title: Activation of Cl- channels by extracellular Ca2+ in freshly isolated rabbit osteoclasts.
abstract: Ionic channels regulated by extracellular Ca2+ concentration ([Ca2+]o) were examined in freshly isolated rabbit osteoclasts. K+ current was suppressed by intracellular and extracellular Cs+ ions. In this condition, high [Ca2+]o evoked an outwardly rectifying current with a reversal potential of about -25 mV. When the concentration of extracellular Cl ions was altered, the reversal potential of the outwardly rectifying current shifted as predicted by the Nernst equation. 4',4-diisothiocyanostilbene-2' 2-disulphonic acid (DIDS) inhibited the outwardly rectifying current. These results indicated that this current was carried through Cl- channels. Cd2+ or Ni2+ caused a transient activation of the Cl- current in contrast to the sustained activation elicited by Ca2+. Intracellular 20 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) inhibited the divalent cation-induced Cl- current. Either when the osmolarity of extracellular medium was increased, or when 100 microM cAMP was dissolved in the patch pipette solution, high [Ca2+]o still elicited the Cl- current, indicating that the divalent cation-induced Cl- current was carried through Ca(2+)-activated Cl- channels. Under perforated whole cell clamp extracellular divalent cations evoked the Cl- current, indicating that the activation of Cl- current did not arise from possible leakage of divalent cations from the extracellular medium under the whole cell clamp condition. This experiment further excluded a possible activation of volume-sensitive Cl- channels under whole cell clamp. Intracellular application of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) activated the Cl current and it was inhibited by intracellular 20 mM EGTA, suggesting that the activation of Cl current was mediated through a G protein, and that an increase in [Ca2+]i was critical for the activation of Cl-channels. A protein phosphatase inhibitor, okadaic acid (100 nM), caused an irreversible activation of the Cl current, suggesting that protein phosphatase 1 or 2A was involved in the regulation of Ca(2+)-activated Cl- channels.
authors.affiliation: Fourth Department of Internal Medicine, University of Tokyo School of Medicine, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Fujita
authors.forename: T
authors.initials: T
authors.lastname: Matsumoto
authors.forename: H
authors.initials: H
authors.lastname: Kawashima
authors.forename: E
authors.initials: E
authors.lastname: Ogata
authors.forename: T
authors.initials: T
authors.lastname: Fujita
authors.forename: N
authors.initials: N
authors.lastname: Yamashita
mesh_terms: D000818:Animals; D002118:Calcium / Q000502:physiology*; D002413:Cations, Divalent / Q000494:pharmacology; D002469:Cell Separation; D018118:Chloride Channels / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D004553:Electric Conductivity; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005110:Extracellular Space / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000502:physiology; D009994:Osmolar Concentration; D010010:Osteoclasts / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D054645:Protein Phosphatase 1; D011817:Rabbits
chemical_list: D002413:Cations, Divalent; D018118:Chloride Channels; D004791:Enzyme Inhibitors; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D019204:GTP-Binding Proteins; D002118:Calcium