Semicarbazide-sensitive Amine Oxidase Inhibitor Research Articles

Effect of benzylamine and its metabolites on the responses of the isolated perfused mesenteric arterial bed of the rat

1. Semicarbazide-sensitive amine oxidase (SSAO) is an enzyme activity which can be found in the plasma membrane of rat vascular smooth muscle cells. We have investigated the possibility that the products of deamination by this enzyme, namely ammonia, hydrogen peroxide and the aldehyde, may be important in the modulation of the responses of vascular smooth muscle to extracellular stimuli. 2. The isolated perfused mesenteric arterial bed of the rat was used and dose-pressure response curves (DRC) to bolus injections of adrenaline (Ad) or ATP were plotted by non-linear curve fitting. The relaxant effects of carbachol (CCh), which releases endothelium dependent relaxing factor (ERDF), were studied by co-administering CCh with Ad. The effects of including the preferred SSAO substrate, benzylamine (BZ; 25 microM), in the perfusion fluid throughout the experiment and of inhibition of SSAO by treatment of rats with (E)-2-(3',4'-dimethoxyphenyl)-3-fluoroallylamine (MDL 72145; 1 mg kg-1) 1 h before dissection, have been studied. 3. Neither BZ nor SSAO inhibition affected the DRC to ATP. BZ shifted Ad responses to the left, inhibition of SSAO increased this shift indicating that the amine, but not its metabolites, were responsible for the potentiation of the responses to Ad. DRC to CCh showed a shift to the left and a significant decrease in the Hill slope with BZ, indicative of a potentiation of low doses of CCh more than high doses. Inhibition of SSAO prevented this change and so the metabolites of BZ deamination appeared to be involved in the potentiation. 4. Ammonia generated by SSAO may contribute to the production of EDRF or hydrogen peroxide may sensitize guanylate cyclase to stimulation by EDRF and so explain these findings.

The influence of amine metabolizing enzymes on the pharmacology of tyramine in the isolated perfused mesenteric arterial bed of the rat.

1. The pressor response to the infusion of tyramine (Tyr) into the isolated perfused mesenteric arterial bed of the rat has been studied at both a low and a high dose (0.2 and 2.0 mumol) and the effect of monoamine oxidase-A (MAO-A) and semicarbazide-sensitive amine oxidase (SSAO) inhibition was examined. Very little MAO-B activity is found in homogenates of this tissue when Tyr is used as substrate. 2. Inhibition of SSAO by treating rats with 1 mg kg-1 (E)-2-(3',4'-dimethoxyphenyl)-3-fluoroally lamine (MDL 72145) 1 h before dissection, had no significant effect on the maximum pressure attained or the area under the curve (AUC) of the response to both low and high doses of Tyr. Inhibition of MAO-A, by inclusion of 10 microM clorgyline in the perfusing fluid, resulted in no significant potentiation at both low or high doses of Tyr. The inhibition of both these enzymes together substantially increased the AUC of the pressor response. 3. Cocaine (3 microM) significantly potentiated the responses to adrenaline (Ad). At this dose, cocaine significantly reduced the peak height and the AUC of the responses to both doses of Tyr. 4. Inhibition of extraneuronal uptake mechanisms with corticosterone (29 microM) did not potentiate the response to Ad and did not significantly alter the response to Tyr (low dose). 5. The effects of MDL 72145 and clorgyline on the directly acting amine, Ad, were studied. MDL 72145 caused a small but significant increase in the EC50 and in the maximum response to Ad, whilst clorgyline (10 microM) increased the EC50 value slightly and decreased the maximum response.

Metabolism of amines in the isolated perfused mesenteric arterial bed of the rat.

