THE EFFECTS OF CHRONIC REGIMENS OF CLORGYLINE AND PARGYLINE ON MONOAMINE METABOLISM IN THE RAT BRAIN

The effects of chronic administration of clorgyline and pargyline on rat brain monoamine metabolism have been examined. The inhibitory selectivity of these drugs towards serotonin deamina-tion (MAO type A) and phenylethylamine deamination (MAO type B) can be maintained over a 21-day period by proper selection of low doses of these drugs (0.5-1.0 mg/kg/24h). The results are consistent with MAO type A catalyzing the deamination of serotonin and norepinephrine and with MAO type B having little effect on these monoamines. Dopamine appears to be dcaminated in vivo principally by MAO type A. Clorgyline administration during a 3-week period was accompanied by persistent elevations in brain norepinephrine concentrations; serotonin levels were also increased during the first 2 weeks, but returned towards control levels by the third week of treatment. Low doses of pargyline did not increase brain monoamine concentrations, but treatment with higher doses for 3 weeks led to elevations in brain norepinephrine and 5-hydroxytryptamine; at this time significant MAO-A inhibition had developed. The changes in monoamine metabolism seen at the end of the chronic clorgyline regimen are not due to alterations in tryptophan hydroxylase activity. At this time tyrosine hydroxylase activity was also unaffected.     

Differences in the methylation of brain histamine in vivo between audiogenic seizure-sensitive and -resistant deermice

We have investigated the catabolism of [³H] histamine (HA), after intraventricular (i.vt.) administration, in brains of the audiogenic seizure susceptible (SS) and resistant (SR) deermouse $\text{Peromyscus}$. Brains of SS mice had lower endogenous HA levels and contained less [³H]-HA 20, 60 and 300 sec after i.vt. [³H]-HA than did brains of SR deermice. Twenty sec after [³H]-HA, brain [³H] methylhistamine (MeHA) levels and the resulting MeHA conversion index were found to be increased in the SS animals while later, at 60 and 300 sec, these parameters were found to be decreased. There were no SS-SR differences in the levels of brain [³H] methylimidazoleacetic acid. The data indicate that SS deermice catabolize exogenous HA, at least initially, more rapidly than their SR counterparts, confirming a like result noted immediately prior to seizure activity elicited by the administration of L-methionine-dl-sulfoximine in $\text{Mus}$.     

Effects of intrastriatal kainic acid injection on [3H]dopamine metabolism in rat striatal slices: evidence for postsynaptic glial cell metabolism by both the type A and B forms of monoamine oxidase

Abstract: Intrastriatal injections of kainic acid (KA) were utilized to investigate the cellular localization of postsynaptic dopamine (DA) metabolism by type A and B monoamine oxidase (MAO) in rat striatum. At 2 days postinjection, maximal degeneration of cholinergic and γ-aminobutyric acid (GABA)ergic neurons was observed and found to be associated with a significant decrease in both type A and B MAO activity. However, over the next 8-day period, when only the process of gliosis appeared to be occurring, a selective return to control of type B MAO activity was seen. When the metabolism of [³H]DA (10^?7 M) was examined in 8-day KA-lesioned rat striatal slices, an increase in [³H]dihydroxyphenylacetic acid (DOPAC) and [³H]homovanillic acid (HVA) formation was observed. The KA-induced elevation of [³H]DOPAC formation (but not [³H]HVA) was abolished by the DA neuronal uptake inhibitor nomifensine. This is consistent with earlier findings suggesting that HVA is formed exclusively within sites external to DA neurons. Experiments with clorgyline and/or deprenyl revealed that the relative roles of type A and B MAO in striatal DA deamination remained unchanged following KA (90% deamination by type A MAO) even though total deamination was substantially enhanced. At high concentrations of [³H]DA (10^?5 M), deamination by type B MAO could be increased to 30% of the total MAO activity; however, this was observed in both control and KA-lesioned striata. These results suggest that KA-sensitive neurons contain type A and/or type B MAO. Moreover, whereas these neurons may metabolize DA, a major portion of postsynaptic DA deamination appears to occur within glial sites of rat striatal tissue. Furthermore, glial cells would appear to contain functionally important quantities of both type A and B MAO.     

