Abstract-Adrenomedullin (AM) is a potent vasodilating peptide having a variety of pharmacological properties mainly in respect to vascular pathophysiology. We have previously demonstrated that angiotensin II (Ang II) or natriuretic peptides have influence on the expression of tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1) in vascular endothelial cells. The aim of this study was to elucidate the effects of AM on TF and PAI-1 mRNA and protein expression in endothelial cells. As a result, AM inhibited Ang II-induced TF and PAI-1 mRNA expression in a doseand time-dependent manner. Because the expression of TF and PAI-1 mRNA induced by Ang II was attenuated by the increase of intracellular concentrations of cAMP by forskolin and 8-bromo-cAMP and because AM increased the intracellular level of cAMP in rat aortic endothelial cells, it was indicated that the inhibitory effect of AM on the expressions of TF and PAI-1 was mainly mediated by the cAMP-dependent signal transduction. Furthermore, the inhibitory effect of AM on TF and PAI-1 expression was partly attenuated by an NO synthase inhibitor, A drenomedullin (AM), a potent 52-amino acid vasodilating peptide, was originally isolated from human pheochromocytoma cells as a substance that increases platelet cAMP concentration. 1,2 In addition to its vasodilatory activity, AM has various biological activities, including diuresis, 3 inhibition of aldosterone secretion, 4 and inhibition of the proliferation and migration of vascular smooth muscle cells (VSMCs). 5 The plasma AM concentration in patients with hypertension or congestive heart failure is significantly higher than that in healthy individuals. 6 -8 This elevated AM concentration is believed to attenuate the effects of activation of the renin-angiotensin system in these diseases. We have previously demonstrated that angiotensin II (Ang II) induces the expression of tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1) 9 in vascular endothelial cells. Enhanced expressions of TF and PAI-1 may promote thrombus formation and the development of ischemia, respectively, by initiating extrinsic blood coagulation and by inhibiting fibrinolytic activation. 10 -14 We also reported that the enhanced expression of TF and PAI-1 by Ang II is inhibited by natriuretic peptides (NPs), 15 which are important endogenous vasodilatory substances. In the present study, we determined the pathophysiological significance of AM, another counterregulator of Ang II, on the coagulation and fibrinolytic systems regulated by vascular endothelial cells.
Methods
ReagentsEndothelial cell growth supplement was obtained from Becton Dickinson Labware. DMEM and heat-inactivated FBS were from GIBCO-BRL Life Technologies Inc. Rat AM, Ang II, pro-AM NH 2 -terminal 20 peptide (PAMP), and AM- were purchased from Peptide Institute, Inc. BSA, forskolin, 8-bromo-cAMP, N Gnitro-L-arginine methyl ester (L-NAME), and antibiotics were purchased from Sigma Chemical Co.
Cell CulturesRat aortic endothelial cells (RAECs) were isolated fr...
