title: The K+-channel opener NS1619 increases endothelial NO-synthesis involving p42/p44 MAP-kinase.
abstract: Ca(2+)-activated K(+) channels with large conductance (BK(Ca)) 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/p44(MAPK)) on nitric oxide (NO) production in human endothelial cells induced by the BK(Ca)-opener NS1619. Using DiBAC-fluorescence imaging a concentration-dependent (2.5-12.5 microM) hyperpolarization induced by NS1619 was observed. A significant increase of intracellular Ca(2+)-concentration by NS1619 was seen using Fura-2-fluorescence-imaging, which was blocked by 2-APB, or reduction of extracellular Ca(2+) (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 Ca(2+), 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 + Ca(2+)-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/p44(MAPK) was examined by immunocytochemistry. Phosphorylation of p42/p44(MAPK) 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 Ca(2+)-influx leading to an increased NO production involving p42/p44(MAPK). This rise of NO formation is responsible for the NS1619 induced reduction of endothelial cell growth.
authors.affiliation: Justus Liebig University of Giessen, Dept. of Cardiology and Angiology, Klinikstr. 36, 35392 Giessen, Germany. Christoph.R.Kuhlmann@innere.med.uni-giessen.de
authors.forename: Christoph Rüdiger Wolfram
authors.initials: CR
authors.lastname: Kuhlmann
authors.forename: Jan Rasmus Friedrich Carl
authors.initials: JR
authors.lastname: Trümper
authors.forename: Yaser
authors.initials: Y
authors.lastname: Abdallah
authors.forename: Dörte
authors.initials: D
authors.lastname: Wiebke Lüdders
authors.forename: Christian Alexander
authors.initials: CA
authors.lastname: Schaefer
authors.forename: Astrid Kerstin
authors.initials: AK
authors.lastname: Most
authors.forename: Ulrich
authors.initials: U
authors.lastname: Backenköhler
authors.forename: Thomas
authors.initials: T
authors.lastname: Neumann
authors.forename: Sabine
authors.initials: S
authors.lastname: Walther
authors.forename: Hans Michael
authors.initials: HM
authors.lastname: Piper
authors.forename: Harald
authors.initials: H
authors.lastname: Tillmanns
authors.forename: Ali
authors.initials: A
authors.lastname: Erdogan
mesh_terms: D001562:Benzimidazoles / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D049109:Cell Proliferation; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism*; D006801:Humans; D051036:Large-Conductance Calcium-Activated Potassium Channels; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III; D010766:Phosphorylation; D024681:Potassium Channels, Calcium-Activated / Q000187:drug effects / Q000502:physiology*; D014471:Umbilical Veins
chemical_list: D001562:Benzimidazoles; D051036:Large-Conductance Calcium-Activated Potassium Channels; D024681:Potassium Channels, Calcium-Activated; C086491:NS 1619; D009569:Nitric Oxide; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D002118:Calcium