Effects of halothane on sarcoplasmic reticulum calcium release channels in porcine airway smooth muscle cells

Background: Volatile anesthetics relax airway smooth muscle (ASM) by altering intracellular Ca²⁺ concentration ([Ca²⁺]₁). The authors hypothesized that relaxation is produced by decreasing sarcoplasmic reticulum Ca²⁺ content via increased Ca²⁺ "leak" through both inositol trisphosphate (IP₃) and ryanodine receptor channels. Methods: Enzymatically dissociated porcine ASM cells were exposed to acetylcholine in the presence or absence of 2 minimum alveolar concentration (MAC) halothane, and IP₃ levels were measured using radioimmunoreceptor assay. Other cells were loaded with the Ca²⁺ indicator fluo-3 and imaged using real-time confocal microscopy. Results: Halothane increased IP₃ concentrations in the presence and absence of acetylcholine. Inhibition of phospholipase C blunted the IP₃ response to halothane. Exposure to 2 MAC halothane induced a transient [Ca²⁺]₁ response, suggesting depletion of sarcoplasmic reticulum Ca²⁺. Exposure to 20 μM Xestospongin D, a cell-permeant IP₃ receptor antagonist, resulted in a 45 ± 13% decrease in the [Ca²⁺)₁ response to halothane compared with halothane exposure alone. In permeabilized cells, Xestospongin D or 0.5 mg/ml heparin decreased the [Ca²⁺]₁ response to halothane by 65 ± 13% and 68 ± 22%, respectively, compared with halothane alone. In both intact and permeabilized cells, 20 μM ryanodine blunted the [Ca²⁺]₁ response to halothane by 32 ± 13% and 39 ± 21%, respectively, compared with halothane alone. Simultaneous exposure to Xestospongin D and ryanodine completely inhibited the [Ca²⁺]₁ response to halothane. Conclusions: The authors conclude that halothane reduces sarcoplasmic reticulum Ca²⁺ content in ASM cells via increased Ca²⁺ leak through both IP₃ receptor and ryanodine receptor channels. Effects on IP₃ receptor channels are both direct and indirect via elevation of IP₃ levels.