Endothelin receptors in teleost fishes: cardiovascular effects and branchial distribution

By observing gill blood flow using epi-illuminating microscopy, in parallel with cardiovascular recordings and immunohistochemistry, we have tried to identify the receptor mediating endothelin (ET) type 1 (ET1)-induced pillar cell contraction in the lamellae of the Atlantic cod (Gadus morhua). Intra-arterial injection of the specific ET(B) receptor agonist BQ-3020 induced dose-dependent increases in ventral aortic blood pressure, gill vascular resistance, and pillar cell area (indicating contraction). The specific ET(A) receptor antagonist BQ-610 did not prevent either pillar cell contraction or increased gill vascular resistance induced by ET-1 injection. The cardiovascular responses were corroborated by the detection of ET(B) receptor-like immunoreactivity (IR) associated with pillar cells in the lamellar region and in neuroendocrine cells. ET(B) receptor-like IR was also found lining the muscle layer of lamellar arterioles and filament arteries. In contrast, strong ET(A) receptor-like IR was found on branchial nerves throughout the filaments. In addition, ET-like IR was concentrated in neuroendocrine cells in the filament and lamellae. We also present data suggesting that ET-mediated pillar cell contraction is widespread among teleost fish, including Atlantic cod, rainbow trout (Oncorhynchus mykiss), sculpin (Myoxocephalus scorpius), and mackerel (Scomber scombrus). Taken together, our results suggest that an ET(B)-like receptor mediates pillar cell contraction in fishes, whereas ET(A)-like receptors may serve another function in the gill, inasmuch as ET(A) receptor-like IR is found on branchial nerves.     

Endothelin A and endothelin B receptors differ in their ability to stimulate ERK1/2 activation

Endothelin-1 (ET-1) acts on two different G protein-coupled receptors, namely the endothelin A (ET(A)) and the endothelin B (ET(B)) receptors. Both receptor subtypes show differences in their tissue expression and signal transduction. In the present study, we compared the ability of ET(A) and ET(B) receptors to stimulate extracellular signal-regulated kinase 1/2 (ERK1/2). In addition, we analyzed the role of the extracellular N terminus for ERK1/2 activation, because the ET(B) receptor undergoes an agonist-dependent N-terminal proteolysis. ET-1 stimulation of HEK293 cells stably expressing the ET(A) receptor induced a monophasic, but sustained ERK1/2 activation, whereas the ET(B) receptor showed a biphasic ERK1/2 activation. A truncated mutant ET(B) receptor, lacking the proteolytically cleaved N terminus (delta2-64 ET(B)) revealed only a monophasic and transient ERK1/2 activation. Treatment of HEK293 delta2-64 ET(B) cell clones with ET-1 and a synthetic NT27-64 peptide, corresponding to the N-terminally cleaved fragment of the ET(B) receptor and ET-1, did not restore the biphasic activation of ERK1/2. A chimeric ET(B) receptor in which the N terminus was replaced by the N terminus of the ET(A) receptor elicited biphasic ERK1/2 activation. The presented data suggest that an intact N terminus of the ET(B) receptor is necessary for the second phase of ERK1/2 activation. However, it appears that the length of the N terminus rather than a specific sequence motif is required for biphasic ERK1/2 activation.     

