Effects of endothelin-1 1-31 on cell viabililty and [Ca2+]i in cultured neonatal rat cardiomyocytes

We previously found that Endothelin-1(1-31) (ET-1(1-31)) exhibited a pro-arrhythmogenic effect in isolated rat hearts. In this study, we further investigated the effects of ET-1(1-31) on a cell viability and observed [Ca(2+)](i) in cultured cardiomyocytes. Cultured neonatal rat cardiomyocytes were treated with 0.1, 1, and 10 nM ET-1(1-31) for 24h in the presence or absence of ET(A) receptor antagonist (BQ(123)) or phosphoramidon, a NEP/ECE inhibitor. Cell injury was evaluated by supernatant lactate dehydrogenase (LDH) assay, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) content. Cell viability was assessed by MTT assay. [Ca(2+)](i) was measured with Fluo-3/AM under a laser confocal microscope. 1) ET-1(1-31) dose-dependently increased LDH release and decreased cell viability. 2) LDH and MDA levels were significantly elevated and SOD activity decreased after administration of 1 nM ET-1(1-31) for 24h, and these changes were markedly attenuated by 1 uM BQ(123). 3) Exposure to 10 nM ET 1(1-31) caused a continuous increase in [Ca(2+)](i) to cultured beating cardiomyocytes and termination of [Ca(2+)](i) transient within 6 min, and this change was reversed by 1 uM BQ(123) and attenuated by 0.5 mM phosphoramidon. These results suggest that ET-1(1-31) could cause cell injury, and that the effect of ET-1(1-31) on [Ca(2+)](i) transients is mainly mediated by ET(A) receptor and partially attributed to the conversion of ET-1(1-31) to ET-1(1-21).     

Effect of endothelin-1(1-31) on intracellular free calcium in cultured human mesangial cells

We found that human chymase selectively produces 31-amino-acid length endothelins (1-31) (ETs(1-31)). We investigated the effect of synthetic ET-1(1-31) on intracellular free Ca2+ concentration ([Ca2+]i) in cultured human mesangial cells. ET-1(1-31) increased [Ca2+]i in a concentration-dependent manner to a similar extent as ET-1. The ET-1 (1-31)-induced [Ca2+]i increase was not influenced by removal of extracellular Ca2+ but was inhibited by thapsigargin. ET-1(1-31)-induced [Ca2+]i increase was not affected by phosphoramidon. It was inhibited by BQ123, but not by BQ788. These results suggest that ET-1(1-31) by itself exhibits bioactive properties probably through endothelin ET(A) or ET(A)-like receptors. Since human chymase has been reported to exist in the kidney, ET-1(1-31) may be a candidate substance for mesangium-relevant diseases.     

Effect of endothelin-1(1-31) on p38 mitogen-activated protein kinase in cultured human mesangial cells

It was reported that human chymase cleaves big endothelins (ETs) at the Tyr31-Gly32 bond and produces 31-amino acid ETs(1-31). In this study, we investigated the effect of ET-1(1-31) on p38 mitogen-activated protein kinase (p38-MAPK) activity in human mesangial cells (HMCs). By measuring the kinase activity, we demonstrated that ET-1 (1-31) activated the p38-MAPK dose-dependently (10(-9) M to 10(-7) M), which was inhibited by SB203580. The p38-MAPK activation induced by ET-1(1-31) peaked at 10 minutes. BQ123 almost abolished ET-1(1-31)-induced p38-MAPK activation, whereas BQ788 failed to inhibit it. These findings suggest that the stimulatory effect of ET-1(1-31) on p38-MAPK activation is mediated through ET(A) or ET(A)-like receptor. In conclusion, ET-1(1-31) induced increase in p38-MAPK activation in cultured HMCs.     

Characterization of the contractile and relaxant action of the endothelin-1 precursor, big endothelin-1, in the isolated rat basilar artery

