ROLE OF MITOGEN-ACTIVATED PROTEIN-KINASE IN THE ANGIOTENSIN-II-INDUCED DNA-SYNTHESIS IN VASCULAR SMOOTH-MUSCLE CELLS

The activation of mitogen-activated protein (MAP) kinase and increase in intracellular free calcium concentration ([Ca2+](i)) are discussed in reference to activation of different protein kinases and growth of vascular smooth muscle cells (VSMCs). The aim of the present study was to investigate the role of angiotensin (Ang) II-induced increase in [ Ca2+](i) for activation of 44-kD/42-kD MAP kinase (p44(mapk)/p42(mapk) ) and DNA synthesis in VSMCs, Experiments were performed by chelation of [Ca2+](i) by the intracellular chelator amino-5-methylphenoxy)ethan e-N,N,N',N'-tetraacetic acid tetraacetoxymethyl eater (MAPTAM). Ca2+ w as measured by the fura 2 method. MAP kinase activation was determined by the Western blotting method. DNA synthesis was determined by measu rement of [H-3]thymidine incorporation into the cell DNA, Treatment of VSMCs with 20 mu mol/L MAPTAM for 30 minutes resulted in a complete a bolishment of the maximal Ang II-induced increase at 10 seconds. Ang I I phosphorylated the p44(mapk)/p42(mapk) in a time-dependent manner, s howing a maximum at 3 minutes. In MAPTAM-treated cells, the maximal ph osphorylation of MAP kinase isoforms was shifted to 5 minutes, and dep hosphorylation was delayed compared with untreated cells. In concordan ce with this finding, the induction of the MAP kinase phosphatase-1 wa s markedly impaired in MAPTAM-treated cells. Ang II induced a 2.3-fold increase in [H-3]thymidine incorporation into DNA synthesis in untrea ted cells. This effect was not reduced in MAPTAM-treated cells. Treatm ent of the cells with PD 98059 (10 mu mol/L), a MAP kinase kinase inhi bitor, caused 85% inhibition of the Ang II-induced activation of MAP k inases but did not inhibit the Ang II-induced DNA synthesis. In conclu sion, the Ang II-induced stimulation of the MAP kinase is a Ca2+-depen dent process. Furthermore, blockade of the Ang II-induced stimulation of the early intracellular events, such as increase in [Ca2+](i) or ph osphorylation of the MAP kinase, is not accompanied by an inhibition o f the Ang II-induced DNA synthesis.