Regulation and intracellular trafficking pathways of the endothelin receptors

The effects of endothelin (ET) are mediated via the G protein-coupled receptors ET(A) and ET(B). However, the mechanisms of ET receptor desensitization, internalization, and intracellular trafficking are poorly understood. The aim of the present study was to investigate the molecular mechanisms of ET receptor regulation and to characterize the intracellular pathways of ET-stimulated ET(A) and ET(B) receptors. By analysis of ET(A) and ET(B) receptor internalization in transfected Chinese hamster ovary cells in the presence of overexpressed betaARK, beta-arrestin-1, beta-arrestin-2, or dynamin as well as dominant negative mutants of these regulators, we have demonstrated that both ET receptor subtypes follow an arrestin- and dynamin/clathrin-dependent mechanism of internalization. Fluorescence microscopy of Chinese hamster ovary and COS cells expressing green fluorescent protein (GFP)-tagged ET receptors revealed that the ET(A) and ET(B) subtypes were targeted to different intracellular routes after ET stimulation. While ET(A)-GFP followed a recycling pathway and colocalized with transferrin in the pericentriolar recycling compartment, ET(B)-GFP was targeted to lysosomes after ET-induced internalization. Both receptor subtypes colocalized with Rab5 in classical early endosomes, indicating that this compartment is a common early intermediate for the two ET receptors during intracellular transport. The distinct intracellular routes of ET-stimulated ET(A) and ET(B) receptors may explain the persistent signal response through the ET(A) receptor and the transient response through the ET(B) receptor. Furthermore, lysosomal targeting of the ET(B) receptor could serve as a biochemical mechanism for clearance of plasma endothelin via this subtype.     

Endothelin-B (ET(B)) receptor distribution in tissues of the lizard Podarcis sicula

Although the structural and pharmacological properties of endothelin (ET) receptors have been studied, little is known concerning their physiological significance, even if each subtype is supposed to have a distinct physiological action. Thus, to further elucidate the physiological function of this receptor, we examined the presence and distribution of the endothelin-B receptor (ET(B)) subtype in tissues of the lizard Podarcis sicula, using immunoblotting and immunohistochemistry. Immunoblotting indicated that, although the ET(B) receptor appears to be ubiquitous, it is present at different levels in the tissues examined. Furthermore, immunohistochemistry showed that this receptor is very abundant in endothelial cells of all tissues, suggesting that there is an ET(B)-mediated autocrine system of endothelin, which plays an important role in the regulation of endothelial cell function. On the other hand, the presence of ET(B) immunoreactivity also in endocrine systems such as adrenal and thyroid glands suggests an involvement also in the paracrine system of these organs.     

Ligand-dependent differences in the internalization of endothelin A and endothelin B receptor heterodimers

Endothelin-1 (ET-1) is a potent vasoactive peptide that acts on endothelin A (ET(A)) and endothelin B (ET(B)) receptors. Although both receptor subtypes are co-expressed in numerous cells, little is known about their ability to form heterodimers. Here we show that both receptors were co-immunoprecipitated with an ET(B)-specific antibody using extracts from HEK293 cells stably co-expressing a fusion protein consisting of a myc-tagged ET(A) receptor and CFP (ET(A)myc.CFP) and a fusion protein consisting of an ET(B) receptor and YFP (ET(B).YFP). Co-immunoprecipitation was also observed with extracts from HEK293 cells transiently co-expressing FLAG-tagged ET(B) and myc-tagged ET(A) receptors, thereby excluding that heterodimerization is mediated by the CFP/YFP moieties. Heterodimerization was further confirmed in fluorescence resonance energy transfer (FRET) analysis of HEK293 cells transiently co-expressing ET(A)myc.CFP and ET(B).YFP receptors. FRET efficiencies were between 12 and 18% in untreated and antagonist- or ET-1-treated cells, indicating constitutive heterodimerization. Prolonged stimulation (30 min) with the ET(B) receptor-selective agonist BQ3020 decreased FRET efficiency by 50%. This decrease was not observed when internalization was inhibited by co-expression of dominant-negative K44A.dynamin I or incubation with 450 mm sucrose. Enzyme-linked immunosorbent assay and laser scanning microscopy of cell clones stably co-expressing ET(A)myc.CFP/ET(B)flag.YFP receptors revealed a slower sequestration of the ET(B)flag.YFP receptors upon stimulation with ET-1 than with BQ3020. No difference in ET-1 or BQ3020-mediated sequestration was observed with cell clones expressing ET(B)flag.YFP receptors alone. The data suggest that ET(A) and ET(B) receptors form constitutive heterodimers, which show a slower sequestration upon stimulation with ET-1 than with BQ3020. Heterodimer dissociation along the endocytic pathway only occurs upon ET(B)-selective stimulation.     

