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.
Highlights
Extracellular ATP induces cation fluxes in thiogly- Investigators have reported various effects of extracellular colate-elicited mouse peritoneal macrophages and the 5774 macrophage cell line apparently due to ligation of a plasma membrane receptor for ATP4
Mouse peritoneal macrophages and the 5774 mouse macrophage-like cell line respond to extracellular ATPby membranedepolarization, Na’ influx, K’ efflux, an increase in cytosolic free Ca”, and inhibition of Fc receptor-mediated cell line resistant to ATP-induced Rb’ efflux did not phagocytosis [11].Cation efflux from 5774 cells is not meditake up lucifer yellowwhen exposed to ATP
ATP ated by MgATP2, which is the prevalent ATP species in permeabilization was reversed within 5 min by re- physiologic solutions, but rather by ATP4, which comprises moval of ATP or by addition of divalent cations
Summary
Chemicals-ATP (special grade), GTP,ITP,and ATP-yS were purchased from Boehringer Mannheim. Lucifer yellow CH, fura-2 pentapotassium salt, and fluorescein-conjugated phalloidin were from Molecular Probes (Eugene, OR), and 6-. Microscopy-To assess the effect of ATP on the uptake of fluorescent dyes, cells plated on coverslips were incubated for 5 min in DMEM or buffered salt solution with or without 5 mM ATP and fluorescent dyes at the indicated concentration: lucifer yellow, 0.5 mg/ml; 6-~arboxyfluorescein 0 pg/ml; fura-2,10 pg/ml; Evans blue, 1.0 mg/ml;trypan blue, 0.05 mg/ml; fluorescein-phalloidin conjugate, 0.04 pg/ml; or fluorescein-dextran 4100, 5.0 mg/ml. Measurement of Fluorescent Dye Uptake-Cell-associated lucifer yellow and fluorescein-dextran fluorescence was measured as previously described [13]. 5774 cells remained impermeable to trypanblue, Evans blue, a fluorescein-phalloidin conjugate, and a 4100-Da fluorescein-dextran cation of the method ofLowry et al [15] on all samples, and dye conjugate. ATP permeabilizes the 5774 plasma uptake was expressed as ng of dye/mg of cell protein
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