Effect of prolonged cold exposure on monoamine oxidase activity and kinetics and on serotonin metabolism in the rat brain

Effects of long-term cold exposure on the content of serotonin and its metabolite 5-hydroxyindolacetic acid (5-HIAA) and monoamine oxidase (MAO) activity and kinetic parameters (Km and Vmax) of oxidative deamination of serotonin in rat brain stem. The increase of 5-HIAA level in the initial period of chronic cold exposure was determined by the blockade of active metabolite transport from the brain. The level of serotonin and the rate of its catalytic deamination by MAO were found to be decreased in cold-adapted rats. The magnitude of the Km of serotonin deamination was unchanged.     

Possible mechanism of the change in the catalytic properties of brain monoamine oxidase in rats

An over two-fold decrease in the affinity of mitochondrial MAO to serotonin was found under hyperoxia, hypoxia and cold stress. At the same time serotonin deaminase and glucosamine deaminase activity was detected in the supernatant obtained after precipitation of the mitochondria. The data obtained indicate that the modification of the catalytic properties of MAO is caused both by alteration of the molecular properties of the enzyme and structural derangement of the mitochondrial membranes.     

Chlorphentermine inhibits pulmonary mitochondrial monoamine oxidase

Oxidation of six amine substrates by rat, rabbit and guinea-pig lung mitochondrial monoamine oxidase (MAO) was investigated polarographically with a Clark oxygen electrode in the presence of chlorphentermine (CP). This amphiphilic drug decreased the deamination of serotonin, norepinephrine, tyramine and dopamine significantly in all three species. However, the oxidation of tryptamine and benzylamine was unchanged. Amine oxidation by MAO in guinea-pig lung mitochondria was much more sensitive to the CP-mediated inhibition than rat or rabbit. A kinetic study of serotonin oxidation in the absence and presence of CP showed that both Vmax and Km were affected. These combined data indicate that CP is a specific inhibitor of pulmonary, mitochondrial monoamine oxidase form A with mixed-type inhibition.     

Deamination of adenosine monophosphate in the rat brain in hyperoxia, hypoxia, and cold stress]

The causes of the adenosine monophosphate (AMP) deamination increase in rat brain mitochondria under conditions of hyperoxia, hypoxia and cold stress were studied. Data from the inhibitory analysis suggest that the increased intensity of AMP deamination under hypoxia is conditioned by the alterations in the substrate specificity of type A monoamine oxidase which acquires the ability to deaminate AMP. The enhancement of AMP deamination under hyperoxia and cold stress is due to the activation of true AMP deaminase in the mitochondrial fraction. The cytoplasmic AMP deaminase activity remains unchanged thereby. The effects of the AMP deaminase specific effectors, ATP and inorganic phosphate, were investigated.     

THE SUBMITOCHONDRIAL LOCALIZATION OF MONOAMINE OXIDASE : An Enzymatic Marker for the Outer Membrane of Rat Liver Mitochondria

Controlled osmotic lysis (water-washing) of rat liver mitochondria results in a mixed population of small vesicles derived mainly from the outer mitochondrial membrane and of larger bodies containing a few cristae derived from the inner membrane. These elements have been separated on Ficoll and sucrose gradients. The small vesicles were rich in monoamine oxidase, and the large bodies were rich in cytochrome oxidase. Separation of the inner and outer membranes has also been accomplished by treating mitochondria with digitonin in an isotonic medium and fractionating the treated mitochondria by differential centrifugation. Treatment with low digitonin concentrations released monoamine oxidase activity from low speed mitochondrial pellets, and this release of enzymatic activity was correlated with the loss of the outer membrane as seen in the electron microscope. The low speed mitochondrial pellet contained most of the cytochrome oxidase and malate dehydrogenase activities of the intact mitochondria, while the monoamine oxidase activity could be recovered in the form of small vesicles by high speed centrifugation of the low speed supernatant. The results indicate that monoamine oxidase is found only in the outer mitochondrial membrane and that cytochrome oxidase is found only in the inner membrane. Digitonin treatment released more monoamine oxidase than cytochrome oxidase from sonic particles, thus indicating that digitonin preferentially degrades the outer mitochondrial membrane.     

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.     

