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.
Nicolas
F
F
INSERM U.272, Université de Nancy, France.
Oillet
J
J
Koziel
V
V
Daval
J L
JL
D000241:Adenosine / Q000031:analogs & derivatives / Q000378:metabolism; D000262:Adenylyl Cyclases / Q000378:metabolism; D000375:Aging / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D000959:Antihypertensive Agents / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004622:Embryo, Mammalian; D005260:Female; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D007700:Kinetics; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D010627:Phenethylamines / Q000378:metabolism; D016548:Prosencephalon / Q000254:growth & development / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D018047:Receptors, Purinergic P1 / Q000378:metabolism*; D014316:Tritium
D000959:Antihypertensive Agents; D010627:Phenethylamines; D018047:Receptors, Purinergic P1; D014316:Tritium; C061282:2-(4-(2-carboxyethyl)phenethylamino)-5'-N-ethylcarboxamidoadenosine; D006165:Guanylyl Imidodiphosphate; C058394:2-chloro-N(6)cyclopentyladenosine; D000262:Adenylyl Cyclases; D000241:Adenosine