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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 A1 adenosine receptor (A1AR) signaling pathway in rat cortical membranes. Membrane treatment with 0.5 mM SIN-1 for various periods of time (0–240 min) decreased specific binding of the radiolabeled A1AR agonist, [3H]N6-cyclohexyladenosine ([3H]CHA), in a time-dependent manner, reaching the steady state after 120 min. The inhibitory effect of SIN-1 was concentration-dependent, with an ec50 value of 0.60±0.30 mM (N=3). Membrane pre-incubation with the superoxide anion (O2•−) scavenger superoxide dismutase (SOD) followed by SIN-1 addition, abolished SIN-1 inhibition of [3H]CHA binding. Membrane treatment with 0.5 mM SIN-1 for 120 min caused a significant 2-fold increase of the KD value for [3H]CHA without changing the Bmax value. Moreover, pre-incubation of membranes with A1AR agonists, CHA or N6-(2-phenylisopropyl)-adenosine (R-PIA) before SIN-1 addition increased the inhibitory effect while the selective A1AR antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) had no activity. Membrane treatment with SIN-1 decreased receptor-stimulated guanosine 5′-O-(γ[35S]thio)triphosphate ([35S]GTPγS) binding in a concentration-dependent manner. This treatment influenced [35S]GTPγS binding affinity for A1AR activated Gi proteins in cortical membranes. These findings suggest that ONOO− modulates A1AR signaling pathways by affecting receptor Gi protein coupling.