Effect of solubilization by methylethyl ketone of mitochondrial monoamine oxidase of the rat liver on the kinetic properties of the enzyme

The values of Km and V for serotonin, tyramine and beta-phenylethylamine deamination by solubilized and partially purified preparations of monoamine oxidase (MAO) from rat liver were determined. As a result of MAO solubilization by methylethylketone, 14% of activity localized in the mitochondria passed into solution. Subsequent chromatography on AH-Sepharose 4B resulted in 27-fold purification of the enzyme with a 9% yield. In experiments with membrane-bound and partially purified MAO, the Km and V values were shown to increase non-competitively with a rise in O2 concentration. In contrast with intact mitochondria, the use of partially purified MAO preparations led to a loss of the enzyme sensitivity to O2 depending on the nature of the amine. The dependence of kinetic properties of MAO on the lipid environment of mitochondrial membranes is discussed.     

On hepatic mitochondrial monoamine oxidase activity in lipid deficiency.

A marked decrease in liver mitochondrial monoamine oxidase activity was noticed in rats fed a fat-free diet as compared with that of their controls. In lipid-deprived rats, the specific activity of this enzyme was very low towards different substrates studied. The activity of kynurenine 3-monooxygenase, which like monoamine oxidase is localized on the mitochondrial outer membrane, was similarly depressed under conditions of lipid deprivation. On the other hand no major changes were observed in the activity of the inner membrane enzyme, kynurenine amino-transferase. Mitochondria from fat-free diet-fed rats were deficient in essential fatty acids whereas no appreciable variations were found in the relative proportions of phospholipids in comparison with those of control mitochondria. Mitochondrial monoamine oxidase activity of the deficient rats retained its sensitivities to inhibitor drugs like clorgyline and deprenyl. No changes were noticeable in the substrate specificity of monoamine oxidase in these rats. When we switched the fat-free diet-fed rats to a diet supplemented with a source of essential fatty acids, there was an elevation in the activities of both monoamine oxidase and kynurenine 3-monooxygenase, their levels approaching those of the control rats.     

Role of monoaminoxidase in the intensification of peroxidation of lipids in mitochondria during experimental myocardial necrosis

The relationship between lipid peroxidation and rat heart mitochondrial monoamine oxidase activity was studied in experimental myocardial necrosis induced by adrenaline injection. It has been established that both the intensity of peroxidation and the activity of monoamine oxidase in mitochondria from adrenaline-injured rat myocardium were essentially increased. The preliminary administration of antioxidants (vitamin E and ionol) was shown to decrease both the intensity of lipid peroxidation and the activity of monoamine oxidase. It is suggested that intensification of lipid peroxidation which is considered to be the main pathogenic factor in ischemic myocardial injury depends on mitochondrial monoamine oxidase activity. Protective effects of antioxidants are realized by the action on two subsequent chains during the formation of active oxygen forms and destruction of lipid peroxidation products.     

Oxidative deamination of biogenic amines by intestinal amine oxidases: histamine is specifically inactivated by diamine oxidase.

The ability of the gut to inactivate various amines by oxidative deamination was tested with a 130-fold purified amine oxidase preparation from dog small intestine. Of 34 amines tested, putrescine, benzylamine, cadaverine, and serotonin were the most favourable substrates. Histamine was inactivated rapidly by this enzyme preparation, too. Histamine derivatives methylated at the imidazole nucleus were also deaminated, whereas Nalpha-methylhistamine was only a poor substrate and Nalpha, Nalpha-dimethylhistamine was not a substrate at all. Using a second procedure for the purification of amine oxidases from gut, the separation of a soluble monoamine oxidase from diamine oxidase was achieved by gel filtration on Sephadex G-200. The diamine oxidase deaminated putrescine (Km = 1.3 x 10(-4)M) and histamine (Km = 6.6 x 10(-5)M), but not serotonin, and was inhibited by aminoguanidine, but not by pargyline. The soluble monoamine oxidase inactivated serotonin (Km = 4.5 x 10(-4)M), but not histamine and putrescine and was inhibited by pargyline, but not by aminoguanidine. It was concluded that in dog small intestine (as well as in rabbit small intestine) only diamine oxidase was capable of inactivating histamine by oxidative deamination.     

Kinetic behaviour of acid phosphatase-albumin co-polymers in homogeneous phase and under gel-immobilized conditions.

1. Antiserum raised to purified human liver monoamine oxidase was used to characterize the monoamine oxidase from human liver, brain cortex, placenta and platelets. 2. Antibodies to monoamine oxidase were purified by adsorption with a mitochondrial preparation. 3. Monoamine oxidase was present in liver particle-free supernatant as measured by enzyme activity and immunodiffusion. 4. Multiple precipitin lines were obtained on immunodiffusion analysis against the purified liver enzyme. It is proposed that this is due to either aggregation or to differential lipid binding. 5. The results suggest that the functionally different enzymes found in liver, brain cortex, platelets and placenta are immunochemically related and may be identical.     

