P2X7 receptors mediate ATP release and amplification of astrocytic intercellular Ca2+ signaling

Abstract

Modulation of synaptic transmission and brain microcirculation are new roles ascribed to astrocytes in CNS function. A mechanism by which astrocytes modify neuronal activity in the healthy brain depends on fluctuations of cytosolic Ca2+ levels, which regulate the release of "gliotransmitters" via an exocytic pathway. Under pathological conditions, however, the participation of other pathways, including connexin hemichannels and the pore-forming P2X7R, have been proposed but remain controversial. Through the use of genetically modified 1321N1 human astrocytoma cells and of spinal cord astrocytes derived from neonatal Cx43- and P2X7R-null mice, we provide strong evidence that P2X7Rs, but not Cx43 hemichannels, are sites of ATP release that promote the amplification of Ca2+ signal transmission within the astrocytic network after exposure to low divalent cation solution. Moreover, our results showing that gap junction channel blockers (heptanol, octanol, carbenoxolone, flufenamic acid, and mefloquine) are antagonists of the P2X7R indicate the inadequacy of using these compounds as evidence for the participation of connexin hemichannels as sites of gliotransmitter release.

Figure 1.

Functional characterization of P2X₇R expressed in 1321N1 human astrocytoma cells. A, Intracellular Ca²⁺ changes induced by bath application (arrow) of 200 μm BzATP in fura-2 AM loaded parental (white triangles) and P2X₇R-expressing 1321N1 (black squares) cells. B, Increase in intracellular Ca²⁺ levels of P2X₇R-expressing 1321N1 cells induced by bath application (arrow) of 100 μm BzATP was prevented by 100 nm BBG (specific r-P2X₇R antagonist). C, YoPro-1 fluorescence intensity measured from parental and from P2X₇R-expressing 1321N1 cells bathed in control (DPBS) and in low divalent cation (LDPBS) solutions in the absence and presence of 200 μm BzATP. Note the characteristic potentiation of BzATP-induced YoPro-1 uptake in cells bathed in low divalent cation solutions. D, BzATP-induced changes in YoPro-1 fluorescence intensity measured from P2X₇R-expressing 1321N1 cells bathed in low divalent cation solutions in the absence and in the presence of 100 nm BBG. The first bar shows background YoPro fluorescence measured from cells bathed in low divalent cation solution before the addition of BzATP. Data are expressed as mean ± SEM (n = 80–120 cells from at least 2 independent experiments; **p < 0.01, ANOVA followed by Newman–Keuls test). Error bars represent SEM.

Figure 2.

Gap junction channel blockers are antagonists of P2X₇R. A, Dose–response for BzATP (squares) obtained from fura-2 AM loaded P2X₇R-expressing 1321N1 cells. Evidence for the nonsurmountable blockade of BzATP-induced Ca²⁺ influx by MFQ (diamonds), FFA (circles), and CBX (triangles) were obtained by using a fixed concentration of BzATP (100 μm) and varying the concentration of gap junction channel blockers. B, Intracellular Ca²⁺ elevations induced by 100 μm BzATP in the absence and in the presence of 1.5 mm OCT or HEP. C and D show examples of the blockade provided by two doses of CBX and MFQ on BzATP-induced Ca²⁺ mobilization, respectively; note that after CBX and MFQ washout (10 min), no response to BzATP (100 μm) was recorded. Arrows indicate BzATP addition. E, Bar histograms showing the mean relative YoPro fluorescence intensity (F/F₀) changes induced by 100 μm BzATP in the absence and presence of 100 μm GCZ, 50 μm CBX, 1 nm MFQ, 100 μm FFA (concentrations that produced at least 80% blockade of BzATP-induced Ca²⁺ influx) (Fig. 1), 1.5 mm OCT, 1.5 mm HEP, and 3 mm HEXA. F, Bar histograms showing the mean values of ATP (nm/10 μg of total protein) present in the bathing solution of parental and P2X₇R-expressing 1321N1 cells exposed for 2 min to control and low divalent cation solutions in the absence and presence of BBG (100 nm) and MFQ (2 nm). Data are expressed as mean ± SEM (n = 100–210 cells from at least 2 independent experiments; *** p < 0.001, ANOVA followed by Newman–Keuls test). Error bars represent SEM.

Figure 3.

ATP mediates ICW amplification induced by low divalent cation solution. Pseudocolored time-lapse images of mechanically induced ICW spread in confluent primary cultures of spinal cord astrocytes exposed to control (DPBS) (A) and low divalent cation solutions (LDPBS) (B). C, Bar histogram showing the mean efficacy values of the Ca²⁺ signal transmission in cells bathed in control and low divalent cation solutions in the absence and presence of MCPG and apyrase. Note that only apyrase (50 U/ml) but not MCPG (1 mm) completely prevented the increase in the efficacy of ICW induced by low divalent cation solution. Error bars correspond to mean ± SEM. n = 25–97 fields; 3–18 experiments; *** p < 0.001 (ANOVA followed by Tukey’s test).

Figure 4.

P2X₇ receptors contribute to ICW amplification induced by low divalent cation solution. Relative number of cells per tier (efficacy per tier) participating in the transmission of Ca²⁺ signals triggered by focal mechanical stimulation of single cells in confluent cultures of WT (A), Cx43- (B), and P2X₇R-null (C) spinal cord astrocytes bathed in control (gray bars) and low divalent cation (black bars) solutions. The increased relative number of participating cells per tier and extent of ICW transmission induced by low divalent cation solution is absent only in P2X₇R-null cells. Data are expressed as mean ± SEM (n = 5–6 fields from one representative experiment). Error bars represent SEM.

Figure 5.

A, Bar histograms showing the mean values of ICW efficacies obtained for WT and Cx43-null astrocytes exposed to control (DPBS) and low divalent cation (LDPBS) solutions in the absence and presence of BBG (100 nm and 1 μm) and MFQ (2 nm) (n = 20–97 fields, 3–18 experiments; p values from ANOVA followed by Tukey’s test). B, Representative Western blot showing the expression levels of P2X₇R, Cx43, and GAPDH obtained from whole-cell lysates of WT, Cx43-, and P2X₇R-null spinal cord astrocytes. C, Intracellular Ca²⁺ changes induced by bath application (arrows) of 300 μm BzATP in fura-2 AM loaded WT spinal cord astrocytes bathed in control (DPBS; closed cirlces) and in low divalent cation solution (LDPBS; open circles). D, Time course of relative YoPro-1 fluorescence intensity changes recorded from WT spinal cord astrocytes induced by 300 μm BzATP, in the absence and presence of BBG (100 nm) and MFQ (2 nm). Error bars represent SEM.