Lysophosphatidylcholine-induced modulation of Ca(2+)-activated K(+)channels contributes to ROS-dependent proliferation of cultured human endothelial cells.
Proliferation of endothelial cells plays a crucial role in the process of atherosclerotic plaque destabilization. The major component of oxidized low-density lipoprotein lysophosphatidylcholine (LPC) has been shown to promote endothelial proliferation by increasing the production of reactive oxygen species (ROS). Since K(+) channels are known to control the cell cycle, we investigated the role of Ca(2+)-activated K(+) channels (BK(Ca)) in the regulation of LPC-induced endothelial proliferation and ROS generation. A significant increase of cell growth induced by LPC (20 micromol/l; cell counts (CCs): +87%, thymidin incorporation: +89%; n = 12, P < 0.01) was observed, which was inhibited by the BK(Ca) inhibitor iberiotoxin (IBX; 100 nmol/l), by the NAD(P)H-oxidase inhibitor diphenyleneiodonium (5 micromol/l) and by transfection with antisense (AS) oligonucleotides against NAD(P)H oxidase, whereas N(G)-monomethyl-l-arginine (l-NMMA) further increased LPC-induced cell growth. Using the patch-clamp technique a significant increase of BK(Ca) open-state probability (control: 0.004 +/- 0.002; LPC: 0.104 +/- 0.035; n = 21, P < 0.05) by LPC was observed. Using dichlorofluorescein fluorescence microscopy a significant increase of ROS induced by LPC was reported, that was blocked by IBX and Ca(2+) antagonists. Intracellular Ca(2+) measurements revealed a capacitative Ca(2+) influx caused by LPC. Bioactivity of nitric oxide (NO) was measured using a [(3)H]-cGMP radioimmunoassay. LPC significantly decreased acetylcholine-induced NO synthesis. LPC significantly increased cGMP levels in endothelial cells transfected with AS, which was blocked by IBX. In conclusion, our results demonstrate that LPC activates BK(Ca) thereby increasing ROS production which induces endothelial proliferation. In addition LPC-induced BK(Ca)-activation contributes to increased cGMP levels, if ROS production is prevented by AS.
Wolfram Kuhlmann
Christoph Rüdiger
CR
Department of Cardiology and Angiology, Justus-Liebig-University of Giessen, Klinikstrasse 36, Giessen 35392, Germany. Chr_Kuhlmann@web.de
Wiebke Lüdders
Dörte
D
Schaefer
Christian Alexander
CA
Kerstin Most
Astrid
A
Backenköhler
Ulrich
U
Neumann
Thomas
T
Tillmanns
Harald
H
Erdogan
Ali
A
D002118:Calcium / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D006706:Homeostasis / Q000187:drug effects; D006801:Humans; D008244:Lysophosphatidylcholines / Q000494:pharmacology*; D019255:NADPH Oxidases / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D009589:Nitrogen Oxides / Q000378:metabolism; D024681:Potassium Channels, Calcium-Activated / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D014470:Umbilical Cord / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*
D008244:Lysophosphatidylcholines; D009589:Nitrogen Oxides; D024681:Potassium Channels, Calcium-Activated; D017382:Reactive Oxygen Species; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D019255:NADPH Oxidases; D006152:Cyclic GMP; D002118:Calcium