PKC and MAPK signalling pathways regulate vascular endothelin receptor expression

Up-regulation of vascular endothelin type A (ET(A)) and type B (ET(B)) receptors are implicated in the pathogenesis of cardiovascular disease. Culture of arteries has been shown to induce similar receptor alterations and has therefore been suggested as a suitable method for in detail delineation of the regulation of endothelin receptors. We hypothesize that protein kinase C (PKC) and mitogen-activated kinases (MAPK) are involved in the regulation of endothelin receptors. Porcine coronary arteries were studied before and after 24 h of culture, using in vitro pharmacology, real-time PCR and immunofluorescence techniques. Sarafotoxin 6c and endothelin ET-1 were used to examine the endothelin ET(A) and ET(B) receptor effects. The involvement of PKC and MAPK in the receptor regulation was examined by culture in the presence of antagonists. Organ culture resulted in increased sarafotoxin 6c and endothelin-1 contractions, endothelin ET(A) and ET(B) receptor immunofluorescence staining intensities and endothelin ET(B), but not ET(A), receptor mRNA levels. The general PKC inhibitors, bisindolylmaleimide I (10 microM) or Ro-32-0432 (10 microM), inhibited these effects. Also, the increase in sarafotoxin 6c contraction, endothelin ET(B) receptor and mRNA levels and endothelin ET(A) and ET(B) immunofluorescence staining intensities were inhibited by MAPK inhibitors for extracellular signal related kinases 1 and 2 (ERK1/2), PD98059 (10 microM), C-jun terminal kinase (JNK), SP600125 (10 microM), but not by p38 MAPK, SB203580 (10 microM). In conclusion, PKC and MAPK seem to be involved in the regulation of endothelin receptor expression in porcine coronary arteries. Inhibiting these intracellular signal transduction pathways may provide a future therapeutic target for hindering the development of vascular endothelin receptor changes in cardiovascular disease.     

Enhanced expression of contractile endothelin ET(B) receptors in rat coronary artery after organ culture

Endothelin-1 is a potent vasoconstrictor mediating its effects via two receptor subtypes, the endothelin type A (ET(A)) preferentially situated on smooth muscle cells, mediating vasoconstriction and endothelin type B (ET(B)) mainly located on endothelial cells, mediating vasodilatation. In cardiovascular disease and in organ culture in vitro, endothelin ET(B) receptors are up-regulated on smooth muscle cells. The objectives of the present study were to characterise the endothelin receptor-induced vasoconstriction and quantify the endothelin receptor mRNA levels and immunoreactivity in fresh and cultured rat coronary arteries. We demonstrate that endothelin-1 induces strong and equal concentration-dependent contractions in fresh and cultured segments from the left anterior descending coronary artery. Sarafotoxin 6c, an endothelin ET(B) receptor agonist, had negligible effect in fresh arteries but produced significant vasoconstriction after organ culture. The endothelin ET(B) receptor mRNA level and the receptor protein immunoreactivity were increased, whereas the level of endothelin ET(A) receptor mRNA was down-regulated but not its receptor protein immunoreactivity after organ culture. Pharmacological inhibition of endothelium-derived dilatory mediators did not influence endothelin ET(A) or ET(B) receptor-mediated vasoconstriction in fresh segments. In cultured arteries, inhibition of endothelial vasodilators potentiated the effect of sarafotoxin 6c. In conclusion, endothelin ET(B) receptor stimulation in cultured coronary arteries elicits vasoconstriction. This is likely not related to endothelial dysfunction with putative loss of its vasodilator components, but rather explained by the up-regulation of contractile endothelin ET(B) receptors on smooth muscle cells.     

