Some factors influencing the metabolism of benzylamine by type A and B monoamine oxidase in rat heart and liver

The ability of MAO-A and MAO-B to metabolize benzylamine in vitro has been investigated in mitochondrial preparations from rat liver and heart. Although under normal circumstances benzylamine appeared to be metabolized exclusively by MAO-B in the rat liver, a contribution by both MAO-A and a clorgyline-resistant enzyme component was revealed when the MAO-B activity was much reduced by pretreatment of the mitochondria with appropriate concentrations of deprenyl. These three enzyme activities also contributed to benzylamine deamination in rat heart mitochondria. However, binding studies with [³ H]pargyline, which provided an estimate of the respective concentrations of MAO-A and MAO-B active centres in heart mitochondria, indicated a ratio between MAO-A and MAO-B, markedly different from that shown by plots of inhibition of benzylamine metabolism by various concentrations of clorgyline. The interpretation of these clorgyline plots is discussed in terms of the kinetic constants of both MAO-A and MAO-B, and the relative amounts of each enzyme. It is proposed that although the turnover rate constant for benzylamine metabolism by MAO-A is much smaller than that shown by MAO-B, in those tissues containing a large ratio of MAO-A:MAO-B content, the metabolism of benzylamine by MAO-A can be detected.     

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.     

The deamination of isoamylamine by monoamine oxidase in mitochondrial preparations from rat liver and heart: A comparison with phenylethylamine

Isoamylamine (IAA) and phenylethylamine (PEA) have been studied as substrates for MAO activity in vitro in rat liver and heart mitochondrial fractions. The metabolism of both IAA and PEA in the liver was brought about by low- and high-K_m activities which, by the use of the inhibitor clorgyline, were found to correspond to MAO-B and MAO-A, respectively. Similar conclusions were reached for IAA metabolism in the rat heart. In contrast, only a single enzyme component of PEA metabolism in the rat heart was detected by Lineweaver-Burk analysis, although inhibition studies revealed that there was a small proportional contribution of MAO-B activity towards this amine. It was concluded from these results that the relative importance of MAO-A and MAO-B for metabolism of these amines depends upon the substrate concentration used. In addition, the possible physiological significance of IAA as a biogenic amine is discussed.     

Substrate and inhibitor selectivity of human heart monoamine oxidase.

The subcellular distribution, inhibitor sensitivity, thermostability and pH profiles of monoamine oxidase (MAO) from samples of human heart obtained at post mortem have been investigated with several substrates. A simple subcellular fractionation showed that, with either tyramine or benzylamine as substrate, about 50 per cent of the MAO activity was found in the mitochondrial fraction, with negligible quantities in the high speed supernatant. From the use of clorgyline, it appears that 5-HT is a substrate for MAO-A, benzylamine and β-phenethylamine are substrates for MAO-B, while tyramine and dopamine are substrates for both forms of the enzyme, d-Amphetamine was shown to be a selective competitive inhibitor of MAO-A, of similar potency to that observed with MAO from rat liver. No significant difference between the thermostability at 50° of the MAO activity towards 5-HT and benzylamine was observed. Preliminary results for the effect of pH on human heart MAO are presented. The results are discussed with respect to similar data obtained for MAO from other human and animal tissues.     

Monoamine oxidase activities of dissociated cell fractions from rat ventricular muscle

