Semicarbazide-sensitive Amine Oxidase Research Articles

Hepatic consequences of vascular adhesion protein-1 expression

The liver is constantly exposed to antigens present in the blood and to particulate antigens delivered from the gut. To maintain effective levels of immune surveillance and yet tolerate food antigens, the hepatic environment has become highly specialised. A low flow environment exists within the hepatic sinusoids that not only facilitates the exchange of toxins and nutrients within the liver parenchyma, but also provides an ideal niche for the recruitment of leukocytes. One such adhesion molecule involved in this process, the vascular adhesion protein-1 (VAP-1), is unusual in the context of the leukocyte adhesion cascade in that it is both an adhesion molecule and a primary amine oxidase. In this review, we examine the biological functions of VAP-1 and examine what role this molecule might play in the establishment and progression of chronic liver disease.

Monoamine Oxidase and Semicarbazide-Sensitive Amine Oxidase Kinetic Analysis in Mesenteric Arteries of Patients With Type 2 Diabetes

Monoamine oxidase (MAO, type A and B) and semicarbazide-sensitive amine oxidase (SSAO) metabolize biogenic amines, however, the impact of these enzymes in arteries from patients with type 2 diabetes remains poorly understood. We investigated the kinetic parameters of the enzymes to establish putative correlations with noradrenaline (NA) content and patient age in human mesenteric arteries from type 2 diabetic patients. The kinetic parameters were evaluated by radiochemical assay and NA content by high-performance liquid chromatography (HPLC). The activity of MAO-A and SSAO in type 2 diabetic vascular tissues was significantly lower compared to the activity obtained in non-diabetic tissues. In the correlation between MAO-A (K(m)) and NA content, we found a positive correlation for both the diabetic and non-diabetic group, but no correlation was established for patient age. In both groups, MAO-B (V(max)) showed a negative correlation with age. The results show that MAO-A and SSAO activities and NA content of type 2 diabetic tissues are lower compared to the non-diabetic tissues, while MAO-B activity remained unchanged. These remarks suggest that MAO-A and SSAO may play an important role in vascular tissue as well as in the vascular pathophysiology of type 2 diabetes.

Properties of a semicarbazide-sensitive amine oxidase in human umbilical artery.

The metabolism of some aromatic amines by amine oxidase activities in human umbilical artery homogenates has been studied. The inhibitory effects of clorgyline showed that 5-hydroxytryptamine (5-HT) and tryptamine, 1 mM, were predominantly substrates for monoamine oxidase (MAO) type A, whereas MAO-A and B were both involved in the metabolism of beta-phenylethylamine (PEA), 100 microM, and tyramine, 1 mM. About 20-30% of tyramine and PEA metabolism was resistant to 1 mM clorgyline, but sensitive to inhibition by semicarbazide, 1 mM, indicating the presence of a semicarbazide-sensitive amine oxidase (SSAO). Benzylamine, 1 mM, appeared to be metabolized exclusively by SSAO with a Km (161 microM) at pH 7.8 similar to that found for SSAO in other human tissues. Tyramine and PEA were relatively poor substrates for SSAO, with very high apparent Km values of 17.6 and 13.3 mM, respectively, when determined in the presence of clorgyline, 10(-3) M, added to inhibit any metabolism of those amines by MAO activities. However, kinetic studies with benzylamine indicated that clorgyline, 10(-3) M, also appears to inhibit SSAO competitively such that the true Km values for tyramine and PEA may be about 60% of those apparent values given above. No evidence for the metabolism of 5-HT or tryptamine by SSAO was obtained. The aliphatic amine methylamine was recently shown to be a specific substrate for SSAO in umbilical artery homogenates. We have used benzylamine and methylamine as SSAO substrates in histochemical studies to localize SSAO in tissue sections.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects in-vitro of procarbazine metabolites on some amine oxidase activities in the rat.

