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.     

Evaluation of endothelin-1-induced pulmonary vasoconstriction following myocardial infarction

Endothelin (ET) levels are elevated in congestive heart failure secondary to myocardial infarction (MI) and correlate well with the severity of pulmonary hypertension (PH), suggesting that the ET peptide could contribute to the pathophysiology of venous PH. Alterations of pulmonary vasoreactivity to ET after MI and the respective roles of the ET(A) and ET(B) receptors (ET(A)-R and ET(B)-R) have never been evaluated, to our knowledge. MI was induced in rats. Three weeks later, small pulmonary resistance arteries were mounted on a microvascular myograph. Cumulative concentration-response curves to ET-1 and sarafotoxin 6c (S6c) were performed. Response to ET was also assessed in the presence of ET-R antagonists. Heterodimerization of receptors was evaluated by immunoprecipitation of the ET(B)-R, followed by western blotting for the expression of the ET(A)-R. Maximal vasoconstriction and sensitivity to ET-1 were similar in sham and MI with values of 88 +/- 3.9% and 80 +/- 3.8%, respectively. The response to S6c was similarly less in both sham (67 +/- 5.7%) and MI groups (60 +/- 6.6%). When administered alone, the ET(A)-R antagonist (10 nM A-147627.1) and the ET(B)-R antagonist (1 microM A-192621.1) had no significant effect. However, their combination markedly reduced vaso-constriction (52 +/- 5.3%; P < 0.001). The endothelial and medial distribution of ET-Rs was similar in sham and MI groups. In vitro studies demonstrated co-immunoprecipitation of the ET(A)-R and ET(B)-R. Vasoconstriction of isolated resistance pulmonary arteries to ET agonists is not altered after MI. Dual antagonism results in optimal blockade of vasoconstriction, possibly because the ET(A)-R and ET(B)-R can form functional heterodimers.     

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.     

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.     

Enhanced endothelin-1 response and receptor expression in small mesenteric arteries of insulin-resistant rats

Hyperinsulinemia, a primary feature of insulin resistance, is associated with increased endothelin-1 (ET-1) activity. This study determined the vascular response to ET-1 and receptor binding characteristics in small mesenteric arteries of insulin-resistant (IR) rats. Rats were randomized to control (C) (n = 32) or IR (n = 32) groups. The response to ET-1 was assessed (in vitro) in arteries with (Endo+) and without (Endo-) endothelium. In addition, arteries (Endo+) were pretreated with the ET(B) antagonist A-192621 or the ET(A) antagonist A-127722. Finally, binding characteristics of [(125)I]ET-1 were determined. Results showed that in Endo+ arteries the maximal relaxation (E(max)) to ET-1 was similar between C and IR groups; however, the concentration at 50% of maximum relaxation (EC(50)) was decreased in IR arteries. In Endo- arteries, the E(max) to ET-1 was enhanced in both groups. Pretreatment with A-192621 enhanced the E(max) and EC(50) to ET-1 in both groups. In contrast, A-127722 inhibited the ET-1 response in all arteries in a concentration-dependent manner; however, a greater ET-1 response was seen at each concentration in IR arteries. Maximal binding of [(125)I]ET-1 was increased in IR versus C arteries although the dissociation constant values were similar. In conclusion, we found the vasoconstrictor response to ET-1 is enhanced in IR arteries due to an enhanced expression of ET receptors and underlying endothelial dysfunction.     

