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First published online 9 September 2008
doi: 10.1242/jcs.029991

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ATP-induced P2X₇-associated uptake of large molecules involves distinct mechanisms for cations and anions in macrophages

¹ Laboratório de Imunobiofísica, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 21941-902, Rio de Janeiro, Brasil
² Departamento de Farmacologia Básica e Clínica, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, 21941-902, Rio de Janeiro, Brasil

View larger version (19K): [in this window] [in a new window]   Fig. 1. ATPe-induced inward currents and Ca2+ signaling in HEK-P2X7 cells and macrophages. (A-C) Whole-cell voltage clamp recordings of macrophages (A), HEK-293 cells (B), and HEK-P2X7 cells (C). Cells were kept at room temperature and ATP (1 mM) was applied (arrows) pneumatically. (D-F) Changes in free cytoplasmic Ca2+ concentration of macrophages (D), HEK-293 cells (E), and HEK-P2X7 cells (F) loaded with Fura-2-AM. Cells were kept at 37°C and 5 mM ATP (final concentration) was applied as indicated by the horizontal bars. Ca2+ concentration was measured for 20-30 cells in the same microscope field using arbitrary fluorescence ratio units (a.u.). (G) Dose-effect curve of ATPe in HEK-P2X7 cells. Cells were exposed to different ATPe concentrations in the same conditions as in F and the fluorescence signal was normalized taking the maximum fluorescence obtained after the addition of 0.1% saponin. (H) Anti-P2X7 western blotting of macrophages (lane 1) HEK-293 cells (lane 2), and HEK-P2X7 cells (lane 3). Each result is representative of at least three independent experiments. In G, the bars represent the mean ± s.d. of at least three measurements per data point.

View larger version (69K): [in this window] [in a new window]   Fig. 2. ATPe-induced ethidium uptake in HEK-P2X7 cells and in macrophages. Fluorescence images of cells incubated for 15 minutes at 37°C in the presence (B,D,F,G) or not (A,C,E) of 5 mM ATP. Ethidium bromide (10 µM) was added in all preparations. (A,B) Macrophages; (C,D) HEK-293 cells; (E,F) HEK-P2X7 cells. (G) HEK-P2X7 cells pre-incubated for 2 hours with 300 µM oxATP before the addition of ATP. Each result is representative of at least three independent experiments. Bar, 100 µm.

View larger version (13K): [in this window] [in a new window]   Fig. 3. Large non-selective channels observed in macrophages but not in HEK-P2X7 cells correlates with ATPe-induced anion uptake. Cell-attached recordings (A-C) and dye uptake assays (D) were made in macrophages (A,C,D) and in HEK-P2X7 cells (B). For patch-clamping experiments, cells were kept at 37°C and recordings were performed with a holding potential of –40 mV. ATP was applied manually with a micropipette (1 mM final concentration). Results are representative of at least ten independent experiments. (A) Cell-attached recordings of macrophages. Upper trace: normal extracellular solution. Lower trace: divalent-free extracellular solution. (B) Cell-attached recording of a HEK-P2X7 cell. (C) I-V curves of the macrophage channels obtained in the same conditions as in the upper trace of A. (D) ATPe-induced uptake of ethidium and LY in macrophages using the same solutions as in A. Data in D was obtained by fluorescence microscopy as described in Materials and Methods. The values were normalized by taking the specific fluorescence, obtained in normal solution in the presence of ATP, as 100%. Data points and bars represent the mean ± s.d. of at least four independent experiments.

View larger version (68K): [in this window] [in a new window]   Fig. 4. ATPe induces the uptake of anionic dyes in macrophages but not in HEK-P2X7 cells. Fluorescence microscopy of cells incubated for 10 minutes at 37°C in the absence (A,C,E,G) or in the presence (B,D,F,H) of 5 mM ATP. LY (3 mM) or MQAE (5 mM) were used as fluorescent dyes. (A,B) Macrophages in the presence of LY; (C,D) HEK-P2X7 cells in the presence of LY; (E,F) Macrophages in the presence of MQAE. (G,H) HEK-P2X7 cells in the presence of MQAE. Each result is representative of at least three independent experiments. Bar, 100 µm.

View larger version (49K): [in this window] [in a new window]   Fig. 5. Uptake of anions but not cations occurs through a diffusion process. Fluorescence microscopy of cells incubated for 10 minutes at 37°C in the absence (A,D,F) or in the presence (B,C,E,G) of 5 mM ATP and cooled on ice in the absence of any dyes. Ethidium bromide (A,B), MQAE (D,E), or LY (F,G) was then added for 15 minutes and the cells were immediately transferred to the stage of the microscope. Temperature was kept all the time below 4°C. In C, cells treated as in B were heated up to 37°C and kept in the presence of ATPe and ethidium bromide for 10 minutes. Experiments using MQAE were performed in a low chloride solution obtained by substituting Cl– for glutamate in the normal extracellular solution. Each image is representative of the results of at least three independent experiments performed in duplicates. Bar, 100 µm.

View larger version (49K): [in this window] [in a new window]   Fig. 6. ATPe induces the efflux of anions but not cations from macrophages. Fluorescence microscopy of macrophages incubated for 10 minutes at 37°C in the absence (A and E) or in the presence (B-D and F-H) of 5 mM ATP. LY (A-D) or SR-B mM (E-H) was added at 37°C and cells were kept at this temperature until transferred to the stage of the microscope. In B and F, cells were loaded with the dye by incubation for 10 minutes in the presence of one of the dyes and ATP. In C and G cells were prepared as in B and F, respectively, washed, and kept in normal solution without the dye or ATP for an additional period of 10 minutes. In D and H cells were prepared as in B and F, respectively, washed, and kept in normal solution in the presence of 5 mM ATP and without the dye for an additional period of 10 minutes. In the LY experiments (A-D), macrophages were pre-incubated for 30 minutes and kept in the presence of probenecid (5 mM) during all incubations. Images in A-H is representative of the results of at least three independent experiments. Bar, 100 µm. (I) A quantitative comparison of the uptake of LY and SR-B by macrophages. Cells were incubated at 37°C with LY or SR-B for 10 minutes in the presence of 0, 1, 3 and 5 mM ATP and the amount of dye taken up by the cells was measured by spectrofluorimetry as described in the Materials and Methods. Data were normalized for a better comparison of the different data sets. Typical absolute values at 0 and 5 mM ATP were 1.2 and 28 mg of SR-B/mg protein and 0.2 and 1.0 mg of LY/mg protein. The bars represent the means of three independent experiments.

View larger version (17K): [in this window] [in a new window]   Fig. 7. Uptake of SR-B in the presence of carbenoxolone, mefloquine and 10Pnx1. Representative experiments showing the uptake of SR-B by macrophages in different conditions. Cells were pre-incubated at 37°C with 50 µM carbenoxolone for 5 minutes (CBX), 100 nM mefloquine (MFQ) for 30 minutes, 100 µM 10Pnx1 for 30 minutes, or vehicle (DMSO) in the case of mefloquine and 10Pnx1. ATP (0, 1, 3, and 5 mM) and SR-B were then added for an additional period of 10 minutes and the amount of dye taken up by the cells was measured by spectrofluorimetry as described in the Materials and Methods. Data were normalized for a better comparison of the different data sets. Each result is representative of at least three independent experiments.

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