OCTOPAMINE IN MAMMALIAN BRAIN: RAPID POST MORTEM INCREASE AND EFFECTS OF DRUGS

A modified enzyme radiochemical assay for octopamine, based upon the N-methylation of octopamine by the enzyme phenylethanolamine N-methyl transferase (S-Adenosyl-1-methionine: phenylcthanolamine N-methyl transferase EC 2.1.28), has been developed. [³H]Methyl-S-adenosyl-l- methionine was used as methyl donor, and the reaction products separated by thin-layer chromatography prior to liquid scintillation counting. The method had a sensitivity of about 100 pg, and was suitable for the measurement of endogenous octopamine levels in mammalian brain. Although the method could be used for the determination of phenylethanolamine with similar sensitivity, concentrations of this amine in brain were too low for routine measurement. Octopamine levels in the brains of a number of mammalian species were determined using this procedure. Concentrations of the amine in mouse brain were lower in animals killed by rapid freczing than in animals killed by decapitation; a further increase in brain octopamine took place post-mortem. Brain octopamine was increased following treatment with MAO inhibitors, p-chlorophenylalanine, phenylalanine, tyrosine or phenylethylamine. The effects of tyrosine and phenylethylamine were greatly increased by pretreatment with a monoamine oxidase inhibitor. The antidepressants imipramine and iprindole gave rise to increased brain octopamine concentrations, possibly through an effect upon monoamine oxidase. Administration of chlordiazepoxide chlorpromazine, thyroxine, or reserpine had no effect upon brain octopamine.     

Contribution of aldehyde oxidizing enzymes on the metabolism of 3,4-dimethoxy-2-phenylethylamine to 3,4-dimethoxyphenylacetic acid by guinea pig liver slices.

BACKGROUND/AIMS: 3,4-Dimethoxy-2-phenylethylamine is catalyzed to its aldehyde derivative by monoamine oxidase B, but the subsequent oxidation into the corresponding acid has not yet been studied. Oxidation of aromatic aldehydes is catalyzed mainly by aldehyde dehydrogenase and aldehyde oxidase. METHODS: The present study examines the metabolism of 3,4-dimethoxy-2-phenylethylamine in vitro and in freshly prepared and cryopreserved guinea pig liver slices and the relative contribution of different aldehyde-oxidizing enzymes was estimated by pharmacological means. RESULTS: 3,4-Dimethoxy-2- phenylethylamine was converted into the corresponding aldehyde when incubated with monoamine oxidase and further oxidized into the acid when incubated with both, monoamine oxidase and aldehyde oxidase. In freshly prepared and cryopreserved liver slices, 3,4-dimethoxyphenylacetic acid was the main metabolite of 3,4-dimethoxy-2- phenylethylamine. 3,4-Dimethoxyphenylacetic acid formation was inhibited by 85% from disulfiram (aldehyde dehydrogenase inhibitor) and by 75-80% from isovanillin (aldehyde oxidase inhibitor), whereas allopurinol (xanthine oxidase inhibitor) inhibited acid formation by only 25-30%. CONCLUSIONS: 3,4- Dimethoxy-2-phenylethylamine is oxidized mainly to its acid, via 3,4-dimethoxyphenylacetaldehyde, by aldehyde dehydrogenase and aldehyde oxidase with a lower contribution from xanthine oxidase.     

In vivo relationship between monoamine oxidase type B and alcohol dehydrogenase: effects of ethanol and phenylethylamine

The role of acute ethanol (2.5 g/kg i.p.) and phenylethylamine (100 mg/kg i.p.) on the brain and platelet monoamine oxidase activities, hepatic cytosolic alcohol dehydrogenase, redox state and motor behaviour were studied in male rats. Ethanol on its own decreased the redox couple ratio, as well as, alcohol dehydrogenase activity in the liver whilst at the same time it increased brain and platelet monoamine oxidase activity due to lower Km with no change in Vmax. The elevation in both brain and platelet MAO activity was associated with ethanol-induced hypomotility in the rats. Co-administration of phenylethylamine and ethanol to the animals, caused antagonism of the ethanol-induced effects described above. The effects of phenylethylamine alone, on the above mentioned biochemical and behavioural indices, are more complex. Phenylethylamine on its own, like ethanol, caused reduction of the cytosolic redox ratio and elevation of monoamine oxidase activity in the brain and platelets. However, in contrast to ethanol, this monoamine produced hypermotility and activation of the hepatic cytosolic alcohol dehydrogenase activity in the animals. The results suggest that some of the toxic actions of ethanol in rats may be mediated through the activation of monoamine oxidase type B, with the consequent depletion of the endogenous levels of phenylethylamine. The data appear to support the concept of phenylethylamine involvement in affective disorders.     

