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'-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. 2. 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. 3. When added at 100 microM the rank order of potency of a series of nucleotide analogues for stimulation of c-Jun phosphorylation was UTP > ATP = UDP = ATP gamma S > 2-methylthio-ATP > beta gamma-imido-ATP = ADP > AMP = UMP = adenosine = uridine. Activation of stress-activated protein kinase activity by ATP and UTP was dose-dependently attenuated by suramin. 4. Down-regulation of protein kinase C-alpha, -delta and -epsilon 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. 5. 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. 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-sensitive G-protein and tyrosine kinase activation.
Huwiler
A
A
Department of Pharmacology, University of Basel, Switzerland.
van Rossum
G
G
Wartmann
M
M
Pfeilschifter
J
J
D000111:Acetylcysteine / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D002478:Cells, Cultured; D004789:Enzyme Activation; D019833:Genistein; D005920:Glomerular Mesangium / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D007529:Isoflavones / Q000494:pharmacology; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D008969:Molecular Sequence Data; D037342:Pertussis Toxin; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D014544:Uridine Triphosphate / Q000494:pharmacology*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases
D007529:Isoflavones; D010566:Virulence Factors, Bordetella; D000255:Adenosine Triphosphate; D019833:Genistein; D037342:Pertussis Toxin; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D014544:Uridine Triphosphate; D000111:Acetylcysteine