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.     

The extracellular N terminus of the endothelin B (ETB) receptor is cleaved by a metalloprotease in an agonist-dependent process

The extracellular N terminus of the endothelin B (ET(B)) receptor is susceptible to limited proteolysis (cleavage at R64 downward arrow S65), but the regulation and the functional consequences of the proteolysis remain elusive. We analyzed the ET(B) receptor or an ET(B)-GFP fusion protein stably or transiently expressed in HEK293 cells. After incubation of cells at 4 degrees C, only the full-length ET(B) receptor was detected at the cell surface. However, when cells were incubated at 37 degrees C, N-terminal cleavage was observed, provided endothelin 1 was present during the incubation. Cleavage was not inhibited by internalization inhibitors (sucrose, phenylarsine oxide). However, in cells incubated with both internalization inhibitors and metalloprotease inhibitors (batimastat, inhibitor of TNFalpha-convertase) or metal chelators (EDTA, phenanthroline), the cleavage was blocked, indicating that metalloproteases cleave the agonist-occupied ET(B) receptor at the cell surface. Functional analysis of a mutant ET(B) receptor lacking the first 64 amino acids ([Delta2-64]ET(B) receptor) revealed normal functional properties, but a 15-fold reduced cell surface expression. The results suggest a role of the N-terminal proteolysis in the regulation of cell surface expression of the ET(B) receptor. This is the first example of a multispanning membrane protein, which is cleaved by a metalloprotease, but retains its functional activity and overall structure.     

Endothelin promotes neurite elongation by a mechanism dependent on c-Jun N-terminal kinase

Endothelin (ET), which is known as a vasoconstrictive peptide, is associated with a lot of biological functions. Although endothelin receptors are expressed in the central nervous system (CNS), little is known about the effects of endothelin on neuronal function. In this study, we reported that endothelins elongate cortical neurites via the endothelin A receptor. All the endothelin isoforms tested, endothelin-1, endothelin-2, and endothelin-3, promoted neurite elongation. ET-1-induced neurite elongation was specifically inhibited by treatment with BQ123, an antagonist for the endothelin A receptor. In addition, inhibition of ET-1-induced c-Jun N-terminal kinase (JNK) activation by treatment with SP600125, a JNK inhibitor, also prevented the ET-1-mediated promotion of neurite elongation. Thus, endothelin induces cortical neurite elongation through the endothelin A receptor by a mechanism dependent on JNK.     

Cardiomyocyte-specific endothelin A receptor knockout mice have normal cardiac function and an unaltered hypertrophic response to angiotensin II and isoproterenol

Even though endothelin is recognized as an important vasoregulatory molecule, the roles of endothelin receptors in specific cell types are not yet fully understood. Mice with a null mutation in endothelin A receptor gene (ET(A)) or in the gene of its ligand (endothelin 1) die neonatally due to craniofacial and cardiac abnormalities. This early lethality has in the past hindered studies on the role of endothelin in cardiovascular physiology and pathophysiology. To overcome this obstacle, we utilized the cre/loxP technology to generate mice in which the ET(A) gene could be deleted specifically in cardiomyocytes. The cre recombinase transgene driven by the alpha-myosin heavy-chain promoter deleted the floxed ET(A) allele specifically in the hearts of these mice, resulting in a 78% reduction in cardiac ET(A) mRNA level compared to wild-type controls. Cardiomyocyte-specific ET(A) knockout animals are viable and exhibit normal growth, cardiac anatomy, and cardiac contractility, as assessed by echocardiography. In addition, these animals exhibit hypertrophic and contractile responses to 10-day infusion of angiotensin II or isoproterenol similar to those observed in control animals. These results indicate that in adult mice cardiac ET(A) receptors are not necessary for either baseline cardiac function or stress-induced response to angiotensin II or isoproterenol.     

