STIMULATION BY EXTRACELLULAR ATP AND UTP OF THE MITOGEN-ACTIVATED PROTEIN-KINASE CASCADE AND PROLIFERATION OF RAT RENAL MESANGIAL CELLS

1 Extracellular ATP and UTP have been reported to activate a nucleotid e receptor that mediates phosphoinositide and phosphatidylcholine hydr olysis by phospholipases C and D, respectively. Here we report that AT P and UTP potently stimulate mesangial cell proliferation. 2 Both nucl eotides stimulate phosphorylation and activation of mitogen-activated protein kinase and a biphasic phosphorylation of the up-stream mitogen -activated protein kinase kinase. 3 When added at 100 mu M, ATP gamma S, UTP and ATP were the most potent activators of mitogen-activated pr otein kinase. beta gamma-imido-ATP was somewhat less active and ADP an d 2-methylthio-ATP caused a weak induction of enzyme activity. Activat ion of mitogen-activated protein kinase by both ATP and UTP is dose-de pendently attenuated by the P-2-receptor antagonist, suramin. 4 The pr otein kinase C activator 12-O-tetradecanoylphorbol 13-acetate, but not the biologically inactive 4 alpha-phorbol 12,13-didecanoate, increase d mitogen-activated protein kinase activity in mesangial cells, sugges ting that protein kinase C may mediate nucleotide-induced stimulation of mitogen-activated protein kinase. 5 Down-regulation of protein kina se C -alpha and-delta isoenzymes by 4 h or 8 h treatment with phorbol ester partially inhibited ATP- and UTP-triggered mitogen-activated pro tein kinase activation. Moreover, a 24 h treatment of mesangial cells with phorbol ester, a regimen that also causes depletion of protein ki nase C-epsilon did not further reduce the level of mitogen-activated p rotein kinase stimulation. 6 The specific protein kinase C inhibitor, CGP 41251, which displayed a selectivity for the Ca2+-dependent isoenz ymes, as compared to the Ca2+-independent isoenzymes did not inhibit n ucleotide-stimulated mitogen-activated protein kinase phosphorylation, thus implicating the involvement of a Ca2+-independent protein kinase C isoform. 7 In summary, these results suggest that ATP and UTP trigg er the activation of the mitogen-activated protein kinase signalling c ascade in mesangial cells and this may be responsible for the potent m itogenic activity of both nucleotides.