The enigma of conditioned taste aversion learning: stimulus properties of 2-phenylethylamine derivatives

The functional aversive stimulus properties of several IP doses of (+/-)-amphetamine (1.25-10 mg.kg-1), 2-phenylethylamine (PEA, 2.5-10 mg.kg-1, following inhibition of monoamine oxidase with pargyline 50 mg.kg-1) and phenylethanolamine (6.25-50 mg.kg-1) were measured with the conditioned taste aversion (CTA) paradigm. A two-bottle choice procedure was used, water vs. 0.1 % saccharin with one conditioning trial and three retention trials. (+/-)-Amphetamine and phenylethanolamine induced a significant conditioned taste aversion but PEA did not. (+/-)-Amphetamine and PEA increased spontaneous locomotor activity but phenylethanolamine had no effects on this measure. Measurement of whole brain levels of these drugs revealed that the peak brain elevation of PEA occurred at approximately 10 min whereas the peak elevations of (+/-)-amphetamine and phenylethanolamine occurred at approximately 20 min. The present failure of PEA to elicit conditioned taste aversion learning is consistent with previous reports for this compound. The differential functional aversive stimulus effects of these three compounds are surprising since they exhibit similar discriminative stimulus properties and both (+/-)-amphetamine and PEA are self-administered by laboratory animals. The present data suggest that time to maximal brain concentrations following peripheral injection may be a determinant of the aversive stimulus properties of PEA derivatives.     

Phenylethanolamine is a specific substrate for type B monoamine oxidase

The characteristics of phenylethanolamine as both a competitive inhibitor and as a substrate for monoamine oxidase (MAO) were studied using rat brain and liver homogenates. Although phenylethanolamine, even at high concentrations (1 mM), produced minimal inhibition of MAO when serotonin (a substrate for type A MAO) was used as the substrate, it was a potent competitive inhibitor (K_i=11 μM) of the deamination of phenylethylamine (a substrate for type B MAO). When phenylethanolamine was used as a substrate, deprenyl, a selective inhibitor of type B MAO, was found to produce a single sigmoid inhibition curve at low concentrations of the inhibitor (pI₅₀=7.5). These results indicate that phenylethanolamine is a specific substrate for type B MAO. Identification of the products formed under the assay conditions show that phenylethanolamine is converted to both mandelic acid and phenylethylene glycol by liver homogenates but only to the latter, neutral metabolite by brain homogenates.     

Characterization of rat pulmonary monoamine oxidase.

The substrate specificities and kinetics of rat lung monoamine oxidase (MAO) have been studied. Utilizing the irreversible MAO inhibitors, clorgyline and deprenyl, rat lung was shown to possess at least two types of MAO, A and B. Tyramine was a substrate for both forms of the enzyme, whereas 5-hydroxytryptamine (5-HT) was a preferred substrate for the A-form. In contrast to most other tissues, 2-phenylethylamine was not solely a B-type substrate for the rat lung MAO and some metabolism by the A-type was apparent $\text{(}\text{B}\text{A}\text{=}\text{80}\text{20}\text{)}$. Using tyramine as substrate the ratio A/B was shown to be $\text{55}\text{45}$. Rat pulmonary MAO-B was inhibited by deprenyl and the kinetics of MAO-A studied. The Km values for the A-form for tyramine, phenylethylamine and 5-HT were relatively similar and were 270, 244 and 170 μM respectively. Whereas, when the A-form was inhibited by clorgyline, the Km values for the B-form were found to differ considerably: 330, 42 and 850 μM for tyramine, phenylethylamine and 5-HT respectively.     

