Dual enzyme-activated irreversible inhibition of monoamine oxidase

(E)-β-Fluoromethylene-m-tyrosine and related amino acids were synthesized from acetophenone derivatives and shown to be dual enzyme-activated inhibitors of monoamine oxidase. These substances are decarboxylated by hog kidney aromatic l-amino acid decarboxylase liberating (E)-β-fluoromethylene-m-tyramine derivatives which, in turn, are enzyme-activated inhibitors of rat brain mitochondrial monoamine oxidase.     

Effects of nutritional stress on brain tyramine concentration and dopamine turnover

This is an investigation of the effect of nutritional stress at various ages on the levels of thep- andm-isomers of tyramine in the caudate nucleus of the rat. For comparison, the effects of nutritional stress on the concentration and turnover of dopamine were also studied. Nutritional stress induced in pre-weaning (3 weeks of age) or post-weaning (up to 9 weeks of age) rats resulted in a decrease in the concentration ofp-tyramine and an increase in the concentration ofm-tyramine in the caudate nucleus. Dopamine concentration or turnover in the caudate nucleus was not affected by pre-weaning undernutrition; in the olfactory tubercles, however, a significant decrease was observed in dopamine turnover, calculated from the decrease in homovanillic acid levels after monoamine oxidase inhibition. The results suggest the changes observed are dependent on the availability of the amino acid precursorsp- andm-tyrosine and their competition towards aromatic-l-amino acid decarboxylase.     

Disaggregation of brain polysomes by L-5-hydroxytryptophan: Mediation by serotonin

In rats, intraperitoneal administration of L-5-hydroxytryptophan (200 mg/kg) causes extensive disaggregation of whole brain polysomes after one hour. Polysome disaggregation is prevented if the conversion of L-5-hydroxytryptophan to serotonin is blocked by pretreatment with an aromatic L-amino acid decarboxylase inhibitor; disaggregation is potentiated by pretreatment with a monoamine oxidase inhibitor. The brain polysome disaggregation induced by L-phenylalanine administration (1 g/kg) is not blocked by decarboxylase inhibition.     

Estimation of the p and m Isomers of Hydroxyphenylacetic Acid in Mouse Brain by a Gas Chromatographic Procedure: Their Regional Distribution and the Effects of Some Drugs

Abstract: The mouse brain contains 12.5 and 4.1 ng/g of p- and m -hydroxyphenylacetic acids, respectively. The hydroxyphenylacetic acids were isolated by chromatography on DEAE-Sephadex A-25 and quantitated as their pentafluoropropionyl and hexafluoropropanol esters by use of a gas chromatograph equipped with an electron-capture detector. The highest concentrations of p- or m -hydroxyphenylacetic acids were observed in the caudate nuclei (27.9 and 8.7 ng/g, respectively) and olfactory tubercles (20.2 and 5.3 ng/g, respectively). The identities of the p- and m -hydroxyphenylacetic acids were further confirmed as a consequence of the reductions observed following monoamine oxidase inhibition or the increases observed in the appropriate acid following the parenteral administration of p- or m -tyramine.     

Production of 2-phenylethanol in roses as the dominant floral scent compound from L-phenylalanine by two key enzymes, a PLP-dependent decarboxylase and a phenylacetaldehyde reductase

We investigated the biosynthetic pathway for 2-phenylethanol, the dominant floral scent compound in roses, using enzyme assays. L-[(2)H8] Phenylalanine was converted to [(2)H8] phenylacetaldehyde and [(2)H8]-2-phenylethanol by two enzymes derived from the flower petals of R. 'Hoh-Jun,' these being identified as pyridoxal-5'-phosphate-dependent L-aromatic amino acid decarboxylase (AADC) and phenylacetaldehyde reductase (PAR). The activity of rose petal AADC to yield phenylacetaldehyde was nine times higher toward L-phenylalanine than toward its D-isomer, and this conversion was not inhibited by iproniazid, a specific inhibitor of monoamine oxidase. Under aerobic conditions, rose petal AADC stoichiometrically produced NH3 together with phenylacetaldehyde during the course of decarboxylation and oxidation, followed by the hydrolysis of L-phenylalanine. Phenylacetaldehyde was subsequently converted to 2-phenylethanol by the action of PAR. PAR showed specificity toward several volatile aldehydes.     

