title: Kinetics of extracellular ATP in mastoparan 7-activated human erythrocytes.
abstract: BACKGROUND
  The peptide mastoparan 7 (MST7) stimulated ATP release in human erythrocytes. We explored intra- and extracellular processes governing the time-dependent accumulation of extracellular ATP (i.e., ATPe kinetics).
  
  
  METHODS
  Human erythrocytes were treated with MST7 in the presence or absence of two blockers of pannexin 1. ATPe concentration was monitored by luciferin-luciferase based real-time luminometry.
  
  
  RESULTS
  Exposure of human erythrocytes to MST7 led to an acute increase in [ATPe], followed by a slower increase phase. ATPe kinetics reflected a strong activation of ATP efflux and a low rate of ATPe hydrolysis by ectoATPase activity. Enhancement of [ATPe] by MST7 required adhesion of erythrocytes to poly-D-lysin-coated coverslips, and correlated with a 31% increase of cAMP and 10% cell swelling. However, when MST7 was dissolved in a hyperosmotic medium to block cell swelling, ATPe accumulation was inhibited by 49%. Erythrocytes pre-exposure to 10μM of either carbenoxolone or probenecid, two blockers of pannexin 1, exhibited a partial reduction of ATP efflux. Erythrocytes from pannexin 1 knockout mice exhibited similar ATPe kinetics as those of wild type mice erythrocytes exposed to pannexin 1 blockers.
  
  
  CONCLUSIONS
  MST7 induced release of ATP required either cell adhesion or strong activation of cAMP synthesis. Part of this release required cell swelling. Kinetic analysis and a data driven model suggested that ATP efflux is mediated by two ATP conduits displaying different kinetics, with one conduit being fully blocked by pannexin 1 blockers.
  
  
  CONCLUSIONS
  Kinetic analysis of extracellular ATP accumulation from human erythrocytes and potential effects on microcirculation.
authors.affiliation: Department of Biological Chemistry, University of Buenos Aires, Buenos Aires, Argentina.
authors.forename: María Florencia
authors.initials: MF
authors.lastname: Leal Denis
authors.forename: J Jeremías
authors.initials: JJ
authors.lastname: Incicco
authors.forename: María Victoria
authors.initials: MV
authors.lastname: Espelt
authors.forename: Sandra V
authors.initials: SV
authors.lastname: Verstraeten
authors.forename: Omar P
authors.initials: OP
authors.lastname: Pignataro
authors.forename: Eduardo R
authors.initials: ER
authors.lastname: Lazarowski
authors.forename: Pablo J
authors.initials: PJ
authors.lastname: Schwarzbaum
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D004285:Dogs; D004912:Erythrocytes / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D036341:Intercellular Signaling Peptides and Proteins; D007700:Kinetics; D051379:Mice; D018345:Mice, Knockout; D010455:Peptides / Q000494:pharmacology*; D015398:Signal Transduction
chemical_list: D036341:Intercellular Signaling Peptides and Proteins; C084070:Mas7 protein, synthetic; D010455:Peptides; D000255:Adenosine Triphosphate
keywords: 3V; ATPases; ATPe; ATPi; CBX; CTZ; Erythrocyte; Extracellular ATP; GEFs; J(R); J(V); MST; PBC; Pannexin 1; Vr; a cAMP activating cocktail containing isoproterenol, forskolin and papaverine; carbenoxolone; cilostazol; extracellular ATP; flux of ATP release; flux of ATPe hydrolysis; guanine exchange factors; intracellular ATP; mastoparan; pannexin 1; pannexin 1 heterozygous mice; pannexin 1 knockout mice, PTX, pertussis toxin; pannexin 1 wild type mice; pnx; pnx(+/+); pnx(+/−); pnx(−/−); probenecid; rbcs; red blood cells; relative cell volume