Regulation by CRAMP of the responses of murine peritoneal macrophages to extracellular ATP |
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Authors: | Michèle Seil Carole Nagant Unai Fontanils Stéphanie Pochet |
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Affiliation: | a Laboratoire de Chimie biologique et médicale et de Microbiologie pharmaceutique, Institut de Pharmacie C.P. 205/3, Université libre de Bruxelles, Bruxelles, Belgium b Laboratoire de Biochimie et d'Immunologie, Unité de Formation et de Recherche des Sciences de la Santé, Université de Ouagadougou, Burkina Faso c Laboratory for the Structure and Function of Biological Membranes, Center for Structural Biology and Bioinformatics, Faculty of Sciences C.P. 206/02, Université libre de Bruxelles, Bruxelles, Belgium d Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad del Pais Vasco, 48080 Bilbao, Spain |
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Abstract: | Peritoneal macrophages were isolated from wild type (WT) mice and from mice invalidated for the P2X7 receptor (KO) which had been pretreated with thioglycolate. In cells from WT mice, 1 mM ATP increased the intracellular concentration of calcium (Ca2+]i), the uptake of ethidium bromide, the production of reactive oxygen species (ROS), the secretion of IL-1β, the release of oleic acid and of lactate dehydrogenase; it decreased the intracellular concentration of potassium (K+]i). In KO mice, ATP transiently increased the Ca2+]i confirming that the P2X7 receptor is a major receptor of peritoneal macrophages. WKYMVm, an agonist of receptors for formylated peptides (FPR) also increased the Ca2+]i in murine macrophages. The slight increase of the Ca2+]i was strongly potentiated by ivermectin confirming the expression of functional P2X4 receptors by murine peritoneal macrophages. CRAMP, the unique antimicrobial peptide derived from cathelin in mouse inhibited all the responses coupled to P2X7 receptors in macrophages from WT mice. Agonists for FPR had no effect on the increase of the Ca2+]i in response to ATP. CRAMP had no effect on the increase of the Ca2+]i evoked by a combination of ATP and ivermectin in macrophages from P2X7-KO mice.In summary CRAMP inhibits the responses secondary to the activation of the murine P2X7 receptors expressed by peritoneal macrophages. This inhibition is not mediated by FPR receptors and is specific since CRAMP has no effect on the response coupled to P2X4 receptors. It can thus be concluded that the interaction between P2X7 receptors and cathelin-derived antimicrobial peptides is species-specific, in some cases (man) positive in others (mouse) negative. |
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Keywords: | a f u arbitrary fluorescence units ATR-FTIR attenuated total reflection-Fourier transform infrared spectroscopy BSA bovine serum albumin CRAMP cathelin-related antimicrobial peptide DCFH-DA 2&prime 7&prime -dichlorodihydrofluorescein diacetate EGTA ethylene glycol-bis-(β-aminoethyl ether)-N N N' N'-tetraacetic acid FBS fetal bovine serum FPR receptors for formylated peptides HEPES N-[2-hydroxyethyl] piperazine-N'-[2-ethanesulfonic acid] KO mice knockout mice ROS reactive oxygen species WT mice wild-type mice |
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