Metabolism of intracerebrally administered histidine, histamine and imidazoleacetic acid in mice and frogs

Abstract— After intracerebral administration of [¹⁴C]histidine to mice the major labelled substance found in the brain extracts was histidine itself; small amounts of labelled carnosine and homocarnosine were detected. No other labelled substances were detected on radio- autographs of two-dimensional TLC's of the extracts. In the case of the frog, radioactive histidine, N-acetylhistidine, carnosine and homocarnosine were found in the brain extracts at various times after intracerebral injection of the labelled histidine. With time, approximately 90 per cent of the radioactivity in the extracts was found in the N-acetylhistidine. In neither the mouse nor frog could we find unequivocal evidence for the formation either of histamine or imidazoleacetic acid from intracerebrally administered histidine, but our analytical procedures may have lacked sufficient sensitivity to pick up extremely low activities of histamine and imidazoleacetic acid. Experiments with [¹⁴C]histamine administered intracerebrally into mice demonstrated the major pathway of metabolism in brain to be histamine → methylhistamine → methylimidazoleacetic acid. No detectable label appeared in inlidazoleacetic acid. In the frog intracerebral administration of the labelled histamine led to the formation of methylhistamine and imidazoleacetic acid, but at most only traces of methylimidazoleacetic acid were found. The injection of [¹⁴C]imidazoleacetic acid intra- cerebrally into mice and frogs resulted in virtually no loss of the label in the form administered in the frog brain over a period of 4 h and in a slow rate of decrease in the mouse brain. No radioactive metabolites of imidazoleacetic acid were found in either species. The limitations of trying to determine natural functions of substances in brain by following the fate of exogenously administered materials is discussed.     

[3H]HARMALINE AS A SPECIFIC LIGAND OF MAO A–II. MEASUREMENT OF THE TURNOVER RATES OF MAO A DURING ONTOGENESIS IN THE RAT BRAIN

The combined measurement of MAO A activity (using [³H]5-HT as a specific substrate) and [³H]harmaline binding capacity indicated that the concentration of MAO A in brain was higher in 14-28 day old rats than in adult animals. The turnover rates of this enzyme in the forebrain and the brain stem of young (14-28 day old) and adult rats were calculated by following the recovery of MAO A activity and of [³H]harmaline binding capacity after an acute treatment with pargyline (75mg/kg i.p.). Both the fractional rate constant for MAO A degradation and its synthesis rate per g of fresh tissue were significantly higher in young animals. However, the calculation of the absolute synthesis rates of MAO A per brain area gave very similar values in young and adult animals: 1.3-1.5 × 10¹³ molecules of MAO A synthesized per day in the forebrain and 2.3-2.9 × 10¹² molecules per day in the brain stern. The results illustrate the validity of using [³H]harmaline binding to evaluate possible changes in the turnover rate of MAO A in tissues.     

4-Fluoro-3-nitrophenyl azide binding sites on purified beef liver monoamine oxidase B

Previous work from this laboratory has shown that 4-fluoro-3-nitrophenyl azide (FNPA) is an effective photoaffinity labeling probe for MAO-B (Chen et al., Biochem. Pharmac.34, 781–785, 1985). The FNPA binding sites have been further studied by using [³H]FNPA. When [³H]FNPA was photolyzed with purified beef liver MAO, then subjected to tryptic and chymotryptic digestion, three radioactive peaks were observed after Sephadex G-25 column chromatography procedure. The extent of [³H]FNPA incorporation varied directly with [³H]FNPA concentration. They could be protected by the presence of the substrate (phenylethylamine) or inhibitors (pargyline and trans-phenylcyclopropylamine) of MAO-B during photolysis. These protections were concentration dependent. Furthermore, the decrease in [³H]FNPA labeling in the presence of inhibitors paralleled the decrease in MAO catalytic activity. These results suggest that the FNPA binding sites were related to the active site of MAO-B. Under the same conditions, the separation profiles of [³H]FNPA labeled and [³H]pargyline labeled tryptic-chymotryptic peptides after Sephadex G-25 column chromatography are distinctly different. This result suggests that FNPA labeling sites may be different from the pargyline binding site. Since pargyline binds to the prosthetic group(-FAD) of MAO, [³H]FNPA may label different domains of the active site. This probe may be useful for the characterization of the active site of MAO-B.     

