title: The induction of NOS2 expression by the hybrid cecropin A-melittin antibiotic peptide CA(1-8)M(1-18) in the monocytic line RAW 264.7 is triggered by a temporary and reversible plasma membrane permeation.
abstract: There is an increasing awareness of immune cell modulation by antimicrobial peptides. While this process often requires specific receptors for the peptides involved, several reports point out to a receptor-independent process. The cecropin A-melittin hybrid peptide CA(1-8)M(1-18) (KWKLFKKIGIGAVLKVLTTGLPALIS-amide) modifies gene expression in the macrophage line RAW 264.7 in the absence of any previous macrophage priming, suggesting a membrane permeation process. To further analyze the initial steps of this mechanism, we have studied the interaction of the peptide with these cells. Below 2 microM, CA(1-8)M(1-18) causes a concentration-dependent membrane depolarization partially reversible with time. At 2 microM, the accumulation of the SYTOX green vital dye is one half of that achieved with 0.05% Triton X-100. The binding level, as assessed by fluorescein-labeled CA(1-8)M(1-18), varies from 7.7+/-1.2 to 37.4+/-3.9 x 10(6) molecules/cell over a 0.5-4.0 microM concentration range. Electrophysiological experiments with 0.5 microM CA(1-8)M(1-18), a concentration that triggers maximal NOS2 expression and minimal toxicity, show a reversible current induction in the RAW 264.7 plasma membrane that is maintained as far as peptide is present. This activation of the macrophage involves the production of nitric oxide, a metabolite lethal for many pathogens that results from unspecific membrane permeation by antimicrobial peptides, and represents a new mode of action that may open new therapeutic possibilities for these compounds against intracellular pathogens.
authors.affiliation: Institute of Pharmacology and Toxicology, CSIC/UCM, School of Medicine, Universidad Complutense, 28040 Madrid, Spain.
authors.forename: Cristina
authors.initials: C
authors.lastname: Arias
authors.forename: Miriam
authors.initials: M
authors.lastname: Guizy
authors.forename: Juan R
authors.initials: JR
authors.lastname: Luque-Ortega
authors.forename: Esther
authors.initials: E
authors.lastname: Guerrero
authors.forename: Beatriz G
authors.initials: BG
authors.lastname: de la Torre
authors.forename: David
authors.initials: D
authors.lastname: Andreu
authors.forename: Luis
authors.initials: L
authors.lastname: Rivas
authors.forename: Carmen
authors.initials: C
authors.lastname: Valenzuela
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D023181:Antimicrobial Cationic Peptides / Q000494:pharmacology*; D002463:Cell Membrane Permeability / Q000187:drug effects*; D002478:Cells, Cultured; D004594:Electrophysiology; D004790:Enzyme Induction / Q000187:drug effects; D005456:Fluorescent Dyes; D008555:Melitten / Q000494:pharmacology*; D051379:Mice; D009000:Monocytes / Q000166:cytology / Q000201:enzymology*; D016328:NF-kappa B / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000096:biosynthesis*; D010455:Peptides / Q000494:pharmacology*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding
chemical_list: D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; D005456:Fluorescent Dyes; D016328:NF-kappa B; D010455:Peptides; D008555:Melitten; C031161:cecropin A; D052247:Nitric Oxide Synthase Type II