A new type of mitochondrial monamine oxidase distinct from type-A and type-B

The characteristics of mitochondrial monoamine oxidase (MAO) in carp liver were studied with MAO inhibitors and substrates. This enzyme was thermolabile, but was stabilized in the presence of bovine serum albumin. With clorgyline and deprenyl, single-sigmoidal curves for inhibition of the activity towards tyramine or 5-hydroxytryptamine were obtained; the sensitivities to the two inhibitors were identical. The activity towards β-phenylethylamine was not completely inhibited by clorgyline or deprenyl, but the remaining activity was inhibited by semicarbazide and the inhibition curves by either clorgyline or deprenyl and semicarbazide were also identical to the curves with the other two substrates. These results suggest that carp liver mitochondria contain “classical” MAO and a clorgyline- and deprenyl-resistant amine oxidase and that the classical MAO does not seem to be MAO-A or MAO-B, which are present in mitochondria of most mammalian tissues.     

2-Chloro-2-phenylethylamine as a mechanistic probe and active site-directed inhibitor of monoamine oxidase from bovine liver mitochondria

The reaction of 2-chloro-2-phenylethylamine with monoamine oxidase B was investigated to study the mechanism of this enzyme and its inactivation by this compound. 2-Chloro-2-phenylethylamine is a substrate with a Km of 30 microM and a turnover number of 80 min-1 at pH 6.5 at 30 degrees C. Incubation of 2-chloro-2-phenylethylamine with the enzyme led to the normal oxidation product, 2-chloro-2-phenylacetaldehyde, but only traces (0.25 mol%) of 2-phenylacetaldehyde, the product anticipated if the oxidation of substrate involved a stabilized carbanion at C-1 and elimination of chloride ion. These data suggest that a carbanion is not a likely intermediate in the oxidation of amines by monoamine oxidase. During the mechanistic studies we noted time-dependent inactivation of monoamine oxidase B by 2-chloro-2-phenylethylamine under both aerobic and anaerobic conditions. Inactivation was not reversible. Aerobically 2-chloro-2-phenylethylamine is oxidized to 2-chloro-2-phenylacetaldehyde which covalently modifies the enzyme (tau 1/2 = 40 min). Benzyl alcohol, a substrate analog, gives substantial protection against inactivation under aerobic conditions (tau 1/2 = 320 min), suggesting that an active site residue is modified. Anaerobic reaction of 2-chloro-2-phenylethylamine with monoamine oxidase B probably proceeds by direct alkylation of an enzyme residue (tau 1/2 = 140 min). Reduction with [3H]NaBH4 of the inactivated enzyme gave from 0 to 0.7 and from 4.5 to 5.6 mol of hydride incorporation for enzyme inactivated anaerobically and aerobically, respectively. The latter results are in agreement with inactivation by unmodified inhibitor and inactivation by oxidized inhibitor for the anaerobic and aerobic reactions, respectively. It is suggested that 2-chloro-2-phenylethylamine or its oxidation product 2-chloro-2-phenylacetaldehyde may serve as an active site affinity reagent for monoamine oxidase.     

DEVELOPMENTAL CHARACTERISTICS OF PHENYLETHANOLAMINE AND OCTOPAMINE IN THE RAT BRAIN

Abstract— Phenylethanolamine and octopamine have been detected in the developing rat brain. Maximum concentration of these amines occurs early in development (16-17 days of gestation). At this developmental stage, the brain concentration of these amines is higher than that of norepinephrine. There is a sharp decline in the phenylethanolamine and octopamine concentrations on day 18 of gestation to approximately those of the adult. This decrease coincides with an increase in-monoamine oxidase activity of fetal brain, with an increase in the activities of tyrosine hydroxylase and dopamine-β-hydroxylase, and with the appearance of a saturable active uptake mechanism for norepinephrine. The administration of iproniazid, a monoamine oxidase inhibitor, to pregnant rats produced an increase in phenylethanolamine, octopamine and norepinephrine concentrations in the fetal rat brain at 16 days of gestation. p -Chlorophenylalanine, an inhibitor of phenylalanine hydroxylase, decreased fetal brain norepinephrine; this drug increased brain levels of phenylethanolamine and octopamine. The combined administration of iproniazid, p -chlorophenylalanine and phenylalanine to pregnant rats resulted in increased concentrations of octopamine and in a several-fold increase of phenylethanolamine levels; norepinephrine concentrations were sharply reduced. The possible significance of these findings in relation to pathological conditions such as phenylketonuria is discussed.     

