QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 25 August 2004
doi: 10.1242/jcs.01345

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fortes, F. S. A. Articles by Goldenberg, R. C. S. Search for Related Content PubMed PubMed Citation Articles by Fortes, F. S. A. Articles by Goldenberg, R. C. S.

Modulation of intercellular communication in macrophages: possible interactions between GAP junctions and P2 receptors

¹ Institute of Biophysics Carlos Chagas Filho, UFRJ, Cidade Universitária, Ilha do Fundão, Rio de Janeiro, RJ 21941-590, Brazil
² Institutes of Biosciences, UNESP, Department of Physics and Biophysics, Rubião Junior, Butucatu, São Paulo, SP 18611-006, Brazil
³ Institute Oswaldo Cruz, FIOCRUZ, Avenue Brasil, Manguinhos - 4635, Rio de Janeiro, Brazil
⁴ Department of Neuroscience, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA

View larger version (17K): [in a new window]   Fig. 1. Expression of P2X7 receptors in macrophages, ATP-sensitive (ATP-s), and ATP-insensitive (ATP-i) J774 cells. (A) ATP-s (second lane) but not ATP-i (fourth lane) cells express P2X7 receptor mRNA. cDNAs obtained from both cells were amplified by PCR using a set of primers specific for either P2X7 (top) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH), used as control of cDNA loading (bottom). RT-PCR product was separated on a 1% agarose gel and stained with ethidium bromide. In mock reactions (first and third lanes), RNA samples obtained from the same preparations were substituted for cDNA as a control for genomic DNA contamination. (B) Western blots showing that peritoneal macrophages (lane 1) and ATP-s cells (lane 2), but not ATP-i cells (lane 3) express P2X7 protein. Each lane was loaded with 100 µg of protein and separated by 10% SDS-PAGE before transfer to nitrocellulose membrane. The bar histograms represent the mean±s.d. (n=5) densitometric values relative to those obtained for macrophages. The asterisk indicates that the intensities of the bands for ATP-s cells are significantly different from those of ATP-i cells (P<0.05). Whole-cell patch-clamp recordings show that ATP-s (C) but not ATP-i (D) cells display a biphasic current after iontophoretic application of ATP (arrows). The spikes in (D) are passive currents sensed by the recording electrode during each iontophoretic application of ATP. Holding potential was maintained at -40 mV. Data are representative of at least ten recordings for each cell type.

View larger version (13K): [in a new window]   Fig. 5. Expression of Cx43 mRNA (A) and protein (B) in J774 cells. (A) Relative expression of Cx43 mRNA in ATP-s and ATP-i cells, obtained by amplified Cx43 and GAPDH RT-PCR products. The graph represents the mean±s.d. values for the ratio between Cx43 and GAPDH bands (n=5). The insert shows the agarose gel of a semiquantitative RT-PCR experiment. Mock is used as a negative control. There were no significant differences between ATP-s and ATP-i cells. (B) Western blot showing that ATP-s and ATP-i cells express Cx43 protein (43 kDa arrow). Liver and brain homogenates were used as negative and positive controls, respectively. Each lane was loaded with 100 µg of protein and separated by 10% SDS-PAGE before transfer to nitrocellulose membrane. The histogram represents the mean±s.d. (n=5) of relative densitometry values for Cx43 labeling taking the rat-brain homogenate as reference. The intensity of the bands in ATP-s and ATP-i cells are not significantly different from each other. The asterisk indicates that murine brain homogenate expressed significantly more Cx43 than the other cells (P<0.05).

View larger version (14K): [in a new window]   Fig. 3. Quantification of the dye-coupling assays. The bar histograms show the proportions of ATP-s and ATP-i cells that were coupled when in the presence and the absence of octanol. Data represent the mean±s.d. number of injected cells that were coupled to at least one neighboring cell. At least 30 cells were injected with Lucifer Yellow in each culture dish and at least five independent dishes were injected on different days. Degree of coupling: a total of 0 cells coupled to the injected cell; 1-3 cells coupled to the injected cell; or 4-6 cells coupled to the injected cell. The degree of coupling between ATP-i cells and octanol-treated ATP-s cells was significantly decreased compared with that obtained for ATP-s cells to the group of the ATP-s cells. The asterisk and double asterisk indicate P<0.05.

