Presence Of MPP Research Articles

Structural Analysis of the Extracellular Entrance to the Serotonin Transporter Permeation Pathway

Neurotransmitter transporters are responsible for removal of biogenic amine neurotransmitters after release into the synapse. These transporters are the targets for many clinically relevant drugs, such as antidepressants and psychostimulants. A high resolution crystal structure for the monoamine transporters has yet to be solved. We have developed a homology model for the serotonin transporter (SERT) based on the crystal structure of the leucine transporter (LeuT(Aa)) from Aquifex aeolicus. The objective of the present studies is to identify the structural determinants forming the entrance to the substrate permeation pathway based on predictions from the SERT homology model. Using the substituted cysteine accessibility method, we identified residues predicted to reside at the entrance to the substrate permeation pathway that were reactive with methanethiosulfonate (MTS) reagents. Of these residues, Gln(332) in transmembrane helix (TMH) VI was protected against MTS inactivation in the presence of serotonin. Surprisingly, the reactivity of Gln(332) to MTS reagents was enhanced in the presence of cocaine. Bifunctional MTS cross-linkers also were used to examine the distances between helices predicted to form the entrance into the substrate and ion permeation pathway. Our studies suggest that substrate and ligand binding may induce conformational shifts in TMH I and/or VI, providing new opportunities to refine existing homology models of SERT and related monoamine transporters.

Estradiol selectively stimulates endothelial prostacyclin production through estrogen receptor-α

Estradiol (E(2)) acts on the endothelium to promote vasodilatation through the release of several compounds, including prostanoids, which are products of arachidonic acid metabolism. Among these, prostacyclin (PGI2) and thromboxane A2 (TXA2) exert opposite effects on vascular tone. The role of different estrogen receptors (ERs) in the PGI2/TXA2 balance, however, has not been fully elucidated. Our study sought to uncover whether E(2) enhances basal production of PGI2 or TXA2 in cultured human umbilical vein endothelial cells (HUVECs), to analyze the enzymatic mechanisms involved, and to evaluate the different roles of both types of ERs (ERalpha and ERbeta). HUVECs were exposed to E(2), selective ERalpha (1,3,5-tris(4-hydroxyphenyl)-4-propyl-1h-pyrazole, PPT) or ERbeta (diarylpropionitrile, DPN) agonists and antagonists (unspecific: ICI 182 780; specific for ERalpha: methyl-piperidino-pyrazole, MPP). PGI2 and TXA2 production was measured by ELISA. Expression of phospholipases, cyclooxygenases (COX-1 and COX-2), PGI2 synthase (PGIS), and thromboxane synthase (TXAS) was analyzed by western blot and quantitative RT-PCR. E(2) (1-100 nM) dose dependently increased PGI2 production (up to 50%), without affecting TXA2 production. COX-1 and PGIS protein and gene expressions were increased, whereas COX-2, phospholipases, and TXAS expression remained unaltered. All these effects were mediated through ERalpha, since they were produced not only in the presence of E(2), but also in that of PPT, while they were abolished in the presence of MPP. In conclusion, E(2), acting through ERalpha, up-regulates COX-1 and PGIS expression, thus directing prostanoid balance toward increased PGI2 production.

In vitro and in vivo evidences that antioxidant action contributes to the neuroprotective effects of the neuronal nitric oxide synthase and monoamine oxidase-B inhibitor, 7-nitroindazole

