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ATP4- permeabilizes the plasma membrane of mouse macrophages to fluorescent dyes
Abstract:
Extracellular ATP induces cation fluxes in thioglycolate-elicited mouse peritoneal macrophages and the J774 macrophage cell line apparently due to ligation of a plasma membrane receptor for ATP4-. We report that ATP permeabilizes the plasma membrane of J774 cells to 6-carboxyfluorescein (376 Da), lucifer yellow (457 Da), and fura-2 (831 Da) but not to trypan blue (961 Da), Evans blue (961 Da), or larger dye conjugates. We employed fluorescence microscopy and quantitative fluorimetry to study entry of lucifer yellow into the cytoplasm of J774 cells. Permeabilization to lucifer yellow appears to be mediated by the same ATP4- receptor that induces cation fluxes because it was inhibited by divalent cations and low pH, was mediated by the nonhydrolyzable analog adenosine 5'-(beta, gamma-imido)triphosphate, and because a variant J774 cell line resistant to ATP-induced Rb+ efflux did not take up lucifer yellow when exposed to ATP. ATP permeabilization was reversed within 5 min by removal of ATP or by addition of divalent cations. ATP also caused a transient increase in lucifer yellow uptake by pinocytosis. These data suggest that ATP4- ligates a receptor on macrophages which induces the formation of a channel admitting molecules less than or equal to 831 daltons into the cytoplasmic matrix and that removal of ATP4- from the medium causes rapid channel closure.
Insights
Extracellular ATP opens channels in macrophage plasma membranes, allowing small molecules up to 831 daltons to enter. This ATP-induced permeabilization is reversible and mediated by a specific ATP receptor.
Area of Science:
- Cell Biology
- Immunology
- Molecular Biology
Background:
- Extracellular ATP (adenosine triphosphate) is known to trigger cation fluxes in macrophages.
- These cation fluxes are thought to be mediated by a plasma membrane receptor for ATP (ATP4-).
Purpose of the Study:
- To investigate the mechanism by which extracellular ATP affects macrophage plasma membrane permeability.
- To determine the size exclusion limit of ATP-induced membrane permeabilization.
- To confirm the role of the ATP4- receptor in this process.
Main Methods:
- Utilized J774 macrophage cell line and thioglycolate-elicited mouse peritoneal macrophages.
- Employed fluorescence microscopy and quantitative fluorimetry to track dye uptake.
- Tested permeability with various molecular weight dyes (e.g., lucifer yellow, trypan blue).
- Investigated the role of the ATP4- receptor using inhibitors, analogs, and resistant cell lines.
Main Results:
- ATP permeabilized the J774 cell plasma membrane to molecules up to 831 Da (e.g., lucifer yellow, fura-2), but not larger molecules.
- This permeabilization was mediated by the same ATP4- receptor involved in cation fluxes, as evidenced by inhibition studies and resistant cell lines.
- ATP-induced permeabilization was rapidly reversible upon ATP removal or addition of divalent cations.
- A transient increase in pinocytosis was also observed.
Conclusions:
- Extracellular ATP binding to a specific macrophage receptor induces a transient channel.
- This channel allows the passage of molecules up to 831 Da into the cytoplasm.
- Channel closure is rapid upon removal of extracellular ATP.