L-type Ca2+ channel sparklets revealed by TIRF microscopy in mouse urinary bladder smooth muscle.
L-type Ca2+ channel sparklets revealed by TIRF microscopy in mouse urinary bladder smooth muscle.
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DOI：
10.1371/journal.pone.0093803
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Teramoto N
中科院分区：
文献类型：
--
作者：
Sidaway P;Teramoto N
钙离子是膀胱平滑肌中普遍存在的第二信使。在这项研究中，小的离散升高的细胞内Ca 2+，被称为Ca 2+火花已检测到在一个完整的逼尿肌平滑肌电合胞体使用TIRF显微镜Ca 2+成像方法。去除细胞外Ca ~（2+）后，火花消失（0.035± 0.01vs.0.23 ± 0.07Hz/mm ~ 2; P<0.05）。用缓慢的Ca 2+螯合剂EGTA-AM（10 mM）共加载平滑肌条证实了Ca 2+火花被限制在细胞膜上。钙通道抑制剂R-（+）-Bay K 8644（1 μM）（0.034±0.02 vs. 0.21±0.08 Hz/mm 2; P<0.05）和地尔硫卓（10 μM）（0.097±0.04 vs. 0.16±0.06 Hz/mm 2; P<0.05）可抑制钙火花。Ca 2+火花不受P2 X1受体α，β-meATP（10 μM）抑制的影响，而阿托品（1 μM）显著降低了火花频率。用Gö 6976（100 nM）抑制PKC显著降低了Ca 2+火花频率（0.030±0.01 vs. 0.30±0.1 Hz/mm 2; P<0.05），表明Ca 2+火花是PKC依赖性的。在CPA（10 μM）的存在下，尽管各UBSM的火花频率在统计学上变得彼此独立（斯皮尔曼等级相关0.2，P>0.05），但Ca 2+火花的总体频率没有明显变化，这意味着Ca 2+库介导的信号调节Ca 2+火花。在对照条件下，使用ML-9（100 μM）抑制钙池操作的Ca 2+内流无显著影响。Ca 2 + sparklets的振幅不受任何激动剂或拮抗剂的影响，表明这些信号是由单个通道或通道复合物的激活引起的量子事件。CPA和ML-9的作用表明，Ca 2+火花调节细胞膜中的事件，并有助于胞浆和肌浆Ca 2+浓度。
Calcium is a ubiquitous second messenger in urinary bladder smooth muscle (UBSM). In this study, small discrete elevations of intracellular Ca2+, 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 P2X1 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.
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