Quercetin-induced induction of the NO/cGMP pathway depends on Ca2+-activated K+ channel-induced hyperpolarization-mediated Ca2+-entry into cultured human endothelial cells

Christoph R. Kuhlmann, Justus-Liebig-University of Giessen, Germany.
Christian A. Schaefer, Justus-Liebig-University of Giessen, Germany.
Christoph Kosok, Justus-Liebig-University of Giessen, Germany.
Yaser Abdallah, Aga Khan University
Sabine Walther, Justus-Liebig-University of Giessen, Germany.
Dörte W. Lüdders, Justus-Liebig-University of Giessen, Germany.
Thomas Neumann, Justus-Liebig-University of Giessen, Germany.
Harald Tillmanns, Justus-Liebig-University of Giessen, Germany.
Claudia Schäfer, Justus-Liebig-University of Giessen, Germany.
Hans M. Piper, Justus-Liebig-University of Giessen, Germany.
Ali Erdogan, Justus-Liebig-University of Giessen, Germany.

This work was published before the author joined Aga Khan University.

Abstract

Quercetin is one of the dietary-derived flavonoids that are held responsible for the beneficial effects of red wine drinking in coronary artery disease known as the "French paradox". We examined whether quercetin modulates endothelial function by influencing Ca2+-activated K+ channels with large conductance (BK(Ca)) in cultured human endothelial cells. Membrane potential and intracellular Ca2+ concentrations of cultured human endothelial cells derived from umbilical cord veins (HUVEC) were measured using the fluorescence dyes DiBAC, and FURA-2, respectively. NO production was examined using a cGMP radioimmunoassay. HUVEC proliferation was analyzed by cell counts and thymidine incorporation. A dose-dependent hyperpolarization of HUVEC was recorded when quercetin was added (5-100 micromol/L). The maximum effect (50 micromol/L) was significantly reduced by the addition of the highly selective BK(Ca) inhibitor iberiotoxin (100 nmol/L), but not by blockers of other Ca2+-activated K+ channels (n = 30; p < 0.05). This BK(Ca)-induced hyperpolarization caused a transmembrane Ca2+ influx, because the quercetin-induced increase of intracellular Ca2+ was blocked by iberiotoxin, or by applying 2-aminoethoxydiphenylborate (100 micromol/L)--an inhibitor of capacitative Ca2+ entry (n = 30; p < 0.05). Quercetin-induced cGMP levels were significantly reduced by the eNOS-inhibitor l-NMMA (300 micromol/L), and by iberiotoxin (n = 10; p < 0.05). Endothelial proliferation was significantly reduced by 56 % when cells were incubated with quercetin (n = 12; p < 0.05). This effect was due to the increased NO production, because it was reversed when the cells were treated with a combination of quercetin and l-NMMA. In conclusion quercetin improves endothelial dysfunction by increasing NO synthesis involving BK(Ca)-dependent membrane hyperpolarization-induced capacitative Ca 2+ entry. Increased NO production is responsible for the quercetin-dependent inhibition of endothelial proliferation.