Effects of Anions on ATP-Induced [Ca2+]i Increase in NG108-15 Cells

Abstract

ABSTRACT-We investigated the effects of anions on different P2 receptors by measuring ATP-induced increase in intracellular Ca2+ concentration ([Ca 2+ ]i) in fura-2-loaded NG108-15 and PC12 cells. In NG108-15 cells, ATP at 100 mM and 1 mM induced a transient and a sustained [Ca 2+ ]i increase, respectively. The former, but not the latter, was inhibited by U-73122, indicating that the former was via the P2Y2 receptor and the latter via the P2X7 receptor. When external Cl -was replaced by other anions, the [Ca 2+ ]i increase mediated by the P2Y2 receptor was not changed, but that mediated by the P2X7 receptor varied in the order of aspartate]i increases mediated by the P2Y2 and P2X2 receptors were not affected by various anions. These results suggest that modulation by anions is unique to the P2X7 receptor and does not occur in P2Y2 and P2X2 receptors. This may be because the mechanism of ATP binding to the P2X7 receptor may be different than that to other P2 receptors.Keywords: ATP, P2X7 receptor, Anion, NG108-15 cell, PC12 cell NG108-15 cells are hybrids of mouse neuroblastoma N18TG-2 and rat glioma C6Bu-1 cells. NG108-15 cells possess two types of functional P2 receptors, P2X7 and P2Y 2 (1 -7). P2X 7 receptors are nonselective cation channels, which cause influx of extracellular Ca 2+ (2 -4, 8, 9). P2Y2 receptors are G-protein coupled receptors that are involved in the production of inositol-1,4,5-trisphosphate (IP3) via activation of phospolipase C (PLC) and induce Ca 2+ release from IP3-sensitive intracellular Ca 2+ stores (1). Using the whole-cell voltage clamp, Kaiho et al. (3) found that the current through the P2X7 receptor was enhanced in NG108-15 cells when extracellular ] i ) via P2X 7 and P2Y 2 receptors (1 -7) in NG108-15 cells. Ca 2+ signals by these two receptors can be distinguished from each other because the P2Y2 receptor is activated by a low concentration of ATP (100 m M), while the P2X7 receptor requires a higher concentration of ATP (>0.3 mM) for activation. In the present study, using fura-2-loaded cells, we investigated whether anions modulate Ca 2+ permeability through the P2X 7 receptors by monitoring ATP-induced [Ca 2+ ]i elevation. We also investigated whether the effects of anions were unique to the P2X7 receptor by comparing them with the effects on P2Y 2 and P2X2 receptors in NG108-15 and PC12 cells. MATERIALS AND METHODS Cell cultureNG108-15 cells were grown in high-glucose Dulbecco's modified Eagle's medium supplemented with 7% (v /v) fetal bovine serum, 100 mM hypoxanthine, 0.4 m M aminopterin and 16 mM thymidine (7). Cells were maintained in 100-mm tissue culture dishes and incubated at 37°C in a humidified atmosphere of 10% CO2 and 90% air.PC12 cells were grown in Dulbecco's modified Eagle's medium supplemented with 5% (v /v) fetal bovine serum, 5% (v /v) heat-inactivated horse serum, and 2 mM Lglutamine (10). Cells were maintained in 100-mm tissue culture dishes and incubated at 37°C in a humidified atmosphere of 5% CO2 and 95% air. Solutions and drugsThe standard Cl ...

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