Properties and functions of the semicarbazide-sensitive amine oxidases.
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Amine Oxidase (Copper-Containing)↗
Biomedical subjects
B A Callingham
Publications and source records attributed to B A Callingham.
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.
Adenosine Triphosphate↗
Evidence for regulatory control of iron uptake from ferric maltol across the small intestine of the rat.
1. 59Fe absorption from the novel iron compound, ferric maltol, was studied in rats pretreated twice daily for two weeks with non-radioactive ferric maltol in oral doses containing 7 mg elemental iron. Tissue accumulation of 59Fe 2 h after administration of radioactive ferric maltol into the stomach was significantly lower in iron pretreated animals than in saline-treated controls. 2. 59Fe uptake from ferric maltol into isolated fragments of ileum and of duodenum was of similar magnitude in control animals but in iron-treated animals, duodenal uptake was significantly lower than that of the ileum. 3. Absorption of 59Fe was also investigated in anaesthetized rats after intestinal perfusion with saline (controls) or with 5 mM chenodeoxycholate to render the intestines more permeable. 4. Changes in permeability of the small intestine were monitored by estimating the amount of [14C]-mannitol absorbed and fluid secreted with reference to the non-absorbable [3H]-inulin in the perfusate effluents. 5. Despite the increased permeability of the intestines after bile salt treatment, there was little difference from control in the tissue accumulation of 59Fe from ferric maltol 2 h after intraduodenal administration. However 59Fe absorption from ferrous sulphate was significantly increased after bile salt treatment. 6. Gel filtration profiles of plasma made 5 and 60 min after intraduodenal administration of [59Fe]-ferric [3H]-maltol demonstrated that metal and ligand do not enter the circulation as the complex even when intestinal permeability is increased. 7. Uptake of 59Fe was investigated in isolated fragments of rat small intestine after saline or bile salt perfusion. Although 59Fe uptake from ferric maltol was somewhat greater in the bile salt-treated intestinal fragments, saturable kinetics were still observed. By contrast, "Fe uptake from ferrous sulphate: ascorbate was greatly enhanced by bile salt pretreatment and a very large diffusional component of uptake was evident. 8. It is concluded that iron uptake from ferric maltol may well be under endogenous regulatory control even in damaged intestines, so it is unlikely that this novel iron compound can bring about iron overload when administered orally.
Animals↗
Dissociation of a ferric maltol complex and its subsequent metabolism during absorption across the small intestine of the rat.
1. The fate and disposition of [59Fe]-ferric [3H]-maltol after intravenous administration were investigated in anaesthetized rats. Immediate dissociation of ferric iron from maltol took place in the circulation even with high doses of ferric maltol (containing 1 mg elemental iron). In plasma samples withdrawn within 1 min of injection and subjected to gel filtration, 59Fe eluted with the high molecular weight proteins whilst the tritium was associated with low molecular weight material. 2. The rates of elimination of 59Fe and of tritium from the plasma and their ultimate fate were very different. The half life for 59Fe in the plasma was around 70 min and 59Fe appeared mainly in the bone marrow and liver. There was an initial rapid exit of tritium from the plasma with a half life of around 12 min. This was followed either by a plateau or by a rise in tritium levels, involving entry of maltol metabolites into the circulation. These metabolites could be recovered in the urine. 3. Entry of 59Fe and of tritium into the blood plasma after intraduodenal administration of [59Fe]-ferric [3H]-maltol was also very different. At low doses of ferric maltol (containing 100 micrograms elemental iron), the tritium appeared in the plasma in highest amounts within seconds and then decreased whilst there was a slow rise in 59Fe levels. At higher doses of ferric maltol (containing 7 mg elemental iron), levels of 59Fe in the plasma were highest at 5 min and then fell whereas tritium levels rose steadily. Mucosal processing of 59Fe prevented further entry of iron at high dose into the circulation. 4. Initial rates of uptake of [3H]-maltol into isolatcd intestinal fragments were measured over a range of concentrations and revealed that maltol alone could diffuse freely into the tissues whereas maltol complexed to iron showed saturable uptake kinetics similar to those seen with the iron itself. 5. After intestinal uptake, 59Fe and tritium were associated with different subcellular fractions, maltol itself being metabolized to the glucuronide conjugate within the intestinal mucosa. 6. It is concluded that dissociation of metal and ligand takes place before entry into the intestinal mucosa. Iron is then taken up on the endogenous carrier and processed in the normal way whilst maltol enters by diffusion, its rate of entry being limited by the degree of dissociation. It is subsequently metabolized by conjugation and eliminated rapidly from the body in the urine.
