EPACs Inhibit ATP Release from Human Erythrocytes (RBCs) in Response to Activation of GPCRs possibly via Activation of PKC.

Abstract

Increases in cAMP are required for ATP release from RBCs and are regulated by phosphodiesterases (PDEs). Selective PDE inhibitors potentiate increases in cAMP and ATP in response to activation of either the prostacyclin or β adrenergic receptor in these cells. Here we demonstrate that non‐selective PDE inhibition attenuates ATP release in response to activation of either receptor. We hypothesize that increases in cAMP not associated with receptor activation stimulate exchange proteins activated by cAMP (EPACs) that inhibit ATP release via PKC activation. 8pCPT2OMecAMP (8pCPT, 10μM), a direct EPAC activator inhibited both iloprost (ILO 10nM, n=6)‐and isoproterenol (1μM, n=6)‐induced ATP release. To demonstrate that this inhibition was not due to activation of PKA, RBCs were incubated with 6BnzcAMP (30μM), a direct PKA activator, followed by ILO. PKA activation potentiated ATP release (n=2). To determine if 8pCPT‐induced inhibition of ATP release was via activation of PKC, RBCs were treated with PMA (1μM) prior to ILO. PMA inhibited ILO‐induced ATP release (n=6). 8pCPT also inhibited ATP release with forskolin (10μM, n=6), a direct activator of adenylyl cyclase, indicating that inhibition of ATP release is downstream of cAMP synthesis. These results support the hypothesis that increases in cAMP within RBCs can activate EPACs which inhibit ATP release, possibly via PKC activation. [NIH grants HL‐64180 & ‐89094]