The effects were examined of four metabolites of the anticancer agent, procarbazine (N-isopropyl-alpha-(2-methyl hydrazino)-p-toluamide hydrochloride) on semicarbazide-sensitive amine oxidase (SSAO) and monoamine oxidase-A and -B (MAO-A and -B) activities in rat brown adipose tissue and liver homogenates, respectively. Azoprocarbazine (AZO) and monomethylhydrazine (MMH) inhibited selectively the deamination of benzylamine by SSAO, when compared with their effects on MAO activities. The IC50 values against SSAO, of 32.7 nM (AZO) and 7.0 nM (MMH), were more than three orders of magnitude lower than those exhibited against MAO. Neither isomer of azoxyprocarbazine was an effective inhibitor of rat amine oxidase activities. The inhibition of SSAO by AZO was reversed very slowly by dialysis, in contrast to results seen for MMH. The non-competitive kinetics of MMH and the ability of B24, a rapidly reversible SSAO inhibitor, to protect SSAO against inhibition by MMH are consistent with the view that this compound binds to the enzyme cofactor at, or near, the active site.

Characteristics of procarbazine as an inhibitor in-vitro of rat semicarbazide-sensitive amine oxidase.

Procarbazine (N-isopropyl-alpha-(2-methyl hydrazino)-p-toluamide hydrochloride) inhibited more powerfully the deamination of benzylamine by semicarbazide-sensitive amine oxidase (SSAO) of rat brown adipose tissue than the deamination of 5-hydroxytryptamine and benzylamine by rat liver monoamine oxidase-A or -B activities, respectively. Inhibition of SSAO, but not monoamine oxidase, was time-dependent. Use of metabolic inhibitors, and an enzyme dilution technique, suggested that any conversion of procarbazine to an active species must be as a result of the action of SSAO itself and not of any other enzyme. The non-competitive kinetics and the time-dependence of inhibition were indicative of a suicide interaction between procarbazine and SSAO. The slow reversal of inhibition by dialysis was evidence in favour of the involvement of tight binding, rather than covalent bonding. High concentrations of benzylamine afforded the enzyme significant protection from the action of procarbazine, indicating that the interaction is at or near the active site. If the properties of procarbazine, evident in in-vitro studies, are retained in-vivo, these data suggest that procarbazine might be suitable for the examination of SSAO activities, both in-vivo and ex-vivo.

Further studies on the ex-vivo effects of procarbazine and monomethylhydrazine on rat semicarbazide-sensitive amine oxidase and monoamine oxidase activities.

Following administration of the anticancer agent, procarbazine, or one of its metabolites, monomethylhydrazine, to rats, activities of monoamine oxidases A and B (MAO A and MAO B) and of semicarbazide-sensitive amine oxidase (SSAO) were measured ex-vivo. Both compounds were found to be potent inhibitors of SSAO in tissue homogenates, exhibiting ID50 values in most tissues of approximately 8 mg kg-1 (procarbazine) and 0.08 mg kg-1 (monomethylhydrazine). Concurrent dose-dependent inhibition of MAO activities did not occur. However, in liver, potentiation of MAO B activity, to 140% of that in controls, was apparent following monomethyl-hydrazine and this effect was independent of the drug dose. Both compounds produced a dose-dependent potentiation of MAO A in brown adipose tissue, the elevation being more pronounced following monomethylhydrazine, with activity rising to 350% of that in control homogenates. In a parallel in-vitro study, monomethylhydrazine was without effect on MAO A in brown adipose tissue homogenates. By perfusing the SSAO substrate, benzylamine, through the isolated mesenteric arterial bed of the rat, it was found that pretreatment of animals with procarbazine or monomethylhydrazine reduced metabolism of this amine by a similar degree as had been determined ex-vivo in blood vessel homogenates. The results presented suggest that these compounds would be suitable for use as selective inhibitors in pharmacological examinations of SSAO function in isolated tissues and organs.

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.

Skin wound healing: some biochemical parameters in guinea-pig.