Animals↗
Some aspects of the pharmacology of semicarbazide-sensitive amine oxidases.
Semicarbazide-sensitive amine oxidase enzymes (SSAO) are found in animals, plants, fungi and bacteria. In vertebrates, their distribution in tissues and blood plasma varies between species. Studies of the SSAO enzymes have concentrated on their biochemical identities separate from those of MAO. Attention is now being paid to their possible physiological and pharmacological significance. These may include, besides the scavenging of circulating amines, functions dependent upon the hydrogen peroxide these enzymes produce. Modulation, by SSAO, of blood vessel tone may be due to the control of amine concentration itself or to actions of released peroxide. In the plasma the activity of SSAO may be susceptible to hormonal control as well as being an indicator of copper status of the animal. However, SSAO may convert xenobiotics to more toxic metabolites. Use of highly selective SSAO inhibitors, such as procarbazine and B24 should enable these preliminary observations to be examined further.
Amine Oxidase (Copper-Containing)↗
The importance of reductive mechanisms for intestinal uptake of iron from ferric maltol and ferric nitrilotriacetic acid (NTA).
Intestinal iron absorption is thought to proceed with iron mainly in the ferrous form, yet the novel iron complex, ferric maltol is an effective oral preparation. Although possessing a high oil: water partition coefficient, ferric maltol does not diffuse across the intestine but donates its iron to the endogenous uptake system. Reduction of the ferric iron in the gut lumen appears to precede iron uptake both from ferric maltol and from ferric nitrilotriacetic acid (NTA) which is a non-penetrating iron ligand. Uptake of radiolabelled iron (59Fe) into isolated fragments of rat small intestine was inhibited by the ferrous chelator, bathophenanthroline sulphonate (BPS) and enhanced at low concentrations by the reducing agent ascorbic acid. Spectrophotometric evidence was obtained that ferrous ions are generated from these ferric complexes in the presence of ascorbic acid and other reducing agents. The rate of ferrous ion formation was independent of ferric maltol concentration at low ascorbic acid levels and decreased with increasing ferric maltol concentration at higher levels of ascorbate. Maltol has a high affinity for ferric ions and may delay reduction at higher concentrations. By contrast, a higher rate of ferrous ion generation was seen with ferric NTA and this increased with iron ligand concentration. Washings from the intestinal lumen also brought about ferrous ion formation from these ferric ligands. Gel filtration revealed these reducing factors to be of low molecular weight. The washings, however, interfered with 59Fe uptake into the isolated fragments, but when reducing fractions only from the filtered washings were used, enhanced iron uptake was seen.(ABSTRACT TRUNCATED AT 250 WORDS)
Acetates↗
Effects of aspirin on xylazine-induced hypoxaemia in sheep.
Aspirin (10 mg kg-1) administered intravenously to conscious sheep four hours before intravenous xylazine injection (50 micrograms kg-1), failed to abolish or attenuate the hypoxaemic effect of xylazine in this species. Serum thromboxane levels measured in one animal revealed that aspirin administered in this way reduced serum thromboxane levels by 95 per cent. Xylazine (3 x 10(-5) M--4 x 10(-3) M) failed to induce platelet aggregation in vitro. It appears that the mechanism whereby xylazine causes arterial hypoxaemia in sheep does not involve a cyclo-oxygenase-dependent aggregation of platelets.
Animals↗
Semicarbazide-sensitive amine oxidase (SSAO) of the rat aorta. Interactions with some naturally occurring amines and their structural analogues.
