Inhibition of human brain aromatic L-amino acid decarboxylase by cooked food-derived 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and other heterocyclic amines

A carcinogenic, food-derived heterocyclic amine, 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), was found to inhibit aromatic L-amino acid decarboxylase isolated from human brainstem. Trp-P-2 inhibited the enzyme activity toward L-DOPA more markedly than that toward 5-hydroxytryptophan. The inhibition was competitive to a cofactor of the enzyme, pyridoxal-5-phosphate, and the Ki value of Trp-P-2 was 163 microM. The enzyme activity could be fully recovered after removal of Trp-P-2 by gel filtration, which indicates that the inhibition was reversible. Among a series of heterocyclic amines examined for their effects on the activity toward L-DOPA, Trp-P-2 was the most potent inhibitor, followed by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, then Trp-P-1. Another heterocyclic amine, 2-amino-3-methyl-9H-pyrido[2,3-b]indole also inhibited the enzyme. The inhibition of the decarboxylase activity by these heterocyclic amines may affect the catecholamine metabolism in human brain.     

Uptake of heterocyclic amines, Trp-P-1 and Trp-P-2, into clonal rat pheochromocytoma PC12h cells by dopamine uptake system

Heterocyclic amines, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), are known to be produced in food by cooking and are carcinogenic. These amines were found to be accumulated in clonal rat pheochromocytoma PC12h cells and to reduce enzyme activity related to catecholamine synthesis. The mechanism of uptake of these heterocyclic amines into PC12h cells was studied. The uptake was dependent on the incubation time, the amount of the cells, and the concentrations of Trp-P-1 and Trp-P-2 in the incubation mixture. The uptake of these amines was saturable with their concentrations, and the uptake velocity followed the Michaelis-Menten equation, indicating that the uptake was mediated by a transporting protein. The uptake was inhibited by dopamine and serotonin, but not by noradrenaline. Involvement of the dopamine uptake system in uptake of the heterocyclic amines was further indicated by the fact that nomifensine and mazindol, specific inhibitors of dopamine uptake, reduced the uptake, but sulpiride, an antagonist of D2 receptor, did not. The significance of the uptake of the carcinogenic heterocyclic amines was discussed in relation to their possible neurotoxicity in the human brain.     

Early and late effects of systemically administered 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on tyrosine hydroxylase activity in vitro and on tyrosine hydroxylation in tissue slices of mouse striatum

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces a parkinsonian-like state in humans and some animals. To compare the early biochemical abnormalities produced by this neurotoxin with late effects, we examined both in vitro tyrosine hydroxylase activity in striatal homogenates and in situ tyrosine hydroxylation in striatal tissue slices after single and repeated systemic injection of MPTP to mice. The acute administration of MPTP (30 mg/kg, s.c., 1 h prior to sacrifice) in mice resulted in a decrease of tyrosine hydroxylation in situ in tissue slices but not in vitro in homogenates. In contrast, repeated treatment of mice with MPTP (30 mg/kg, s.c. daily for 8 days) caused a decrease of tyrosine hydroxylase activity both in vitro in homogenates and in situ in tissue slices. These results suggest that MPTP inhibits tyrosine hydroxylation in dopaminergic neurons in an early stage and causes reduction of tyrosine hydroxylase itself after repeated administration.     

Substance P inhibits the acute stimulation of ganglionic tyrosine hydroxylase activity by a nicotinic agonist

Incubation of rat superior cervical ganglia with dimethylphenylpiperazinium (30 microM) for 30 min resulted in a two-fold increase in tyrosine hydroxylase activity. This effect was completely inhibited by substance P (30 microM) but not by substance P-free acid, kassinin or physalaemin. Neither of these four peptides alone produced any change in the activity of tyrosine hydroxylase. The IC50 for the inhibitory effect of substance P was approximately 3 microM. Substance P did not inhibit the stimulatory effects of bethanechol or vasoactive intestinal peptide on this enzyme activity. Thus substance P, acting at a site which has a different pharmacology than previously characterized substance P receptors, selectively inhibits nicotinic stimulation of tyrosine hydroxylase activity. These data raise the possibility that substance P may modulate the nicotinic regulation of catecholamine synthesis in sympathetic ganglia in vivo.     

Thyroid cold acclimation influences on norepinephrine metabolism in brown fat.