药物保护肺毛细血管内皮细胞 降低SARS发病率

急性呼吸窘迫综合征(ARDS)的致病因素很多，但最终均形成急性肺损伤(ALI)，主要由肺毛细血管内皮细胞损害所介导，虽采用医疗措施，包括吸氧及气管插管进行呼吸机呼吸等，病人死亡率仍高。传染性非典型肺炎(SARS)引起ALI时，出现肺动脉高压及肺纤维化，最终临床出现ARDS。ARDS的病理核心是肺毛细血管内皮细胞的损害，血管内皮细胞可作为治疗ARDS及SARS的靶点。SARS病毒感染后，引起细胞因子的释放，其中包括内皮素-1(ET-1)，ET-1可强烈收缩血管及致炎症反应。本文讨论在致病因子作用下，ET-1的增多与形成ARDS、败血症、休克的关系，致病机制包括ET-1诱导iNOS生成、使细胞膜Ca^2 内流增多、促使氧化应激而造成ALI。ET-1致肺损伤主要由于激活ETB受体，而ALI时肺内纤维化，ET-1亦起着重要的作用。过多的ET-1可使ARDS病人形成肺动脉高压，以ET-1及其所参与的SARS及ARDS发病的各个环节为靶点，应用抗病毒药、抑制炎症的糖皮质激素及阻断冠状病毒侵入细胞的大分子药物，使用内皮素受体拮抗剂和多离子通道阻断剂CPU86017，可阻断感染冠状病毒后形成肺损伤、肺纤维化、肺动脉痉挛及肺动脉高压等，有可能明显降低SARS及ARDS的发病率及致死率。     

Oxidative stress mediates sodium arsenite-induced expression of heme oxygenase-1, monocyte chemoattractant protein-1, and interleukin-6 in vascular smooth muscle cells.

Arsenic exposure is associated with an increased risk of vascular disorders, and results in increased oxidative stress in endothelial cells and vascular smooth muscle cells (VSMCs). Since oxidative stress is involved in regulating the expression of genes related to atherogenesis, we investigated its involvement in the enhanced expression of three atherosclerosis-related genes coding for heme oxygenase-1 (HO-1), monocyte chemoattractant protein-1 (MCP-1), and interleukin-6 (IL-6) in VSMCs treated with inorganic sodium arsenite (iAs). In human VSMCs (hVSMCs) and rat VSMCs (rVSMCs), HO-1, MCP-1, and IL-6 mRNA levels were significantly increased by iAs treatment. An increase in HO-1 protein levels in hVSMCs was confirmed by Western blotting technique, while increased MCP-1 and IL-6 secretion by hVSMCs was demonstrated by enzyme-linked immunosorbent assay. Although modulators of oxidative stress inhibited this iAs-induced increase in the expression of these three genes, different modulators had differential effects. In iAs-treated rVSMCs, catalase, dimethylsulfoxide, and L-omega-nitro-L-arginine significantly inhibited the increase in expression of all three genes, allopurinol inhibited the increase in MCP-1 and IL-6 expression, but had no effect on HO-1 expression, while superoxide dismutase had no significant effect on HO-1 expression, but had an inhibitory effect on IL-6 expression and a stimulatory effect on MCP-1 expression. Therefore, iAs may enhance the expression of HO-1, MCP-1, and IL-6 in VSMCs via different reactive oxygen molecules. Furthermore, using tin protoporphyrin IX (SnPP) and anti-MCP-1 antibody to abolish iAs-induced HO-1 and MCP-1 activity, respectively, shows that HO-1 has protective effect against iAs-induced injury in VSMCs and MCP-1 is chemoattractive to human monocytes, THP-1.     

Induction of oxidative stress and inhibition of plasminogen activator inhibitor-1 production in endothelial cells following exposure to organic extracts of diesel exhaust particles and urban fine particles

Endothelial cells play important roles in anticoagulant and fibrinolytic systems. Recent studies suggest that increases in ambient particulate matter (PM) levels have been associated with an increase in mortality rate from cardiovascular diseases. We examined the production of heme oxygenase-1 (HO-1) and factors related to the fibrinolytic function by rat heart microvessel endothelial cells exposed to organic extracts of diesel exhaust particles (OE-DEP) and urban fine particles (OE-UFP) to investigate the direct effects of these soluble organic fractions in these PM on the fibrinolytic function of endothelial cells. The cell monolayer exposed to 10 μg/ml OE-DEP produced a larger amount of HO-1 than cells exposed to 10 μg/ml OE-UFP. OE-DEP and OE-UFP exposure reduced plasminogen activator inhibitor-1 (PAI-1) production by the cells but did not affect the production of thrombomodulin, tissue-type plasminogen activator, or urokinase-type plasminogen activator. Increased PAI-1 synthesis in response to treatment with 1.0 ng/ml tumor necrosis factor-α or 0.5 ng/ml transforming growth factor-β1 was reduced by OE-DEP exposure. Suppression of PAI-1 production by OE-DEP exposure was mediated through oxidative stress and was independent of HO-1 activity. These results suggest that exposure to the soluble organic fraction of PM and DEP induced oxidative stress and reduced the PAI-1 production of endothelial cells.     

