Factors affecting SOCE activation in mammalian skeletal muscle fibers.
Enzymatically dissociated mouse FDB muscle fibers, loaded with Fura-2 AM, were used to study the effect of mitochondrial uncoupling on the capacitative Ca(2+) entry, SOCE. Sarcoplasmic reticulum (SR) Ca(2+) stores were depleted by repetitive exposures to high K(+) or 4-chloro-m-Cresol (4-CmC) in the absence of extracellular Ca(2+). SR Ca(2+) store replenishment was substantially reduced using 5 microM cyclopiazonic acid (CPA). Readmission of external Ca(2+) (5 mM) increased basal [Ca(2+)](i) under two modalities. In mode 1 [Ca(2+)](i) initially increased at a rate of 0.8 +/- 0.1 nM/s and later at a rate of 12.3 +/- 2.6 nM/s, reaching a final value of 477.8 +/- 36.8 nM in 215.7 +/- 25.9 s. In mode 2, [Ca(2+)](i) increased at a rate of 0.8 +/- 0.1 nM/s to a value of 204.9 +/- 20.6 nM in 185.4 +/- 21.1 s. FCCP, 2 microM, reduced this Ca(2+) entry. In nine FCCP-poisoned fibers, the initial rate of Ca(2+) increase was 0.34 +/- 0.1 nM/s (mean +/- SEM), reaching a plateau of 149.2 +/- 14.1 nM in 217 +/- 19 s. The results may likely be explained by the hypothesis that SOCE is inhibited by mitochondrial uncouplers, pointing to a possible mitochondrial role in its activation. Using time-scan confocal microscopy and the dyes CaOr-5N AM or Rhod-2 AM to label mitochondrial Ca(2+), we show that during depletion [Ca(2+)](mito) initially increases and later diminishes. Finally, we show that the increase in basal [Ca(2+)](i), associated with SOCE activation, diminishes upon external Na(+) withdrawal. Na(+) entry through the SOCE pathway and activation of the reversal of Na(+)/Ca(2+) exchanger could explain this SOCE modulation by Na(+).
Laboratorio de Fisiología Celular, Centro de Biofísica y Bioquímica, Instituto Venezolano de Investigaciones Científicas IVIC, Apartado 20632, Caracas 1020A, Venezuela. pbolanos@ivic.ve
Pura
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Bolaños
Alis
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Guillén
Reinaldo
R
DiPolo
Carlo
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Caputo
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000633:toxicity; D066298:In Vitro Techniques; D007211:Indoles / Q000494:pharmacology; D051379:Mice; D018613:Microscopy, Confocal; D008931:Mitochondria, Muscle / Q000187:drug effects / Q000378:metabolism; D008954:Models, Biological; D018485:Muscle Fibers, Skeletal / Q000187:drug effects / Q000378:metabolism*; D019831:Sodium-Calcium Exchanger / Q000378:metabolism
D015220:Calcium Channels; D007211:Indoles; D019831:Sodium-Calcium Exchanger; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D002118:Calcium; C000543:cyclopiazonic acid