[3H]HARMALINE AS A SPECIFIC LIGAND OF MAO A—I. PROPERTIES OF THE ACTIVE SITE OF MAO A FROM RAT AND BOVINE BRAINS

A high-affinity (K_d= 5.9 nM) specific binding site for [³H]harmaline was detected in membranes from rat and bovine brains. Studies of the regional and subcellular distributions of this binding indicated its close association with monoamine oxidase type A activity (MAO A) measured with [³H]serotonin ([³H]5-HT) as the substrate. Maximal binding capacity and MAO A activity were found in mitochondrial enriched fractions. Mitochondria of synaptosomal or extra-synaptosomal origin exhibited very similar properties with respect to [³H]harmaline binding characteristics and MAO A activity. Among psychoactive drugs, only monoamine oxidase inhibitors (MAO I) prevented the specific binding of [₃H]harmaline. Logit-log inhibition curves of binding by MAO I gave only one slope which was not significantly different from 1.0, suggesting the existence of only 1 category of specific sites for [³H]harmaline in the membrane preparations from rat and bovine brains. Consistent with the preferential inhibition of MAO A by harmaline, other MAO I of this class, i.e. clorgyline and Lilly 51641, were 10²-2 × 10³ times more efficient than deprenyl and pargyline, two inhibitors of MAO type B, in displacing [³H]harmaline from its specific binding site. K_i and IC₅₀ values for the inhibition of [³H]harmaline binding by MAO I and MAO substrates (tryptamine, 5-HT, norepinephrine) were almost identical with those characterizing their action on MAO A activity with [³H]5-HT as the substrate. In conclusion, the specific binding site for [³H]harmaline exhibited all the expected properties of the active site of MAO A. Like the technique of precipitation with a specific antibody, binding of [³H]harmaline should be of great help for studying the structural characteristics of the active site of MAO A and determining the number of MAO molecules in tissues under various physiological conditions.     

Isotopic microassay of histamine, histidine, histidine decarboxylase and histamine methyltransferase in brain tissue

Abstract— Microassays are described for histamine, histidine, and the activities of the enzymes histidine decarboxylase (EC 4.1.1.22) and histamine niethyltransferase (EC 2.1.1.8) in brain tissue. The enzymic-isotopic microassay for histamine is based on the methylation of tissue histamine by added histamine methyl-transferase and [¹⁴C]- or [³H]-labelled S-adenosyl-l -methionine. In a double-isotopic form of the assay, a tracer of [³H]histamine is employed along with [¹⁴C]S-adenosyl-l -methionine, and the ratio [¹⁴C]:[³H] reflects the amount of histamine in the sample. Because the methylation of histamine is uniform in brain samples studied, a single isotopic assay with [³H]S-adenosyl-l -methionine as the methyl donor is possible and increases sensitivity, so that 10 pg of tissue histamine can be estimated reliably. The assay for histidine involves decarboxylation of histidine by a bacterial histidine decarboxylase and measurement of the histamine formed by the enzymicisotopic procedure. In the histidine decarboxylase assay, histamine synthesized from added histidine is measured. The assay for histamine methyltransferase involves measuring the formation of [¹⁴C]methylhistamine with [¹⁴C]S-adenosyl-l -methionine serving as the methyl donor.     