Norepinephrine turnover rates and tyrosine hydroxylase activities were determined in the interscapular brown fat pad of the rat during cold acclimation, hyperthyroxinism, and after thyroidectomy. Rats were cold acclimated by placement in a cold room, one rat to a cage, for a period of 6 wk. Hyperthyroxinism was induced by daily subcutaneous injections of L-thyroxine (1 mg/kg) for 6 days. Norepinephrine turnover rate and enzyme activity were determined at the end of each experimental period and at 8 wk after thyroidectomy. The rate of norepinephrine turnover increased during cold acclimation and hyperthyroxinism and decreased after thyroidectomy. Cold acclimation resulted in a significant increase in tyrosine hydroxylase activity, whereas no significant effect on enzyme activity was observed in hyperthyroxinism or after thyroidectomy. None of the conditions produced a change compared to controls in the apparent Km of tyrosine hydroxylase for L-tyrosine. Cold acclimation resulted in a significant decrease in the apparent Km of tyrosine hydroxylase for pterin cofactor, whereas thyroxine treatment and thyroidectomy had no effect.     

Functional aspects of serotonin transmission in the basal ganglia: a review and an in vivo approach using the push-pull cannula technique

Peripheral deafferentation of the rodent olfactory bulb results in loss of dopamine content, tyrosine hydroxylase activity and immunocytochemical staining for tyrosine hydroxylase in juxtaglomerular dopamine neurons. Reinnervation of the bulb by afferent neurons results in the return of all parameters to control levels suggesting that the dopamine neurons did not degenerate but that the expression of tyrosine hydroxylase enzyme was transneuronally regulated in a static population of juxtaglomerular cells. To evaluate this possibility, we determined the activity and immunocytochemical localization of the second enzyme in the dopamine biosynthetic pathway, DOPA decar?ylase. At a time when tyrosine hydroxylase activity was reduced to 25% of control values, DOPA decar?ylase activity in the lesioned bulb was maintained at about 65% of that in the unlesioned bulb. Immunocytochemical staining with antibodies to both enzymes, performed sequentially in the same sections, demonstrated that in the unlesioned bulb tyrosine hydroxylase and DOPA decar?ylase are co-localized in the same population of juxtaglomerular neurons. Similar results were obtained in adjacent sections each stained with one of the two antibodies. In contrast, in the deafferented bulb, about three times as many neurons were stained with DOPA decar?ylase as with tyrosine hydroxylase antibodies. The DOPA decar?ylase activity measurements and immunocytochemistry argue for the continued presence, in the lesioned olfactory bulb, of a population of tyrosine hydroxylase deficient dopamine neurons.The data suggest that olfactory receptor cell innervation transneuronally regulates the expression of tyrosine hydroxylase by mechanisms separate from those controlling the levels of DOPA decar?ylase.     

The structural basis of phenylketonuria.

The human phenylalanine hydroxylase gene (PAH) (locus on human chromosome 12q24.1) contains the expressed nucleotide sequence which encodes the hepatic enzyme phenylalanine hydroxylase (PheOH). The PheOH enzyme hydroxylates the essential amino acid l-phenylalanine resulting in another amino acid, tyrosine. This is the major pathway for catabolizing dietary l-phenylalanine and accounts for approximately 75% of the disposal of this amino acid. The autosomal recessive disease phenylketonuria (PKU) is the result of a deficiency of PheOH enzymatic activity due to mutations in the PAH gene. Of the mutant alleles that cause hyperphenylalaninemia or PKU 99% map to the PAH gene. The remaining 1% maps to several genes that encode enzymes involved in the biosynthesis or regeneration of the cofactor ((6R)-l-erythro-5,6,7,8-tetrahydrobiopterin) regenerating the cofactor (tetrahydrobiopterin) necessary for the hydroxylation reaction. The recently solved crystal structures of human phenylalanine hydroxylase provide a structural scaffold for explaining the effects of some of the mutations in the PAH gene and suggest future biochemical studies that may increase our understanding of the PKU mutations.     

Protection against Trp-P-2 mutagenicity by purpurin: mechanism of in vitro antimutagenesis

