Endothelin-converting enzyme-1 expression in acute and chronic liver injury in fibrogenesis

Endothelin-1 (ET-1) induces contraction, proliferation, and collagen synthesis of activated hepatic stellate cells and is a potent mediator of portal hypertension. Endothelin-converting enzyme-1 (ECE-1) generates ET-1 from the inactive precursor big-endothelin-1. The cellular distribution and activity of ECE-1 in the liver is unknown. Hepatic fibrogenesis was induced in rats by CCl₄ administration and secondary biliary cirrhosis after 6 weeks of complete bile duct occlusion (BDO). The tissue ET-1 and ET receptor protein levels were quantified, the ECE-1 isoform mRNAs were measured by RNase protection assay and ECE-1 activity was analyzed. ECE-1a and -b mRNA were upregulated in biliary cirrhosis and in CCl₄-injured livers, whereas ECE-1c mRNA remained unchanged. ECE-1 activity was increased after BDO and peaked at 12?h after acute CCl₄-intoxication. Tissue levels of ET-1, ET_A- and ET_B receptors were elevated 7-, 5-, and 4.6-fold in cirrhotic rats, respectively. ECE-1 activity increased following BDO and acute CCl₄-intoxication. In conclusion, ECE-1a and -b RNAs are upregulated in fibrogenesis, indicating that these isoforms play a central role in ET-1 generation during fibrogenesis and portal hypertension.     

Cardiac myofibroblasts isolated from the site of myocardial infarction express endothelin de novo

Recently it was demonstrated that treatment with a nonselective endothelin (ET) receptor antagonist significantly reduces myocardial infarct size, which suggests a major role for ET in tissue repair following myocardial infarction (MI). Tissue repair and remodeling found at the site of MI are mainly attributed to myofibroblasts (myoFbs), which are phenotypically transformed fibroblasts that express alpha-smooth muscle actin. It is unclear whether myoFbs generate ET peptides and consequentially regulate pathophysiological functions de novo through expression of the ET-1 precursor (prepro-ET-1), ET-converting enzyme-1 (ECE-1), a metalloprotease that is required to convert Big ET-1 to ET-1 and ET receptors. To address these intriguing questions, we used cultured myoFbs isolated from 4-wk-old MI scar tissue. In cultured cells, we found: 1) expression of mRNA for ET precursor gene (ppET1), ECE-1, and ETA and ETB receptors by semiquantitative RT-PCR; 2) phosphoramidon-sensitive ECE-1 activity, which converts Big ET-1 to biologically active peptide ET-1; 3) expression of ETA and ETB receptors; 4) elaboration of Big ET-1 and ET-1 peptides in myoFb culture media; and 5) upregulation of type I collagen gene expression and synthesis by ET, which was blocked by bosentan (a nonselective ETA- and ETB receptor blocker). These studies clearly indicated that myoFbs express and generate ET-1 and receptor-mediated modulation of type I collagen expression by ET-1. Locally generated ET-1 may contribute to tissue repair of the infarcted heart in an autocrine/paracrine manner.     

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.     

Effect of eicosapentaenoic acid on the different endothelin system components in endothelin-1-induced hypertrophied cardiomyocytes

