Brian A. Callingham

Monoamine oxidase A and B: A useful concept?

The use of a number of inhibitors of monoamine oxidasc (monoamine:O, oxidoreductase, EC 1.4.3.4) has shown that many mam~lian tissues contain two major forms of the enzymic activity that differ in their substrate specificities and sensitivities to inhibitors. Although several pieces of evidence indicate that these two forms may not represent two distinct enzymes but result from a single enzyme species existing in different membrane-bound environments [see eg., 1.21. it has frequently been assumed that their specificities will be broadly similar in a variety of organs and tissues and also in different animal species. If such an assumption were true it should be possible to extrapolate results found in any convenient laboratory animal to the situation in man. It would seem however, that this general assumption may be an oversimplification. This review is therefore an attempt to assess the usefulness of the concept of the existence of two functionally distinct forms of the enzyme, without exploring the possible nature of any such forms, since this aspect has recently been reviewed in detail elsewhere f-?].

Evidence for a clorgyline-resistant monoamine metabolizing activity in the rat heart.

When benzylamine was used as substrate, a component of the total monoamine oxidase (MAO) activity in the rat heart was found to be resistant to inhibition by clorgyline. The proportion of the total activity represented by this component, decreased as the rat grew. It was also inhibited by both semicarbazide and isoniazid but not by potassium cyanide. Inhibitor studies with MAO in subcellular fractions showed that this component was more concentrated in the microsomal and soluble fractions. However, it could not be concluded that the activity was entirely a soluble enzyme. Determination of quasi‐Michaelis constants (“Km”) for total benzylamine oxidizing activity revealed a high (“Km” of approximately 10−5M) and low (“Km” of approximately 5 times 10−4M) affinity component. The high affinity component was inhibited by semicarbazide and the low affinity component by clorgyline. In the presence of 10−3M clorgyline, the high affinity component showed substrate inhibition at higher substrate concentrations. The possibility is discussed that the clorgyline‐resistant activity is due to an amine‐oxidizing activity distinct from mitochondrial MAO.

Studies on the nature of the increased monoamine oxidase activity in the rat heart after adrenalectomy

The increased activity, induced by adrenalectomy, of the enzyme monoamine oxidase (MAO) in the rat heart was found to resemble closely that present in the hearts of control animals. No significant differences were observed in the nature of the response to heat denaturation, changes in pH or to inhibition by pargyline or clorgyline. The relative activities using the substrates tyramine, 5‐hydroxytryptamine, dopamine or benzylamine were the same. No evidence was found to suggest the presence of a heat‐stable or dialysable inhibitor of enzyme activity. In young rats there was an increase in the relative enzyme activity using benzylamine as substrate, compared with the activity using tyramine, in the first few days after adrenalectomy. No effect of adrenalectomy could be detected upon the MAO activity in the rat brain or liver. It is concluded that the increase in rat heart MAO following adrenalectomy cannot be due to the synthesis of an enzyme with different catalytic properties, nor to the transformation of the existing enzyme into one of increased catalytic ability but with different properties.

The acetylenic monoamine oxidase inhibitors clorgyline, deprenyl, pargyline and J‐508: their properties and applications

The article presents a short review of some of the properties of the acetylenic inhibitors of monoamine oxidase currently under investigation: clorgyline, (—)‐deprenyl, pargyline and J‐508. Their substrate‐selective inactivation, mechanism of inhibition, titration and pharmacology with respect to monoamine oxidase are critically discussed.

Monoamine oxidase activities in brown adipose tissue of the rat: Some properties and subcellular distribution

Amine oxidase activity towards 5-hydroxytryptamine (5HT), tyramine (TYR), 2-phenylethylamine (PEA) and benzylamine (BZ) was studied in homogenates of interscapular brown adipose tissue of the rat. By the use of clorgyline, an irreversible inhibitor of MAO, it was established that 5HT was deaminated solely by MAO-A, and TYR and PEA mainly by MAO-A and clorgyline-resistant semicarbazide-sensitive amine oxidase (CRAO). BZ appeared to be oxidized almost entirely by CRAO. A very small amount of MAO-B activity was detectable with PEA and BZ as substrates. A variety of amines, amino acids and known amine oxidase inhibitors were tested for their ability to inhibit the deamination of BZ by CRAO. BZ metabolism by the enzyme was not affected by any secondary amines, unlike enzymes of the flavin type, but it was inhibited by carbonyl reagents, like the pyridoxal phosphate and copper-dependent amine oxidases described in plasma and connective tissue. Unlike these enzymes, however, CRAO in brown adipose tissue was resistant to KCN and unaffected by the amines, histamine, mescaline and some polyamines but it was inhibited by cuprizone. It was found to have a low Km (<5 μM) for BZ and showed the greatest similarity to a clorgyline-resistant enzyme described in rat blood vessels. Cell fractionation studies revealed that CRAO, being associated with the particulate fractions, was mainly membrane-bound. The distribution of CRAO activity between various cell fractions was different from that of the mitochondrial enzymes assayed and was more like that of either the plasma membrane or microsomal enzymes. When microsomal and plasma membrane vesicles were separated CRAO activity appeared distributed equally between the two fractions, suggesting that the enzyme may have a dual location within the cell. The specific activity of CRAO was higher in brown adipose tissue from obese animals than in tissue from lean animals. The significance of these findings is discussed in relation to the possible physiological function of this enzyme.