Hairs were removed from the dorsal skin of guinea-pigs and 5-6 wounds (7 x 7 mm) were surgically induced by totally removing the epidermal and part of the dermal surface. They were then allowed to heal. The newly formed wound tissues were dissected at different times during the process and analysed by biochemical and histological methods. Hydroxyproline, proteins, DNA and semicarbazide-sensitive amine oxidase (SSAO) were measured, as were [14C]leucine and [3H]thymidine incorporation in some samples. The peroxidase-like activity of plasma albumin and the histology of wounds stained with haematoxylin-eosin were also studied. It was shown that SSAO enzymes, which are present in normal guinea-pig skin and have a high affinity for benzylamine are localized in fibroblasts. During skin healing in the newly formed tissue there was an increase in protein content which reached a maximum after 4-6 days; DNA content also increased. The rate of incorporation of [3H]thymidine and [14C]leucine paralleled DNA and protein content, respectively. The content of hydroxyproline had greatly decreased with respect to that in normal skin after 2-10 days. SSAO activity increased much less than DNA after 4 days whereas after 10-11 days it increased more than DNA, thus indicating that at this time it was probably produced by fibroblasts. No significant increase in the peroxidase-like activity of albumin was observed 4, 8 or 11 days after surgery. Treatment of the animals with methylprednisolone acetate (20 mg kg-1, i.m.) two days before surgery decreased the rate of skin healing but did not alter the level of albumin peroxidase activity of the plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

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.

The Enhanced Daily Excretion of Urinary Methylamine in Rats Treated with Semicarbazide or Hydralazine May Be Related to the Inhibition of Semicarbazide-sensitive Amine Oxidase Activities

The effects of amine oxidase inhibitors upon the daily urinary excretion of monomethylamine (MMA), dimethylamine (DMA), trimethylamine (TMA) and ammonia in the rat have been examined. Administration of hydralazine (5 mg kg-1) or semicarbazide (100 mg kg-1), drugs which irreversibly inhibit semicarbazide-sensitive amine oxidases (SSAO) but not monoamine oxidase (MAO), enhanced MMA excretion by around three- to six-fold above pretreatment levels, whereas no effect of pargyline (25 mg kg-1), a selective irreversible inhibitor of MAO was found. No apparent changes in DMA or TMA excretion in response to drug-treatment were observed. Ammonia excretion also was generally unchanged except for an apparent marked increase (approximately four-fold) over the 24 h following semicarbazide, a result which might be explained if ammonia is a degradation product of semicarbazide metabolism in the rat. With recent evidence that MMA is a substrate in-vitro for SSAO activities, results here may indicate that SSAO or related enzymes are involved in endogenous MMA turnover.

Age-related changes in the activities of the amine metabolizing enzymes of rat eye

Eye MAO-A, MAO-B, semicarbazide-sensitive amine oxidase (SSAO) and aldehyde reductase (AR) activities were measured in young and old rats. When enzyme activity is expressed as nmol (mg protein)-1 min-1, a significant decrease (18-23%) of SSAO activity in the eye of old rats was found, whereas there was no significant difference in MAO-A and MAO-B activities. A significant increase of AR activity with D-xylose (67%), DL-glyceraldehyde (64%), D-glucuronate (43%) and D-glucose (21%) was found in the eye of old rats. These results suggest that changes in the activities of the amine metabolizing enzymes of rat eye with age might have consequences for their function in senescence; particularly, the increase of AR activity might be involved in cataract formation.

Monoamine oxidase activities in dissociated cell fractions from rat skeletal muscle.

Collagenase was used to dissociate rat skeletal muscle (gastrocnemius) into its constituent cells, from which a myocyte fraction enriched in striated muscle cells, and a non-myocyte fraction containing cells of connective tissue and vascular origin, were prepared. The activities of amine oxidase enzymes and alkaline phosphatase (AP) were then assayed in these fractions, and also in homogenates prepared from corresponding samples of non-dissociated tissue. The specific activities of the semicarbazide-sensitive amine oxidase (SSAO) and AP were considerably higher (30 to 35-fold) in non-myocyte than in myocyte fractions. AP is generally considered to be present predominantly in vascular cells of skeletal muscle, with little, if any, in skeletal muscle cells themselves. Thus, the results obtained may indicate a similar localization for SSAO activity. Support for this came from histochemical studies, which showed staining for SSAO primarily over the walls of larger blood vessels in the muscle. Unlike SSAO and AP, were marked differences in MAO-A activity between myocyte and non-myocyte fractions were not found, suggesting that MAO-A is more probably a constituent of cells within both fractions.

