Methylamine metabolism to formaldehyde by vascular semicarbazide-sensitive amine oxidase.

The capacity of the vascular enzyme, semicarbazide-sensitive amine oxidase (SSAO), to metabolize methylamine to the potentially toxic product, formaldehyde, was tested using rat aortic homogenates and purified porcine aortic SSAO. Formaldehyde production in incubations of enzyme source with methylamine (1 mM) was detected by high performance liquid chromatography and product was confirmed by desorption chemical ionization mass spectrometry (DCI-MS). Inhibitor studies using the specific SSAO inhibitor semicarbazide and the monoamine oxidase inhibitor pargyline indicate that SSAO is responsible for metabolism of methylamine to formaldehyde. These results suggest the possibility that elevated methylamine found in several pathologic states (such as uremia and diabetes mellitus), or generated from exogenous sources, could result in overproduction of formaldehyde in tissues with high SSAO activity, especially blood vessels.     

Semicarbazide-sensitive amine oxidase from the smooth muscles of dog aorta and trachea: activation by the MAO-A inhibitor clorgyline.

Semicarbazide-sensitive amine oxidase (SSAO) has been identified in the dog trachea and aorta smooth muscles. The dog SSAO is blocked by hydrazine inhibitors. SSAOs from several different vascular smooth muscle sources, such as the rat and bovine aorta, and human umbilical artery, as well as the bovine plasma, are insensitive to the MAO-A inhibitor clorgyline; the dog SSAO on the other hand is significantly activated by clorgyline. Two methods, i.e. radioenzymatic and fluorometric methods, have been applied to substantiate this clorgyline-induced activation. The activation was detected with respect to the deamination of different substrates, such as benzylamine, beta-phenylethylamine and longer carbon chain aliphatic amines, but not with respect to methylamine. The clorgyline effect is reversible, non-competitive and time-independent; it depends on electrostatic and hydrophobic interactions between clorgyline and hydrophobic regions of the dog SSAO enzyme.     

2-bromoethylamine, a suicide inhibitor of semicarbazide-sensitive amine oxidase, increases hydralazine hypotension in rats

Previous work has shown that inhibitors of the predominantly vascular enzyme semicarbazide-sensitive amine oxidase (SSAO) potentiate the hypotensive response to hydralazine, itself a SSAO inhibitor, in anesthetized rats. The present study was carried out to determine whether the recently described suicide SSAO inhibitor 2-bromoethylamine shares this effect. Hypotensive responses to hydralazine, 0.1 mg/kg IV, were obtained in chloralose-urethane-anesthetized rats, either unpretreated or receiving bromoethylamine at different doses and pretreatment intervals. Parallel experiments were run with semicarbazide, the prototypical hydrazine SSAO inhibitor. Both inhibitors potentiated hydralazine hypotension, bromoethylamine having a longer latency and a shorter duration of action than semicarbazide. High doses of bromoethylamine did not produce potentiation, a phenomenon attributed to SSAO inactivation by excess substrate and decreased formation by the enzyme of the inhibitor product. Experiments with combined administration of both inhibitors were also carried out. When semicarbazide was administered before bromoethylamine, potentiaton was prevented, apparently by a mechanism similar to the above; when it was given after the amine, potentiation was increased. This was attributed to enzyme inhibition by interaction with 2 different active sites. The charactertistics of hydralazine potentiation by bromoethylamine were considered compatible with the mechanism of SSAO inhibition by the amine.     

Inhibition of a type B monoamine oxidase inhibitor, (E)-2-(4-fluorophenethyl)-3-fluoroallylamine (MDL-72974A), on semicarbazide-sensitive amine oxidases isolated from vascular tissues and sera of different species.

(E)-2-(4-Fluorophenethyl)-3-fluoroallylamine hydrochloride (MDL-72974A) has been discovered recently to be a very potent and highly selective type B monoamine oxidase inhibitor. We have found that this inhibitor is also capable of inhibiting semicarbazide-sensitive amine oxidases (SSAOs) obtained from vascular tissues and sera of different species. The inhibition of SSAO by MDL-72974A was irreversible and time dependent. It was competitive without preincubation of the enzyme with the inhibitor and demonstrated a mixed-type of inhibition when the enzyme was preincubated with the inhibitor. The IC50 values were estimated to be 2 x 10(-9) M, 5 x 10(-9) M, 8 x 10(-8) M and 2 x 10(-8) M for SSAO from dog aorta, rat aorta, bovine aorta and human umbilical artery, respectively. SSAO obtained from bovine serum was relatively insensitive to MDL-72974A (IC50 = 3 x 10(-7) M. Following intraperitoneal administration of MDL-72974A, rat brain MAO-B was inhibited with the ED50 value being about 0.2 mg/kg. Rat aorta SSAO was also inhibited and to a similar extent by the same dose. MDL-72974A is the most potent SSAO inhibitor that has been described thus far.     

