Endothelins (ET) produce endothelium-dependent vasodilation through nitric oxide (NO) synthesis. The present study was designed to elucidate the cellular mechanism by which ET induces synthesis and release of endothelium-derived NO by cultured bovine endothelial cells (EC). Binding studies revealed that bovine EC membrane had the binding sites of a novel agonist (BQ3020) for non-isopeptide-selective receptor subtype (ETB). Affinity labeling studies showed a major labeled band with the apparent molecular mass of 50 kD. Northern blot analysis demonstrated the expression of mRNA for ETB receptor. BQ3020 rapidly and dose dependently induced formation of inositol-1,4,5-triphosphate and increased intracellular Ca2+ concentrations in fura-2-loaded cells. Concomitantly, BQ3020 dose dependently stimulated production of both nitrate/nitrite (NOx) and cyclic GMP, a highly significant correlation existed between NOx and cGMP production.The stimulatory effect on NOx and cGMP production by ETB agonist was inhibited by NO synthase inhibitor monomethyl-Larginine; this effect was reversed by coaddition of L-arginine, but not D-arginine. NOx and cGMP production stimulated by BQ3020 was inhibited by pretreatment with pertussis toxin.ETB agonist-induced NOx production was blocked by a calmodulin inhibitor and an intracellular Ca2" chelator, but not by an extracellular Ca2" chelator or a Ca2+ channel blocker. These data suggest that endothelins stimulate ETB receptor-mediated phosphoinositide breakdown via pertussis toxin-sensitive Gprotein(s), which triggers release of intracellular Ca2+, thereby activating Ca2+/calmodulin-dependent NO synthase in EC. (J. Clin. Invest. 1993. 91:1367-1373.) Key words: endothelin * receptor subtype B * nitric oxide -Ca2`/calmodulinendothelial cell

To elucidate the cellular mechanism of endothelin-1 biosynthesis induced by angiotensin and vasopressin, we first cloned and sequenced full-length bovine preproendothelin-1 complementary DNA (cDNA) from a cultured bovine carotid artery endothelial cell cDNA library. The predicted bovine preproendothelin-1 consists of 202 amino acid residues and has a high percentage of homology to human, porcine, and rat preproendothelin-1 (70%, 81%, and 77%, respectively). ). The predicted 21-residue mature endothelin-1 is identical to human, porcine, rat, canine, and mouse endothelin-1. Northern blot analysis with the cloned cDNA as a probe demonstrated that a single 2.3-kb preproendothelin-1 messenger RNA (mRNA) is expressed not only in endothelial cells, but also in various bovine tissues, including lung, brain, heart, intestine, kidney, ovary, and urinary bladder. Angiotensin II and arginine vasopressin immediately and dose-dependently induced expression of preproendothelin-1 mRNA, whose effects were abolished by specific receptor antagonists. These findings suggest that stimulation of endothelin-1 secretion from endothelial cells by both agonists may be principally due to induction of preproendothelin-1 mRNA. The induction of preproET-1 (ppET-1) messenger RNA (mRNA) in porcine endothelial cells is augmented by several agents, such as thrombin, 1 Ca 2+ ionophore, 2 phorbol ester, 2 transforming growth factor-P, 3 cytokines, 4 and shear stress. 5 Our recent studies have shown that two vasoactive hormones, angiotensin II (Ang II) and arginine vasopressin (AVP), stimulate the release of immunoreactive ET-1 from cultured bovine carotid artery endothelial (BCAE) cells through receptor-mediated mobilization of intracellular Ca 2+ and activation of protein kinase C (PKC). 67Here, we successfully cloned and sequenced fulllength bovine ppET-1 complementary DNA (cDNA) from the BCAE cell cDNA library. By Northern blot analysis with the cloned cDNA as a probe, we investigated whether ppET-1 mRNA is expressed in various bovine tissues and whether ppET-1 mRNA expression is induced by Ang II and AVP in cultured BCAE cells.