Purpurin (1,2,4-trihydroxy-9,10-anthraquinone) is a natural pigment isolated from madder root (Rubia tinctorum) which inhibits the mutagenicity of a number of heterocyclic amines in the Ames mutagenicity test. Two effects were observed in the presence of purpurin. The rate of degradation of 3-hydroxyamino-1-methyl-5H-pyrido?4,3-b?ndole ?Trp-P-2(NHOH) at neutral pH was increased. The major product of this purpurin-dependent degradation was identified as the parent amine 3-amino-1-methyl-5H-pyrido?4,3-b?ndole (Trp-P-2). Secondly, the rate of Trp-P-2 N-hydroxylation, the major route of bioactivation, by PCB-treated rat hepatic microsomes was markedly decreased. Cytochrome P450-dependent O-dealkylation of methoxy-, ethoxy- and pentoxyresorufin by these microsomes was also significantly inhibited by purpurin. The nature of this inhibition was competitive. Spectrophotometric investigations suggest no direct interaction between Trp-P-2 and purpurin. Furthermore, no evidence for Trp-P-2 binding was observed with carminic acid, a structural analog of purpurin, when it was immobilized on omega-aminohexyl agarose. Therefore, in vitro the proposed mechanism by which purpurin protects against heterocyclic amine-induced mutagenesis involves competitive inhibition of cytochrome P450-dependent bioactivation and accelerated degradation of the N-hydroxylamine to the parent amine.     

Immunohistochemical localization of choline acetyltransferase using a monoclonal antibody: a radioautographic method

Monoclonal antibodies to rat striatal choline acetyltransferase were produced by fusion of sensitized mouse lymphocytes with murine plasmacytoma (NS1) cells. Two stable anti-choline acetyltransferase lines were established by limiting dilution cloning. Specificity of antibody was established by the following criteria: (1) on an enzyme linked immunosorbant assay, antibodies reacted against choline acetyltransferase which was highly purified; (2) by immunoprecipitation, monoclonal antibody bound to its antigen and precipitated choline acetyltransferase activity from solution, when used in conjunction with rabbit antimouse IgG; and (3) monoclonal antibody was shown to specifically localize cholinergic neurons. The monoclonal antibody to choline acetyltransferase was radiolabeled in culture by incubating hybridomas in medium containing 3H-labeled amino acids. This 3H-labeled antibody was used for radioautography on cryostat sections of rat peripheral and central nervous systems. In a sampling of areas, highly specific labeling of cholinergic structures was afforded at both light and electron microscopic levels. Double labeling of tyrosine hydroxylase, a catecholaminergic marker, and choline acetyltransferase was carried out by reacting sections first with the 3H-labeled antibody to choline acetyltransferase and then with rabbit antibody to tyrosine hydroxylase. The choline acetyltransferase label was radioautographically processed and tyrosine hydroxylase was visualized by the peroxidase-antiperoxidase method. The combined techniques of peroxidase and radioautographic histochemistry provide permanent electron dense labels which can be examined simultaneously within a single histologic section.     

Absence of tyrosine hydroxylase activity and dopamine beta-hydroxylase immunoreactivity in intrinsic nerves of the guinea-pig ileum.

The activity of tyrosine hydroxylase and the localization of dopamine β-hydroxylase were determined in the myenteric and submucous plexuses of the normal guinea-pig ileum and in these plexuses after extrinsic denervation. In the normal ileum, the distribution of axons showing immunoreactivity for dopamine β-hydroxylase was not distinguishable from the distribution of noradrenergic axons determined by the fluorescence histochemical localization of catecholamines. The distribution of tyrosine hydroxylase in the different layers of the intestine correlated well with the distribution of dopamine β-hydroxylase and noradrenaline, tyrosine hydroxylase activity being most concentrated in the myenteric and submucous plexuses. Extrinsic denervation resulted in the complete disappearance of both biochemically detectable tyrosine hydroxylase and immunohistochemically demonstrable dopamine β-hydroxylase.It is concluded that if the amine-handling neurons which are known to be intrinsic to the intestine synthesize an aromatic amine, it is almost certainly not a catecholamine, and is probably an indoleamine.     

Effects of repeated systemic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on striatal tyrosine hydroxylase activity in vitro and tyrosine hydroxylase content

We examined both in vitro tyrosine hydroxylase (TH) activity and TH content determined by a new enzyme immunoassay in the mouse striatum after repeated systemic injection of MPTP. Repeated systemic administration of MPTP to mice (30 mg/kg per day, subcutaneously for 8 days) caused an approximately 65% decrease of both TH activity and TH content in the striatum. The intensity of immunohistochemical staining of TH protein in the striatum was also reduced in MPTP-treated mice. These results indicate that the reduction of TH activity in vitro after the repeated administration of MPTP is due to reduction of TH protein as a result of nerve degeneration.     

