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.     

Stobadine is a potent modulator of endogenous endothelin-1 in human fibroblasts

Binding of endothelin (ET) peptides to their respective receptors with resulting proliferation of vascular smooth muscle has been implicated in the pathogenesis of arterial hypertension and atherosclerosis. Recently it was hypothesized that endothelin- (ET-1) bound to its two membrane receptors (ET(A) and ET(B)) continues to activate signal transducing proteins in cells. It was also shown that pyridoindole stobadine stabilized lysosomal membranes in myocardium in early ischemia. Therefore we decided to study the effects of stobadine on specific, subtype-selective binding and subsequent degradation of human, synthetic [125I]-ET-1 in human fibroblasts (HF). Our results indicate that stobadine significantly potentiated ET-1 binding by reductive ET(B) selective degradation of ET-1 in HF. Hence, it is very plausible that stobadine may modulate endogenous endothelin and its intracellular mitogenic and chemotactic factors, principally by affecting two presumably related processes, participating in the proliferative and mitogenic response, (1) potentiation of signal trasduction from ET(A) receptors, and (2) subtype-ET(B) selective intracellular processing.     

Effect of the endothelin receptor antagonist bosentan on chronic hypoxia-induced morphological and physiological changes in rat carotid body

Previous experiments have repeatedly demonstrated that exposure to chronic hypoxia (CH) elicits remarkable structural changes and chemosensory hypersensitivity in the mammalian carotid body. Moreover, recent studies have shown that CH upregulates the neuroactive peptide, endothelin (ET), in oxygen-sensitive type I cells. The present study examines the possible involvement of ET in adaptation by concurrently exposing rats to hypobaric CH (B(P) = 380 Torr) and bosentan, a potent nonpeptide antagonist that blocks ET(A) and ET(B) receptors. Carotid body weight indicated that 14 days of CH induced organ enlargement, a response that was blunted in bosentan-treated rats (CH: 2.54 +/- 0.19-fold increase; CH plus bosentan: 1.92 +/- 0.14-fold increase; P < 0.05). Morphometric studies revealed that bosentan substantially eliminated CH-induced hyperplasia of chemosensory cell lobules as well as expansion of the connective tissue matrix. Vascular dilation associated with CH was not altered by the drug. In untreated animals exposed to 3 days of CH, expression of proliferating cell nuclear antigen (PCNA), a marker of mitosis, was increased in lobules of oxygen-sensitive type I cells and in extralobular vascular and connective tissue cells. The incidence of PCNA expression was significantly (P < 0.05) reduced in bosentan-treated animals. In vitro assessments of carotid sinus nerve (CSN) activity showed that enhancement of basal and hypoxia-evoked chemosensory activity following 9 days of CH was significantly (P < 0.001) blunted by concurrent treatment with bosentan. Collectively, our data are consistent with the hypothesis that CH-induced adaptation in the carotid body is at least partially mediated by signaling pathways involving ET receptors.     

17beta-estradiol inhibits cyclic strain-induced endothelin-1 gene expression within vascular endothelial cells

It has been well documented previously that 17beta-estradiol (E2) exerts a protective effect on cardiovascular tissue. The possible role of E2 in the regulation of endothelin (ET)-1 production has been previously reported, although the complex mechanisms by which E2 inhibits ET-1 expression are not completely understood. The aims of this study were to examine whether E2 was able to alter strain-induced ET-1 gene expression and also to identify the putative underlying signaling pathways that exist within endothelial cells. For cultured endothelial cells, E2 (1-100 nM), but not 17alpha-estradiol, inhibited the level of strain-induced ET-1 gene expression and also peptide secretion. This inhibitory effect elicited by E2 was able to be prevented by the coincubation of endothelial cells with the estrogen receptor antagonist ICI-182,780 (1 microM). E2 also inhibited strain-enhanced NADPH oxidase activity and intracellular reactive oxygen species (ROS) generation as measured by the redox-sensitive fluorescent dye 2',7'-dichlorofluorescin diacetate and the level of extracellular signal-regulated kinase (ERK) phosphorylation. Furthermore, the presence of E2 and antioxidants such as N-acetylcysteine and diphenylene iodonium were able to elicit a decrease in the level of strain-induced ET-1 secretion, ET-1 promoter activity, ET-1 mRNA, ERK phosphorylation, and activator protein-1 binding activity. In summary, we demonstrated, for the first time, that E2 inhibits strain-induced ET-1 gene expression, partially by interfering with the ERK pathway via the attenuation of strain-induced ROS generation. Thus this study delivers important new insight regarding the molecular pathways that may contribute to the proposed beneficial effects of estrogen on the cardiovascular system.     