Octopamine metabolism in the cephalic ganglion of Locusta migratoria L.

Octopamine contents in the cephalic ganglia of the locust Locusta migratoria have been determined in male and female late larval and in adult stages. The animals have been pretreated with intra-abdominal administration of p-tyrosine or p-tyramine alone or following the administration of reserpine.1. p-Octopamine levels were higher at the 7th day than at the 5th day in the fourth larval stage. At this stage a significant difference was observed in male and female cephalic ganglia.2. The administration of p-tyrosine and p-tyramine resulted in ganglia.3. The pretreatment with reserpine resulted in an almost complete depletion in p-octopamine.4. This depletion was partially abolished by p-tyrosine or more efficiently by p-tyramine administration.5. Eventually, the p-octopamine levels were considerably decreased by the administration of the decarboxylase inhibitor, R04-4602, of the dopamine β-hydroxylase inhibitor, fusaric acid, and of the false neurotransmitter 6-hydroxydopamine.     

Characteristics of Dihydroxyphenylalanine/5-Hydroxytryptophan Decarboxylase Activity in Brain and Liver of Cat

Abstract: Dihydroxyphenylalanine/5-hydroxytryptophan (DOPA/5-HTP) decarboxylase activity varied widely in different parts of the CNS, being highest in the neostriatum and lowest in the frontal cortex. The addition of 2.5 μ m -pyridoxal 5'-phosphate (PLP), the coenzyme, increased enzyme activity in brainstem and liver, while higher concentrations led to a decrease in activity. In brainstem, the addition of 1000 μ m PLP shows activity similar to that obtained without exogenous PLP. The effects of different monoamine oxidase (MAO) inhibitors on decarboxylase activity were demonstrated. Iproniazid phosphate and harmaline significantly decreased the decarboxylation in liver and brainstem, while pargyline inhibited only liver decarboxylation. Some decarboxylase inhibitors such as RO4–4602 and α-methyl DOPA, as well as piribedil, a dopaminergic receptors agonist, were added in vitro to measure their action on decarboxylase with or without exogenous PLP or with double concentrations of substrate (5-HTP). Piribedil (5000 μ m ) affected the enzymic reaction and triggered a higher inhibition in liver. Inhibition in brainstem needed less RO4–4602 (50 μ m ) than in liver (300 μ m ). Addition of PLP did not reverse this inhibition, while doubling the concentration of 5-HTP nullified the inhibitory effect in liver only. Inhibition induced by α-methyl DOPA (5 μ m ) was easily reversed by doubling the concentration of substrate. However, the presence of exogenous PLP restored the enzymic activity in liver only. We conclude from this work thus that the enzyme can decarboxylate its substrate without exogenous PLP, that MAO inhibitors might inhibit decarboxylase activity, and that decarboxylase inhibitors react differently when brain and liver are used as enzymic source. PLP seems to act as a protective agent on the active site of the enzyme in the brainstem and preferentially with the substrate in the liver.     

Effect of dopamine agonists and antagonists on DOPA formation in the substantia nigra

Abstract: The effect of different psychotropic drugs on the rate of DOPA accumulation after administration of a decarboxylase inhibitor (NSD 1015) was compared in the substantia nigra (SN) and caudate nucleus (CN) by a new radioenzymatic method. Inhibition of monoamine oxidase with pargyline or stimulation of dopamine (DA) receptors with apomorphine, N -n-propyl-norapomorphine or d -amphetamine reduced DOPA formation in the CN and SN to the same extent. Vice versa, both inhibition of DA receptors with haloperidol or (-)sulpiride and depletion of DA concentration with reserpine enhanced DOPA formation to a greater extent in the CN than in the SN. Apomorphine antagonized not only the effect of haloperidol and (-)sulpiride, but also, and even more effectively, that of reserpine. The results indicate that DA synthesis in the SN is controlled by both end-product inhibition and DA receptor-mediated mechanisms.     

