Bradykinin- and thrombin-induced increases in endothelial permeability occur independently of phospholipase C but require protein kinase C activation

We determined whether activation of phosphatidylinositol-specific phospholipase C (PI-PLC] and a subsequent increase in cytosolic calcium concentration ([Ca²⁺](i)) was an obligatory signaling event mediating the increase in transendothelial permeability induced by bradykinin (BK) and α-thrombin (α-T). Both BK and α-T (each at a concentration range of 0.01-1 μM) caused dose-dependent increases in transendothelial ¹²⁵I-albumin permeability in cultured bovine pulmonary artery endothelial cell monolayers. Both agonists also produced a rise in inositol (1,4,5)-trisphosphate [Ins(1,4,5)P₃] by 10 sec that was followed by a prolonged increase in [Ca²⁺](i). Pretreatment of endothelial cells with the PLC inhibitor, 1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino) hexyl)-1H-pyrrole-2,5-dion [(U73122) at 10 μM for 15 min], prevented the increases in [Ins[1,4,5)P₃ and [Ca²⁺](i) induced by both BK and α-T. However, inhibition of PLC with (U73122 or another PLC inhibitor, neomycin, did not prevent the increase in endothelial permeability induced by either agonist. In contrast, depletion of cellular protein kinase C (PKC) with phorbol-12-myristate 13-acetate (0.01 μM for 20 hr) increased both BK- and α-T-induced phosphoinositide turnover but inhibited the agonist-induced increase in permeability. A PKC inhibitor, staurosporine (5 μM) likewise inhibited the BK-induced increase in endothelial cell permeability to albumin. We conclude that increases in endothelial permeability induced by the inflammatory mediators, BK and thrombin, can occur independently of PLC activation and increased [Ca²⁺](i) but that a PKC-dependent pathway is required for the permeability response.