Effect of Halothane on cADP-Ribose-induced Ca²⁺Release System in Tracheal Smooth Muscle

Porcine tracheal smooth muscle was quickly dissected, chilled, and minced in ice-cold solution containing 0.25 m sucrose, 20 mm Tris-HCl (pH 7.2), and 20 μg/ml leupeptin. Microsomes were prepared by differential centrifugation as described before. 8Ca²⁺uptake and release were measured in a medium containing 250 mm N -methyl glucamine, 250 mm potassium gluconate, 20 mm HEPES buffer (pH 7.2), 1 mm MgCl², 2 U/ml creatine kinase, 4 mm phosphocreatine, 1 mm adenosine triphosphate (ATP), 4 mm Pi, 25 μg/ml leupeptin, 20 μg/ml aprotinin, and 100 μg/ml soybean trypsin inhibitor and 3 μm fluo-3 was added. Fluo-3 fluorescence was monitored at 490 nm excitation and 535 nm emission in a 250-μl cuvette at 37°C with a circulation water bath and continuously mixed with a magnetic stirring bar, using a Hitachi spectrofluorometer (F-2000) (San Jose, CA). The addition of stock solutions of various substances did not exceed 2% of the homogenate volume in the cuvette. Changes in fluorescence were calibrated to known Ca²⁺additions using separate samples of the same microsomal preparation.

Fluo-3 was purchased from Molecular Probes (Eugene, OR); IP³, oligomycin, and antimycin were from Calbiochem (San Diego, CA). All other reagents, of the highest purity grade available, were supplied from Sigma Chemical (St. Louis, MO).

The reported experiments were repeated at least three to six times. When appropriate, data are expressed as mean ± SD. The unpaired t  test was used to evaluate statistical significance;P  values < 0.05 were considered significant.

It has been previously shown that the RyR-cADPR system is present and functional in smooth muscle cells. 10–12Furthermore, cADPR is able to activate the RyR in tracheal smooth muscle cells. 10Tracheal smooth muscle cell microsomes supplemented with an ATP-regenerative system sequester added Ca²⁺into vesicular stores in an ATP-dependent manner and release Ca²⁺in response to μm concentrations of cADPR (fig. 1). The cADPR-induced Ca²⁺release was inhibited by several inhibitors of the RyR such as spermine, ruthenium red, and the specific cADPR inhibitor 8-Br-cADPR (fig. 1). 13However, Ca²⁺release induced by cADPR was not inhibited by 1 mg/ml heparin, a specific antagonist of the IP³channel. 13These observations confirmed the evidence that cADPR activates Ca²⁺release through the RyR in tracheal smooth muscle.

We investigated the effect of 350 μm halothane on the cADPR induced Ca ²⁺release. Figure 2demonstrates the effect of halothane on cADPR-induced Ca²⁺release, addition of 350 μm halothane did not produce any significant Ca²⁺release by itself; however, it sensitized the Ca²⁺release system to cADPR (fig. 2). The half-maximal concentration of cADPR was decreased more than fourfold by pretreatment of the microsomes with 350 μm halothane (fig. 2B), although the maximum Ca²⁺release response to cADPR was not enhanced by halothane. Thus halothane increased the apparent affinity of the Ca²⁺-induced Ca²⁺release to stimulation by cADPR. We also observed that 350 μm halothane had no effect steady-state Ca²⁺levels in the microsomal preparations. The effect of halothane on the cADPR-induced Ca²⁺release was abolished by the cADPR antagonist 8-Br-cADPR (fig. 3). Furthermore, the endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin was not able to potentiate Ca²⁺induced by agonists of the RyR (data not shown). These observations further support the hypothesis that halothane at the concentration tested sensitizes the RyR.

In conclusion, we present evidence that halothane can interact with the new second messenger system modulated by cADPR in tracheal smooth muscle cells. It is possible that the effect of halothane on cADPR may play an important role in the complex effect of volatile anesthetics on intracellular Ca²⁺homeostasis in these cells. Halothane can promote depletion of the intracellular Ca²⁺stores by a mechanism that appears to involve leakage of Ca²⁺through both the IP³and RyR. 6Our current results indicate that halothane-induced Ca²⁺leakage may involve sensitization of the RyR to endogenous levels of intracellular cADPR. Increased sensitivity of the RyR to endogenous cADPR induced by halothane may lead to depletion of sarcoplasmic reticulum intravesicular Ca²⁺levels. This decrease in SR Ca²⁺will decrease the amount of Ca²⁺available for SR Ca²⁺release during agonist stimulation leading to decreased contraction.