Kinetics of extracellular ATP in mastoparan 7-activated human erythrocytes

MARÍA FLORENCIA LEAL DENIS; JUAN JEREMÍAS INCICCO; MARÍA VICTORIA ESPELT; SANDRA V. VERSTRAETEN; PIGNATARO OMAR P.; LAZAROWSKI EDUARDO R.; PABLO JULIO SCHWARBAUM

BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS

ELSEVIER SCIENCE BV

Lugar: Amsterdam; Año: 2013 vol. 1830 p. 4692 - 4707

0304-4165

Background: The peptide mastoparan 7 (MST7) stimulated ATP release in human erythrocytes.We explored intraand 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 10 μM 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 drivenmodel suggested that ATP efflux is mediated by two ATP conduits displaying different kinetics, with one conduit being fully blocked by pannexin 1 blockers. General significance: Kinetic analysis of extracellular ATP accumulation from human erythrocytes and potential effects on microcirculation.