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.     

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.     

Hydrogen Peroxide Production by Rat Brain In Vivo

Abstract: H₂ O₂ production by rat brain in vivo was observed with a method based on the measurement of brain catalase. The administration to the rat of 3-amino-1, 2, 4-triazole, an H₂ O_2- dependent inhibitor of catalase, caused progressive inhibition of brain catalase activity in both the supernatant and pellet fractions of homogenates of the striatum and prefrontal cortex. The prevention of catalase inhibition by prior administration of ethanol confirmed that catalase inhibition in vivo was dependent upon H₂ O₂. A significant portion of the catalase (30-33%) appeared in the supernatant fraction from a slow-speed homogenization procedure and was not significantly contaminated by either erythrocytes or capillaries. In the whole homogenate, less than 6% of the catalase activity was attributed to erythrocytes. Modification of intracellular monoamine oxidase activity by either pargyline or reserpine did not change the rate of inhibition of catalase by aminotriazole. A probable interpretation of these data is that H₂ O₂ generated by mitochondrial monoamine oxidase does not reach the catalase compartment; the catalase is contained in particles described by other investigators as the microperoxisomes of brain. In studies in vitro , the production of H₂ O₂ by rat brain mitochondria with either dopamine or serotonin as substrate was confirmed.     

Different roles of dopamine and serotonin in conditioned passive avoidance response of rats

Deamination of dopamine and serotonin by monoamine oxidase was studied in the prefrontal cortex, striatum, hippocampus and amygdaloid complex of the brain of rats during retrieval of conditioned passive avoidance response. Changes in the dopamine and serotonin metabolism were observed in different brain structures. A decrease in dopamine-deaminating activity of monoamine oxidase was found in the hippocampus, striatum and prefrontal cortex. At the same time, serotonin-deaminating activity of the enzyme was decreased in the striatum and increased in the amygdaloid complex, whereas it did not change in the prefrontal cortex and hippocampus. The observed changes in dopamine metabolism in the prefrontal cortex and hippocampus and serotonin metabolism in the amygdaloid complex indicate that dopamine and serotonin are involved in the regulation of two different processes mediating the memory trace retrieval. Dopamine is involved in neuronal mechanisms of information processes providing the strategy of behavior, whereas serotonin is related to emotional mechanisms of memory.     

Early changes in mitochondrial monoamine oxidase activity of rat liver during 2-acetylaminofluorene feeding

The activities of mitochondrial type A and B monoamine oxidase were determined in the liver of rats fed a diet containing 2-acetylaminofluorene (AAF). Three days after the initiation of AAF-feeding, there was a significant decrease of type B monoamine oxidase activity without affect on type A enzyme. The decreased activity of type B monoamine oxidase, which reached a minimum after three weeks, was sustained for as long as AAF-feeding was continued. Sex-related difference in response to AAF was seen in the rat with respect to the onset and the intensity of the decreased type B monoamine oxidase activity, male rats being more sensitive to the carcinogen than female rats. In contrast to the in vivo effect, AAF showed a potent inhibitory effect on type A monoamine oxidase, rather than on type B enzyme, when added in vitro. The pI₅₀ values were estimated to be 7.5 against type A monoamine oxidase and 4.1 against type B enzyme, respectively. The in vitro inhibition of both types of monoamine oxidase by AAF was competitive. The K_i values for AAF were calculated to be 9.51 · 10^?9 M for type A monoamine oxidase and 1.30 · 10^?5 M for type B enzyme, respectively. In accordance with the potent inhibitory effect of AAF on type A monoamine oxidase in vitro, a single administration of the carcinogen, at a dose of 50 mg/kg, resulted in a marked and temporal decrease of the enzyme activity in the mitochondria of male rat liver. Recovery of the decreased type B monoamine oxidase activity was slow, and the enzyme activity did not return to control levels, even if rats were fed the basal diet for 2 or 4 weeks after the cessation of AAF-feeding.     