Differences in sensitivity to nerve growth factor of axon formation and tyrosine hydroxylase induction in cultured sympathetic neurons

Superior cervical ganglia from 2-day-old and 3-week-old rats were maintained in vitro for up to 2 weeks in the presence of a range of concentrations of nerve growth factor up to 100 micrograms/ml. Nerve fibre length and density were measured and tyrosine hydroxylase activity of these cultures assayed after various times. Ganglia were also examined for catecholamines and neuronal numbers using fluorescence histochemistry and histology respectively. In cultures maintained without nerve growth factor, or in those containing low concentrations of nerve growth factor (3 ng/ml), tyrosine hydroxylase decreased to 5-10% of the initial levels by 14 days in vitro. The presence of the high concentration of 1 microgram/ml nerve growth factor in the culture medium or the addition of such a concentration during the culture period did not prevent an initial decrease in tyrosine hydroxylase but subsequently increased the enzyme activity. The maximal effect of nerve growth factor on nerve fibre density was at low concentrations whereas its maximal effect on neuronal survival, tyrosine hydroxylase activity or nerve fibre elongation was at high concentrations. After 2 days in culture, maximum neurite production occurred in cultures containing 10 ng/ml, while maximum nerve fibre elongation and tyrosine hydroxylase activity occurred in cultures containing 100 micrograms/ml nerve growth factor. We conclude that low concentrations of nerve growth factor, as occur in plasma, cause maximum axon formation while high concentrations of nerve growth factor, as occur in effector organs, induce maximum tyrosine hydroxylase activity and cell survival. The former process may be mediated via cell surface receptors and the latter via retrograde axonal transport of nerve growth factor to the cell body, following uptake by the terminal regions of the axons.     

Short-term effects of 3,4-methylenedioximethamphetamine on noradrenergic activity in locus coeruleus and hippocampus of the rat

This study was undertaken to investigate the effects of the administration of 3,4-methylenedioxymethamphetamine (MDMA) on the locus coeruleus firing rate, on the sensitivity of the alpha(2)-adrenoceptors which regulate neuronal activity and on the in vivo tyrosine hydroxylase activity in hippocampus. The basal firing rate was not modified by either a single dose or repeated doses of MDMA, although the latter produced a shift to the right in the dose-response curve for clonidine-induced inhibition of the firing rate (ED(50) increased by 59%) and a reduction in tyrosine hydroxylase activity (20%) in the hippocampus. However, 8 days after the final dose alpha(2)-adrenoceptor sensitivity and tyrosine hydroxylase activity had returned to control values. Our results show a desensitization of alpha(2)-adrenoceptors in locus coeruleus and the existence of short-term changes in the noradrenergic system.     

Lesions of nigrostriatal pathway reduce expression of tyrosine hydroxylase gene in residual dopaminergic neurons of substantia nigra.

The effects of unilateral mechanical transection of the nigrostriatal bundle of rat brain on the level of tyrosine hydroxylase (TH) mRNA and on the activity of TH enzyme in the substantia nigra (SN) were examined. Lesions resulted, by 14 days, in reductions of TH mRNA level to 10% of control and of TH enzyme activity to 39% of control in the ipsilateral SN. The percentage of TH mRNA is lower than either the percentage of surviving dopaminergic neurons or the remaining TH enzyme activity. In situ hybridization analyses also demonstrated the reduction of TH mRNA concentration in surviving dopaminergic neurons in the ipsilateral SN.     

Murine tyrosine hydroxylase maps to the distal end of chromosome 7 within a region conserved in mouse and man

The locus of the structural gene encoding tyrosine hydroxylase, Th, the rate limiting enzyme for catecholamine biosynthesis, was mapped to the distal end of mouse Chromosome (Chr) 7. A DNA probe of genomic origin of rat tyrosine hydroxylase was used to detect restriction fragment length variants among 8 inbred mouse strains. The strain distribution pattern of Th allelic variants in 3 sets of recombinant inbred mouse strains was determined. Comparison of the strain distribution patterns of Th alleles with those of previously typed loci suggested Th was located on Chr 7. The Chr 7 assignment for Th was confirmed by analyzing 108 mice produced from an (NZB X SM)F1 X NZB backcross. Moreover, the Th locus was positioned distally on Chr 7. Mouse Chr 7 and human Chr 11p (the location of the human tyrosine hydroxylase gene) are known to share several homologous loci. With the addition of Th, the homology between the distal 2/3 of mouse Chr 7 and human Chr 11p appears extensive.     

