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.

General significance: Kinetic analysis of extracellular ATP accumulation from human erythrocytes and potential effects on microcirculation.

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.