Inhibition of neuronal noradrenaline uptake (uptake1) and desipramine binding by N-ethylmaleimide (NEM).

The inhibition of N-ethylmaleimide (NEM) of uptake1 and desipramine binding was studied on clonal rat phaeochromocytoma cells (PC12 cells) in different experimental settings: (1) 3H-noradrenaline uptake into intact PC12 cells; (2) 3H-noradrenaline uptake into isolated PC12 plasma membrane vesicles; (3) 3H-desipramine binding to isolated PC12 plasma membrane vesicles. In plasma membrane vesicles, NEM inhibited 3H-desipramine binding and 3H-noradrenaline uptake with similar potency (the IC50's were 1.36 mmol/l and 1.04 mmol/l, respectively). However, in intact cells, NEM was about 75 times more potent in inhibiting 3H-noradrenaline uptake (IC50 = 0.014 mmol/l). The increased potency of NEM in intact cells is probably due to an inhibition of the Na+/K+-ATPase and not to a direct interaction with the noradrenaline carrier. The inactivation by NEM of 3H-desipramine binding to PC12 plasma membrane vesicles was irreversible. Both an inhibitor (cocaine, 1 mmol/l) and a substrate of uptake1 (amezinium, 1 mmol/l) protected desipramine binding from inactivation. These results are compatible with the hypothesis of a common binding site for substrates and inhibitors of the neuronal noradrenaline carrier.