The neuronal nitric oxide synthase (nNOS) inhibitor, 7-nitroindazole (7-NI) is neuroprotective against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced parkinsonism. Monoamine oxidase (MAO)-B inhibitory action partially contributes to this effect. We tested the hypothesis that 7-NI could be a powerful hydroxyl radical ( OH) scavenger, and interferes with oxidative stress caused by MPTP. We measured OH, reduced glutathione (GSH), as well as superoxide dismutase (SOD) and catalase activities in the nucleus caudatus putamen and substantia nigra of Balb/c mice following MPTP and/or 7-NI administration. The nNOS inhibitor caused dose-dependent inhibition in the production of OH in (i) Fenton-like reaction employing ferrous citrate in a cell-free system in test tubes, (ii) in isolated mitochondrial preparation in presence of MPP +, and (iii) in the striatum of mice systemically treated with MPTP. An MPTP-induced depletion of GSH in both the nuclei was blocked by 7-NI, which was dose-dependent (10–50 mg/kg), but independent of MAO-B inhibition. The nNOS-mediated recovery of GSH paralleled attenuation of MPTP-induced depletion of striatal dopamine. MPTP-induced increase in the activities of striatal or nigral SOD and catalase were significantly attenuated by 7-NI treatment. These results suggest potent antioxidant action of 7-NI in its neuroprotective effects against MPTP-induced neurotoxicity.

RNA interference‐mediated knockdown of α‐synuclein protects human dopaminergic neuroblastoma cells from MPP+ toxicity and reduces dopamine transport

The critical observation in the pathology of Parkinson's disease (PD) is that neurodegeneration is largely restricted to dopaminergic neurons that develop cytoplasmic inclusions called Lewy bodies. These aggregations contain the protein alpha-synuclein. Furthermore, it is becoming apparent that alpha-synuclein expression levels are a major factor in PD pathogenesis. Patients with additional copies of the alpha-synuclein gene develop PD with a severity proportional to levels of alpha-synuclein overexpression. Similarly, overexpression of alpha-synuclein in in vitro and in vivo models has been shown to be toxic. However, little is known about the effects of reducing alpha-synuclein expression in human neurons. To investigate this, we have developed a system in which levels of alpha-synuclein can be acutely suppressed by using RNA interference (RNAi) in a physiologically relevant human dopaminergic cellular model. By using small interfering RNA (siRNA) molecules targeted to endogenous alpha-synuclein, we achieved 80% protein knockdown. We show that alpha-synuclein knockdown has no effect on cellular survival either under normal growth conditions over 5 days or in the presence of the mitochondrial inhibitor rotenone. Knockdown does, however, confer resistance to the dopamine transporter (DAT)-dependent neurotoxin N-methyl-4-phenylpyridinium (MPP(+)). We then demonstrate for the first time that alpha-synuclein suppression decreases dopamine transport in human cells, reducing the maximal uptake velocity (V(max)) of dopamine and the surface density of its transporter by up to 50%. These results show that RNAi-mediated alpha-synuclein knockdown alters cellular dopamine homeostasis in human cells and may suggest a mechanism for the increased survival in the presence of MPP(+), a toxin used extensively to model Parkinson's disease.

6-Hydroxydopamine but Not 1-Methyl-4-phenylpyridinium Abolishes α-Synuclein Anti-apoptotic Phenotype by Inhibiting Its Proteasomal Degradation and by Promoting Its Aggregation

We established previously that alpha-synuclein displayed a protective anti-apoptotic phenotype in neurons, mainly by down-regulating p53-dependent caspase-3 activation (Alves da Costa, C., Ancolio, K., and Checler, F. (2000) J. Biol. Chem. 275, 24065-24069; Alves da Costa, C., Paitel, E., Vincent, B., and Checler, F. (2002) J. Biol. Chem. 277, 50980-50984). This function was abolished by Parkinson disease-linked pathogenic mutations and by the dopaminergic toxin, 6-hydroxydopamine (6OH-DOPA) (Alves da Costa, C., Paitel, E., Vincent, B., and Checler, F. (2002) J. Biol. Chem. 277, 50980-50984). However, the mechanisms by which 6OH-DOPA interfered with alpha-synuclein function remained unclear. Here we showed that 6OH-DOPA prevents alpha-synuclein-mediated anti-apoptotic function by altering its degradation. Thus, 6OH-DOPA treatment of TSM1 neurons and SH-SY5Y neuroblastoma cells enhances endogenous alpha-synuclein-like immunoreactivity and inhibits the catabolism of endogenous and recombinant alpha-synucleins by purified 20 S proteasome. Furthermore, we demonstrated that 6OH-DOPA directly inhibits endogenous proteasomal activity in TSM1 and SH-SY5Y cells and also blocks purified proteasome activity in vitro. This inhibitory effect can be prevented by the anti-oxidant phenyl-N-butylnitrone. We also established that 6OH-DOPA triggers the aggregation of recombinant alpha-synuclein in vitro. Therefore, we conclude that 6OH-DOPA abolishes alpha-synuclein anti-apoptotic phenotype by inhibiting its proteasomal degradation, thereby increasing its intracellular concentration and potential propensity to aggregation, the latter phenomenon being directly exacerbated by 6OH-DOPA itself. Interestingly, 1-methyl-4-phenylpyridinium (MPP(+)), another toxin inducer of Parkinson disease-like pathology, does not affect alpha-synuclein protective function and fails to trigger aggregation of recombinant alpha-synuclein. Furthermore, MPP(+) does not alter cellular proteasomal activity, and only high concentrations of the toxin affect purified 20 S proteasome by a mechanism that remains insensitive to phenyl-N-butylnitrone. The drastically distinct effects of 6OH-DOPA and MPP(+) on alpha-synuclein function are discussed with respect to Parkinson disease pathology and animal models mimicking this pathology.

