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We determined the role of endothelin ET(B) receptor in the renal hemodynamic and excretory responses to big endothelin-1, using A-192621, a selective endothelin ET(B) receptor antagonist and the spotting-lethal (sl) rat, which carries a naturally occurring deletion in the endothelin ET(B) receptor gene. An intravenous injection of big endothelin-1 produced a hypertensive effect, which is greater in wild-type (+/+) rats pretreated with A-192621 and in homozygous (sl/sl) rats. Big endothelin-1 markedly increased urine flow, urinary excretion of sodium and fractional excretion of sodium in wild-type rats treated with the vehicle. These excretory responses to big endothelin-1 were markedly reduced by pharmacological endothelin ET(B) receptor blockade. On the other hand, big endothelin-1 injection to the endothelin ET(B) receptor-deficient homozygous animals resulted in a small diuretic effect. When renal perfusion pressure was protected from big endothelin-1-induced hypertension by an aortic clamp, the excretory responses in vehicle-treated wild-type rats were markedly attenuated. In homozygous or A-192621-treated wild-type rats, there was a small but significant decreasing effect in urine flow. In addition, big endothelin-1 significantly elevated nitric oxide (NO) metabolite production in the kidney of wild-type rats but not in the homozygous rats. We suggest that the diuretic and natriuretic responses to big endothelin-1 consist of pressure-dependent and pressure-independent effects and that the increased NO production via the activation of endothelin ET(B) receptors in the kidney is closely related to the big endothelin-1-induced excretory responses.

We evaluated the effects of cilnidipine, a long-acting Ca(2+) channel antagonist, on endothelial nitric oxide synthase (eNOS), preproendothelin-1 and endothelin ETA receptor expression in the left ventricle, and evaluated the relations between these effects and coronary microvascular remodeling and extracellular signal-regulated kinases belonging to one subfamily of mitogen-activated protein kinases in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Cilnidipine (DOCA-cilnidipine, 1 mg/kg/day, subdepressor dose) or vehicle (DOCA-vehicle) was given after induction of DOCA-salt hypertension for 5 weeks. The eNOS mRNA and protein expression in the left ventricle was significantly lower in DOCA-vehicle than in control rats and significantly higher in DOCA-cilnidipine than in DOCA-vehicle rats. Preproendothelin-1 and endothelin ETA receptor expression levels and phospho-p42/p44 extracellular signal-regulated kinase activities were significantly increased in DOCA-vehicle compared with control rats and significantly suppressed in DOCA-cilnidipine compared with DOCA-vehicle rats. DOCA-vehicle rats showed a significant increase in the wall-to-lumen ratio, perivascular fibrosis and myocardial fibrosis, with all these parameters being significantly improved by cilnidipine. These results led us to conclude that phospho-p42/p44 extracellular signal-regulated kinase activities may contribute to the coronary microvascular remodeling of DOCA rats and that protective effects of cilnidipine on cardiovascular remodeling may be at least in part mediated by an increased eNOS expression and a decreased endothelin-1 and endothelin ETA receptor expression in the left ventricle.

Interactions between hepatic stellate cells and endothelin-1 are implicated in liver fibrosis. We determined endothelin-1, its receptors and its effects on the synthesis of a fibrogenic agent transforming growth factor (TGF)-beta1 and collagen in stellate cells from control and CCl(4)-induced cirrhotic rats. The basal synthesis of endothelin-1, TGF-beta1 and collagen was much higher in cirrhotic stellate cells than in control cells. Endothelin-1 stimulated TGF-beta1 and collagen synthesis via endothelin ET(A) and endothelin ET(B) receptors, respectively, in control stellate cells, but did not elicit these effects in the cirrhotic cells despite increased density of the respective receptor subtypes in them. These results indicate that the actions of endothelin-1 on stellate cells may be an important physiological mechanism in maintenance of hepatic architecture. However, inability of endothelin-1 to stimulate TGF-beta1 and collagen synthesis in cirrhotic stellate cells suggests that it does not influence fibrogenic activity by direct action on them probably because the processes are already maximally activated.

The characteristics of specific binding of human [125I]Tyr(13)-endothelin-(1-21), [125I]-Tyr(13)-Suc-[Glu(9),Ala(11, 15)]-endothelin-(8-21), ([125I]IRL-1620) and endothelin ET(A) receptor antagonist [125I]Tyr(3)-(N-[(hexahydro-1H-azepin-1-yl)carbonyl]-L-Leu]-1Me )-D-Trp ([125I]PD151242) (number of sites and their affinity) and proliferation responses to exogenous endothelin receptor agonists (endothelin-1 and the endothelin ET(B) receptor-selective, truncated N-acetyl-[Ala(11,15)]-endothelin-(6-21) analogue BQ3020) were determined in cultured human fibroblasts and in tumorigenic HeLa cells. The cells were pre-incubated with equimolar concentrations of human endothelin-1 or its truncated analogue BQ3020. After pre-incubation (2 h), both peptides induced down-regulation of surface-membrane endothelin-1 receptors. This process was specific for endothelin ET(B) receptors and was much more intensive in tumorigenic cells. BQ3020, acting mostly through its C-terminus, induced nearly maximal endothelin ET(B) receptor down-regulation in HeLa cells. Staurosporine, a wide spectrum protein kinase inhibitor, significantly reduced, and N-[N-[N-[2, 6-dimethyl-1piperidinyl)carbonyl]-4-Me-L-Leu]-1-(methoxycarbonyl)- D-t ryptophanyl]-D-norleucine (BQ788), an endothelin ET(B) receptor antagonist, attenuated the down-regulation of endothelin receptors induced by endothelin receptor agonists. The down-regulation of endothelin ET(B) receptors was prevented by pre-incubation of the cells with the lysosomal enzyme blocker chloroquine. The endothelin-1-induced cell proliferation was attenuated by pre-incubation of the cells with the non-selective endothelin receptor antagonist Ac-D-10,11-dihydro-5H-dibenzo[a,d] cycloheptene-glycine-3,3-D-diphenyl-Ala-Leu-Asp-Ile-Ile-Trp (PD142893) and it was only partially reduced by the endothelin ET(A) receptor-selective endothelin antagonist PD151242.

