Kinetics of extracellular ATP in mastoparan 7-activated human erythrocytes

• Published in: Biochimica et biophysica acta 2013-10, Vol.1830 (10), p.4692-4707
• Main Authors: Leal Denis, María Florencia, Incicco, J. Jeremías, Espelt, María Victoria, Verstraeten, Sandra V., Pignataro, Omar P., Lazarowski, Eduardo R., Schwarzbaum, Pablo J.
• Format: Article
• Language: English
• Subjects: adenosine triphosphate; Adenosine Triphosphate - metabolism; adhesion; Animals; ATP; ATPases; cell adhesion; cyclic AMP; data analysis; Dogs; Erythrocyte; erythrocytes; Erythrocytes - drug effects; Erythrocytes - metabolism; Extracellular ATP; Humans; Hydrolysis; Kinetics; knockout mutants; Mice; Mice, Knockout; Pannexin 1; Peptides - pharmacology; Signal Transduction
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2013.05.033

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). 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. 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. 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. Kinetic analysis of extracellular ATP accumulation from human erythrocytes and potential effects on microcirculation. •Kinetic analysis of extracellular ATP regulation in human erythrocytes•Mastoparan 7 induced two pathways of ATP release.•Cell adhesion, cAMP and swelling modulate mastoparan 7 induced ATP efflux.•A data driven-model account for the extracellular ATP kinetics