The presence of functional endothelin converting enzyme (ECE) activity in basilar artery ring segments was investigated by measuring the contractile and relaxant effects of big endothelin (ET)-1. Under resting tension conditions cumulative application of big ET1-1 elicited a concentration-related contraction with the concentration-effect curve (CEC) shifted to the right against ET-1 by a factor of 31 and 29 in segments with the endothelium intact or mechanically removed, respectively. Preincubation with the ET(A) receptor antagonist, BQ123, induced an apparently parallel rightwards shift without affecting the maximum contraction. This shift was more pronounced for ET-1 than for big ET-1. With the putative ECE inhibitor phosphoramidon (10(-3) M) in the bath a small rightwards shift of the CEC for big ET-1 was observed in control segments and a more marked one in de-endothelialized segments. In segments precontracted with prostaglandin (PG) F(2alpha) big ET-1 induced a significant although transient relaxation whereas ET-1 did not. However, in the presence of BQ123 both ET-1 and big ET-1 elicited concentration-related relaxation with a significantly higher maximum effect obtained with big ET-1. The potency was 13 fold higher for ET-1, which is markedly less than that found for contraction. The results, therefore, suggest 1) the presence of functional ECE-activity in the rat basilar artery wall, and 2) differences in the functional ECE activity located in the endothelium and media.     

NLRP3 activation contributes to endothelin-1-induced erectile dysfunction

In the present study, we hypothesized that endothelin (ET) receptors (ET_A and ET_B) stimulation, through increased calcium and ROS formation, leads to Nucleotide Oligomerization Domain-Like Receptor Family, Pyrin Domain Containing 3 (NLRP3) activation. Intracavernosal pressure (ICP/MAP) was measured in C57BL/6 (WT) mice. Functional and immunoblotting assays were performed in corpora cavernosa (CC) strips from WT, NLRP3^−/− and caspase^−/− mice in the presence of ET-1 (100 nM) and vehicle, MCC950, tiron, BAPTA AM, BQ123, or BQ788. ET-1 reduced the ICP/MAP in WT mice, and MCC950 prevented the ET-1 effect. ET-1 decreased CC ACh-, sodium nitroprusside (SNP)-induced relaxation, and increased caspase-1 expression. BQ123 an ET_A receptor antagonist reversed the effect. The ET_B receptor antagonist BQ788 also reversed ET-1 inhibition of ACh and SNP relaxation. Additionally, tiron, BAPTA AM, and NLRP3 genetic deletion prevented the ET-1-induced loss of ACh and SNP relaxation. Moreover, BQ123 diminished CC caspase-1 expression, while BQ788 increased caspase-1 and IL-1β levels in a concentration-dependent manner (100 nM–10 μM). Furthermore, tiron and BAPTA AM prevented ET-1-induced increase in caspase-1. In addition, BAPTA AM blocked ET-1-induced ROS generation. In conclusion, ET-1-induced erectile dysfunction depends on ET_A- and ET_B-mediated activation of NLRP3 in mouse CC via Ca²⁺-dependent ROS generation.     

Proliferative effects of angiotensin II and endothelin-1 on guinea pig gingival fibroblast cells in culture

We investigated whether phenytoin (PHT) and nifedipine (NIF) induce angiotensin II (Ang II) and endothelin-1 (ET-1) generation by cultured gingival fibroblasts derived from guinea pigs and whether Ang II and ET-1 induce proliferation of these cells. Immunohistochemical experiments showed that PHT (250 nM) and NIF (250 nM) increased the immunostaining intensities of immunoreactive Ang II and ET-1 (IRET-1) in these cells. Captopril (3 microM), an angiotensin-converting enzyme inhibitor, reduced these enhanced intensities to control levels. Ang II (100 nM) enhanced the immunostaining intensity of IRET-1. PHT (250 nM) and NIF (250 nM)-induced cell proliferation. Both PHT- and NIF-induced proliferation was inhibited by captopril (3 microM). Ang II (100 nM) and ET-1 (100 nM) also induced cell proliferation. Ang II-induced proliferation was inhibited by CV11974 (1 microM), an AT(1) receptor antagonist and saralasin (1 microM), an AT(1)/AT(2) receptor antagonist, but not by PD123,319 (1 microM), an AT(2) receptor antagonist. ET-1-induced proliferation was inhibited by BQ123 (10 microM), an ET(A) receptor antagonist, but not by BQ788 (1 microM), an ET(B) receptor antagonist. These findings suggest that PHT- and NIF-induced gingival fibroblast proliferation is mediated indirectly through the induction of Ang II and ET-1 and probably mediated through AT(1) and ET(A) receptors present in or on gingival fibroblasts.     

Profibrotic effects of endothelin-1 via the ETA receptor in cultured human cardiac fibroblasts.

