Quercetin-Induced Induction of the NO/cGMP Pathway Depends on Ca²⁺-Activated K⁺ Channel-Induced Hyperpolarization-Mediated Ca²⁺-Entry into Cultured Human Endothelial Cells

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 μmol/L). The maximum effect (50 μmol/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 μmol/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 μmol/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 Ca²⁺ entry. Increased NO production is responsible for the quercetin-dependent inhibition of endothelial proliferation.

Key words

Quercetin - membrane potential - nitric oxide - calcium - endothelial cell growth - cardiovascular disease

References

• 1 Hertog M G, Kromhout D, Aravanis C, Blackburn H, Buzina R, Fidanza F. et al . Flavonoid intake and long-term risk of coronary heart disease and cancer in the seven country study.  Arch Intern Med. 1995;  155 381-6
• 2 Hertog M G, Feskens E J, Hollman P C, Katan M B, Kromhout D. Dietary antioxidant flavonoids and risk of coronary heart disease: the Zutphen Elderly Study.  Lancet. 1993;  342 1007-11
• 3 Criqui M H, Ringel B L. Does diet or alcohol explain the French paradox?.  Lancet. 1994;  344 1719-23
• 4 Cook N C, Samman S. Flavonoids: chemistry, metabolism, cardioprotective effects, and dietary sources.  J Nutr Biochem. 1996;  7 66-76
• 5 Kobuchi H, Roy S, Sen C K, Nguyen H G, Packer L. Quercetin inhibits inducible ICAM-1 expression in human endothelial cells through the JNK pathway.  Am J Physiol. 1999;  277 403-11
• 6 Fan P S, Gu Z L, Liang Z Q. Effect of quercetin adhesion of platelets to microvascular endothelial cells in vitro .  Acta Pharmacol Sin. 2001;  22 857-60
• 7 Perez-Vizcaino F, Ibarra M, Cogoluddo A L, Duarte J, Zaragoza-Arnaez L, Moreno L. et al . Endothelium-independent vasodilatator effects of the flavonoid quercetin and its methylated metabolites in rat conductance and resistance arteries.  J Pharmacol Exp Ther. 2002;  302 66-72
• 8 Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s.  Nature. 1993;  362 801-9
• 9 Modifi R, Crotty T B, McCarthy P, Sheehan S J, Mehigan D, Keaveny T V. Association between plaque instability, angiogenesis and symptomatic carotid occlusive disease.  Br J Surg. 2001;  88 941-4
• 10 Bredt D S, Snyder S H. Isolation of nitric oxide synthase; a calmodulin-requiring enzyme.  Proc Natl Acad Sci USA. 1990;  87 682-5
• 11 Nilius B, Droogmans G. Ion channels and their functional role in vascular endothelium.  Physiol Rev. 2001;  81 1415-59
• 12 Kuhlmann C R, Gast C, Li F, Schäfer M, Tillmanns H, Waldecker B. et al . Cerivastatin activates endothelial calcium-activated potassium channels and thereby modulates endothelial NO production and cell proliferation.  J Am Soc Nephrol. 2004;  15 868-75
• 13 Kuhlmann C R, Lüdders D W, Schaefer C A, Most A K, Backenköhler U, Neumann T ,. et al . Lysophosphatidylcholine induced modulation of Ca²⁺-activated K⁺ channels contributes to ROS dependent proliferation of cultured human endothelial cells.  J Mol Cell Cardiol. 2004;  36 675-82
• 14 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
• 15 Bishara N B, Murphy T V, Hill M A. 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
• 16 Cermak R, Kuhn G, Wolffram S. The flavonol quercetin activates K(+) channels in distal colon epithelium.  Br J Pharmacol. 2002;  135 1183-90
• 17 Rouzaire-Dubois B, Gerard V, Dubois J M. Involvement of K⁺ channels in the quercetin induced inhibition of neuroblastoma cell growth.  Pflugers Arch. 1993;  423 202-5
• 18 Saponara S, Sparagli G, Fusi F. Quercetin as a novel activator of L-type Ca(2+) channels in rat tail artery smooth muscle cells.  Br J Pharmacol. 2002;  135 1819-27
• 19 Adams D J, Barakeh J, Laskey R, van Breemen C. Ion channels and regulation of intracellular calcium in vascular endothelial cells.  FASEB J. 1989;  3 2389-400
• 20 Kubota Y, Tanaka N, Umegaki K, Takenaka H, Mizuno H, Nakamura K. et al . Ginko biloba extract-induced relaxation of rat aorta is associated with increase in endothelial intracellular calcium level.  Life Sci. 2001;  69 2327-36
• 21 Flesch M, Schwarz A, Bohm M. Effects of red and white wine on endothelium-dependent vasorelaxation of rat aorta and human coronary arteries.  Am J Physiol. 1998;  275 1183-90
• 22 Igura K, Ohta T, Kuroda Y, Kaji K. Resveratrol and quercetin inhibit angiogenesis in vitro .  Cancer Lett. 2001;  28 11-6
• 23 Heller R, Polack T, Grabner R, Till U. Nitric oxide inhibits proliferation of human endothelial cells via a mechanism independent of cGMP.  Atherosclerosis. 1999;  144 49-57
• 24 Papapetropoulos A, Garcia-Cardena G, Madri J A, Sessa W C. Nitric oxide production contributes to the angiogenic properties of vascular endothelial growth factor in human endothelial cells.  J Clin Invest. 1997;  100 3131-9
• 25 Agullo P, Gamet-Payrastre L, Manenti S, Viala C, Remesy C, Chap H. et al . Relationship between flavonoid structure and inhibition of phosphatidylinositol 3-kinase: a comparison with tyrosine kinase and protein kinase C inhibition.  Biochem Pharmacol. 1997;  53 1649-57