Central endothelin ET(B) receptors mediate IL-1-dependent fever induced by preformed pyrogenic factor and corticotropin-releasing factor in the rat

Blockade of central endothelin ET(B) receptors inhibits fever induced by LPS in conscious rats. The contribution of ET(B) receptor-mediated mechanisms to fever triggered by intracerebroventricular IL-6, PGE2, PGF(2alpha), corticotropin-releasing factor (CRF), and preformed pyrogenic factor derived from LPS-stimulated macrophages (PFPF) was examined. The influence of natural IL-1 receptor antagonist or soluble TNF receptor I on endothelin (ET)-1-induced fever was also assessed. The selective ET(B) receptor antagonist BQ-788 (3 pmol icv) abolished fever induced by intracerebroventricular ET-1 (1 pmol) or PFPF (200 ng) and reduced that caused by ICV CRF (1 nmol) but not by IL-6 (14.6 pmol), PGE2 (1.4 nmol), or PGF(2alpha) (2 nmol). CRF-induced fever was also attenuated by bosentan (dual ET(A)/ET(B) receptor antagonist; 10 mg/kg iv) but unaffected by BQ-123 (selective ET(A) receptor antagonist; 3 pmol icv). alpha-Helical CRF(9-41) (dual CRF1/CRF2 receptor antagonist; 6.5 nmol icv) attenuated fever induced by CRF but not by ET-1. Human IL-1 receptor antagonist (9.1 pmol) markedly reduced fever to IL-1beta (180 fmol) or ET-1 and attenuated that caused by PFPF or CRF. Murine soluble TNF receptor I (23.8 pmol) reduced fever to TNF-alpha (14.7 pmol) but not to ET-1. The results of the present study suggest that PFPF and CRF recruit the brain ET system to cause ET(B) receptor-mediated IL-1-dependent fever.     

ET-1 induces cortical spreading depression via activation of the ETA receptor/phospholipase C pathway in vivo

Recently, it has been shown that brain topical superfusion of endothelin (ET)-1 at concentrations around 100 nM induces repetitive cortical spreading depressions (CSDs) in vivo. It has remained unclear whether this effect of ET-1 is related to a primary neuronal/astroglial effect, such as an increase in neuronal excitability or induction of interastroglial calcium waves, or a penumbra-like condition after vasoconstriction. In vitro, ET-1 regulates interastroglial communication via combined activation of ET(A) and ET(B) receptors, whereas it induces vasoconstriction via single activation of ET(A) receptors. We have determined the ET receptor profile and intracellular signaling pathway of ET-1-induced CSDs in vivo. In contrast to the ET(B) receptor antagonist BQ-788 and concentration dependently, the ET(A) receptor antagonist BQ-123 completely blocked the occurrence of ET-1-induced CSDs. The ET(B) receptor antagonist did not increase the efficacy of the ET(A) receptor antagonist. Direct stimulation of ET(B) receptors with the selective ET(B) agonist BQ-3020 did not trigger CSDs. The phospholipase C (PLC) antagonist U-73122 inhibited CSD occurrence in contrast to the protein kinase C inhibitor G?-6983. Our findings indicate that ET-1 induces CSDs through ET(A) receptor and PLC activation. We conclude that the induction of interastroglial calcium waves is unlikely the primary cause of ET-1-induced CSDs. On the basis of the receptor profile, likely primary targets of ET-1 mediating CSD are either neurons or vascular smooth muscle cells.     

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.     

Hypoxia-induced pulmonary endothelin-1 expression is unaltered by nitric oxide.

Nitric oxide (NO) attenuates hypoxia-induced endothelin (ET)-1 expression in cultured umbilical vein endothelial cells. We hypothesized that NO similarly attenuates hypoxia-induced increases in ET-1 expression in the lungs of intact animals and reasoned that potentially reduced ET-1 levels may contribute to the protective effects of NO against the development of pulmonary hypertension during chronic hypoxia. As expected, hypoxic exposure (24 h, 10% O(2)) increased rat lung ET-1 peptide and prepro-ET-1 mRNA levels. Contrary to our hypothesis, inhaled NO (iNO) did not attenuate hypoxia-induced increases in pulmonary ET-1 peptide or prepro-ET-1 mRNA levels. Because of this surprising finding, we also examined the effects of NO on hypoxia-induced increases in ET peptide levels in cultured cell experiments. Consistent with the results of iNO experiments, administration of the NO donor S-nitroso-N-acetyl-penicillamine to cultured bovine pulmonary endothelial cells did not attenuate increases in ET peptide levels resulting from hypoxic (24 h, 3% O(2)) exposure. In additional experiments, we examined the effects of NO on the activity of a cloned ET-1 promoter fragment containing a functional hypoxia inducible factor-1 binding site in reporter gene experiments. Whereas moderate hypoxia (24 h, 3% O(2)) had no effect on ET-1 promoter activity, activity was increased by severe hypoxic (24 h, 0.5% O(2)) exposure. ET-1 promoter activity after S-nitroso-N-acetyl-penicillamine administration during severe hypoxia was greater than that in normoxic controls, although activity was reduced compared with that in hypoxic controls. These findings suggest that hypoxia-induced pulmonary ET-1 expression is unaffected by NO.     

