Cultured neurons from embryonic rat brain display central type benzodiazepine receptors characterized by high-affinity binding of [3H]flunitrazepam which is allosterically enhanced in the presence of γ-aminobutyric acid (GABA). A 48 h treatment of the cultured neurons with 1 μM diazepam, 0.1 μM clonazepam or 0.1 μM β-carboline ester derivatives did not change either Bmax or KD values of the [3H]flunitrazepam specific binding. A 48 h incubation in the presence of GABA (1 mM) or muscimol (0.1 mM) induced a 30% decrease of the Bmax value of [3H]flunitrazepam specific binding without change of the KD value. The down-regulation was dependent on GABA concentrations and temperature, and was partially inhibited by bicuculline but not by the benzodiazepine antagonist Ro 15–1788. The other subunits of the benzodiazepine-GABA-chloride channel receptor complex also seemed to be down-regulated by GABA since there was a decrease of the specific binding of [3H]muscimol and [35S]t-butylbicyclophosphorothionate (TBPS) to the GABAA and choride channel sites respectively. The GABA-induced down-regulation of the GABA-benzodiazepine receptor seems to be selective since the specific binding of ligands to other receptors was not affected. Our results suggests that activation of the low-affinity GABA subunit which is involved in cellular electrophysiological responses, induced the receptor down-regulation.

Benzodiazepine receptors have been studied on primary neuronal culture of foetal rat brain using different labelled ligands. The agonists [(3)H]flunitrazepam and [(3)H]diazepam, the inverse agonist [(3)H]methyl-?-carboline-3-carboxylate and the antagonist [(3)H]Ro 15-1788 were used. The binding properties of these ligands to homogenate from cultured neurons are very similar to those determined on receptors mediating the pharmacological effects of benzodiazepines in the brain. The [(3)H]ligands behaved differently when the binding was performed with intact cells. Among them, [(3)H]Ro 15-1788 offers two advantages making it fitted for binding to intact cells: it shows a low non-specific binding and a specificity for "central type" of benzodiazepine receptors. On the contrary, [(3)H]diazepam is useless because of its high non-specific binding. In cultured neurons the benzodiazepine receptors are coupled to GABA receptors: this is shown by the enhancement of [(3)H]flunitrazepam binding by GABA and its reduction by bicuculline.

Binding properties of [3H] dexetimide , L-quinuclidinyl[phenyl-4-3H] benzilate and [3H]methylscopolamine were compared with intact 108 CC 15 cells and membrane preparations of those. The ability of the three ligands to label specifically muscarinic receptors on membrane fractions was quite similar. By contrast, when performed with intact cells, [3H] dexetimide and L-quinuclidinyl [phenyl-4-3H]benzilate revealed higher nonspecific binding which was prevented by methylamine, suggesting a trapping of the ligands within the cells presumably in the lysosomes. To the contrary, such nonspecific 'binding' or trapping was not detectable when [3H]methylscopolamine was used as ligand, a fact which makes this ligand particularly appropriate for labelling cell surface muscarinic receptors. It is concluded that more caution is needed in binding studies when performed with intact cells; indeed, besides specific binding on receptor sites, [3H]ligand can be entrapped within the cell and can even sometimes give the illusion of specific binding. The use of lysosomal agents which do not interfere with specific receptors on membrane preparations should allow one, in most cases, to discard the possibility of a trapping phenomenon in intact cells.

Binding on/in whole cells seems to be a more appropriate approach for studying receptor sites in physiological conditions. However, certain difficulties encountered throughout the characterization of [3H]spiperone binding in human lymphocytes led us to reconsider this problem. The IC50 values of [3H]spiperone binding to human lymphocytes did not correlate with those found in rat striatum; domperidone was inactive in lymphocytes whereas it is one of the most potent dopamine antagonists in rat striatal preparations in vitro. In contrast, chloroquine, a lysosomotropic drug, displaced [3H]spiperone at low concentration in intact lymphocytes but did not in the striatum. [3H]Spiperone binding was not displaceable in the membrane preparation of lymphocytes. Similar results were obtained with other intact cells, fibroblasts, hepatocytes and neuroblastoma cells using [3H]spiperone and other ligands, such as [3H]haloperidol, [3H]pyrilamine and [3H]ketanserin. Here again, displaceable binding was only present in intact cells but not in membrane fractions. Such a 'displaceable' binding was not related to receptor sites but may be regarded as non-specific binding which should correspond to a trapping phenomenon presumably in the lysosomes. Binding studies on intact cells need more caution than when performed on membrane preparations; indeed, permeation or trapping of ligands in the nanomolar range represents a serious drawback which, sometimes, can give the illusion of specific binding.

A rapid carbachol‐induced disappearance of muscarinic cell surface receptors was shown using [ 3 H]methyl scopolamine as ligand on intact 108CC15 hybrid cells or rat cerebellar cells. This phenomenon is temperature‐dependent, correlated to agonist stimulation and reversible. In these short time periods (⩽30 min), no change was observed in the total receptor amount measured on membrane preparations. This disappearance of cell surface receptors could represent the first event in cell desensitization which could be followed by receptor recycling in physiological conditions or by receptor degradation if the stimulation by agonists persists, as in long‐term regulation.