A comparison of cardiac and vascular clorgyline-resistant amine oxidase and monoamine oxidase: Inhibition by amphetamine, mexiletine and other drugs

Abstract Clorgyline-resistant amine oxidase (CRAO) and monoamine oxidase (MAO) were studied in homogenates of rat heart and aorta, using benzylamine and tyramine as substrates. In heart, benzylamine at 0.001 mM was deaminated solely by CRAO. With higher concentrations of benzylamine (0.01, 0.1 and 1.OmM), an increasing involvement of MAO-A and MAO-B became apparent in the deamination of benzylamine such that, at 1.0 mM benzylamine, deaminated products resulted equally from MAO-A, MAO-B and CRAO. In aorta, benzylamine was deaminated solely by CRAO irrespective of the concentration used. Tyramine (0.01, 0.1, 1.0 and 5.0 mM) was deaminated entirely by MAO-A in heart, whereas in the aorta both MAO-A and CRAO participated. In aorta the ratio of product formation from MAO-A and CRAO did not vary with changes in the concentration of tyramine, indicating similar Km values for both enzymatic activities. Further studies with tyramine (0.1 mM) and clorgyline showed biphasic inhibition curves suggestive of two distinct MAO-A components in both heart and aorta. The two components showed different properties in the heart when compared with aorta. When homogenates of hearts were heated at 50° for 1 hr, their sensitivity to inhibition by clorgyline increased, while in homogenates of aorta sensitivity to clorgyline decreased. CRAO was investigated further with benzylamine as substrate. Kinetic studies gave similar Km values for both heart and aorta (4–6 μM at pH 7.8), and these values were not altered by flushing the assay tubes with oxygen. However, flushing with nitrogen caused uncompetitive inhibition in the heart and noncompetitive inhibition in aorta. These results suggest a difference in the catalytic mechanism between CRAO of heart and aorta. In both heart and aorta, CRAO was inhibited by semicarbazide, (+)-amphetamine, phenelzine and (+)- and (−)-mexiletine, with the (+)-form being more potent. Straight-chain diamine and polyamine compounds failed to inhibit in concentrations up to 10−4 M. Thus, CRAO is not a typical diamine or polyamine oxidase. The results show differences between heart and aortic CRAO and MAO-A, and the possibility exists for heterogeneity within each of these two distinct forms of amine oxidase. Additionally, drugs known to inhibit MAO-(+)-amphetamine, phenelzine and mexiletine also inhibit CRAO. However, the biological significance of since the physiological role of CRAO is unknown.

The effects of thyroid hormones on monoamine oxidase in the rat heart.

The administration of thyroxine to young male rats produced an increase in the specific activity of their cardiac monoamine oxidase (MAO). A reduction in the circulating concentrations of thyroid hormones, brought about by 2‐thiouracil, led to a decrease. The relative change in activity produced was greater with tyramine than with benzylamine as substrate. By following the time‐course of the return of enzyme activity, with tyramine as substrate, after a single injection of pargyline in vivo, it was concluded that both excess and lack of thyroid hormones cause their effects on MAO activity by changing the rate of synthesis of the enzyme and not its degradation rate constant. The degradation rate constant did change with the age of the animal. The MAO activity, which increased towards tyramine as substrate in hyperthyroid rat hearts, behaved in the same way as that of controls to heat treatment, irreversible inhibition by pargyline or by clorgyline and also in Km determinations. The pattern for benzylamine oxidation was similar, except for the effect of the inhibitor clorgyline which shifted the plateau region of the double sigmoid inhibition curve significantly using enzyme from hyperthyroid rat hearts. The plateau region was also shown to be affected by the age of the animal. The possibility is discussed that the increased cardiac MAO activity produced by thyroid hormones and by the growth of the animal is mediated by that form of the enzyme primarily responsible for the oxidation of tyramine. Mixed substrate experiments suggested that tyramine oxidation could be inhibited competitively by benzylamine.

Effects of the pyrones, maltol and ethyl maltol, on iron absorption from the rat small intestine

