title: Endothelin-1-induced proliferation of human endothelial cells depends on activation of K+ channels and Ca+ influx.
abstract: OBJECTIVE
  Endothelin-1 (ET-1) promotes endothelial cell growth. Endothelial cell proliferation involves the activation of Ca2+-activated K+ channels. In this study, we investigated whether Ca2+-activated K+ channels with big conductance (BK(Ca)) contribute to endothelial cell proliferation induced by ET-1.
  
  
  METHODS
  The patch-clamp technique was used to analyse BK(Ca) activity in endothelial cells derived from human umbilical cord veins (HUVEC). Endothelial proliferation was examined using cell counts and measuring [3H]-thymidine incorporation. Changes of intracellular Ca2+ levels were examined using fura-2 fluorescence imaging.
  
  
  RESULTS
  Characteristic BK(Ca) were identified in cultured HUVEC. Continuous perfusion of HUVEC with 10 nmol L(-1) ET-1 caused a significant increase of BK(Ca) open-state probability (n = 14; P < 0.05; cell-attached patches). The ET(B)-receptor antagonist (BQ-788, 1 micromol L(-1)) blocked this effect. Stimulation with Et-1 (10 nmol L(-1)) significantly increased cell growth by 69% (n = 12; P < 0.05). In contrast, the combination of ET-1 (10 nmol L(-1)) and the highly specific BK(Ca) blocker iberiotoxin (IBX; 100 nmol L(-1)) did not cause a significant increase in endothelial cell growth. Ca2+ dependency of ET-1-induced proliferation was tested using the intracellular Ca2+-chelator BAPTA (10 micromol L(-1)). BAPTA abolished ET-1 induced proliferation (n = 12; P < 0.01). In addition, ET-1-induced HUVEC growth was significantly reduced, if cells were kept in a Ca2+-reduced solution (0.3 mmol L(-1)), or by the application of 2 aminoethoxdiphenyl borate (100 micromol L(-1)) which blocks hyperpolarization-induced Ca2+ entry (n = 12; P < 0.05).
  
  
  CONCLUSIONS
  Activation of BK(Ca) by ET-1 requires ET(B)-receptor activation and induces a capacitative Ca2+ influx which plays an important role in ET-1-mediated endothelial cell proliferation.
authors.affiliation: Department of Cardiology and Angiology, Justus-Liebig-University of Giessen, Giessen, Germany.
authors.forename: C R W
authors.initials: CR
authors.lastname: Kuhlmann
authors.forename: A K
authors.initials: AK
authors.lastname: Most
authors.forename: F
authors.initials: F
authors.lastname: Li
authors.forename: B M
authors.initials: BM
authors.lastname: Münz
authors.forename: C A
authors.initials: CA
authors.lastname: Schaefer
authors.forename: S
authors.initials: S
authors.lastname: Walther
authors.forename: T
authors.initials: T
authors.lastname: Raedle-Hurst
authors.forename: B
authors.initials: B
authors.lastname: Waldecker
authors.forename: H M
authors.initials: HM
authors.lastname: Piper
authors.forename: H
authors.initials: H
authors.lastname: Tillmanns
authors.forename: J
authors.initials: J
authors.lastname: Wiecha
mesh_terms: D002118:Calcium / Q000378:metabolism / Q000502:physiology; D002452:Cell Count; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D003470:Culture Media; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D004553:Electric Conductivity; D042783:Endothelial Cells / Q000187:drug effects / Q000502:physiology*; D065131:Endothelin B Receptor Antagonists; D019332:Endothelin-1 / Q000502:physiology*; D006801:Humans; D008564:Membrane Potentials / Q000502:physiology; D009842:Oligopeptides; D010455:Peptides / Q000494:pharmacology; D010880:Piperidines; D024681:Potassium Channels, Calcium-Activated / Q000037:antagonists & inhibitors / Q000502:physiology*
chemical_list: D002614:Chelating Agents; D003470:Culture Media; D065131:Endothelin B Receptor Antagonists; D019332:Endothelin-1; D009842:Oligopeptides; D010455:Peptides; D010880:Piperidines; D024681:Potassium Channels, Calcium-Activated; C086539:BQ 788; D004533:Egtazic Acid; C064719:iberiotoxin; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium