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

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 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.

Endogenous Expression of Adenosine A1, A2 and A3 Receptors in Rat C6 Glioma Cells

“…Similar results for adenosine A 1 receptor have also been described in cerebellar granule cells [49] and primary neuronal culture [10]. Since all assays in intact cells were performed in the presence of dipyridamole, a potent inhibitor of adenosine uptake, the increase detected in B max value from intact cells cannot be due to the binding to a transporter protein [9].…”

“…However, binding parameters were higher than in plasma membranes. The decrease in total receptor numbers (B max ) detected in membrane preparations versus intact cells has been reported for [ 3 H]CCPA and [ 3 H]CGS 21680 binding to culture neurons from fetal rat forebrain and could be explained by an alteration of adenosine receptors following cell homogenization [9]. Similar results for adenosine A 1 receptor have also been described in cerebellar granule cells [49] and primary neuronal culture [10].…”

“…A 2A receptors are highly expressed in striatum [7] while A 2B receptors are more diffusely distributed in the brain, as well as being highly expressed in the small intestine and colon [8]. Cell cultures have been widely used to study adenosine receptors, which have been described in primary cultures of neurons [9,10,11] and astrocytes [12].…”

A Simple Method for Evaluation of Superoxide Radical Production in Neural Cells under Various Culture Conditions: Application to Hypoxia

“…Whole embryos were placed in cul ture medium previously equilibrated at 37°C, and forebrains were carefully collected. Neuronal cell suspensions were then obtained as described previ ously (Daval et al, 1991;Nicolas et al, 1994). Brain tissues were dissected free of meninges and gently dispersed in a mixture of Dulbecco modified Ea gle' s medium (DMEM) and Ham' s F12 medium (50:50) supplemented with 5% inactivated fetal calf serum.…”

Adenosine A1 Receptor in Cultured Neurons from Rat Cerebral Cortex

“…This problem can be overcome by specific primary cell cultures. A 1 R has been studied in primary cultures of rat cerebellar granule cells (Hettinger‐Smith et al, 1996 ; Sanz et al, 1996 ; Vendite et al, 1998) and rat forebrain (Nicolas and Daval, 1993 ; Nicolas et al, 1994). In the present work, due to the importance of the neuroprotective effects of adenosine in CNS, we have characterized A 1 Rs in primary cultures from rat cortical neurons.…”