Search

When allyl formate (AF) was administered to rats, the marked elevation of B-form MAO activity in plasma was found with beta-PEA as a substrate. In contrast, in the case of carbon tetrachloride (CCl4), A-form MAO activity elevated predominantly. The deaminations of 5-HT and beta-PEA in these plasma treated with AF or CCl4 were not inhibited completely by a high concentration of MAO inhibitor, deprenyl or clorgyline. These results indicate that there may be two or more distinct amine oxidases released from the liver and other organs in response to CCl4 or AF.

The extraction and partial purification of endogenous "monoamine oxidase (MAO) inhibitor-like" material from the monkey brain are described. The endogenous material (F-1 and F-2) obtained after Bio-Gel P-2 gel filtration and silica column chromatography inhibited MAO in the monkey brain mitochondria toward 5-hydroxytryptamine (5-HT), beta-phenylethylamine (beta-PEA), tyramine and dopamine as substrates. The inhibitory effects of F-1 and F-2 were non-linear concentration dependent, and F-1 non-competitively inhibited A-form MAO, while F-2 inhibited A-form MAO competitively and inhibited B-form MAO non-competitively. These substances were more potent inhibitors of A-form than of B-form MAO. F-2 was heat stable but liable to the treatment with pepsin and trypsin. F-1 was not inactivated by heat treatment and digestion with pepsin and trypsin. F-1 may be a low molecular weight (less than 1350) compound, including certain monoamines or their metabolites or other unidentified compounds, while F-2 was a low molecular weight (about 2500) peptide.

Effects of NKK-105 against lipid peroxidation of microsomes by CCl4 are investigated, in vitro. NKK-105 had no effect on microsomal lipid peroxidation. When the microsomal fraction was preincubated with NKK-105 before adding CCl4, the lipid peroxidation induced with CCl4 was almost prevented by NKK-105. Effects of NKK-105 administration on aminopyrine N-demethylase, cyt.b5, cyt.p-45O and lipid peroxidation in CCl4-induced liver damage were investigated, in vivo. NKK-105 had no effect on the activity of aminopyrine N-demethylase and the content of cyt.b5, cyt.p-450 and peroxide. The content of cyt.b5, cyt.p-450 and the activity of demethylase remarkably decreased with CCl4 administration, but the content of cyt. p-450 and the demethylase activity were restored about 75% of the control values and cyt.b5 content was completely restored with previous administration of NKK-105. The amount of peroxide in the microsomal fraction increased with CCl4 administration was restored to the control level with previous administration of NKK-105.

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.

We examined neurochemically the effects of bifemelane (BF) on muscarinic ACh (mACh-R) and beta-adrenergic receptors (beta-AdR) and imipramine binding sites and the activities of acetylcholinesterase (AChE), choline acetyltransferase (CAT) and monoamine oxidase (MAO) in the P2 fractions of rat brain, ex vivo and in vitro. Male rats were given daily injections of 10, 30 mg/kg BF, p.o., for a period of 4 weeks. The Kd and Bmax values for mACh-R in the rat forebrain by administration of 10, 30 mg/kg BF decreased significantly compared with that of the control, although the Kd and Bmax values for beta-AdR and imipramine binding sites were almost identical. The Km and Vmax values of A- and B-form MAO decreased in rats that had been administered 30 mg/kg BF for 4 weeks. The binding of 3H-QNB (quinuclidinyl benzilate) on mACh-R, 125I-CYP (iodocyanopindolol) on beta-AdR and 3H-imipramine on imipramine binding sites decreased by 60, 20 and 70% in the presence of 1 microM BF, respectively, while the addition of 1 microM BF inhibited MAO activity by about 50%. However, CAT and AChE activities were not inhibited by BF.

The type of inhibition of the different forms of monoamine oxidase by clorgyline (preferentially ‘A’ form-inhibiting) and deprenyl (preferentially ‘B’ form-inhibiting) has been investigated. For both inhibitors a reversible phase of inhibition was found to precede the irreversible reaction. When incubated at 25° for 20 min, clorgyline inhibited the ‘A’ form in an irreversible fashion, while 4 hr at 37° was needed to inhibit the ‘B’ form irreversibly. In contrast, deprenyl inhibited the ‘A’ form reversibly even when incubated at 37° for 4 hr, whereas the ‘B’ form was irreversibly inhibited under these conditions. The selectivity of both inhibitors was considerably lower with longer incubation times. The implications of the results for the interpretation of previous findings on multiple forms of monoamine oxidase (an ‘A’ and a ‘B’ form) and for chronic treatment with the inhibitors in vivo is discussed.

