REGULATED ATP RELEASE FROM HUMAN ERYTHROCYTES

LEAL DENIS MF,MONTALBETTI N, SCHWARZBAUM PJ

Congreso; XXV Reunião Anual Federação de Sociedades de Biologia Experimental (FeSBE); 2010

In circulating human erythrocytes (RBCs) a large steady pool of intracellular ATP is generated by glycolysis. Release of intracellular ATP is reported to occur in response to several physiological stimuli such as hipoxia, acidosis or mechanical stress. Previous reports have shown that cAMP is an important mediator for this non-lytic ATP efflux by participating in a signaling pathway involving heterotrimeric G proteins Gi and Gs, adenylate cyclase and protein kinase A. In this study we evaluate the kinetics of Gi-induced ATP release by quantitating the kinetic of extracellular ATP (ATPe) concentration of RBCs. Exposure of cells to 10 µM mastoparan-7 (Mas-7), that stimulates Gi, led to a bi-exponential increase of [ATP]e to a constant steady value. Lack of a post-activation decrease of [ATP]e is consistent with an ectoATPase activity being extremely low (≈0.3 nmol Pi/1010cells/min at 300 µM ATP). Mas-7 induced release of ATP was inhibited 86% by pertussis toxin (which blocks Gi activation). and 58% by a peptide (10Panx1) known to selectively block the ATP channel candidate Pannexin 1. A comparison with ATP release caused by Ga activation (by isoproterenol-forskolin-papaverin treatment) shows that in RBCs both Ga and Gi activation lead to a regulated ATP release partly mediated by Pannexin 1. Irrespective of the mechanisms enabling ATP transmembrane transport, the fact that the Mas-7 induced increase in [ATP]e to a constant [ATP]e maximum value is consistent with transient activation of a non lytic ATP release mechanism.