Studies of monoamine oxidase and semicarbazide-sensitive amine oxidase. I. Inhibition by a selective monoamine oxidase-B inhibitor, MD 780236

In vitro studies of the effect of MD 780236, a selective monoamine oxidase (MAO)-B inhibitor, on a semicarbazide-sensitive amine oxidase (SSAO) in rat testis and lung showed that this compound dose-dependently inhibited SSAO activity. The extents of inhibition of MAO-A, -B and SSAO in these two rat tissues by this compound after 30 min of preincubation were found to be MAO-B greater than MAO-A greater than SSAO. This selectivity was also evident in preparations without preincubation. Degree of inhibition of SSAO was not significantly influenced by pretreatment with either 10(-3) M clorgyline, I-deprenyl or 10(-4) M SKF 525A. Inhibition of SSAO was not enhanced by varying the time of preincubation of the enzyme and the compound, indicating direct action on and reversible inhibition of SSAO. The inhibition of SSAO by MD 780236 was non-competitive with or without preincubation, with a K1 value of 110 muM. Although MD 780236 is a selective and "suicide substrate" inhibitor of MAO-B, these present results indicate that this compound may also inhibit SSAO activity, but by a mechanism different from that for MAO-B. These findings confirm an earlier hypothesis that compounds that inhibit both MAO and SSAO have totally different modes of action on these two different amine oxidases.     

Prolonged treatment with aminoguanidine strongly inhibits adipocyte semicarbazide-sensitive amine oxidase and slightly reduces fat deposition in obese Zucker rats.

Beneficial effects of aminoguanidine (AG) on diabetic vascular complications result from prevention of protein glycation, inhibition of inductible NO synthase, and inhibition of vascular semicarbazide-sensitive amine oxidase (SSAO). However, influence of AG on adipose tissue deposition has been poorly investigated in obesity. Considering that SSAO is highly expressed in fat cells, and that a SSAO blocker has been recently reported to reduce body weight gain in obese mice, this work aimed to investigate the influence of AG on adipose tissue functions. First, AG was shown to directly inhibit SSAO activity in cultured adipocytes. Although AG did not directly alter lipolytic activity in human adipocytes, it inhibited benzylamine-induced antilipolysis via SSAO (but not NO synthase) inhibition. When AG was i.p. administered to obese Zucker rats (270 micromol kg(-1)day(-1) for 3 weeks), treated rats lost their capacity to oxidize benzylamine in a SSAO-dependent manner in adipose tissues and in cerebral vessels. Monoamine oxidase activity was unmodified in liver, skeletal muscles or adipose tissues and tended to increase in brain vessels. AG-treatment did not change body weight gain or hyperinsulinemic state of obese rats but slightly reduced subcutaneous fat deposition. AG did not modify insulin responsiveness in adipocytes but impaired the effects of SSAO substrates, such as glucose transport activation and lipolysis inhibition by methylamine or benzylamine plus vanadate. These results show that complete impairment of SSAO activity produced by AG-treatment in obese rats was likely responsible for a weak limitation of fat deposition. Previously proposed for prophylaxis in diabetes, AG may be useful for treating obesity via its SSAO blocking properties.     

Characterization of semicarbazide-sensitive amine oxidase in human subcutaneous adipocytes and search for novel functions