Endothelin B receptor deficiency potentiates ET-1 and hypoxic pulmonary vasoconstriction

Endothelin (ET)-1 contributes to the regulation of pulmonary vascular tone by stimulation of the ET(A) and ET(B) receptors. Although activation of the ET(A) receptor causes vasoconstriction, stimulation of the ET(B) receptors can elicit either vasodilation or vasoconstriction. To examine the physiological role of the ET(B) receptor in the pulmonary circulation, we studied a genetic rat model of ET(B) receptor deficiency [transgenic(sl/sl)]. We hypothesized that deficiency of the ET(B) receptor would predispose the transgenic(sl/sl) rat lung circulation to enhanced pulmonary vasoconstriction. We found that the lungs of transgenic(sl/sl) rats are ET(B) deficient because they lack ET(B) mRNA in the pulmonary vasculature, have minimal ET(B) receptors as determined with an ET-1 radioligand binding assay, and lack ET-1-mediated pulmonary vasodilation. The transgenic(sl/sl) rats have higher basal pulmonary arterial pressure and vasopressor responses to brief hypoxia or ET-1 infusion. Plasma ET-1 levels are elevated and endothelial nitric oxide synthase protein content and nitric oxide production are diminished in the transgenic(sl/sl) rat lung. These findings suggest that the ET(B) receptor plays a major physiological role in modulating resting pulmonary vascular tone and reactivity to acute hypoxia. We speculate that impaired ET(B) receptor activity can contribute to the pathogenesis of pulmonary hypertension.     

AM reverses pressor response to ET-1 independently of NO in rat coronary circulation

Endothelin-1 (ET-1) elicits a vasoconstrictor response via ET(A) receptors, whereas simultaneous activation of ET(B) receptors triggers the release of nitric oxide (NO), which may limit the constrictor effect of ET-1. Recently, stimulation of ET(B) receptors has been shown to increase the secretion of adrenomedullin (AM), a newly identified vasorelaxing peptide. The present study was designed to see whether AM can oppose the vasoconstrictor response to ET-1. In the isolated perfused paced rat heart preparation, infusion of ET-1 at concentrations of 1 nmol/l for 30 min induced a significant coronary vasoconstriction, whereas it had no effect on perfusion pressure at a dose of 0.08 nmol/l. N(omega)-nitro-L-arginine methyl ester (L-NAME; 300 micromol/l), a potent inhibitor of NO synthase (NOS), did not change the perfusion pressure when added alone to the perfusion fluid but it unmasked the constrictor effect of ET-1 at both concentrations. In the presence of L-NAME, AM (0.03 to 1 nmol/l) markedly reversed the pressor response to ET-1 at both concentrations. Administration of AM (0.03 and 1 nmol/l) alone resulted in a dose-dependent decrease in perfusion pressure, which was not modified in the presence of L-NAME. In conclusion, the coronary vasoconstrictor response to ET-1 is markedly augmented in the presence of a NOS inhibitor. This constrictor response is substantially reversed by AM. Our results indicate that AM may serve as a paracrine modulator of ET-1-induced vasoconstriction independently of the NO pathway.     

