Na+ dependence of extracellular Ca2+-sensing mechanisms leading to activation of an outwardly rectifying Cl- channel in murine osteoclasts

Abstract

An elevation in the extracellular Ca(2+) concentration ([Ca(2+)](o)) is a key signal for bone remodeling by inhibiting the resorbing activity of osteoclasts. The [Ca(2+)](o)-sensing responses include a variety of morphological and functional changes, but the underlying mechanisms are yet to be defined. This study was aimed at investigating the [Ca(2+)](o)-sensing mechanisms leading to the activation of the Cl(-) channel in murine osteoclasts. A rise in either Ca(2+) or Gd(3+) activated an outwardly rectifying Cl(-) (OR(cl)) channel reversibly and dose-dependently, which was characterized by rapid activation kinetics, little inactivation, and blockage by DIDS. The concentration required for a half-maximal response was estimated to be >20-30 mmol/L for Ca(2+). Intracellular dialysis with an ATP-free pipette solution or application of an actin destabilizer, cytochalasin D, decreased the [Ca(2+)](o)-activated OR(cl) current. Substitution of extracellular Na(+) by an impermeable cation, N-methyl-D-glucamine(+), inhibited the [Ca(2+)](o)-activated OR(cl) channel, suggesting that the activation depended on extracellular Na(+). A blocker for the Na(+)-Ca(2+) exchanger, 2'4'-dichlorobenzamil hydrochloride (DCB), inhibited the [Ca(2+)](o)-activated OR(cl) channel as well. Although 10 mmol/L Ca(2+) activated the OR(cl) current only slightly at a standard intracellular pH (7.3), decreasing pH by dialyzing cells with an acidic pipette solution (pH 6.6) enhanced the [Ca(2+)](o)-activated OR(cl) current. This potentiation by cell acidosis was eliminated by amiloride, a blocker for the Na(+)-H(+) exchanger. Zinc ion (0.1 mmol/L) and a polycation, neomycin (0.2 mmol/L), activated the OR(cl) current at intracellular pH 6.6, whereas the effects of those cations were negligible at intracellular pH 7.3. These results suggest that [Ca(2+)](o)-sensing mechanisms, leading to activation of the OR(cl) channel in murine osteoclasts, are regulated by ATP and actin cytoskeletal organization, and are sensitized greatly by cell acidosis. Contributions of Na(+)-dependent transporters in this activating process are examined in the context of a possible intermediate signal of cell swelling caused by Na(+) influx.