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 Ca(2+) 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 Ca(2+) (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 [(3)H]-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 micromol/l). VEGF caused an increase in transmembrane Ca(2+) influx (n=30, p<0.05) that was sensitive to MTX and the blocker of transmembrane Ca(2+) entry 2-aminoethoxydiphenyl borate (APB, 100 micromol/l). VEGF-induced NO production was significantly reduced by MTX, APB and a reduction in extracellular Ca(2+) (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 micromol/l: CC: -86%, TI --164%).


CONCLUSIONS
VEGF caused an MTX-sensitive hyperpolarization which results in an increased transmembrane Ca(2+) entry that is responsible for the effects on endothelial proliferation and NO production.
Erdogan
Ali
A
Department of Cardiology and Angiology, Justus Liebig University of Giessen, Giessen, Germany.
Schaefer
Christian Alexander
CA
Schaefer
Matthias
M
Luedders
Doerte Wiebke
DW
Stockhausen
Florian
F
Abdallah
Yaser
Y
Schaefer
Claudia
C
Most
Astrid Kerstin
AK
Tillmanns
Harald
H
Piper
Hans Michael
HM
Kuhlmann
Christoph Ruediger Wolfram
CR
D002118:Calcium / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D016764:Cell Polarity / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004730:Endothelium, Vascular / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D051664:Kv1.3 Potassium Channel; D009498:Neurotoxins / Q000494:pharmacology*; D009569:Nitric Oxide / Q000378:metabolism*; D024642:Potassium Channels, Voltage-Gated / Q000378:metabolism; D012604:Scorpion Venoms; D014471:Umbilical Veins / Q000166:cytology; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism; D019323:omega-N-Methylarginine / Q000494:pharmacology
D004791:Enzyme Inhibitors; C496973:KCNA3 protein, human; D051664:Kv1.3 Potassium Channel; D009498:Neurotoxins; D024642:Potassium Channels, Voltage-Gated; D012604:Scorpion Venoms; D042461:Vascular Endothelial Growth Factor A; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; C077910:margatoxin; D040301:Vascular Endothelial Growth Factor Receptor-2; D006152:Cyclic GMP; D002118:Calcium