Alteration of the cerebrovascular function of endothelin B receptor after subarachnoidal hemorrhage in the rat

The substantial role of endothelin-1 (ET-1) in the development of cerebral vasospasm (CVS) after subarachnoidal hemorrhage (SAH) has been demonstrated by numerous experimental and, recently, clinical investigations. Whether the expression or function of the ET(B) receptor is altered in CVS is still unclear, however. The aim of the present study was, therefore, to characterize the cerebroarterial ET(B) receptor function during CVS. Experimental CVS was induced by the rat double-hemorrhage model. Reduction of the cerebral blood flow (CBF) was confirmed by magnetic resonance perfusion-weighted imaging. Animals were sacrificed on days 3 (d3) and 5 (d5) after CVS induction. The basilar arteries (BA) were dissected, cut into ring segments, and prepared for measurement of isometric force in an organ bath. Concentration-effect curves (CECs) were constructed by cumulative application of ET-1, acetylcholine (Ach), or sarafotoxin S6c (S6c). Segments with (E+) endothelial function were used. CECs were compared by the maximum effect (E(max)), the pD2, and the shift calculated on the pD2 level. The pD2 is the negative decadic logarithm of the concentration producing the half maximal effect (-log10EC50). After SAH, the relative regional CBF in the d3 and d5 groups was reduced to 63% and 32%, respectively, of the CBF in controls. ET-1 induced a dose-dependent contraction of segments with and segments without CVS. In E+ segments, the E(max) for ET-1 was not significantly changed after SAH (mean values [ +/- SEM] of 104% +/- 4% for the control group, 106% +/- 4% for the d3 group, and 104% +/- 3% for the d5 group). The CECs, however, were significantly shifted to the left versus the control by factors of 2.4 in the d3 group and 3.6 in the d5 group. Relaxation by S6c was significantly reduced after SAH (E(max:) 73% +/- 11% in the control group, 21% +/- 13% in the d3 group, and 13% +/- 8% in the d5 group), whereas relaxation associated with Ach was not significantly changed (E(max): 45% +/- 7% in the control group, 56% +/- 6% in the d3 group, and 43% +/- 6% in the d5 group). Significant contraction by S6c was not observed in E+ and E - segments in any of the study groups. The present data indicate the loss of the ET(B) receptor-mediated relaxation of the cerebral arteries in cases of CVS, which is independent of the endothelial nitric oxide synthase level.     

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.     

Endothelin-1-mediated vasoconstriction at rest and during dynamic exercise in healthy humans

It is now generally accepted that alpha-adrenoreceptor-mediated vasoconstriction is attenuated during exercise, but the efficacy of nonadrenergic vasoconstrictor pathways during exercise remains unclear. Thus, in eight young (23 +/- 1 yr), healthy volunteers, we contrasted changes in leg blood flow (ultrasound Doppler) before and during intra-arterial infusion of the alpha(1)-adrenoreceptor agonist phenylephrine (PE) with that of the nonadrenergic endothelin A (ET(A))/ET(B) receptor agonist ET-1. Heart rate, arterial blood pressure, common femoral artery diameter, and mean blood velocity were measured at rest and during knee-extensor exercise at 20%, 40%, and 60% of maximal work rate (WR(max)). Drug infusion rates were adjusted for blood flow to maintain comparable doses across all subjects and conditions. At rest, PE infusion (8 ng x ml(-1) x min(-1)) provoked a rapid and significant decrease in leg blood flow (-51 +/- 3%) within 2.5 min. Resting ET-1 infusion (40 pg x ml(-1) x min(-1)) significantly decreased leg blood flow within 5 min, reaching a maximal vasoconstriction (-34 +/- 3%) after 25-30 min of continuous infusion. Compared with rest, an exercise intensity-dependent attenuation to PE-mediated vasoconstriction was observed (-18 +/- 5%, -7 +/- 2%, and -1 +/- 3% change in leg blood flow at 20%, 40%, and 60% of WR(max), respectively). Vasoconstriction in response to ET-1 was also blunted in an exercise intensity-dependent manner (-13 +/- 3%, -7 +/- 4%, and 2 +/- 3% change in leg blood flow at 20%, 40%, and 60% of WR(max), respectively). These findings support a significant contribution of ET-1 and alpha-adrenergic receptors in the regulation of skeletal muscle blood flow in the human leg at rest and suggest a similar, intensity-dependent "lysis" of peripheral ET and alpha-adrenergic vasoconstriction during dynamic exercise.     