Small interfering RNA targeting the PINK1 induces apoptosis in dopaminergic cells SH-SY5Y

PTEN-induced kinase 1 ( PINK1) is a recently identified gene, mutations of which cause levodopa-responsive parkinsonism. An over-expression of wild-type PINK1 protects neurons from stress-induced mitochondrial dysfunction and apoptosis. We studied the effects of PINK1 suppression using small interfering RNA (siRNA), which can inhibit PINK1 mRNA expression up to 87%, and decrease PINK1 protein up to 80% in human dopaminergic cell line SH-SY5Y. Incubation with PINK1 siRNA decreased SH-SY5Y cell viability and significantly increased MPP + or rotenone-induced cytotoxicity. Our results indicate that reduction in PINK1 expression can trigger apoptotic process that can be exacerbated by the presence of MPP + or rotenone. These findings support the hypothesis that PINK1 participates in the protection of dopaminergic neurons.

Prosaptide D5, a retro-inverso 11-mer peptidomimetic, rescued dopaminergic neurons in a model of Parkinson's disease.

SPECIFIC AIMSOur aim was to evaluate the efficacy of ProsaptideTM D5, a bioactive protease-resistant 11-mer retro-inverso trophic peptidomimetic of prosaposion, in a model of Parkinson’s disease (PD). Experiments were devised to test the efficacy of ProsaptideTM D5 both in vitro and in vivo in the rescue of dopaminergic (DA) neurons from MPP+/MPTP toxicity, a specific toxin for DA neurons.PRINCIPAL FINDINGS1. ProsaptideTM D5 promoted neurite outgrowth and rescued DA neuron from MPP+ toxicity in the primary culture of DA neuronsThe addition of MPP+ (48 h incubation at 20 μM) significantly inhibited neurite outgrowth of primary DA neurons; after addition of MPP+, the average number of neurites per neuron decreased to 55% (primary neurites) and 34% (secondary neurites) of controls, respectively, and the neurites were also shorter in length (Fig. 1B⤻ ). ProsaptideTM D5 treatment (1 ng/ml) promoted neurite outgrowth in the presence of MPP+ (Fig. 1C⤻ , D⤻ ); the numbers of both primary and secondary neurites we...

Metabolic effects of 1-methyl-4-phenylpyridinium (MPP(+)) in primary neuron cultures.