Cell fractions enriched in cardiac muscle cells (myocytes), on the one hand, and in non-myocytes, on the other, were prepared by dissociation of rat ventricular tissue with collagenase. Amine oxidase activities in homogenates of these cell fractions and also in homogenates of the corresponding undissociated ventricular tissue were compared. In addition, the activity of alkaline phosphatase (AP), an enzyme found predominantly associated in the heart with non-myocytes, particularly capillary endothelial cells, was also measured. No significant difference in the activity of MAO-A (assayed with 1 mM 5-hydroxytryptamine) was found between myocyte and non-myocyte fractions. In contrast, the activities of alkaline phosphatase (AP) and also the semicarbazide-sensitive amine oxidase (SSAO), assayed with 1 microM benzylamine (BZ), were both significantly higher in non-myocytes, by several-fold, than in myocyte fractions. Studies of the inhibition by clorgyline of 1 mM BZ metabolism confirmed that both MAO-A and MAO-B can also contribute to BZ oxidation in the rat heart. These experiments indicated different ratios of MAO-A: MAO-B in the various cell fractions. The ratios of the percentage contributions of MAO-A and MAO-B, respectively, to the total metabolism of 1 mM BZ were 78:20 (myocytes), 43:52 (non-myocytes) and 57:32 (undissociated tissue). These results suggest that MAO-B, in addition to AP and SSAO, may be associated preferentially with non-myocyte constituents of the rat heart. Although cardiac myocytes appear to contain predominantly MAO-A, this enzyme form is also localized, with high activity, to the non-myocyte fraction. However, since the non-myocyte fraction is heterogeneous in its cell content, containing vascular components of the coronary microcirculation, as well as other cells of connective tissue origin, the exact cellular localization of the enzyme activities within this fraction has not yet been defined.     

Effect of lignocaine on monoamine oxidase activity of brain and liver

In vivo administration of a single dose (100-150 mg/kg, i.p.) of lignocaine produces no change in MAO activity, while long-term treatment (50 mg/kg/day for 15 and 30 consecutive days, i.p.) produces a slight but appreciable inhibition of MAO activity with tyramine or serotonin but not with benzylamine as substrate in both rat brain and liver mitochondria. Lignocaine (2-20 mM) inhibits (in vitro) both brain and liver mitochondrial MAO activity, using tyramine, serotonin and benzylamine as substrates, in a concentration-dependent manner. Furthermore, lignocaine produces a marked in vitro inhibition of serotonin and tyramine oxidation in MAO-A and not in MAO-B preparation of rat brain. Ackermann-Potter plots of MAO indicate that lignocaine-induced inhibition of MAO activity is reversible in nature. Lineweaver-Burk plots show that lignocaine (2-10 mM) produces a significant increase in Km and decrease in Vmax of MAO for tyramine and serotonin in both brain and liver. Similarly Km and Vmax values are changed using benzylamine as substrate in the presence of relatively higher concentrations of lignocaine (5-20 mM). These results suggest that lignocaine-induced inhibition of mitochondrial membrane-bound MAO activity of both neuronal and non-neuronal tissues is associated with its conformational change.     

Concentration dependence of the oxidation of tyramine by the two forms of rat liver mitochondrial monoamine oxidase

The two forms of monoamine oxidase in rat liver mitochondria were shown to have different K_m and maximum velocity values with tyramine as the substrate. K_m values of 107±15 μM and 579±45μM were determined for the A- and B-form respectively at pH 7.2 in air-saturated buffer. The maximum velocity of the A-form was found to be approximately half of that of the B-form under these conditions. A consequence of these differences is that the ratio of activities of MAO-A:MAO-B determined from clorgyline inhibition curves will be dependent upon the concentration of tyramine used to assay for enzyme activity. As a fixed concentration of tyramine, the height of the plateau in a clorgyline inhibition curve will also be affected by the presence of a selective competitive inhibitor. Procaine, an MAO-A selective competitive inhibitor, was found to increase the K_m value of the MAO-A towards tyramine to 505 ± 172 μM and under these conditions the plateau-height of the clorgyline inhibition curve was not significantly affected by variations of the tyramine concentration over a 20-fold range.     

Species differences in the deamination of dopamine and other substrates for monoamine oxidase in brain

