John F. Reinhard

A rapid and sensitive assay for tyrosine-3-monooxygenase based upon the release of 3H2O and adsorption of [3H]-tyrosine by charcoal.

A rapid, simple and sensitive assay has been developed for tyrosine-3-monooxygenase, the enzyme catalyzing the rate-limiting step in catecholamine biosynthesis. The assay is based upon the release of 3H2O from 3H-[3,5]-L-tyrosine with adsorption of the isotopic substrate (and its metabolites) by an aqueous slurry of activated charcoal. This method routinely yields low blank values and is simpler than the procedure requiring the use of cation exchange columns to separate the isotopic substrate from the 3H2O formed during the hydroxylation reaction.

Characterization of the origins of astrocyte response to injury using the dopaminergic neurotoxicant, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

We used the dopaminergic neurotoxicant, 1-methyl-1,2,3,6-tetrahydropyridine (MPTP), as a tool to characterize the origins of astroglial response to injury. Radioimmunoassay of the astrocyte protein, glial fibrillary acidic protein (GFAP), was used to quantify the astrocyte reaction to MPTP. Assays of neuron-localized proteins and of dopamine were used to assess neuronal damage caused by MPTP. A single administration of MPTP (12.5 mg/kg, s.c.) to the C57BL/6J mouse resulted in more than a 3-fold increase in striatal GFAP within 48 h, followed by a decline to baseline at 3 weeks. A decrease in the amount of striatal tyrosine hydroxylase (TH), a marker of dopaminergic neurons, preceded the rise in GFAP. The concentration of striatal DARPP-32, a phosphoprotein enriched in neurons receiving dopaminergic input, was not affected by MPTP. Protecting the dopaminergic neurons from the neurotoxic metabolite of MPTP, 1-methyl-4-phenylpyridinium (MPP+), either by blocking its formation or by preventing its uptake into dopaminergic neurons, completely blocked the increase in GFAP. MPTP did not appear to disrupt the blood-brain barrier, therefore, blood-borne elements probably did not mediate the increase in GFAP. In addition, immunoblot data indicated that brain-derived interleukin 1, an astrocyte growth factor, also did not play a role in MPTP-induced gliosis. Together, these findings suggest that diffusible factors derived from damaged dopaminergic neurons initiate the astrocyte response to MPTP and that large increases in GFAP can be induced without the participation of serum-derived growth factor.(ABSTRACT TRUNCATED AT 250 WORDS)

Elevation of the neurotoxin quinolinic acid occurs following spinal cord trauma

Excitatory amino acid neurotoxicity and the inflammatory response are suspected as mediators of some of the pathological sequelae occurring as a result of spinal cord injury. Here we report temporal and regional increases of the NMDA receptor agonist, quinolinic acid (QUIN), in an experimental model of spinal contusion injury. These changes occurred at a time when the blood-brain barrier is known to be dysfunctional and the activation state and density of microglia and macrophages are increased. Thus, alterations in tissue QUIN levels may occur as a result of secondary activation of CNS inflammatory cells or from peripherally derived sources across a damaged blood-brain barrier.

The neurotoxicant MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) increases glial fibrillary acidic protein and decreases dopamine levels of the mouse striatum: Evidence for glial response to injury

The neurotoxicant MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), administered to male or female mice, decreased striatal dopamine content and increased the levels of the astrocyte intermediate filament protein, glial fibrillary acidic protein (GFAP). The rise in GFAP was evident as early as two days following the last dose of MPTP, was maximal 7 days after the toxicant and returned to control levels by two months, post MPTP. Striatal dopamine content was decreased post-MPTP administration, showing a slight recovery between one and two months after the toxicant. No differences were observed among male and female mice in their responses to the toxicant. Hippocampal noradrenaline content was not affected by the toxicant, neither was the GFAP content altered by MPTP in this structure. Additionally, pargyline pretreatment prevented both the rise in GFAP and the decrease in dopamine in striatum. MPTP produced a smaller elevation in GFAP levels within a midbrain section of tissue containing the substantia nigra, without significantly decreasing the dopamine content of this structure, suggesting neurotoxic involvement at the level of dopamine perikarya. The toxicant did not affect the molecular radius of the protein detected by the antibody to GFAP, as determined by immunoblot analysis.

Quinolinic Acid in Neurological Disease: Opportunities for Novel Drug Discovery

Publisher Summary Quinolinic acid is found throughout nature in organisms ranging from bacteria to humans. Chemically this compound is known as pyridine-2,3-dicarboxylic acid. The biosynthetic pathways for quinolinic acid and nicotinamide adenine dinucleotide (NAD) appear to be conserved evolutionarily. The neurotoxicity of quinolinic acid has been demonstrated in vivo and in vitro. Quinolinic acid toxicity has been demonstrated most often through its direct intrastriatal injection. considerable reasons exist to suspect that quinol in k acid may be an important mediator in the primary phase of inflammatory brain disease and may be important in the secondary phase of brain injury following a variety of insults to the central nervous system (CNS). However, the real test for the importance of quinolinic acid will be the application of quinolinate synthesis inhibitors, possibly at the level of kynurenine hydroxylase or kynureninase, to human disease. However, such inhibitors are not presently available. Several advantages will aid us in that task. First, the enzymes of the macrophage pathway are largely present in the liver and should be amenable to testing potential inhibitors. Second, cultures of human macrophages are readily obtained and flux can be determined on a relevant cell type. Third, animal models of elevated quinolinate biosynthesis are available in which the in vivo efficacy of inhibitors can be evaluated.