The cardiovascular benefit of fish oil, including eicosapentaenoic acid (EPA), in humans and experimental animals has been reported. The role of endothelin-1 (ET-1) in cardiac hypertrophy is well known. Endothelin-1 stimulates prepro-ET-1 mRNA expression in cardiomyocytes, and the autocrine/paracrine system of ET-1 is important for cardiomyocyte hypertrophy. Although many studies link EPA to cardiac protection, the effect of EPA on cardiac hypertrophy has yet to be clarified. Recently, we demonstrated that ET-1-induced cardiomyocytic change could be prevented by pretreatment with EPA. The present study investigated the changes of different components of the ET system at the mRNA level in ET-1-administered cardiomyocytes, and examined the effect of EPA pretreatment. Ventricular cardiomyocytes were isolated from 2-day-old Sprague-Dawley rats, cultured in Dulbecco's modified Eagle's medium and Ham F12 supplemented with 0.1% fatty acid-free bovine serum albumin for 3 days. At Day 4 of culture, the cardiomyocytes were divided into 3 groups: control group, ET-1-treated (0.1 nM) group, and ET-1-treated group pretreated with EPA (10 microM). Twenty-four hours after treatment, the gene expressions of different components of the endothelin system in three experimental groups were evaluated by real-time polymerase chain reaction. Prepro-ET-1 mRNA expression was 53% upregulated in ET-1-induced hypertrophied cardiomyocytes and suppressed in the EPA-pretreated group. Endothelin-converting enzyme-1 (ECE-1) was also increased in ET-1-administered cardiomyocytes by 42% compared with the control group and was reversed in the EPA-pretreated group. The two receptors of ET system, ET(A) and ET(B), tended to be increased in the ET-1-treated group, but no statistical significance was seen among study groups. Endothelin-1 increased prepro-ET-1 and ECE-1 mRNA expression in hypertrophied-neonatal cardiomyocytes, and this was reversed with EPA pretreatment. Thus, EPA may play a crucial role in the regression of ET-1-induced cardiomyocyte hypertrophy, partly through the suppression of ET-1 and ECE-1 expression.     

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.     

ETA and ETB receptors are expressed in vascular adventitial fibroblasts

The adventitia has been recognized to play important roles in vascular oxidative stress, remodeling, and contraction. We recently demonstrated that adventitial fibroblasts are able to express endothelin (ET)-1 in response to ANG II. However, it is unclear whether ET-1 receptors are expressed in the adventitia. We therefore investigated the expression and roles of both ET(A) and ET(B) receptors in collagen synthesis and ET-1 clearance in adventitial fibroblasts. Adventitial fibroblasts were isolated and cultured from the mouse thoracic aorta by the explant method. Cultured cells were treated with ANG II (100 nmol/l) or ET-1 (10 pM) in the presence or absence of the ANG II type 1 receptor antagonist losartan (100 μM), the ET-1 receptor antagonists BQ-123 (ET(A) receptor, 1 μM) and BQ-788 (ET(B) receptor, 1 μM), and the ET(B) receptor agonist sarafotoxin 6C (100 nM). ET-1 peptide levels were determined by ELISA, whereas ET(A), ET(B), and collagen levels were determined by Western blot analysis. ANG II increased ET-1 peptide levels in a time-dependent manner. ANG II increased ET(A) and ET(B) receptor protein levels as well as collagen in a similar fashion. ANG II-induced collagen was reduced while in the presence of BQ-123, suggesting a role for the ET(A) receptor in the regulation of the extracellular matrix. ANG II treatment in the presence of BQ-788 significantly increased ET-1 peptide levels. Conversely, the ET(B) receptor agonist sarafotoxin 6C significantly decreased ET-1 peptide levels. These data implicate a role for the ET(B) receptor in the clearance of the ET-1 peptide. In conclusion, both ET(A) and ET(B) receptors are expressed in adventitial fibroblasts, which paves the ground for the biological significance of adventitial ET-1. The ET(A) receptor subtype mediates collagen I expression, whereas the ET(B) receptor subtype may play a protective role through increasing the clearance of the ET-1 peptide.     

Affinity profile of the novel muscarinic antagonist, guanylpirenzepine

The study reports the functional affinity of an amidino derivative of pirenzepine, guanylpirenzepine, for muscarinic receptors mediating relaxation of rat duodenum, inhibition of rabbit vas deferens twitch contraction (both receptors previously classified as M1), guinea pig negative inotropism (M2) and ileal contraction (M3). Unlike pirenzepine, guanylpirenzepine discriminated between duodenum and vas deferens receptors, with a 30-fold greater affinity for the former subtype. The unique selectivity pattern of guanylpirenzepine (duodenum greater than vas deferens greater than ileum greater than atrium) renders it a promising tool for the classification of muscarinic receptor subtypes.     