Cortex and caudate specimens from human, non-human primate and rodent brains were examined for their ability to deaminate dopamine and for their sensitivity to irreversible monoamine oxidase (MAO) inhibitors. Using inhibition curves obtained with clorgyline, deprenyl and pargyline to estimate the relative proportions of MAO-A and MAO-B activity, dopamine was found to be deaminated predominantly by MAO-A in rat cortex and caudate. In contrast, dopamine was primarily an MAO-B substrate in human and vervet cortex and caudate. When clorgyline inhibition curves with tyramine or dopamine as substrate were compared in human, vervet and rat cortex, more pronounced species differences were found with dopamine than with tyramine. In all three species caudate tended to be more sensitive to inhibition by low concentrations of clorgyline than was cortex, suggesting a higher proportion of MAO-A activity in caudate. Similar species differences were also found when MAO-A activities were estimated using serotonin (5-HT): -phenylethylamine (PEA) ratios (5-HT/5-HT + PEA). These ratios with selective substrates were highly correlated with clorgyline inhibition curves obtained with tyramine as substrate across 29 brain regions and tissues from different rodent and primate species (r=0.85, P<0.001). Data from both the substrate ratios and the clorgyline inhibition curves confirmed the relative predominance of MAO-B activity in primate brain regions (70–85%) as compared to rat brain regions (45%). Smaller species differences were observed in liver. Species differences in the proportion of brain MAO-A and B activities and in the deamination of dopamine and other substrates for MAO may have important implications in regard to the widespread use of rodent rather than primate models in the study of biogenic amine metabolism and of drugs affecting amine function.     

Substrate- and inhibitor-related characteristics of human platelet monoamine oxidase

Human platelet monoamine oxidase (MAO) preferentially deaminated benzylamine and phenylethylamine, two substrates relatively specific for type B MAO, in comparison to 5-hydroxytryptamine, a substrate specific for type A MAO. In studies comparing human platelet and rat brain MAO specific activities, benzylamine and 5-hydroxytryptamine deamination by platelets was approximately 90 and 2 per cent, respectively, that of brain, while platelet deamination of dopamine, tryptamine and tyramine was 20 per cent or less than that of brain. Among sixteen drugs studied, platelet MAO activity was selectively inhibited by low concentrations of the MAO-B inhibitors, deprenyl and pargyline, and was relatively insensitive to the MAO-A inhibitors, clorgyline and Lilly 51641. These observations, in addition to the simple sigmoid inhibition curves obtained with increasing concentrations of either clorgyline or deprenyl, suggest that platelet MAO consists of essentially one distinguishable form of MAO which most closely resembles the MAO type B found in other tissues.     

High-throughput screening for monoamine oxidase-A and monoamine oxidase-B inhibitors using one-step fluorescence assay

AIM: To develop high-throughput screening (HTS) assays for monoamine oxidase (MAO)-A and MAO-B inhibitors. METHODS: A fluorescence probe based method measuring MAO-A and MAO-B activity was established and optimized, with its sensitivity, stability and specificity evaluated. Reaction conditions including enzyme sources, substrate concentrations, incubation volume and reaction time in 384-well format were optimized to achieve sensitive and low consumptive goal. RESULTS: In optimized conditions, dynamic parameters of MAO-A and MAO-B were obtained. The K(m) value of serotonin to MAO-A was 1.66 micromol/L, while that of benzylamine to MAO-B was 0.80 micromol/L. The IC(50) value of clorgyline to MAO-A was 2.99 nmol/L, and that of deprenyl to MAO-B was 7.04 nmol/L, matching those obtained from traditional spectrometric assays. Among tested samples, one compound exerted an inhibitory effect on MAO-A activity with IC(50) as 0.36 micromol/L, and three compounds had an inhibitory effect on MAO-B activity with IC(50) as 0.13, 0.19, and 0.13 micromol/L. The Z' factor was 0.71+/-0.03 and 0.75+/-0.03 in MAO-A-inhibitor and MAO-B-inhibitor HTS system, respectively. CONCLUSION: The established assays can be well applied to MAO-A and MAO-B inhibitor screening with high quality, precision and reproducibility.     

Observations on the inhibition of rat liver monoamine oxidase by clorgyline

Two forms of monoamine oxidase (MAO) denned as MAO A and B by others differ in their specificities to substrates and their sensitivities to the irreversible inhibitor clorgyline. From studies using the substrates 5-HT, tyramine and benzylamine, the presence of both MAO forms in rat liver mitochondria has been confirmed and some characteristics of their inhibition by varying concentrations of clorgyline investigated. Although both MAO forms showed time-dependent inhibition, this process occurred, in general, at a qualitatively slower rate for MAO B, despite the fact that this enzyme form requires higher concentrations of clorgyline than MAO A for inhibition of its activity. However, factors such as the concentration of enzyme, the concentration of clorgyline and the enzyme: drug ratio employed in the assay all influence the resultant time-course and the final degree of the inhibition observed. The possible importance of the lipid environment of the outer mitochondrial membrane in generating multiple MAO forms and in regulating the inhibition kinetics of these forms is discussed. The results indicate that the effects of pre-incubation time and the enzyme: drug ratio on inhibition of MAO by clorgyline should be fully recognized when using the drug to indicate multiple forms in animal tissues.     

