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 vario us nucleotides in the endothelial cell line EAhy 926. 2 Adenosine 5'-t riphosphate (ATP) in the concentration range 0.1-100 mu M stimulated t he rapid and transient tyrosine phosphorylation and activation of the 42 kDa isoform of MAP kinase in EAhy 926 endothelial cells which peake d at 2 min and returned to basal values by 60 min. ATP also stimulated a similar response in primary cultured bovine aortic endothelial cell s. 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 m u M for UTP; 2.9 +/- 0.8 mu M for ATP), whilst the selective P-2Y-puri noceptor 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 l oss of the alpha and epsilon isoforms of protein kinase C (PPC) and vi rtual abolition of nucleotide-stimulated MAP kinase activity (>90% inh ibition). 5 Preincubation for 30 min with the PKC inhibitor, Re-31 822 0 (10 mu M) reduced MAP-kinase activation at 2 min but potentiated the response at 60 min. 6 Removal of extracellular calcium in the presenc e of EGTA reduced the MAP kinase activation in response to UTP by appr oximately 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 kin ase C-dependent manner through interaction with a P-2U-purinoceptor.