Stimulation by extracellular ATP and UTP of the stress‐activated protein kinase cascade in rat renal mesangial cells

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Abstract
Extracellular adenosine 5′‐triphosphate (ATP) and uridine 5′‐triphosphate (UTP) have been shown to activate a nucleotide receptor (P2U receptor) in rat mesangial cells that mediates phosphoinositide and phosphatidylcholine hydrolysis by phospholipases C and D, respectively. This is followed by an increased activity of the mitogen‐activated protein kinase cascade and cell proliferation. Here we show that ATP and UTP potently stimulate the stress‐activated protein kinase pathway and phosphorylation of the transcription factor c‐Jun. Both nucleotides stimulated a rapid (within 5 min) and concentration‐dependent activation of stress‐activated protein kinases as measured by the phosphorylation of c‐Jun in a solid phase kinase assay. When added at 100 μm the rank order of potency of a series of nucleotide analogues for stimulation of c‐Jun phosphorylation was UTP>ATP=UDP=ATPγS>2‐methylthio‐ATP>βγ‐imido‐ATP= ADP>AMP=UMP=adenosine=uridine. Activation of stress‐activated protein kinase activity by ATP and UTP was dose‐dependently attenuated by suramin. Down‐regulation of protein kinase C‐α, ‐δ and ‐ε isoenzymes by 24 h treatment of the cells with 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 inhibit nucleotide‐stimulated c‐Jun phosphorylation, suggesting that protein kinase C is not involved in ATP‐ and UTP‐triggered stress‐activated protein kinase activation. Pretreatment of the cells with pertussis toxin or the tyrosine kinase inhibitor, genistein, strongly attenuated ATP‐ and UTP‐induced c‐Jun phosphorylation. Furthermore, N‐acetyl‐cysteine completely blocked the activation of stress‐activated protein kinase in response to extracellular nucleotide stimulation. 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 – sensitive G‐protein and tyrosine kinase activation. British Journal of Pharmacology (1997) 120, 807–812; doi:10.1038/sj.bjp.0700979