Comparative effects of inhaled diesel exhaust and ambient fine particles on inflammation,atherosclerosis, and vascular dysfunction

Ambient air PM_2.5 (particulate matter less than 2.5 μm in diameter) has been associated with cardiovascular diseases (CVDs), but the underlying mechanisms affecting CVDs are unknown. The authors investigated whether subchronic inhalation of concentrated ambient PM_2.5 (CAPs), whole diesel exhaust (WDE), or diesel exhaust gases (DEGs) led to exacerbation of atherosclerosis, pulmonary and systemic inflammation, and vascular dysfunction; and whether DEG interactions with CAPs alter cardiovascular effects. ApoE^?/? mice were simultaneously exposed via inhalation for 5 hours/day, 4 days/week, for up to 5 months to one of five different exposure atmospheres: (1) filtered air (FA); (2) CAPs (105 μg/m³); (3) WDE (DEP = 436 μg/m³); (4) DEG (equivalent to gas levels in WDE group); and (5) CAPs+DEG (PM_2.5: 113 μg/m³; with DEG equivalent to WDE group). After 3 and 5 months, lung lavage fluid and blood sera were analyzed, and atherosclerotic plaques were quantified by ultrasound imaging, hematoxylin and eosin (H&E stain), and en face Sudan IV stain. Vascular functions were assessed after 5 months of exposure. The authors showed that (1) subchronic CAPs, WDE, and DEG inhalations increased serum vascular cell adhesion molecule (VCAM)-1 levels and enhanced phenylephrine (PE)-induced vasoconstriction; (2) for plaque exacerbation, CAPs > WDE > DEG?=?FA, thus PM components (not present in WDE) were responsible for plaque development; (3) atherosclerosis can exacerbated through mechanistic pathways other than inflammation and vascular dysfunction; and (4) although there were no significant interactions between CAPs and DEG on plaque exacerbation, it is less clear whether the effects of CAPs on vasomotor dysfunction and pulmonary/systemic inflammation were enhanced by the DEG coexposure.     

Reduction of bFGF-induced smooth muscle cell proliferation and endothelin receptor mRNA expression by mevastatin and atorvastatin

The anti-atherosclerosis mechanisms of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) occur via both cholesterol-dependent and cholesterol-independent mechanisms. The present study used aortic and cerebral vascular smooth muscle cells (SMC) from rat to investigate whether atorvastatin and mevastatin affect basic fibroblast growth factor (bFGF)-induced SMC proliferation and the mRNA expression of endothelin A (ET(A)) and endothelin B (ET(B)) receptors. Cell proliferation was assessed by MTT and real-time PCR was used to quantify ET(A) and ET(B) receptor mRNA. bFGF-induced concentration and time dependent SMC proliferation and up-regulation of the mRNA expression of ET(A) and ET(B) receptors. The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors inhibited bFGF-induced proliferation of SMC (P<0.01). In aortic SMC atorvastatin and mevastatin significantly inhibited bFGF-induced mRNA expression of endothelin ET(A) and ET(B) receptors (P<0.05). Although in cerebral SMC the inhibitory effect of the statins was comparable in size with that seen in aortic SMC, only reached borderline significance (P=0.06) for ET(A) receptor mRNA but not for ET(B). The findings suggested a direct effect of statins on the vascular wall beyond their well-known lipid lowering effect in anti-atherosclerosis. Furthermore, the specific antagonists of ET(A) and ET(B) receptors (FR139317 and BQ788, respectively) significantly inhibited bFGF-induced SMC proliferation (P<0.001). The results suggested that endothelin receptors and the mevalonate pathway were involved in bFGF-induced SMC proliferation.     

