Solubilization and characterization of the3H-desipramine binding site of rat phaeochromocytoma cells (PC12-cells)

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Abstract
3H-Desipramine binding sites of the plasma membranes of rat phaeochromocytoma cells (PC12-cells) were solubilized with the nonionic detergent digitonin (0.5%). With the method described here, the binding characteristics of the desipramine binding site were essentially unaltered by solubilization. Binding of3H-desipramine to the solubilized binding site showed the following characteristics: (1)3H-desipramine bound with high affinity (KD=16.6 nmol/l) to a single class of noninteracting (Hill-coefficient=1.01) binding sites; (2) binding was reversible; (3) binding of unlabelled desipramine had the same dissociation constant as had3H-desipramine; (4) increasing concentrations of sodium- and chloride-ions stimulated the binding of3H-desipramine; (5) binding was inhibited by various inhibitors and substrates of neuronal uptake of noradrenaline; and (6) inhibition of binding by the optical isomers of cocaine, oxaprotiline, and amphetamine showed marked stereoselectivity (with preference for (−)cocaine, (+)oxaprotiline, and (+)amphetamine). The finding that the binding of3H-desipramine to the solubilized binding site was dependent on sodium and chloride, as the neuronal uptake of noradrenaline is, and the finding that all substrates of uptake1 inhibited the binding of3H-desipramine, is consistant with the view that desipramine binds to the substrate recognition site of the neuronal carrier for noradrenaline.

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