Clorgyline-resistant Amine Oxidase Research Articles

The effect of DSP-4 (N-[2-chloroethyl]-N-ethyl-2-bromobenzylamine) on monoamine oxidase activities in tissues of the rat.

The in vitro inhibition of monoamine oxidase (MAO) in rat liver, and of the clorgyline-resistant amine oxidase (CRAO) in rat heart and aorta, by DSP-4 (N-[2-chloroethyl]-N-ethyl-2-bromobenzylamine) has been studied. Inhibition of each enzyme activity was independent of prolonged preincubation, was reversed by dialysis, and also Ackermann-Potter plots were consistent with reversible inhibition. Simple linear competitive inhibition of MAO-A and MAO-B was observed, with Ki values of 6 x 10(-6) and 8 x 10(-5) M, respectively. CRAO was inhibited in a mixed, non-competitive manner (Ki of 3.2 x 10(-5) and 7.8 x 10(-6) M in heart and aorta, respectively) which conformed to a kinetic model in which the binding of DSP-4 to CRAO increased the affinity of substrate binding, but prevented product formation. The possible significance of these results for the in vivo actions of the drug is discussed.

Hydralazine is an irreversible inhibitor of the semicarbazide-sensitive, clorgyline-resistant amine oxidase in rat aorta homogenates.

Hydralazine is an irreversible inhibitor of the semicarbazide-sensitive, clorgyline-resistant amine oxidase in rat aorta homogenates.

The deamination of n-pentylamine by monoamine oxidase and a semicarbazide-sensitive amine oxidase of rat heart.

n-Pentylamine is deaminated by homogenates of rat heart. Clorgyline inhibition curves at 10 and 100 microM n-pentylamine indicated that this substrate was deaminated by MAO-A, -B and a clorgyline-resistant amine oxidase sensitive to inhibition by semicarbazide. These results have been compared with two other commonly used monoamine substrates, beta-phenethylamine and benzylamine.

Amine oxidase activities in brown adipose tissue of the rat: identification of semicarbazide-sensitive (clorgyline-resistant) activity at the fat cell membrane

Amine oxidase activity, previously described in homogenates of brown adipose tissue of the rat, has now been investigated in preparations of isolated fat cells. It was found that the specific activities of both monoamine oxidase A (MAO) and of the semicarbazide-sensitive clorgyline-resistant amine oxidase (SSAO) were higher in isolated fat cells than in the original whole tissue. Brown adipocytes therefore represent a major source of both these enzymes. In plasma membranes prepared from these isolated brown fat cells by borate extraction there was a similar enrichment of activity of SSAO and of the plasma membrane marker enzyme, phosphodiesterase I. However in preparations of cell membranes made by binding the cells to polycation-coated beads, enrichment of phosphodiesterase I activity was much greater than that of SSAO. It is suggested that the disposition of the enzyme within the cell membrane may account for the discrepancy in these results, i.e. the sidedness of the membrane may be important. Histochemical visualization of enzyme activity in whole tissue at the ultrastructural level was undertaken. Positive staining of mitochondria was achieved in the presence of the MAO substrate, tryptamine. Staining around the edges of the brown fat cells was observed with the SSAO substrates, tyramine and benzylamine. Staining was largely absent when substrate was omitted or after pretreatment with the irreversible SSAO inhibitor, hydralazine and the slowly reversible inhibitor, semicarbazide. It is not definitely proven that this staining represents sites of enzyme activity but the results are consistent with evidence from other studies indicating that SSAO in brown adipose tissue of the rat may be found predominantly at the fat cell surface.(ABSTRACT TRUNCATED AT 250 WORDS)

On the presence of a clorgyline resistant benzylamine oxidase activity in mouse kidney

In the crude homogenates of three different strains (C3H, Swiss, C57b) of mouse kidney the exposure to a millimolar concentration of clorgyline reveals a benzylamine oxidative deaminating activity that is due to a clorgyline resistant amine oxidase (CRAO). In the three strains this CRAO activity shows a low sensitivity to semicarbazide and to alpha- amiguanidine. In the C3H mouse kidney the glycoprotein nature of the CRAO was established from affinity chromatography with immobilized concanavalin A. Studies of this activity in the C3H mouse kidney revealed the presence of a reversible inhibitor which is precipitated by ammonium sulphate between 35-55% of saturation. The presence of this inhibitor precludes accurate measurement of the CRAO distribution in the different subcellular fractions. The enzyme purified by affinity chromatography, is semicarbazide insensitive. This is the first observation of the presence of a CRAO-semicarbazide insensitive activity in a tissue. The fact that "original homogenate" and the subcellular fractions show some sensitivity to semicarbazide also indicates that a semicarbazide-sensitive amine oxidase (SSAO) is present in the C3H mouse kidney.

Solubilization and some properties of a semicarbazide-sensitive amine oxidase in brown adipose tissue of the rat.