5-HYDROXYTRYPTAMINE CATABOLISM IN THE RAT BRAIN DURING ONTOGENESIS

Although the serotoninergic innervation is immature in the brains of young rats, the 5-HIAA content is similar to that found in adults. As indicated by the ratio of 5-HIAA to 5-HT levels in the brain stem and the forebrain, the catabolism of the indolamine was more rapid during the first 3 postnatal weeks than in adults. This was contirmed by measuring the total formation of [³H]5-HIAA from [³H]5-HT newly synthesized from L-[³H]tryptophan in brain stem slices of young and adult rats. Electrolytic lesions of midbrain raphe nuclei (B7 and B8) performed on the 5th postnatal day resulted in parallel decreases in brain 5-HT and 5-HIAA levels; this ruled out the possibility that 5-HIAA might be formed from 5-HT synthesized outside serotoninergic neurons, using peripheral 5-hydroxytryptophan. Inhibition of 5-HT storage by reserpine pretreatment did not alter the higher capacity of newborn tissues to catabolize exogenous [³H]5-HT. Therefore, possible differences in 5-HT binding in serotoninergic neurons between newborn and adult rats were not likely to account for the differences in 5-HT catabolism. Estimation of the rate of 5-HIAA efflux from the brain after MAO inhibition did not reveal marked changes with age. The activity of MAO type A, the enzyme involved in 5-HT catabolism, was higher during early life than later on. This could be shown by using 5-HT as substrate and clorgyline as a selective inhibitor. An opposite pattern of development was seen for MAO B, measured with benzylamine as substrate and deprenyl as selective inhibitor. These results suggest that the high 5-HIAA levels found in the brains of young rats can be attributed mainly to the presence of high MAO A activity during early life.     

Isoelectric analysis of 3H-pargyline-labelled monoamine oxidase in rat and carp

1. After selective binding of [3H]pargyline to either monoamine oxidase (MAO) A or MAO B in the rat liver, MAO B alone in the rat brain and MAO in carp brain and liver, molecular weight and isoelectric points (pI) of these MAO were determined by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis and isoelectric focusing and results obtained were compared. 2. For all tissues tested, SDS-polyacrylamide gel electrophoresis of [3H]pargyline-bound samples revealed a labelled protein band of an apparent mol. wt of 60,000 da. 3. Estimation of radioactivity of [3H]pargyline bound after isoelectric focusing revealed a single protein band with acidic pI values of about 5.5 for rat brain and liver MAO B. 4. Moreover, the pI values of about 7.5 were obtained for carp brain and liver MAO. This basic value was also found for MAO A in the rat liver MAO A.     

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.     

[3H]Tryptamine Binding Sites Are Not Identical to Monoamine Oxidase in Rat Brain

Competition binding studies, subcellular distribution, and in vitro autoradiography were employed to compare the binding in rat brain of [3H]tryptamine with two radioligands for monoamine oxidase (MAO), [3H]pargyline, and [3H]1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine ([3H]MPTP). The MAO inhibitors pargyline, clorgyline, and deprenyl all yielded biphasic competition curves versus [3H]tryptamine. At low concentrations, these drugs stimulated binding by protecting the radioligand from MAO oxidation; at considerably higher concentrations, they inhibited binding by direct competition at the [3H]tryptamine binding site. In subcellular distribution studies, [3H]tryptamine was localized preferentially to the synaptosomal fraction, whereas [3H]pargyline showed greater binding to the mitochondrial fraction. Equilibrium binding studies revealed that the potencies of a series of seven compounds at inhibiting [3H]tryptamine binding were completely different from their potencies at inhibiting [3H]MPTP binding. Finally, the autoradiographic distribution of [3H]tryptamine binding in rat brain was different from that of [3H]MPTP and [3H]pargyline. We conclude that the [3H]tryptamine binding site in rat brain is not equivalent to MAO.     