Numerous studies have characterized semicarbazide-sensitive amine oxidase activity (SSAO) in rat fat cells but this oxidase is scarcely documented in human adipose tissue. Our aim was to further characterize SSAO in human adipose tissue (activity, mRNA and protein abundance) and to investigate whether SSAO activity can interplay with glucose and lipid metabolism in human adipocytes via the hydrogen peroxide it generates. Polyclonal antibodies directed against bovine lung SSAO allowed the detection of a substantial amount of immunoreactive protein (apparent molecular mass 100 kDa) in human subcutaneous adipocytes from either mammary or abdominal fat depots. A 4-kb mRNA was detected in fat depots using a cDNA probe designed from the placenta SSAO sequence. Almost all the oxidation of benzylamine found in adipose tissue homogenates was due to fat cells and was located in the adipocyte membrane fraction. The oxidation of benzylamine and methylamine were similar and totally inhibited by semicarbazide or hydralazine but resistant to pargyline. Histamine was poorly oxidized. Benzylamine and methylamine dose-dependently stimulated glucose transport in intact adipocytes. This insulin-like effect of amines did not increase in the presence of 0.1 mM vanadate but was inhibited by semicarbazide and antioxidants. Benzylamine and methylamine also exhibited antilipolytic effects, with complete inhibition of lipolysis at 1 mM. These results show that fat cells from non-obese subjects express a membrane-bound SSAO which readily oxidizes exogenous amines, generates hydrogen peroxide and exerts short-term insulin-like actions on glucose and lipid metabolism.     

An allylamine derivative (MDL 72145) with potent irreversible inhibitory actions on rat aorta semicarbazide-sensitive amine oxidase

(E)-2-(3,4-dimethoxyphenyl)-3-fluoroallylamine (MDL 72145) was found to be an extremely potent inhibitor of the semicarbazide-sensitive amine oxidase (SSAO) in rat aorta homogenates. Considerable inhibition, which was not reversed by dialysis, could be produced under appropriate in-vitro conditions at drug concentrations around 10 nM. The pseudo first order kinetics for time-dependent inhibition by MDL 72145 (10-100 nM) were found to be consistent with a bimolecular reaction between enzyme and inhibitor with a rate constant for this step of 2 X 10(6) min-1 M-1. A similar rate of inhibition under an oxygen atmosphere to that obtained under nitrogen was produced upon incubation of enzyme with inhibitor, suggesting that oxidation of the inhibitor to an active metabolite was not required for its activity. Incubation of homogenates for very short periods (1 min) with inhibitor (0.05-0.5 microM) and benzylamine (1-10 microM) as substrate indicated non-competitive kinetics for the early interaction of enzyme with the drug. Benzylamine (50 microM), but not pyridoxal phosphate (100 microM), was able to protect SSAO from inhibition by 10 nM MDL 72145. However, pyridoxal phosphate alone appeared to produce some irreversible inhibition of the enzyme. Dialysis against buffer containing 50 microM or 1 mM benzylamine was unable to reactivate SSAO inhibited by 10 nM MDL 72145. It is concluded that MDL 72145 irreversibly inhibits SSAO by acting at, or near, the substrate binding site, but the exact nature of the complex formed remains to be identified.     

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)     

Various mammalian tissues contain cell membrane-bound amine oxidase termed semicarbazide-sensitive amine oxidase (SSAO,EC 1.4.3.6)

Recently it has been demonstrated a role of fat-cell SSAO on glucose transport and GLUT4 translocation to the cell surface. Many compounds have been identified as relatively selective SSAO inhibitors, but those currently available also inhibit monoamine oxidase (MAO, EC 1.4.3.4). In this study, inhibitory properties of a haloamine, 2-bromoethylamine (2-BEA), to guinea pig and rat lung-bound SSAO have been studied. 2-BEA could not inhibit both forms of MAO, but it competitively inhibited rat lung SSAO activity with a Ki value of 2.5 microM without preincubation and after preincubation, the mode of inhibition changed to be non-competitive. Dialysis and dilution experiments with 2-BEA-pretreated preparations resulted in no recovery of SSAO activity. A decreased rate of SSAO inhibition under N2 atmosphere to that obtained under O2 was produced after 2-BEA treatment, suggesting that oxidised intermediate was necessary for its inhibition. The plot of 1/k' vs 1/2-BEA intersected on the y-axis indicate that the inhibition by 2-BEA is not affinity-labeling agent, but a suicide inhibitor of SSAO.     

