L-type Ca2+ channel sparklets revealed by TIRF microscopy in mouse urinary bladder smooth muscle.
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 Ca(2+) sparklets have been detected in an intact detrusor smooth muscle electrical syncytium using a TIRF microscopy Ca(2+) imaging approach. Sparklets were virtually abolished by the removal of extracellular Ca(2+) (0.035 ± 0.01 vs. 0.23 ± 0.07 Hz/mm(2); P<0.05). Co-loading of smooth muscle strips with the slow Ca(2+) chelator EGTA-AM (10 mM) confirmed that Ca(2+) sparklets are restricted to the cell membrane. Ca(2+) sparklets were inhibited by the calcium channel inhibitors R-(+)-Bay K 8644 (1 μM) (0.034 ± 0.02 vs. 0.21 ± 0.08 Hz/mm(2); P<0.05), and diltiazem (10 μM) (0.097 ± 0.04 vs. 0.16 ± 0.06 Hz/mm(2); P<0.05). Ca(2+) sparklets were unaffected by inhibition of P2X1 receptors α,β-meATP (10 μM) whilst sparklet frequencies were significantly reduced by atropine (1 μM). Ca(2+) sparklet frequency was significantly reduced by PKC inhibition with Gö6976 (100 nM) (0.030 ± 0.01 vs. 0.30 ± 0.1 Hz/mm(2); P<0.05), demonstrating that Ca(2+) sparklets are PKC dependant. In the presence of CPA (10 μM), there was no apparent change in the overall frequency of Ca(2+) 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 Ca(2+) store mediated signals regulate Ca(2+) sparklets. Under control conditions, inhibition of store operated Ca(2+) entry using ML-9 (100 μM) had no significant effect. Amplitudes of Ca(2+) 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 Ca(2+) sparklets regulate events in the cell membrane, and contribute to cytosolic and sarcoplasmic Ca(2+) concentrations.
Department of Pharmacology, Faculty of Medicine, Saga University, Saga City, Japan.
Peter
P
Sidaway
Department of Pharmacology, Faculty of Medicine, Saga University, Saga City, Japan.
Noriyoshi
N
Teramoto
D000818:Animals; D020746:Calcium Channels, L-Type / Q000502:physiology*; D020013:Calcium Signaling / Q000502:physiology*; D008297:Male; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D009130:Muscle, Smooth / Q000502:physiology*; D011493:Protein Kinase C / Q000378:metabolism; D001743:Urinary Bladder / Q000502:physiology*
D020746:Calcium Channels, L-Type; D011493:Protein Kinase C