Total internal reflection fluorescence imaging of Ca2+-induced Ca2+ release in mouse urinary bladder smooth muscle cells

Abstract

In smooth muscles (SMs), cytosolic Ca2+ ([Ca2+]cyt) dynamics during an action potential are triggered by Ca2+ influx through voltage-dependent Ca2+ channels (VDCCs) in the plasma membrane. The physiological significance of Ca2+ amplification by subsequent Ca2+ 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 Ca2+ dynamics in Ca2+ 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 [Ca2+]cyt was followed by larger [Ca2+]cyt increase with propagation occurred in a limited TIRF zone within ∼200nm from cell surface. The depolarization-evoked [Ca2+]cyt increase in a TIRF zone was abolished or greatly reduced by the pretreatment with Cd2+ or ryanodine, respectively. The initial local [Ca2+]cyt increases were mediated by Ca2+ influx through single or clustered VDCCs as Ca2+ sparklets, and the following step was elicited by Ca2+-induced Ca2+ release (CICR) through RyR from SR. The depolarization-induced outward currents, mainly due to large-conductance Ca2+-activated K+ channel activation, were also markedly reduced by Cd2+ 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 Ca2+ events upon depolarization in Ca2+ microdomain of UBSMCs; the initial Ca2+ influx as Ca2+ sparklets through discrete VDCC or their clusters and the following CICR via the activation of loosely coupled RyRs in SR located in the Ca2+ microdomains.