Naoko Ozawa

Adrenomedullin is an autocrine/paracrine growth factor for rat vascular smooth muscle cells.

Adrenomedullin is a potent vasodilator peptide secreted by vascular endothelial and smooth muscle cells. Adrenomedullin stimulates the proliferation of quiescent rat vascular smooth muscle cells (VSMCs) via p42/p44 ERK/MAP kinase activation. Recently, receptor-activity-modifying proteins (RAMPs) have been shown to transport calcitonin-receptor-like-receptor (CRLR) to the cell surface to present either as CGRP receptor or adrenomedullin receptor. We investigated whether adrenomedullin acts as an autocrine/paracrine growth factor for cultured rat VSMCs and whether coexpressions of RAMP isoform and CRLR may mediate p42/p44 ERK/MAP kinase activation by adrenomedullin. Adrenomedullin dose-dependently stimulated the proliferation of quiescent rat VSMCs, and this effect was inhibited by an adrenomedullin receptor antagonist, a MAP kinase kinase inhibitor and phosphatidylinositol 3-kinase inhibitors. Addition of either CGRP(8-37) or anti-adrenomedullin antibody to exponentially growing rat VSMCs inhibited the serum-induced cell proliferation, suggesting its role as an autocrine/paracrine growth factor. Cotransfection of RAMP2 or RAMP3 with CRLR into rat VSMCs potentiated activation of cAMP activity, but not of p42/p44 ERK/MAP kinase activity in response to adrenomedullin. Our results suggest that adrenomedullin is an autocrine/paracrine growth factor for rat VSMCs via p42/p44 ERK/MAP kinase and phosphatidylinositol 3-kinase pathways and that it is not mediated by human RAMP-CRLR receptors.

Endothelin-1 induces cyclooxygenase-2 expression and generation of reactive oxygen species in endothelial cells.

Since both endothelin-1 (ET-1) and aldosterone have been shown to induce expression of several pro-inflammatory genes, including cyclooxygenase-2 (COX-2), in the vasculature as a cardiovascular risk hormone, the present study was undertaken to examine the effects of ET-1 and aldosterone on COX-2 gene expression as measured by a real-time reverse transcriptasepolymerase chain reaction in aortic endothelial cells. Treatment with ET-1(10-7 M) markedly upregulated COX-2 mRNA levels in rat endothelial cells, whereas aldosterone (10-7 M) did not show any effect. The ET-1-induced COX-2 upregulation was inhibited by pretreatment with a non-selective endothelin receptor antagonist (TAK044), a protein kinase C inhibitor (GF109203X), and a MEK inhibitor (PD98059). Furthermore, ET-1 increased intracellular reactive oxygen species generation as estimated by the measurement of dichlorofluorescein fluorescence, whose effect was blocked by a COX-2 inhibitor (NS398). Our data show that ET-1 induces COX-2 upregulation in rat endothelial cells via a protein kinase C-dependent and extracellular signal-regulated kinasedependent pathway, which may largely contribute to the generation of intracellular reactive oxygen species.