ATP-induced P2X7-associated uptake of large molecules involves distinct mechanisms for cations and anions in macrophages.
Macrophages express the P2X(7) receptor and other nucleotide (P2) receptors, and display the phenomenon of extracellular ATP (ATP(e))-induced P2X(7)-dependent membrane permeabilization, which occurs through a poorly understood mechanism. We used patch-clamp recordings, cytoplasmic Ca(2+) measurements and fluorescent dye uptake assays to compare P2X(7)-associated transport phenomena of macrophages and HEK-293 cells transfected with P2X(7) receptors (HEK-P2X(7) cells). Both cell types showed inward currents, increase of free cytoplasmic Ca(2+) concentration and the uptake of cationic dyes upon exposure to ATP(e), as previously described. However, in contrast to the macrophages, HEK-P2X(7) cells did not take up anionic dyes and did not display the 440 pS channels (Z pores) under cell-attached patch-clamping conditions. In addition, the transport mechanism of anionic dyes displayed by macrophages was also able to support dye efflux and, once activated at 37 degrees C, it remained active at 4 degrees C, whereas uptake of cationic dyes was temperature-dependent and unidirectional. Our results indicate that the mechanism of ATP(e)-induced dye uptake, usually called a ;permeabilization phenomenon' and associated with a ;permeabilization pore' can be ascribed to at least two distinct mechanisms in macrophages: a diffusional pathway, possibly associated with the 440 pS Z pores, and a cation uptake mechanism that is not diffusional and should be ascribed to an, as yet, unidentified transport mechanism.
Schachter
Julieta
J
Laboratório de Imunobiofísica, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
Motta
Andressa Piedade
AP
de Souza Zamorano
Aliane
A
da Silva-Souza
Hercules Antônio
HA
Guimarães
Marilia Zaluar P
MZ
Persechini
Pedro Muanis
PM
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D000838:Anions / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects; D002412:Cations / Q000378:metabolism*; D002460:Cell Line; D004396:Coloring Agents / Q000378:metabolism; D004058:Diffusion / Q000187:drug effects; D004996:Ethidium / Q000378:metabolism; D005110:Extracellular Space / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects; D017136:Ion Transport / Q000187:drug effects; D008264:Macrophages / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D051379:Mice; D009419:Nerve Tissue Proteins / Q000378:metabolism; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058486:Receptors, Purinergic P2X7; D012235:Rhodamines
D000838:Anions; D002412:Cations; D004396:Coloring Agents; D009419:Nerve Tissue Proteins; C546468:P2RX7 protein, human; C546469:P2rx7 protein, mouse; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; D012235:Rhodamines; C022027:lissamine rhodamine B; D000255:Adenosine Triphosphate; D004996:Ethidium