Up-regulated inflammatory factors endothelin, NFkappaB, TNFalpha and iNOS involved in exaggerated cardiac arrhythmias in l-thyroxine-induced cardiomyopathy are suppressed by darusentan in rats

The exaggerated cardiac arrhythmias in cardiomyopathy induced by L-thyroxine treatment are related to ion channelopathies and to an abnormal endothelin (ET) pathway. It was hypothesized that an increased incidence of ventricular fibrillation (VF) could be mediated by inflammatory factors including the ET pathway, nuclear factor kappa B (NFkappaB), tumor necrosis factor-alpha (TNFalpha) and inducible nitric oxide synthase (iNOS). Abnormal expression of NFkappaB, TNFalpha, iNOS and enhanced VF are linked with the activated ET pathway and a significant reversion could be achieved by the selective endothelin A receptor antagonist darusentan. Cardiomyopathy in rats was produced by L-thyroxine treatment (0.3 mg kg(-1) d(-1), sc) for 10 days. The mRNA expression of the ET pathway, NFkappaB, TNFalpha, iNOS and the activity of the redox system were assayed in association with the incidence of VF produced by coronary ligation/reperfusion. Darusentan was administered on days 6-10 of L-thyroxine treatment. The VF incidence, which was higher in the l-thyroxine cardiomyopathy group, was suppressed by darusentan. The mRNA levels of preproET-1, endothelin converting enzyme, endothelin receptor A (ET(A)R), endothelin receptor B (ET(B)R), NFkappaB, TNFalpha and iNOS in left ventricle were up-regulated in the cardiomyopathic heart. There was significant oxidative stress in this cardiomyopathy model. Darusentan suppressed the up-regulated mRNA levels of ET(A)R, ET(B)R, NFkappaB, TNFalpha, and iNOS. These results indicate that the high incidence of VF which is related to up-regulation of inflammatory factors in the cardiomyopathic myocardium is significantly suppressed by selective ET(A)R blockade.     

Characteristic defects in neural crest cell-specific Galphaq/Galpha11- and Galpha12/Galpha13-deficient mice

The endothelin/endothelin receptor system plays a critical role in the differentiation and terminal migration of particular neural crest cell subpopulations. Targeted deletion of the G-protein-coupled endothelin receptors ET(A) and ET(B) was shown to result in characteristic developmental defects of derivatives of cephalic and cardiac neural crest and of neural crest-derived melanocytes and enteric neurons, respectively. Since both endothelin receptors are coupled to G-proteins of the G(q)/G(11)- and G(12)/G(13)-families, we generated mouse lines lacking Galpha(q)/Galpha(11) or Galpha(12)/Galpha(13) in neural crest cells to study their roles in neural crest development. Mice lacking Galpha(q)/Galpha(11) in a neural crest cell-specific manner had craniofacial defects similar to those observed in mice lacking the ET(A) receptor or endothelin-1 (ET-1). However, in contrast to ET-1/ET(A) mutant animals, cardiac outflow tract morphology was intact. Surprisingly, neither Galpha(q)/Galpha(11)- nor Galpha(12)/Galpha(13)-deficient mice showed developmental defects seen in animals lacking either the ET(B) receptor or its ligand endothelin-3 (ET-3). Interestingly, Galpha(12)/Galpha(13) deficiency in neural crest cell-derived cardiac cells resulted in characteristic cardiac malformations. Our data show that G(q)/G(11)- but not G(12)/G(13)-mediated signaling processes mediate ET-1/ET(A)-dependent development of the cephalic neural crest. In contrast, ET-3/ET(B)-mediated development of neural crest-derived melanocytes and enteric neurons appears to involve G-proteins different from G(q)/G(11)/G(12)/G(13).     

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.     

