Characterization of adenosine receptors in a model of cultured neurons from rat forebrain |
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Authors: | F Nicolas J Oillet V Koziel J L Daval |
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Institution: | (1) INSERM U.272, Université de Nancy I, 30 rue Lionnois, B.P. 3069, 54013 Nancy Cédex, France |
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Abstract: | 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 forkolin 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. |
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Keywords: | Adenosine receptors forskolin G-proteins binding cultured neurons rat forebrain |
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