A semicarbazide-sensitive clorgyline-resistant amine oxidase (SSAO) was solubilized from membrane fractions of rat brown adipose tissue by the non-ionic detergent, Triton X-100. Alteration of ionic strength or addition of chelating agents alone failed to release the enzyme from its membrane. Lipid-depletion led to loss of enzyme activity and alteration of substrate affinity. Over 80% of the activity of the solubilized enzyme was found in gel filtration fractions corresponding to an Mr of between 160 000 and 180 000. The glycoprotein nature of SSAO was established from affinity chromatography with either immobilized concanavalin A or Lens culinaris lectin. Elution of over 50% SSAO activity from the lentil lectin was achieved with 0.25M-alpha-methyl D-mannoside to give 80-90-fold purification of the enzyme. Irradiation inactivation gave a value for Mr of around 183 000 for both soluble and membrane-bound SSAO. Substrate affinity and inhibitor sensitivity of the enzyme were unaltered by solubilization. The copper-chelating agent, diethyldithiocarbamate, did not affect the enzyme, shedding doubt on the suggestion that SSAO is a copper-requiring enzyme. The significance of these findings in relation to the nature of SSAO and to its disposition within the cell membrane is discussed.

Properties of Monoamine Oxidase in Monkey Heart

The activities of monoamine oxidase (MAO) in a homogenate and the mitochondrial fraction of monkey (Macaca facicularis) heart were measured with labelled benzylamine, serotonin (5-HT), tyramine and β-phenylethylamine (PEA) as substrates. The effects of clorgyline, deprenyl and semicarbazide on MAO were also investigated. Benzylamine deamination was more sensitive to deprenyl than to clorgyline, although it was not completely inhibited by a high concentration of either, and the remaining activity was completely inhibited by pretreatment with semicarbazide. The activities of 5-HT and tyramine were both more sensitive to clorgyline than to deprenyl, and both inhibitors gave single-sigmoidal inhibition curves. Experiments with increasing concentrations of PEA as substrate and clorgyline and deprenyl as inhibitors indicated that at higher concentrations, PEA was a substrate for MAO-A as well as MAO-B in monkey heart. These results suggest that monkey heart mitochondria contain not only MAO-A and MAO-B, but also clorgyline resistant amine oxidase (CRAO). The ratio of MAO-A, MAO-B and CRAO was determined from plots of inhibitions with each substrate. The kinetic constants of MAO in monkey heart were compared with those of MAO in hearts of other an mals. The enzymic properties of MAO in monkey heart were discussed on the basis of these results.

Inhibitory actions of hydralazine upon monoamine oxidizing enzymes in the rat

The inhibition by hydralazine of the clorgyline-resistant amine oxidase (CRAO) and monoamine oxidase (MAO) activities in various rat tissues has been studied. Hydralazine was a potent, time-dependent inhibitor of rat heart CRAO activity in vitro. The inhibition was not reversed by dialysis for 18 hr at 4°, and only partially reversed by dialysis at 37°. Dialysis at 4° in the presence of pyridoxal phosphate (10−4 M) also did not reverse the inhibition. Ex vivo inhibition of CRAO was found in heart and aorta homogenates in a dose-dependent manner after administration of hydralazine (1–40 mg/kg i.p.) to rats. In contrast, MAO-A activity was unaffected or, in some cases, significantly increased in these tissue homogenates from drug-treated animals. However, in vitro inhibition by hydralazine of both MAO-A and B activities of rat liver mitochondrial fractions was found, and these effects were fully reversible by dialysis for 18 hr at 4°. Inhibition of MAO-A was competitive (Ki, of 2.5 × 10−6 M), while inhibition of MAO-B showed complex mixed non-competitive kinetics. These results indicate that hydralazine possesses different inhibitory properties towards the various amine oxidases in rat tissues, and these actions are discussed in relation to the clinical use of the drug as an anti-hypertensive agent.

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

Benserazide ( d, l-serine 2-[2,3,4-trihydroxybenzyl]-hydrazide) has 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 timedependent inhibition of CRAO in heart and aorta which was reversed by dialysis for 18hr. At high concentrations (10 −4−10 −3M) 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 m 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 Parkinson's Disease, although the paucity of information about the physiological function of CRAO makes the significance of its inhibition by benserazide unclear.

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

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 K m 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 K m 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.

Short- and long-term effects of L-DOPA treatment upon monoamine oxidase: A comparative study in several rat tissues

The influence of L-DOPA administration (250 mg/kg/day) to the rat for up to 26 days, upon the oxidative deamination of 5-hydroxytryptamine, dopamine and benzylamine, has been investigated in homogenates from heart (atria and ventricles separately), aorta, brain, liver and kidneys. In the cardiovascular tissues and the kidneys, the metabolism of these amines was generally increased after L-DOPA treatment, although these changes were only maintained over the time course of the experiment in kidney homogenates. In contrast, relatively few changes were obtained in brain and liver. At least three enzyme activities, called MAO-A, MAO-B and a clorgyline-resistant amine oxidase, have previously been identified as being responsible in varying degrees for the metabolism of these substrates in the different tissues studied. The present results are discussed in the context of possible selective influences of L-DOPA upin these activities. Similar considerations have been applied to various developmental changes in amine metabolism, observed in some tissues of growing control animals during the course of these experiments. The results reveal a considerable variation among different rat tissues in the ability of L-DOPA administration to modify amine metabolism. This variation is dependent upon the particular deaminating enzyme studied, and the period of L-DOPA administration.