Developmental vasculotoxicity associated with inhibition of semicarbazide-sensitive amine oxidase

The endogenous substrate(s) and physiological function(s) of semicarbazide-sensitive amine oxidase (SSAO), a group of enzymes exhibiting highest activity in vascular smooth muscle cells of the mammalian aortic wall, remain undetermined. This study examines the pathophysiological effects in the thoracic aortic wall resulting from specific in vivo SSAO inhibition. Weanling Sprague-Dawley rats were treated acutely or chronically with either semicarbazide hydrochloride or the allylamine derivatives MDL-72274 or MDL-72145 (Marion Merrell Dow Research Institute, Cincinnati, OH). Treatment with these compounds produced acute (6 and 24 h) and chronic (21 day) lowering of SSAO activity in aorta and lung with little effect on the activity of the vital matrix-forming enzyme, lysyl oxidase, in aortas of chronically treated animals. Chronic SSAO inhibition produced lesions consisting of striking disorganization of elastin architecture within the aortic media accompanied by degenerative medial changes and metaplastic changes in vascular smooth muscle cells. No significant difference in the total weight of dry, lipid-extracted aortic elastin and collagen components were observed between chronically SSAO inhibited and control animals. However, the amount of mature elastin was lowered and mature collagen was raised in the aortas of animals treated chronically with semicarbazide. Descending thoracic aortic rings isolated from chronically SSAO-inhibited animals had larger cross-sectional diameters (i.e., exhibited dilation) when compared to corresponding rings from control animals. This study demonstrates that developmental toxicity, characterized by striking vascular lesions and dilated thoracic aortas, can result from specific in vivo SSAO inhibition, suggesting a role for SSAO in connective tissue matrix development and maintenance, and specifically in the development of normal elastin.     

Semicarbazide-sensitive amine oxidase: role in the vasculature and vasodilation after in situ inhibition

1. The characteristics of semicarbazide-sensitive amine oxidase (SSAO) are reviewed and the unknown physiological or pathological role of this enzyme emphasized. 2. The various mechanisms of action proposed for the vasodilator drug hydralazine are considered. In particular, the inhibitory action on various enzymes, related or not to cardiovascular function, are discussed. 3. Studies linking inhibition of SSAO to hydralazine hypotension are reviewed and a general hypothesis relating both actions is presented. The hypothesis postulates that (a). vascular SSAO is involved in the regulation of vascular tone, and (b). hydralazine vasodilation is the consequence of vascular SSAO inhibition. 4. Evidence supporting these postulates is presented and vascular SSAO inhibition is proposed as a novel mechanism of vasodilation.     

Acrolein generation stimulates hypercontraction in isolated human blood vessels

Increased risk of vasospasm, a spontaneous hyperconstriction, is associated with atherosclerosis, cigarette smoking, and hypertension-all conditions involving oxidative stress, lipid peroxidation, and inflammation. To test the role of the lipid peroxidation- and inflammation-derived aldehyde, acrolein, in human vasospasm, we developed an ex vivo model using human coronary artery bypass graft (CABG) blood vessels and a demonstrated acrolein precursor, allylamine. Allylamine induces hypercontraction in isolated rat coronary artery in a semicarbazide-sensitive amine oxidase activity (SSAO) dependent manner. Isolated human CABG blood vessels (internal mammary artery, radial artery, saphenous vein) were used to determine: (1) vessel responses and sensitivity to acrolein, allylamine, and H(2)O(2) exposure (1 microM-1 mM), (2) SSAO dependence of allylamine-induced effects using SSAO inhibitors (semicarbazide, 1 mM; MDL 72274-E, active isomer; MDL 72274-Z, inactive isomer; 100 microM), (3) the vasoactive effects of two other SSAO amine substrates, benzylamine and methylamine, and (4) the contribution of extracellular Ca(2+) to hypercontraction. Acrolein or allylamine but not H(2)O(2), benzylamine, or methylamine stimulated spontaneous and pharmacologically intractable hypercontraction in CABG blood vessels that was similar to clinical vasospasm. Allylamine-induced hypercontraction and blood vessel SSAO activity were abolished by pretreatment with semicarbazide or MDL 72274-E but not by MDL 72274-Z. Allylamine-induced hypercontraction also was significantly attenuated in Ca(2+)-free buffer. In isolated aorta of spontaneously hypertensive rat, allylamine-induced an SSAO-dependent contraction and enhanced norepinephrine sensitivity but not in Sprague-Dawley rat aorta. We conclude that acrolein generation in the blood vessel wall increases human susceptibility to vasospasm, an event that is enhanced in hypertension.     

