Synergetic activation of outwardly rectifying Cl − currents by hypotonic stress and external Ca 2+ in murine osteoclasts

1 An outwardly rectifying Cl − (OR Cl ) current of murine osteoclasts was activated by hypotonic stimulation. The current was characterized by rapid activation, little inactivation, strong outward rectification, blockage by DIDS and permeability to organic acids (pyruvate and glutamate). 2 The hypotonically activated OR Cl current was inhibited by intracellular dialysis with an ATP‐free pipette solution, but not by replacement of ATP with a poorly hydrolysable ATP analogue adenosine 5′ ‐O‐ (3‐thiotriphosphate). The current amplitude was reduced when intracellular alkalinity increased over the pH range 6.6–8.0. 3 Intracellular application of cytochalasin D occasionally activated the OR Cl current without hypotonic stress, but inhibited activation of the OR Cl current by hypotonic stimulation. The hypotonically activated OR Cl current was unaffected by a non‐actin‐depolymerizing cytochalasin, chaetoglobosin C, but partially inhibited by deoxyribonuclease I. 4 Removal of extracellular Ca 2+ inhibited activation of the OR Cl current by hypotonic shock, but did not reduce the current once activated. The hypotonically activated OR Cl current was partially decreased by intracellular dialysis with 20 m m EGTA. 5 With 10 m m Ca 2+ in the extracellular medium, the OR Cl current was activated in response to more minor decreases in osmolarity than with 1 m m Ca 2+ . The increased sensitivity to hypotonicity was mimicked by increasing the intracellular Ca 2+ level (pCa 6.5). 6 These results suggest that hypotonic stimulation and a rise in the extracellular Ca 2+ level synergistically activate the OR Cl channel of murine osteoclasts, and that the activating process is modified by multiple intracellular factors (pH, ATP and actin cytoskeletal organization).