Ca2+ influx during agonist and Ins(2,4,5)P3-evoked Ca2+ oscillations in HeLa epithelial cells.

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1. Histamine-stimulated [Ca2+]i oscillations were studied in > 162 HeLa cells using the techniques of Ca2+ imaging, patch clamp and single-cell indo-1 fluorescence. 2. [Ca2+]i oscillations in HeLa cells were acutely dependent on extracellular Ca2+ and were also blocked by the extracellular addition of Cd2+ (100 microM). The Mn2+ quench technique, using fura-2 fluorescence, demonstrated that agonist-stimulated Ca2+ oscillations were associated with an increase in plasma membrane Mn2+ permeability. However, no cyclic fluctuations in Mn2+ influx were resolved over the period of [Ca2+]i spiking. 3. In whole-cell patch clamped cells an imposed potential of +80 mV was shown to block the thapsigargin-induced Ca2+ influx. Histamine and Ins(2,4,5)P3-induced [Ca2+]i oscillations were tested for the phase dependence on extracellular Ca2+ by rapidly switching the membrane potential to +80 mV, reversibly blocking Ca2+ influx. Ca2+ spikes were abolished by steps to +80 mV made at the point of spike initiation but not by steps made after development of the rapid rising phase of the spike. Steps of membrane potential to +80 mV, for increasing periods of time during the interspike period, increased the latency to the next [Ca2+]i spike by up to a maximum of approximately 150% of the control interspike interval. 4. It is concluded that the extracellular Ca2+ dependence of the histamine-induced [Ca2+]i oscillations is due to a crucial role of Ca2+ influx during spike initiation and an additional important role in setting the interspike interval. The results obtained can be interpreted in terms of a constant stimulated Ca2+ influx during [Ca2+]i spiking.

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