Kinetics of extracellular ATP in mastoparan 7-activated human erythrocytes

Abstract

SUMMARY 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 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 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.

Dynamic Regulation of Cell Volume and Extracellular ATP of Human Erythrocytes

“…Under certain conditions fluorescence intensity of fluorophore loaded cells decreases with the increase in fluorophore concentration. This quenching property can be used to measure water volume changes in various cell types by fluorescence microscopy [ 40 ], and has been employed by our group to assess volume regulation of human erythrocytes loaded with BCECF [ 29 , 32 ].…”

“…Accordingly, the kinetics of cell volume of rbcs before and after addition of MST7 was estimated by the quenching method using BCECF loaded cells [ 2 , 29 , 32 ]. Rbcs (3 10 6 cells) were attached to 0.001% poly-D-lysine-coated coverslips and incubated with isosmotic medium containing 5 μM BCECF-AM during 60 min at room temperature.…”

“…In hypotonic conditions, the relationship between ATPe (and byproducts) and cell volume has been almost exclusively studied in the context of a volumetric response termed volume regulatory decrease or RVD, which is present in most animal cells. Several reports showed that ATPe, and or its metabolic products generated by ectonucleotidase activity, interact with P receptors to mediate RVD [ 26 – 29 ]. It then became clear that, not only swelling could activate ATP efflux, but that the subsequent increase of [ATPe] could active P receptors and induce RVD.…”

“…That is, erythrocytes can swell or shrink as dictated only by their osmotic environment. Still, rbcs can swell under hypotonicity imposed by either a decrease of extracellular osmolarity, or an increase of intracellular osmolarity, a condition where ATP release was verified [ 29 , 32 ].…”

“…Activation of one or more ATP release pathways depends on the nature of the stimulus [ 2 , 29 ]. Under a robust hypotonic treatment rbcs were able to release ATP primarily through pannexin 1 [ 34 ], whereas mice rbcs responded to a hypotonic- depolarizing stimulus by triggering two different ATP release pathways [ 35 ].…”