Disruption of mitochondrial function has been proposed as an action of 1-methyl-4-phenylpyridinium (MPP(+)) that is responsible for its toxicity. In order to characterize effects of MPP(+) on energy metabolism in primary culture neurons, we monitored levels of several metabolites in cultured rat cerebellar granule cells exposed to MPP(+). The toxin produced a rapid concentration-dependent reduction in intracellular phosphocreatine (PCr), amounting to a 50-80% decrease within 30-60 min at 50 microM, that was maintained through the 1 week exposure interval examined. In contrast, ATP levels remained comparable to those of untreated neurons for approximately 4 days, at that time a 50% reduction in ATP was observed in association with a decrease in cell viability. Acute decreases in PCr were accompanied by increases in creatine such that the total creatine levels were maintained. Lactate levels in the culture medium were significantly increased (from 4.5 to 6.0 mM) within 6 hr after addition of MPP(+), with a concentration dependence similar to that observed for the reduction in PCr. Increased lactate production in the presence of MPP(+) coincided with a more rapid depletion of glucose in the culture medium. MPP(+) induced a rapid and sustained decrease in intracellular pH calculated from the creatine kinase equilibrium, and this acidification is considered primarily responsible for the observed decrease in PCr. These studies provide direct evidence that toxic concentrations of MPP(+) have acute effects on energy metabolism in primary culture neurons, consistent with an increased dependence on glycolysis to meet metabolic demand, but indicate that toxicity is not associated with overt, immediate failure to maintain cellular ATP.

Impaired Glutamate Clearance as a Consequence of Energy Failure Caused by MPP+ in Astrocytic Cultures

Astrocytes are the site of bioactivation of the parkinsonism-inducing agent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) into its toxic 1-methyl-4-phenylpyridinium (MPP+) metabolite. The mechanism by which MPP+ is capable of decreasing astrocytic glutamate uptake was evaluated in this study using primary cultures of astrocytes. Addition of glutamate to these cultures was followed by its efficient clearance from the extracellular space. However, when astrocytes were preincubated with MPP+, glutamate clearance was significantly impaired. This effect was concentration-dependent, became more pronounced by prolonging the incubation in the presence of MPP+ and occurred at a time when cell membrane integrity was still preserved. No evidence was found that reactive oxygen species contributed to MPP+-induced decrease in glutamate clearance. Indeed, neither the spin trapping agent α-phenyl-tert-butyl nitrone, the lazaroid antioxidant U-74389G, nor the disulfide-reducing agent dithiothreitol was capable of restoring glutamate net uptake. The effect of MPP+ on glutamate clearance: (i) was accompanied by a decrease in cellular ATP; (ii) could be enhanced by withdrawing glucose from the incubation medium or by inhibiting glycolysis with 2-deoxyglucose, and (iii) could be reproduced using the mitochondrial complex I inhibitor rotenone. Taken together, these results indicate that, by acting as a mitochondrial poison, MPP+ impairs energy metabolism of astrocytes and significantly reduces their ability to maintain low levels of extracellular glutamate.

Sonic Hedgehog Promotes the Survival of Specific CNS Neuron Populations and Protects These Cells from Toxic InsultIn Vitro

Sonic hedgehog (Shh), an axis-determining secreted protein, is expressed during early vertebrate embryogenesis in the notochord and ventral neural tube. In this site it plays a role in the phenotypic specification of ventral neurons along the length of the CNS. For example, Shh induces the differentiation of motor neurons in the spinal cord and dopaminergic neurons in the midbrain. Shh expression, however, persists beyond this induction period, and we have asked whether the protein shows novel activities beyond phenotype specification. Using cultures derived from embryonic day 14.5 (E14. 5) rat ventral mesencephalon, we show that Shh is also trophic for dopaminergic neurons. Interestingly, Shh not only promotes dopaminergic neuron survival, but also promotes the survival of midbrain GABA-immunoreactive (GABA-ir) neurons. In cultures derived from the E15-16 striatum, Shh promotes the survival of GABA-ir interneurons to the exclusion of any other cell type. Cultures derived from E15-16 ventral spinal cord reveal that Shh is again trophic for interneurons, many of which are GABA-ir and some of which express the Lim-1/2 nuclear marker, but it does not appear to support motorneuron survival. Shh does not support the survival of sympathetic or dorsal root ganglion neurons. Finally, using the midbrain cultures, we show that in the presence of MPP+, a highly specific neurotoxin, Shh prevents dopaminergic neuron death that normally would have occurred. Thus Shh may have therapeutic value as a protective agent in neurodegenerative disease.

