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Extracellular ATP4- promotes cation fluxes in the J774 mouse macrophage cell line
Abstract:
Extracellular ATP stimulates transmembrane ion fluxes in the mouse macrophage cell line J774. In the presence of Mg2+, nonhydrolyzable ATP analogs and other purine and pyrimidine nucleotides do not elicit this response, suggesting the presence of a specific receptor for ATP on the macrophage plasma membrane. One candidate for such a receptor is the ecto-ATPase expressed on these cells. We, therefore, investigated the role of this enzyme in ATP-induced 86Rb+ efflux in J774 cells. The ecto-ATPase had a broad nucleotide specificity and did not hydrolyze extracellular ATP in the absence of divalent cations. 86Rb+ efflux was not blocked by inhibition of the ecto-ATPase and did not require Ca2+ or Mg2+. In fact, ATP-stimulated 86Rb+ efflux was inhibited by Mg2+ and correlated with the availability of ATP4- in the medium. In the absence of divalent cations, the slowly hydrolyzable ATP analogs adenosine 5'-(beta, gamma-imido)triphosphate (AMP-PNP) and adenosine 5'-O-(3-thio)triphosphate (ATP-gamma-S) also stimulated 86Rb+ efflux, albeit at higher concentrations than that required for ATP4-. Exposure of J774 cells to 10 mM ATP for 45 min caused death of 95% of cells. By this means we selected variant J774 cells that did not exhibit 86Rb+ efflux in the presence of extracellular ATP but retained ecto-ATPase activity. These results show that the ecto-ATPase of J774 cells does not mediate the effects of ATP on these cells; that ATP4- and not MgATP2- promotes 86Rb+ efflux from these cells; and that hydrolysis of ATP is not required to effect this change in membrane permeability. These findings suggest that J774 cells possess a plasma membrane receptor which binds ATP4-, AMP-PNP, and ATP-gamma-S, and that the ecto-ATPase limits the effects of ATP on these cells by hydrolyzing Mg-ATP2-.
Insights
Extracellular ATP triggers ion fluxes in macrophages, but not via the ecto-ATPase enzyme. The study identifies a specific ATP receptor, highlighting the role of the ATP4- form in this cellular response.
Area of Science:
- Cellular Physiology
- Molecular Biology
- Immunology
Background:
- Extracellular ATP (adenosine triphosphate) is known to influence cellular functions, including ion transport, in various cell types.
- Mouse macrophage cell line J774 exhibits transmembrane ion fluxes in response to extracellular ATP, suggesting a specific ATP-sensing mechanism.
- The ecto-ATPase, an enzyme on the cell surface, was a potential candidate for mediating ATP's effects due to its nucleotide-binding and hydrolyzing capabilities.
Purpose of the Study:
- To investigate the role of the ecto-ATPase in mediating ATP-induced 86Rubidium (86Rb+) efflux in J774 macrophages.
- To determine the specific form of ATP (ATP4- vs. MgATP2-) responsible for stimulating ion fluxes.
- To identify the mechanism by which extracellular ATP affects macrophage membrane permeability.
Main Methods:
- Measurement of 86Rb+ efflux from J774 cells stimulated with extracellular ATP and its analogs.
- Inhibition of ecto-ATPase activity using specific inhibitors.
- Selection of variant J774 cells resistant to ATP-induced cell death and ion efflux, while retaining ecto-ATPase activity.
Main Results:
- ATP-stimulated 86Rb+ efflux was not blocked by ecto-ATPase inhibition and did not require divalent cations (Ca2+, Mg2+).
- Magnesium ions (Mg2+) inhibited ATP-induced 86Rb+ efflux, which correlated with the availability of the ATP4- form.
- Slowly hydrolyzable ATP analogs (AMP-PNP, ATP-gamma-S) stimulated 86Rb+ efflux in the absence of divalent cations, indicating a receptor-mediated process.
- Variant cells lacking ATP-induced 86Rb+ efflux but retaining ecto-ATPase activity confirmed the enzyme's non-involvement in the primary response.
Conclusions:
- The ecto-ATPase enzyme does not mediate the effects of extracellular ATP on 86Rb+ efflux in J774 cells.
- The ATP4- anion, not the MgATP2- complex, is the primary species that promotes 86Rb+ efflux from J774 macrophages.
- ATP hydrolysis is not required for ATP to alter macrophage membrane permeability, suggesting a direct receptor-ligand interaction.
- J774 cells possess a plasma membrane receptor that binds ATP4- and its analogs, with ecto-ATPase activity potentially regulating the availability of Mg-ATP2-.