Binding of3H-desipramine to the neuronal noradrenaline carrier of rat phaeochromocytoma cells (PC-12 cells)

Summary The specific (i.e., nisoxetine-sensitive) binding of³H-desipramine was studied in purified plasma membranes of PC-12 cells (rat phaeochromocytoma cells).³H-desipramine bound reversibly and with high affinity (K _D=4.5 nmol/l) to a single, non-interacting site (Hill coefficient=1.04); the maximal number of binding sites (B _max) was 19.6 pmol/mg protein.Like the uptake of noradrenaline (by uptake₁), the binding of³H-desipramine was dependent on both sodium and chloride. The stimulation of binding by chloride and sodium was characterized by a Hill coefficient of about 1 and 2, respectively. Both, chloride and sodium, slowed the rate of dissociation of bound³H-desipramine. Increasing concentrations of sodium decreased theK _D of³H-desipramine binding without altering theB _max.The binding of³H-desipramine was inhibited by tricyclic antidepressants and other noradrenaline uptake blockers. There was a highly significant correlation between the potencies of a series of drugs for the inhibition of³H-desipramine binding and for the inhibition of³H-noradrenanne uptake into intact PC-12 cells. Both, binding of³H-desipramine and uptake of³H-noradrenaline, were stereoselectively inhibited by the enantiomers of cocaine and oxaprotiline. However, for most of the substrates of uptake₁ the IC₅₀ for inhibition of³H-desipramine binding was much higher than that for inhibition of³H-noradrenaline uptake. Nevertheless, noradrenaline competitively inhibited³H-desipramine binding and unmasked dissociation of bound³H-desipramine. Thus,³H-desipramine probably binds to the substrate recognition site.From theB _max of³H-desipramine binding to PC-12 membranes and from theV _max of³H-noradrenaline uptake into PC-12 cells, a duration of about 400 ms for the transport cycle for a single noradrenaline molecule was calculated. In addition, from theB _max the maximum number of³H-desipramine binding sites (carriers) for a single PC-12 cell was calculated to be 55,000.A part of this study was presented at the IUPAHR 9th International Congress of Pharmacology (Bönisch et al. 1984)This study was supported by the Deutsche Forschungsgemeinschaft (Bo 521 and SFB 176)