Inhibition of human mitochondrial aldehyde dehydrogenase by 4-hydroxynon-2-enal and 4-oxonon-2-enal

Previous studies found the lipid peroxidation product 4-hydroxynon-2-enal (4HNE) to be both a substrate and an inhibitor of mitochondrial aldehyde dehydrogenase (ALDH2). Inhibition of the enzyme by 4HNE was demonstrated kinetically to be reversible at low micromolar aldehyde but may involve covalent modification at higher concentrations. Structurally analogous to 4HNE is the lipid peroxidation product 4-oxonon-2-enal (4ONE), which is more reactive than 4HNE toward protein nucleophiles. The goal of this work was to determine whether 4ONE is a substrate or inhibitor of human ALDH2 (hALDH2) and elucidate the mechanism of enzyme inhibition by 4HNE and 4ONE. Both 4ONE and its glutathione conjugate were found to be substrates for the enzyme in the presence of NAD. At low concentrations of 4ONE (< or = 10 microM), hALDH2 catalyzed the oxidation of 4ONE to 4-oxonon-2-enoic acid (4ONEA) with a maximal yield of 5.2 mol 4ONEA produced per mol of enzyme (monomer). However, subsequent analysis of hALDH2 activity toward propionaldehyde revealed that both 4ONE and the oxidation product, 4ONEA, were potent, irreversible inhibitors of the enzyme. In contrast, inhibition of hALDH2 by a high concentration of 4HNE (i.e., 50 microM) was primarily reversible. The reactivity of 4ONEA toward glutathione was measured and found to be comparable to that of 4HNE, indicating that the 4ONE-oxidation product is a reactive electrophile. hALDH2/NAD was incubated with 4HNE, 4ONE, and 4ONEA, and mass spectral analysis of tryptic peptides revealed covalent modification of an hALDH2 active site peptide by both 4ONE and 4ONEA. These data demonstrate that hALDH2 catalyzes the oxidation of 4ONE to 4ONEA; however, the product 4ONEA is a reactive electrophile. Furthermore, both 4ONE and 4ONEA are potent, irreversible inhibitors of the enzyme.     

甲胺对兔心血管内皮的慢性毒性(英文)

目的探讨甲胺在体内慢性分布是否导致氨基脲敏感胺氧化酶(SSAO)活性上调从而诱导心血管内皮损伤。方法新西兰兔灌胃给予甲胺100mg.kg-1,每日1次,连续6个月,隔周称量体重调整给药量。分析动脉血中循环内皮细胞数、血清NO浓度和主动脉内皮细胞的超微结构。高效液相色谱法检测血浆SSAO活性及甲醛浓度。结果甲胺组兔动脉血中内皮细胞数、血清NO浓度、血浆SSAO活性及甲醛浓度明显增高。主动脉内皮细胞超微结构显示核内大量包函体、核浓缩和核分裂等病理形态学改变。结论低剂量甲胺在体内慢性分布能够诱导SSAO活性上调而引起心血管内皮损伤。     

Contribution of serum and cellular semicarbazide-sensitive amine oxidase to amine metabolism and cardiovascular toxicity.

Semicarbazide-sensitive amine oxidase (SSAO) plays a role in the in vivo and in vitro toxicity of several environmental and endogenous amines. We investigated the role of SSAO as a component of cell culture medium (through addition of fetal calf serum (FCS)) compared to intracellular SSAO in the in vitro cytotoxicity of three amines and metabolites. Smooth muscle cells and beating cardiac myocytes were grown in 96-well plates and exposed to various concentrations and combinations of FCS in medium, amines (allylamine, AA; benzylamine, BZA; and methylamine, MA), and amine metabolites (aldehydes: acrolein, benzaldehyde, and formaldehyde; hydrogen peroxide, H2O2; ammonia, NH3). Amine and amine metabolite cytotoxicity was quantified by monitoring cell viability. SSAO activity was measured in FCS, cardiovascular cells, or rat plasma by a radioenzymatic assay using [14C]BZA. Our data show that AA and its aldehyde metabolite, acrolein, were the most toxic compounds to both cell types. However, AA toxicity was FCS-dependent in both cell types, while BZA, MA, and amine metabolite (i.e., aldehydes, H2O2, and NH3) cytotoxicity showed little FCS dependence. In these experiments, medium containing 10% FCS had a calculated amine metabolic capacity that was 30- to 50-fold that of the cultured smooth muscle cellular content in a single well of a 96-well plate. Our study demonstrates that SSAO in FCS contributes to amine metabolism and cytotoxicity to rat cardiovascular cells in vitro and how critical it is to evaluate serum for its role in mechanisms of amine toxicity in vitro and in vivo.     