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.     

2-Arylthiomorpholine derivatives as potent and selective monoamine oxidase B inhibitors

2-Arylthiomorpholine and 2-arylthiomorpholin-5-one derivatives, designed as rigid and/or non-basic phenylethylamine analogues, were evaluated as rat and human monoamine oxidase inhibitors. Molecular docking provided insight into the binding mode of these inhibitors and rationalized their different potencies. Making the phenylethylamine scaffold rigid by fixing the amine chain in an extended six-membered ring conformation increased MAO-B (but not MAO-A) inhibitory activity relative to the more flexible α-methylated derivative. The presence of a basic nitrogen atom is not a prerequisite in either MAO-A or MAO-B. The best K_i values were in the 10^?8 M range, with selectivities towards human MAO-B exceeding 2000-fold.     

The effect ofl-deprenyl on behavior,cognitive function,and biogenic amines in the dog

Behavioral and pharmacological effects of oral administration ofl-deprenyl in the dog are described. Spontaneous behavior is unaffected at doses below 3 mg/kg while at higher doses there was stereotypical responding. There was evidence of improved cognitive function in animals chronically treated with a 1 mg/kg dose but the effectiveness varied considerably between subjects. Chronic administration produced a dose dependent inhibition in brain, kidney and liver monoamine oxidase B, and had no effect on monoamine oxidase A. There were also dose dependent increases in brain phenylethylamine and in plasma levels of amphetamine. Dog platelets did not have significant levels of MAO-B. Brain dopamine and serotonin metabolism were unaffected byl-deprenyl at doses up to 1 mg/kg. It appears that for the dog, deamination of catecholamines is controlled by MAO-A. Nevertheless, it is suggested thatl-deprenyl serves as a dopaminergic agonist, and there is also evidence that it affects adrenergic transmission. These catecholaminergic actions may account for the effects ofl-deprenyl on behavior and cognitive function.     

Evidence for a single catalytic binding site on human brain type B monoamine oxidase

The tricyclic antidepressant drug, amitriptyline, inhibited the B form of human brain mitochondrial monoamine oxidase (MAO) under normal atmospheric conditions in a noncompetitive manner when phenylethylamine (PEA) was used as substrate and competitively when benzylamine (BzNH2) was employed as substrate. In addition, it was also found that PEA and BzNH2 inhibited each other's degradation noncompetitively. Similar results have previously been reported with human platelet MAO. These data suggest that the catalytic binding sites for PEA and BzNH2 on the B form of human brain MAO may be different. Attempts were made to further distinguish these catalytic binding sites on the brain oxidase using the irreversible MAO inhibitors, pargyline and clorgyline. Though these drugs have considerably different affinities for the B form of the oxidase, the degree to which either compound inhibited PEA or BzNH2 deamination was essentially identical. When incubations were performed at elevated oxygen concentrations PEA and BzNH2 became mutually competitive inhibitors of each other's metabolism. Also at the higher levels of oxygen, amitriptyline inhibition of PEA deamination approached a competitive fashion. These results suggest that PEA and BzNH2 share a common catalytic binding site on the B form of MAO and, in addition, bind to an inhibitory site on the reduced form of the oxidase. Accordingly, the data indicate that amitriptyline also binds to both the oxidized and reduced forms of this human brain oxidase.     

