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

Christoph Rüdiger Wolfram Kuhlmann; Jan Rasmus Friedrich Carl Trümper; Yaser Abdallah; Dörte Wiebke Lüdders; Christian Alexander Schaefer; Astrid Kerstin Most; Ulrich Backenköhler; Thomas Neumann; Sabine Walther; Hans Michael Piper; Harald Tillmanns; Ali Erdogan

doi:10.1160/TH04-03-0196

Thrombosis and Haemostasis, Inhaltsverzeichnis

Thromb Haemost 2004; 92(05): 1099-1107
DOI: 10.1160/TH04-03-0196

Endothelium and Vascular Development

Schattauer GmbH

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

Christoph Rüdiger Wolfram Kuhlmann

¹Justus Liebig University of Giessen, Department of Cardiology and Angiology, Germany

,

Jan Rasmus Friedrich Carl Trümper

¹Justus Liebig University of Giessen, Department of Cardiology and Angiology, Germany

,

Yaser Abdallah

¹Justus Liebig University of Giessen, Department of Cardiology and Angiology, Germany

,

Dörte Wiebke Lüdders

¹Justus Liebig University of Giessen, Department of Cardiology and Angiology, Germany

,

Christian Alexander Schaefer

¹Justus Liebig University of Giessen, Department of Cardiology and Angiology, Germany

,

Astrid Kerstin Most

¹Justus Liebig University of Giessen, Department of Cardiology and Angiology, Germany

,

Ulrich Backenköhler

¹Justus Liebig University of Giessen, Department of Cardiology and Angiology, Germany

,

Thomas Neumann

¹Justus Liebig University of Giessen, Department of Cardiology and Angiology, Germany

,

Sabine Walther

²Justus Liebibg University of Giessen, Department of Physiology, Germany

,

Hans Michael Piper

²Justus Liebibg University of Giessen, Department of Physiology, Germany

,

Harald Tillmanns

¹Justus Liebig University of Giessen, Department of Cardiology and Angiology, Germany

,

Ali Erdogan

¹Justus Liebig University of Giessen, Department of Cardiology and Angiology, Germany

› Institutsangaben

Abstract

Summary

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.

Keywords

Calcium-activated potassium channel - nitric oxide - MAP-kinase - endothelial cells - calcium

