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Single-channel and Fura-2 analysis of internal Ca2+ oscillations in HeLa cells: contribution of the receptor-evoked Ca2+ influx and effect of internal pH.

Publication Type:

Journal Article

Source:

Pflugers Arch, Volume 416, Issue 1-2, p.43-52 (1990)

Abstract:

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.

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