We explored the intra-and extracellular processes governing the kinetics of extracellular ATP (ATPe) in human erythrocytes stimulated with agents that increase cAMP. Using the luciferinluciferase reaction in off-line luminometry we found both direct adenylyl cyclase activation by forskolin and indirect activation through ␤-adrenergic stimulation with isoproterenol-enhanced [ATP]e in a concentration-dependent manner. A mixture (3V) containing a combination of these agents and the phosphodiesterase inhibitor papaverine activated ATP release, leading to a 3-fold increase in [ATP]e, and caused increases in cAMP concentration (3-fold for forskolin ؉ papaverine, and 10-fold for 3V). The pannexin 1 inhibitor carbenoxolone and a pannexin 1 blocking peptide ( Both for soluble luciferase and proA-luc, ATP efflux was fully blocked by carbenoxolone, pointing to a 3V-induced mechanism of ATP release mediated by pannexin 1. Ecto-ATPase activity was extremely low (ϳ28 fmol ؋ (10 6 cells min) ؊1 ), but nevertheless physiologically relevant considering the high density of erythrocytes in human blood.All cell types appear to possess mechanisms that enable a controlled, nonlytic release of ATP, that is, a release not involving cell membrane rupture, and which occurs in response to osmotic, mechanical, and/or neurohormonal stimuli (1). Specifically human erythrocytes release ATP following exposure to ␤-adrenergic stimulation, mechanical deformation, reduced oxygen tension, or acidosis (2). All of these represent physiological conditions to which erythrocytes are exposed in the vasculature, e.g. when passing through constricted vessels or in the contracting striated muscle (3). Once in the extracellular medium, extracellular ATP (ATPe) 5 can trigger different cellular responses by interacting with P receptors on the cell surface while at the same time its concentration is controlled by the activities of one or more ectonucleotidases (4, 5). Several reports published over recent years have shown that an increase in intracellular cyclic AMP (cAMP) concentration triggered ATP release from human erythrocytes (6, 7). Receptor-mediated ATP release in human erythrocytes involves activation of heterotrimeric G proteins G s or G i/o (3, 8, 9). Regarding the G s pathway, activation of ␤-adrenergic receptors by various agonists was reported to stimulate adenylyl cyclase, with concomitant increases in cAMP levels and protein kinase A activity (6, 10). Moreover, direct activation of adenylyl cyclase by forskolin resulted in both ATP release and cAMP increases in human and rabbit erythrocytes (6).Despite the accumulated knowledge regarding the intracellular signaling events mediating ATP release, comparatively little is known regarding the processes governing the kinetics of ATPe accumulation at the surface in animal cells (11,12), with rates of intracellular ATP release and extracellular ATP hydrolysis being the main actors. The human non-nucleated erythrocyte is an excellent model in this respect, because it lacks intracellular compartments and...

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.

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