The effects of some antipsychotic drugs, D-amphetamine, and reserpine on the concentration and rate of accumulation of tryptamine and 5-hydroxytryptamine in the mouse striatum

The mouse striatum contains about 2 ng/g of tryptamine and 600 ng/g of 5-hydroxytryptamine. No significant changes in mouse striatal tryptamine were observed after the administration of chlorpromazine, haloperidol, spiperone, or alpha-flupenthixol. The levels of 5-hydroxytryptamine were moderately reduced by chlorpromazine, spiperone, and alpha-flupenthixol but not by haloperidol. The administration of antipsychotic drugs to mice pretreated with a monoamine oxidase inhibitor (pargyline) produced an increase in the rate of accumulation of striatal tryptamine compared with that of pargyline-treated mice. In contrast, the rate of accumulation of 5-hydroxytryptamine after monoamine oxidase inhibition was reduced by chlorpromazine, spiperone, and alpha-flupenthixol but not haloperidol. D-Amphetamine administration did not change either tryptamine or its 5-hydroxyderivative while reserpine increased tryptamine and reduced 5-hydroxytryptamine. The results suggest that changes in striatal tryptamine may be controlled by the availability of tryptophan, the amino acid precursor of tryptamine.     

Nucleotide and bivalent cation specificity of the insulin-granule proton translocase

3-(4-[(3-Chlorophenyl)methoxy]phenyl)-5-[(methylamino)methyl]-2- oxazolidinone methanesulphonate (compound MD 780236) is a selective inhibitor of the B-form of monoamine oxidase. Inhibition involves an initial non-covalent interaction between enzyme and inhibitor followed by a time-dependent process resulting in irreversible inhibition. The initial, reversible, phase of inhibition was found to be competitive with respect to phenethylamine and 5-hydroxytryptamine, and a comparison of the Ki values indicated the affinity of the inhibitor for the B-form of the enzyme to be some 7-fold greater than its affinity for the A-form. This selectivity was considerably enhanced by preincubation of the enzyme and inhibitor. Time courses showed that complete inhibition was not achieved under conditions where the inhibitor concentration was over 100-fold greater than that of the enzyme. Assay of the activity of monoamine oxidase by determining the release of hydrogen peroxide fluorometrically showed compound MD 780236 to be a substrate for, as well as an inhibitor of, monoamine oxidase, and kinetic analysis revealed that the rate of product formation was some 530-fold greater than that of the process leading to irreversible inhibition of the B-form of the enzyme.     

Inhibition of uroporphyrinogen decarboxylase activity. The role of cytochrome P-450-mediated uroporphyrinogen oxidation.

It was previously shown that uroporphyrinogen oxidation is catalysed by a form of cytochrome P-450 induced by 3-methylcholanthrene [Sinclair, Lambrecht & Sinclair (1987) Biochem. Biophys. Res. Commun. 146, 1324-1329]. We have now measured uroporphyrinogen oxidation and uroporphyrinogen decarboxylation simultaneously in 10,000 g supernatants from the livers of methylcholanthrene-treated mice and chick embryos incubated with an NADPH-generating system. We found that uroporphyrinogen oxidation is associated with inhibition of uroporphyrinogen decarboxylase activity. The decreased uroporphyrinogen decarboxylase activity was not due to depletion of substrate, since decarboxylase activity was not increased by a 2.6-fold increase in uroporphyrinogen. Uroporphyrinogen oxidation and the associated inhibition of decarboxylase activity were also observed with liver supernatant from methylcholanthrene-treated chick embryo; both actions required the addition of 3,3',4,4'-tetrachlorobiphenyl. Uroporphyrinogen oxidation catalysed by microsomes from a methylcholanthrene-treated mouse inhibited the uroporphyrinogen decarboxylase activity in the 100,000 g supernatant. Ketoconazole, an inhibitor of cytochrome P-450, prevented both uroporphyrinogen oxidation and the inhibition of uroporphyrinogen decarboxylation. The addition of ketoconazole to mouse supernatant actively oxidizing uroporphyrinogen inhibited the oxidation and restored decarboxylation. The latter finding suggested that a labile inhibitor was formed during the oxidation. These results suggest uroporphyrinogen oxidation may be important in the mechanism of chemically induced uroporphyria.     