Endothelin receptors in human and guinea-pig gallbladder muscle

We measured contraction of muscle strips caused by endothelin (ET) isopeptides and binding of (125)I-ET-1 to muscle cell membranes prepared from human and guinea-pig gallbladders. Visualization of (125)I-ET-1 binding sites in tissue was performed by autoradiography. Results in human were similar to those in guinea-pig. ET-1 caused tetrodotoxin and atropine-insensitive contraction. The relative potencies for ET isopeptides to cause contraction were ET-1=ET-2>ET-3. ET-1 caused contraction was only slightly inhibited by BQ-123 (potent ET(A) receptor antagonist) and not by BQ-788 (potent ET(B) receptor antagonist). It was inhibited by the combination of both. Autoradiography localized (125)I-ET-1 binding to the smooth muscle layer. Binding of (125)I-ET-1 to muscle cell membranes was saturable and specific. Analysis of dose-inhibition curves demonstrated the presence of two classes of receptors. One class (ET(A) receptor) had a high affinity for ET-1 and ET-2 but a low affinity for ET-3, and the other (ET(B) receptor) a high affinity for ET-1, ET-2 and ET-3. These results demonstrate that similar to guinea-pig, human gallbladder possesses both ET(A) and ET(B) receptors cooperating to mediate muscle contraction.     

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.     

Cell-autonomous and nonautonomous actions of endothelin-A receptor signaling in craniofacial and cardiovascular development

Craniofacial and cardiac development relies on the proper patterning of the neural crest-derived ectomesenchyme of the pharyngeal arches, from which many craniofacial and great vessel structures arise. One of the intercellular signaling molecules that is involved in this process, endothelin-1 (ET-1), is expressed in the arch epithelium and influences arch development by binding to its cognate receptor, the endothelin A (ET(A)) receptor, found on ectomesenchymal cells. We have previously shown that absence of ET(A) signaling in ET(A)(-/-) mouse embryos disrupts neural crest cell development, resulting in craniofacial and cardiovascular defects similar in many aspects to those in mouse models of DiGeorge syndrome. These changes may reflect a cell-autonomous requirement for ET(A) signaling during crest cell development because the ET(A) receptor is an intracellular signaling molecule. However, it is also possible that some of the observed defects in ET(A)(-/-) embryos could arise from the absence of downstream signaling that act in a non-cell-autonomous manner. To address this question, we performed chimera analysis using ET(A)(-/-) embryonic stem cells. We observe that, in almost all early ET(A)(-/-) --> (+/+) chimeric embryos, ET(A)(-/-) cells are excluded from the caudoventral aspects of the pharyngeal arches, suggesting a cell-autonomous role for ET(A) signaling in crest cell migration and/or colonization. Interestingly, in the few embryos in which mutant cells do reach the ventral arch, structures derived from this area are either composed solely of wild type cells or are missing, suggesting a second cell-autonomous role for ET(A) signaling in postmigratory crest cell differentiation. In the cardiac outflow tract and great vessels, ET(A)(-/-) cells are excluded from the walls of the developing pharyngeal arch arteries, indicating that ET(A) signaling also acts cell-autonomously during cardiac neural crest cell development.     

Differences in endothelin receptor types in the vasculature of Bothrops jararaca (Viperidae) and Oxyrhopus guibei (Colubridae) snakes

Endothelins (ETs) are vasoactive peptides evolutionary well conserved that exert their effects through two specific receptors (ET(A) and ET(B)) widely distributed in all vertebrates. In snakes, the presence and function of endothelins and their receptors are still scarcely described. We have recently demonstrated the presence of ET(A) and ET(B2) receptors in the snake Bothrops jararaca (Bj). In the present work we showed that distinctively from Bj, the vascular contraction induced by endothelin in Oxyrhopus guibei (Og) snake is mediated only by ET(A) receptors. Selective ET(B) agonists (SRTX-c and IRL(1620)) and antagonists (IRL(1038) and BQ(788)) were ineffective in Og preparations of isolated aorta. We also showed that ET-1 response on Og arterial blood pressure was monophasic hypertensive as opposed to biphasic (hypotension followed by hypertension) in Bj. Furthermore, we characterized the relaxing properties of endothelin receptor ET(B1) in pre-contracted aorta preparations. We showed that IRL(1620) induced relaxation of pre-contracted Bj aorta but was ineffective in relaxing Og preparations. IRL(1620) relaxing effect on Bj aorta was abolished by l-NAME, indicating involvement of NO release, and was reduced by selective ET(B) antagonists. Our findings suggest that Og snake has a more primitive spectrum of ET receptors (only ET(A) receptor) than Bj (presence of ET(A), ET(B1) and ET(B2) receptors).     

