The Biological Effects And Mechanisms Of Adrenomedullin On Vascular Endothelial Cells

Research Background and Aim:Adrenomedullin (ADM),which was discovered as a vasodilating peptide,has been originally isolated from the extract of human pheochromocytoma. Subsequent study revealed that ADM regulates not only vascular tone but also cell proliferation and defferentiation in an autocrine/paracrine manner. It has been shown to be a multifunctional regulatory peptide. But most of those effects exhibited a cell type specific manner. In Swiss 3T3 cells, ADM increased DNA synthesis in a dose-dependent manner by a mechanism involving specific ADM receptors and increased cAMP/PKA. In human vascular smoothe muscle cells and glial cell tumors, ADM suppressed cell growth and increased intracellular cAMP. The growth of human normal glial cells and human glioblastomas, as well as cultured glioblastoma-derived cell lines, was also inhibited by ADM. However. Moody et al. reported that ADM exerted mitogenic effects on cultured C6 gliomal cells that correlated with increases in cAMP. To evaluate the mechanism of ADM function, a principal question is how a stimulus at the cell surface can be transmitted to the nucleus to initiate a process of gene expression that results in the creation of an appropriate physiological response to changes in the cellular environment. There are a number of studies about the signal pathways and biological effects activated by ADM. cAMP has been found to be the second messenger in the majority of ADM actions. However, the effects of ADM are not fully mimicked by forskolin, possibly suggesting that a role for an additional second messenger must be involved.ADM transcripts are expressed in various tissues and cells, vascular endothelial cells(VECs) are the most important one. In the vascular smooth muscle cells including human, ADM is a much more efficacious activator of adenylate cyclase and a much more effective relaxant than calcitonin gene-related peptide (CGRP). These findings indicate that ADM may be involved in modulating the roles of vascular endothelial cells in physiological and pathological processes.VECs plays a vital role in cardiovascular diseases. The apoptosis, degeneration and proliferation of VECs are responsible for the developments of a variety of blinding diseases such as coronary heart diseases, hypertension,heart failure et al .Agents that significantly increase intracellular levels of cAMP are also inhibitors of VECs proliferation. Recent studies have shown that ADM inhibited Platelet-derived growth factor (PDGF)-BB and fetal calf serum (FCS)-induced human VECs proliferation in a concentration-dependent manner. The findings of previous studies raise the possibility that ADM is related to the pathophysiology of heart diseases related with VECs. But we have little information about the signal transduction pathways activated by ADM in VECs. The physiological roles and mechanism of pharmacological effects of ADM on VECs are still unclear.Here we investigated whether ADM could suppress the proliferation of serum-stimulated and quiescent rabbit VECs; whether ADM acted on VECs through two independent signal transduction pathways, cAMP accumulation and Ca2+ fluxion; We also characterized whether nuclear transcription factor- K B (NF- K B) is activated in rabbit VECs in response to ADM; whether ADM has effects on the apoptosis on rabbit VECs. whether ADM and ADMR mRNA are expressed in primary cultured rabbit VECs and if so, whether lipopolysaccharide (EPS) could upregulate the expression of ADM and ADMR mRNA in cultured rabbit VECs.Methods: (1) Rabbit VECs were incubated in DMEM medium with or without serum containing ADM(10~10 to 10~7M);on the other hand VECs were also cultured in DMEM with PDGF-BB and 1%FCS Proliferation in VECs was evaluated using the 3H-TdR incorporation assay. (2) Cultured rabbit VECs were incubated in PBS buffer with or without ADM for 15 minutes. Intracellular cAMP and cGMP levelswere measured by enzyme immunoassay (ElA). To determine the cytoplasmic free Ca2+ concentration ([Ca2+]i) response to ADM, the fluo-3 AM loaded cells wer...