Voltage-sensitive Ca2+ channels, intracellular Ca2+ stores and Ca2+-release-activated Ca2+ channels contribute to the ATP-induced [Ca2+]i increase in differentiated neuroblastoma x glioma NG 108-15 cells

Abstract

Activation of ionotropic P2X7 purinoreceptors in NG108-15 cells directly opens non-selective cation channels, leading to an increase in intracellular Ca2+ concentration ([Ca2+]i) and membrane depolarization and, hence, by indirect opening of voltage-stimulated Ca2+ channels (VSCC) to further increases of [Ca2+]i, whereas activation of the metabotropic P2Y receptor causes intracellular Ca2+ release. The quantitative contribution of Ca2+ entry and release to ATP-induced [Ca2+]i increase in differentiated NG108-15 cells is not known. Here we have investigated the Ca2+ influx and Ca2+ release components by studying [Ca2+]i in Fura-2-loaded cells and by using the following tools: nifedipine to block L-type VSCC, omega-conotoxin GVIa (omegaCT) to block N-type VSCC and thapsigargin to deplete intracellular Ca2+ stores. With 1.8 mM Ca2+ in the medium, ATP (600 microM) increased [Ca2+]i by 656 +/- 50 nM (n = 11). This response was reduced to 72% by nifedipine (50 microM), to 63% by omegaCT (1 microM), and to 31% by nifedipine and omegaCT in combination. Since nifedipine and omegaCT completely block VSCC in our model, the remaining 31% of [Ca2+]i increase could be caused by influx via P2X7-activated non-selective channels or by intracellular release mediated by P2Y receptors. When Ca2+-free medium was used to exclude Ca2+ influx, ATP (600 microM) increased [Ca2+]i by only 34 +/- 4 nM (n = 4), indicating that the majority of [Ca2+]i increase depends on Ca2+ influx. A similar rise by 37 +/- 4 nM (n = 4) was observed with the selective P2Y agonist UTP (150 microM). This small response was sensitive to thapsigargin and hence represents Ca2+ release. The remainder (i.e. total [Ca2+]i increase minus nifedipine-, omegaCT- and thapsigargin-sensitive [Ca2+]i increases) should, therefore, represent Ca2+ influx via P2X7 non-selective cation channels.