Sarcoplasmic Reticulum Ca 2+ Depletion by Caffeine and Changes of [Ca 2+] i During Refilling in Bovine Airway Smooth Muscle Cells

Abstract

Background In airway smooth muscle (ASM), Ca 2+ influx in response to the Ca 2+ depletion of the sarcoplasmic reticulum (SR) seems to play a role in the regulation of intracellular free Ca 2+ concentrations ([Ca 2+] i). This study evaluates some possible Ca 2+ entry pathways activated during SR-Ca 2+ depletion induced by 10 mM caffeine. Methods Enzymatically dispersed bovine ASM cells were loaded with Fura-2/AM to permit measurement of [Ca 2+] i changes in single cells. Results Caffeine (10 mM) induced a transient increase in [Ca 2+] i that depleted SR-Ca 2+ content. After caffeine washout, a decrease in basal [Ca 2+] i (undershoot) was invariably observed, followed by a slow recovery. This phenomenon was inhibited by cyclopiazonic acid (5 μM). External Ca 2+ removal in depolarized and nondepolarized cells induced a decrease in basal [Ca 2+] i that continued until depletion of the SR-Ca 2+ content. The decrease in [Ca 2+] i induced by Ca 2+-free physiological saline solution (PSS) was accelerated in caffeine-stimulated cells. Recovery from undershoot was not observed in Ca 2+-free PSS. Depolarization with KCl and addition of D600 (30 μM) did not modify recovery. Similar results were obtained when the Na +/Ca 2+ exchanger was blocked by substituting NaCl with KCl in normal PSS (Na +-free PSS) or by adding benzamil amiloride (25 μM). Conclusions SR-Ca 2+ content plays an important role in the Ca 2+ leak induced by Ca 2+-free medium, and does not depend on membrane potential. Additionally, recovery from undershoot after caffeine depends on extracellular Ca 2+, and neither voltage-dependent Ca 2+ channels nor the Na +/Ca 2+ exchanger are involved.