Inhibition of Aromatic L-Amino Acid Decarboxylase and Tyrosine Aminotransferase by the Monoamine Oxidase Inhibitor Phenelzine

The concentration of p-tyramine in the rat striatum was increased significantly by intraperitoneal injection of phenelzine (5 or 100 mg/kg). Unlike other monoamine oxidase (MAO) inhibitors, phenelzine had no effect on p-tyramine levels in the first 1-2 h following injection. The high dose of phenelzine increased the p-tyramine levels much more than the low dose. In addition, the high dose of phenelzine increased striatal p-tyrosine levels significantly 12 h after injection. Further studies showed that phenelzine inhibited the tyrosine aminotransferase activity of rat liver homogenates; the IC50 was 50 microM. Phenelzine also inhibited the aromatic L-amino acid decarboxylase activity of rat brain homogenate with an IC50 of 25 microM. Following intraperitoneal injection of 100 mg/kg phenelzine, the initial concentration of phenelzine in the striatum appears to be high enough to inhibit aromatic L-amino acid decarboxylase. It is suggested that the multiple enzyme inhibition caused by administration of high doses of phenelzine accounts for its unusual effects on striatal p-tyramine levels compared with other MAO inhibitors, i.e., its initial lack of effect on p-tyramine levels followed later by very large increases in p-tyramine levels.     

Conjugated Dopamine in Superfusates of Slices of Rat Striatum

Abstract: An acid-hydrolyzable conjugate of 3,4-dihydroxyphenylethylamine (dopamine, DA) was detected in superfusates from slices from rat striatum. The concentrations of endogenous free and conjugated DA, and of the acid metabolites (3,4-dihydroxyphenylacetic acid [DOPAC] and homovanillic acid [HVA]) in superfusates were measured using HPLC with electrochemical detection. Conjugated DA in superfusates represented 10–20% of the free DA under basal conditions and during release evoked by p -tyramine (5 × 10⁻⁶ M to 5 × 10⁻⁴ M ); much smaller amounts of conjugated DA overflowed into superfusate when DA was released by equimolar concentrations of β-phenylethyl-amine. Surprisingly, inhibition of monoamine oxidase by the inhibitors N -methyl- N -propargyl-3-(2,4-dichlorophenoxy)propylamine hydrochlo-ride (clorgyline) or N -methyl- N -2-propynylbenylamine (pargyline) had little effect on the amounts of conjugated DA present in superfusate. Under basal conditions, the amounts of conjugated DA in superfusate were always less than the amounts of DOPAC but quite similar to the amounts of HVA. However, during release of DA evoked by p -tyramine the concentrations of conjugated DA in superfusate showed much more pronounced increases than those of the acidic metabolites.     

ENZYMATIC ISOTOPIC ASSAY FOR AND PRESENCE OF β-PHENYLETHYLAMINE IN BRAIN

Abstract— An enzymatic isotopic assay for the measurement of β-phenylethylamine in brain, with a sensitivity of 100-200 pg, has been developed. With this assay, the endogenous β-phenylethylamine content (1.5 ng/g) in the rat brain has been determined. Phenylalanine administration increases the brain levels of this amine; inhibition of monoamine oxidase causes a 40-fold increase in brain β-phenylethylamine. After a combined treatment with a monoamine oxidase inhibitor and phenylalanine, the β-phenylethylamine content in the brain increases to about 400-fold. This increase can be blocked by the central decarboxylase inhibitor NSD-1055. p-Chlorophenylalanine also increases β-phenylethylamipe concentration in the brain, and this effect is potentiated by a simultaneous administration of phenylalanine.     

