The antibiotic polymyxin B modulates P2X7 receptor function.
The natural peptide polymyxin B (PMB) is a well-known and potent antibiotic that binds and neutralizes bacterial endotoxin (LPS), thus preventing its noxious effects among LPS-mediated endotoxin shock in animal models. We have investigated the effect of PMB on responses mediated by the P2X(7)R in HEK293 and K562 cells transfected with P2X(7) cDNA and in mouse and human macrophages. In addition, in view of the potential exploitation of P2X(7)-directed agonists in antitumor therapy, we also investigated the effect of PMB in B lymphocytes from patients affected by chronic lymphocytic leukemia. PMB, at an optimal concentration dependent on the given cell type, greatly potentiated the effect of nucleotide-mediated P2X(7) stimulation. In particular, ATP-mediated Ca(2+) influx, plasma membrane permeabilization, and cytotoxicity were enhanced to an extent that, in the presence of PMB, cells were killed by otherwise ineffective nucleotide concentrations. The synergistic effect due to the combined application of ATP and PMB was prevented by incubation with the irreversible P2X blocker oxidized ATP (oATP), but not with the reversible antagonist 1-(N,O-bis(1,5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl)-4-phenilpiperazine (KN-62). Cells lacking P2X(7) were fully insensitive to the combined stimulation with PMB and ATP. Furthermore, PMB at the concentrations used had no untoward effects on cell viability. These results point to PMB as a useful tool for the modulation of P2X(7)R function and suggest that care should be used in the evaluation of ATP-stimulated immune cell responses in the presence of PMB as they may not solely be affected by removal of contaminating LPS.
Ferrari
Davide
D
Department of Experimental and Diagnostic Medicine, Sections of General Pathology, University of Ferrara, Italy. dfr@unife.it
Pizzirani
Cinzia
C
Adinolfi
Elena
E
Forchap
Sylvia
S
Sitta
Barbara
B
Turchet
Laura
L
Falzoni
Simonetta
S
Minelli
Mattia
M
Baricordi
Roberto
R
Di Virgilio
Francesco
F
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002118:Calcium / Q000037:antagonists & inhibitors / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D004996:Ethidium / Q000378:metabolism; D006801:Humans; D007424:Intracellular Fluid / Q000187:drug effects / Q000378:metabolism; D020014:K562 Cells; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011112:Polymyxin B / Q000494:pharmacology*; D058919:Purinergic P2 Receptor Antagonists; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D058486:Receptors, Purinergic P2X7; D014162:Transfection
D000900:Anti-Bacterial Agents; C546468:P2RX7 protein, human; C546469:P2rx7 protein, mouse; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; C017199:2',3'-dialdehyde ATP; C063302:KN 62; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000255:Adenosine Triphosphate; D004996:Ethidium; D011112:Polymyxin B; D002118:Calcium