Gordienko D V, Bolton T B
Department of Pharmacology and Clinical Pharmacology, St George's Hospital Medical School, Cranmer Terrace, London SW17 0RE, UK.
J Physiol. 2002 Aug 1;542(Pt 3):743-62. doi: 10.1113/jphysiol.2001.015966.
In smooth muscle cells freshly isolated from rabbit portal vein, there was only one site discharging the majority of spontaneous Ca(2+)-release events; the activity of this single site was studied using laser scanning confocal imaging after loading the cells with the fluorescent Ca(2+) indicator fluo-4 acetoxymethyl ester. Localised spontaneous Ca(2+)-release events visualised by line-scan imaging revealed two predominant spatiotemporal patterns: (i) small-amplitude, fast events similar to Ca(2+) sparks in cardiomyocytes and (ii) larger and slower events. The sum of two Gaussian profiles was well fitted to the amplitude histogram (peak frequencies at 1.8 and 3.2 F/F(0)) and spatial spread (full width at half-maximal amplitude) histogram (peak frequencies at 2 and 3.8 microm) for the 230 localised Ca(2+)-release events analysed. The existence of two populations of Ca(2+)-release events was also supported by the histograms of the rise times and half-decay times, which revealed modes at 38 and 65 ms, respectively. Shifting the scan line along the z-axis during imaging from a single discharge site suggested that the appearance of two populations of Ca(2+)-release events is not due to out-of-focus imaging. Both small and large events persisted upon 3-5 min exposure to 1-5 microM nicardipine, but were abolished after 10-15 min exposure to 50-100 microM ryanodine, 0.1 microM thapsigargin or 10 microM cyclopiazonic acid. Only small-amplitude, fast events persisted in the presence of inhibitors of inositol 1,4,5-trisphosphate (IP(3))-induced Ca(2+) release, 10 microM xestospongin C or 30 microM 2-aminoethoxy-diphenylborate (2-APB), or in the presence of 2.5 microM U-73122 (a phospholipase C (PLC) inhibitor). Coupling between neighbouring Ca(2+)-release domains giving rise to spontaneous Ca(2+) waves was abolished in the presence of 2-APB. Examination of the saltatory propagation of the waves suggested that the critical factor that determines propagation between domains is a time-dependent change in the sensitivity of ryanodine receptors and/or IP(3) receptors to Ca(2+), which can give rise to 'loose coupling' between release sites. These results suggest that activation of IP(3) receptors (due to the tonic activity of PLC and ongoing production of IP(3)) recruits neighbouring domains of ryanodine receptors, leading to larger Ca(2+) releases and saltatory propagation of Ca(2+) waves in portal vein myocytes.
在从兔门静脉新鲜分离的平滑肌细胞中，只有一个位点释放大部分自发钙释放事件；在用荧光钙指示剂氟-4 乙酰氧基甲酯加载细胞后，使用激光扫描共聚焦成像研究了这个单一位点的活性。通过线扫描成像可视化的局部自发钙释放事件揭示了两种主要的时空模式：（i）类似于心肌细胞中钙火花的小幅度、快速事件，以及（ii）幅度更大、速度更慢的事件。对于分析的 230 个局部钙释放事件，两个高斯分布的总和很好地拟合了幅度直方图（峰值频率在 1.8 和 3.2 F/F(0)）和空间扩散（半最大幅度处的全宽）直方图（峰值频率在 2 和 3.8 微米）。钙释放事件的两个群体的存在也得到了上升时间和半衰减时间直方图的支持，这两个直方图分别显示出 38 和 65 毫秒的模式。在从单个放电位点成像期间沿 z 轴移动扫描线表明，钙释放事件的两个群体的出现不是由于离焦成像。在暴露于 1 - 5 microM 尼卡地平 3 - 5 分钟后，小事件和大事件都持续存在，但在暴露于 50 - 100 microM 兰尼碱、0.1 microM 毒胡萝卜素或 10 microM 环匹阿尼酸 10 - 15 分钟后被消除。只有小幅度、快速事件在存在肌醇 1,4,5 - 三磷酸（IP(3)）诱导的钙释放抑制剂（10 microM 西司他丁 C 或 30 microM 2 - 氨基乙氧基 - 二苯基硼酸（2 - APB））时持续存在，或者在存在 2.5 microM U - 73122（一种磷脂酶 C（PLC）抑制剂）时持续存在。在存在 2 - APB 时，相邻钙释放域之间产生自发[Ca(2+)]i 波的耦合被消除。对波的跳跃传播的检查表明，决定域之间传播的关键因素是兰尼碱受体和/或 IP(3)受体对钙的敏感性的时间依赖性变化，这可能导致释放位点之间的“松散耦合”。这些结果表明，IP(3)受体的激活（由于 PLC 的紧张活性和 IP(3)的持续产生）招募了相邻的兰尼碱受体域，导致更大的钙释放和门静脉肌细胞中[Ca(2+)]i 波的跳跃传播。