1. Semicarbazide-sensitive amine oxidase (SSAO) activity has been demonstrated in the isolated mesenteric arterial bed of the rat in vitro by studying the metabolism of benzylamine (Bz) and tyramine (Tyr) added to the perfusing fluid. 2. Pretreatment of rats with (E)-2-(3',4'-dimethoxyphenyl)-3-fluoroallylamine (MDL72145), a potent inhibitor of SSAO in rat mesenteric blood vessels, reduced the amount of metabolites, following the addition of Bz (25 microM) or Tyr (100 microM) to the perfusing fluid, by 83% and 52% respectively. Inactivation of monoamine oxidase type A (MAO-A) by the addition of clorgyline (10 microM) to the perfusing fluid, had little effect on the appearance of metabolites from Tyr. 3. The presence of 3 microM cocaine in the perfusing fluid increased the amount of metabolites produced from Tyr. 4. The metabolites of Tyr appearing in the perfusion fluid from control preparations were 85% p-hydroxyphenylacetic and the remainder consisted of a mixture of p-hydroxyphenylacetaldehyde and, possible, p-hydroxyphenylethanol. 5. The metabolism of Tyr by homogenates of the rat mesenteric vascular bed was carried out by SSAO (60%) and MAO-A (40%) with very little contribution from MAO-B. Homogenates from rats pretreated with MDL 72145 showed metabolism of Tyr by MAO-A only. 6. These data indicate that SSAO is capable of metabolizing amines present in the fluid perfusing blood vessels to metabolites that are readily released. Histochemical evidence has shown that whereas MAO-A is present in the mitochondria of smooth muscle cells and nerve endings, SSAO is located in the plasma membrane of the smooth muscle cells. This subcellular distribution may explain the differences found between metabolites released from intact vessels and the metabolism seen in homogenates. The identity of the Tyr metabolizing activity in intact vessels that is resistant to both MDL 72145 and clorgyline remains to be determined.

In-vivo Effects of (E)-2-(3′,4′-dimethoxyphenyl)-3-fluoroallylamine (MDL 72145) on Amine Oxidase Activities in the Rat. Selective Inhibition of Semicarbazide-sensitive Amine Oxidase in Vascular and Brown Adipose Tissues

One hour after MDL 72145 ((E)-2-(3',4'-dimethoxyphenyl)-3-fluoroallylamine) (2.5 mg kg-1) was given by intraperitoneal injection, the semicarbazide-sensitive amine oxidase (SSAO) activity of rat aorta and brown adipose tissue measured in-vitro was reduced by more than 95% of its control value, whereas the monoamine oxidase (MAO-A) activity remained virtually unaffected. The action of this drug on amine oxidases in the liver at this dose was less selective. The in-vitro effect of MDL 72145 on the soluble enzyme diamine oxidase from rat intestine was 100 fold less potent than that of semicarbazide but about equipotent with semicarbazide on sheep plasma amine oxidase. Overall MDL 72145 was selectively more active against membrane bound SSAO enzymes that deaminate primary monoamines. Although MDL 72145 does inhibit MAO-B activity these results suggest that this compound may be used to study the effect of selective inhibition of SSAO activity on the pharmacological responses of appropriate preparations in-vitro.

Allylamine-induced vascular toxicity in vitro: Prevention by semicarbazide-sensitive amine oxidase inhibitors

The present studies were designed to evaluate the role that metabolic activation plays in allylamine (AAM)-induced vascular toxicity. The effects of AAM were evaluated in primary cultures of rat vascular endothelial (VEC) and smooth muscle cells (SMC). Semicarbazide (SC) and diethyldithiocarbamate (DDC) were used as inhibitors of semicarbazide-sensitive amine oxidase (SSAO). Clorgyline and pargyline were used as inhibitors of monoamine oxidase (MAO) A and B, respectively. The effect of catalase, a hydrogen peroxide scavenger, on AAM-induced cytotoxicity was also evaluated. Lactate dehydrogenase (LDH) release and morphological alterations were chosen as indicators of cytotoxicity. Confluent cultures of VEC and SMC were exposed to various concentrations of AAM (2-200 microM) in the absence and presence of serum for 4, 12, or 24 hr. High concentrations of AAM (200 microM) alone produced a time-dependent increase in LDH release and morphologic alterations in cultures of both cell types. Lower concentrations of AAM did not compromise the structural integrity of the cells. Semicarbazide (200 microM) or DDC (2 mM), but not clorgyline (10 microM) or pargyline (10 microM), prevented the toxicity of AAM (200 microM). Allylamine-induced cytotoxicity was partially prevented by catalase (2500 U/ml). The presence of fetal bovine serum in the medium was not essential for the manifestation of cytotoxicity. Single cell suspensions of VEC or SMC formed acrolein (ACR) when incubated in the presence of AAM. The formation of ACR mediated by SMC was inhibited by SC (20 microM), but not clorgyline (10 microM). These results support the concept that AAM is oxidatively deaminated by an SSAO present in vascular cells to generate toxic metabolic by-products capable of causing extensive cellular injury.