Selective MPP+ uptake into synaptic dopamine vesicles: possible involvement in MPTP neurotoxicity

1. In the present study we provide evidence for a saturable, Mg2+/ATP- and temperature-dependent, tetrabenazine-, dopamine-, and amphetamine-sensitive uptake of 1-methyl-4-phenylpyridinium ion (MPP+) in synaptic vesicles from mouse striatum. 2. Similarity in the properties of the vesicular uptake suggests that in the striatum dopamine and MPP+ share the vesicular carrier. 3. The presence of MPP+ vesicular uptake in dopamine-rich regions such as striatum, olfactory, tubercles and hypothalamus, as well as its absence in cerebellum, cortex and pons-medulla, suggest that monoamine vesicular carriers differ between highly and poorly dopamine-innervated regions. 4. The restriction of active MPP+ uptake to the dopaminergic regions, which reflects the previously shown distribution of [3H]-MPP+ binding sites in mouse brain membranes, indicates MPP+ as a marker of the vesicular carrier for dopamine in dopaminergic neurones. 5. A role in MPP+ neurotoxicity is suggested for this region-specific, vesicular storage of the toxin.

The glutamate antagonist, MK-801, does not prevent dopaminergic cell death induced by the 1-methyl-4-phenylpyridinium ion (MPP +) in rat dissociated mesencephalic cultures

The neuroprotective effects of MK-801, a non-competitive antagonist of the N- methyl- d-aspartate (NMDA) receptor/channel, were assessed in a culture model which reproduces in vitro the selectìve degeneration of mesencephalic dopaminergic neurons seen in parkinsonian brains. Dissociated mesencephalic cells derived from rat embryonic brains were subjected for 24 h to intoxication by the 1-methyl-4-phenylpyridinium (MPP +), the active metabolite of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPP + at 3 and 10 μM produced selective and dose-dependent damages to dopaminergic neurons as quantified by the loss of the number of TH immunoreactive cells and the loss of [ 3H]DA uptake whereas other cell types remained unaffected. MK-801 at 3 and 10 μM failed to rescue degenerating dopaminergic neurons in presence of MPP +. At 50 μM, i.e. the highest concentration that is not toxic by itself in this culture system, MK-801 was also found ineffective. Furthermore, degree of dopaminergic cell damage was not reduced when repeated additions of the glutamate antagonist (10 μM/6 h for 24 h) were performed during exposure to MPP + or when mesencephalic cultures were left after intoxication for up to 2 days in a culture medium still supplemented with MK-801 but free of toxin. In accordance with these results, MK-801 did not affect significantly the uptake of [ 3H]DA in control cultures, thereby suggesting that this compound cannot prevent intracellular accumulation of MPP + within dopaminergic neurons. At higher concentrations of MPP + (100 μM) tested, toxic effects were seen toward dopaminergic neurons and non-dopaminergic cells as quantified by Trypan blue dye accumulation and loss of [ 3H]GABA uptake. Similarly, widespread degenerative changes were observed with high levels of glutamate (4 mM), the brain endogenous agonist of the NMDA receptor/channel. Yet, degree of cell injury was not reduced in presence of MK-801 in both situations. Meanwhile, to establish that MK-801 was pharmacologically active in our incubation conditions, we showed that the different batches of this compound which have been found ineffective in mesencephalic cultures were indeed capable of preventing neurotoxic effects of glutamate toward dissociated cortical cells. Altogether, these results indicate that MPP + detrimental effects in vitro are not related to excitotoxicity and/or that MK-801 does not interfere with MPP + toxic scenario. They do not support the idea that glutamate antagonists could be used to prevent dopaminergic cell degeneration in Parkinson's disease.

The acute convulsant effect of MPTP is dependent on intracerebral MPP +

The administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to C57 black mice causes an acute seizure syndrome the severity of which is dose dependent; there is also a good correlation between the seizure inducing potential of MPTP and the neostriatal dopamine (DA) depletion caused by MPTP. The simultaneous administration of MPTP and MAO B inhibitors attenuates both epileptiform phenomena and neostriatal DA depletion. On the contrary diethyldithiocarbamate (DDC) exacerbates both responses. All these pharmacological manipulations are known to affect the accumulation of 1-methyl-4-phenylpyridinium ion (MPP +) the main metabolite of MPTP. Thus the present data support the hypothesis of a strict dependence of the epileptiform phenomena on the presence of MPP +. Furthermore the tight correlation existing between the severity of epileptic events and DA depletion suggests that the acute excitotoxic syndrome may contribute to the long-term toxicity of MPTP. On leave from the Department of Neurology, University of Pisa, Pisa, Italy.