Anatomically distinct activation of endothelin-3 and the L-arginine/nitric oxide pathway in the kidney with advanced aging

Aging is associated with spontaneous degenerative changes of renal function and structure. The aim of this study was to determine changes of the endothelin (ET) system and NO tissue bioactivity during the physiological aging process. Renal protein expression of ET-1 and ET-3, ETA, and ETB receptor mRNA expression, ET receptor binding and distribution, and tissue NO metabolite content were determined in adult, middle-aged, and senescent normotensive female Wistar rats. In senescent animals, medullary ET-3 content increased 3.4-fold (p<0.05 vs. adult), whereas aging did not affect ET-3 levels in the cortex. Local NO bioavailability, determined by NO metabolite tissue measurements, decreased in the cortex only. ET receptor binding capacity--predominantly due to ETB receptor binding--was lower in medulla than in cortex. Aging had no effect on ET-1 binding capacity or ET receptor distribution, whereas with advanced age gene expression of both receptors decreased. In conclusion, aging causes distinctive expressional changes of the renal endothelin system in otherwise healthy rats. The pronounced increase of endothelin-3 in the renal medulla is associated with preservation of local NO metabolite levels, changes not observed in the cortex. These findings could be important for pathologies and possibly therapy associated with renal aging.     

Endothelin antagonism suppresses plasma and cardiac endothelin-1 levels in SHRSPs at the typical hypertensive stage

Endothelin-1 (ET-1) has been implicated in hypertension, heart failure, atherosclerosis, and pulmonary hypertension. In all these conditions, plasma immunoreactive ET-1 levels are elevated, and tissue ET-1 expression is increased. Clinical trials have demonstrated potentially important benefits of ET antagonism among patients with essential hypertension, pulmonary hypertension, and heart failure. It is unknown whether ET antagonism affects the production of ET-1 in stroke-prone spontaneously hypertensive rat (SHRSP) heart at the typical hypertensive stage. The objective of this study was to investigate the effects of ET blockade on the expression levels of plasma and cardiac ET-1 in SHRSPs. SHRSPs were treated for 3 months with SB209670 (ET(A)/ET(B) dual receptor antagonist) or with saline (vehicle) commencing at the prehypertensive stage (age 6 weeks). Plasma and left ventricular ET-1 peptide levels were measured using enzyme-linked immunoabsorbent assay. Compared with age-matched control Wistar-Kyoto rats, peptide levels of ET-1 were significantly upregulated in vehicle-treated SHRSP heart; this upregulation was reversed by long-term ET antagonism. Plasma ET-1 levels were also significantly increased in vehicle-treated SHRSPs and were normalized by ET antagonism. mRNA expression of preproET-1, which is the source of ET-1 peptide production, was significantly increased in vehicle-treated SHRSP heart and was normalized by ET antagonism. Marked cardiac hypertrophy and fibrosis at the histologic level in SHRSPs were ameliorated by ET antagonism, and left ventricular hypertrophy as seen on echocardiography in SHRSPs was suppressed by ET blockade. After ET antagonism, systolic blood pressures were reduced in SHRSPs; diastolic blood pressures were unchanged. The reversal effect of the upregulated ET system in SHRSP heart by ET antagonism might be independent of blood pressure change. By suppressing the upregulated ET system, ET antagonism might be beneficial in arresting cardiac remodeling.     

Subacute reserpine treatment reveals preferential coupling between the M3 muscarinic receptor subtype and phosphatidylinositol turnover.