Multiplicity of monoamine oxidase: inhibition of mitochondrial monoamine oxidase activity by isopropylhydrazide of D,L-serine]

Isopropylhydrazide of D,L-serine (IHS) inhibits by 50% (at 37 degrees for 10 min) deamination of serotonin or beta-phenylethylamine by monoamine oxidases from bovine brain stem mitochondrial membranes at the 2.6 X X 10(-5) M or 9 X 10(-5) M, respectively. In order to inhibit by 50% the deamination of tyramine under the same conditions a considerably lower (2.5 X X 10(-6) M) concentration of IHS is required. Kinetic studies of inhibition of enzymatic deamination of all the three biogenic monoamines by IHS showed that the irreversible blocking of the monoamine oxidase activity is preceeded by formation of dissociating enzyme-inhibitor complexes. Values of the dissociation constants of these complexes measured (at 37 degrees) with serotonin, phenylethylamine or tyramine as substrates for estimation of the residual monoamine oxidase activity are 0.47; 0.13 or 0.023 mM, respectively. Significant differences are also found between thermodynamic and activation parameters characterizing both both steps of interaction between IHS and the monoamine oxidases of mitochondrial membranes in the experiments with serotonin, phenylethylamine or tyramine as substrates. The data obtained suggest the existence of different monoamine oxidases (or their active sites) catalyzing oxidative deamination of serotonin, phenylethylamine or tyramine in the fragments of mitochondrial membranes from bovine brain stem.     

Multiple catalytic sites of rat brain mitochondrial monoamine oxidase

We compared the inhibitory and catalytic effects of various monoamines on forms A and B of monoamine oxidase (MAO) on mitochondrial preparations from rat brain in mixed substrate experiments. MAO activity was determined by a radioisotopic assay. MAO showed lower K_m values for tryptamine and β-phenylethylamine than for tyramine and serotonin. The K_m values of the untreated preparation for tyramine, tryptamine, and β-phenylethylamine obtained were the same as those of the form B enzyme and the K_m value for serotonin was the same as that of the form A enzyme. Tyramine and tryptamine were competitive inhibitors of serotonin oxidation and β-phenylethylamine did not bind with form A enzyme or inhibit the oxidation of serotonin, while tyramine and tryptamine were competitive inhibitors of β-phenylethylamine oxidation. Although serotonin was not oxidized by form B enzyme, serotonin was a competitive inhibitor of β-phenylethylamine oxidation. It is suggested that rat brain mitochondrial MAO is characterized by two kinds of binding sites.     

Substrates and inhibitors of hepatic amine oxidase inhibit dimethylnitrosamine-induced mutagenesis in Salmonella typhimurium

The mutagenic effect of dimethylnitrosamine in Salmonella typhimurium TA100, in the presence of a rat-liver homogenate derived from animals treated with Aroclor 1254, was inhibited by substrates and inhibitors of monoamine oxidase. Substrates of diamine oxidase did not inhibit dimethylnitrosamine mutagenesis and, furthermore, monoamine oxidase inhibitors had no effects on mutagenesis by benzo[a]pyrene or aflatoxin B₁. The results suggest that monoamine oxidase participates in the activation of dimethylnitrosamine to a mustagen.     

Kinetic properties of membrane-bound and Triton X-100-solubilized human brain monoamine oxidase

The kinetic properties of membrane-bound and Triton X-100-solubilized human brain mitochondrial type A and B monoamine oxidase were examined. These studies reveal that the K_m values for phenylethylamine and benzylamine, type B monoamine oxidase substrates, were only slightly increased by the solubilization procedure. The K_m value for 5-hydroxytryptamine, a type A monoamine oxidase substrate, was similarly increased by treatment with Triton X-100. The K_m values for oxygen with all three amine substrates were unaffected by solubilization of the oxidase. Similarly, the optimum pH for deamination of substrates for the B isoenzyme was essentially unaltered in the solubilized preparation as compared to the membrane-bound enzyme whereas that for 5-hydroxytryptamine metabolism was decreased from pH 8.5 to approximately 7.75 on solubilization. The energy of activation with all three substrates was altered on solubilization of the oxidases with Triton X-100. The energy of activation for the B monoamine oxidase substrates increased whereas that for 5-hydroxytryptamine decreased. These data support the contention that the lipid environment surrounding the two forms of monoamine oxidase controls, in part, the activity and kinetic properties of the enzymes.     

