Sauvé R, Diarra A, Chahine M, Simoneau C, Garneau L, Roy G
Départment de Physiologie, Université de Montréal, Canada.
Pflugers Arch. 1990 Apr;416(1-2):43-52. doi: 10.1007/BF00370220.
Patch-clamp and Fura-2 experiments were performed in order to investigate the calcium oscillations due to H1 receptor stimulation in HeLa cells. The cytosolic calcium fluctuations occurring directly at the plasma membrane inner face were detected by measuring the activity of calcium-dependent potassium channels. This method also allowed measurement of changes in intracellular potential using as indicator the amplitude of the channel current jump. The average internal calcium concentration was obtained from Fura-2 experiments carried out at either the single-cell level or from a small population of cells in monolayer. The results indicate that the internal calcium oscillations in HeLa cells arise from a biphasic process with an initial phase independent of the presence of external calcium. External calcium was found, however, to become essential once the regular oscillatory process has been established. Removing external calcium after this initial phase produced a rapid decay in the burst frequency and eventually a complete abolition of the oscillations. In addition, the calcium oscillations occurring during the external-calcium-dependent phase could be blocked by calcium entry blockers such as Co2+ or La3+, or abolished by perfusing the external medium with a high-K+ solution. Experiments were also performed in which the cell internal pH (pHi) was changed by removing the external bicarbonate or by adding NH4Cl to the bathing solution. The results obtained under these conditions indicate that an increase in internal pH abolishes selectively the appearance of calcium spikes without increasing the basal calcium level, while a cellular acidification maintains or stimulates the calcium oscillatory process. It was also observed that the inhibitory effect of alkaline pH was independent of external calcium, and that calcium oscillations could always be seen at alkaline pH during the initial phase of histamine stimulation. On the basis of these results, it is proposed that the internal calcium oscillations in HeLa cells depend on the release of calcium from internal pools, which are reloaded via a pH-dependent mechanism. Part of the calcium sequestration occurring during the oscillatory process would be carried out, however, by pH-insensitive calcium compartments.
为了研究海拉细胞中H1受体刺激引起的钙振荡，进行了膜片钳和Fura-2实验。通过测量钙依赖性钾通道的活性来检测直接发生在质膜内表面的胞质钙波动。该方法还允许使用通道电流跳跃幅度作为指标来测量细胞内电位的变化。平均细胞内钙浓度是通过在单细胞水平或单层中的一小群细胞上进行的Fura-2实验获得的。结果表明，海拉细胞中的细胞内钙振荡源于一个双相过程，其初始阶段与外部钙的存在无关。然而，一旦建立了规则的振荡过程，外部钙就变得至关重要。在这个初始阶段之后去除外部钙会导致爆发频率迅速下降，最终振荡完全消失。此外，在外部钙依赖性阶段发生的钙振荡可以被钙进入阻滞剂如Co2+或La3+阻断，或者通过用高钾溶液灌注外部介质而消除。还进行了实验，其中通过去除外部碳酸氢盐或向浴液中添加氯化铵来改变细胞内pH（pHi）。在这些条件下获得的结果表明，细胞内pH升高会选择性地消除钙尖峰的出现，而不会增加基础钙水平，而细胞酸化则维持或刺激钙振荡过程。还观察到碱性pH的抑制作用与外部钙无关，并且在组胺刺激的初始阶段，在碱性pH下总能看到钙振荡。基于这些结果，有人提出海拉细胞中的细胞内钙振荡取决于从内部钙库释放钙，这些钙库通过pH依赖性机制重新加载。然而，在振荡过程中发生的部分钙螯合将由对pH不敏感的钙区室进行。