BACKGROUND/AIMS: Endothelin-1 (ET-1) has been implicated in pathologic remodelling and tissue repair processes in the heart. We investigated the effects of ET-1 on growth and collagen synthesis responses in cardiac fibroblasts isolated from human hearts. We also studied the receptor subtype(s) mediating such responses and the factors regulating their expression. METHODS: Fibroblasts were isolated from cardiac transplant recipient hearts and characterised by immunocytochemistry. Serum-starved cells were exposed to ET-1 and incorporation of [3H]proline and thymidine were measured as indexes of collagen and DNA synthesis respectively. Blocking experiments utilised the selective ETA receptor antagonist BQ123 and the ETB antagonist BQ788. RESULTS: ET-1 elicited a potent collagen synthesis response in cardiac fibroblasts, with a maximum 29+/-5% increase that was abolished by BQ123. Cardiac fibroblasts responded to ET-1 with a concentration-dependent decrease in DNA synthesis rate. The effects of ET-1 were similar to those of TGF-beta. Radioligand binding studies revealed the presence of high-affinity ET-1 binding sites on these cells, which were upregulated by treatment with the growth factors PDGF and EGF but downregulated by TGF-beta. CONCLUSIONS: These results therefore implicate ET-1 as a trophic agent in the human heart with the ability to influence the development of cardiac fibrosis.     

Molecular modeling of the complex of endothelin-1 (ET-1) with the endothelin type A (ET(A)) receptor and the rational design of a peptide antagonist

ET-1 is the most potent vasoconstrictor known to date, causing vasoconstriction when bound to the ET(A) receptor. Inhibitors of the binding of ET-1 to the ET(A) receptor would be of immense value as potential therapeutic agents in the treatment of cardiovascular disorders such as angina and hypertension. We present here the rational design of such an inhibitor, which is arrived at on the basis of a model of the ET-1/ET(A) receptor complex proposed by us. The model is found to be consistent with binding and mutagenesis studies of ET-1 as well as of BQ123, a known, potent ET(A)-selective antagonist which competes with ET-1 for receptor binding. BQ123 is a peptidic antagonist which is constrained to adopt a definite conformation on account of its cyclic nature. The noncyclic peptide antagonist designed by us also has a unique conformation because it contains two dehydro-Alanine (deltaAla) residues which, on account of their planarity, cause the peptide backbone to bend in a specific and predictable manner. The folding rules for peptides containing deltaAla were derived in our earlier studies. Energy minimization and modelling of the complex of the designed peptide with the ET(A) receptor indicate that the antagonist is ET(A)-selective and the binding is more stable and more specific as compared to that of BQ123.     

Exaggerated coronary reactivity to endothelin-1 in diabetes: reversal with bosentan

We previously demonstrated that chronic endothelin receptor blockade (with bosentan) improved functional cardiac performance in streptozotocin-diabetic rats, suggesting a novel role of endothelin-1 (ET-1) in modulating diabetic heart dysfunction. To gain insight into the mechanism(s) underlying this effect, we examined the coronary vascular responses to ET-1 in hearts from diabetic and control rats treated with or without bosentan. Rats were divided into control, control-treated, diabetic, and diabetic-treated groups. The control-treated and diabetic-treated groups received bosentan (100 mg x kg(-1) x d(-1)) for 8 weeks. Following treatment, hearts were isolated and perfused, and coronary reactivity to ET-1 was assessed by measuring the changes in coronary perfusion pressure in response to ET-1 (50 and 100 pM). Additionally, maximal coronary blood flow (assessed with 10(-5) M adenosine) was measured in isolated perfused hearts. The key observation is that coronary reactivity to ET-1 was significantly higher in the diabetic than the control rats. This effect was normalized in diabetic rats chronically receiving bosentan. Maximal coronary vasodilation did not differ between the four groups. In conclusion, the reactivity of ET-1 is altered in the isolated perfused coronary vascular bed from diabetic rats, and chronic ET receptor blockade restores this reactivity to control values. These observations provide a possible mechanism for the improvement in diabetic heart function observed after chronic bosentan treatment.     