Inhibition of rat aorta semicarbazide-sensitive amine oxidase by 2-phenyl-3-haloallylamines and related compounds

The inhibition of semicarbazide-sensitive amine oxidase (SSAO) in rat aorta homogenates by some 2-phenyl-3-haloallylamines has been studied. Derivatives containing a fluorine atom were approximately three times more potent than the corresponcling 3-chloroallylamines. These halogencontaining compounds were irreversible inhibitors of SSAO after preincubation with aorta homogenates; kinetic evidence for an initial competitive, reversible interaction ( K i, around 0.4–0.6 μM) was found with two compounds (MDL 72145 and 72274). A similar K i (approx. 0.7 μM) was obtained with 2-phenylallylamine (MDL 72200). However, this compound which lacks a halogen atom was a reversible inhibitor, even after preincubation. The use of a spectrophotometric assay to measure H 2O 2 production from amine metabolism demonstrated that MDL 72200 was a substrate ( K m = 1.4 μM) for SSAO, with a V max. approximately five times smaller than that of benzylamine ( K m = 8.1 μM). Of particular interest in this study is the fincling that (E)-2-phenyl-3-chloroallylamine (MDL 72274) is highly selective as an inhibitor of SSAO, compared with MAO-A or B activities, and may be a useful compound for investigating the importance of SSAO in animal tissues.

Some aspects of the enzymic inactivation of sympathomimetic amines.

This review seeks to discuss the possible importance of monoamine oxidase (MAO) and semicarbazide-sensitive amine oxidase (SSAO) in terminating the effects of released sympathetic transmitters and in the inactivation of endogenous or administered sympathomimetic amines with particular reference to some aspects of the cardiovascular system. Use of in vitro preparations of blood vessels and of other smooth muscles, such as vas deferens and anococcygeus, has thrown light on possible roles for these deaminating enzymes, even in the inactivation of noradrenaline, while the new reversible inhibitors of MAO-A show promise as antidepressants with a reduced risk of hypertensive crises following the ingestion of tyramine-containing food. However, the role of SSAO in the in inactivation of amines remains an enigma, even though much is now known of its biochemical nature. While the pharmacological responses to amine substrates can be potentiated by inhibition of SSAO, there is a suspicion that it is product rather than substrate that is more important.

Studies of Monoamine Oxidase and Semicarbazide-Sensitive Amine Oxidase: I. Inhibition by a Selective Monoamine Oxidase-B Inhibitor, MD 780236

AbstractIn 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-dependency inhibited SSAO activity. The extents of inhibition of MAO-A, -B and SSAO in these two rat tissues by this compound after 30 mm of preincubation were found to be MAO-B>MAO-A>SSAO. This selectivity was also evident in preparations without preincubation. Degree of inhibition of SSAO was not significantly influenced by pretreatment with either 10−3M clorgyline, l-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 μM. 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.