cDNA cloning and chromosomal assignment of the endothelin 2 gene: vasoactive intestinal contractor peptide is rat endothelin 2.

Four members of the endothelin family of vasoactive and mitogenic peptides have been identified: human endothelins 1, 2, and 3 (ET1, ET2, and ET3, respectively) and mouse vasoactive intestinal contractor (VIC). To characterize the mRNA encoding ET2, a 192-bp fragment of the ET2 gene, amplified by the polymerase chain reaction from human genomic DNA, was used to screen cell lines and tissues for ET2 gene expression. ET2 mRNA was detected in a cell line (HTB119) derived from a human lung small cell carcinoma, and an ET2 cDNA was cloned from a cDNA library prepared from HTB119 mRNA. DNA prepared from human-mouse somatic hybrid cell lines was used to assign the gene encoding ET2 (EDN2) to the 1p21----1pter region of chromosome 1, demonstrating that EDN2 is not linked to genes encoding ET1 (EDN1; chromosome 6) and ET3 (EDN3; chromosome 20). Southern blot hybridization revealed a single gene in human and rat genomes that hybridized with the ET2 gene fragment, and the rat gene was cloned. The endothelin peptide encoded by the rat gene differed from ET2 at 1 of 21 residues and was identical to mouse VIC. We conclude that VIC is the mouse and rat analogue of the human ET2 gene.     

Arrestins 2 and 3 differentially regulate ETA and P2Y2 receptor-mediated cell signaling and migration in arterial smooth muscle

Overstimulation of endothelin type A (ET(A)) and nucleotide (P2Y) Gα(q)-coupled receptors in vascular smooth muscle causes vasoconstriction, hypertension, and, eventually, hypertrophy and vascular occlusion. G protein-coupled receptor kinases (GRKs) and arrestin proteins are sequentially recruited by agonist-occupied Gα(q)-coupled receptors to terminate phospholipase C signaling, preventing prolonged/inappropriate contractile signaling. However, these proteins also play roles in the regulation of several mitogen-activated protein kinase (MAPK) signaling cascades known to be essential for vascular remodeling. Here we investigated whether different arrestin isoforms regulate endothelin and nucleotide receptor MAPK signaling in rat aortic smooth muscle cells (ASMCs). When intracellular Ca(2+) levels were assessed in isolated ASMCs loaded with Ca(2+)-sensitive dyes, P2Y(2) and ET(A) receptor desensitization was attenuated by selective small-interfering (si)RNA-mediated depletion of G protein-coupled receptor kinase 2 (GRK2). Using similar siRNA techniques, knockdown of arrestin2 prevented P2Y(2) receptor desensitization and enhanced and prolonged p38 and ERK MAPK signals, while arrestin3 depletion was ineffective. Conversely, arrestin3 knockdown prevented ET(A) receptor desensitization and attenuated ET1-stimulated p38 and ERK signals, while arrestin2 depletion had no effect. Using Transwell assays to assess agonist-stimulated ASMC migration, we found that UTP-stimulated migration was markedly attenuated following arrestin2 depletion, while ET1-stimulated migration was attenuated following knockdown of either arrestin. These data highlight a differential arrestin-dependent regulation of ET(A) and P2Y(2) receptor-stimulated MAPK signaling. GRK2 and arrestin expression are essential for agonist-stimulated ASMC migration, which, as a key process in vascular remodeling, highlights the potential roles of GRK2 and arrestin proteins in the progression of vascular disease.