REGULATED ATP RELEASE FROM HUMAN ERYTHROCYTES.

LEAL DENIS MARÍA FLORENCIA; MONTALBETTI NICOLÁS; SCHWARZBAUM PABLO JUIO

Aguas de Lindoia, San Pablo. Brasil

Congreso; 1° Encontro do CLUBE BRASILEIRO DAS PURINAS: SINALIZACAO PURINÉRGICA E IMPLICACOES TERAPÊUTICAS; 2010

In circulating human red blood cells (RBCs), as in most cell types, intracellular ATP concentrations generated via a glycolytic pathway are present in the millimolar range. Release of intracellular ATP into the surrounding environment 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 signalling pathway involving heterotrimeric G proteins Gi and Gs, adenylate cyclase and protein kinase A (PKA). In this study we evaluate the kinetics of Gi-induced ATP release by quantitating the kinetic of extracellular ATP (ATPe) accumulation 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. Pretreatment of RBCs with pertussis toxin, which catalyzes the mono-ADP-ribosylation of the α-subunit of Gi/s preventing its dissociation from the βγ-subunit complex, led to a 86% inhibition of Mas-7-induced ATP release. On the other hand as is well known, erythrocytes express the gap junction protein pannexin 1 (Panx-1), which can form a mechanosensitive and ATP-permeable channel in the nonjunctional plasma membrane, consistent with a role of Panx-1 as an ATP release channel, ATP release by RBCs was attenuated in a 58% when they were pretreated with the Panx-1-selective blocking peptide. A comparison with ATP release caused by Gs activation (by isoproterenol-forskolin-IBMX treatment) shows that in RBCs both Gs and Gi activation lead to a regulated ATP release mediated by Panx-1.