Activation of particulate guanylyl cyclase by endothelins in cultured SV-40 transformed cat iris sphincter smooth muscle cells

We investigated the effects of endothelins (ETs) on cGMP production in cultured SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells. ET-3 increased cGMP formation in a concentration-dependent manner (EC50 = 98nM), which was 2.5 times higher than that of ET-1. The ET(B)receptor agonists sarafotoxin-S6c and IRL 1620 also increased cGMP production, mimicking the effects of the ETs. The ET(B) receptor antagonist BQ 788, but not the ET(A) receptor antagonist BQ610, dose-dependently blocked ET-3-stimulated cGMP formation (IC50=10nM). The phorbol ester, Phorbol 12, 13-dibutyrate (PDBu), which inhibits particulate guanylyl cyclase in smooth muscle, dose-dependently inhibited ET-3-stimulated cGMP accumulation (IC50=66nM). LY83583 and ODQ, inhibitors of soluble guanylyl cyclases, as well as inhibitors of the nitric oxide cascade and of intracellular Ca2+ elevation had no appreciable effect on ET-3-induced cGMP production. ET-3 markedly inhibited carbachol-induced intracellular Ca2+ mobilization. We conclude that ET-3 increases intracellular cGMP levels in SV-CISM-2 cells through activation of the ET(B) receptor subtype and subsequent stimulation of the membrane-bound guanylyl cyclase. Elevation of cGMP by ET and the subsequent inhibition of muscarinic stimulation of intracellular Ca2+ mobilization by the cyclic nucleotide could serve to modulate the contractile effects of Ca2+-mobilizing agonists in the iris sphincter smooth muscle.     

Obligatory role of the endocardial endothelium in the increase of myocardial distensibility induced by endothelin-1

This study investigated how the endocardial endothelium (EE) and particularly endothelial type B (ET(B)) receptors influence the effects of endothelin-1 (ET-1) on diastolic distensibility. ET-1 (0.1, 1, and 10 nM) was tested in rabbit papillary muscles (Krebs-Ringer; 1.8 mM CaCl2, 35 degrees C) (i) with intact EE (n = 10), (ii) with damaged EE (0.5% Triton X-100, n = 11), and (iii) in the presence of RES-701-1 (selective endothelial ET(B1) receptor antagonist, 1 microM, n = 6). Additionally, increasing doses (0.1 nM to 1 microM) of Sarafotoxin S6c (SRTXc, a selective ET(B) receptor agonist) and IRL-1620 (a selective endothelial ET(B1) agonist) were studied (i) in muscles with intact EE (n = 7 and n = 6, respectively) and (ii) after damaging the EE (n = 8 and n = 7, respectively). In papillary muscles with intact EE, ET-1 induced dose-dependent positive inotropic and lusitropic effects. At 10 nM, active tension (AT) increased 78% +/- 17%, maximum velocity of tension rise (dT/dt(max)) increased 82% +/- 10%, and maximum velocity of tension decline (dT/dt(min)) increased 77% +/- 17%. These effects were maintained when ET-1 was given after damaging the EE (AT increased 70% +/- 12%, dT/dt(max) increased 93% +/- 14%, and dT/dt(min) increased 56% +/- 14%), but were significantly reduced in the presence of RES-701-1 (AT increased 30% +/- 6%, dT/dt(max) increased 37% +/- 7%, and dT/ dt(min) increased 29% +/- 9%). ET-1 reduced resting tension (RT) and increased diastolic distensibility by 3% +/- 1%, 5% +/- 1%, and 9% +/- 2% (at 0.1, 1, and 10 nM, respectively) in muscles with intact EE. This effect was abolished after damaging the EE or in the presence of RES-701-1. In muscles with intact EE, SRTXc had no significant effects, whereas, when given after damaging the EE, SRTXc (1 microM) increased inotropy and lusitropy (AT increased 116% +/- 24%, dT/dt(max) 110% +/- 28%, and dT/dt(min) 88% +/- 19%) without affecting RT. IRL-1620 dose-dependently decreased AT, dT/dt(max), and dT/dt(min) in muscles with intact EE-effects that were abolished after EE damage. No significant effects were elicited by IRL-1620 in RT. ET-1-induced increase in myocardial distensibility, previously shown to be mediated by ET(A) receptor stimulation, requires an intact EE and active endothelial ET(B1) receptors.     

