamics. IL-1beta increased the release of ET-1 by primary endothelial cells in a dose-dependent manner; as well, it reduced expression of the endothelin (ET) type B (ET(B)) receptor on endothelial cells and increased expression of the ET type A (ET(A)) receptor on vascular smooth muscle cells. IL-1beta increased endothelial cell endothelial nitric oxide (NO) synthase (eNOS) expression but did not enhance eNOS activity as evidenced by release of NO(x) into conditioned medium in response to acetylcholine or shear stress. The effects of IL-1beta on flow-induced dilation were evaluated in terminal mesenteric arteries in vitro. Pretreatment with IL-1beta (1 ng; 4 h) significantly attenuated vasodilation in response to flow rates of 100 and 200 microl/min. This effect was mediated, in part, by the endothelin ET(A) receptor; thus selective blockade of ET(A) receptors with BQ610 nearly restored flow-induced dilation. In contrast, exogenous ET-1 only shifted the diameter-flow curve downward without altering the percent vasodilation in response to flow. The effects of IL-1beta on ileal oxygenation were then studied using in vivo gut loops. Intramesenteric artery infusion of IL-1beta upstream of the gut loop caused ileal vasoconstriction and reduced the arterial-venous O(2) difference across the gut loop; consequently, it reduced ileal oxygenation by 60%. This effect was significantly attenuated by pretreatment with BQ610. These data support a linkage between the proinflammatory cytokine IL-1beta and vascular dysfunction within the intestinal circulation, mediated, at least in part, by the ET system.     

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.     

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.     

Upregulation of ET-1 and its receptors and remodeling in small pulmonary veins under hypoxic conditions

Pulmonary veins show greater sensitivity to endothelin (ET)-1-induced vasoconstriction than pulmonary arteries, and remodeling was observed in pulmonary veins under hypoxic conditions. We examined, using an immunohistochemical method, the expression of Big ET-1, ET-converting enzyme (ECE), and ET(A) and ET(B) receptors in rat pulmonary veins under normoxic and hypoxic conditions. In control rats, Big ET-1 and ECE were coexpressed in the intima and media of the pulmonary veins, with an even distribution along the axial pathway. ET(A) and ET(B) receptors were expressed in the pulmonary veins, with a predominant distribution in the proximal segments. The expression of Big ET-1 was more abundant in the pulmonary veins than in the pulmonary arteries. After exposure to hypoxia for 7 or 14 days, the expression of Big ET-1, ECE, and ET receptors increased in small pulmonary veins. Increases in the medial thickness, wall thickness, and immunoreactivity for alpha-smooth muscle actin were also observed in the small pulmonary veins under hypoxic conditions. The upregulation of ET-1 and ET receptors in the small pulmonary veins is associated with vascular remodeling, which may lead to the development of hypoxic pulmonary hypertension.     

The role of endogenous lung neuropeptides in regulation of the pulmonary circulation

Vascular resistance in the mammalian pulmonary circulation is affected by many endogenous agents that influence vascular smooth muscle, right ventricular myocardium, endothelial function, collagen and elastin deposition, and fluid balance. When the balance of these agents is disturbed, e.g. by airway hypoxia from high altitude or pulmonary obstructive disorders, pulmonary hypertension ensues, as characterized by elevated pulmonary artery pressure (P(PA)). Among neuropeptides with local pulmonary artery pressor effects are endothelin-1 (ET-1), angiotensin II (AII), and substance P, and among mitigating peptides are calcitonin gene-related peptide (CGRP), adrenomedullin (ADM), atrial natriuretic peptide (ANP), vasoactive intestinal peptide (VIP) and ET-3. Moreover, somatostatin28 (SOM28) exacerbates, whereas SOM14 decreases P(PA) in hypoxic rats, with lowering and increasing of lung CGRP levels, respectively. Pressure can also be modulated by increasing or decreasing plasma volume (VIP and ANP, respectively), or by induction or suppression of vascular tissue remodeling (ET-1 and CGRP, respectively). Peptide bioavailability and potency can be regulated through hypoxic up- and down- regulation of synthesis or release, activation by converting enzymes (ACE for AII and ECE for ET-1), inactivation by neutral endopeptidase and proteases, or by interaction with nitric oxide (NO). Moreover, altered receptor density and affinity can account for changed peptide efficacy. For example, upregulation of ET(A) receptors and ET-1 synthesis occurs in the hypoxic lung concomitantly with reduced CGRP release. Also, receptor activity modifying protein 2 (RAMP2) has been shown to confer ADM affinity to the pulmonary calcitonin-receptor-like receptor (CRLR). We recently detected the mRNA encoding for RAMP2, CRLR, and the CGRP receptor RDC-1 in rat lung. The search for an effective, lung selective treatment of pulmonary hypertension will likely benefit from exploring the imbalance and restoring the balance between these native modulators of intrapulmonary pressure. For example, blocking of the ET-1 receptor ET(A) and vasodilation by supplemental CGRP delivered i. v. or via airway gene transfer, have proven to be useful experimentally.     