血红素氧合酶-1的表达水平同冠心病的相关性分析

目的探讨血红素氧合酶-1(HO-1)的表达水平与冠心病的相关性。方法急性心肌梗死(AMI)组32例,稳定型心绞痛(AP)组35例,不稳定型心绞痛(UAP)组31例,体检不存在心脑血管疾病患者40例列入对照组,分析4组HO-1表达水平。结果各组冠心病患者HO-1表达水平与对照组存在明显差异(P<0.05),随着患病程度的减轻,HO-1表达平均面积、绝对灰度和蛋白带灰度峰值逐渐降低(P<0.05)。结论 HO-1的表达水平与冠心病的程度密切相关,随着粥样斑块的破裂和血栓的形成,氧化应激反应逐渐加重,HO-1呈现出越来越高的表达。     

Role of mitogen-activated protein kinases in endothelin ETB receptor up-regulation after organ culture of rat mesenteric artery

Organ culture of isolated arteries results in increased levels of endothelin ET(B) (ET(B)) receptor mRNA and in enhanced ET(B) receptor mediated contraction. The present study was designed to pinpoint the mitogen-activated protein kinase (MAPK) subtype involved in up-regulation of ET(B) receptors after organ culture of rat mesenteric arteries. Western blot and selective antibodies towards constitutional and phosphorylated MAPKs revealed the appearance of phosphorylated MAPK of the extracellular signal-regulated kinases (ERK) 1/2 type at 3 h of organ culture. The functional ET(B) receptor and its mRNA expression were up-regulated after 24 h of organ culture. Following incubation with the MEK 1/2 specific inhibitor SB408039 or the raf inhibitor SB386023b the up-regulation was attenuated both for ET(B) receptor responses and in ET(B) receptor mRNA expression in the vessel segments. Neither Western blot nor myograph or mRNA analysis showed involvement of the other MAPKs studied. Our results suggest that the ERK1/2 MAPKs are involved in the endothelin ET(B) receptor up-regulation following organ culture.     

Endothelin receptor-mediated vasodilatation: effects of organ culture

Culture of intact arteries is a frequently employed experimental model for investigating the mechanisms governing the regulation of vascular endothelin receptors. Endothelin type A (ET(A)) and type B (ET(B)) receptors on vascular smooth muscle cells are up-regulated in organ culture and the enhanced vasoconstriction mimics the changes that occur in cardiovascular disease. The effect of organ culture on endothelial dilatory endothelin ET(B) receptors is not known. We hypothesize that organ culture decreases the endothelin receptor-mediated dilatation and that this is one possible mechanism by which the effects of the endothelin in blood vessels are altered during culture. Porcine coronary arteries were studied before and after 24 h of culture, using in vitro pharmacology and immunofluorescence. Sarafotoxin 6c and endothelin-1 were used to examine the endothelin ET(A) and ET(B) receptor effects, and the antagonists, Nomega-nitro-l-arginine (l-NOARG) for nitric oxide (NO), indomethacin for prostaglandins and charybdotoxin in combination with apamin for endothelium-derived hyperpolarizing factor (EDHF), were used to study the endothelium-derived dilatory mediators. Organ culture induced up-regulation of the sarafotoxin 6c (ET(B) receptor agonist) and endothelin-1 (ET(A) receptor agonist) elicited vasoconstriction. The sarafotoxin 6c contraction was stronger after endothelium denudation, suggesting endothelium-dependent dilatation. The endothelin-1 contraction was not affected by endothelium denudation. The increase in sarafotoxin 6c contraction after removal of the endothelium was more pronounced before than after organ culture, suggesting down-regulated endothelial endothelin ET(B) receptors. Also, the immunofluorescence staining intensities for endothelial endothelin ET(B) receptors were higher before than after organ culture. Pre-incubation with inhibitors for dilatory mediators suggested that both NO and EDHF play a vasodilatory role, while prostaglandins are not involved. In conclusion, endothelial endothelin ET(B) receptors induce NO and EDHF mediated vasodilatation in porcine coronary arteries. In organ culture, endothelial endothelin ET(B) receptors are down-regulated, mimicking the changes that occur in cardiovascular disease. Down-regulation of endothelial endothelin ET(B) receptors may in part explain the increased endothelin ET(B) receptor-mediated vasoconstriction frequently studied in organ culture.     