Potentiation by reserpine and tetrabenazine of brain catecholamine depletions by MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) in the mouse; evidence for subcellular sequestration as basis for cellular resistance to the toxicant.

Administration to mice of the neurotoxicant MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) decreased striatal dopamine and, to a lesser extent, hippocampal noradrenaline levels when measured 2 weeks after the last dose of MPTP. Reserpine and tetrabenazine, inhibitors of catecholamine vesicular transporter, potentiated the catecholamine depletions produced by MPTP in the hippocampus and striatum, respectively. These results are compatible with our hypothesis that sequestration of the toxic MPTP metabolite MPP+ (1-methyl-4-phenylpyridinium) in the catecholamine storage vesicle retards the catecholaminergic toxicity of MPTP.

A radiometric assay for kynurenine 3-hydroxylase based on the release of 3H2O during hydroxylation of L-[3,5-3H]kynurenine.

A rapid and sensitive assay for kynurenine 3-hydroxylase (KH) has been developed. This radiometric assay is based on the enzymatic synthesis of tritiated water from L-[3,5-3H]kynurenine during the hydroxylation reaction. Radiolabeled water is quantified following selective adsorption of the isotopic substrate and its metabolite with activated charcoal. The assay is suitable for detecting 0.1 pmol enzyme activity per minute per milligram protein in tissues displaying low levels of the enzyme. The amount of water produced in the reaction, as calculated from the tritium released, was stoichiometric with the 3-hydroxykynurenine product detected by HPLC. Rat liver KH was characterized by cofactor specificity and kinetic parameters. NADPH was preferred over NADH as coreductant in the reaction. Tetrahydrobiopterin was not a cofactor. The tissue distribution of KH activity in the rat suggested that the majority of active enzyme is located in liver and kidney. Detectable amounts were found in several other tissues, including brain which had low but significant levels of activity in every region assayed.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)- induced damage of striatal dopaminergic fibers attenuates subsequent astrocyte response to MPTP

Acute administration of the dopaminergic neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to the C57BL/6 mouse caused a rapid decrease in the amount of striatal tyrosine hydroxylase (TH), a marker of the nigrostriatal dopaminergic pathway, followed by a large increase in the astrocyte protein, glial fibrillary acidic protein (GFAP). The astrocyte (GFAP) response declined to baseline three weeks after administration of MPTP. Administration of a second dosage of MPTP at this time evoked a second GFAP response. The magnitude of the second response, however, was decreased in comparison to the response seen after only a single exposure to MPTP. Increasing the initial dosage of MPTP resulted in greater reductions of the second GFAP response. These data indicate that MPTP-induced damage or loss of striatal dopaminergic neurons reduces the signal available for initiating astrogliosis and thereby reduces the astrocyte response to a second exposure to MPTP.

Cofactor activity of dihydroflavin mononucleotide and tetrahydrobiopterin for murine epididymal indoleamine 2,3-dioxygenase

Dihydroflavin mononucleotide (FMNH2) and tetrahydrobiopterin (BH4) serve as cofactors for indoleamine 2,3-dioxygenase isolated from mouse epididymis. The optimal pH was between 7 and 8, and FMNH2-dependent activity was 4 to 5-fold higher than activity with methylene blue as the electron donor. Using FMNH2 with a FMN reductase system, the enzyme exhibited higher efficiency and specificity for L-Trp (an apparent Km of 1 X 10(-5)M and an apparent Vmax of 182 nmol/min/mg of protein). The apparent Km and Vmax for D-Trp were 6.2 X 10(-5)M and 31 nmole/min/mg, respectively. Consequently, these observations appear to present the first evidence for a flavin-dependent mammalian dioxygenase.

Carrier-independent entry of 1-methyl-4-phenylpyridinium (MPP+) into adrenal chromaffin cells as a consequence of charge delocalization.

The administration of 1-methyl-4-phenylpyridinium (MPP+) to cultures of adrenal medullary chromaffin cells resulted in time and concentration-dependent increases in the cellular content of MPP+. Co-incubation of cells with MPP+, in the presence of desmethylimipramine (DMI), reduced but did not prevent the accumulation of the pyridinium in these cells. Similarly, DMI and MPP+ co-administration reduced but did not prevent the neurotoxicant-induced release of a cytosolic marker, lactate dehydrogenase, into the media. Molecular orbital calculations reveal that the positive charge of MPP+ is highly delocalized throughout the pyridinium ring and consequently MPP+ may be able to diffuse down concentration or charge gradients. Thus, these data provide a basis for the entry of MPP+ into cells and subcellular organelles that lack a catecholamine transporter, e.g. mitochondria.