Mechanisms underlying enhanced contractile response to endothelin-1 in diabetic rat basilar artery

We investigated the influence of streptozotocin-induced diabetes on the responsiveness of the rat basilar artery to endothelin-1 (ET-1) and nitric oxide (NO), which is known to counteract ET-1. In basilar arteries isolated from diabetic rats: (a) the ET-1-induced contraction was enhanced, (b) the contraction induced by N(G)-nitro-l-arginine [a nitric oxide synthase (NOS) inhibitor] was weaker, and (c) the levels of the mRNAs for ET(A)/ET(B) receptors and prepro-ET-1, but not for NOS, were significantly elevated (all versus age-matched controls). These data indicate that ET-1-induced vasoconstriction may be increased in the diabetic rat basilar artery, and that this hyper-reactivity to ET-1 may be due to an overproduction of ET-1, an up-regulation of ET(A)/ET(B) receptors, and a defect in the bioavailability of 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.     

The NO donor molsidomine reduces endothelin-1 gene expression in chronic hypoxic rat lungs

We investigated the effects of the nitric oxide (NO) donor molsidomine and the nitric oxide synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME) on pulmonary endothelin (ET)-1 gene expression and ET-1 plasma levels in chronic hypoxic rats. Two and four weeks of hypoxia (10% O2) significantly increased right ventricular systolic pressure, the medial cross-sectional vascular wall area of the pulmonary arteries, and pulmonary ET-1 mRNA expression (2-fold and 3.2-fold, respectively). ET-1 plasma levels were elevated after 4 wk of hypoxia. In rats exposed to 4 wk of hypoxia, molsidomine (15 mg x kg(-1) x day(-1)) given either from the beginning or after 2 wk of hypoxia significantly reduced pulmonary hypertension, pulmonary vascular remodeling, pulmonary ET-1 gene expression, and ET-1 plasma levels. L-NAME administration (45 mg x kg(-1) x day(-1)) in rats subjected to 2 wk of hypoxia did not modify these parameters. Our findings suggest that in chronic hypoxic rats, exogenously administered NO acts in part by suppressing the formation of ET-1. In contrast, inhibition of endogenous NO production exerts only minor effects on the pulmonary circulation and pulmonary ET-1 synthesis in these animals.     

Endothelin-mediated vasoconstriction in postischemic newborn intestine

We previously suggested that the profound, sustained vasoconstriction noted in 3-day-old swine intestine after a moderate episode of ischemia-reperfusion (I/R) reflects the unmasking of underlying constrictor tone consequent to a loss of endothelium-derived nitric oxide (NO). In this study, we sought to determine whether endothelin-1 (ET-1) was the unmasked constrictor and whether selective loss of endothelial ET(B) receptors, which mediate NO-based vasodilation, participated in the hemodynamic consequences of I/R in newborn intestine. Studies were performed in innervated, autoperfused intestinal loops in 3- and 35-day-old swine. Selective blockade of ET(A) receptors with BQ-610 had no effect on hemodynamics under control conditions; however, when administered before and during I/R, BQ-610 significantly attenuated the post-I/R vasoconstriction and reduction in arteriovenous O(2) difference in the younger group. In 3-day-old intestine, reduction of intestinal O(2) uptake to a level similar to that noted after I/R by lowering tissue temperature had no effect on the response to BQ-610 or ET-1, indicating that the change in response to BQ-610 noted after I/R was not simply consequent to the reduction in tissue O(2) demand. In studies in mesenteric artery rings suspended in myographs, we observed a leftward shift in the dose-response curve for ET-1 after selective blockade of ET(B) receptors with BQ-788 in 3- but not 35-day-old swine. Rings exposed to I/R in vivo behaved in a manner similar to control rings treated with BQ-788 or endothelium-denuded non-I/R rings.     

