Wu H M, Yuan Y, Zawieja D C, Tinsley J, Granger H J
Departments of Medical Physiology and Surgery, Texas A & M University System Health Science Center, Temple, Texas 76504, USA.
Am J Physiol. 1999 Feb;276(2):H535-42. doi: 10.1152/ajpheart.1999.276.2.H535.
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 NG-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 (Pa) and endothelial cytosolic 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 Pa, 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 (10(-6) M) and ionomycin (10(-6) 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-gamma and serine phosphorylation of endothelial constitutive NO synthase (ecNOS). The hyperphosphorylation of PLC-gamma 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.
我们先前证明，一氧化氮（NO）合酶抑制剂NG-单甲基-L-精氨酸（L-NMMA）可阻断血管内皮生长因子（VEGF）引起的冠状小静脉通透性增加。本研究的目的是更详细地描述VEGF诱导的小静脉通透性增加过程中NO产生上游的信号通路。使用荧光显微镜在完整灌注的猪冠状小静脉中测量白蛋白的表观通透系数（Pa）和内皮细胞胞质Ca2+浓度（[Ca2+]i）。VEGF（10(-10) M）使Pa增加了两到三倍，这被针对VEGF受体Flk-1/KDR的单克隆抗体、磷脂酶C（PLC）拮抗剂U-73122或蛋白激酶C（PKC）拮抗剂双吲哚马来酰亚胺（BIM）所阻断。在12条对缓激肽（10(-6) M）和离子霉素（10(-6) M）显示出[Ca2+]i反应的小静脉中，只有4条血管对VEGF有[Ca2+]i的短暂增加反应。此外，对培养的人脐静脉内皮细胞的蛋白质印迹分析表明，VEGF增加了PLC-γ的酪氨酸磷酸化和内皮型组成型NO合酶（ecNOS）的丝氨酸磷酸化。KDR受体抗体和U-73122可大大减弱PLC-γ的过度磷酸化，但BIM或L-NMMA则不能。相反，U-73122和BIM能够抑制VEGF引起的ecNOS丝氨酸磷酸化。结果表明，VEGF通过KDR受体介导的PLC激活诱导小静脉通透性增加。反过来，ecNOS通过PLC介导的PKC和/或胞质Ca2+升高刺激而被激活。
