HYPEROSMOTIC MODULATION OF THE CYTOSOLIC CALCIUM-CONCENTRATION IN A RAT OSTEOBLAST-LIKE CELL-LINE

1. The effects of hyperosmotic stress on cytosolic calcium concentration ([Ca2+](i)) were studied by ratio image analysis in single cells of an osteoblast-like bone cell line (RCJ 1.20) loaded with fura-2 AM. 2. The ratio (340 nm/380 nm) of steady-state [Ca2+](i) in resting osteoblasts kept in Hepes-buffered medium was 0.82 +/- 0.04. A hyperosmotic stimulus (200 mosmol l(-1) sucrose) produced a [Ca2+](i) transient with a peak ratio of 1.28 +/- 0.09, which decayed with an apparent half-life (t(1/2)) of 42.7 +/- 2.6 s. 3. The hyperosmotically induced [Ca2+](i) transients were insensitive to verapamil, diltiazem or nifedipine, which excludes the involvement of dihydropyridine-sensitive Ca2+ channels in the process. Non-specific Ca2+ channel blockers (Mn2+, Ni2+, La3+ or Gd3+) partially abolished the hyperosmotically induced [Ca2+](i) elevation, indicating the contribution of extracellular Ca2+ influx. 4. A hyperosmotic stimulus applied in Ca2+-free medium (0.5 mM EGTA) lowered the [Ca2+](i) peak to a ratio of 0.96 +/- 0.08 (P < 0.001) compared with a Ca2+-containing medium. This suggests that the [Ca2+](i) increase is due to extracellular influx, as well as release from an intracellular Ca2+ pool. 5. Application of thapsigargin (0.5 mu M), a specific inhibitor of endoplasmic reticulum Ca2+-ATPase, in Ca2+-free medium caused transient [Ca2+](i) elevation to peak ratios of 1.33 +/- 0.09, and completely abolished the [Ca2+](i) response to a hyperosmotic stimulus. This implies the existence of a thapsigargin-sensitive intracellular pool of Ca2+ that is mobilized by hyperosmotic stimulus. 6. Complete inhibition of the hyperosmotic Ca2+-induced changes was obtained following exposure of cells to dihydrocytochalasin B (5 mu M), whereas cell pretreatment with colchicine (5 mu M) did not affect [Ca2+](i) increase. 7. The results suggest that a hyperosmotic stimulus generates a [Ca2+](i) transient through the mobilization of Ca2+ from an intracellular thapsigargin-sensitive pool and via bivalent and trivalent cation-sensitive Ca2+ influx. The integrity of microfilaments is essential for this [Ca2+](i) response.