Platelet‐Activating Factor Effects on Bovine Pulmonary Artery Endothelial Cells
作者:
George Grigorian,
Una Ryan,
期刊:
Circulation Research
(OVID Available online 1987)
卷期:
Volume 61,
issue 3
页码: 389-395
ISSN:0009-7330
年代: 1987
出版商: OVID
关键词: platelet-activating factor;endothelium;phorbol esters;β-adrenergic receptors;protein kinase C;prostacyclin;thromboxane
数据来源: OVID
摘要:
Endothelial cells (ECs) were isolated from bovine pulmonary artery and maintained in long-term culture. On reaching confluency, ECs formed a characteristic "cobblestone" monolayer. One hour after addition of 1 nM platelet-activating factor (PAF) to the growth medium, ECs underwent dramatic changes in shape from their normal polygonal morphology to more elongated spindle-shaped forms. More pronounced effects were evident in the presence of 0.1 nM phorbol-12-myristate-13-acetate (PMA), a potent activator of C kinase. It was found that at concentrations from 10<SUP>-11</SUP>-10<SUP>-7</SUP>M, PAF stimulates the phosphoinositide turnover in EC. The half-maximal activation in the release of inositol phosphates was at 10<SUP>-9</SUP>M. This finding suggested that an increase in intracellular Ca<SUP>2+</SUP>concentration and activation of protein kinase C were involved in the mechanism of action of PAF on EC. The metabolic responses of EC were evaluated by measuring the activity of β-adrenergic receptor-coupled adenylate cyclase (AC) in a crude membrane fraction and by assay of prostacyclin and thromboxane released by cultured EC. AC from control membranes was activated by isoproterenol in a dose-dependent manner (EC<SUB>50</SUB>= 30 nM) from 0.8–5.5 pmol cAMP/min/mg protein. If the membranes were isolated after preincubation of ECs with 1 nMPAF or 0.l nM PMA, the AC activity was decreased by 70 and 90%, respectively; in both cases, affinity for isoproterenol was lowered threefold (EC<SUB>50 </SUB>= 100 nM). Our data suggest that PAF interaction with EC leads to an apparent β-adrenergic receptor desensitization that probably acts via a phosphorylation mechanism involving C kinase. Incubation of EC for 30 minutes with 0.1–1.0 nM PAF caused inhibition of both prostacyclin and thromboxane production (55 and 75%, respectively) indicating that PAF acts at a level common to both pathways of arachidonate metabolism. Similar results were obtained using PMA (0.1 nM) but not with phorbol-12,13-didecanoate, an inactive analogue of PMA. Taken together, these data indicate that C kinase is involved in PAF-induced alteration in receptor sensitivity at the plasma membrane level as well as in intracellular enzymes responsible for prostacyclin and thromboxane synthesis by EC. This down-regulation of metabolic activity of EC is accompanied by concomitant shape changes.
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