Platelets release a soluble factor into blood and conditioned medium (PCM) that decreases vascular endothelial permeability. The objective of this study was to determine the signal-transduction pathway that elicits this decrease in permeability. Permeability-decreasing activity of PCM was assessed by the real-time measurement of electrical resistance across cell monolayers derived from bovine pulmonary arteries and microvessels. Using a desensitization protocol with cAMP/protein kinase A (PKA)-enhancing agents and pharmacological inhibitors, we determined that the activity of PCM is independent of PKA and PKG. Genistein, an inhibitor of tyrosine kinases, prevented the increase in endothelial electrical resistance. Because lysophosphatidic acid (LPA) has been proposed to be responsible for this activity of PCM and is known to activate the Giprotein, inhibitors of the G protein pertussis toxin and of the associated phosphatidylinositol 3-kinase (PI3K) wortmannin were used. Pertussis toxin and wortmannin caused a 10- to 15-min delay in the characteristic rise in electrical resistance induced by PCM. Inhibition of phosphorylation of extracellular signal-regulated kinase with the mitogen-activated kinase kinase inhibitors PD-98059 and U-0126 did not prevent the activity of PCM. Similar findings with regard to the cAMP protocols and inhibition of Giand PI3K were obtained for 1-oleoyl-LPA. These results demonstrate that PCM increases endothelial electrical resistance in vitro via a novel, signal transduction pathway independent of cAMP/PKA and cGMP/PKG. Furthermore, PCM rapidly activates a signaling pathway involving tyrosine phosphorylation, the Giprotein, and PI3K.
Signal transduction pathway analysis in fibromatosis: receptor and nonreceptor tyrosine kinases
