Role of phospholipase C, protein kinase C, and calcium in VEGF-induced venular hyperpermeability

We previously demonstrated that vascular endothelial growth factor (VEGF)-e licited increase in the permeability of coronary venules was blocked by the nitric oxide (NO) synthase inhibitor N-G-monomethyl-L-arginine (L-NMMA). T he aim of this study was to delineate in more detail the signaling pathways upstream from NO production in VEGF-induced venular hyperpermeability. The apparent permeability coefficient of albumin (P-a) and endothelial cytosol ic Ca2+ concentration ([Ca2+](i)) were measured in intact perfused porcine coronary venules using fluorescence microscopy. VEGF (10(-10) M) induced a two- to threefold increase in P-a, which was blocked by a monoclonal antibo dy directed against the VEGF receptor Flk-1/KDR, the phospholipase C (PLC) antagonist U-73122, or the protein kinase C (PKC) antagonist bisindolylmale imide (BIM). In 12 venules that displayed the [Ca2+](i) response to bradyki nin (10(-6) M) and ionomycin (10(-6) M), only 4 vessels responded to VEGF w ith a transient increase in [Ca2+](i). Furthermore, Western blot analysis o f cultured human umbilical vein endothelial cells showed that VEGF increase d tyrosine phosphorylation of PLC-gamma and serine phosphorylation of endot helial constitutive NO synthase (ecNOS). The hyperphosphorylation of PLC-ga mma was greatly attenuated by the KDR receptor antibody and U-73122, but no t by BIM or L-NMMA. In contrast, U-73122 and BIM were able to inhibit VEGF- elicited serine phosphorylation of ecNOS. The results suggest that VEGF ind uces venular hyperpermeability through a KDR receptor-mediated activation o f PLC. In turn, ecNOS is activated by PLC-mediated PKC and/or cytosolic Ca2 + elevation stimulation.