We previously demonstrated that vascular endothelial growth factor (VEGF)-elicited increase in the permeability of coronary venules was blocked by the nitric oxide (NO) synthase inhibitor N^G-monomethyl-L-arginine (L-NMMA). The 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 cytosolic Ca²⁺ concentration ([Ca²⁺]_i) were measured in intact perfused porcine coronary venules using fluorescence microscopy. VEGF (10¹⁰ M) induced a two- to threefold increase in P_a, which was blocked by a monoclonal antibody directed against the VEGF receptor Flk-1/KDR, the phospholipase C (PLC) antagonist U-73122, or the protein kinase C (PKC) antagonist bisindolylmaleimide (BIM). In 12 venules that displayed the [Ca²⁺]_i response to bradykinin (10⁶ M) and ionomycin (10⁶ M), only 4 vessels responded to VEGF with a transient increase in [Ca²⁺]_i. Furthermore, Western blot analysis of cultured human umbilical vein endothelial cells showed that VEGF increased tyrosine phosphorylation of PLC- and serine phosphorylation of endothelial constitutive NO synthase (ecNOS). The hyperphosphorylation of PLC- was greatly attenuated by the KDR receptor antibody and U-73122, but not 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 induces venular hyperpermeability through a KDR receptor-mediated activation of PLC. In turn, ecNOS is activated by PLC-mediated PKC and/or cytosolic Ca²⁺ elevation stimulation.

endothelial barrier; cytosolic calcium; nitric oxide; vascular endothelial growth factor

This article has been cited by other articles:

				F. A. Sanchez, D. D. Kim, R. G. Duran, C. J. Meininger, and W. N. Duran Internalization of eNOS via caveolae regulates PAF-induced inflammatory hyperpermeability to macromolecules Am J Physiol Heart Circ Physiol, October 1, 2008; 295(4): H1642 - H1648. [Abstract] [Full Text] [PDF]

				L. Guo, C. Ye, W. Chen, H. Ye, R. Zheng, J. Li, H. Yang, X. Yu, and D. Zhang Anti-Inflammatory and Analgesic Potency of Carboxyamidotriazole, a Tumorostatic Agent J. Pharmacol. Exp. Ther., April 1, 2008; 325(1): 10 - 16. [Abstract] [Full Text] [PDF]

				J. R. Klinger, J. D. Murray, B. Casserly, D. F. Alvarez, J. A. King, S. S. An, G. Choudhary, A. N. Owusu-Sarfo, R. Warburton, and E. O. Harrington Rottlerin causes pulmonary edema in vivo: a possible role for PKC{delta} J Appl Physiol, December 1, 2007; 103(6): 2084 - 2094. [Abstract] [Full Text] [PDF]

				Z.-Y. Chen, G.-G. Feng, K. Nishiwaki, Y. Shimada, Y. Fujiwara, T. Komatsu, and N. Ishikawa Possible roles of neuropeptide Y Y3-receptor subtype in rat aortic endothelial cell proliferation under hypoxia, and its specific signal transduction Am J Physiol Heart Circ Physiol, August 1, 2007; 293(2): H959 - H967. [Abstract] [Full Text] [PDF]

				H.-W. Cheng, A.F. James, R.R. Foster, J.C. Hancox, and D.O. Bates VEGF Activates Receptor-Operated Cation Channels in Human Microvascular Endothelial Cells Arterioscler Thromb Vasc Biol, August 1, 2006; 26(8): 1768 - 1776. [Abstract] [Full Text] [PDF]

				H. Aramoto, J. W. Breslin, P. J. Pappas, R. W. Hobson II, and W. N. Duran Vascular endothelial growth factor stimulates differential signaling pathways in in vivo microcirculation Am J Physiol Heart Circ Physiol, October 1, 2004; 287(4): H1590 - H1598. [Abstract] [Full Text] [PDF]