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.

Semicarbazide-sensitive amine oxidase activity in white adipose tissue of the insulin-deficient rat.

We have investigated whether the effects in white adipose tissue due to insulin deficiency might also be related to an alteration of histamine levels which are regulated by semicarbazide-sensitive amine oxidase. The lack of circulating insulin induced by streptozotocin produced, in rat white adipose tissue, a loss of affinity of the semicarbazide-sensitive amine oxidase for histamine oxidation. In parallel, a decrease of cell transformation, measured by glycerol-3-phosphate dehydrogenase activity and an augmented sensitivity to histamine lipolysis were observed. These findings could contribute to the understanding of histamine metabolism and function in diabetic rats and to the knowledge concerning amine oxidases in this animal pathology.

Further Studies on the Metabolism of Methylamine by Semicarbazide-sensitive Amine Oxidase Activities in Human Plasma, Umbilical Artery and Rat Aorta

An ion exchange radiochemical assay has been developed to study the deamination of [14C]methylamine (MA) in homogenates of rat aorta and human umbilical artery, as well as in samples of human plasma. MA metabolism was found to be inhibited almost completely by 1 mM semicarbazide, but virtually unaffected by 0.1 mM clorgyline, suggesting that MA is a substrate for the semicarbazide-sensitive amino oxidase (SSAO) activities which also metabolize benzylamine (BZ) in these sources. Mean Km values for MA metabolism by aorta, umbilical artery and plasma were 182, 832 and 516 microM, respectively, with corresponding Vmax values in aorta and umbilical artery of 100 and 590 nmol (mg prot.)-1 h-1, and in plasma of 48 nmol (mL serum)-1 h-1. Kinetic constants determined for [14C]BZ metabolism in plasma (by an organic solvent extraction assay) and in umbilical artery (by the ion exchange assay) yielded mean Km values of 225 microM (plasma), 222 microM (umbilical artery), and Vmax values of 28 nmol (mL serum)-1 h-1 (plasma) and 377 nmol (mg prot.)-1 h-1 (umbilical artery). The deamination of [14C]MA was inhibited competitively by unlabelled BZ, with Ki values in umbilical artery and plasma of 220 and 172 microM, respectively. Also, metabolite formation from mixtures of [14C]BZ (200 microM) and [14C]MA (800 microM) was extremely close to that predicted for a single enzyme capable of metabolizing two alternative substrates in a competitive fashion.(ABSTRACT TRUNCATED AT 250 WORDS)

Some histamine-related compounds interacting with the benzylamine-oxidizing activity of rat white adipocytes.

In rat white adipocytes histamine is oxidized by a semicarbazide-sensitive amine oxidase which has benzylamine or preferential substrate (Bz-SSAO). To determine whether Bz-SSAO could control the extracellular levels of histamine and other histamine-related compounds active in lipid mobilization, a series of histaminergic compounds was screened as possible substrates or inhibitors of Bz-SSAO activity. Histaminergic compounds with imidazolo or thiazolo groups are oxidized by rat white-adipocyte Bz-SSAO whereas S-isothiourea derivatives, with two- or three-carbon-atom alkyl chains between the isothiourea and the N,N-dimethyl residue are, instead, inhibitors of the enzyme. Amtamine has been identified as a selective, high affinity substrate for rat white adipocyte Bz-SSAO. This enzymatic degradation might represent a catabolic pathway for the drug. These results show that the histaminase property of the rat white-adipocyte enzyme Bz-SSAO also extends to other histamine derivatives active at histamine receptors.

Oxidative deamination of aliphatic amines by rat aorta semicarbazide-sensitive amine oxidase

Rat aorta semicarbazide-sensitive amine oxidase (SSAO) exhibits very high affinity in the deamination of an homologous series of aliphatic amines of 1 to 18 straight chain carbon atoms. The Km value decreases substantially as the chain length of these amines increases. The Vmax values are higher for the short chain amines. Diamines are poor substrates for SSAO or are not acted upon by the enzyme. The substrate preference for SSAO differs from that for monoamine oxidase.

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)