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

Open Access
Publisher Website
Google Scholar
Abstract
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 μm 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 (EC50 values 5.1 ± 0.2 μm for UTP; 2.9 ± 0.8 μm for ATP), whilst the selective P2Y‐ purinoceptor agonist, 2‐methylthioATP (2‐meSATP) was without effect up to concentrations of 100 μm. 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 α and ε isoforms of protein kinase. C (PKC) and virtual abolition of nucleotide‐stimulated MAP kinase activity (> 90% inhibition). 5 Preincubation for 30 min with the PKC inhibitor, Ro‐31 8220 (10 μm) 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 P2U‐purinoceptor.