View larger version (18K): [in a new window]   Fig. 4. Short exposures to extracellular ATP or UTP do not interfere with dye coupling. The histogram shows the proportion of dye-coupled ATP-s cells when treated with 100 µM and 500 µM ATP or UTP. The drugs were applied 5 minutes before the experiment and maintained in the culture during the injection procedure. Data represent the mean±s.d. number of injected cells that displayed dye coupling in an individual Petri-dish culture. At least 30 cells per dish were injected with Lucifer Yellow, for a total of 90 cells injected. No difference was observed between ATP-s cells treated or untreated with extracellular ATP and UTP (P>0.05).

View larger version (94K): [in a new window]   Fig. 2. ATP-s but not ATP-i cells are dye coupled. ATP-s (A,B) and ATP-i (C,D) cells were injected with Lucifer Yellow and observed under phase-contrast (A,C) and fluorescence (B,D) microscopy. In ATP-s cells (B), dye spreads to at least three adjacent cells, whereas, in ATP-i cells (D), dye remained restricted to the injected cells. Arrow and asterisk indicate the injected cells. Bar, 50 µm.

View larger version (152K): [in a new window]   Fig. 6. Spatial distribution of Cx43 in ATP-s and ATP-i cells. Phase-contrast (A,C) and immunofluorescence (B,D) micrographs were taken from ATP-s (A,B) and ATP-i (C,D) cells cultivated on glass cover slips. Notice the Cx43-specific labeling at membrane appositional areas in ATP-s cells (B, arrow). By contrast, labeling is absent from the appositional areas in ATP-i cells and concentrates in the interior of the cells (D, arrow). Bar, 20 µm.

View larger version (102K): [in a new window]   Fig. 7. Co-localization and immunoprecipitation of Cx43 and P2X7 proteins in ATP-s cells. (A) Cx43 immunolabeling, (B) P2X7 immunolabeling and (C) TOPRO3 staining showing the nuclei of the J774 cells. Bar, 20 µm. (D) Reconstruction of 28 optical slices of 0.8 µm thickness. The green line indicates an orthogonal section from (D), projected laterally in (E); the red line indicates an orthogonal section from (D), projected laterally in (F); the blue line indicates the level of the 15th optical slice in (D). Confocal microscopy demonstrates co-localization of the Cx43 and P2X7 proteins (yellow) to the membrane of the ATP-s cells (D-F, arrows). Notice the Cx43 (green) and P2X7 (red) specific labeling located at membrane apposition areas (A,B, arrows). (G) Lane 1 shows a 69 kDa band corresponding to P2X7 receptor obtained after immunoprecipitation of ATP-s cell lysates with Cx43 antibodies, and lane 2 shows the corresponding band obtained from whole ATP-s cell lysate after probing with the P2X7 antibody.

View larger version (100K): [in a new window]   Fig. 8. Co-localization and immunoprecipitation of Cx43 and P2X7 proteins in peritoneal macrophages. (A) Cx43 immunolabeling, (B) P2X7 immunolabeling and (C) TOPRO3 staining showing the nuclei of the J774 cells. Bar, 20 µm. (D) Reconstruction of 49 optical slices of 0.8 µm thickness. The green line indicates an orthogonal section from (D), projected laterally in (E); the red line indicates an orthogonal section from (D), projected laterally in (F); the blue line indicates the level of the 25th optical slice in (D). Confocal microscopy demonstrates co-localization of the Cx43 and P2X7 proteins (yellow) to the membrane of the macrophages (D-F, arrows). Notice the Cx43 (green) and P2X7 (red) specific labeling located at membrane apposition areas (A,B, arrows). (G) Lane 1 shows a 69 kDa band corresponding to P2X7 receptor obtained after immunoprecipitation of peritoneal macrophage lysates with Cx43 antibodies, and lane 2 shows the corresponding band obtained from whole peritoneal macrophage cell lysates probed with the P2X7 antibody.