Ca2+ signaling by distinct endothelin peptides in glomerular mesangial cells.

Ca2+ signaling by peptides of the endothelin (ET) gene family was studied in cultured glomerular mesangial cells. In addition to the increase in cytosolic free [Ca2+] ([Ca2+]i) previously described for ET-1, we also observed that ET-2, ET-3, and sarafotoxin S6b generate similar [Ca2+]i waveforms but with dissimilar potencies and kinetics. The prepro form of ET-1 was inactive, suggesting that mature ET peptides are constrained in an inactive conformation within the preproET species. ET isopeptides caused both release of Ca2+ from intracellular stores and Ca2+ influx via a voltage- and dihydropyridine-insensitive pathway. ET-mediated Ca2+ influx was independent of the increase in [Ca2+]i. Activation of protein kinase C inhibited ET-induced Ca2+ signaling, whereas addition of ET to protein kinase C-depleted cells resulted in enhanced [Ca2+]i waveforms. Mesangial cells also demonstrated a marked adaptive desensitization response to ET. These data demonstrate that Ca2+ signaling is a common response to different ET peptides in glomerular mesangial cells and that activation of protein kinase C down-regulates these Ca2+ signals.     

Endothelin induces rapid, dynamin-mediated budding of endothelial caveolae rich in ET-B

Clathrin-independent trafficking pathways for internalizing G protein-coupled receptors (GPCRs) remain undefined. Clathrin-mediated endocytosis of receptors including ligand-engaged GPCRs can be very rapid and comprehensive (<10 min). Caveolae-mediated endocytosis of ligands and antibodies has been reported to be much slower in cell culture (≫10 min). Little is known about the role of physiological ligands and specific GPCRs in regulating caveolae trafficking. Here, we find that one receptor for endothelin, ET-B but not ET-A, resides on endothelial cell surfaces in both tissue and cell culture primarily concentrated within caveolae. Reconstituted cell-free budding assays show that endothelins (ETs) induce the fission of caveolae from endothelial plasma membranes purified from rat lungs. Electron microcopy of lung tissue sections and tissue subcellular fractionation both show that endothelin administered intravascularly in rats also induces a significant loss of caveolae at the luminal surface of lung vascular endothelium. Endothelial cells in culture show that ET stimulates very rapid internalization of caveolae and cargo including caveolin, caveolae-targeting antibody, and itself. The ET-B inhibitor BQ788, but not the ET-A inhibitor BQ123, blocks the ET-induced budding of caveolae. Both the pharmacological inhibitor Dynasore and the genetic dominant negative K44A mutant of dynamin prevent this induced budding and internalization of caveolae. Also shRNA lentivirus knockdown of caveolin-1 expression prevents rapid internalization of ET and ET-B. It appears that endothelin can engage ET-B already highly concentrated in caveolae of endothelial cells to induce very rapid caveolae fission and endocytosis. This transport requires active dynamin function. Caveolae trafficking may occur more rapidly than previously documented when it is stimulated by a specific ligand to signaling receptors already located in caveolae before ligand engagement.     

Competency of embryonic cardiomyocytes to undergo Purkinje fiber differentiation is regulated by endothelin receptor expression

Purkinje fibers of the cardiac conduction system differentiate from heart muscle cells during embryogenesis. In the avian heart, Purkinje fiber differentiation takes place along the endocardium and coronary arteries. To date, only the vascular cytokine endothelin (ET) has been demonstrated to induce embryonic cardiomyocytes to differentiate into Purkinje fibers. This ET-induced Purkinje fiber differentiation is mediated by binding of ET to its transmembrane receptors that are expressed by myocytes. Expression of ET converting enzyme 1, which produces a biologically active ET ligand, begins in cardiac endothelia, both arterial and endocardial, at initiation of conduction cell differentiation and continues throughout heart development. Yet, the ability of cardiomyocytes to convert their phenotype in response to ET declines as embryos mature. Therefore, the loss of responsiveness to the inductive signal appears not to be associated with the level of ET ligand in the heart. This study examines the role of ET receptors in this age-dependent loss of inductive responsiveness and the expression profiles of three different types of ET receptors, ET(A), ET(B) and ET(B2), in the embryonic chick heart. Whole-mount in situ hybridization analyses revealed that ET(A) was ubiquitously expressed in both ventricular and atrial myocardium during heart development, while ET(B) was predominantly expressed in the atrium and the left ventricle. ET(B2) expression was detected in valve leaflets but not in the myocardium. RNase protection assays showed that ventricular expression of ET(A) and ET(B) increased until Purkinje fiber differentiation began. Importantly, the levels of both receptor isotypes decreased after this time. Retrovirus-mediated overexpression of ET(A) in ventricular myocytes in which endogenous ET receptors had been downregulated, enhanced their responsiveness to ET, allowing them to differentiate into conduction cells. These results suggest that the developmentally regulated expression of ET receptors plays a crucial role in determining the competency of ventricular myocytes to respond to inductive ET signaling in the chick embryo.     

