Evidence for requirement of tyrosine phosphorylation in endothelial P_2y‐ and P_2u‐ purinoceptor stimulation of prostacyclin release

Abstract

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.

Protein kinase C isoforms in bovine aortic endothelial cells: role in regulation of P2Y‐ and P2U‐purinoceptor‐stimulated prostacyclin release

“…The lack of effect at 6 h is surprising in view of the report by Carter et al (1989) Burch & Axelrod, 1987;Felder et al, 1990 In a related study we have shown that stimulation of these P2-purinoceptors enhances tyrosine phosphorylation and provided evidence that this is required for agonist-stimulated PGI2 production (Bowden et al, 1995). These observations, taken together with those in the present report, require re-evaluation of the position that endothelial PGI2 production is essentially controlled by elevated cytosolic Ca2+ and only modulated by phosphorylating events.…”

“…Many other intracellular events would also be expected to correlate with PGI2 production following agonist stimulation. The observations reported here and elsewhere (Bowden et al, 1995) showing a dissociation between the Ins(1,4,5)P3 response and the PGI2 response, and an obligate role for both PKC and tyrosine kinases, give some indication of which of these events may be important in this endothelial response. Despite this, a causal role for Ca2" in the endothelial PGI2 response does seem likely in view of observations that depletion and buffering of intracellular Ca2+ attenuates the stimulation of 6-keto PGFIc accumulation (Hallam et al, 1988).…”

“…We have recently shown (Patel V. & Boarder M.R., in preparation) that stimulation of either P2Y-or P2u-purinoceptors on cultured endothelial cells leads to MAPK activation. It is interesting to speculate, therefore, that the observations on PKC described here, and those on endothelial P2-purinoceptor stimulated tyrosine phosphorylations reported independently (Bowden et al, 1995), may converge at the level of MAPK and may represent different ways of controlling the MAPK regulation of PLA2 activity and thus endothelial PGI2 production.…”

“…Phosphorylating activities shown to be significant for PLA2 stimulation and PGI2 release are tyrosine kinases (e.g. Kast et al, 1993;Bowden et al, 1995), mitogen activated protein kinases (MAPK) (Lin et al, 1993;Sa et al, 1995), and protein kinase C (PKC) (Carter et al, 1988;Zavoico et al, 1990).…”

Activation of UTP-Sensitive P2Y2Receptor Induces the Expression of Cholinergic Genes in Cultured Cortical Neurons: A Signaling Cascade Triggered by Ca2+Mobilization and Extracellular Regulated Kinase Phosphorylation

“…Activation of P2Y 2 R can stimulate PLC and increase Ca 21 from intracellular stores (Van Kolen and Slegers, 2006), and the UTP-sensitive P2Y 2 R can trigger downstream MAP kinase-dependent signaling in neuronal PC12 cells and endothelial cells (Bowden et al, 1995). Such a UTP-mediated cell signaling cascade has also been reported at the NMJ (Tung et al, 2004).…”