Stretch-induced endothelin B receptor-mediated apoptosis in vascular smooth muscle cells.

Growing evidence suggests that a pressure-induced increase in the synthesis of endothelin (ET-1) is involved in arterial remodeling and, as a consequence, in the manifestation of chronic hypertension. To study potential stretch-induced changes in gene expression and their functional consequences, we have cultured rat aortic smooth muscle cells (raSMC) and porcine aortic endothelial cells (PAEC) on flexible elastomer membranes. The cells were periodically stretched (up to 20% elongation, 0.5 Hz, 6 h) and the expression of prepro-ET-1 and that of the endothelin A and B receptors (ET(A)-R and ET(B)-R) were analyzed by semi-quantitative RT-PCR analysis and ELISA (ET-1). In contrast to PAEC where ET-1 synthesis was up-regulated up to eightfold on exposure to cyclic stretch, ET-1 synthesis in raSMC was decreased by more than 80% under these conditions. ET(A) R -mRNA expression in stretched raSMC declined to 50% whereas ET(B) R -mRNA levels were increased up to 10-fold. One functional consequence of this apparent shift in receptor abundance was an apoptosis-promoting action of exogenous ET-1 (10 nM), as judged by the appearance of subdiploid peaks during FACS analysis, caspase-3 activation and chromatin condensation. This ET-1-induced apoptosis appeared to be ET(B)-R mediated, as it was completely suppressed by the ET(B)-R antagonist BQ 788 but not by the ET(A)-R antagonist BQ 123. Moreover, raSMC derived from homozygous spotting lethal rats, which lack a functional ET(B)-R, showed no signs of apoptosis after exposure to cyclic strain and exogenous ET-1. These findings suggest a central role for the endothelin system in the onset of hypertension-induced remodeling in conduit arteries, which may proceed via an initial stretch-induced apoptosis of the smooth muscle cells.     

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.     

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.     

Phosphoramidon inhibits the vasoconstrictor effects evoked by big endothelin-1 but not the elevation of plasma endothelin-1 in vivo

Intravenous injections of big endothelin (ET)-1 (700 pmol/kg) in the pig increased arterial plasma levels of ET-1-like immunoreactivity (ET-1-LI) from 11.1 +/- 0.7 pM to 46.3 +/- 6.7 pM in the control situation and from 11.5 +/- 0.4 pM to 58.2 +/- 17 pM in the presence of the neutral endopeptidase inhibitor phosphoramidon (3 mg/kg). Big ET-1 increased splenic vascular resistance by 29% in the control situation. The vasoconstriction evoked by big ET-1 in the spleen was reduced after phosphoramidon treatment whereas the elevation of arterial ET-1-LI was not influenced. Furthermore the splenic vasoconstriction evoked by ET-1 was reduced after phosphoramidon without influencing plasma ET-1-LI. Also in rats the pressor effect of big ET-1 (1 nmol/kg) was inhibited by phosphoramidon (5 mg/kg) whereas the elevation of plasma ET-1 was not influenced. It is concluded that the vasoconstrictor effects of both big ET-1 and ET-1 are inhibited, but the increase in plasma ET-1 is unaffected by phosphoramidon.