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+](i)). 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+](i) changes in single cells. Results. Caffeine (10 mM) induced a transient increase in [Ca2+](i) that depleted SR-Ca2+ content. After caffeine washout, a decrease in basal [Ca2+](i) (undershoot) was invariably observed, followed by a slow recovery. This phenomenon was inhibited by cyclopiazonic acid (5 muM). External Ca2+ removal in depolarized and nondepolarized cells induced a decrease in basal [Ca2+](i) that continued until depletion of the SR-Ca2+ content. The decrease in [Ca2+](i) 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 muM) 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 muM). 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. (C) 2001 IMSS. Published by Elsevier Science Inc.