Modulation of A1 adenosine receptor signaling by peroxynitrite

Nitric oxide (NO) is a gaseous free radical involved in many pathophysiological processes. During oxidative stress, NO, its derivatives and adenosine are released. Considering adenosine neuroprotective role in the central nervous system (CNS) and toxicity of NO, we investigated the effect of a NO/peroxynitrite (ONOO(-)) donor, 3-morpholinosydnonimine (SIN-1), on A(1) adenosine receptor (A(1)AR) signaling pathway in rat cortical membranes. Membrane treatment with 0.5mM SIN-1 for various periods of time (0-240min) decreased specific binding of the radiolabeled A(1)AR agonist, [3H]N(6)-cyclohexyladenosine ([3H]CHA), in a time-dependent manner, reaching the steady state after 120min. The inhibitory effect of SIN-1 was concentration-dependent, with an EC(50) value of 0.60+/-0.30mM (N=3). Membrane pre-incubation with the superoxide anion (O(2)z.rad;(-)) scavenger superoxide dismutase (SOD) followed by SIN-1 addition, abolished SIN-1 inhibition of [3H]CHA binding. Membrane treatment with 0.5mM SIN-1 for 120min caused a significant 2-fold increase of the K(D) value for [3H]CHA without changing the B(max) value. Moreover, pre-incubation of membranes with A(1)AR agonists, CHA or N(6)-(2-phenylisopropyl)-adenosine (R-PIA) before SIN-1 addition increased the inhibitory effect while the selective A(1)AR antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) had no activity. Membrane treatment with SIN-1 decreased receptor-stimulated guanosine 5'-O-(gamma[35S]thio)triphosphate ([35S]GTPgammaS) binding in a concentration-dependent manner. This treatment influenced [35S]GTPgammaS binding affinity for A(1)AR activated G(i) proteins in cortical membranes. These findings suggest that ONOO(-) modulates A(1)AR signaling pathways by affecting receptor G(i) protein coupling.