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Am. J. Respir. Cell Mol. Biol., Volume 26, Number 4, April, 2002 491-498

Regulation of Capacitative and Noncapacitative Receptor-Operated Ca2+ Entry by Rho-Kinase in Tracheal Smooth Muscle

Satoru Ito, Hiroaki Kume, Kenichi Yamaki, Hideki Katoh, Haruo Honjo, Itsuo Kodama, and Hideharu Hayashi

Second Department of Internal Medicine, School of Medicine, Nagoya University, Nagoya, Japan; and Departments of Humoral Regulation and Circulation, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan

To determine the mechanisms of Ca2+ mobilization induced by receptor agonists, we examined the role of Rho-kinase on the sarcoplasmic reticulum (SR) Ca2+ stores-dependent and -independent Ca2+ influx in guinea pig tracheal smooth muscle (TSM). Isometric tension and intracellular Ca2 + concentration ([Ca2+]i) were simultaneously measured using fura-2-loaded tissues. Depletion of the SR Ca2+ stores by thapsigargin caused an increase in [Ca2+]i and contraction, demonstrating capacitative Ca2+ entry (CCE). Because CCE was not inhibited by nifedipine, voltage-operated Ca2+ channels are not involved in CCE. Under the condition that CCE is fully activated, methacholine (MCh) and histamine caused further increases in [Ca2+]i and tension, demonstrating noncapacitative receptor-operated Ca2+ entry (non-CCE). The Ca2+ influx and contraction via non-CCE was inhibited by Y-27632, a Rho-kinase inhibitor, in a concentration-dependent fashion. In contrast, Y-27632 did not affect thapsigargin-induced CCE. Cytochalasin D, which disrupts actin cytoskeleton, inhibited contraction induced by CCE or MCh with no change in [Ca2+]i. Our results indicate that not only CCE but also non-CCE exist in TSM and that the latter is regulated by Rho-kinase, independent of actin cytoskeleton. In conclusion, Ca2+ influx regulated by the RhoA/Rho-kinase pathway may play a functional role in contraction by agonists.

Abbreviations: airway smooth muscle, ASM; intracellular Ca2+ concentration, [Ca2+]i; extracellular Ca2+ concentration, [Ca2+]o; capacitative Ca2+ entry, CCE; GTP-binding protein, G protein; inositol, 1,4,5-trisphosphate, IP3; methacholine, MCh; noncapacitative receptor-operated Ca2+ entry, non-CCE; protein kinase C, PKC; sarcoplasmic reticulum, SR; tracheal smooth muscle, TSM; transient receptor potential, TRP; voltage-operated Ca2+ channels, VOCC.




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