Using receptor-selective agonists and antagonists, the possible presence of both A2a and A2b adenosine receptor subtypes coupled to activation of adenylyl cyclase was investigated in NG108-15 neuroblastoma x glioma hybrid cells. The relatively non-selective adenosine receptor agonist 5'-(N-ethyl carboxamido)-adenosine (NECA; 1 nM-300 microM) produced a biphasic increase in adenylyl cyclase activity in cell homogenates, best fitted to two components with high (EC50 0.7 microM) and low (EC50 16.0 microM) potency, respectively. The selective adenosine A2a receptor agonist CGS-21680 (1 nM-300 microM) also produced a biphasic increase in adenylyl cyclase. The NECA-dependent increase in adenylyl cyclase activity was almost completely inhibited by the non-selective adenosine receptor antagonist xanthine amine congener (XAC; 30 microM), but only partially inhibited by the selective A2a adenosine antagonist 8-(3-chlorostyryl)caffeine (CSC; 1 microM). Experiments were also performed to investigate the time course of NECA-induced desensitization of putative A2a and A2b receptor responses. The A2a-response was quantified using 10 microM CGS-21680, whilst the A2b response was quantified using 100 microM NECA in the presence of 1 microM CSC. The t0.5 for desensitization for each subtype was found to be around 20 min. Neither activation (with dibutyryl cAMP; 1 mM) nor inhibition (with H-89; 10 microM) of cyclic AMP-dependent protein kinase altered the ability of NECA pretreatment to desensitize A2a or A2b receptor-activated adenylyl cyclase. However zinc (200 microM), an inhibitor of G-protein coupled receptor kinase 2 (GRK2), significantly reversed the agonist-induced desensitization of A2a and A2b receptor-activated adenylyl cyclase. These experiments suggest the co-existence of A2a and A2b receptors coupled in a stimulatory fashion to adenylyl cyclase in NG108-15 cells. Furthermore desensitization of A2a and A2b responses occurs at the same rate and may involve a G-protein-coupled receptor kinase.