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

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

Endothelium-derived reactive oxygen species and endothelin-1 attenuate NO-dependent pulmonary vasodilation following chronic hypoxia

Vasodilatory responses to exogenous nitric oxide (NO) are diminished following exposure to chronic hypoxia (CH) in isolated, perfused rat lungs. We hypothesized that both endothelium-derived reactive oxygen species (ROS) and endothelin-1 (ET-1) mediate this attenuated NO-dependent pulmonary vasodilation following CH. To test this hypothesis, we examined vasodilatory and vascular smooth muscle (VSM) Ca2+ responses to the NO donor spermine NONOate in UTP-constricted, isolated pressurized small pulmonary arteries from control and CH rats. Consistent with our previous findings in perfused lungs, we observed attenuated NO-dependent vasodilation following CH in endothelium-intact vessels. However, in endothelium-denuded vessels, responses to spermine NONOate were augmented in CH rats compared with controls, thus demonstrating an inhibitory influence of the endothelium on NO-dependent reactivity following CH. Whereas both the ROS scavenger tiron and the ETA receptor antagonist BQ-123 augmented NO-dependent reactivity in endothelium-intact vessels from CH rats, neither fully restored vasodilatory responses to those observed following endothelium denudation in vessels from CH rats. In contrast, the combination of tiron and BQ-123 or the nonselective ET receptor antagonist PD-145065 enhanced NO responsiveness in endothelium-intact vessels from CH rats similar to that observed following endothelium denudation. We conclude that both endothelium-derived ROS and ET-1 attenuate NO-dependent pulmonary vasodilation following CH. Furthermore, CH augments pulmonary VSM reactivity to NO.     

Inhalation of endothelin receptor blockers in pulmonary hypertension

Endothelin 1 (ET-1) is a potent pulmonary vasoconstrictor and mediator of lung diseases. Antagonism of the ET-1-mediated effects has become an important therapeutic approach. ET-1 (A and B) receptors are differentially distributed in the lung vasculature. Whereas the ET(A) receptors mainly mediate vasoconstriction, the endothelial ET(B) receptor seems to have vasodilative properties. We sought to determine if antagonism of ET receptors can be achieved by inhalation of specific blockers in a model of ET-1-mediated pulmonary hypertension.     

Hypoxia-induced reactive oxygen species downregulate ETB receptor-mediated contraction of rat pulmonary arteries

Production of reactive oxygen species (ROS) may be increased during hypoxia in pulmonary arteries. In this study, the role of ROS in the effect of hypoxia on endothelin (ET) type B (ETB) receptor-mediated vasocontraction in lungs was determined. In rat intrapulmonary (approximately 0.63 mm ID) arteries, contraction induced by IRL-1620 (a selective ETB receptor agonist) was significantly attenuated after 4 h of hypoxia (30 mmHg Po2) compared with normoxic control (140 mmHg Po2). The effect was abolished by tiron, a scavenger of superoxide anions, but not by polyethylene glycol (PEG)-conjugated catalase, which scavenges H2O2. The hypoxic effect on ETB receptor-mediated vasoconstriction was also abolished by endothelium denudation but not by nitro-L-arginine and indomethacin. Exposure for 4 h to exogenous superoxide anions, but not H2O2, attenuated the vasoconstriction induced by IRL-1620. Confocal study showed that hypoxia increased ROS production in pulmonary arteries that were scavenged by PEG-conjugated SOD. In endothelium-intact pulmonary arteries, the ETB receptor protein was reduced after 4 h of exposure to hypoxia, exogenous superoxide anions, or ET-1. BQ-788, a selective ETB receptor antagonist, prevented these effects. ET-1 production was stimulated in endothelium-intact arteries after 4 h of exposure to hypoxia or exogenous superoxide anions. This effect was blunted by PEG-conjugated SOD. These results demonstrate that exposure to hypoxia attenuates ETB receptor-mediated contraction of rat pulmonary arteries. A hypoxia-induced production of superoxide anions may increase ET-1 release from the endothelium and result in downregulation of ETB receptors on smooth muscle.