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

1 Extracellular adenosine 5'-triphosphate (ATP) and uridine 5'-triphos phate (UTP) have been shown to activate a nucleotide receptor (P-2U re ceptor) in rat mesangial cells that mediates phosphoinositide and phos phatidylcholine hydrolysis by phospholipases C and D, respectively. Th is is followed by an increased activity of the mitogen-activated prote in kinase cascade and cell proliferation. Here we show that ATP and UT P potently stimulate the stress-activated protein kinase pathway and p hosphorylation of the transcription factor c-Jun. 2 Both nucleotides s timulated a rapid (within 5 min) and concentration-dependent activatio n of stress-activated protein kinases as measured by the phosphorylati on of c-Jun in a solid phase kinase assay. 3 When added at 100 mu M th e rank order of potency of a series of nucleotide analogues for stimul ation of c-Jun phosphorylation was UTP>ATP=UDP=ATP gamma S>2-methylthi o-ATP>beta gamma-imido-ATP=ADP>AMP=UMP=adenosine=uridine. Activation o f stress-activated protein kinase activity by ATP and UTP was dose-dep endently attenuated by suramin. 4 Down-regulation of protein kinase C- alpha, -delta and -epsilon isoenzymes by 24 h treatment of the cells w ith 12-O-tetradecanoylphorbol 13-acetate did not inhibit ATP- and UTP- induced activation of c-Jun phosphorylation. Furthermore, the specific protein kinase C inhibitors, CGP 41251 and Ro 31-8220, did not inhibi t nucleotide-stimulated c-Jun phosphorylation, suggesting that protein kinase C is not involved in ATP- and UTP-triggered stress-activated p rotein kinase activation. 5 Pretreatment of the cells with pertussis t oxin or the tyrosine kinase inhibitor, genistein, strongly attenuated ATP- and UTP-induced c-Jun phosphorylation. Furthermore, N-acetyl-cyst eine completely blocked the activation of stress-activated protein kin ase in response to extracellular nucleotide stimulation. 6 In summary, these results suggest that ATP and UTP trigger the activation of the stress-activated protein kinase module in mesangial cells by a pathway independent of protein kinase C but requiring a pertussis toxin - sen sitive G-protein and tyrosine kinase activation.