The effects of a single and repeated administration of methamphetamine (MP) in vivo in rats on its own metabolism in vitro were investigated. In both cases, the p-hydroxylation of MP to p-hydroxymethamphetamine by a microsomal fraction from rat liver was inhibited for a period of 16 hr after the last injection of MP. This inhibition was diminished by dialysis of the microsomal preparations. In contrast, the reduced level of cytochrome P-450 in hepatic microsomes from rats pretreated with the SKF 525-A did not revert to the control value after dialysis. When microsomes were preincubated with N-hydroxymethamphetamine, which is the metabolite of MP and a potent substrate for the formation of a metabolic intermediate (MI) complex with cytochrome P-450, the content of the MI was increased and the MP-hydroxylation activity decreased in direct proportion to the length of the preincubation. These results suggest that the inhibition of MP-hydroxylation may be due to reduction of the level of cytochrome P-450 that accompanies the formation of the MI complex. Furthermore, it appears that the complex can be dissociated by dialysis.

Monoamine oxidase was investigated histochemically in tissues of the mouse by incubating freeze-dried, whole-body sections with tryptamine, serotonin, tyramine, beta-phenylethylamine, or benzylamine as substrate and Nitroblue tetrazolium as the final electron acceptor. The most intense staining with tryptamine was exhibited by intestinal epithelium and adrenal cortex; moderate staining was noted in the epithelium of the nose, bronchi, oesophagus, and upper stomach and in preputial gland, pancreas, nerve, spinal cord and brain. Weak staining was seen in the lung, spleen, liver and kidney. The distribution of the formazan deposition was similar, but much less intense, when serotonin and tyramine were used as the substrates. Only very weak staining was observed when beta-phenylethylamine was the substrate; no staining was seen with benzylamine. Monoamine oxidase activities with tryptamine were greatly inhibited by pretreatment with clorgyline (10 microM), while deprenyl (10 microM) slightly inhibited activities in all tissues except liver. This staining technique should be useful in further studies on the identification of the multiple forms of monoamine oxidase in tissues of the mouse. Nicotine and nitrosonornicotine were not substrates in any of the tissues; consequently, this enzyme system does not appear to produce the proximal carcinogen from this nitrosamine.

It is known that platelet MAO appears to behave more like the B-form enzyme than the A-form enzyme based on inhibitor sensitivity and substrate specificity. However, dog platelets showed a different substrate specificity such as high activity with 5-HT and beta-PEA as substrates. Moreover, dog platelet MAO was sensitive to the drugs clorgyline and harmaline with 5-HT as the substrate, while it was sensitive to the drug deprenyl with beta-PEA as the substrate. These results also indicate the existence of two forms of MAO in dog platelets unlike in other platelets such as those from humans. A-form MAO from dog platelets was more stable against heat treatment at 55 degrees C than A-form MAO from dog liver and brain. On the other hand, there was no difference in the heat resistance of the three enzymes with beta-PEA as the substrate. After digestion with trypsin at 37 degrees C for 30 min, it was found that MAO from dog platelets, brain and liver were mostly inhibited with 5-HT as the substrate. In contrast, MAO in brain and liver were inhibited about 10%, but platelet MAO was inhibited about 50% with beta-PEA as the substrate. From these results, it is considered that dog platelet MAO exists as the two forms of the enzyme and has different enzymic properties in comparison with those of MAO from dog liver and brain mitochondria.

