Evidence for co-expression and desensitization of A2a and A2b adenosine receptors in NG108-15 cells

Using receptor-selective agonists and antagonists, the possible presence of both A(2A) and A(2b) 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 mu M) produced a biphasic increase in adenylyl cyclase activity in cell homogenates, best fitted to two components with high (EC50, 0.7 mu M) and low (EC50 16.0 mu M) potency, respectively. The selective adenosine A,, receptor agonist CGS-21680 (1 nM-300 mu M) 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 mu M), but only partially inhibited by the selective Az, adenosine antagonist 8-(3-chlorostyryl)caffeine (CSC; 1 mu M) Experiments were also performed to investigate the time course of NECA-induced desensitization of putative A(2a) and A(2b), receptor responses. The A(2a)-response was quantified using 10 mu M CGS-21680, whilst the A(2b) response was quantified using 100 mu M NECA in the presence of 1 mu M CSC The t(0.5) 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 mu M) Of cyclic AMP-dependent protein kinase altered the ability of NECA pretreatment to desensitize A,, or A,, receptor-activated adenylyl cyclase. However zinc (200 mu M), an inhibitor of G-protein coupled receptor kinase 2 (GRK2), significantly reversed the agonist-induced desensitization of A(2b) and A(2b) receptor-activated adenylyl cyclase. These experiments suggest the co-existence of A(2a) and A(2b) receptors coupled in a stimulatory fashion to adenylyl cyclase in NG108-15 cells. Furthermore desensitization of A(2a) and A(2b) responses occurs at the same rate and may involve a G-protein-coupled receptor kinase. (C) 1998 Elsevier Science Inc.