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1 Molecular and Cellular Pharmacology, Graduate Shool of Pharmaceutical Sciences, Nagoya City University, Nagoya, Aichi, Japan
2 Molecular and Cellular Pharmacology, Graduate Shool of Pharmaceutical Sciences, Nagoya City University, Nagoya, Aichi, Japan; Pharmacology, Faculty of Medicine, Toyama Medical and Pharmaceutical University, Toyama, Toyama, Japan
3 Molecular and Cellular Pharmacology, Graduate Shool of Pharmaceutical Sciences, Nagoya City University, Nagoya, Aichi, Japan; Cell Signaling & Ion Channel Research Group, Cellular Pharmacology, School of Pharmacy, Aichigakuin University, Nagoya, Aichi, Japan
* To whom correspondence should be addressed. E-mail: yimaizum{at}phar.nagoya-cu.ac.jp.
The relative contributions of Ca2+-induced Ca2+-release (CICR) versus Ca2+ influx through voltage-dependent Ca2+ channels (VDCC) to excitation-contraction (E-C) coupling has not been defined in most smooth muscle cells (SMCs). The present study was undertaken to address this issue in mouse urinary bladder smooth muscle cell (UBSMC). Confocal Ca2+ images were obtained under voltage- or current-clamp conditions. When UBSMCs were activated by a 30 ms depolarization to 0 mV, intracellular Ca2+ concentration ([Ca2+]i) increased in several discrete small areas just beneath the cell membrane. These Ca2+ hot spots then spread slowly in myoplasm as Ca2+ waves, which continued even after repolarization. Shorter depolarizations (5 ms) elicited only a few Ca2+ sparks, which declined quickly. The number of Ca2+ sparks/hot spots was closely related to the depolarization duration in the range 5-20 ms. There was an apparent threshold for depolarization duration approximately 10 ms to induce enough Ca2+ transients to spread globally and then induce a contraction. Application of 100 µM ryanodine to the pipette solution did not change the resting [Ca2+]i nor the VDCC current, but abolished Ca2+ hot spots elicited by depolarizations. Application of 3 µM xestospongin C reduced acetylcholine-induced Ca2+ release but did not affect depolarization-induced Ca2+ events. Addition of 100 µM ryanodine to tissue segments markedly reduced the amplitude of contractions triggered by direct electrical stimulation. In conclusion, global [Ca2+]i rise triggered by a single action potential is not mainly due to Ca2+ influx through VDCCs but it is attributable to the subsequent two step CICR.
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