Enzymic and molecular characteristics of a new form of monoamine oxidase, distinct from form-A and form-B

The present study was undertaken to clarify the enzymic and molecular properties of monoamine oxidase (MAO) in carp brain. In particular, its sensitivities to selective MAO inhibitors, kinetic properties and molecular weight were compared with those of the enzyme in carp liver. The selective and potent MAO-A and MAO-B inhibitors FLA 788(+), FLA 336(+), MD 780236 and benzylcyanide caused dose-dependent inhibitions of MAO activity in both carp brain and liver; the inhibition curves were all single-sigmoidal, and the degrees of inhibition of the activities towards 5-hydroxytryptamine (5-HT, selective MAO-A substrate), tyramine (substrate for both forms of MAO) and beta-phenylethylamine (PEA, selective MAO-B substrate) were similar. This was also the case for inhibition of activity in carp brain by the irreversible and selective MAO-A and MAO-B inhibitors clorgyline and I-deprenyl, indicating the presence in both preparations of a single MAO which differs from either form of MAO. Studies on the substrate specificities and Km values for these three substrates and the inhibitory effects of some compounds suggested that the enzymic characters of MAO in carp preparations were similar and that these enzymes might be FAD-containing enzymes, like MAO in various mammals. By labelling the preparations with radioactive pargyline and then subjecting them to sodium dodecyl sulfate electrophoresis, the apparent molecular weights of carp brain and liver MAO were estimated as 60,000 daltons. The same value was also obtained for rat brain and liver mitochondrial MAO-B. These results indicate that by the present definitions of MAO-A and MAO-B, MAO in carp brain and liver is similar to, but distinct from, both these forms of MAO.     

Drug-induced changes in motor activity after selective MAO inhibition

The increase in motor activity produced in mice by phenylethylamine (PEA), L-DOPA and amphetamine was evaluated after selective inhibition of MAO Type A (by clorgyline) or Type B (by low doses of pargyline). PEA-induced motor stimulation was intensified in the presence of MAO-B inhibition, but not when MAO-A was inhibited. This was paralleled by higher concentrations of brain and plasma PEA (after injection) in mice in which there was inhibition of MAO-B compared with control or MAO-A inhibition. Conversely, L-DOPA produced significant stimulation only when MAO-A was inhibited. The clorgyline pretreatment resulted in larger increases in brain dopamine concentrations (in the striatum, olfactory tubercles and in the area containing the substantia nigra) than did MAO-B inhibition. This effect occurred both in mice receiving L-DOPA + inhibitor and in mice receiving the inhibitor alone. Amphetamine-induced stimulation was increased following the inhibition of either form of MAO, and this was not the result of changes in the distribution or metabolism of amphetamine. These results support the concept that MAO-A and MAO-B deaminate different substrates in the rodent CNS and that amphetamine may utilize either dopamine or PEA in producing its stimulant effects.     

Selectivity of clorgyline and pargyline as inhibitors of monoamine oxidases A and B in vivo in man

During 4 weeks of treatment with clorgyline, a selective MAO-A inhibitor, platelet monoamine oxidase (MAO) activity was unchanged. During a similar 4-week crossover treatment period with pargyline, a selective MAO-B inhibitor, platelet MAO activity was essentially completely inhibited in the same individuals. The differential effects of the two drugs on platelet MAO, which consists exclusively of the MAO-B form, suggests that the in vitro selectivity of clorgyline, and possibly of pargyline, on MAO-A and MAO-B may be maintained in vivo during long-term administration in man. Reductions in blood pressure, heart rate, and plasma amine oxidase activity were generally similar in magnitude during treatment with both drugs, however, suggesting that either these effects are nonspecific consequences of both MAO-A and MAO-B inhibition, or that pargyline also inhibited MAO-A activity.     

