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

These data suggest that urinary hUII is derived mainly from a renal source, and that hUII functions as an autocrine/paracrine vasoactive factor not only in the cardiovascular system, but also in the kidney, with an as yet unspecified function.

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.

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

We studied whether a novel vasoconstrictor peptide, endothelin-i (ET-I), is synthesized by and released from human carcinoma cell lines, and whether ET-1 stimulates proliferation of these tumor cells. ET-1-like immunoreactivity was released from both HeLa and HEp-2 cells as a function of time. Reverse-phase HPLC of the conditioned media from HeLa cells revealed a major peak coeluting with standard ET-1. Northern blot analysis demonstrated the expression of mRNA for ET-1 precursor in both tumor cell lines. Both cell lines contained a single class of specific binding sites for ET-1. ET-1 dose-dependently induced increases in cytosolic free Ca2" concentration in fura-2-loaded tumor cells, whose effect was completely abolished by chelating extracellular Ca2" or by Ca2"-channel blocker. ET-1 stimulated proliferation of the quiescent cell lines in a dose-dependent manner, whose effect was inhibited by Ca2-channel blocker. Polyclonal antibody for ET-1 inhibited proliferation of these cell lines, whereas nonimmune serum had no effect. These results demonstrate that ET-1 is synthesized by and released from human epithelial carcinoma cell lines, and that exogenous and endogenous ET-1 stimulates proliferation of the cells possibly through Ca2" influx, suggesting its role as an autocrine/paracrine growth factor for certain tumor cells. (J.

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By measurements of NO /NO (NOx) production and Northern blot analysis, we studied the effects of a membranepermeable cAMP derivative, 8-bromo-cAMP, on the expression of inducible nitric oxide synthase (iNOS) gene and the synthesis of NOx in cultured rat vascular smooth muscle cells (VSMCs). 8-bromo-cAMP stimulated NOx production and increased steady-state levels of iNOS mRNA in rat VSMC in a time-and dose-dependent manner. NG-monomethyl-L-arginine, a NOS inhibitor, completely blocked the 8-bromocAMP-induced NOx production, whose effect was partially, but significantly reversed by an excess L-arginine, but not by D-arginine. Compounds that increase intracellular cAMP levels (cholera toxin, forskolin, and 3-isobutyl-1-methylxanthine), all stimulated NOx production. Dexamethasone inhibited the stimulated NOx production, as well as the induction of iNOS mRNA by cAMP. Both actinomycin D and cycloheximide completely blocked the stimulated NOx production by cAMP. Actinomycin D abolished the cAMP-induced iNOS mRNA, whereas cycloheximide remarkably increased iNOS mRNA levels in the presence and absence of 8-bromo-cAMP (superinduction). Actinomycin D, but not dexamethasone, completely abolished the cycloheximide-induced iNOS mRNA. The half-life of cAMP-induced iNOS mRNA was -2 h, whereas no decay in the cycloheximide-induced iNOS mRNA was observed during 12 h. These results demonstrate that iNOS gene is upregulated by cAMP and the superinduction of iNOS mRNA is attributable to increased mRNA stability in rat VSMC. (J. Clin. Invest. 1994. 93:543-549.)

1. The vascular endothelium produces endothelium-derived relaxing factor (EDRF) or nitric oxide (NO), which exerts vasodilation through cyclic guanosine monophosphate (cGMP) as a second messenger. To determine whether EDRF has any vasodilating and natriuretic action in man, the present study examined the effects of L-arginine (L-Arg), a substrate for NO, on the responses of mean blood pressure (MBP) and heart rate (HR); plasma concentrations of cGMP, atrial natriuretic factor (ANF) and nitrite/nitrate (NOx); urinary excretion of sodium, cGMP and NOx; and urinary flow in eight normal male subjects. These parameters were compared with those following saline infusion in the same subjects. Clearance of para-aminohippuric acid (PAH) and inulin was studied in five normal subjects. 2. Infusion of L-Arg (30 g) caused a significant fall in MBP (-8 mmHg) with a concomitant rise in HR (10 beats/min), while saline infusion had no effects on these parameters. 3. Neither L-Arg nor saline infusion caused appreciable changes in plasma concentrations of ANF or NOx. Plasma cGMP concentrations increased significantly during (1.7-fold) and after (1.9-fold) L-Arg infusion, but only slightly (1.3-fold) during saline infusion. 4. Urine flow increased more remarkably following L-Arg infusion than that following saline infusion. Remarkable increases in urinary excretion of sodium and fractional excretion of sodium were observed after L-Arg infusion compared with those after saline infusion. Natriuresis was associated with enhanced urinary excretion of cGMP and Nox. Urinary Nox excretion showed positive correlations with urinary flow (r = 0.69, P less than 0.001) and with urinary cGMP excretion (r = 0.60, P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)