Studies of Monoamine Oxidase and Semicarbazide-Sensitive Amine Oxidase: II. Inhibition by α-Methylated Substrate-Analogue Monoamines, α-Methyltryptamine, a-Methylbenzylamine and Two Enantiomers of α-Methylbenzylamine

AbstractThe α-methylated substrate-analogue monoamines, dl-α-methyltryp-tamine. dl-α-methylbenzylamine and two optical isomers of α-methylbenzylamine, were shown to be inhibitors of rat lung semicarbazide-sensitive amine oxidase (SSAO). with dl-α-methyltryptamine being the most potent and d-α-methyl-benzylamine, the least. The three compounds, dl-α-methyltryptamine and the two Isomers of α-methylbenzylamine also inhibited rat brain monoamine oxidase (MAO)-A and -B with a greater selectivity towards MAO-ॅ. Preincubation of rat lung and brain homogenates with either of these compounds revealed that the inhibition of MAO and SSAO is reversible. The modes of inhibition of MAO-A and -B were competitive with the substrates tested. However, inhibition of SSAO by dl-α-methyltryptamine was found to be a mixed type (with a K1 value of 47 μM) and those by the racemic form and two isomers of α-methylbenzylamine were noncompetitive (with K1 values of 90 μM for the racemic compound, 1070 μM for the d-isomer and 72 μM for the l-isomer). The present results indicate that SSAO can recognize optical isomers and that some α-methylated monoamines tested in the present study inhibit SSAO with properties different from those as MAO inhibitors.

The interaction of hydralazine with a semicarbazide-sensitive amine oxidase in brown adipose tissue of the rat. Its use as a radioactive ligand for the enzyme.

Hydralazine is a potent irreversible inhibitor of the semicarbazide-sensitive amine oxidase (SSAO) found in brown fat. Initially it may act on the enzyme as a competitive inhibitor, but irreversible inhibition occurs rapidly in a concentration- and temperature-dependent manner. The presence of primary amines known to be substrates for the enzyme, but not of secondary amines, which are not metabolized by SSAO, diminishes this rate of inactivation, whereas removal of O2 is without effect. The kinetic pattern of inactivation of SSAO by hydralazine is consistent with an active-site-directed site-saturable binding followed by the development of an irreversible enzyme-inhibitor complex. [3H]Hydralazine, used as an affinity label for SSAO, shows saturable binding to brown-fat membranes. This binding is inhibited by other inhibitors of SSAO. The rate of binding to membrane pellets containing SSAO is not affected by substrates for the enzyme. However, if solubilized partially purified SSAO preparations are used instead, the rate of binding is lowered in the presence of the SSAO substrate benzylamine. 3H-labelled material solubilized from [3H]hydralazine-treated membrane pellets by Triton X-100 at that detergent/protein ratio which releases SSAO from membranes shows the same gel-filtration characteristics as SSAO and appears by lentil lectin-agarose affinity chromatography to contain similar carbohydrate moieties. 3H-labelled material, partially purified by gel filtration and affinity chromatography, produces predominantly a single band of radioactivity on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The position of this band corresponds to an Mr of about 94 000, almost exactly half the Mr already estimated for the functional unit of SSAO. Radiolabelled hydralazine may thus be used as a label for purified SSAO, but it is not specific enough to be suitable as a ligand in vivo.

Vascular smooth muscle cells: a major source of the semicarbazide-sensitive amine oxidase of the rat aorta.

Several methods have been used to study the distribution of the semicarbazide-sensitive amine oxidase (SSAO) within the wall of the rat aorta. After separation of the smooth muscle-containing layers of the tunica media from the connective tissue of the tunica adventitia, much higher specific enzyme activity (measured with 1 microM benzylamine) was found in homogenates of the media than of adventitia. Similar results were obtained for MAO-A (with 1 mM 5-HT as substrate). SSAO activity was also considerably higher in homogenates of cells (predominantly smooth muscle) isolated from medial tissue by enzymatic dissociation with collagenase and elastase compared with homogenates of cells (mostly of connective tissue origin) from the adventitia. Histochemical staining resulting from SSAO activity (with benzylamine as substrate) occurred predominantly and intensely over the tunica media in rat aortic sections, although some occasional staining of adventitial sites was also observed. Staining was prevented by the SSAO inhibitors hydroxylamine (1 microM) and semicarbazide (1 mM), but not by the MAO inhibitor, clorgyline (1 mM). These results indicate that SSAO is associated predominantly, although not exclusively, with the smooth muscle cells in the rat aorta. Our findings that beta-aminopropionitrile (BAPN) is a reversible, competitive inhibitor (Ki around 2 X 10(-4)M) of SSAO, in contrast to the irreversible inhibition of the connective tissue lysyl oxidase by BAPN reported by others, provides further evidence that these enzymes are not identical.

