Characterization of adenosine receptors in a model of cultured neurons from rat forebrain

The neuromodulator adenosine is acting through specific receptors coupled to adenylate cyclase via G-proteins. The expression of both adenosine receptors A1 and A2 as well as forskolin binding sites was investigated by radioligand binding techniques in 8-day-old neurons isolated from fetal rat forebrain and cultured in chemically-defined medium. Adenosine A1 receptors were specifically labeled with [3H]chloro-N6-cyclopentyladenosine (CCPA), whereas [3H]CGS 21680 was used for the analysis of A2 receptors. Cultured neurons exhibited high affinity binding sites for CCPA (Bmax = 160 fmol/mg protein; Kd = 2.9 nM), and for CGS 21680 (Bmax = 14 fmol/mg protein; Kd = 1.7 nM). These data correlate well with those obtained in crude membranes isolated from the newborn rat forebrain. The incubation of culture membranes in the additional presence of guanylyl-5'-imidodiphosphate (Gpp(NH)p, a GTP analogue) led to significantly increased Kd-values, suggesting the association of adenosine receptors with G-proteins. Finally, cultured neurons also bound specifically [3H]forskolin with characteristics close to those found in the newborn brain, indicating that cultured neurons appear as an appropriate model for studying the neuromodulatory properties of adenosine.