Muscarinic receptors in the rat cerebral cortex, cardiac atria and vas deferens were identified, quantitated, and characterized relative to phosphatidylinositol (PI) turnover as the functional response to stimulation of specific receptor subtypes. Receptor densities as determined by 3H-QNB binding were ranked: cerebral cortex greater than vas deferens greater than heart. Using displacement of 3H-QNB binding by the selective M1 and M2 muscarinic receptor antagonists pirenzepine and 11[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro- 6H-pyrido [2,3-b] [1,4] benzodiazepine-6-one (AF-DX 116) respectively, heterogeneous populations were found in the cerebral cortex and vas deferens. The M1 receptor subtype predominated in the former and the M2 predominated in the latter. An homogeneous M2 receptor population was present in the heart. Methacholine-stimulated accumulation of 3H inositol-1-phosphate was greater in the vas deferens than in the cerebral cortex, whereas PI turnover was not enhanced in cardiac atria. Reserpine treatment of rats (0.5 mg kg-1 day-1 for 7 days) increased muscarinic receptor density in the vas deferens coincident with a shift in the low affinity pKi for AF-DX 116 to a value comparable to high affinity binding, and abolished the enhanced PI hydrolysis. In the cerebral cortex, reserpine treatment shifted only the early portion of the methacholine dose-response curve to the right. These results are judged to be supportive of preferential coupling between the M3 muscarinic receptor subtype and PI turnover.     

Effect of endothelin receptor antagonists on ventricular susceptibility in postinfarcted rats

This study investigated whether selective endothelin (ET) type A (ET(A)) or nonselective ET(A)/ET(B) receptor blockade exerted antiarrhythmic effects through attenuated sympathetic reinnervation after infarction. Twenty-four hours after ligation of the left anterior descending artery, male Wistar rats received either vehicle, ABT-627 (selective ET(A) receptor antagonist), bosentan (nonselective ET(A)/ET(B) receptor antagonist), or hydralazine for 4 wk. The measurement of myocardial ET-1 levels at the remote zone revealed a significant increase in vehicle-treated infarcted rats compared with sham-operated rats, consistent with increased activities of ET-1 after infarction. Sympathetic nerve function changes assessed by the norepinephrine content of myocardium and the dialysate and plasma dihydroxyphenylglycol levels were parallel to ET-1 levels. Immunohistochemical analysis for tyrosine hydroxylase, growth-associated protein 43, and neurofilament also confirmed the change of nerve function. This was accompanied with a significant upregulation of nerve growth factor protein expression and mRNA in the vehicle-treated infarcted rats, which reduced after the administration of either ET(A) or ET(A)/ET(B) blockade to a similar extent. The beneficial effects of ET receptor antagonists on sympathetic nerve function and structures were dissociated from their blood pressure-lowering effect because ET receptor antagonists and hydralazine reduced arterial pressure similarly. Arrhythmic severity during programmed stimulation in ET receptor antagonists-treated rats was significantly lower than that in vehicle-treated infarcted rats. Our data indicate that the ET system, especially via ET(A) receptors, plays an important role in attenuating sympathetic reinnervation after infarction. Independent of their hemodynamic effects, a chronic use of either ET(A) or ET(A)/ET(B) antagonists may modify the arrhythmogenic response to programmed electrical stimulation.     

Absence of endothelin receptors and receptor mRNA in mammalian fibroblasts transformed with SV40 or ras oncogene

Endothelin-1 (ET-1), a peptide isolated from the culture medium of endothelial cells, mediates a variety of physiological and pathological responses including mitogenesis. We have compared the expression of ET receptors in untransformed versus ras-transformed NIH-3T3 murine fibroblasts and in untransformed versus SV40-transformed Wl38 (VA13) human fibroblasts by ligand binding and Northern analysis. NIH-3T3 and Wl38 cells displayed high affinity (200 and 220 pM) and high density (23,000 sites/cell and 14,000 sites/cell for NIH-3T3 and Wl38 cells, respectively) ET receptors. Competition binding experiments using subtype-selective ligands identified these receptors as the ETA subtype. Addition of ET-1 to the cells produced a concentration-dependent increase in intracellular calcium release. Both ras-transformed NIH-3T3 cells and SV40-transformed Wl38 cells (VA13) completely lacked [125I]ET-1 binding and failed to release calcium when exposed to ET-1. Northern analysis of the polyadenylat ed RNA (polyA RNA) isolated from untransformed and transformed cells revealed that the steady-state level of ETA receptor RNA was 90-95% less in transformed cells compared to untransformed cells. Thus, the loss of ET receptors as well as the receptor-mediated responses in transformed cells can be explained by down-regulation of ET receptor mRNA.