title: Modulation of P2X7 receptor functions by polymyxin B: crucial role of the hydrophobic tail of the antibiotic molecule.
abstract: OBJECTIVE
  P2X7 is a membrane receptor for extracellular ATP which is highly expressed in dendritic cells, macrophages and microglia where it mediates pro-inflammatory responses. The antibiotic polymyxin B, which binds to and neutralizes the toxic residue of bacterial lipopolysaccharide, greatly amplifies cellular responses mediated by the P2X7 receptor. However, the molecular mechanism involved is so far unknown.
  
  
  METHODS
  We investigated the effects of polymyxin B and polymyxin B nonapeptide (PMBN) which is the deacylated amino derivative of polymyxin B lacking the N-terminal fatty amino acid 6-methylheptanoic/octanoic-Dab residue, in human macrophages and HEK293 cells stably expressing the human P2X7 receptor (HEK293-hP2X7). Differences between the two antibiotics were assessed by monitoring the following: nucleotide-induced cytoplasmic free Ca2+ concentration changes, plasma membrane permeability changes, lactate dehydrogenase activity, cell morphology changes. Western blot and microscopic analyses of P2X7GFP-expressing cells were also performed.
  
  
  RESULTS
  In contrast to polymyxin B, the polymyxin B nonapeptide was unable to potentiate: a) the ATP-induced Ca2+ increase, b) pore formation and consequently ATP-mediated plasma membrane permeabilization; c) ATP-dependent cytotoxicity. Moreover, in contrast to polymyxin B, polymyxin B nonapeptide did not affect aggregation of the P2X7 receptor subunits and it did not potentiate P2X7-dependent cell fusion.
  
  
  CONCLUSIONS
  The effects of polymyxin B depended on the presence of its N-terminal fatty amino acid 6-methylheptanoic/octanoic-Dab residue as deletion of this residue abolished polymyxin B-dependent modulation of ATP-triggered responses. These findings are important in the search for allosteric modulators of the P2X7 receptor.
authors.affiliation: Department of Experimental and Diagnostic Medicine, Section of General Pathology, and Interdisciplinary Center for the Study of Inflammation (ICSI), University of Ferrara, Ferrara, Italy. dfr@unife.it
authors.forename: D
authors.initials: D
authors.lastname: Ferrari
authors.forename: C
authors.initials: C
authors.lastname: Pizzirani
authors.forename: S
authors.initials: S
authors.lastname: Gulinelli
authors.forename: G
authors.initials: G
authors.lastname: Callegari
authors.forename: P
authors.initials: P
authors.lastname: Chiozzi
authors.forename: M
authors.initials: M
authors.lastname: Idzko
authors.forename: E
authors.initials: E
authors.lastname: Panther
authors.forename: F
authors.initials: F
authors.lastname: Di Virgilio
mesh_terms: D000255:Adenosine Triphosphate / Q000502:physiology; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology*; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002459:Cell Fusion; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects; D048430:Cell Shape / Q000187:drug effects; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008264:Macrophages / Q000187:drug effects; D011112:Polymyxin B / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology*; D016062:Porosity; D018048:Receptors, Purinergic P2 / Q000187:drug effects*; D058486:Receptors, Purinergic P2X7; D013329:Structure-Activity Relationship
chemical_list: D000900:Anti-Bacterial Agents; C546468:P2RX7 protein, human; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; D049452:Green Fluorescent Proteins; D000255:Adenosine Triphosphate; D007770:L-Lactate Dehydrogenase; D011112:Polymyxin B; D002118:Calcium