Upregulation of leptin pathway correlates with abnormal expression of SERCA2a,phospholamban and the endothelin pathway in heart failure and reversal by CPU86017

Emerging evidence indicates that leptin may be a potential new target in chronic heart failure (CHF) treatment. We hypothesized that hyperleptinemia may correlate with abnormal expression of SERCA2a, PLB (phospholamban), and the endothelin (ET) pathway in CHF. An activated ET pathway is involved in CHF that is suppressed by CPU86017 (p-chlorobenzyltetrahydroberberine chloride), a complex class III antiarrhythmic agent with an antioxidant effect. Thus, relief of CHF may be mediated by a reversal of abnormalities of the leptin system, the ET-reactive oxygen species (ROS) pathway, SERCA2a, and PLB by CPU86017. CHF was produced by coronary artery ligation for 6 weeks in rats. The rats were divided into 3 groups: sham, CHF untreated, and CHF+CPU86017 (4 mg/kg per day, s.c.). Hemodynamic changes, cardiac morphology, serum biochemistry, messenger ribonucleic acid (mRNA) and protein expression of the leptin pathway, ET pathway, and redox were measured. In CHF rats, hemodynamic abnormalities, cardiac remodeling, and histological changes with features of cardiac failure were associated with hyperlipidemia accompanied by oxidative stress and upregulated OB-Rb, ECE, pp-ET-1, ET(A)R, and ET(B)R mRNA expression in the myocardium. Protein expression of leptin and ET(A)R in the myocardium was markedly increased in CHF rats. An activated leptin pathway was associated with downregulation of SERCA2a and upregulation of PLB in mRNA and protein expression in CHF. CPU86017 downregulated the leptin system and reversed the above changes in the myocardium. An activated leptin pathway correlates with abnormal expression of SERCA2a and PLB and an activated ET-ROS system in the affected myocardium. The multi-ion-channel-blocking and antioxidative effects of CPU86017 downregulate the leptin pathway and ET system, resulting in reversal of the abnormalities of expression of SERCA2a and PLB and cardiac performance in CHF.     

The therapeutic potential of endothelin-1 receptor antagonists and endothelin-converting enzyme inhibitors on the cardiovascular system

Clinical trials have established bosentan, an orally active non-selective endothelin (ET) receptor antagonist, as a beneficial treatment in pulmonary hypertension. Trials have also shown short-term benefits of bosentan in systemic hypertension and congestive heart failure. However, bosentan also increased plasma levels of ET-1, probably by inhibiting the clearance of ET-1 by endothelin type B (ET_B) receptors, and this may mean its effectiveness is reduced with long-term clinical use. Preliminary data suggests that selective endothelin type A (ET_A) receptor antagonists (BQ-123, sitaxsentan) may be more beneficial than the non-selective ET receptor antagonists in heart failure, especially when the failure is associated with pulmonary hypertension. Experimental evidence in animal disease models suggests that non-selective ET or selective ET_A receptor antagonism may have a role in the treatment of atherosclerosis, restenosis, myocarditis, shock and portal hypertension. In animal models of myocardial infarction and/or reperfusion injury, non-selective ET or selective ET_A receptor antagonists have beneficial or detrimental effects depending on the conditions and agents used. Thus clinical trials of the non-selective ET or selective ET_A receptor antagonists in these conditions are not presently warranted. Several selective endothelin-converting enzyme inhibitors have been synthesised recently, and these are only beginning to be tested in animal models of cardiovascular disease, and thus the clinical potential of these inhibitors is still to be defined.     

Different roles of two types of endothelin receptors in partial ablation-induced chronic renal failure in rats.

