THE ROLE OF PLATELET-DERIVED GROWTH FACTOR-BB-INDUCED INCREASE IN CYTOSOLIC-FREE CA2-ACTIVATED PROTEIN-KINASE AND DNA-SYNTHESIS IN VASCULARSMOOTH-MUSCLE CELLS( IN ACTIVATION OF MITOGEN)

Background Platelet derived growth factor (PDGF)-BB is an important va scular smooth muscle cell (VSMC) mitogen. PDGF-BB induces an increase in intracellular free calcium concentration ([Ca2+](i)), an activation of mitogen-activated protein (MAP) kinase and an increase in DNA synt hesis. The increase in [Ca2+](i) is thought to be an important second messenger in the intracellular signalling cascade, leading to growth o f VSMC. Objective The aim of the present study was to elucidate the ro le of the PDGF-BB-induced increase in [Ca2+](i) in the activation of M AP kinase and increase in DNA synthesis. Binding of [Ca2+](i) was perf ormed by the intracellular chelator bis-(2-amino-5-methylphenoxy) etha ne-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (MAPTAM). Metho ds Ca2+ levels were measured by the Fura-2 method. MAP kinase activati on was determined by Western blotting. DNA synthesis was determined by measurement of incorporation of [H-3]-thymidine into the cell DNA. Re sults Administration of 50 ng/ml PDGF-BB induced an increase in [Ca2+] (i), an activation of MAP kinase and an increase in DNA synthesis. In bis-(2-amino-5-methylphenoxy) ethane-N,N,N'N'-tetraacetic acid tetraac etoxymethyl ester (MAPTAM)-treated cells the PDGF-BB-induced effect on [Ca2+](i) was totally blunted, whereas no effect on MAP kinase activa tion and DNA synthesis could be observed. Conclusions These findings s how that the effect of PDGF-BB on MAP kinase activation is independent of calcium level. [Ca2+](i) might be implicated in the PDGF-BB-induce d mitogenic process only in conjugation with other signalling componen ts. (C) Rapid Science Publishers ISSN 0263-6352.