Mechanism of endothelin-1-induced cytosolic Ca(2+) mobility in cultured H9c2 myocardiac ventricular cells

The effect of endothelin-1 (ET-1) on the intracellular free Ca(2+) ([Ca(2+)](i)) mobility in cultured H9c2 myocardiac ventricular cells was studied after loading with fura-2-AM. In Ca(2+)-containing buffer, ET-1 induced [Ca(2+)](i) rise from 10(-7) to 10(-9) M. ET-1 induced [Ca(2+)](i), which was composed of a first small peak and a secondary persistent plateau. In Ca(2+)-free buffer, pretreatment with 10(-7) M ET-1 inhibited the thapsigargin and carbonylcyanide m-chlorophenylhydrazone (CCCP)-induced [Ca(2+)](i) increase. Meanwhile, pretreatment with thapsigargin and CCCP also inhibited ET-1-induced [Ca(2+)](i) rise. In Ca(2+)-containing buffer, the ET(A) receptor antagonist (BQ123) completely abolished the secondary rising peak and plateau. Conversely, the ET(B) receptor antagonist (BQ788) completely inhibited the first small peak and secondary peak plateau. Nifedipine and La(3+) also abolished the 10(-7) M ET-1-induced [Ca(2+)](i) in the first rising peak. The internal Ca(2+) release induced by ET-1 was inhibited by U73122 (phospholipase C inhibitor), propranolol (phospholipase D inhibitor) and aristolochic acid (phospholipase A2 inhibitor). After incubation of 10(-7) M ET-1 in Ca(2+)-free buffer, the addition of 5 mM CaCl(2) increased Ca(2+) influx, implying that release of Ca(2+) from internal stores further induces capacitative Ca(2+) entry. Taken together, these results suggest that both ET(A) and ET(B) receptors are involved in ET-1-induced [Ca(2+)](i) rise in H9c2 myocardiac ventricular cells. Whereas ET(B) receptor seems to mediate the initial Ca(2+) influx via L-type Ca(2+) channel, ET(A) receptor appears to be involved in the subsequent Ca(2+) release from endoplasmic reticulum and mitochondria Ca(2+) stores.     

Production of nitric oxide from endothelial cells by 31-amino-acid-length endothelin-1, a novel vasoconstrictive product by human chymase

Human chymase selectively converts big endothelin (ET)-1 to 31-amino-acid-length ET-1 [ET-1(1-31)]. In this study we examined effect of ET-1(1-31) on endothelial function. ET-1(1-31) evoked contraction in a concentration-dependent manner at > 10(-8) M, which was about 10 times weaker than that of conventional ET-1 [ET-1(1-21)]. BQ485, an ETA receptor antagonist, completely abolished ET-1(1-31)-induced contraction, but BQ788, an ETB receptor antagonist, slightly enhanced it, suggesting that ET-1(1-31) relaxes artery via endothelium. On endothelial cells, ET-1(1-21) and ET-1(1-31) increased [Ca2+]i and produced NO, both of which were significantly inhibited by BQ788 and not by BQ485. These results indicate that ET-1(1-31) increased [Ca2+]i and produced NO in endothelial cells through ETB receptor similarly with ET-1(1-21), although slight difference in effect on smooth muscle cells.     

Both endothelin-A and endothelin-B receptors are present on adult rat cardiac ventricular myocytes

Endothelin-A (ET(A)) and endothelin-B (ET(B)) receptors have been demonstrated in intact heart and cardiac membranes. ET(A) receptors have been demonstrated on adult ventricular myocytes. The aim of the present study was to determine the presence of ET(B) and the relative contribution of this receptor subtype to total endothelin-1 (ET-1) binding on adult ventricular myocytes. Saturation binding experiments indicated that ET-1 bound to a single population of receptors (Kd = 0.52 +/- 0.13 nM, n = 4) with an apparent maximum binding (Bmax) of 2.10 +/- 0.25 sites (x 10(5))/cell (n = 4). Competition experiments using 40 pM [125I]ET-1 and nonradioactive ET-1 revealed a Ki of 660 +/- 71 pM (n = 10) and a Hill coefficient (nH) of 0.99 +/- 0.10 (n = 10). A selective ET(A) antagonist, BQ610, displaced 80% of the bound [125I]ET-1. No displacement was observed by concentrations of an ET(B)-selective antagonist, BQ788, up to 1.0 microM. However, in the presence of 1.0 microM BQ610, BQ788 inhibited the remaining [125I]ET-1 binding. Similarly, in the presence of 1.0 microM BQ788, BQ610 inhibited the remaining specific [125I]ET-1 binding. Binding of an ET(B1)-selective agonist, [125I]IRL-1620, confirmed the presence of ET(B). ET(B) bound to ET-1 irreversibly, whereas binding to ET(A) demonstrated both reversible and irreversible components, and BQ610 and BQ788 bound reversibly. Reducing the incubation temperature to 0 degrees C did not alter the irreversible component of ET-1 binding. Hence, both ET(A) and ET(B) receptors are present on intact adult rat ventricular myocytes, and the ratio of ET(A):ET(B) binding sites is 4:1. Both receptor subtypes bind to ET-1 by a two-step association involving the formation of a tight receptor-ligand complex; however, the kinetics of ET-1 binding to ET(A) versus ET(B) differ.     

