Volatile anesthetic-induced efflux of calcium from IP₃-gated stores in clonal (GH₃) pituitary cells

Background: Many hormones and neurotransmitters produce their effects by stimulating the generation of inositol 1,4,5-trisphosphate (IP₃), a chemical second messenger that releases Ca²⁺ from intracellular stores. Interruption of this pathway is a potential mechanism through which volatile anesthetics might inhibit chemically mediated communication between cells. This study used GH₃ cells (a clonal cell line) as a model system in which to characterize the effects of volatile anesthetics on IP₃-induced mobilization of Ca²⁺ from intracellular stores. Methods: Intracellular Ca²⁺ concentration ([Ca²⁺](i)) was continuously monitored in suspensions of GH₃ cells at 37°C using the fluorescent Ca²⁺ indicator Fura-2. Thyrotropin releasing hormone (TRH) was used to discharge IP₃-sensitive intracellular Ca²⁺ stores. The effects of halothane, isoflurane, and octanol on TRH- induced Ca²⁺ mobilization were assessed as a function of time and anesthetic concentration. To distinguish between anesthetic effects on Ca²⁺ uptake and Ca²⁺ release, experiments were performed using thapsigargin (a Ca²⁺-ATPase inhibitor) to inhibit Ca²⁺ uptake into IP₃-sensitive stores. Results: Halothane increased resting [Ca²⁺](i) and caused a time- and concentration-dependent inhibition of TRH-induced increases in [Ca²⁺](i) (IC₅₀ = 0.6 mM). Thapsigargin, in concentrations that completely inhibit Ca²⁺ uptake by IP₃-sensitive stores, also caused a time-dependent reduction in the [Ca²⁺](i) response to TRH; the time constant of this decay describes the rate of spontaneous leak of Ca²⁺ from IP₃-sensitive stores (τ = 98 ± 9 s). In the presence of thapsigargin, halothane produced concentration-dependent increases in the rate of leak from IP₃-sensitive stores (τ = 74 ± 12 and 46 ± 6 s at 0.5 and 1.0 mM halothane, respectively). Isoflurane and octanol also produced concentration-dependent inhibition of the [Ca²⁺](i) response to TRH. Conclusions: Halothane causes a concentration-dependent leak of Ca²⁺ from IP₃-sensitive stores, leading to depletion of the stores and inhibition of IP₃-induced increases in [Ca²⁺](i). This effect occurs at clinically relevant concentrations of halothane (as well as isoflurane and octanol) and may be an important mechanism underlying some of the physiologic effects of volatile anesthetics.