ATP induces the death of developing avian retinal neurons in culture via activation of P2X7 and glutamate receptors

Abstract

Previous data suggest that nucleotides are important mitogens in the developing retina. Here, the effect of ATP on the death of cultured chick embryo retina cells was investigated. In cultures obtained from retinas of 7-day-old chick embryos (E7) that were cultivated for 2 days (E7C2), both ATP and BzATP induced a ∼30 % decrease in cell viability that was time- and dose-dependent and that could be blocked by 0.2 mM oxidized ATP or 0.3 μM KN-62. An increase in cleaved caspase-3 levels and in the number of TUNEL-positive cells was observed when cultures were incubated with 3 mM ATP and immunolabeling for cleaved-caspase 3 was observed over neurons but not over glial cells. ATP-dependent cell death was developmentally regulated, the maximal levels being detected by E7C2-3. Nucleotides were able to increase neuronal ethidium bromide and sulforhodamine B uptake in mixed and purified neuronal cultures, an effect that was blocked by the antagonists Brilliant Blue G and oxidized ATP. In contrast, nucleotide-induced cell death was observed only in mixed cultures, but not in purified cultures of neurons or glia. ATP-induced neuronal death was blocked by the glutamatergic antagonists MK801 and DNQX and activation of P2X7 receptors by ATP decreased the uptake of [(3)H]-D-aspartate by cultured glial cells with a concomitant accumulation of it in the extracellular medium. These results suggest that ATP induces apoptosis of chick embryo retinal neurons in culture through activation of P2X7 and glutamate ionotropic receptors. Involvement of a P2X7 receptor-mediated inhibition of the glial uptake of glutamate is suggested.

Fig. 1

Effect of ATP and BzATP on cell survival as determined by MTT viability assays. Retinal cultures at E7C2 cultivated in complete medium were incubated with increasing concentrations of ATP or BzATP a, c for 3 h or with 3 mM ATP and 0.1 mM BzATP for increasing periods of time (b, d). After this period, MTT assays were performed as described in “Methods.” Data represent the mean ± SEM of at least three separate experiments performed in triplicate. **P < 0.01 compared with control

Fig. 2

ATP and BzATP induced a decrease in the viability of retinal cells in culture. a Effect of P2X7 receptor antagonists. Retinal cell cultures at E7C2 were incubated with 3 mM ATP or 0.1 mM BzATP for 3 h in the absence or presence of 0.2 mM oxiATP or 0.3 μM KN-62 that were added 2 h or 30 min before the agonists, respectively. b ATP-induced decrease in cell viability as a function of the developmental stage of the cultures. Retinal cells obtained from 7-day-old embryos were cultured for several days and incubated with 3 mM ATP for 3 h at the indicated stage (C1 to C7). c ATP-induced decrease in retinal cell viability as a function of the developmental stage of the chick embryo. Retinas from chick embryos at different stages were dissociated and retinal cells cultured for 2 days. Cultures were incubated wht 3 mM ATP for 3 h and cell viability determined by MTT. The results in a and b represent the mean ± SEM of three or four separate experiments performed in duplicate or triplicate. Results in c represent the mean ± SEM of 2 (E6,E9) or 4 (E7,E8) separate experiments performed in duplicate or triplicate. In a, **p < 0.01 and ***p < 0.001, compared with ATP- or BzATP-treated cultures; in b and c, *p < 0.05 and **p < 0.01, compared with control, non-treated cultures. SEM values of control cultures were less than 10 %

Fig. 3

ATP induces cell apoptosis in chick embryo retinal cultures. a Representative photomicrographs showing mixed retinal cultures at E7C2, treated or not with 3 mM ATP for 3 h and labeled with TUNEL (green) or propidium iodide (red). b Quantification of the number of TUNEL-positive cells in control and ATP-treated cultures. Labeled cells in each culture were counted in ten micrographs randomly obtained. Data represent the mean ± SEM of three independent experiments performed in duplicate. c Detection by Western blotting of cleaved caspase-3 in protein extracts of retinal cell cultures at E7C2 stimulated with 3 mM ATP for 5 or 10 min. Experiments were replicated three times with similar results. CT = cultures incubated without ATP. d Representative photomicrographs showing cells immunolabeled for cleaved caspase-3 (arrows) in retinal cell cultures at E7C2 incubated with 3 mM ATP for 10 min. Cell nuclei staining was performed with DAPI. Bars represent 20 and 10 μm in a and d, respectively. *p < 0.05, versus control

Fig. 4

Immunoreactivity for cleaved caspase-3 in ATP-treated retinal cultures. Retinal cultures at E7C2 were stimulated with 3 mM ATP for 10 min, fixed, and immunolabeled with anti-cleaved caspase-3 (red) and anti-β-tubulin III or anti-2 M6 (green) to label neurons and glial cells, respectively. Note that only neurons were labeled for cleaved-caspase 3 (short arrows at upper panels). No double-labeled glial cells were found (long arrows at lower panels). Experiments were replicated twice with similar results. Bar = 10 μm

Fig. 5

Uptake of ethidium bromide induced by BzATP in cultured retinal cells. Retinal cultures at E7C2 were stimulated with 0.1 mM BzATP for 10–15 min in the presence of 5 μM ethidium bromide in Hanks’ balanced salt solution without Ca⁺⁺/Mg⁺⁺. The P2X7 receptor antagonists oxiATP (0.2 mM) and BBG (5 μM) were added 2 h or 15 min before BzATP, respectively. After stimulation, cells were washed and visualized under fluorescence microscopy. In each culture, labeled cells were counted in ten micrographs randomly obtained. Data represent the mean ± SEM of five separate experiments performed in duplicate. ***p < 0.001, compared with control cultures. ^###p < 0.001 and ^##p < 0.01, compared with BzATP-treated cultures