Receptor subtype mediating the action of circulating endothelin on glucose metabolism and hemodynamics in perfused rat liver.

The subtype of endothelin receptor that mediates metabolic and hemodynamic effects of circulating endothelin was explored using perfused rat liver. Infusion of endothelin (ET)-1 or ET-3 into the portal vein at a concentration of 0.3 nM increased glucose and lactate output and decreased perfusion flow, although ET-3 was less effective than ET-1. The metabolic effects of ET-1 were observed even under costant-flow perfusion. Infusion of either sarafotoxin S6b or S6c, an ET(A)- or ET(B)-receptor agonist, mimicked the actions of ET-1 to an equal extent. The flow reduction and glucose production induced by ET-1 were partly attenuated by the ET(A)-receptor antagonist BQ485. By contrast, ET(B)-receptor antagonist BQ788 enhanced glucose production caused by ET-1 and ET-3 without affecting the hemodynamic change. The effects of ET-1 and ET-3 were almost totally inhibited by the combination of BQ485 and BQ788. These results suggest that both ET(A) and ET(B) receptors are involved in the metabolic and hemodynamic effects of circulating endothelin in rat liver, while the ET(A)-receptor-mediated action appears to be dominant.     

Role of protein kinases in mediating diabetes-induced augmented vasoconstriction to endothelin-1 in the renal arteries of STZ-diabetic rats

Diabetes is associated with increased reactivity of the renal vascular bed to endothelin-1 (ET-1). It has been observed that diabetes is associated with over-expression of ET(A)- and ET(B)-receptors in the rat renal cortex. However it is not known if these receptors are over-expressed in the renal artery. The objectives of this study were to determine changes in ET-1 receptors and signalling pathways in diabetic renal arteries, to determine the relative roles of protein kinase C and tyrosine kinase activation in mediating these responses and to investigate the role of Rho-kinase activity in mediating the vasoconstrictor responses to ET-1. This study was performed on isolated renal artery segments obtained from STZ-diabetic rats. Results from this study showed that the vasoconstrictor response to ET-1 was potentiated in the diabetic renal artery segments compared to the control animals. Using selective ET-1 receptor antagonists, BQ123 and BQ788, the enhanced ET-1-induced vasoconstriction was shown in this study not to be related to changes in receptor affiinity or receptor subtype distribution. However, the augmented vasoconstrictor response to ET-1 in the diabetic renal artery preparations may be related to increased influx of Ca(2+) through L-type channels and also to increased tyrosine kinase activity.     

Hemodynamic and proinflammatory actions of endothelin-1 in guinea pig small intestine submucosal microcirculation

The hemodynamic and proinflammatory effects of endothelin-1 (ET-1) in proximal (1st/2nd order) and terminal (3rd/4th order) arterioles and venules were examined in small intestine submucosa of anesthetized guinea pigs. Vessel diameter (D), red blood cell velocity, and blood flow (Q) were determined in eight proximal and eight terminal microvessels before and at 20 min of ET-1 suffusion (10(-10), 10(-9), and 10(-8) M) and then with endothelin-A (ET(A))-receptor blockade with BQ-123 (10(-5) M). This protocol was repeated with platelet-activating factor (PAF) inhibition (WEB-2086, 1.0 mg/kg iv; n = 16). The ET-1-mediated microvascular responses were also examined with endothelin-B (ET(B))-receptor blockade using BQ-788 (10(-5) M; n = 11) alone or with ET(A+B)-receptor blockade with BQ-123 + BQ-788 (n = 10). Microvascular permeability was assessed by FITC-albumin (25 mg/kg iv) extravasation in seven series: 1) buffered modified Krebs solution suffusion (n = 6), 2) histamine suffusion (HIS; 10(-3) M, n = 5), 3) ET-1 suffusion (10(-8) M, n = 5), 4) BQ-123 (10(-5) M) plus ET-1 suffusion (n = 5), 5) PAF inhibition before ET-1 suffusion (n = 5), 6) histamine-1 (H1)-receptor blockade (diphenhydramine, 20 mg/kg iv) before ET-1 suffusion (n = 5), and 7) ET(B)-receptor blockade before (BQ-788 10(-5) M; n = 3) or with ET-1 suffusion (n = 3). D and Q decreased at 10(-8) M ET-1 and returned to control values with BQ-123 and BQ-123+BQ788 but not with BQ-788 in proximal microvessels. D did not change in terminal microvessels with ET-1 (10(-8) M) but decreased with BQ-788 and increased with BQ-123. PAF inhibition did not affect the D and Q responses of proximal microvessels to ET-1 but prevented the fall in Q in terminal microvessels with ET-1. ET-1 increased vascular permeability to approximately 1/3 of that with HIS; this response was prevented with BQ-123 and WEB-2086 but not with H1-receptor blockade. This is the first evidence that submucosal terminal microvessel flow is reduced with ET-1 independent of vessel diameter changes and that this response is associated with increased microvascular permeability mediated via ET(A)-receptor stimulation and PAF activation.     

