Direct visualization of sarcoplasmic reticulum regions discharging Ca2+ sparks in vascular myocytes
Direct visualization of sarcoplasmic reticulum regions discharging Ca2+ sparks in vascular myocytes
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DOI：
10.1054/ceca.2000.0180
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发表时间：
2001-01-01
期刊：
影响因子：
4
通讯作者：
Bolton, TB
中科院分区：
文献类型：
--
作者：
Gordienko, DV;Greenwood, IA;Bolton, TB
局部的钙释放事件，即钙火花，被认为是所有类型肌肉中钙信号系统的基本构件。在横纹肌中，它们沿着与肌节结构相对应的纤维以规则的间隔出现，而肌节结构在平滑肌肉中不存在。我们先前的研究表明，在内脏和血管心肌细胞中，钙火花更多地出现在某些部位(频繁放电部位[FDS])。在本文中，我们将FDS的位置与肌浆网在同一活着的心肌细胞中的分布联系起来。用Dioc(6)和/或BODIPY tr-X ryanodine染色后，用高分辨率共聚焦成像技术观察SR在新鲜分离的兔门静脉心肌细胞中的三维分布。两种荧光染色显示相似的染色模式，表明发育良好的浅表SR呈螺旋排列，约占细胞体积的6%。用Fluo-4荧光检测到的自发钙火花频率的计算表明，在大约70%的心肌细胞中，只有一个主要的FDs位于浅表SR网络的突出部分，通常位于核膜的1-2微米内，尽管少数火花出现在其他部位，一般散布在整个细胞的浅表位置。极化的线粒体很容易通过积累四甲基罗丹明乙酯(TMRE)来识别。这些与核外区域的SR网络密切相关。然而，TMRE染色未能在FDS相关的SR元件附近发现任何线粒体。当观察到时，在FDSS处开始传播[Ca~(2+)](I)波和相关的心肌细胞收缩。本研究首次揭示了活体平滑肌细胞中肌浆网的三维排列，并将肌细胞结构组织的特殊性与亚细胞水平上的钙信号特征联系起来。(C)2001哈考出版有限公司。
Localized Ca2+-release events, Ca2+ sparks, have been suggested to be the 'elementary building blocks' of the calcium signalling system in all types of muscles. In striated muscles these occur at regular intervals along the fibre corresponding to the sarcomeric structures which do not exist in smooth muscle. We showed previously that in visceral and vascular myocytes Ca2+ sparks occurred much more frequently at certain sites (frequent discharge sites [FDSs]). In this paper, we have related the position of FDSs to the distribution of the sarcoplasmic reticulum in the same living myocyte. The three-dimensional distribution of the SR in freshly isolated rabbit portal vein myocytes was visualized by means of high-resolution confocal imaging after staining with DiOC(6) and/or BODIPY TR-X ryanodine. Both fluorochromes revealed a similar staining pattern indicating a helical arrangement of well-developed superficial SR which occupied about 6% of the cell volume. Computing the frequency of spontaneous Ca2+ sparks detected by means of fluo-4 fluorescence revealed that in about 70% of myocytes there was only one major FDS located on a prominent portion of superficial SR network usually within 1-2 mum of the nuclear envelope, although a few sparks occurred at other sites scattered generally in superficial locations throughout the cell. Polarized mitochondria were readily identified by accumulation of tetramethylrhodamine ethyl ester (TMRE). These were closely associated with the SR network in extra-nuclear regions. TMRE staining, however, failed to reveal any mitochondria near the FDS-related SR element. When observed, propagating [Ca2+](i) waves and associated myocyte contractions were initiated at FDSs. This study provide first insight into the three-dimensional arrangement of the SR in living smooth muscle cells and relates the peculiarity of the structural organization of the myocyte to the features of Ca2+ signalling at subcellular level. (C) 2001 Harcourt Publishers Ltd.