L-Type Ca2+ channel sparklets revealed by TIRF microscopy in mouse urinary bladder smooth muscle

Abstract

Calcium is a ubiquitous second messenger in urinary bladder smooth muscle (UBSM). In this study, small discrete elevations of intracellular Ca 2+, referred to as Ca2+ sparklets have been detected in an intact detrusor smooth muscle electrical syncytium using a TIRF microscopy Ca2+ imaging approach. Sparklets were virtually abolished by the removal of extracellular Ca2+ (0.035±0.01 vs. 0.23±0.07 Hz/mm2; P<0.05). Co-loading of smooth muscle strips with the slow Ca2+ chelator EGTA-AM (10 mM) confirmed that Ca2+ sparklets are restricted to the cell membrane. Ca2+ sparklets were inhibited by the calcium channel inhibitors R-(+)-Bay K 8644 (1 μM) (0.034±0.02 vs. 0.21±0.08 Hz/mm2; P<0.05), and diltiazem (10 μM) (0.097±0.04 vs. 0.16±0.06 Hz/mm2; P<0.05). Ca2+ sparklets were unaffected by inhibition of P2X 1 receptors α,β-meATP (10 μM) whilst sparklet frequencies were significantly reduced by atropine (1 μM). Ca2+ sparklet frequency was significantly reduced by PKC inhibition with Gö6976 (100 nM) (0.030±0.01 vs. 0.30±0.1 Hz/mm2; P<0.05), demonstrating that Ca2+ sparklets are PKC dependant. In the presence of CPA (10 μM), there was no apparent change in the overall frequency of Ca2+ sparklets, although the sparklet frequencies of each UBSM became statistically independent of each other (Spearman's rank correlation 0.2, P>0.05), implying that Ca2+ store mediated signals regulate Ca2+ sparklets. Under control conditions, inhibition of store operated Ca2+ entry using ML-9 (100 μM) had no significant effect. Amplitudes of Ca2+ sparklets were unaffected by any agonists or antagonists, suggesting that these signals are quantal events arising from activation of a single channel, or complex of channels. The effects of CPA and ML-9 suggest that Ca2+ sparklets regulate events in the cell membrane, and contribute to cytosolic and sarcoplasmic Ca2+ concentrations. © 2014 Sidaway, Teramoto.