EFFECTS OF LESIONS AND DRUGS ON BRAIN TRYPTAMINE

Abstract— The effects of various drugs and lesions on rat brain 5-hydroxytryptamine and tryptamine were determined. Monoamine oxidase inhibition caused a proportionately greater increase in tryptamine than in 5-hydroxytryptamine, reserpine depleted 5-hydroxytryptamine but had no effect on tryptamine while p -chlorophenylalanine lowered 5-hydroxytryptamine but increased tryptamine. α-Methyl- p -tyrosine reduced striatal dopamine with no effect on either 5-hydroxytryptamine or tryptamine. Increasing brain tryptophan by amphetamine administration. 24 h food deprivation or giving L-tryptophan did not increase brain tryptamine. However a high dose of ^L-tryptophan (100 or 200mg/kg) together with a monoamine oxidase inhibitor caused a proportionately much greater increase in tryptamine than in 5-hydroxytryptamine. Raphe lesions reduced 5-hydroxytryptamine by 64 per cent and tryptamine by only 29 per cent while intraventricular 6-hydroxydopamine lowered striatal dopamine (56 per cent), had no effect on 5-hydroxytryptamine but reduced tryptamine by 24 per cent, suggesting that tryptamine can be formed in both 5-HT and catecholaminergic neurones.
The results are discussed in relation to the formation, distribution, storage and possible transmitter function of tryptamine in rat brain.     

α-DIFLUOROMETHYL DOPA, A NEW ENZYME-ACTIVATED IRREVERSIBLE INHIBITOR OF AROMATIC L-AMINO ACID DECARBOXYLASE

DL-x-Difluoromethyl DOPA (DFMD, RMI 71801), an enzyme-activated irreversible inhibitor of aromatic L-amino acid decarboxylase in vitro, produces a rapid, long-lasting and dose-dependent inhibition of aromatic L-amino acid decarboxylase in peripheral tissues of mice when administered i.p. or orally. Doses of 500 mg/kg i.p. produce only very slight inhibition of the enzyme activity in mouse brain whilst inhibiting the enzyme activity of peripheral tissues by more than 90%. With L-[³H]-DOPA co-administration brain concentrations of L-[³H]DOPA and ³H-catecholamines are increased 3- to 8-fold concomitant with a decrease in the peripheral decarboxylation of L-[³H]DOPA. Under these conditions it is clear that the slight inhibition of enzyme activity in the brain is totally inadequate to inhibit the decarboxylation of L-DOPA in this organ. Similarly, the decarboxylation of exogenously supplied 5-hydroxytryptophan is inhibited peripherally with a consequent increase in brain serotonin concentrations. DFMD is another example of an enzyme-activated irreversible inhibitor which due to its novel and specific mechanism of action, may offer advantages over existing decarboxylase inhibitors.     

Microdialysis Monitoring of 3,4-Dihydroxyphenylalanine Accumulation After Decarboxylase Inhibition: A Means to Estimate In Vivo Changes in Tyrosine Hydroxylase Activity of the Rat Locus Ceruleus

Abstract: An on-line microdialysis approach was developed to estimate changes in tyrosine hydroxylase activity in the locus ceruleus noradrenergic neurons of anesthetized rats by measuring the 3,4-dihydroxyphenylalanine (DOPA) acumulation in the extracellular fluid during perfusion of an aromatic amino acid decarboxylase inhibitor through a dialysis probe. The aromatic amino acid decarboxylase inhibitor used was difluoromethyl-DOPA, which was shown to be more stable than NSD 1015 or Ro 4-4602 in the perfusion fluid. A 1-h perfusion of a 10⁻⁴ mol/L of difluoromethyl-DOPA solution induced a linear increase in DOPA concentration in the locus ceruleus dialysates that achieved a steady state within 1 h. The identity of DOPA accumulated in dialysates during aromatic amino acid decarboxylase inhibition was confirmed by the disappearance of the chromatographic peak when DOPA formation was blocked by the administration of α-methyl- p -tyrosine. Systemic administration of the α₂-antagonist piperoxane before difluoromethyl-DOPA perfusion markedly increased the DOPA concentration during both the accumulation and the steady-state periods, showing that the present technique is a suitable in vivo approach to monitor changes in tyrosine hydroxylase activity occurring in the locus ceruleus neurons.     

