Platelet-conditioned medium increases endothelial electrical resistance independently of cAMP/PKA and cGMP/PKG

Platelets release a soluble factor into blood and conditioned medium (PCM) that decreases vascular endothelial permeability. The objective of this stu dy was to determine the signal-transduction pathway that elicits this decre ase in permeability. Permeability-decreasing activity of PCM was assessed b y the real-time measurement of electrical resistance across cell monolayers derived from bovine pulmonary arteries and microvessels. Using a desensiti zation protocol with cAMP/protein kinase A (PKA)-enhancing agents and pharm acological inhibitors, we determined that the activity of PCM is independen t of PKA and PKG. Genistein, an inhibitor of tyrosine kinases, prevented th e increase in endothelial electrical resistance. Because lysophosphatidic a cid (LPA) has been proposed to be responsible for this activity of PCM and is known to activate the G(i) protein, inhibitors of the G protein pertussi s toxin and of the associated phosphatidylinositol 3-kinase (PI3K) wortmann in were used. Pertussis toxin and wortmannin caused a 10- to 15-min delay i n the characteristic rise in electrical resistance induced by PCM. Inhibiti on of phosphorylation of extracellular signal-regulated kinase with the mit ogen-activated kinase kinase inhibitors PD-98059 and U-0126 did not prevent the activity of PCM. Similar findings with regard to the cAMP protocols an d inhibition of G(i) and 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/P KG. Furthermore, PCM rapidly activates a signaling pathway involving tyrosi ne phosphorylation, the G(i) protein, and PI3K.