Extracellular ATP4- promotes cation fluxes in the J774 mouse macrophage cell line

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-.