Kinetics of intraventricularly injected trace amines and their deuterated isotopomers

Intraventricular injection into the rat brain of four trace amines and a catecholamine resulted in rapid exponential loss of the amines in the first 30 minutes after injection. The half-lives were: phenylethylamine 3.8 min,para-tyramine 5.1 min,meta-tyramine 7.4 min and dopamine 8.0 min. Tryptamine showed a biphasic loss with half-lives of 4.7 min (over the 5 to 10 min period) and 14.1 min (10 to 30 min). The half-lives were substantially increased by deuterium labeling at the alpha carbon position: phenylethylamine 4.8 min,para-tyramine 8.8 min,meta-tyramine 14.1 min, dopamine 13.0 min and tryptamine 6.0 min (5 to 10 min period) and 28.7 min (10 to 20 min). The loss of the amines was reduced by monoamine oxidase inhibition by pargyline hydrochloride and the deuterium isotope effect was abolished. It is noteworthy that the half-life of dopamine was similar to those of the trace amines in this time period and that the trace amine half-lives after i.v. injection was longer than those obtained from measurements of increases of concentrations of endogenous amines after MAOI in vivo and that of dopamine shorter than values calculated from turnover measurements.     

Adrenergic enzymes in cultured mouse neuroblastoma: absence of detectable aromatic-L-amino-acid decarboxylase.

The relative activities of tyrosine hydroxylase, aromatic-l -amino-acid decarboxylase and dopamine beta-hydroxylase were established in a number of clones of neuroblastoma cells isolated from the uncloned mouse C-1300 tumor. One clone, NBD-2, was chosen for further analysis on the basis of its relatively high activities of tyrosine hydroxylase and dopamine beta-hydroxylase. The levels of these enzymes, and monoamine oxidase and catechol O-methyltransferase, were at least 20-80 fold lower in the neuroblastoma culture than in mouse superior cervical ganglion. More importantly, aromatic-l -amino-acid decarboxylase activity was not even detectable in any neuroblastoma clone examined. Based on the relative sensitivities of the tyrosine hydroxylase and aromatic-l -amino-acid decarboxylase assays and on the ratio of these two enzymes in the mouse ganglion, decarboxylase activity is more than 10 fold lower in the cultured cells than would be predicted on the basis of tyrosine hydroxylase activity. Dialysis and mixing studies with neuroblastoma extracts and partially purified aromatic-l -amino-acid decarboxylase did not reveal the presence of any endogenous inhibitors that could account for the low level of decarboxylase activity in the cultured cells. During growth of the neuroblastoma cells to confluency, only one enzyme, monoamine oxidase, exhibited an elevated specific activity on the basis of cell number. However, when based on the amount of protein, the specific activity of all measurable enzymes increased in culture-because cell protein decreased 5 fold during growth to confluency. These findings are discussed with respect to individual cell function.     

BRAIN SEROTONIN CHANGES IN PHENYLALANINE-FED RATS: SYNTHESIS STORAGE AND DEGRADATION1

The brain serotonin levels of rats maintained on a 5 % phenylalanine diet rose more slowly (0.18 μ g/g brain/hr) after administration of a monoamine oxidase inhibitor than did serotonin levels of controls (0.41 μ g/g brain/hr). The rate of brain serotonin decline following reserpine or dimethylaminobenzoyl methyl reserpate was the same for both groups as was basal monoamine oxidase activity. Brain uptake of monoamine oxidase inhibitor was also the same for both groups. It was concluded that the decrease in brain serotonin levels in phenylalanine-fed animals was due to decreased serotonin formation rather than enhanced degradation. On the basis of available data it was concluded that both hydroxylase inhibition and inhibited precursor transport were involved.