Ca2+ influx during agonist and Ins(2,4,5)P3-evoked Ca2+ oscillations in HeLa epithelial cells.
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.
Thorn
P
P
Babraham Institute, Cambridge, UK.
D002118:Calcium / Q000502:physiology*; D015220:Calcium Channels / Q000502:physiology; D002463:Cell Membrane Permeability / Q000502:physiology*; D004594:Electrophysiology; D004848:Epithelium / Q000378:metabolism / Q000502:physiology; D006367:HeLa Cells; D006632:Histamine / Q000378:metabolism*; D006801:Humans; D007295:Inositol Phosphates / Q000494:pharmacology*; D008274:Magnesium / Q000378:metabolism
D015220:Calcium Channels; D007295:Inositol Phosphates; D006632:Histamine; C062616:inositol 2,4,5-trisphosphate; D008274:Magnesium; D002118:Calcium