A novel 31-amino-acid-length endothelin, ET-1(1-31), can act as a biologically active peptide for vascular smooth muscle cells

Human chymase produces a novel endothelin-1 with 31 amino-acid length ?ET-1(1-31)?, which is longer than conventional ET-1, ?ET-1(1-21)?. The aim of our study was to investigate the role of ET-1(1-31) on porcine coronary vascular smooth muscle cell (VSMC). Although the increase in [Ca(2+)](i) by ET-1(1-31) was 10 times weaker than that of ET-1(1-21), ET-1(1-31) showed equivalent potency in VSMC proliferation, c-fos/c-myc mRNA expression and cell cycle analysis with ET-1(1-21). ET-1(1-31) significantly induced expression of cyclin D1 but not those of cyclin D2 or D3. These effects were specifically inhibited by BQ485, an ET(A) receptor antagonist, although that of ET-1(1-21) was not specific to BQ485, suggesting different receptor specificity from ET-1(1-21). These results indicate that ET-1(1-31) also can involve a VSMC proliferation process such as atherosclerosis, although it has weaker vasoconstricting potency and different receptor subtypes on VSMC from those of ET-1(1-21).     

内皮素-1对大鼠血管平滑肌细胞产生MCP-1的影响及其机制

目的：观察内皮素-1（ET-1）对大鼠血管平滑肌细胞（VSMCs）产生单核细胞趋化蛋白-1（MCP-1）的影响及其机制。方法：培养大鼠血管平滑肌细胞（VSMCs）。细胞分为2组：ET-1刺激组：以不同浓度ET-1刺激VSMCs不同时间;阻断剂干预组：VSMCs分别与不同阻断剂[ET_AR、ET_BR阻断剂BQ123、BQ788,抗氧化剂N-乙酰半胱氨酸（NAC）,ERK、p38MAPK、JNK及NF-κB抑制剂PD98059、SB203580、SP600125及PDTC]预先孵育30 min,再加入ET-1刺激24 h。在预定时间,以酶联免疫吸附（ELISA）法、逆转录聚合酶链反应（RT-PCR）法分别测定不同因素下VSMCs MCP-1蛋白质及mRNA表达量。VSMCs分别与不同阻断剂（BQ123、BQ788、NAC、PD98059、SB203580及SP600125预先孵育20 min,再加入ET-1刺激5 min,免疫印迹（WB）法测定VSMCs胞浆中细胞外调节蛋白激酶（ERK）、p38丝裂原活化蛋白激酶（p38MAPK）、c-Jun氨基末端激酶（JNK）及其各自磷酸化蛋白质的水平。各项检测均重复3次。结果：ET-1能刺激VSMCs MCP-1蛋白质及mRNA表达,其表达量随ET-1浓度及刺激时间的增加呈升高趋势（P<0.05,P<0.01）;BQ123、NAC、PD98059、SB203580及PDTC能显著抑制ET-1诱导的大鼠VSMCs MCP-1蛋白质及mRNA表达（P<0.01）,而BQ788及SP600125对此作用无明显影响。BQ123、NAC与PD98059或SB203580能分别抑制ET-1刺激后VSMCs胞浆内ERK及p38MAPK的磷酸化（P<0.05,P<0.01）,而ET-1对JNK的磷酸化无明显激活作用。结论：ET-1通过ET_AR、ROS、ERK、p38MAPK及NF-κB诱导大鼠VSMCs产生MCP-1。     