The pyrones, 3‐hydroxy‐2‐methyl‐4‐pyrone (maltol) and 3‐hydroxy‐2‐ethyl‐4‐pyrone (ethyl maltol) chelate iron with a high affinity and selectivity. The resulting 1:3 (metal‐igand) complexes, being neutral, are able to partition readily across cell membranes and thus may facilitate iron transport across the intestinal wall. Absorption of radioactive iron (59Fe) in the presence of these pyrones was investigated in male rats 1, 2, 4 and 6 h after intraduodenal administration of a 7 μg dose and compared with that of 59Fe given as the sulphate, gluconate, fumarate or complexed to EDTA. Total body absorption and distribution were calculated from the 59Fe content of various tissue samples. With all the iron preparations used, blood levels of 59Fe were highest 1 h after injection whilst the 59Fe content at the major site of deposition, i.e. the bone marrow, increased up to 6 h. No 59Fe was found in the urine. Total body absorption of 59Fe was significantly higher from the pyrones than from the other four preparations. Over the dose range 0.7–700 μg, the proportion of 59Fe absorbed from both iron maltol and iron sulphate decreased with increasing dose. Enhanced 59Fe uptake from maltol was evident at 0.7–70 μg but not at 700 μg suggesting that use of these pyrones will not result in iron overload. Absorption of 59Fe given into the stomach was slower in onset but was sustained longer presumably via a steady delivery of iron to the duodenum from the gastric reservoir. The presence of excess maltol to maintain the integrity of the neutral 1:3 iron‐maltol complex under a variety of physiological conditions did not appear critical in‐vivo for effective iron absorption. There was no difference in 59Fe uptake between maltol, ethyl maltol or a mixture of ethyl maltol and maltol. 59Fe uptake was significantly enhanced in iron‐deficient animals. Within 1 min of i.v. injection of iron complexed to maltol or ethyl maltol, 59Fe became associated with a plasma protein of molecular ratio similar to that of transferrin. With EDTA this process was much slower, i.e. up to 60 min. The half‐life of 59Fe in the blood was similar whether administered as the maltol, ethyl maltol, sulphate or EDTA; at the 110 μg iron dose it was 133 ± 8 min and at 1 μg it was 74 ± 10 min in iron‐replete animals and 44 ± 5 min in iron‐deficient animals. The rate of absorption from the duodenum of 59Fe given as maltol, ethyl maltol or as iron sulphate increased with increasing dose but the shape of the absorption curve depended on the rate of movement of iron along the intestine. It is concluded that the pyrones, maltol and ethyl maltol, are able to enhance the initial stages of iron uptake from the intestinal lumen, possibly by holding the iron in a readily absorbable form, but do not influence subsequent iron distribution and so may provide safe and palatable alternatives to those iron preparations presently available for the treatment of iron deficiency.

The effect of lipophilic compounds upon the activity of rat liver mitochondrial monoamine oxidase-A and -B

Abstract The effect of various lipophilic compounds on the activity of monoamine oxidase (MAO) was determined. The local anaesthetics procaine, procainamide, tetracaine and lignocaine were all MAO-A selective inhibitors, whereas benzyl alcohol, butan-l-ol, hexan-l-ol and octan-l-ol inhibited MAO-B selectively. Procaine was found to be a competitive inhibitor of the deamination of 5-hydroxy-tryptamine (5-HT), tyramine, β-phenethylamine and benzylamine. Benzyl alcohol was competitive towards β-phenethylamine and benzylamine, but a mixed-type inhibitor towards 5-HT and tyramine. The same patterns of inhibition for both drugs were found when the activity was assayed under atmospheres of either oxygen or air. The inhibition produced by both compounds was fully reversible. Triton X-100 appeared to inhibit the activity of MAO-A selectively when preincubated with the enzyme for 30 min at 30°. This selectivity was lost when the preincubation temperature was raised to 37°. The inhibition of MAO activity by Triton X-100 after preincubation at 37° was found to be irreversible. Sodium deoxycholate and SDS were also found to inhibit the activity of MAO after preincubation with the enzyme at 37°. The significance of these results is discussed.

In vitro and in vivo inhibition by benserazide of clorgyline-resistant amine oxidases in rat cardiovascular tissues

Bernserazide (D,L-serine 2-[2,3,4-trihydroxybenzyl]-hydrazide) as been shown to inhibit the clorgyline-resistant amine oxidase (CRAO) activities which metabolize benzylamine in homogenates of rat aorta, heart and brown adipose tissue. In vitro studies showed a concentration- and time-dependent inhibition of CRAO in heart and aorta which was reversed by dialysis for 18hr. At high concentrations (10(-4)-10(-3)M) benserazide appeared to increase enzyme activity towards and occasionally above control value. These increases became more prominent after long periods of preincubation (especially in the presence of saturating benzylamine concentrations) and remained after dialysis of those homogenates preincubated with benserazide. The administration of benserazide for one or seven days in daily doses of 5-150 mg/kg also inhibited CRAO activity in vivo in a dose-dependent manner, with greater inhibition after seven days treatment. Reversal of inhibition, by dialysis of tissue homogenates from benserazide-treated rats, was much slower than was found with homogenates incubated in vitro with the drug. After benserazide administration to rats, MAO-A activity towards 5-hydroxytryptamine was generally not inhibited, and in fact was significantly increased in some cases. The administration of L-DOPA (250 mg/kg) together with benserazide (40 mg/kg) resulted in a similar degree of CRAO inhibition in aorta and heart to that seen after benserazide alone. These findings are discussed with regard to the use of these drugs in the therapy of Parkinsons Disease, although the paucity of information about the physiological function of CRAO makes the significance of its inhibition by benserazide unclear.