Capacitative Ca2+ Entry During Ca2+ Undershoot in Bovine Airway Smooth Muscle

Abstract

In numerous cells, Ca²⁺ undershoot is commonly observed after withdrawing stimulus that release Ca²⁺ from intracellular stores. In airway smooth muscle (ASM), the fast intracellular Ca²⁺ concentration ([Ca²⁺]_i) drop during undershoot is produced by sarcoplasmic reticulum (SR) reloading, but the mechanisms involved in the long lasting basal [Ca²⁺]_i recovery are unknown. We investigated the post-caffeine Ca²⁺ undershoot recovery in ASM isolated cells from bovine trachea. [Ca²⁺]_i determination was done by a ratiometric method by incubating cells with Fura-2/AM. After inducing a transient response, caffeine withdrawn generated a Ca²⁺ undershoot. SR-Ca²⁺ content during maximum undershoot drop was ∼40% of SR caffeine-releasable Ca²⁺ (SR-Ca²⁺ load). Undershoot recovery rate increased in presence of cyclopiazonic acid (CPA, a SR-Ca²⁺ ATPase inhibitor), but SR-Ca²⁺ load was reduced. Genistein (a tyrosine kinase inhibitor) slowed down the Ca²⁺ undershoot drop and the SR-Ca²⁺ load but did not affect the undershoot recovery rate. Ni²⁺ (a capacitative Ca²⁺ inhibitor), but neither SKF-96365 (a passive Ca²⁺ entry inhibitor) nor econazole (a capacitative Ca²⁺ inhibitor in non-excitable cells), inhibited Ca²⁺ undershoot recovery and SR-Ca²⁺ load. Our data suggest that capacitative Ca²⁺ entry is involved in bovine ASM Ca²⁺ undershoot recovery, and that changes in Ca²⁺ undershoot have an impact on SR-Ca²⁺ loading which might affect in turn ASM excitability.