Differential effects of clorgyline on catecholamine and indoleamine metabolites in the cerebrospinal fluid of rhesus monkeys

The effects of acute and chronic administration of clorgyline, an irreversible inhibitor of monoamine oxidase type A (MAO-A), on the deaminated metabolites of norepinephrine, dopamine and serotonin were examined in rhesus monkey cerebrospinal fluid (CSF). Acute clorgyline treatment resulted in highly significant, dose-dependent reductions in 3-methoxy-4-hydroxyphenylglycol (MHPG) of 50% (1 mg/kg) and 68% (2 mg/kg) compared to pretreatment values. Chronic clorgyline administration (0.25 to 0.5 mg/kg X 24 days) resulted in a 67% reduction in CSF MHPG. In contrast, the concentrations of 5-hydroxyindoleacetic acid (5-HIAA) and homovanillic acid (HVA) were less affected by acute clorgyline administration, being reduced significantly only after the 2 mg/kg dose, which lowered 5-HIAA 27% and HVA 48%. Chronic clorgyline treatment had no significant effect on the CSF concentrations of HVA and 5-HIAA. These data, which suggest that MAO-A inhibition by clorgyline in vivo is more closely associated with changes in the noradrenergic than the serotonergic or dopaminergic systems in nonhuman primates, are in general agreement with the effects of clorgyline on CSF and urinary biogenic amine metabolites in man. They differ from several in vitro studies which indicate a primary role of MAO-A in the metabolism of serotonin and of MAO-B in norepinephrine degradation in primate brain. The discrepancies may reflect modulating effects of synaptic feedback mechanisms on the actions of clorgyline in vivo or perhaps a failure of CSF metabolites to adequately reflect brain amine metabolism changes. The lack of change in platelet MAO-B activity during clorgyline treatment together with the minimal changes in HVA concentrations indicate that the selective inhibitory effects of clorgyline on MAO-A were maintained during chronic administration of low drug doses.     

A monoamine oxidase-B inhibitor, MD 780236, metabolized essentially by the A form of the enzyme in the rat

In-vivo studies on the metabolism of [14C]MD 780236 a short-acting selective type B MAO inhibitor in the rat showed the acid to be the major metabolite in plasma and urine, whereas it was minor in brain, where the alcohol was the major metabolite. Pretreatment with SKF 525-A did not modify the metabolite profile in brain, but benserazide decreased the alcohol. Pretreatment with (-)-selegiline had no effect, but clorgyline or clorgyline with (-)-selegiline significantly decreased the alcohol and increased the primary amine metabolite in brain. In-vivo results suggest that MAO-A is the enzyme responsible for the metabolism of MD 780236. This was confirmed by in-vitro studies. Rat brain homogenates extensively metabolized the drug, with the aldehyde being the major metabolite formed (28% of the total radioactivity in the incubation mixture after 60 min incubation). The acid (12%) was more important than the alcohol (4%) in-vitro. The addition of all metabolites originating from possible MAO activity gave 46% when the incubation was carried out at pH 7.4 and 82% at pH 8.8. The presence of NADPH or NAD+ did not alter the relative amounts of metabolites formed. Total metabolites originating from MAO activity in the presence of (-)-selegiline accounted for 40% of total radioactivity, whereas in the presence of clorgyline they accounted for 8% and in the presence of both clorgyline and (-)-selegiline they were reduced to 3%, compared with 45% in controls. As a further proof of the importance of MAO-A in the metabolism of MD 780236, rats were pretreated with clorgyline 1 h before the drug and MAO-B inhibition measured at different times ex-vivo in brain and liver. The short-lasting phase of inhibition of MAO-B disappeared after pretreatment with clorgyline, and inhibition at 24 h was as high as that at 1 h. These results demonstrate the importance of the A form of MAO for the metabolism of MD 780236.     