Comparative enzymologic study of catalytical properties of liver monoamine oxidases of sturgeon fish

We carried out the comparative study of the substrate and inhibitory specificity of liver monoamine oxidases (MAO) of the giant sturgeon Huso huso, the starred sturgeon Acipenser stellatus, the Persian sturgeon Acipenser persicus, and the Russian sturgeon Acipenser gueldenstaedtii. Results of the substrate-inhibitor analysis with use of inhibitors chlorgilin and deprenil, as well as five specific substrates indicate homogeneity of these enzymes. All studied MAO have the several orders higher sensitivity to chlorgilin than to deprenil, with essential interspecies differences being observed. There are determined kinetic parameters of enzymatic deamination (K _M and V) of tyramine, serotonin, noradrenalin, benzylamine, β-phenylethylamine, and N-methylhistamine. All studied enzymes have been established to have the higher activity toward serotonin and noradrenalin-substrates of the MAO A form as compared with benzylamine, β-phenylethylamine, and N-methylhistamine-substrate of the mammalian MAO B form, the maximal activity being characteristic of the giant sturgeon.     

ESTIMATION OF NORADRENALINE AND ITS MAJOR METABOLITES SYNTHESIZED FROM [3H]TYROSINE IN THE RAT BRAIN

Abstract— A new procedure is described for the estimation of [³H]noradrenaline (NA) and its major metabolites free and conjugated 3-methoxy-4-hydroxyphenylglycol (MOPEG) and free and conjugated 3,4-dihydroxyphenylglycol (DOPEGI in the rat brain. The procedure involves adsorption on to alumina, cation exchange chromatography. enzymatic hydrolysis of conjugates and thin-layer-chromatography after intraventricular (IVT) or intravenous injection of [³H]tyrosine. In a time-course study the formation and accumulation of the metabolites have been measured from 15min to 23h after IVT injection of [³H]tyrosine. [³H]MOPEG and [³H]DOPEG were found in almost equal amounts during the synthesis phase of [³H]NA as well as during the storage and disappearance phase of [³H]NA. The maximum levels of conjugated [³H]MOPEG and conjugated [³H]DOPEG were found 2 h after IVT [³H]tyrosine. At this time interval the levels of free [³H]MOPEG and free [³H]DOPEG amounted to 25% and 11%, respectively of the corresponding conjugates. Increasing doses of IVT injected [³H]tyrosine (10-90 °Ci) revealed that the accumulation of [³H]NA and metabolites was linear up to about 50 °Ci. Following intravenous instead of IVT injection of [³H]tyrosine. much higher doses (325 °Ci) were needed to obtain measurable amounts of total [³H]MOPEG and [³H]DOPEG-SO₄ in the rat brain. The formation of labelled NA metabolites from [³H]NA in the rat brain in vim measured as total [³H]MOPEG and [³H]DOPEG-SO₄ was influenced by drugs affecting [³H]NA synthesis, release and metabolism. Synthesis inhibition with a-methyltyrosine (250mg-kg^?1) or FLA-63 (30mg-kg^?1) and inhibition of monoamine oxidase with pargyline (75mg-kg^?1) or clorgyline (2mg-kg^?1) strongly decreased the accumulation of total [³H]MOPEG and [³H]DOPEG-SO₄. Noradrenaline receptor blockade with phenoxybenzamine (20mg-kg^?1) increased both total [³H]MOPEG and [³H]DOPEG-SO₄ to about 160% of the control values. NA release and uptake inhibition induced by d-amphetamine (10mg-k^?1) or phenylethylamine (two doses of 80mg-kg^?1) decrease strongly the levels of [³H]NA and [³H]DOPEG-SO₄. whereas total [³H]MOPEG was only very slightly decreased or even increased as compared to controls.     

Studies on the pargyline-binding site of different types of monoamine oxidase

[3H]Pargyline has been covalently linked to active sites of both type A and type B monoamine oxidase (MAO) obtained from various tissues. Rat heart and human placenta were chosen to represent predominantly type A MAO, pig and bovine livers to represent type B MAO, and rat liver and brain to represent mixed type A and type B MAO's. The [3H]pargyline-MAO adducts were isolated and hydrolyzed by proteolytic enzymes, and the labelled peptides (pargyline-binding sites) separated and compared by paper chromatography and by paper electrophoresis at various pH values. Only one common pargyline peptide was obtained from all the different MAO's. The alternative A and B sites were assessed after preincubation of rat liver MAO with the selective inhibitors deprenyl (to block the B site) and clorgyline (to block the A site). Following proteolysis of the [3H]pargyline of both type A and type B MAO from this pretreated rat liver, MAO has been purified by a series of chromatographic and electrophoretic procedures. Micro-Edman degradation, followed by dansylation, revealed the amino acid sequence to be Ser-Gly-Gly-Cys(X)-Tyr. It is concluded that the primary structures immediately surrounding the pargyline-binding sites are identical for both type A and type B MAO in these tissues.     