Inhibition of rat brainstem monoamine oxidase activity by CGP 6085 A

CGP 6085 A [4-(5,6-dimethyl-2-benzofuranyl)piperidine] HCl, a known serotonin inhibitor, also inhibits rat brainstem monoamine oxidase A (MAO-A) and monoamine oxidase B (MAO-B) in both in vivo and in vitro experiments. Serotonin (5-HT) deamination by MAO-A is inhibited 35% at a dose of 100 mg/kg i.p. in vivo. Similar experiments show a maximal 20% decrease in phenylethylamine (PEA) deamination by MAO-B at a dosage of 30 mg/kg i.p. Over the range of 0.1 to 10 mg/kg i.p., CGP 6085 A decreases 5-HIAA levels in the brainstem. This in vivo inhibition of MAO activity is confirmed by in vitro experiments. In vitro studies in rat brainstem mitochondrial preparations show a dose-dependent, reversible, inhibition of MAO using tyramine as the substrate for the enzyme reaction. With an in vitro IC50 of 2-3 microM, the potency of CGP 6085 A is comparable to pargyline.     

Development of serotonergic neurons of dorsal raphe nuclei in mice with knockout of monoamine oxidase a and 5-HT1A and 5-HT1B autoreceptor

The morphological changes in the development of serotonergic neurons of the dorsal raphe nuclei in the medulla oblongata was studied by immunocytochemistry in mice with knockout of 1A and 1B serotonin autoreceptors as well as monoamine oxidase A. Serotonin autoreceptors regulate electric activity of serotonergic neurons as well as the synthesis and release of the neurotransmitter, while monoamine oxidase A catalyzes its degradation. These genetic modifications proved to have no effect on the number of serotonergic neurons in the medulla oblongata but induced morphofunctional changes. Decreased cell size and increased intracellular serotonin level were observed in the case of monoamine oxidase A deficiency, while excessive cell size and decreased intracellular serotonin level were observed in the case of autoreceptor deficiency. The data obtained confirm the hypothesis of autoregulation of serotonergic neurons in development.     

Functional groups of mitochondrial monoamine oxidase]

Functional groups of mitochondrial monoamine oxidase critical for monoamine oxidase activity were investigated by chemical modification of highly purified monoamine oxidase preparations from pig liver by specific inhibitors. The substrate and inhibitory properties of synthesized derivatives of beta-phenylethylamine, containing various acylating and alkylating groups in the p-position of the benzene ring, were studied. It was shown that 4-carbmethoxy-beta-phenylethylamine (I) is readily deaminated by monoamine oxidase, whereas 4-O-acetyl-beta-phenylethylamine (II) is not affected by the enzyme. 4-O-acetyl-beta-phenylethylamine (II) and 4-ethyl-O-chloroacethyl phenol (III) inhibit deamination of tyramine, 4-amino-beta-phenylethylamine, beta-phenylethylamine, 4-chloro-beta-phenylethylamine and serotonin in different degrees. The kinetic studies demonstrated that this inhibition is probably due to the acylating properties of the compounds obtained. Selectivity in inhibition may be accounted for by acylation of the group of monoamine oxidase which is located in the nearest proximity to the nucleophynoamine oxidase which is located in the nearest proximity to the nucleophylic site of monoamine oxidase active centre important for binding of tyramine. This group is neither the imidazole group of histidyl, nor the SH-group of cysteinyl residues of monoamine oxidase protein molecule. Its nature is discussed in the light of the data obtained.     

The insertion of monoamine oxidase A into the outer membrane of rat liver mitochondria.

Human monoamine oxidase A that had been synthesized in a reticulocyte lysate translation system was capable of binding to and inserting into either rat liver mitochondria or isolated mitochondrial outer membranes. The inserted form was as resistant to proteinase K as endogenous mitochondrial monoamine oxidase A. The insertion, but not the binding, of monoamine oxidase A was prevented by depleting the reaction mixture of either ATP (with apyrase) or ubiquitin (with purified antibodies against this polypeptide). Addition of ATP or ubiquitin, respectively, to these depleted mixtures restored the insertion of the enzyme. In the absence of mitochondria, in vitro synthesized monoamine oxidase A did not catalyze its own alkylation by the mechanism-based inhibitor, [3H]clorgyline. However, both monoamine oxidase A that had been membrane-inserted in vitro and monoamine oxidase A that had been bound to the mitochondria under conditions of ATP depletion catalyzed adduct formation. Furthermore, reaction of either clorgyline or another mechanism-based inhibitor, pargyline, with the membrane-bound enzyme during ATP depletion inhibited the insertion of monoamine oxidase A when ATP was restored. These observations indicate that monoamine oxidase A acquired a catalytically active conformation on interaction with the mitochondrial outer membranes prior to its ATP and ubiquitin-dependent insertion into the membrane.