Characteristics and regulation of high affinity [ 3H]imipramine binding to rat hippocampal membranes

The imipramine binding sites located in the membrane of the rat hippocampus have been characterized in order to obtain a better understanding of their role in the treatment of mental depression. The binding of [ 3H]imipramine to hippocampal membranes has a K d of 8.3 ± 1.5 nM, B max of 800 ± 60 fmol/mg protein and a Hill coefficient of 1.0. The number of specific binding sites for [ 3H]imipramine was rapidly and irreversibly reduced when the incubation temperature was raised from 0 to 37°C. Experiments using leupeptin and EGTA suggest that this temperature-dependent inactivation is due to a destruction of the binding sites by Ca 2+-dependent proteases released upon disruption of the cells. The binding of [ 3H]imipramine to the hippocampal membrane is also sensitive to phospholipase A 2 as well as pronase, suggesting the complexity of the regulation of this binding. The [ 3H]imipramine binding sites appear to have been solubilized from membranes by treatment with Triton X-100. However, these solubilized binding sites also display a high level of nonspecific binding of this ligand. Studies of tissue distribution and subcellular localization of the imipramine binding sites suggest that these sites are located in neurons. This possibility is strengthened by the finding that the high affinity imipramine binding sites are also detected in the membranes of neuroblastoma N4TG1 cells.