Sarcoplasmic reticulum Ca2+ depletion by caffeine and changes of [Ca2+](i) during refilling in bovine airway smooth muscle cells.

BACKGROUND

In airway smooth muscle (ASM), Ca2+ influx in response to the Ca2+ depletion of the sarcoplasmic reticulum (SR) seems to play a role in the regulation of intracellular free Ca2+ concentrations (Ca2+). This study evaluates some possible Ca2+ entry pathways activated during SR-Ca2+ depletion induced by 10 mM caffeine.

METHODS

Enzymatically dispersed bovine ASM cells were loaded with Fura-2/AM to permit measurement of Ca2+ changes in single cells.

RESULTS

Caffeine (10 mM) induced a transient increase in [Ca2+ that depleted SR-Ca(2)+ content. After caffeine washout, a decrease in basal Ca2+ (undershoot) was invariably observed, followed by a slow recovery. This phenomenon was inhibited by cyclopiazonic acid (5 microM). External Ca(2)+ removal in depolarized and nondepolarized cells induced a decrease in basal Ca2+ that continued until depletion of the SR-Ca2+ content. The decrease in Ca2+ induced by Ca2+-free physiological saline solution (PSS) was accelerated in caffeine-stimulated cells. Recovery from undershoot was not observed in Ca2+-free PSS. Depolarization with KCl and addition of D600 (30 microM) did not modify recovery. Similar results were obtained when the Na(+)/Ca2+ exchanger was blocked by substituting NaCl with KCl in normal PSS (Na(+)-free PSS) or by adding benzamil amiloride (25 microM).

CONCLUSIONS

SR-Ca2+ content plays an important role in the Ca2+ leak induced by Ca2+-free medium, and does not depend on membrane potential. Additionally, recovery from undershoot after caffeine depends on extracellular Ca2+, and neither voltage-dependent Ca2+ channels nor the Na(+)/Ca2+ exchanger are involved.