2-Bromoethylamine, a suicide inhibitor of tissue-bound semicarbazide-sensitive amine oxidase

Various mammalian tissues contain plasma membrane-bound amine oxidase, termed semicarbazide-sensitive amine oxidase (SSAO). In the present study, 2-bromoethylamine has been studied with regard to inhibitory properties towards tissue-bound SSAO in rat lung. Without preincubation, 2-bromoethylamine was a competitive and reversible SSAO inhibitor with a Ki value of 2.5 microM. After preincubation, it time-dependently and non-competitively inhibited SSAO activity, probably by forming the covalently-bound enzyme-inhibitor adduct. The data presented suggest that 2-bromoethylamine may act as a suicide inhibitor of SSAO.     

The kinetics of monoamine oxidase inhibition by three 2-indolylmethylamine derivatives

The inhibition of bovine brain mitochondrial MAO-A and MAO-B by three acetylenic and non-acetylenic derivatives of 2-indolylmethylamine, chosen among more than 100 new compounds, were studied. The non-acetylenic derivative N-methyl-2(5-hydroxy-1-methylindolyl)methylamine (1) was a weak non-selective inhibitor which was shown to act in a reversible and competitive manner towards the deamination of tyramine. The two acetylenic derivatives N-methyl-N-(2-propynyl)-2-(5-benzyloxy-1-methylindolyl)methylamine (2) and N-methyl-N-(2-propynyl)-2-(5-hydroxy-1-methylindolyl)methylamine (3) were potent MAO inhibitors, one of them non-selective (compound 2) and the other MAO-A selective inhibitor (compound 3). Both of them were irreversible and competitive inhibitors, compound 2 towards the deamination of tyramine and compound 3 towards the deamination of serotonin and beta-phenylethylamine. A mechanism for the inhibition of the enzyme by both irreversible inhibitors is proposed and the inhibition parameters are determined.     

Inhibition of monoamine oxidase A-form and semicarbazide-sensitive amine oxidase by selective and reversible monoamine oxidase-A inhibitors, amiflamine and FLA 788(+)

In vitro studies demonstrated that two selective monoamine oxidase (MAO)-A inhibitors, amiflamine and FLA 788(+), have been shown to inhibit semicarbazide-sensitive amine oxidase (SSAO) in rat testis and lung homogenates in a concentration-dependent way. The inhibition was not greatly influenced by pretreatment of the preparations with either clorgyline (10(-3) mol/l), l-deprenyl (10(-3) mol/l) or SKF 525A (10(-4) mol/l). The two compounds showed a time-dependent inhibition of SSAO, and for the initial phase of the inhibition, amiflamine is a competitive inhibitor with a Kislope of 135 mumol/l, but FLA 788(+) is a noncompetitive inhibitor with a Ki value of 180 mumol/l. After preincubation for 60 min at 37 degrees C, however, inhibition by amiflamine was found to be essentially irreversible whereas that produced by FLA 788(+) was still noncompetitive and reversible. These two compounds also reversibly and competitively inhibited rat testis MAO-A with FLA 788(+) being much more selective towards this MAO (Kislope = 0.26 mumol/l for FLA 788(+) and 7 mumol/l for amiflamine, respectively). The present results indicate that both MAO-A-selective inhibitors also inhibit SSAO in vitro, but their properties as SSAO inhibitors differ from those as MAO-A inhibitors.     

Further evidence for suicide inhibition of semicarbazide-sensitive amine oxidase in guinea pig lung by 2-bromoethylamine

The inhibitory effects of 2-bromoethylamine (2-BEA), a derivative of ethylamine, on guinea pig lung semicarbazide-sensitive amine oxidase (SAO) have been studied. Preincubation with 2-BEA time-dependently inhibited SSAO activity. The mode of the initial phase of inhibition was competitive, with a Ki value of 52 microM. After preincubation at 37 degrees C for 2 h, the inhibition was noncompetitive and irreversible, as there was no recovery of SSAO activity by dilution of the inhibited samples. Kinetic analyses confirmed previous results with rat lung SSAO that 2-BEA is a suicide SSAO inactivator with a dissociation constant of 42 microM. This latter value is similar to that of the Ki value (52 microM) for the reversible phase of inhibition by 2-BEA. Addition of the nucleophilic compound 2-mercaptoethanol could not reduce the SSAO inhibition, indicating that inactivation could not be prevented by trapping the enzymatic reaction product from 2-BEA. This finding clearly indicates that the reaction product should not diffuse away from its site of genesis and agrees with one of the characteristics of suicide inhibitors. This conclusively excludes the possibility of an affinity-labeling mechanism.