Volltext

Referenzen

References
1 Inagami T, Naruse M, Hoover R. Endothelium as an endocrine organ. Annu. Rev Physiol 1995; 57: 171-89.
2 Beny J. Electrical coupling between smooth muscle cells and endothelial cells in pig coronary arteries. Pflugers Arch 1997; 433: 364-7.
3 Haynes WG, Hand MF, Johnstones HA. et al. Direct and sympathetically mediated vasoconstriction in essential hypertension. Enhanced responses to endothelin-1. J Clin Invest 1994; 94: 1359-64.
4 Michel T, Smith TW. Nitric oxide synthases and cardiovascular signaling. Am J Cardiol 1993; 72: 33-8.
5 Bredt DS, Snyder SH. Isolation of nitric oxide synthase; a calmodulin-requiring enzyme. Proc Natl Acad Sci USA 1990; 87: 682-5.
6 Papapetrpoulos A, Garcia-Cardena G, Madri JA. et al. Nitric oxide production contributes to the angiogenic properties of Vascular Endothelial Growth Factor in human endothelial cells. J Clin Invest 1997; 100: 3131-9.
7 Oike M, Gericke M, Droogmans G. et al. Calcium entry activated by store depletion in human umbilical vein endothelial cells. Cell Calcium 1994; 16: 367-76.
8 Luckhoff A, Busse R. Calcium influx into endothelial cells and formation of endothelium-derived relaxing factor is controlled by the membrane potential. Pflugers Arch 1990; 416: 305-11.
9 Nilius B, Droogmans G. Ion channels and their functional role in vascular endothelium. Physiol Rev 2001; 81: 1415-59.
10 Gauthier KM, Liu C, Popovic A. et al. Freshly isolated endothelial cells do not express the BKCa channel gene. J Physiol 2002; 54: 829-36.
11 Begg M, Mo FM, Offertaler L. et al. G protein-coupled endothelial receptor for atypical cannabinoid ligands modulates a Ca2+-dependent K+ current. J Biol Chem 2003; 278: 46188-94.
12 Brakemeier S, Eichler I, Knorr A. et al. Modulation of Ca2+-activated K+ channel in renal artery endothelium in situ by nitric oxide and reactive oxygen species. Kidney Int 2003; 64: 199-207.
13 Wyatt AW, Steinert JR, Wheeler-Jones CPD. et al. Early activation of the p42/p44MAPK pathway mediates adenosine-induced nitric oxide production in human endothelial cells: a novel calcium-insensitive mechanism. FASEB J 2002; 16: 1584-94.
14 Schäfer M, Ewald N, Schafer C. et al. Signaling of hypoxia induced autonomous proliferation of endothelial cells. FASEB J 2003; 17: 449-51.
15 Kuhlmann CRW, Schafer M, Li F. et al. Modulation of endothelial Ca(2+)-activated K(+) channels by oxidized LDL and its contribution to endothelial proliferation. Cardiovasc Res 2003; 69: 626-34.
16 Langheinrich U, Daut J. Hyperpolarization of isolated capillaries from guinea pig heart induced by K+ channel openers and glucose deprivation. J Physiol 1997; 502: 397-408.
17 Wiecha J, Munz BM, Wu Y. et al. Blockade of Ca2+-activated K+ channels inhibits proliferation of human endothelial cells induced by basic fibroblast growth factor. J Vasc Res 1998; 35: 363-71.
18 Wedgwood S, Mitchell CJ, Fineman JR. et al. Developmental differences in the shear stressinduced expression of endothelial NO synthase: changing role of AP-1. Am J Physiol 2003; 284: 650-62.
19 Latorre R, Oberhauser A, Labarca P. et al. Varieties of calcium-activated potassium channels. Annu Rev Physiol 1989; 51: 385-99.
20 Adams DJ, Barakeh J, Laskey R. et al. Ion channels and regulation of intracellular calcium in vascular endothelial cells. FASEB J 1989; 03: 2389-400.
21 Colden-Stanfield M, Schilling WP, Ritchie AK. et al. Bradykinin-induced increases in cytosolic calcium and ionic currents in cultured bovine aortic endothelial cells. Circ Res 1987; 61: 632-40.
22 Li L, Bressler B, Prameya R. et al. Agonist stimulated calcium entry in primary cultures of human microvascular endothelial cells. Microvasc Res 1999; 57: 211-26.
23 Cheng GF, Cheung DW. Characterization of acetylcholine-induced membrane hyperpolarization in endothelial cells. Circ Res 1992; 70: 257-63.
24 Bishara NB, Murphy TV, Hill MA. Capacitative Ca2+ entry in vascular endothelial cells is mediated via pathways sensitive to 2 aminoethoxydiphenyl borate and xestospongin C. Br J Pharmacol 2002; 135: 119-28.
25 Demirel E, Rusko J, Laskey RE. et al. TEA inhibits Ach-induced EDRF release: endothelial Ca(2+)-dependent K+ channels contribute to vascular tone. Am J Physiol 1994; 267: 1135-41.
26 Busse R, Pohl U, Luckhoff A. Mechanisms controlling the production of endothelial autacoids. Z Kardiol 1989; 78: 64-9.
27 Luckhoff A, Pohl U, Mulsch A. et al. Differential role of extra- and intracellular calcium in the release of EDRF and prostacyclin from cultured endothelial cells. Br J Pharmacol 1988; 95: 189-96.
28 Mineo C, Yuhanna IS, Quon MJ. et al. High Density Lipoprotein-induced endothelial nitric-oxide synthase activation is mediated by Akt and MAP Kinase. J Biol Chem 2002; 278: 9142-9.
29 Chen Z, Yuhanna IS, Galcheva-Gargova Z. et al. Estrogen receptor alpha mediates the nongenomic activation of endothelial nitric oxide synthase by estrogen. J Clin Invest 1999; 103: 401-6.
30 Lorenz M, Wessler S, Follmann E. et al. A constituent of green tea, epigallocatechin-3-gallate, activates endothelial nitric oxide synthase by a phosphatidylinositol-3-OHkinase-, cAMP-dependent protein kinase-, and Akt-dependent pathway and leads to endothelial-dependent vasorelaxation. J Biol Chem 2004; 279: 6190-5.
31 Touyz RM. Molecular and cellular mechanisms regulating vascular function and structure – implications in the pathogenesis of hypertension. Can J Cardiol 2000; 16: 1137-46.
32 Nilius B, Droogmanns G. A role for K+ channels in cell proliferation. NIPS 1994; 09: 105-10.
33 Heller R, Polack T, Grabner R. et al. Nitric oxide inhibits proliferation of human endothelial cells via a mechanism independent of cGMP. Atherosclerosis 1999; 144: 49-57.
34 Babaei S, Teichert-Kuliszewska K, Monge JC. et al. Role of nitric oxide in the angiogenic response in vitro to basic fibroblast growth factor. Circ Res 1998; 82: 1007-15.
35 Bychkov R, Gollasch M, Steinke T. et al. Calcium-activated potassium channels and nitrate-induced vasodilatation in human coronary arteries. J Pharmacol Exp Ther 1998; 285: 293-8.