				N. Kurimoto, Y.-S. Nan, Z.-Y. Chen, G.-G. Feng, T. Komatsu, N. Kandatsu, J. Ko, N. Kawai, and N. Ishikawa Effects of specific signal transduction inhibitors on increased permeability across rat endothelial monolayers induced by neuropeptide Y or VEGF Am J Physiol Heart Circ Physiol, July 1, 2004; 287(1): H100 - H106. [Abstract] [Full Text] [PDF]

				J. H. Tinsley, N. R. Teasdale, and S. Y. Yuan Myosin light chain phosphorylation and pulmonary endothelial cell hyperpermeability in burns Am J Physiol Lung Cell Mol Physiol, April 1, 2004; 286(4): L841 - L847. [Abstract] [Full Text] [PDF]

				H. Ashrafpour, N. Huang, P. C. Neligan, C. R. Forrest, P. D. Addison, M. A. Moses, R. H. Levine, and C. Y. Pang Vasodilator effect and mechanism of action of vascular endothelial growth factor in skin vasculature Am J Physiol Heart Circ Physiol, March 1, 2004; 286(3): H946 - H954. [Abstract] [Full Text] [PDF]

				J. W. Breslin, P. J. Pappas, J. J. Cerveira, R. W. Hobson II, and W. N. Duran VEGF increases endothelial permeability by separate signaling pathways involving ERK-1/2 and nitric oxide Am J Physiol Heart Circ Physiol, January 1, 2003; 284(1): H92 - H100. [Abstract] [Full Text] [PDF]

				P. M. Becker, A. D. Verin, M. A. Booth, F. Liu, A. Birukova, and J. G. N. Garcia Differential regulation of diverse physiological responses to VEGF in pulmonary endothelial cells Am J Physiol Lung Cell Mol Physiol, December 1, 2001; 281(6): L1500 - L1511. [Abstract] [Full Text] [PDF]

				K.-Y. Kim, S.-Y. Jeong, J. Won, P.-D. Ryu, and M.-J. Nam Induction of Angiogenesis by Expression of Soluble Type II Transforming Growth Factor-beta Receptor in Mouse Hepatoma J. Biol. Chem., October 12, 2001; 276(42): 38781 - 38786. [Abstract] [Full Text] [PDF]

				K. Bove, P. Neumann, N. Gertzberg, and A. Johnson Role of ecNOS-derived NO in mediating TNF-induced endothelial barrier dysfunction Am J Physiol Lung Cell Mol Physiol, May 1, 2001; 280(5): L914 - L922. [Abstract] [Full Text] [PDF]

				T. Ferro, P. Neumann, N. Gertzberg, R. Clements, and A. Johnson Protein kinase C-alpha mediates endothelial barrier dysfunction induced by TNF-alpha Am J Physiol Lung Cell Mol Physiol, June 1, 2000; 278(6): L1107 - L1117. [Abstract] [Full Text] [PDF]

				B.-Q. Shen, D. Y. Lee, and T. F. Zioncheck Vascular Endothelial Growth Factor Governs Endothelial Nitric-oxide Synthase Expression via a KDR/Flk-1 Receptor and a Protein Kinase C Signaling Pathway J. Biol. Chem., November 12, 1999; 274(46): 33057 - 33063. [Abstract] [Full Text] [PDF]

				S. Blum, K. Issbruker, A. Willuweit, S. Hehlgans, M. Lucerna, D. Mechtcheriakova, K. Walsh, D. von der Ahe, E. Hofer, and M. Clauss An Inhibitory Role of the Phosphatidylinositol 3-Kinase-signaling Pathway in Vascular Endothelial Growth Factor-induced Tissue Factor Expression J. Biol. Chem., August 31, 2001; 276(36): 33428 - 33434. [Abstract] [Full Text] [PDF]

				D. Thuringer, L. Maulon, and C. Frelin Rapid Transactivation of the Vascular Endothelial Growth Factor Receptor KDR/Flk-1 by the Bradykinin B2 Receptor Contributes to Endothelial Nitric-oxide Synthase Activation in Cardiac Capillary Endothelial Cells J. Biol. Chem., January 11, 2002; 277(3): 2028 - 2032. [Abstract] [Full Text] [PDF]