Cytotoxic effect of 1-methyl-4-phenylpyridinium ion on human melanoma cell lines, HMV-II and SK-MEL-44, is dependent on the melanin contents and caused by inhibition of mitochondrial electron transport

MPP+, an oxidative metabolite of a neurotoxin, MPTP, was found to be cytotoxic to human melanoma cell lines, HMV-II and SK-MEL-44. After 3 days of culture in the presence of MPP+, a larger amount of MPP+ was accumulated in HMV-II cells than in SK-MEL-44 cells, which correlated well with the melanin contents; HMV-II cells contain larger amounts of melanin than SK-MEL-44 cells. After 6 days of culture in the presence of MPP+, the cytotoxicity of MPP+ on these cell types was evaluated by counting cell numbers with the dye exclusion test and double-layer soft agar clonogenic assay. It was found that exposure to MPP+ reduced the survival of HMV-II cells more significantly than that of SK-MEL-44 cells. In HMV-II cells, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay was used to elucidate the mechanism of MPP+ lethality. The formazan formation was reduced markedly by the presence of MPP+ at concentrations much lower than those required for cell death. These results suggest that cytotoxicity of MPP+ may be ascribed to its accumulation due to high affinity for melanin, and to inhibition of the enzymes utilizing ubiquinone in the mitochondrial respiratory chain.

Characterization of cellular transport, subcellular distribution, and secretion of the neurotoxicant 1-methyl-4-phenylpyridinium in bovine adrenomedullary cell cultures.

Cultures of bovine adrenomedullary chromaffin cells accumulated 1-[methyl-3H]methyl-4-phenylpyridinium ([3H]MPP+) in a time- and concentration-dependent manner with an apparent Km of 0.7 microM and a Vmax of 3 pmol/min/10(6) cells. The uptake was sodium dependent and sensitive to inhibitors of the cell-surface catecholamine transporter. At low concentrations of MPP+, the subcellular distribution was identical to that of endogenous catecholamines in the catecholamine-containing chromaffin vesicles. However, at a higher concentration of MPP+, a larger proportion of the toxicant was recovered in the cytosolic fraction, with less in the chromaffin vesicle fractions. When cells were prelabeled with [3H]MPP+, at 1 and 300 microM, and then permeabilized with digitonin in the absence of Ca2+, there was a proportionally greater release of MPP+ from the cells labeled at the higher concentration of the toxicant. In the presence of Ca2+, cell permeabilization induced a time-dependent secretion of catecholamines and a parallel secretion of MPP+. Under these conditions, the secretion of endogenous catecholamines was unaffected by the presence of MPP+. When the permeabilization studies were carried out in the presence of tetrabenazine, a massive release of MPP+ was observed in the absence of Ca2+ and was not further increased by Ca2+. In intact cells prelabeled with 300 microM [3H]MPP+, the secretagogues nicotine and veratridine elicited a Ca2+ -dependent secretion of catecholamines and MPP+ from the cells in similar proportions to their cellular contents. Barium-induced release of both species was independent of external Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduction of aromatic l-amino acid decarboxylase activity in clonal pheochromocytoma PC12h cells by culture in the presence of N-methyl-4-phenylpyridinium ion (MPP +)

Rat clonal pheochromocytoma PC12h cells were cultured in the presence of N-methyl-4-phenylpyridinium ion (MPP +) and the activity of aromatic l-amino acid decarboxylase (AADC) was reduced after 3 and 6 days of culture. AADC activity in control cells was increased markedly by addition of pyridoxal 5-phosphate (PLP) to the reaction mixture, but that in the cells cultured in the presence of MPP + was not increased by addition of PLP. After 6 days culture, AADC activity was almost negligible in the cells cultured in the presence of 1 mM MPP +, but PLP concentration in the cells was not reduced. AADC in the cells cultured in the presence of MPP + reduced the affinity to PLP, but the affinity to a substrate, l-DOPA, did not change. Intracellular concentration of AADC protein was not reduced, as shown by an immunobinding assay with anti-AADC antibody. These data indicate that MPP + may induce conformational changes in AADC protein and reduces its affinity to the cofactor, PLP.