As there are significant amounts of functional endothelin receptors in the mammalian urinary tract, we examined the effect of experimental diabetes on the expression of endothelin receptors and their mRNAs in the rat bladder dome. The density of endothelin receptors in the rat bladder dome was higher (8 and 16 weeks following the onset of diabetes) than in age-matched controls. Insulin treatment, started 8 weeks after the induction of diabetes, partially reversed the endothelin receptor alterations. The pharmacological profile of the endothelin receptors in the bladder dome was similar in all groups and was consistent with the predominance of the endothelin ET(A) receptor subtype (ET(A):ET(B)=approximately 4:1). Autoradiographic studies demonstrated that the endothelin receptors were located in all tissue components of the bladder, including epithelial and muscular layers. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) data indicated that diabetes increased the expression level of gene transcripts for both endothelin receptor subtypes and that insulin treatment reversed the mRNA upregulation.

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.

Using a reverse transcription-polymerase chain reaction (RT-PCR) based assay the ratio of mRNA for the human endothelin ET(A) and ET(B) receptors in bronchial biopsies was assessed. In patients with diagnoses like bronchial cancer, endothelin ET(A) mRNA was the dominating subtype (ratio 3.74 +/- 0.99). Subjects with the diagnosis of asthma or chronic obstructive pulmonary disease showed significantly higher levels (ratio 0.81 +/- 0.04) of endothelin ET(B) receptor mRNA compared to endothelin ET(A) receptor mRNA. Our results indicate alterations in the endothelin receptor balance in these states.

The aim of the present study was to investigate the level of regulation of the contractile endothelin ET(B) receptor which appears spontaneously after organ culture of vascular segments. Endothelin-1 elicited a strong contraction while the selective endothelin ET(B) receptor agonist, sarafotoxin 6c, had a negligible effect on fresh ring segments of rat mesenteric artery. After organ culture in serum-free Dulbecco's modified Eagle's medium at 37 degrees C (for 1 or 2 days) the endothelin-1-induced contraction was unchanged, whereas sarafotoxin 6c induced, after 1 day, a marked contraction which was further increased at day 2. The contraction induced by sarafotoxin 6c was significantly attenuated by the transcriptional inhibitor, actinomycin D, or the translational inhibitor, cyclohexamide, while the endothelin-1-induced contraction was much less affected. mRNA for endothelin ET(A) and endothelin ET(B) receptors was present in fresh human omental arteries denuded of endothelium. However, after organ culture, endothelin ET(B) mRNA was more prominent than endothelin ET(A) mRNA. Furthermore, the mRNA for both receptors was decreased after treatment with actinomycin D but not with cyclohexamide. This suggests that the endothelin ET(A) receptor is the dominating contractile receptor in fresh arteries while organ culture induces transcription and subsequent translation of contractile endothelin ET(B) receptors.

Receptor binding and antagonist properties of endothelin-1 analogues, [Thr18,gamma-methylleucine19]endothelin-1, [Thr18,Leu19]endothelin-1 and [Thr18,cyclohexylalanine19]endothelin-1, were investigated using cloned human endothelin ET(A) and ET(B) receptors expressed in Chinese hamster ovary cells. Among them, [Thr18,gamma-methylleucine19]endothelin-1 had a high affinity for endothelin ET(A) and ET(B) receptors with respective Kd values of 300 and 110 pM and had no agonist activity on the stimulation of arachidonic acid release in endothelin ET(A) and ET(B) receptor-expressing cells. [Thr18,gamma-methylleucine19]Endothelin-1 had potent antagonist activity in endothelin-1-induced arachidonic acid release in endothelin ET(A) and ET(B) receptor-expressing cells with respective pA2 values of 8.2 and 8.5. In an inositol phosphates accumulation assay, [Thr18,gamma-methylleucine19]endothelin-1 also exhibited potent antagonist activity for endothelin ET(A) and ET(B) receptors with respective pA2 values of 8.0 and 8.4. In conclusion, [Thr18,gamma-methylleucine19]endothelin-1 acts as a potent and nonselective antagonist with no agonist activity for cloned human endothelin ET(A) and ET(B) receptors.

To investigate mechanisms for the receptor-mediated inhibition of a rat cardiac K+ channel clone (KV1.2), we coexpressed KV1.2 with a subtype of endothelin receptors (ETA) in Xenopus oocytes. Effects of endothelin ETA receptor stimulation were mimicked by application of PMA (4-beta-phorbol 12-myristate 13-acetate; 0.1 microM) or intracellular injection of CaCl2 (estimated concentration of 1 microM). These effects diminished in the presence of staurosporine (1 microM) or EGTA (estimated concentration of 5 mM). These results suggest that both activation of protein kinase C and an increase in intracellular Ca2+ contribute to the suppression.