Characterization of Xylamine Binding to Proteins of PC12 Pheochromocytoma Cells

PC12 pheochromocytoma cells take up 3,4‐dihydroxyphenylethylamine (dopamine) and norepinephrine by a Na + ‐dependent, cocaine‐sensitive system. The kinetics suggest that the same transporter functions for both substrates. Xylamine, a nitrogen mustard that blocks catecholamine uptake into neurons, irreversibly inhibited norepinephrine uptake into PC12 (IC 50 = 15 μ M ). Pretreatment with 10 μ M xylamine did not inhibit norepinephrine transport if 10 μ M cocaine or 100 μ M norepinephrine was also present during the pretreatment period or if Na + was absent. These results indicate that xylamine must interact with the norepinephrine transporter to inhibit norepinephrine uptake. PC12 accumulated [ 3 H]xylamine; this uptake had Na + ‐dependent and Na + ‐independent components. The Na + ‐dependent uptake was saturable ( K m = 13 μ M ), and it was inhibited by cocaine (IC 50 = 0.6 μ M ), desipramine (IC 50 < 1 n M ), and norepinephrine (IC 50 = 1 μ M ). Several proteins became prominently labeled when intact PC12 cells were incubated with [ 3 H]xylamine; these proteins were enriched in a plasma membrane fraction and have molecular weights of 17,000, 24,000, 31,000, 33,000, 41,000, 42,000, 52,000, and 80,000. Other proteins were labeled less prominently. The labeling of all proteins was markedly decreased when the incubation with [ 3 H]xylamine occurred in the presence of cocaine, desipramine, gramicidin D, or in a Na + ‐free buffer. These results indicate that xylamine must be transported into the cells for covalent binding to proteins to occur. [ 3 H] Xylamine labeled essentially the same proteins when incubated with cell homogenates, but competition experiments with bretylium, desipramine, and cocaine failed to reveal which of the [ 3 H]xylamine‐labeled proteins is associated with the norepinephrine transporter.