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 (t1/2) 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 Ca(2+)‐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 Ca(2+)‐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 microM), a specific inhibitor of endoplasmic reticulum Ca(2+)‐ATPase, in Ca(2+)‐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.(ABSTRACT TRUNCATED AT 250 WORDS)