Monkey liver monoamine oxidase (MAO) was predominantly the B-form enzyme from the observed differences in substrate specificities and differences in sensitivities to MAO inhibitors. It is known that a MAO inhibitor, pargyline, binds to MAO irreversibly in the molar ratio of 1:1. 3H-pargyline was used as a marker to determine the existence of MAO. The molecular weight of MAO in monkey liver mitochondria was investigated by SDS-polyacrylamide gel electrophoresis after solubilization of 3H-pargyline binding mitochondria with 6% sodium dodecyl sulphate (SDS). The subunit molecular weight was found to be 60,000. The molecular weight determined from the electrophoretic mobility on several concentrations of gels by disc gel electrophoresis in the absence of SDS was found to be 120,000. These results indicate that monkey liver mitochondrial MAO exists as a dimer. Isoelectric focusing of the enzyme after solubilization with 0.1% Triton X-100 and 0.75% Triton X-100 and 0.75% Lubrol showed that it had a pI value near 6.5. Similar pI values were obtained for enzyme preparations solubilized with 0.75% Triton X-100 after treatment with phospholipase A or methylethylketone. These results suggest that the pI value of MAO in monkey liver mitochondria does not depend on the properties of detergents used to solubilize the enzyme preparation.

The multiple forms of monoamine oxidase (MAO) in monkey brain were investigated using an electrofocusing technique. When beta-phenylethylamine (beta-PEA) was used as substrate, two peaks (peak I and peak II) could be clearly distinguished from the profile; the isoelectric point (pI) values were near 7.8 and 6.3, respectively. When serotonin (5-HT) was used, MAO activity was observed in peak I enzyme. The peak I enzyme with a pI value of 7.8 contains AB-form MAO and oxidizes 5-HT and beta-PEA, while peak II enzyme with a pI value of 6.3 contains B-form MAO and oxidizes beta-PEA, respectively. However, when peak II enzyme was incubated in a pH 8.8 reaction medium, MAO activity toward 5-HT in peak II enzyme was seen.

gamma-Hexachlorocyclohexane (gamma-HCH) was metabolized by dehydrochlorination, dehydrogenation and dechlorination in rat liver microsomes and these initial metabolites of gamma-HCH were identified as gamma-pentachlorocyclohexene (gamma-PCCH), gamma-hexachlorocyclohexene (gamma-HCCH) and gamma-tetrachlorocyclohexene (gamma-TCCH) by gas chromatography-mass spectrometry (GC/MS). The dehydrochlorination and dehydrogenation were performed in incubation media containing NADPH and p,p'-tetramethyldiaminodiphenyl methane (TPD, 30 nmol) which is known to inhibit the degradation of initial metabolites formed during an aerobic incubation of gamma-HCH with microsomes at 25 degrees C. The dechlorination was found to proceed well under anaerobic conditions. The dehydrogenation was inhibited by SKF 525-A, CO, piperonyl butoxide, N2 and the absence of NADPH, but not by cyanide. Additionally, pretreatment of rats with phenobarbital (PB), but not with 3-methylcholanthrene (3-MC), induced the dehydrogenation of gamma-HCH. These results suggest that cytochrome P-450 is involved in this reaction. The cytochrome b5 system may not be involved. The dehydrochlorination was inhibited by N2, CO, piperonyl butoxide, KCN and the absence of NADPH, but not by SKF 525-A. This reaction was enhanced by pretreatment of rats with SKF 525-A, CoCl2 and piperonyl butoxide. Pretreatment with PB and 3-MC did not show a significant effect on the dehydrochlorination activity. Thus, the results suggest that the dehydrochlorination could be catalyzed by a specific species of cytochrome P-450 and cytochrome b5 system and/or other microsomal enzyme systems.

After mice were given methamphetamine (MA) at a dose of 25 mg/kg i.p. twice daily (9:00 a.m. and 5:00 p.m.) for 4 days, the behavioral effects and releases of brain catecholamines induced by MA were investigated. The behavioral effects which were estimated according to the rating system of Peachey et al. were a predominant increase in locomotor activity at 2.5 mg/kg MA, a predominant stereotyped behavior at 10 mg/kg MA and the mixed type at 5 mg/kg MA in the saline control. Following repeated administration of MA, the rating scores of each effect at 2.5 and 5 mg/kg MA were not significantly different from those of the saline control. At 10 mg/kg MA, the behavioral effects were of the mixed type. However, when locomotor activity was measured by Automex II 2SD, significant increases in locomotor activity were observed over 2 hr. Following repeated administration of MA, the brain NE level did not alter, while the brain DA level was significantly decreased. The increased accumulation of brain MHPG-S0i+ induced by MA was not significantly different from the saline control. The accumulation of HVA, however, did not alter at high doses of MA, which caused the increased accumulation of HVA in the saline control.