1 The release of prostacylin (PGI2) from vascular endothelial cells is stimulated by ATP acting at G protein-coupled P2-purinoceptors. Here we investigate the hypothesis that tyrosine protein phosphorylations are involved in this response. 2 The use of Western blots with anti-phosphotyrosine antibodies showed that 30 gM 2MeSATP (selective for P2Y-purinoceptors), 300 gM UTP (selective for P2u-purinoceptors) and 300 gM ATP (effective at both these purinoceptors), each stimulate the tyrosine phosphorylation of proteins in bovine cultured aortic endothelial cells. Each of these agonists also stimulates 6-keto PGFI,, accumulation in the medium (an index of PGI2 release) in these cells in the same period.3 The tyrosine kinase inhibitor, genistein, inhibits the 6-keto PGFIe response with the same concentration-dependency (1 -100 gM) as the tyrosine phosphorylation response. 4 Tyrphostin, a structurally and functionally distinct tyrosine kinase inhibitor, is also a potent inhibitor (0.1-10 JM) of the 6-keto PGFI,, response.5 Neither tyrphostin nor genistein inhibit the phospholipase C response to P2-purinoceptor stimulation.Furthermore, these inhibitors do not affect the 6-keto PGFI,, response to ionomycin. 6 These results show that the regulation of vascular endothelial cells by ATP acting at both P2y-and P2u-purinoceptors involves the stimulation of tyrosine phosphorylation, and suggest that this is a necessary event for the purinoceptor-mediated stimulation of PGI2 production.
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...
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