The K+-channel opener NS1619 increases endothelial NO-synthesis involving p42/p44 MAP-kinase

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Abstract
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 (2.5-12.5 µM) 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.