Selective influences of age and thyroid hormones on Type A monoamine oxidase of the rat heart

The specific activity of rat heart MAO, towards both tyramine and benzylamine as substrates, was found to increase with the age of the animal, and also after administration of (?)-thyroxine to young male rats. Conversely, enzyme activity was decreased in animals made hypothyroid by including 2-thiouracil in their diet. However, with both age and altered thyroid status, relatively greater changes in the deamination of tyramine rather than in that of benzylamine, were obtained. Clorgyline and deprenyl, used as inhibitors of rat heart MAO, indicated that tyramine is metabolized solely by MAO-A, whereas benzylamine is a substrate for both MAO-A and -B, and also a clorgyline- and deprenyl-resistant enzymic activity. The proportional contribution of MAO-A, -B and the clorgyline-resistant enzyme towards the total benzylamine deamination in the rat heart was found to vary with the age and with altered thyroid status of the animal in such a way that selective changes in the activity of MAO-A appear to be largely responsible for the overall changes in the specific activity of rat heart MAO which occur in response to these developmental factors.     

Selective influences of age and thyroid hormones on type A monoamine oxidase of the rat heart.

The specific actiivty of rat heart MAO, towards both tyramine and benzylamine as substrates, was found to increase with the age of the animal, and also after administration of (-)-thyroxine to young male rats. Conversely, enzyme activity was decreased in animals made hypothyroid by including 2-thiouracil in their diet. However, with both age and altered thyroid status, relatively greater changes in the deamination of tyramine rather than in that of benzylamine, were obtained. Clorgyline and deprenyl, used as inhibitors of rat heart MAO, indicated that tyramine is metabolized solely by MAO-A, whereas benzylamine is a substrate for both MAO-A and -B, and also a clorgyline- and deprenyl-resistant enzymic activity. The proportional contribution of MAO-A, -B and the clorgyline-resistant enzyme towards the total benzylamine deamination in the rat heart was found to vary with the age and with altered thyroid status of the animal in such a way that selective changes in the activity of MAO-A appear to be largely responsible for the overall changes in the specific activity of rat heart MAO which occur in response to these developmental factors.     

Inhibition of rat brain monoamine oxidase activity by cerebral anti-ischemic agent, ifenprodil

Ifenprodil, which is clinically used as a cerebral vasodilator, inhibited rat brain type A (MAO-A) and type B (MAO-B) monoamine oxidase activity. It did not, however, affect rat lung semicarbazide-sensitive amine oxidase. The degree of inhibition of either form of MAO was not changed by 30 min preincubation of the enzyme preparations at 37 degrees C with ifenprodil. Modes of inhibition of MAO-A and MAO-B by ifenprodil were competitive towards oxidation of their respective substrates, 5-hydroxytryptamine and benzylamine, with Ki values of 75 microM for inhibition of MAO-A and 110 microM for inhibition of MAO-B.     

Monoamine oxidase activities of porcine vascular endothelial and smooth muscle cells

Amine uptake by cultured vascular cells was studied under conditions minimizing nonenzymic oxidation. 5-Hydroxytryptamine (5HT) was accumulated only very poorly; detailed kinetic analysis couid not be performed, but there was no evidence for a saturable high affinity process. Comparison of β-phenylethylamine (PEA) and 5HT metabolism in intact cells and lysed cells demonstrated that the rates of entry of the amines into cells usually limited their metabolism especially at low (μM) concentrations. Primary cultures of aortic endothelial cells metabolised 5HT and PEA substantially faster than did subcultured endothelium. Subcultured aortic vascular smooth muscle cells and endothelial cells metabolised PEA and 5HT with comparable specific enzyme activities to those found in aortic medial tissue. Inhibition by clorgyline of PEA, 5HT and benzylamine (BZA) metabolism reveaied, however, that while aortic tissue possessed monoamine oxidase (MAO) types A and B and a comparable amount of a clorgyline resistant amine oxidase(s) (CRAO), cultured vascular cells possessed MAO-A, but little or no CRAO or MAO-B. Cultured venous endothelium, and smooth muscle from several vascular sites, metabolised PEA and 5HT at similar rates to those found in aortic cells. the studies demonstrate that although cultured porcine endothelial and smooth muscle cells from large blood vessels contain MAO, they do not apparently possess the amine transport process present in the lung. Additionally, conditions of culture can affect both the extent of amine metabolism and the pattern of amine oxidase present.