Monoamine oxidase-dependent histamine catabolism accounts for post-ischemic cardiac redox imbalance and injury

Monoamine oxidase (MAO), a mitochondrial enzyme that oxidizes biogenic amines generating hydrogen peroxide, is a major source of oxidative stress in cardiac injury. However, the molecular mechanisms underlying its overactivation in pathological conditions are still poorly characterized.Here, we investigated whether the enhanced MAO-dependent hydrogen peroxide production can be due to increased substrate availability using a metabolomic profiling method. We identified N¹-methylhistamine -the main catabolite of histamine- as an important substrate fueling MAO in Langendorff mouse hearts, directly perfused with a buffer containing hydrogen peroxide or subjected to ischemia/reperfusion protocol. Indeed, when these hearts were pretreated with the MAO inhibitor pargyline we observed N¹-methylhistamine accumulation along with reduced oxidative stress. Next, we showed that synaptic terminals are the major source of N¹-methylhistamine. Indeed, in vivo sympathectomy caused a decrease of N¹-methylhistamine levels, which was associated with a marked protection in post-ischemic reperfused hearts. As far as the mechanism is concerned, we demonstrate that exogenous histamine is transported into isolated cardiomyocytes and triggers a rise in the levels of reactive oxygen species (ROS). Once again, pargyline pretreatment induced intracellular accumulation of N¹-methylhistamine along with decrease in ROS levels. These findings uncover a receptor-independent mechanism for histamine in cardiomyocytes.In summary, our study reveals a novel and important pathophysiological causative link between MAO activation and histamine availability during pathophysiological conditions such as oxidative stress/cardiac injury.     

Substrate-dependent activation energy of the reaction catalyzed by monoamine oxidase

The effect of temperature on the deamination of 5-hydroxytryptamine, tyramine, and phenethylamine by monoamine oxidase (MAO) of human placenta, beef liver, and rat liver has been studied. Both MAO A and MAO B activities are influenced by the lipid-phase transition and, in some cases, another type of transition. The estimates of activation energy (E_act) for the deamination of 5-hydroxytryptamine, phenethylamine, tyramine, dopamine, and pentylamine at 5–20 °C show that a given substrate is associated with a particular value irrespective of the source of MAO acting upon it. The substrate dependence of E_act is explained by the differences in lipophilicity of the various substrates. The interaction of enzyme and the lipids in the environment of its active site would differ with each substrate, and would give rise to different activated complexes, each corresponding to a given substrate. The E_act values are presumably related to these complexes, rather than to enzyme alone.     

Neurotransmitter-induced changes in arachidonate incorporation into rat brain phospholipids: pharmacological manipulations of receptors,presynaptic uptake,mao activity and deacylation-reacylation processes

