Stimulation by the nucleotides, ATP and UTP of mitogen-activated protein kinase in EAhy 926 endothelial cells

1 We have investigated the characteristics of activation of the 42kDa isoform of mitogen-activated protein (MAP) kinase in response to various nucleotides in the endothelial cell line EAhy 926. 2 Adenosine 5'-triphosphate (ATP) in the concentration range 0.1-100 mu M stimulated the rapid and transient tyrosine phosphorylation and activation of the 42 kDa isoform of MAP kinase in EAhy 926 endothelial cells which peaked at 2 min and returned to basal values by 60 min. ATP also stimulated a similar response in primary cultured bovine aortic endothelial cells. 3 Uridine 5' triphosphate (UTP) also stimulated the 42 kDa isoform of MAP kinase with similar potency to ATP (EC(50) values 5.1 +/- 0.2 mu M for UTP; 2.9 +/- 0.8 mu M for ATP), whilst the selective P-2Y-purinoceptor agonist, 2-methylthioATP (2-meSATP) was without effect up to concentrations of 100 mu M. In bovine aortic endothelial cells however, UTP and 2-meSATP both stimulated MAP kinase. 4 Pretreatment of cells for 24 h with 12-O tetradecanoyl phorbol 13-acetate resulted in the loss of the alpha and epsilon isoforms of protein kinase C (PPC) and virtual abolition of nucleotide-stimulated MAP kinase activity (>90% inhibition). 5 Preincubation for 30 min with the PKC inhibitor, Re-31 8220 (10 mu M) reduced MAP-kinase activation at 2 min but potentiated the response at 60 min. 6 Removal of extracellular calcium in the presence of EGTA reduced the MAP kinase activation in response to UTP by approximately 30-50%. 7 Pretreatment with pertussis toxin (18 h, 50 ng ml(-1)) did not significantly affect the UTP-mediated activation of pp42 MAP kinase. 8 These results show that in the EAhy 926 endothelial cell line, nucleotides stimulate activation of MAP kinase in a protein kinase C-dependent manner through interaction with a P-2U-purinoceptor.