Margatoxin inhibits VEGF-induced hyperpolarization, proliferation and nitric oxide production of human endothelial cells

Background: Vascular endothelial growth factor ( VEGF) induces proliferation of endothelial cells (EC) in vitro and angiogenesis in vivo. Furthermore, a role of VEGF in K+ channel, nitric oxide (NO) and Ca2+ signaling was reported. We examined whether the K+ channel blocker margatoxin (MTX) influences VEGF-induced signaling in human EC. Methods: Fluorescence imaging was used to analyze changes in the membrane potential (DiBAC), intracellular Ca2+ (FURA-2) and NO (DAF) levels in cultured human EC derived from human umbilical vein EC ( HUVEC). Proliferation of HUVEC was examined by cell counts (CC) and [H-3]- thymidine incorporation ( TI). Results: VEGF (5 - 50 ng/ml) caused a dose-dependent hyperpolarization of EC, with a maximum at 30 ng/ml ( n = 30, p < 0.05). This effect was completely blocked by MTX (5 mu mol/ l). VEGF caused an increase in transmembrane Ca2+ influx ( n = 30, p < 0.05) that was sensitive to MTX and the blocker of transmembrane Ca2+ entry 2-aminoethoxydiphenyl borate (APB, 100 mu mol/ l). VEGF-induced NO production was significantly reduced by MTX, APB and a reduction in extracellular Ca2+ ( n = 30, p < 0.05). HUVEC proliferation, examined by CC and TI, was significantly increased by VEGF and inhibited by MTX ( CC: - 58%, TI - 121%); APB ( CC - 99%, TI - 187%); N-monomethyl-L - arginine ( 300 mu mol/ l: CC: - 86%, TI - 164%). Conclusions: VEGF caused an MTX-sensitive hyperpolarization which results in an increased transmembrane Ca2+ entry that is responsible for the effects on endothelial proliferation and NO production. Copyright (C) 2005 S. Karger AG, Basel.