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)
Dascalu
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Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel-Aviv University, Ramat Aviv, Israel.
Oron
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Y
Nevo
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Korenstein
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R
D000818:Animals; D002118:Calcium / Q000502:physiology*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002460:Cell Line; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D016257:Fura-2 / Q000031:analogs & derivatives; D009994:Osmolar Concentration; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin
D002121:Calcium Channel Blockers; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D013729:Terpenes; C049925:fura-2-am; D019284:Thapsigargin; D000252:Calcium-Transporting ATPases; D002118:Calcium; D016257:Fura-2