Total internal reflection fluorescence imaging of Ca(2+)-induced Ca(2+) release in mouse urinary bladder smooth muscle cells

Abstract

In smooth muscles (SMs), cytosolic Ca(2+) ([Ca(2+)](cyt)) dynamics during an action potential are triggered by Ca(2+) influx through voltage-dependent Ca(2+) channels (VDCCs) in the plasma membrane. The physiological significance of Ca(2+) amplification by subsequent Ca(2+) release through ryanodine receptors (RyRs) from the sarcoplasmic reticulum (SR) is still a matter of topics in SMs. In the present study, depolarization-evoked local Ca(2+) dynamics in Ca(2+) microdomain were imaged using total internal reflection fluorescence (TIRF) microscopy in mouse urinary bladder SM cells (UBSMCs). Upon depolarization under whole-cell voltage-clamp, the rapid and local elevation of [Ca(2+)](cyt) was followed by larger [Ca(2+)](cyt) increase with propagation occurred in a limited TIRF zone within ~200nm from cell surface. The depolarization-evoked [Ca(2+)](cyt) increase in a TIRF zone was abolished or greatly reduced by the pretreatment with Cd(2+) or ryanodine, respectively. The initial local [Ca(2+)](cyt) increases were mediated by Ca(2+) influx through single or clustered VDCCs as Ca(2+) sparklets, and the following step was elicited by Ca(2+)-induced Ca(2+) release (CICR) through RyR from SR. The depolarization-induced outward currents, mainly due to large-conductance Ca(2+)-activated K(+) channel activation, were also markedly reduced by Cd(2+) and ryanodine. In addition, TIRF analyses showed that the fluorescent signals of individual or clustered VDCC distributed in relatively uniform fashion and that a subset of RyRs in the subplasmalemmal SR also located in TIRF zone. In conclusion, fast TIRF imaging successfully demonstrated two step Ca(2+) events upon depolarization in Ca(2+) microdomain of UBSMCs; the initial Ca(2+) influx as Ca(2+) sparklets through discrete VDCC or their clusters and the following CICR via the activation of loosely coupled RyRs in SR located in the Ca(2+) microdomains.