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Christoph Rdiger Wolfram Kuhlmann, Jan Rasmus Friedrich Carl Trmper, Yaser Abdallah, Drte Wiebke Ldders, Christian Alexander Schaefer, Astrid Kerstin Most, Ulrich Backenkhler, Thomas Neumann, Sabine Walther, Hans Michael Piper, Harald Tillmanns, Ali Erdog
The K+-channel opener NS1619 increases endothelial NO-synthesis involving p42/p44 MAP-kinase
Ca2+-activated K+ channels with large conductance (BKCa) have been shown to play an important role in the regulation of vascular tone. We examined the role of the p42/p44 MAP-kinase (p42/p44MAPK) on nitric oxide (NO) production in human endothelial cells induced by the BKCa-opener NS1619. Using DiBAC-fluorescence imaging a concentration-dependent (2.512.5 M) hyperpolarization induced by NS1619 was observed. A significant increase of intracellular Ca2+-concentration by NS1619 was seen using Fura-2-fluorescence-imaging, which was blocked by 2-APB, or reduction of extracellular Ca2+ (n=30; p<0.05). A cGMP-radioimmunoassay was used to examine NO synthesis. NS1619 significantly increased cGMP levels which was inhibited by LNMMA, iberiotoxin, BAPTA, 2-APB, reduction of extracellular Ca2+, PD 98059, or U0126 (cGMP (pmol/mg protein): NS1619 3.25 0.85; NS1619 + L-NMMA 0.86 0.02; NS1619 + iberiotoxin 0.99 0.09; NS1619 + BAPTA 0.93 0.29; NS1619 + 2-APB 0.99 0.31; NS1619 + Ca2+-reduction 1.17 0.06; NS1619 + PD98059 1.06 0.49; NS1619 + U0126 1.10 0.24; n=10; p<0.05). The phosphorylation of eNOS and p42/p44MAPK was examined by immunocytochemistry. Phosphorylation of p42/p44MAPK was significantly increased after 10 minutes of NS1619 stimulation, whereas eNOS phosphorylation was not changed over a period of 1 to 30 minutes. NS1619-induced hyperpolarization was not affected by treatment with PD 98059 or U0126. Additionally, NS1619 inhibited endothelial proliferation involving a NO-dependent mechanism. Our data demonstrate that NS1619 causes a transmembrane Ca2+-influx leading to an increased NO production involving p42/p44MAPK. This rise of NO formation is responsible for the NS1619 induced reduction of endothelial cell growth.
Thrombosis and Haemostasis, Schattauer
Print ISSN: 0340-6245
Volume: 92, 11/2004
Pages: 1099 - 1107