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Uptake of N-methyl-4-phenylpyridinium ion (MPP(+)) into PC12h pheochromocytoma cells
T Takahashi1, M Naoi, T Nagatsu
1Department of Biochemistry, Nagoya University School of Medicine, Nagoya, Japan.
Abstract:
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 ?M, 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.
Insights
Neurotoxin N-methyl-4-phenylpyridinium ion (MPP(+)) enters PC12h cells via two distinct carrier-mediated transport systems. This uptake is influenced by dopamine transporters and metabolic processes, with MPP(+) accumulating in the cytosol and mitochondria.
Area of Science:
- Neuroscience
- Cell Biology
- Toxicology
Background:
- N-methyl-4-phenylpyridinium ion (MPP(+)) is a neurotoxin formed from MPTP oxidation.
- PC12h pheochromocytoma cells are a model system for studying neuronal processes.
Purpose of the Study:
- To investigate the uptake and accumulation mechanisms of MPP(+) in PC12h cells.
- To characterize the kinetic properties of MPP(+) transport systems.
- To identify the cellular localization of accumulated MPP(+).
Main Methods:
- Uptake studies using varying concentrations of MPP(+).
- Kinetic analysis to determine Michaelis-Menten constants (K(m)) and maximal velocities (Vmax).
- Inhibition studies with specific transporter blockers and metabolic inhibitors.
- Subcellular fractionation to determine MPP(+) localization.
Main Results:
- MPP(+) uptake is mediated by two saturable, carrier-dependent transport systems: high-affinity/low-capacity and low-affinity/high-capacity.
- The high-affinity system shares carriers with dopamine and noradrenaline transporters, inhibited by nomifensine and mazindol.
- Uptake is sensitive to metabolic inhibitors (e.g., cyanide, dinitrophenol) and ionophores (ouabain, nigercin).
- MPP(+) primarily localizes to the cytosol, with a significant portion also found in mitochondria.
Conclusions:
- PC12h cells possess distinct, carrier-mediated systems for MPP(+) uptake.
- The high-affinity transport is linked to monoamine transporters, suggesting a mechanism relevant to Parkinson's disease pathogenesis.
- MPP(+) accumulation is an energy-dependent process, with mitochondrial localization potentially contributing to neurotoxicity.
