CHARACTERIZATION OF ADENOSINE RECEPTORS IN A MODEL OF CULTURED NEURONS FROM RAT FOREBRAIN

The neuromodulator adenosine is acting through specific receptors coup led to adenylate cyclase via G-proteins. The expression of both adenos ine receptors A(1) and A(2) as well as forkolin binding sites was inve stigated by radioligand binding techniques in 8-day-old neurons isolat ed from fetal rat forebrain and cultured in chemically-defined medium. Adenosine A(1) receptors were specifically labeled with [H-3]chloro-N -6-cyclopentyladenosine (CCPA), whereas [H-3]CGS 21680 was used for th e analysis of A, receptors. Cultured neurons exhibited high affinity b inding sites for CCPA (Bmax = 160 fmol/mg protein; Kd = 2.9 nM), and f or CGS 21680 (Bmax = 14 fmol/mg protein; Kd = 1.7 nM). These data corr elate well with those obtained in crude membranes isolated from the ne wborn rat forebrain. The incubation of culture membranes in the additi onal presence of guanylyl-5'-imidodiphosphate (Gpp(NH)p, a GTP analogu e) led to significantly increased Kd-values, suggesting the associatio n of adenosine receptors with G-proteins. Finally, cultured neurons al so bound specifically [H-3]forskolin with characteristics close to tho se found in the newborn brain, indicating that cultured neurons appear as an appropriate model for studying the neuromodulatory properties o f adenosine.