Homeostasis of Extracellular ATP in Human Erythrocytes

MONTALBETTI NICOLÁS*; LEAL DENIS MARÍA FLORENCIA*; PIGNATARO OMAR P.; KOBATAKE EIRY; LAZAROWSKI EDUARDO R.; SCHWARZBAUM PABLO JUIO

JOURNAL OF BIOLOGICAL CHEMISTRY (ONLINE)

American Society for Biochemistry and Molecular Biology.

Año: 2011 vol. 286 p. 38397 - 38407

1083-351X

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 (10Panx1) decreased [ATP]e by 75%. The residual efflux of ATP resulted from unavoidable mechanical perturbations stimulating a novel, carbenoxolone-insensitive pathway. In real-time luminometry experiments using soluble luciferase, addition of 3V led to an acute increase in [ATP]e to a constant value of 1 pmol (106 cells)1. A similar treatment using a surface attached luciferase (proA-luc) triggered a rapid accumulation of surface ATP levels to a peak concentration of 2.4 pmol(106 cells)1, followed by a slower exponential decay (t1⁄2 = 3.7 min) to a constant value of 1.3 pmol (106 cells)1. 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 (28 fmol (106 cells min)1), but nevertheless physiologically relevant considering the high density of erythrocytes in human blood.