Comparative study of catalytic properties of mink and rat liver monoamine oxidase

Comparative substrate-inhibitor analysis of catalytic properties of mitochondrial monoamine oxidase (MAO) of liver of the American mink Mustela vison Schreber and of liver of Wistar rat has been performed. It has been found that MAO of mink, like MAO of rat, has properties of classic mammalian MAO: it deaminates tyramine, tryptamine, serotonin, benzylamine, β-phenylethylamine and does not deaminate histamine as well as does not have sensitivity to semicarbazide. Study of kinetics of the monoamine oxidase deamination revealed both qualitative and quantitative differences between these enzymes. Specificity of action on MAO-A form of four irreversible inhibitors—acridine derivatives—has been shown; this specificity was several times higher for the mink liver MAO than for the rat liver MAO. It is suggested that the liver MAO of both species of the studied animals has several isoenzyme forms or several centers of the substrate binding.     

Subcellular localization, inhibiting specificity and catalytic properties of several aminooxidases from the human placenta

Human placenta was shown to contain practically all known types of aminooxidase, i.e., Membrane-bound and soluble monoamine oxidases A that predominantly oxidize serotonin (Km approximately 0.05 and 0.2 mM) and tyramine (Km approximately 0.03 and 0.085 mM), partly oxidize phenylethylamine (Km approximately 0.013 and 0.1 mM) and slightly oxidize benzylamine; Monoamine oxidase B and its intermediate form, B', with equal sensitivity towards the inhibitors, Lilly 51641 and deprenyl. The main substrates for these enzymes are phenylethylamine (Km = 0.011 mM for the membrane-bound and 0.019 mM for the soluble enzymes); Membrane-bound and soluble benzylamine oxidases that are stable to MAO inhibitors but are highly labile towards semicarbazide and aminoguanidine and that predominantly oxidize benzylamine. The Km value for the soluble enzyme is 0.19 mM, its specific activity is 0.058 nmol aldehyde/min/mg protein, which markedly exceeds that for serum benzylamine oxidase (i.e., 0.014 nmol/min/mg) and thus excludes its serum origin; Diamine oxidase that oxidizes putrescine (Km = 0.025 mM), histamine and cadaverine and only slightly oxidizes benzylamine. One characteristic feature of the placenta is the presence of soluble MAO as well as MAO incorporated into the endoplasmic reticulum membrane (microsomes). In all probability, these enzymes are precursors of the mitochondrial enzyme. The concentration of MAO A in the mitochondria is approximately 1.3%, that in microsomes--approximately 1%, kcat = 270 and 320 min-1, respectively.     

Electrical Stimulation of the Substantia Nigra and Changes of 2-Phenylethylamine Synthesis in the Rat Striatum

In rats pretreated with deprenyl (2 mg/kg), electrical stimulation of the left substantia nigra produced an increase in the concentrations of 3,4-dihydroxyphenylacetic acid and homovanillic acid in the left striatum by 57 and 45%, but the levels of 2-phenylethylamine and p-tyramine decreased by 22 and 41%, respectively, as compared with those in the right striatum. The administration of alpha-methyl-p-tyrosine (1.25 mg/kg, i.p.), a tyrosine hydroxylase inhibitor, 1 h before nigral stimulation, did not affect the concentration of 2-phenylethylamine in unstimulated striata but prevented the stimulation-induced decrease in the concentration of 2-phenylethylamine. Neither stimulation nor alpha-methyl-p-tyrosine affected the activity of monoamine oxidase A or B, and stimulation did not produce any change in striatal blood flow, a finding demonstrating that the changes in the rate of accumulation of 2-phenylethylamine were not due to changes in catabolism or removal of 2-phenylethylamine from the brain. These experiments demonstrate that the rate of synthesis of striatal 2-phenylethylamine is decreased following nigral stimulation and that this effect is blocked after partial inhibition of tyrosine hydroxylase. This suggests that 2-phenylethylamine is present in tyrosine hydroxylase-containing neurons and therefore supports the coexistence of 2-phenylethylamine and dopamine in the nigrostriatal pathway.