Endothelin-l is a novel endothelium-derived vasoconstrictive peptide. Using a highly specific and sensitive radioimmunoassay for endothelin-l, plasma levels of immunoreactive endothelin-I were measured in 32 research subjects with normal renal function (21 normal subjects and II patients with essential hypertension), 24 patients with nondialyzed chronic renal failure, and 51 patients undergoing maintenance hemodialysis. Although there was no significant difference in plasma immunoreactive endothelin-l levels among the three groups, patients with essential hypertension had significantly higher plasma endothelin-l levels than normal subjects (2.29±1.09 vs. 1.41+0.50 pg/ml,p<0.025). When nondialyzed and hemodialyzed patients were divided into hypertensive and normotensive groups, the nondialyzed hypertensive group (n=17) had higher plasma endothelin-l levels than the comparable normotensive group (n=7) (3.08±3.43 vs. 0.73±0.34 pg/ml, p<0.05), and the hemodialyzed hypertensive group (n=18) had higher plasma endothelin-l levels than the comparable normotensive group (n=33) (2.66±1.92 vs. 1.35±0.73 pg/ml,p<0.005). Plasma atrial natriuretic factor, arginine vasopressin, renin activity, and aldosterone concentration did not show significant differences between hypertensive and normotensive individuals or a correlation with plasma endothelin-l levels. These data suggest that circulating endothelin-l may be partly involved in the development or maintenance of hypertension in humans. We recently demonstrated the presence of ET-1-like immunoreactivity (ET-LI) in normal human plasma with a highly sensitive and specific radioimmunoassay (RIA), 8 suggesting its potential role as a circulating vasoconstrictor. However, no information From the Second Department of Internal Medicine, Tokyo Medical and Dental University, Kitasato Biochemical Laboratories, SMI-Bristol Ltd. Sagamihara, Kanagawa, Tokyo Metropolitan Tama Geriatric Medical Center, Tokyo, and Shimoochiai Clinic, Shinjuku-ku, Tokyo, Japan.Address for correspondence: Yukio Hirata, MD, Endocrine Division, Second Department of Internal Medicine, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113, Japan.Received October 6, 1989; accepted in revised form January 23, 1990.is yet available as to its pathophysiological role in hypertension. Therefore, the present study was designed to determine plasma ET-LI in normal subjects and in patients with essential hypertension as well as those with chronic renal failure with or without hypertension. Methods SubjectsThe study population consisted of 21 normal individuals (11 men and 10 women), 11 patients with essential hypertension (seven men and four women), 24 patients with nondialyzed chronic renal failure (18 men and six women), and 51 patients undergoing maintenance hemodialysis (34 men and 17 women). Informed consent was obtained from each subject. Essential hypertension was defined as elevated blood pressure while in a sitting position, exceeding 160/95 mm Hg, for three consecutive measurements over a pe...

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Release of endothelin-1, a novel potent vasoconstrictor peptide originally isolated from endothelial cells, from cultured bovine endothelial cells has been shown to be stimulated by arginine vasopressin and angiotensin II. To elucidate the cellular mechanism by which endothelin-1 is released by these vasoconstrictors, we tested the effects of several compounds on the agonist-induced endothelin-1 release and studied the changes of cytosolic free Ca peptide with 21-amino acid residues, was originally isolated and sequenced from the supernatant of cultured porcine endothelial cells (ECs).1 ET-1 has not only strong and sustained vasoconstrictive action on a variety of blood vessels from various species 1 -2 but also a wide spectrum of pharmacological effects such as release of eicosanoids and endothelium-derived relaxing factor from perfused vascular beds, 3 aldosterone from cultured calf adrenal zona glomerulosa cells, 4 atrial natriuretic peptide from cultured rat atrial myocytes, 5 and mitogenic action in cultured vascular smooth muscle cells.6 These effects are dependent on extracellular Ca 2+ and inhibited by Ca 2+ channel antagonists, suggesting the importance of Ca 2+ influx in the mechanism of its actions. The induction of preproET-1 messenger RNA (mRNA) in porcine EC is augmented by several agents, such as epinephrine, thrombin, Ca 2+ ionophore, 1 phorbol ester, 8 transforming growth factor (TGF)-/3, 9 cytokines, 10 and shear stress. 11 Our recent studies have shown that two vasoconstrictive hormones, arginine vasopressin (AVP) and angiotensin (Ang) II, also stimulate the release of immunoreactive ET-1 from cultured bovine ECs, and these effects are mimicked by phorbol ester and Ca 2+ ionophore. 12These findings raise the possibility that the AVP-and Ang II-induced ET-1 release from EC may involve a common mechanism (i.e., phosphoinositide breakdown with the resultant mobilization of intracellular Ca 2+ and activation of protein kinase C [PKC]. To address this question, the present study was designed to elucidate the cellular mechanism of AVP and Ang II responsible for ET-1 release in bovine EC. Methods Cell Culture and IncubationECs from bovine carotid artery were prepared by digestion with collagenase and elastase, 13 and culDownloaded from http://ahajournals.org by on April 27, 2019