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

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Abstract
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.

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