All-or-nothing Ca²⁺ mobilization from the intracellular stores of single histamine-stimulated HeLa cells

Bootman, Martin D.; Berridge, Michael J. and Taylor, Colin W. (1992). All-or-nothing Ca²⁺ mobilization from the intracellular stores of single histamine-stimulated HeLa cells. Journal of Physiology, 450 pp. 163–178.

URL: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC117611...

Abstract

1. Histamine-stimulated mobilization of intracellular Ca²⁺ stores was monitored in intact and permeabilized populations of HeLa cells using both the fluorescent Ca²⁺ indicator Fura-2 and ⁴⁵Ca²⁺ measurements. Digital video imaging of Fura-2-loaded cells was used to measure the intracellular calcium concentration ([Ca²⁺]_i) of single cells.

2. In populations of HeLa cells, histamine caused a concentration-dependent increase in cytoplasmic [Ca²⁺]. The initial transient increase was independent of extracellular Ca²⁺ (Ca²⁺_o) and was followed by a sustained increase that was abolished by removal of Ca²⁺_o.

3. In Ca²⁺-free medium ([Ca²⁺]_o < 1 μM), a maximal histamine concentration (25 μM) caused a transient increase in [Ca²⁺]_i, and a subsequent challenge with histamine failed to evoke a further response indicating that the inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca²⁺ stores had been completely emptied. Lower concentrations of histamine (0.5-10 μM) caused smaller, concentration-dependent increases in [Ca²⁺]_i that were also transient. After exposure to these low histamine concentrations, where [Ca²⁺]_i returned to baseline within 2 min, addition of a higher histamine concentration evoked a further increase in [Ca²⁺]_i. The second increase in [Ca²⁺]_i was inversely proportional to the increase caused by the first exposure to histamine, indicating that Ca²⁺ released in the initial response was not substantially resequestered into histamine-sensitive stores.

4. Single HeLa cells challenged with low concentrations of histamine in Ca²⁺-free medium responded with transient increases in [Ca²⁺]_i, but individual cells differed in their sensitivity with 51% of cells responding to 1 μM, and 98% responding to 25 μM-histamine.

5. When single cells in Ca²⁺-free medium were challenged with stepwise increases in histamine concentration, they responded to each step with a transient [Ca²⁺]_i increase after which [Ca²⁺]_i returned to baseline within 1 min. Prolonging the interval between histamine additions by up to 25 min did not affect the [Ca²⁺]_i increase evoked by a subsequent histamine addition.

6. Unidirectional ⁴⁵Ca²⁺ efflux from saponin-permeabilized HeLa cells showed that, under conditions that prevented Ca²⁺ resequestration, submaximal concentrations of InsP₃ rapidly emptied only a fraction of the InsP₃-sensitive Ca²⁺ stores. The failure of low InsP₃ concentrations to fully mobilize the InsP₃-sensitive Ca²⁺ stores was not a consequence of InsP₃ degradation.

7. We conclude that within single HeLa cells, intracellular Ca²⁺ stores are heterogeneous in their sensitivity to InsP₃, and the fraction of Ca²⁺ stores mobilized by InsP₃ increases as the InsP₃ concentration increases.

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