Osmotic Membrane Stretch Increases Cytosolic Ca2+ and Inhibits Bone Resorption Activity in Rat Osteoclasts.

Abstract

Although the importance of mechanical stress on bone metabolism is well known, the intracellular mechanisms involved are not well understood. To evaluate the role of mechanical stress on osteoclastic function, we investigated the effects of membrane stretch induced by osmotic cell swelling on cytosolic Ca(2+) and bone resorption activity in freshly isolated rat osteoclasts. The intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured by fura-2 microspectrofluorimetry. Exposure to hypotonic solution (211-151 mOsm) caused cell swelling and reversibly increased [Ca(2+)](i) in the osteoclasts. This [Ca(2+)](i) increase was abolished by the omission of extracellular Ca(2+), but was not affected by the depletion of intracellular Ca(2+) stores. Gd(3+) and La(3+) inhibited the swelling-induced [Ca(2+)](i) increase, while nifedipine and Bay K 8644 did not. Neither protein kinase A inhibitors (Rp-cAMP, H-89) nor protein kinase C inhibitors (staurosporine, chelerythrine) affected the [Ca(2+)](i) increase. Membrane depolarization was not essential for the [Ca(2+)](i) increase either. To assess the effects of membrane stretch on the bone resorption activity of osteoclasts, we investigated actin ring formation, the intracellular structure responsible for bone resorption in osteoclasts. Hypotonic stimulation acutely disrupted actin ring formation in an extracellular Ca(2+)-dependent manner, and this disruption was prevented by Gd(3+). Moreover, Ca(2+) ionophore (ionomycin) also induced disruption of the actin rings. These results indicate that mechanical stress inhibits osteoclastic bone resorption activity, possibly via the elevation of [Ca(2+)](i) through stretch-activated, non-selective cation channels.