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Involvement of Ca²⁺ Mobilization in Tachyphylaxis to ß-Adrenergic Receptors in Trachealis

Division of Respiratory Medicine, Department of Medicine, Nagoya University Graduate School of Medicine, Showa-ku, Nagoya, Japan; and Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York

Address correspondence to: Hiroaki Kume, M.D., Ph.D., Division of Respiratory Medicine, Department of Medicine, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466–8550, Japan. E-mail: hkume{at}med.nagoya-u.ac.jp

We examined the mechanisms underlying tachyphylaxis to ß-adrenergic receptor agonists (ß-agonists) in tracheal smooth muscle. Simultaneous measurements of isometric tension and intracellular Ca²⁺ concentration ([Ca²⁺]_i) using fura-2–loaded guinea pig tracheas showed that the inhibitory effects of isoproterenol (ISO) on tension and increases in [Ca²⁺]_i induced by methacholine exhibited marked tachyphylaxis with repeated exposure to ISO at intervals of 15 min. Similarly, the activation of single Ca²⁺-activated K⁺ (K_Ca) channels in on-cell patches by 1 µM ISO was gradually attenuated after repeated extracellular application of ISO to single smooth cells of porcine tracheas. Desensitization of ß-adrenergic receptor/K_Ca channel stimulatory coupling and relaxation responses was prevented by separately antagonizing the voltage-dependent Ca²⁺ channel (VDCC) with verapamil, suggesting a surprising relationship between Ca²⁺ influx through VDCC and ß-adrenergic desensitization. Conversely, repeated exposure of 10 U/ml protein kinase A to inside-out patches did not result in desensitization of channel activation, and repeated exposure to 10 µM forskolin modestly augmented the inhibitory effects of forskolin on tension and [Ca²⁺]_i by methacholine, indicating that the mechanism of desensitization is mediated by the ß-adrenergic receptor/G protein complex. These results indicate that an uncoupling of ß-adrenergic receptor from K_Ca channels augments Ca²⁺ mobilization through VDCC and stimulates tachyphylaxis.

Abbreviations: intracellular Ca²⁺ concentration, [Ca²⁺]_i • dimethyl sulfoxide, DMSO • ethyleneglycol-bis-(ß-aminoethyl ether)-N,N',-tetraacetic acid, EGTA • isoproterenol, ISO • Ca²⁺-activated K⁺, K_Ca • open probability, nP_o • voltage-dependent Ca²⁺ channel, VDCC