Fig. 6

Uptake of ethidium bromide induced by ATP in three types of retinal monolayer cultures. Mixed and neuronal retinal cultures at E7C2 or glial cultures were treated with 3 mM ATP for 10–15 min in the presence of 5 μM ethidium bromide (EtBr) in Hanks’ balanced salt solution without Ca⁺⁺/Mg⁺⁺. After incubation, cells were washed and visualized under fluorescence illumination. a Representative photomicrographs of the three types of cultures labeled for β-tubulin III and the glial marker 2 M6. b Representative micrographs showing EtBr-labeled cells in the three types of cultures. c Quantification of EtBr-positive cells in mixed and neuronal retinal cultures. d Positive control showing the uptake of EtBr induced by 3 mM ATP in rat macrophage cultures. Labeled cells in each culture were counted in ten different micrographs randomly obtained. Data represent the mean ± SEM of five separate experiments performed in duplicate. ***p < 0.01 versus control. Bars = 20 μm

Fig. 7

Uptake of sulforhodamine B (SRB) induced by ATP in the three types of retinal monolayer cultures. Mixed and neuronal retinal cultures at E7C2 or glial cultures were treated with 3 mM ATP for 10–15 min in the presence of 5 μM SRB in Hanks’ balanced salt solution without Ca⁺⁺/Mg⁺⁺. After incubation, cells were washed and visualized under fluorescence illumination. a Representative micrograph showing SRB labeled cells in mixed, purified neuronal, glial retinal cultures, and cultured macrophages. b High magnification of the cells shown in the red square in a. Note the presence of labeled neuronal processes. c Quantification of SRB-positive cells in mixed and neuronal retinal cultures. Labeled cells in each culture were counted in ten different micrographs randomly obtained. Data represent the mean ± SEM of five separate experiments performed in duplicate. ***p < 0.001, compared with control cultures. Bars = 20 μm

Fig. 8

a Effect of ATP on the viability of retinal cells in the three types of retinal cultures. Mixed, neuronal, or glial purified retinal cultures were incubated with 3 mM ATP for 3 h. Cell viability was assessed by the MTT assay. b Effect of BzATP and glutamate on the viability of retinal neurons. Neuronal cultures at E7C2 were incubated with 0.1 mM BzATP or 1 mM glutamate for 3 h and cell viability assessed by MTT. c Effect of the NMDA and non-NMDA receptor antagonists MK801 and DNQX on the decrease in cell viability induced by ATP. Mixed cultures at E7C2 were incubated with 3 mM ATP for 3 h in the absence or presence of 50 μM MK801 or 50 μM DNQX. Antagonist was added 10 min prior to ATP. d Effect of ATP on the viability of retinal progenitors. Mixed cultures at E7C2 were incubated for 90 min with 0.5 μCi [³H]-thymidine to label proliferating progenitors, washed, and cultured for 4 h. Cells were then treated with 3 mM ATP or 0.1 mM BzATP for an additional 3-h period and processed for the detection of radioactivity as described in “Methods.” Data represent the mean ± SEM of three separate experiments performed in duplicate or triplicate (a) or three to four separate experiments performed in duplicate or triplicate (b, c, d). ***p < 0.001 and *p < 0.05, versus control cultures. ^##p < 0.01, versus ATP-treated cultures. NS not statistically significant

Fig. 9

Effect of ATP on the uptake of [³H]-d-aspartate in mixed and purified glial retinal cultures. a Effect of ATP on the uptake of [³H]-d-aspartate in mixed cultures. Cultures at E7C2 were pre-incubated for 3 h in the absence or presence of 3 mM ATP and incubated for the indicated periods with 0.3 μCi/mL of [³H]-d-aspartate. At the end of each period, cells were lysed and the intracellular radioactivity determined as described in “Methods.” c Effect of TBA, BBG, and MgCl₂ on the uptake of [³H]-d-aspartate. Retinal cultures were incubated for 3 h in the presence of 100 μM TBA, 3 mM ATP in the presence or not of 10 μM BBG or 5 mM MgCl₂. Cultures were then incubated for 30 min with 0.3 μCi/mL [³H]-d-aspartate, cells were lysed and intracellular radioactivity determined. c Purified glial cultures at E8C20 were incubated for 3 h in the presence or absence of 100 μM TBA or 3 mM ATP and processed for [³H]-d-aspartate uptake as described for mixed cultures. Oxidized ATP was added 2 h before ATP. d Effect of ATP on the efflux of pre-incorporated [³H]-d-aspartate. Retinal cultures were loaded with 0.3 μCi/mL [³H]-d-aspartate, washed, and incubated for the indicated periods of time in the absence or presence of 3 mM ATP. Radioactivity present in the extracellular medium was estimated as described in “Methods.” Data represent the mean ± SEM of three or four separate experiments performed in triplicate (a, b, c). In d, data represent the mean ± SEM of two or three separate experiments performed in triplicate. ***p < 0.001 and **p < 0.01, compared with control cultures. ^##p < 0.01 and ^###p < 0.001, compared with ATP-treated cultures

Fig. 10

Effect of TBA on cell viability in retinal cultures. Mixed cultures at E7C2 were incubated for 3 h with 3 mM ATP or 100 μM TBA, in the absence or presence of 200 μM AP5. Data represent the mean ± SEM of three independent experiments performed in triplicate or quadruplicate. ***p < 0.001, versus control; *p < 0.05, versus ATP-treated cultures; **p < 0.01, versus TBA-treated cultures