Rong-Sen Shen

Serotonergic conversion of MPTP and dopaminergic accumulation of MPP

[3H]MPP+ had lower K m and higher V max values for its accumulation in rat brain synaptosomes than did [3H]MPTP. The kinetic parameters favored the uptake of [3H]MPP+ in the striatum to that in hypothalamus, whereas they were equally favorable for the uptake of [3H]MPTP in both regions. Hypothalamic uptake of [3H]MPTP and [3H]MPP+ was inhibited by desipramine, imipramine, norepinephrine, and serotonin. Striatal uptake of [3H]MPP+ and [3H]MPTP was blocked by nomifensine and dopamine. These results support the concept that MPTP accumulates in serotonergic neurons where it is oxidized by monoamine oxidase B to MPP+, which is released and then is selectively accumulated in dopaminergic neurons via the dopamine uptake system.

Further insight into the mode of action of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

Chemical reactions of MPDP+, a recognized intermediate in the metabolic conversion of the neurotoxin MPTP by monoamine oxidase B into its major metabolite MPP+ were studied.Addition of cyanide to MPDP+ bromide in aqueous solutions afforded cyano‐compound 5 which isomerized in the presence of silica gel into compound 6. Both 5 and 6 when heated yielded a third isomer 7. MPDP+ bromide disproportionated into MPTP and MPP+ in aqueous solution near neutral or slightly alkaline pH, a reaction which also occurred when MPDP+ bromide was treated with an amine in dichloromethane solution. Disproportionation of MPDP+ at physiological pH may be of biochemical significance, since formation of MPP+ from MPDP+ can occur non‐enzymatically. MPTP, MPDP+, and MPP+ inhibited dopamine uptake in rat synaptosomal preparations with I50 values of 30, 37, and 3.4 μM, respectively. The competition of these compounds with dopamine for uptake sites in the membrane may contribute in part to the reduced levels of dopamine observed in animals treated with MPTP.

MPTP metabolites inhibit rat brain glutathione S-transferases

1-Methyl-4-phenyl-2,3-dihydropyridinium and 1-methyl-4-phenyl-pyridinium species, metabolites of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, non-competitively inhibit glutathione S-transferases of rat brain in vitro. The Ki values for 1-methyl-4-phenyl-2,3-dihydropyridinium bromide and 1-methyl-4-phenyl-pyridinium bromide are 0.67 and 0.3 mM, respectively. Inhibition of these enzymes may lead to impairment of cellular defense mechanisms.

Evaluation of the PaT Stat Kinetic UV Test set for the determination of phenylalanine and tyrosine in serum or plasma.

We have evaluated the PaT Stat Kinetic UV Test Kit for the simultaneous determination of phenylalanine (Phe) and tyrosine (Tyr) in plasma or serum. The Phe and Tyr concentrations measured with the kit show a coefficient of variation of about 15% for both within- and between-day determinations. The assays for both Phe and Tyr are linear to concentrations of at least 18 mg/dL without predilution of the specimen. Concentration differences of as little as 0.5 mg/dL are distinguishable. No significant interference was found from either phenylpyruvate or phenyllactate at levels up to 0.5 mM, nor from bilirubin, haemoglobin, or triglycerides at levels well above those generally found in clinical specimens. A comparative study of 70 clinical specimens, using the kit method and an amino acid analyzer (AAA) showed a linear relationship. Least-squares analysis of the data yielded the following parameters (AAA as reference): slope = 0.93 to 1.00, intercept = 0.04 to 0.21, correlation coefficient = 0.97 to 0.98. We conclude that the kit is suitable for the determination of Phe and Tyr in plasma or serum and can enable any laboratory equipped with a recording UV-spectrophotometer to assay these two amino acids for the dietary management of PKU.

REGULATION OF GTP CYCLOHYDROLASE I AND DIHYDROPTERIDINE REDUCTASE IN RAT PHEOCHROMOCYTOMA PC 12 CELLS

The addition of 8-bromo cyclic AMP, forskolin, theophylline, and 3-isobutyl-1-methylxanthine to the medium of PC 12 cells resulted in an increase in GTP cyclohydrolase I activity, but had no effect on dihydropteridine reductase activity, except theophylline which caused a decrease in dihydropteridine reductase activity at 96 h. GTP cyclohydrolase I activity peaked at 24 h and returned to normal 96 h after drug treatment. Cycloheximide decreased GTP cyclohydrolase I activity at 48 and 96 h, but had little effect on dihydropteridine reductase activity. The addition of reserpine selectively increased only GTP cyclohydrolase I activity. The addition of tetrahydrobiopterin and sepiapterin, however, coordinately inhibited both GTP cyclohydrolase I and dihydropteridine reductase activities. It appears that GTP cyclohydrolase I activity in PC 12 cells is regulated by cyclic AMP stimulation and by end-product inhibition, whereas dihydropteridine reductase activity is only subject to pterin inhibition.

Effects of nomifensine and its metabolites on dihydropteridine reductase

Nomifensine and three of its metabolites were studied as potential inhibitors of dihydropteridine reductase. Purified enzyme preparations from human liver and the P2 fraction of rat striatal synaptosomes were used as enzyme sources. Nomifensine and its 3−hydroxyl derivative inhibit this enzyme from both sources at 1·3 to 3·5 times 10−4 m (150 values). 4−Hydroxylated nomifensines, however, non‐competitively inhibited this enzyme with Ki values of 2·8 to 4·4 times 10−5 m. Dihydropteridine reductase regenerates tetrahydrobiopterin, the required cofactor for the hydroxylation of tyrosine and tryptophan, from quinonoid dihydrobiopterin. Inhibition of this enzyme could reduce the availability of the biopterin cofactor for the synthesis of dopamine and 5−hydroxytryptamine.

Rat Striatal Synaptosomes as a Model System for Studying the Inhibition of Dihydropteridine Reductase Activity

The distribution of dihydropteridine reductase between soluble and particulate fractions in synaptosomes parallels that of lactate dehydrogenase, but not monoamine oxidase. Ki and I50 values for inhibitors obtained with the enzyme-rich P2 fraction and its twice-washed fraction (P2W2) were essentially the same, and were similar to those obtained with highly purified human liver enzyme. Dihydropteridine reductase inhibitory potency of multi-ring compounds containing a catechol-moiety was greater than that of single ring catecholic compounds, which in turn was greater than that of p-hydroxy-phenolic compounds. The P2 fraction of rat striatal synaptosomal preparations may serve as a convenient source of dihydropteridine reductase for studying the inhibition of this enzyme.

An enzyme immunoassay for the quantitation of dihydropteridine reductase.

A competitive, enzyme-linked immunosorbent assay for the quantitative determination of dihydropteridine reductase (DHPR) is described. This highly sensitive method can determine the content of DHPR protein in tissue preparations independently of the enzymatic activity of the protein molecule. The method involves initial incubation of samples containing soluble enzyme in microtiter plates coated with purified goat antibodies to rat DHPR and further incubation with DHPR conjugated to alkaline phosphatase. The assay is used to study the ontogeny of DHPR in rat liver.