Recent work has drawn attention to endothelin as a likely contributor to renal pathogenesis. To elucidate the mechanism of progressive renal disease, we investigated the mRNA expression of endothelin and endothelin receptors, and the effect of endothelin ET(A), and/or ET(B) receptor antagonists on disease progression in the remnant kidney model. Proteinuria progressively increased in rats subjected to 5/6 nephrectomy (Nx) after 8 weeks (from 25+/-3 to 221+/-28 microg min(-1) kg(-1)). Creatinine clearance (Ccr) after renal ablation gradually decreased by 8 weeks (from 5.04+/-0.42 to 2. 68+/-0.26 ml min(-1) kg(-1)). Together with maximal proteinuria and decreased renal function, there was an increase in cortical mRNA expression of prepro endothelin-1 and endothelin ET(A) receptor expression, but a decrease in endothelin ET(B) receptor expression and in urinary excretion of endothelin-1. Administration (1-3 mg/day) of S-0139, (+)-disodium 27-[(E)-3-[2-[(E)-3-carboxylatoacryloylamino]-5-hydroxyphenyl]a crylay loxy]-3-oxoolean-12-en-28-oate, an endothelin ET(A) receptor-specific antagonist, had a beneficial effect on the evolution of the disease, preventing the appearance of intense proteinuria (113+/-11) and decreased Ccr (3.97+/-0.33). High blood pressure was observed in rats with 5/6 Nx and was decreased by S-0139 administration. To examine whether treatment modalities that decrease endothelin ET(B) receptor signaling have a deleterious effect on the kidney remnant, the effect of 97-618, an endothelin ET(B) receptor-specific antagonist, 4-tert-butyl-N-[5-(2-methoxyphenoxy)-6-(4-oxobutoxy)pyromidine+ ++-4-yl]b enzenesulfonamide, was also examined on the action of S-0139. Concomitant administration of S-0139 and 97-618 reversed the beneficial effect of S-0139 alone in the remnant kidney on proteinuria and renal functional impairment. These findings indicate that endothelin participates in the pathogenesis of proteinuria and glomerular injury and that an endothelin ET(A) receptor-specific antagonist could be useful in the treatment of some forms of human nephritis. The loss of endothelin ET(B) receptor seems to be important in the progression of renal disease.     

Modulation of keratinocyte expression of antioxidants by 4-hydroxynonenal,a lipid peroxidation end product

4-Hydroxynonenal (4-HNE) is a lipid peroxidation end product generated in response to oxidative stress in the skin. Keratinocytes contain an array of antioxidant enzymes which protect against oxidative stress. In these studies, we characterized 4-HNE-induced changes in antioxidant expression in mouse keratinocytes. Treatment of primary mouse keratinocytes and PAM 212 keratinocytes with 4-HNE increased mRNA expression for heme oxygenase-1 (HO-1), catalase, NADPH:quinone oxidoreductase (NQO1) and glutathione S-transferase (GST) A1-2, GSTA3 and GSTA4. In both cell types, HO-1 was the most sensitive, increasing 86–98 fold within 6 h. Further characterization of the effects of 4-HNE on HO-1 demonstrated concentration- and time-dependent increases in mRNA and protein expression which were maximum after 6 h with 30 μM. 4-HNE stimulated keratinocyte Erk1/2, JNK and p38 MAP kinases, as well as PI3 kinase. Inhibition of these enzymes suppressed 4-HNE-induced HO-1 mRNA and protein expression. 4-HNE also activated Nrf2 by inducing its translocation to the nucleus. 4-HNE was markedly less effective in inducing HO-1 mRNA and protein in keratinocytes from Nrf2 −/− mice, when compared to wild type mice, indicating that Nrf2 also regulates 4-HNE-induced signaling. Western blot analysis of caveolar membrane fractions isolated by sucrose density centrifugation demonstrated that 4-HNE-induced HO-1 is localized in keratinocyte caveolae. Treatment of the cells with methyl-β-cyclodextrin, which disrupts caveolar structure, suppressed 4-HNE-induced HO-1. These findings indicate that 4-HNE modulates expression of antioxidant enzymes in keratinocytes, and that this can occur by different mechanisms. Changes in expression of keratinocyte antioxidants may be important in protecting the skin from oxidative stress.     

Vascular endothelin ET(B) receptor-mediated contraction requires phosphorylation of ERK1/2 proteins

In cardiovascular diseases, endothelin type B (ET(B)) receptors in arterial smooth muscle cells are upregulated. The present study revealed that organ culture of rat mesenteric artery segments enhanced endothelin ET(B) receptor-mediated contraction paralleled with increase in the receptor mRNA and protein expressions. The endothelin ET(B) receptor-mediated contraction was associated with increase in phosphorylation of extracellular regulation kinase 1 and 2 (ERK1/2) proteins and elevated levels of intracellular calcium. The elevation curve of intracellular calcium consisted of two phases: one rapid and one sustained. Inhibition of ERK1/2 phosphorylation by SB386023 or blockage of calcium channels by nifedipine significantly reduced the endothelin ET(B) receptor-mediated contraction (P<0.05) and decreased the sustained phase of intracellular calcium level, but not the rapid phase. Thus, phosphorylation of ERK1/2 proteins and elevation of intracellular calcium level are required for endothelin ET(B) receptor-mediated contraction in rat mesenteric artery.