Predominant activation of endothelin-dependent cardiac hypertrophy by norepinephrine in rat left ventricle

Locally released endothelin (ET)-1 has been recently identified as an important mediator of cardiac hypertrophy. It is still unclear, however, which primary stimulus specifically activates ET-dependent signaling pathways. We therefore examined in adult rats (n = 51) the effects of a selective ET(A) receptor antagonist in experimental models of cardiac hypertrophy, in which myocardial growth is predominantly initiated by a single primary stimulus. Rats were exposed to mechanical overload (ascending aortic stenosis), increased levels of circulating ANG II (ANG II infusion combined with hydralazine), or adrenergic stimulation (infusion of norepinephrine in a subpressor dose) for 7 days. All experimental treatments significantly increased left ventricular weight/body weight ratios compared with untreated rats, whereas systolic left ventricular peak pressure was increased only after ascending aortic stenosis. ET(A) receptor blockade exclusively reduced norepinephrine-induced cardiac hypertrophy and atrial natriuretic peptide gene expression. Blood pressure levels and heart rates remained unaffected during ET(A) receptor blockade in all experimental groups. These data indicate that in rat left ventricle, the ET-dependent signaling pathway leading to early development of cardiac hypertrophy and fetal gene expression is primarily activated by norepinephrine.     

Identification and endothelin-induced activation of multiple extracellular signal-regulated kinases in aortic smooth muscle cells.

In order to explore intracellular signaling pathways of the mitogenic action of endothelin (ET), we examined the effect of ET on activities of extracellular signal-regulated kinases (ERKs) in rat aortic smooth muscle cells (SMCs). Treatment of rat aortic SMCs with ET-1 increased kinase activities toward myelin basic protein (MBP). Both 43- and 41-kDa proteins were activated when kinase assays were done in MBP-containing polyacrylamide gels after SDS-PAGE. These proteins were identified as ERK1 and ERK2 with immunoprecipitation and immunoblotting using antipeptide antibodies, respectively. These results indicate that ERKs mediate signal transduction by ET.     

Leptin increases endothelin type A receptor levels in vascular smooth muscle cells

Leptin, one of the adipocyte-secreted peptides, is involved in the control of appetite and body weight. Several studies have demonstrated that plasma leptin levels are elevated in obese subjects and are positively correlated with body weight. The arterial endothelin (ET) system plays an important role in the regulation of vascular tone, and ET-1 overexpression may be involved in the pathogenesis of the hypertension associated with insulin resistance. This study was performed to explore the regulatory effects of leptin on ET receptor expression and ET binding in A10 vascular smooth muscle cells (VSMCs) by use of Northern blotting, immunoblotting, and a (125)I-labeled ET-1 binding assay. The effect of leptin on ET receptor-mediated cell proliferation was also tested. The results showed that leptin caused a significant increase in [(125)I]-ET-1 binding, which was time- and dose-dependent. Immunoblotting showed that expression of the ET type A receptor (ET(A)R) in leptin (10(-7) M)-treated cells was increased by up to 2.3-fold compared with controls. Levels of ET(A)R mRNA measured by Northern blotting were also increased by up to 2.2-fold in leptin (10(-7) M)-treated cells. Pretreatment with an ERK inhibitor, PD-98059 (2.5 x 10(-5) M), blocked the leptin-induced increase in (125)I-ET-1 binding. Finally, ET-1 (10(-7) M)-stimulated cell proliferation was enhanced by leptin (10(-7) M) pretreatment, with a maximal increase of twofold compared with controls. In conclusion, leptin increases ET(A)R expression in VSMCs in a time- and dose-dependent manner. This effect is ERK dependent and is associated with increased ET-1-stimulated cell proliferation. These findings provide support for roles for leptin and the ET system in the pathogenesis of obesity-associated hypertension.     