The incorporation of [³H]arachidonic acid ([³H]AA) into phospholipids (PL) of rat brain, was studied in cerebral cortex slices in the presence and absence of norepinephrine (NE), serotonin (5-HT) and carbamylcholine (CCH). Both NE and 5-HT produced a concentration-dependent effect of stimulating [³H]AA incorporation into phosphatidylinositol (PI) while attenuating incorporation into other PL. Addition of CCH had no apparent effect. The β-adrenergic agonist, isoproterenol, had an effect similar to that seen with equimolar concentrations of NE, whereas the α₁ agonist, phenylephrine, or the α₂ agonist, clonidine, did not produce significant changes. However, application of the NE-receptor blockers, propranolol or prazosin, in the presence of NE, did not modify the NE-induced effects. Similarly, the 5-HT-receptor blockers, methysergide or ketanserine, failed to modify the 5-HT-induced effects, indicating that the neurotransmitter-produced changes may not be receptor mediated. Manipulations of the NE or 5-HT reuptake systems by imipramine (IMI) or desipramine (DMI) had a small additive effect on the neurotransmitter-produced changes in [³H]AA incorporation, suggesting that a functional presynaptic reuptake system is not required for the NE or 5-HT-produced effects. The possibility that the NE or 5-HT effects involve the oxidative metabolism of the monoamines by MAO was also investigated. The MAO inhibitors tranylcypromine and pargyline had no appreciable effect on the neurotransmitter-induced changes in [³H]AA incorporation whereas clorgyline clearly reduced the increase in [³H]AA incorporation into PI seen in the presence of NE or 5-HT, but this clorgyline effect may not be related to its activity as MAO inhibitor. The phospholipase A₂ inhibitor mepacrine had no significant effect on the NE-produced increase in [³H]AA incorporation into PI, but it antagonized the NE-produced decrease in [³H]AA incorporation into PC. Delta-9-Tetrahydrocannabinol, which acts as acyltransferase inhibitor, antagonized the NE-produced increase in [³H]AA incorporation into PI without appreciably influencing the NE-produced decrease in [³H]AA incorporation into PC. These findings suggest that the neurotransmitter-produced increase in [³H]AA incorporation into PI is mediated by stimulation of a specific lyso-PI arachidonyl transferase. The neurotransmitter effects on arachidonate incorporation may have physiological significance in view of the importance of processes of deacylation and reacylation of membrane PL in regulating the function of neuronal membranes.     

The study of ubiquitin-dependent increase in monoamine oxidase sensitivity to proteolysis and specific inhibitor, pargyline

Insertion of exogenous ubiquitin into rat brain mitochondria in the presence of ATP and the ATPregenerating system (creatine phosphate/creatine phosphokinase) results in the increase in: sensitivity of mitochondrial monoamine oxidases (MAO) A and B to inhibition by mechanism based inhibitor and incorporation of [³H]-pargyline, which was especially notable in the fraction obtained by immunoprecipitation of mitochondrial proteins with anti-ubiquitin antiserum and protein A Sepharose. This suggests that MAO is a potential substrate for ubiquitination in vitro. However, the content of the tritium label in this fraction was less than 0.1 % and not more than 0.25% of total radioactivity of [³H]-pargyline bound to control and ATP-ubiquitin treated mitochondria, respectively. Insertion of ubiquitin into mitochondria did not influence molecular masses of [³H]-pargyline labeled proteins. These results suggest that direct ubiquitination of MAO insignificantly contributes to marked changes in the sensitivity of MAO A and MAO B to proteolysis and specific inhibition found under these experimental conditions. It is possible that more complex processes are involved into realization of these effects during ATP-dependent ubiquitin incorporation into mitochondria.     

燕麦幼苗单胺氧化酶的热稳定性及催化特性研究

含黄素单胺氧化酶(MAO)在生物体内通过对单胺类物质的氧化脱氨作用生成相应的醛、氨气和过氧化氢。MAO在植物中的研究较少,通过对燕麦幼苗MAO的研究发现,暗条件下生长的燕麦幼苗匀浆内所含MAO活性均高于光照条件,且发芽三天左右的幼苗体内MAO的活性达到峰值(2.5pKat/mg),同时测定不同组织中MAO的活性为:幼芽>幼根>种子。对纯化后的燕麦MAO的热稳定性和催化特性研究表明:燕麦MAO的热稳定性较差,常温下易失活,37℃和50℃下水浴90min后,活性损失分别为50%和75%;燕麦MAO对底物的选择性较强,只对低浓度的苄胺和苯乙胺的氧化具有催化效果,Km分别为265μmol/L和705μmol/L;在对底物的特异性方面与人类MAO B有一定的相似性,但体外催化效率低于黑曲霉MAO和人类MAO B。