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 (IC50 = 15 microM). Pretreatment with 10 microM
xylamine did not inhibit
norepinephrine transport if 10 microM
cocaine or 100 microM
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 [3H]
xylamine; this uptake had Na+-dependent and Na+-independent components. The Na+-dependent uptake was saturable (
Km = 13 microM), and it was inhibited by
cocaine (IC50 = 0.6 microM),
desipramine (IC50 less than 1 nM), and
norepinephrine (IC50 = 1 microM). Several
proteins became prominently labeled when intact PC12 cells were incubated with [3H]
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 [3H]
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. [3H]
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 [3H]
xylamine-labeled
proteins is associated with the
norepinephrine transporter.