Accumulation of 1-methyl-4 phenylpyridinium (MPP +) into bovine chromaffin granules results in a large restriction of its molecular motion: A 13C and 31P NMR study

13C labelled 1-methyl-4-phenylpyridinium ([13C-methyl]MPP+) is taken up by chromaffin granules and a signal from the methyl group within the granular matrix appears at 49.8 ppm. No shift of the [13C-Me] signal is observed upon incorporation into the granules. However the linewidth of the signal is significantly increased suggesting a restriction in the molecular motion of MPP+. Confirmation of the above derives from the determination of the T1 relaxation time of the [13C-Me] carbon of MPP+ which shows a four fold decrease upon incorporation of MPP+ into the granule. 31P NMR spectra of ATP in the presence of MPP+ reveals that the toxicant has low affinity for the nucleotide, suggesting that it is the viscosity of the granular matrix that restricts MPP+ mobility rather than a specific interaction with intragranular ATP.

Accumulation of N-methyl-4-phenylpyridinium ion (MPP +) in human melanoma cell line, HMV-I and -II

N-Methyl-4-phenylpyridinium ion (MPP +) was found to be accumulated in human melanoma cell lines, HMV-I and -II, which originate from human melanoma and differentiate into subclones, HMV-I and -II. HMV-II cells can produce a large amount of melanin, while the other cells produce less. After 3 day culture of these cells in the presence of 1–100 μM MPP +, MPP + was accumulated in both types of cells and a much larger amount of MPP + was accumulated in melanin-rich HMV-II cells than in HMV-I cells, even though the uptake velocity of MPP + into both types of cells was almost the same. In addition, both types of cells could survive, even with MPP + accumulation. Protein amounts, a non-specific enzyme, β-galactosidase activity, and intracellular DOPA concentrations in both types of cells, were not affected by the presence of MPP +. These results suggest that MPP + is accumulated in non-dopaminergic cells and the accumulation is enhanced by the presence of melanin.

Effect of 1-methyl-4-phenylpyridinium ion (MPP +) on catecholamine levels and activity of related enzymes in clonal rat pheochromocytoma PC12h cells

1-Methyl-4-phenylpyridinium ion (MPP +), a metabolite of a neurotoxin, 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine, was found to reduce dopamine (DA) level and the activity of enzymes related to its metabolism in clonal rat pheochromocytoma PC12h cells. After 6 days' culture in the presence of 1 mM and 100 μM MPP +, DA content in PC12h cells was reduced markedly, but with MPP + at concentrations lower than 10 μM, DA levels in the cells did not change. The amounts of 3, 4-dihydrophenylacetic acid (DOPAC), a metabolite of DA were reduced markedly in culture medium and in PC12h cells cultured with MPP + at concentrations higher than 1 μM. MPP + was found to reduce the enzyme activity of tyrosine hydroxylase (TH), monoamine oxidase (MAO) and aromatic L-aminoacid decarboxylase (AADC). In the presence of MPP + at concentrations higher than 10 μM, reduction of TH activity in the cells was more pronounced than reduction of cell protein or of the activity of a non-specific enzyme, β-galactosidase. With 1 mM and 100 μM MPP +, MAO activity was reduced to about 30% of that in control cells. Reduction was observed with MPP + at concentrations higher than 1 μM. AADC was the most sensitive to MPP + and its activity was reduced markedly in the cells cultured with 100 nM MPP +. These results indicate that MPP + inhibits not only the biosynthesis of catecholamines, but also the enzyme participating in their catabolism in cells, and may thus perturb catecholamine levels in the brain.