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OBJECTIVE The present study was designed to examine whether adrenomedullin affects fetal calf serum (FCS)-stimulated proliferation in cultured rat vascular smooth muscle cells (VSMCs). METHODS Rat VSMCs were grown from explants of Sprague-Dawley rat aorta and were grown using the standard cell culture method. After incubation for 24 h with various concentrations of adrenomedullin in the presence of 5% FCS, trichloroacetic acid-insoluble tritiated thymidine was measured in a liquid scintillation counter. After incubation for 48 h, cell counts were performed. Cyclic adenosine 3',5'-monophosphate (AMP) levels were determined by radioimmunoassay. RESULTS Rat adrenomedullin exhibited concentration-dependent inhibition of the FCS-stimulated increase in thymidine incorporation between 10(-7) and 10(-9) mol/l and of cell number at 10(-7) mol/l. However, the calcitonin generelated peptide (CGRP) receptor antagonist human CGRP(8-37) abolished these antiproliferative effects of rat adrenomedullin. Inhibition by adrenomedullin of FCS-stimulated cellular proliferation was paralleled by an increase in the cellular level of cyclic AMP. 8-Bromocyclic AMP, a cyclic AMP analogue, and forskolin, an activator of adenylate cyclase, inhibited the FCS-stimulated increase in thymidine incorporation and cell number. CONCLUSIONS These results suggest that adrenomedullin inhibits FCS-stimulated proliferation in cultured rat VSMCs, probably through a cyclic AMP-dependent process. Taken together with the finding that adrenomedullin is synthesized in and secreted from vascular endothelial cells, adrenomedullin may play a role as an antiproliferative factor for VSMCs in a paracrine fashion.

The effects of endothelin-1 on cytosolic Ca2+ and inositol 1,4,5-triphosphate (IP3) levels were investigated in cultured, untreated human endothelial cells and in endothelial cells pretreated with anti-endothelin-1 serum for 24 h to exclude the effect of endogenous endothelin-1. Endothelin-1 was found to increase the intracellular Ca2+ level, either in the presence or absence of extracellular Ca2+, in endothelial cells pretreated with antiserum by the fura-2 fluorescence technique. IP3 levels immediately started to rise following endothelin-1 stimulation. Resting intracellular Ca2+ levels were significantly lower when the cells were pretreated with antiserum than without antiserum pretreatment. Following stimulation by endothelin-1, intracellular Ca2+ and IP3 levels in endothelial cells pretreated with antiserum increased significantly compared to those in untreated endothelial cells. Endothelin-1 also increased 45Ca influx from the extracellular space. These results suggest that endothelin-1 increases intracellular Ca2+ in endothelial cells through extracellular Ca2(+)-dependent mechanisms and by the release of Ca2+ from intracellular stores, this presumably being induced by IP3 formation.

The effect of glucocorticoid on the prostaglandin E1 (PGE1)-mediated cyclic AMP (cAMP) formation by vascular smooth muscle cells (VSMC) from renal arteries (RA) was studied in rats. Dexamethasone (DEX) at concentrations ranging from 10(-10) to approximately 10(-8) mol/l dose-dependently potentiates the PGE1-mediated response. This facilitation began at 6 h and reached its maximum after 24 h of DEX administration. Aldosterone (10(-6) mol/l) did not affect the dose-response curve of PGE1. Inhibitors of protein and RNA synthesis blocked this glucocorticoid effect. The basal activity of adenylate cyclase in DEX-treated cells was twice as high as in control cells. Treatment of VSMC with DEX increased cholera toxin- and pertussis toxin-stimulated adenylate cyclase activity. DEX treatment also augments forskolin-stimulated adenylate cyclase activity. These results suggest that DEX increases PGE1-mediated cAMP formation of VSMC from RA through a mechanism that involves the induction of protein synthesis, and that the activation of the catalytic unit may play some role in this facilitating process.

In seven patients with mild to moderate essential hypertension, the effect of a new angiotensin converting enzyme (ACE) inhibitor, cilazapril, on atrial natriuretic factor (ANF) was examined at rest and during exercise. The exercise protocol consisted of three fixed workloads (25, 50 and 75W) every 4 min with the use of a sitting bicycle ergometer. Cilazapril reduced blood pressure without increasing the heart rate, both at rest and during exercise. The resting pre-exercise left ventricular ejection fraction was not altered. The plasma ANF level was increased by exercise, but at the 75-W workload it was decreased by cilazapril; the resting plasma ANF value was not altered. At the 75-W workload the plasma ANF level was inversely correlated to the left ventricular ejection fraction (n = 14, r = -0.64, P less than 0.01). These results suggest that the observed decrease in the plasma ANF level during exercise induced by cilazapril is, in part, related to an improvement in the cardiac overload, and that cilazapril may be effective in physically active hypertensive patients.

Cultured porcine endothelial cells derived from aortas spontaneously released immunoreactive endothelin into the medium in a time-dependent manner. This release was completely inhibited by cycloheximide and is, therefore, directly related to de novo protein synthesis. The endothlin-induced release was further stimulated by adrenaline. Adrenaline-induced stimulation was completely inhibited by the alpha-adrenergic blocker phentolamine and was not inhibited by the beta-adrenergic blocker propranolol. Cycloheximide completely prevented the adrenaline-stimulated as well as the basal release. These results suggest that cultured endothelial cells release endothelin slowly but continuously and that this release can be stimulated by adrenaline via alpha-adrenergic receptors. We speculate that the endothelium generates vasoconstrictor signals through endothelin production, thus contributing to the regulation of vascular tone.