title: Role of cGMP in sildenafil-induced activation of endothelial Ca2+-activated K+ channels.
abstract: Intracellular cGMP is an important second messenger in endothelial cells. Because Ca(2+)-activated K(+) channels with large conductance (BK(Ca)) have been shown to regulate endothelial cell functions, the aim of the present study was to examine whether sildenafil modulates BK(Ca) activity in cultured human endothelial cells. Changes of the endothelial cell membrane potential were analyzed using the fluorescence dye DiBAC. The patch-clamp technique was used to study BK(Ca) in human endothelial cells of umbilical cord veins (HUVEC). Intracellular Ca(2+) levels were analyzed using Fura-2 fluorescence imaging. Sildenafil caused a dose-dependent (0.05-5 micromol/l) hyperpolarization of the endothelial cells with a maximum at a concentration of 1 micromol/l. A significant increase of BK(Ca) activity was induced by sildenafil (1 micromol/l) perfusion. BK(Ca) open state-probability (NPo) was also increased by the cGMP-analogue 8-bromo-cGMP (0.5 mmol/l), whereas inhibition of the cGMP-dependent kinase (PKG) had no effect on NPo. PKG-inhibition abolished 8-bromo-cGMP induced BK(Ca) activation, and reduced sildenafil induced NPo. Furthermore, sildenafil caused a significant increase of intracellular calcium that was blocked by the BK(Ca) inhibitor iberiotoxin (100 nmol/l). In conclusion sildenafil activates BK(Ca) by a mechanism, which involves cGMP. The activation of the BK(Ca) is responsible for the sildenafil-induced increase of intracellular Ca(2+).
authors.affiliation: Justus-Liebig-University of Giessen, Department of Cardiology and Angiology, Germany.
authors.forename: Doerte Wiebke
authors.initials: DW
authors.lastname: Luedders
authors.forename: Benedikt Manuel
authors.initials: BM
authors.lastname: Muenz
authors.forename: Fang
authors.initials: F
authors.lastname: Li
authors.forename: Sebastian
authors.initials: S
authors.lastname: Rueckleben
authors.forename: Harald
authors.initials: H
authors.lastname: Tillmanns
authors.forename: Bernd
authors.initials: B
authors.lastname: Waldecker
authors.forename: Johannes
authors.initials: J
authors.lastname: Wiecha
authors.forename: Ali
authors.initials: A
authors.lastname: Erdogan
authors.forename: Christian Alexander
authors.initials: CA
authors.lastname: Schaefer
authors.forename: Christoph Ruediger Wolfram
authors.initials: CR
authors.lastname: Kuhlmann
mesh_terms: D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000037:antagonists & inhibitors*; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000502:physiology*; D017869:Cyclic GMP-Dependent Protein Kinases / Q000502:physiology; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology*; D010879:Piperazines / Q000494:pharmacology*; D024681:Potassium Channels, Calcium-Activated / Q000187:drug effects*; D011687:Purines; D000068677:Sildenafil Citrate; D013450:Sulfones
chemical_list: D010726:Phosphodiesterase Inhibitors; D010879:Piperazines; D024681:Potassium Channels, Calcium-Activated; D011687:Purines; D013450:Sulfones; D000068677:Sildenafil Citrate; D017869:Cyclic GMP-Dependent Protein Kinases; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; C517381:PDE5A protein, human; D006152:Cyclic GMP; D002118:Calcium