Endothelin-1 chronically inhibits Na/H exchanger-3 in ET(B)-overexpressing OKP cells

Endothelin-1 (ET-1) acutely increases Na/H antiporter activity in OKPET(B)6 cells, an opossum kidney proximal tubule cell line transfected with ET(B) receptor cDNA. The purpose of the present study was to examine the chronic effect of ET-1 on Na/H antiporter activity in OKP cells and to examine whether Na/H exchanger (NHE)-3 mRNA and protein abundance are regulated by ET-1. Quiescent OKPET(B)6 cells were treated with 10 nM ET-1 for 3, 6 or 24 h and Na/H antiporter activity was assayed. The Na/H antiporter activity in 3-h ET-1-treated cells was not different from controls. However, Na/H antiporter activity was significantly decreased by 29% at 6 h and 72% at 24 h. The effect of ET-1 on Na/H antiporter activity was blocked by BQ788, an ET(B) receptor antagonist, but not BQ123, an ET(A) receptor antagonist. The NHE-3 mRNA abundance in ET-1-treated cells was not different from controls at 3 h. However, there was a significant decrease in NHE-3 mRNA abundance at 6 and 24 h. There was also a significant decrease in NHE-3 protein abundance at 6 and 24 h. In summary, ET-1 chronically inhibits NHE-3 in OKPET(B)6 cells.     

Dual role of endothelin-1 via ETA and ETB receptors in regulation of cardiac contractile function in mice

An increase in coronary perfusion pressure leads to increased cardiac contractility, a phenomenon known as the Gregg effect. Exogenous endothelin (ET)-1 exerts a positive inotropic effect; however, the role of endogenous ET-1 in the contractile response to elevated load is unknown. We characterized here the role of ETA and ETB receptors in regulation of contractility in isolated, perfused mouse hearts subjected to increased coronary flow. Elevation of coronary flow from 2 to 5 ml/min resulted in 80 +/- 10% increase in contractile force (P < 0.001). BQ-788 (ETB receptor antagonist) augmented the load-induced contractile response by 35% (P < 0.05), whereas bosentan (ETA/B receptor antagonist) and BQ-123 (ETA receptor antagonist) attenuated it by 34% and 56%, respectively (P < 0.05). CV-11974 (ANG II type 1 receptor antagonist) did not modify the increase in contractility. These results show that endogenous ET-1 is a key mediator of the Gregg effect in mouse hearts. Moreover, ET-1 has a dual role in the regulation of cardiac contractility: ETA receptor-mediated increase in contractile force is suppressed by ETB receptors.     

Effects of chronic endothelin-1 stimulation on cardiac myocyte contractile function

Endothelin-1 (ET-1) has acute positive inotropic effects, but consequences of chronically increased ET-1 on contractile function of cardiac myocytes are largely unknown. In the present study, effects of long-term treatment with ET-1 (10 nM) for 5 days on both force development [force of contraction (FOC)] and kinetics of contraction were determined in heart tissue reconstituted from rat cardiac cells. Isometric force was measured in response to cumulative concentrations of Ca(2+) and isoprenaline. ET-1 augmented basal FOC by 64 +/- 11% (P < 0.05), which was associated with a significantly blunted contractile response to Ca(2+) and isoprenaline. Moreover, ET-1 significantly prolonged relaxation (62 +/- 3 vs. 53 +/- 2 ms). Selective ET(A) (BQ-123) and ET(B) receptor blockade (BQ-788) demonstrated that effects of ET-1 on contractile function were mediated through the ET(A) receptor subtype. Effects of ET-1 were prevented by cotreatment with either Ro31-8425, a PKC inhibitor, or dimethylamiloride, an inhibitor of the Na(+)/H(+) exchanger. In contrast to long-term ET-1 treatment, no changes in contractile parameters were observed after ET-1 treatment for 3 h before force measurement. These data suggest that chronic ET-1 stimulation has dual effects on contractility: improvement of basal force but impairment of twitch kinetics and inotropic responsiveness to beta-adrenoceptor stimulation. The signaling pathways involved include ET(A) receptors, PKC, and the Na(+)/H(+) exchanger. The present in vitro findings raise the possibility that ET-1 may exert both adaptive and maladaptive effects in the failing myocardium in which local accumulation of ET-1 is present.