Endothelin-stimulated Ca(2+)signaling and endothelin receptor expression are decreased by parathyroid hormone treatment in UMR-106 osteoblastic osteosarcoma cells

Modulation of endothelin (ET-1)-induced [Ca(2+)](i)transients and receptor expression by parathyroid hormone (PTH) was studied in UMR-106 osteoblastic osteosarcoma cells. Ca(2+)signaling was assessed with Fura-2, and ET receptor mRNA expression was determined using ET(A)- and ET(B)-specific primers and RT-PCR amplification. ET-1 binding in UMR-106 cell membranes was also measured. PTH pretreatment for 8 h decreased the [Ca(2+)](i)transients elicited by ET-1 and by the ET(B)-selective agonist sarafotoxin 6c (S6c). When ET(B)receptors were desensitized by pretreatment with S6c or blocked with the ET(B)-selective antagonist BQ-788, the remaining ET(A)component of the signal was also decreased by PTH pretreatment. In contrast, [Ca(2+)](i)transients elicited by PGF(2alpha)and ionomycin were increased following PTH pretreatment, indicating that the effect of PTH to decrease ET-1-stimulated transients was selective. PTH pretreatment also decreased [(125)I]ET-1 binding and ET(A)and ET(B)mRNA, with maximal effects at approximately 8 h. ET-1 was not detectable in medium from either control or PTH treated UMR-106 cultures, suggesting that the decreased expression of ET receptors was not due to enhanced ET production and subsequent homologous desensitization. The downregulation of ET receptors in osteoblasts by PTH pretreatment may serve as a homeostatic mechanism in bone.     

肺内调节肽对兔支气管上皮细胞与嗜酸性粒细胞粘附功能的影响及机制探讨

为了探讨肺内调节肽在各类过敏性炎症发生,发展中的作用,我们观察了血管活性肠肽（vasoactive intestinal peptide,VIP)、表皮生长因子（epidermal growth factor,EGF)、内皮素-1（endothelin-1,ET-1)、降钙素基因相关肽（calcitonin gene-related peptide,CGRP)在未受应激与臭氧应激两种条件下对支气管上皮细胞（bronchial epithelial cell,BEC)与嗜酸性粒细胞（eosinophil,EOS)粘附的影响,结果发现,VIP、EGF可使O3应激的BEC与EOS的粘附率下降,下调气道上皮炎症反应：ET-1、CGRP可使未受应激的BEC与EOS的粘附率增加,诱发炎症损伤反应;CGRP还能加重臭氧的应激反应;ET-1、CGRP的效应可被W7、H7阻断,抗细胞间粘附分子-1（intercel-lular adhesion molecule,ICAM-1)抗体能阻断BEC与EOS的粘附,提示介导BEC与EOS粘附的粘附分子可能是ICAM-1。     

Chemokine receptor binding and signal transduction in native cells of the central nervous system

Chemokine receptors belong to the superfamily of seven-transmembrane-spanning, G-protein-coupled receptors, and their expression by central nervous system cells is clearly documented. As this gene family has become the target of novel therapeutic development, the analysis of these receptors requires radioligand binding techniques as well as methods that entail assessing receptor stimulation of signal transduction pathways. Herein, we describe specific protocols for measuring radiolabeled chemokine binding to their cognate receptors on cultured glial cells as well as to receptors expressed in heterologous cell systems. Multiple downstream signaling pathways, including intracellular calcium influx and receptor-dependent kinase activation, are associated with chemokine receptor stimulation. Protocols for measuring these signaling events in chemokine-receptor-expressing cells are also presented.     

Direct evidence for multiple endothelin receptors.

Competition binding experiments and peptide mapping techniques were employed in order to directly address the possible existence of endothelin (ET) receptor subtypes in the atria. Competition binding assays for 125I-labeled ET-1 or 125I-labeled ET-3 to bovine atrial membrane preparations suggest the existence of two ET receptor subtypes, one of which binds ET-1 and ET-3 with a similar affinity while the other shows preference for ET-3. However, cross-linking experiments of both peptides to this tissue resulted in the identification of a single 50-kDa protein. To identify directly the existence of multiple ET receptors, peptide mapping of cross-linked 125I-labeled ET-1 or 125I-labeled ET-3 receptors was conducted. Different peptide maps were obtained only under conditions that preferentially label one receptor subtype. These results indicate, for the first time, the existence of two ET receptor subtypes in the atria which differ from each other in both their binding characteristics and primary structure.