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First published online 27 January 2009
doi: 10.1242/jcs.034207

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Sphingosine-1-phosphate receptors stimulate macrophage plasma-membrane actin assembly via ADP release, ATP synthesis and P2X7R activation

¹ EMBL, Meyerhofstr. 1, 69117 Heidelberg, Germany
² Institute for Biological Sciences, University of Rostock, Albert-Einstein-Str. 3, 18051 Rostock, Germany
³ Julius-Bernstein-Institute for Physiology, Martin-Luther-University Magdeburger Str. 6, 06097 Halle, Germany
⁴ Department of Internal Medicine, Washington University School of Medicine, St Louis, MO 63110, USA

View larger version (22K): [in this window] [in a new window]   Fig. 1. (A) Quantification of F-actin changes in RAW cells stimulated with depleted or `old' (control) or fresh fetal calf serum (serum). (B) Corresponding concentration of ATP in the supernatant of RAW cells estimated with the luciferase assay. (C) Effect on the F-actin assembly induced by medium containing fresh serum or serum plus 30 U/ml apyrase. (D,E) F-actin levels 5 seconds after stimulation in response to the indicated amounts of ATP (D) or ADP (E). Statistics: P values for significant effects are given for experiments done in triplicate. See Table 1 for a summary of the effects of different effectors on actin assembly by RAW macrophages 5 seconds after stimulation.

View larger version (20K): [in this window] [in a new window]   Fig. 2. (A) Comparison of F-actin assembly in RAW cells in response to fresh serum or 10 nM S1P. (B) F-actin levels and extracellular ATP concentration in RAW cells in response to 10 nM S1P. (C,D) Effects of different concentration of S1P on actin assembly (C) and ATP release (D) in RAW cells as seen by luciferase fluorescence assay. Mock, solvent control without lipid. Statistical significance for differences relative to the mock treatment analysed using the Student's t-test are indicated.

View larger version (27K): [in this window] [in a new window]   Fig. 3. (A) HPLC quantification of 3H-labeled AMP, ADP and ATP released into supernatants of RAW cells after stimulation with serum or 10 nM S1P compared with cells treated with medium without serum or S1P (control). (B) Cell supernatants were collected at indicated times and incubated in the presence of 15,000 c.p.m. [3H]ADP. After 1 minute of incubation, nucleotides were detected by HPLC. Controls are supernatants without serum, S1P or Ad2P5.

View larger version (27K): [in this window] [in a new window]   Fig. 4. Functional expression of P2X7 receptors in mouse macrophages. (A) Currents induced by ATP (time indicated by horizontal bar) in a RAW macrophage. (B) Normalized concentration-response for P2X7 receptor agonists ATP4– and BzATP4–. (For approximation, see supplementary material Fig. S3.) (C) Effect of oxidized ATP. Currents were activated by application of 1 mM ATP4– for 3 seconds and normalized to the cell capacitance. (D) Effect of extracellular Mg2+. Currents were activated by 0.6 mM total ATP without or with additional 1 mM MgCl2 (0.1 or 0.035 mM free ATP4–, respectively). (E) Effect of co-application of Zn2+ on ATP4–-dependent ion currents. Current amplitudes were normalized to the respective values before Mg2+ or Zn2+ application. Means and s.d. from 5-50 cells are given. (F) F-actin assembly in response to 50 µM ATP in wild-type (+/+) and P2X7R-knockout (–/–) primary bone marrow macrophages. Columns represent means of three individual experiments with s.d. Statistical significance was determined by the Student's t-test. (G) Electrophysiological currents induced by 50 µM ATP in wild-type and P2X7R-knockout bone marrow macrophages. Means and s.d. from 5-50 cells are given.

View larger version (38K): [in this window] [in a new window]   Fig. 5. (A) Immunofluorescence localization of P2X7R and the membrane marker CD14 in wild-type and P2X7R-knockout (def) J774 cells. Arrows indicate the prominent plasma-membrane labeling for both markers only in the wild-type cells. (B) F-actin assembly in P2X7R wild-type and P2X7R knockout (def) J774 cells, with and without S1P or serum stimulation. (C) F-actin assembly in the two J774 cell lines in response to 50 mM ATP. (D) Luciferase assay quantification of extracellular ATP in the two J774 cell lines in response to S1P. Values represent means and s.d. of three individual experiments. *P<0.01.

View larger version (22K): [in this window] [in a new window]   Fig. 6. Schematic model of the data presented in this study. On the plasma membrane we leave open the question as to whether the adenylate-kinase-like activity is exported from the cytoplasm or whether a plasma-membrane-bound form is activated or released by S1P receptor activation. It is also not clear whether ADP crosses the plasma membrane directly, as shown, or whether it crosses an intracellular vesicle membrane and is externalized by exocytosis. The summary scheme of the data from the phagosome system (Kuehnel et al., 2009) is also shown, for comparison.

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