Stimulation of prostacyclin release by endothelial ATP receptors requires activation of protein kinase C and tyrosine kinases

Abstract

Bovine aortic endothelial (BAE) cells contain two coexisting receptors for extracellular ATP [I], which have been designated the P2y and P2u purinoceptors. Agonist stimulation of these receptors leads to the release of the vasodilator substances prostacyclin (PGI2) and nitric oxide [2]. The aim of this study was to investigate the release of PG12 in BAE cells mediated by stimulation of these receptors.BAE cells were prepared from fresh bovine aortae by collagenase digestion essentially according to the method of Booyse et ul, 1975 [3]. Cells were grown to confluence on 24 well multiplates and used between passage 2 -9.The accumulation of 6-keto PGF 1 a in the medium of stimulated cells was used as an index of PGI2 release.Cells were washed twice in balanced salt solution (BSS), and then preincubated for lOmin in lml BSS. After this time 730pl of the preincubate was removed, and 30pl of agonist at 1Ox concentrate was added to begin the stimulation period. Stimulations were terminated by rapid removal of the supernatants which were stored at -2OOC prior to assay.were used these were included during the 1Omin preincubation as well as the stimulation period. Tyrosine kinase inhibitors when used were included for a 3(hnin preincubation as well as during the stimulation period. Measurement of 6-keto PGFla levels in the samples was by radioimmunoassay. Phosphotyrosine western blots were performed using anti-phosphotyrosine monoclonal antibodies and developed with antimouse Ig peroxidaselinked antibody and the Amersham enhanced chemiluminescence system. Visualisation was by autoradiography followed by laser densitometty. Agonists used were 2MeSATP at P2y purinoceptors and UTP at P2u purinoceptors, ATP was active at both receptors.Each agonist stimulated the accumulation of 6-keto PGFl a in the medium of cells over a time course which reached a maximum between 3.5 and 5min for all three agonists. The response to ATP was approximately equivalent to the sum of the responses to 2MeSATP and UTP. Concentration response studies showed that 2MeSATP was more potent than UTP (log EC50 -1.45 f 0.08pM (n=4), and -0.85 f 0.125pM (n=4) for 2MeSATP and UTP respectively). However, both agonists induced similar maximal responses.Where activators or inhibitors of protein kinase C (PKC) Accumulationof 6-keto PGF 1 a induced by either agonkt was enhanced in the presence of the phorbol ester TPA (1OOnM) ( 163 * 37 YO and 209 f 32 % of the response in the absence of TPA for 2MeSATP and UTP respectively), and in the presence of the PKC inhibitor Ro 3 1-8220 ( 1 Op M) the stimulation induced by either agonist was completely abolished. However, neither activation nor inhibition of PKC had any significant effect on basal levels of 6-keto PGFla.Western blots performed using anti-phosphotyrosine antibodies on homogenates of cells stimulated for 5min with either ATP (300pM), 2MeSATP (30pM) or UTP (300pM) showed enhanced levels of tyrosine phosphoproteins in multiple bands, indicating that activation of both P2y and P2u purinoceptors involves tyrosin...