Effects of tyrosine hydroxylase mutants on locomotor activity in Drosophila: a study in functional genomics

The brain of the adult fruit fly, Drosophila melanogaster, contains tyrosine hydroxylase, the rate-limiting enzyme required for catecholamine biosynthesis, as well as dopa decarboxylase. Catecholamines, principally dopamine, are also present. We have previously shown that pharmacological inhibition of tyrosine hydroxylase with -methyl-p-tyrosine results in a dose-related inhibition of locomotor activity in adult organisms. Similar results were found with reserpine, a well-known inhibitor of catecholamine uptake into storage granules. The drug-induced inhibition could be prevented in each case by the concomitant administration of l-dopa. The single-copy gene coding for tyrosine hydroxylase in Drosophila is pale (ple). Both null and temperature-sensitive loss of function mutant alleles of ple are recessive embryonic lethals. Heterozygous null mutant flies have normal locomotor activity demonstrating that only a single dose of the wild type form of ple is required to support normal function. Both hemizygous and homozygous temperature-sensitive ple mutants (ple ^ts1 ) also show normal locomotor activity at the permissive temperature for this mutant allele (18°C), which progressively declines as the temperature is increased to its restrictive level (29°C). These abnormal locomotor effects are reversible by l-dopa. Thus the effects on locomotor activity resulting from the pharmacological inhibition of catecholamine synthesis or storage are the same as those resulting from lack of tyrosine hydroxylase expression. These findings indicate that brain catecholamine loss decreases locomotor activity in the fly, as it does in mammals, and demonstrate the ability of functional genomic studies to mimic that of pharmacological inhibition of enzyme function or other similar processes.     

Selective induction by glucocorticoids of tyrosine hydroxylase in organ cultures of rat pheochromocytoma

Addition of physiological concentrations of glucocorticoids to organ cultures of a transplantable rat pheochromocytoma resulted in an increase in the specific activity of tyrosine hydroxylase. The increase was delayed, beginning after 12 h and reached its peak after 36 h, when the specific activity was twice that of controls. The activities of dopamine β-hydroxylase and dopa decar?ylase showed no significant changes. The tumour contains choline acetyltransferase and the specific activity of this enzyme was reduced by 25% following culture in the presence of glucocorticoids. Addition of cholinergic agonists and/or nerve growth factor to the culture medium had no effect on the specific activity of tyrosine hydroxylase. The effect of glucocorticoids on this enzyme was entirely abolished by inhibitors of ribonucleic acid or protein synthesis.These results are discussed in relation to the modulatory role of glucocorticoids in trans-synaptic and nerve growth factor-mediated enzyme induction in the peripheral sympathetic nervous system.     

Murine tyrosine hydroxylase maps to the distal end of chromosome 7 within a region conserved in mouse and man

The locus of the structural gene encoding tyrosine hydroxylase, Th, the rate limiting enzyme for catecholamine biosynthesis, was mapped to the distal end of mouse Chromosome (Chr) 7. A DNA probe of genomic origin of rat tyrosine hydroxylase was used to detect restriction fragment length variants among 8 inbred mouse strains. The strain distribution pattern of 77i allelic variants in 3 sets of recombinant inbred mouse strains was determined. Comparison of the strain distribution patterns of Th alleles with those of previously typed loci suggested Th was located on Chr 7. The Chr 7 assignment for Th was confirmed by analyzing 108 mice produced from an (NZB × SM)F1 × NZB backcross. Moreover, the Th locus was positioned distally on Chr 7. Mouse Chr 7 and human Chr 1 lp (the location of the human tyrosine hydroxylase gene) are known to share several homologous loci. With the addition of Th, the homology between the distal 2/3 of mouse Chr 7 and human Chr lip appears extensive.     

Reserpine increases opiate-like peptide content and tyrosine hydroxylase activity in adrenal medullary chromaffin cells in culture

Primary cultures of adrenal medullary chromaffin cells maintained in a serum-free medium retain high levels of both catecholamines and opiate-like peptides. Addition of reserpine (100nM) to the culture medium results in the exponential loss of cellular catecholamines (t_½ = 1.5days) and in an elevation of opiate-like peptide content and tyrosine hydroxylase activity without altering total cell protein content. The maximum increase in opioid activity as a result of reserpine treatment averaged 230% of untreated cell levels and was reached by 2–3 days after initiation of treatment. A similar time-course was observed for the elevation of tyrosine hydroxylase activity. The increases of opiate-like peptides and tyrosine hydroxylase induced by reserpine are blocked by inclusion of actinomycin D or cycloheximide in the culture medium, suggesting that both messenger ribonucleic acid and protein synthesis are required for the induction.These data suggest that synaptic activation is not the only long-term regulator of opiate-like peptide and catecholamine biosynthesis in the adrenal medulla and that the biosynthesis of opiate-like peptides, which are components of chromaffin vesicles, and tyrosine hydroxylase, the cytoplasmic, rate-limiting enzyme in catecholamine biosynthesis, are coordinately regulated.