Inhibition of semicarbazide-sensitive amine oxidase by monoamine oxidase B inhibitors from the oxazolidinone series.

The purpose of the present work was to study the semicarbazide-sensitive amine oxidase (SSAO) inhibitory properties of MD 240931 and MD 240928 (the two enantiomers of MD 780236) as well as those of the corresponding primary amines, MD220662 and MD220661, in rat heart and aorta. MD240928 and MD240931 are rather weak SSAO inhibitors, MD 240931 being more potent than MD 240928. Of the four compounds studied, the most potent inhibitor of SSAO is MD 220662, its IC50 value ranging from 2.10(-6) to 6.10(-6)M. The SSAO inhibitory potency of this compound does not change significantly with the time of preincubation in both the absence and presence of clorgyline (10(-4)M). MD 220661 is also an inhibitor of SSAO; however, its SSAO inhibitory potency, which without preincubation is comparable to that of MD 220662, does decrease with the time of preincubation to the same extent in both the absence and presence of clorgyline (10(-4)M). These results suggest that MD 220661 is not only an inhibitor of SSAO, but is also a substrate of the enzyme.

Monoamine oxidase and semicarbazide-sensitive amine oxidase in spontaneously hypertensive and in normotensive control rats

The aim of the present work was to compare monoamine oxidase (MAO) and semicarbazide sensitive amine oxidase (SSAO) activity in several tissues from spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto rats (WKY). Contribution of MAO-A, -B and SSAO to the metabolism of each substrate in each tissue was defined from experiments where the decrease of oxidative deamination of each substrate at a given concentration was measured as a function of increasing concentrations of a selective MAO-A, -B or SSAO inhibitor. In the heart, aorta and, to a lesser extent, the femoral arteries MAO-A activity was higher in SHR than in WKY. Similarly in the liver the enzyme activity was higher in SHR than in WKY but was due to the -B form of MAO. In all the other tissues studied (duodenum, brain, lungs, adrenals and kidneys) no difference in MAO-A, MAO-B or SSAO activity was found between SHR and WKY, except for the kidneys and brain, if the differences in the weights of these organs in SHR are taken into account.

Stereoselective inhibition of monoamine oxidase and semicarbazide-sensitive amine oxidase by 4-dimethylamino-2,alpha-dimethylphenethylamine (FLA 336).

The in vitro inhibition by amiflamine [FLA 336(+)] and related compounds of the activity of rat monoamine oxidase (MAO) -A and -B, rat semicarbazide sensitive amine oxidase (SSAO) and human platelet poor plasma benzylamine oxidase was studied. Amiflamine was an MAO-A selective inhibitor, but also inhibits SSAO with both a reversible (competitive, Ki = 200 mumol/l) and a small time-dependent component which was irreversible in nature. The optical isomer FLA 336(-) was ten times less potent towards MAO-A. However, this compound was much more potent an inhibitor of SSAO (competitive, Ki = 4.6 mumol/l). The amiflamine metabolites FLA 788(+) and FLA 668(+) inhibited SSAO, but only at concentrations considerably higher than required for MAO-A inhibition. Ex vivo experiments indicated that there was no significant irreversible inhibition of rat heart and lung SSAO after both single and repeated administration of amiflamine at doses up to 20 times higher than required for inhibition of MAO-A within central serotoninergic neurones.

P-315) - Mechanisms of inhibition of semicarbazide-sensitive amine oxidase by selective MAO inhibitors, amiflamine, FLA 788(+) and MD 780236(±)

P-315) - Mechanisms of inhibition of semicarbazide-sensitive amine oxidase by selective MAO inhibitors, amiflamine, FLA 788(+) and MD 780236(±)