Uptake of N-methyl-4-phenylpyridinium ion (MPP+) into PC12h pheochromocytoma cells

The uptake and accumulation of N-methyl-4-phenylpyridinium ion (MPP⁺), a neurotoxin produced by oxidation of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), into PC12h pheochromocytoma cells were examined. Concentration gradients of MPP⁺ were established at its low concentrations of 10 to 100 nM. Uptake of MPP⁺ into PC12h cells was mediated by saturable, carrier mediated transport systems with two different kinetic properties; a high-affinity and low-capacity system and a low-affinity and high-capacity system. The apparent K_m values of these two systems were obtained to be 254.4 ± 96.5 nM and 23.1 ± 6.9 μM, respectively, and the maximal uptake velocity was obtained to be 8.47 ± 1.72 and 28.6 ± 5.2 pmol/min/mg protein, respectively. The uptake by a high-affinity system was mediated by a carrier system common to dopamine and noradrenalin and MPTP itself proved to be taken up by this system, which was further confirmed by the inhibition of the MPP⁺ uptake by nomifensine and mazindol. The uptake was inhibited by metabolic inhibitors, such as carbonyl cyanide m-chlorophenyl hydrazone, sodium cyanide and 2,4-dinitrophenol, and the uptake was inhibited by ouabain and nigercin. By subcellular fractionation, MPP⁺ taken up was found to be localized mainly in cytosol fraction, but a definite amount of MPP⁺ was found also in mitochondrial fraction.