The influence of a number of naturally occurring amines and their structural analogues has been examined on the metabolism of radiolabelled benzylamine (BZ) by the membrane bound semicarbazide-sensitive amine oxidase (SSAO) of the rat aorta. Only primary monoamines were effective in reducing the deamination of BZ. In the phenylethylamine series, addition of hydroxyl groups to the benzene ring decreased their potency as inhibitors while addition of a hydroxyl group at the beta position increased the inhibitory potency. Stereoselectivity of action was shown with octopamine, the L-isomer being the more active form. Kinetic analysis of these interactions showed predominantly competitive inhibition and kynuramine had the lowest Ki of 5.4 microM. The aliphatic monoamines, isoamylamine and isobutylamine both competed with BZ. 5-Hydroxytryptamine (5-HT) was the only amine that inhibited non-competitively. Direct evidence for metabolism by SSAO of some of the competing amines such as isoamylamine, phenylethylamine, tyramine and tryptamine was obtained by fluorimetric or radiochemical assays. The inhibitors clorgyline and (E)-2-(3',4'-dimethoxyphenyl)-3-fluoroallylamine (MDL 72145) were used to characterise the amine oxidase activity responsible for the deamination. Octopamine and phenylethanolamine (PeOH) were not SSAO substrates and inhibited BZ metabolism in the fluorimetric assay. It is possible that the activity of SSAO is controlled by octopamine released from sympathetic nerve endings or 5-HT released from platelets.
Allosteric Regulation↗
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.
Animals↗
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.
Animals↗
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.
Adipose Tissue, Brown↗
Biochemical aspects of the pharmacology of moclobemide. The implications of animal studies.
Administration of moclobemide--a relatively short-acting, reversible inhibitor of monoamine oxidase-A (MAO-A)--to experimental animals potentiates the pressor responses to intravenously injected tyramine, but such effects are moderate, short-lived, and much less apparent when the tyramine is given orally. The ability of moclobemide to potentiate the pharmacological actions of amine substrates for MAO-A in in vitro preparations is very weak, unless the drug is 'activated' by prior incubation with the tissues. However, animal experiments, both in vitro and in vivo, can give realistic indications not only of the mode of action of inhibitors of MAO and any toxicity, but also, in the case of MAO inhibitors, of the chance of potentially life-threatening hypertensive crises following inadvertent ingestion of amine-containing food or medicaments. Animal tests may also reveal any influence MAOIs have on amines released from non-neuronal stores.
Animals↗
Characteristics of iron(III) uptake by isolated fragments of rat small intestine in the presence of the hydroxypyrones, maltol and ethyl maltol.
Accumulation of radioactive iron (59Fe) into isolated fragments of rat small intestine in the presence of two hydroxypyrones, maltol and ethyl maltol, was compared with that in the presence of another chelator of iron(III), nitrilotriacetic acid (NTA). The characteristics of uptake were similar with all three ligands. Between 10(-6) and 10(-4) M, iron uptake showed saturable kinetics. The uptake was partially inhibited by metabolic inhibitors. Above 10(-4) M a non-saturable uptake, unaffected by metabolic inhibitors became evident in the presence of the pyrones. The distribution of 59Fe after uptake was determined by gel filtration. At low iron concentrations (10(-6) M), 35-40% of absorbed iron was associated with proteins of molecular weights similar to those of ferritin and transferrin. At high concentrations (10(-3) M), the majority of 59Fe was found in a low molecular weight fraction. At each concentration, a small amount of 59Fe was bound to a membrane fraction. 5% Polyethylene glycol, which reduces glycocalyx viscosity enhanced uptake at low iron concentrations (10(-6) M) but did not affect the non-saturable diffusion seen at higher concentrations (10(-3) M). The iron(II) chelator, bathophenanthroline sulphonate (10(-3) M), decreased uptake at low iron concentrations but did not affect the non-saturable uptake. It is suggested that conversion of iron(III) to iron(II) may take place at the mucosal cell surface before uptake via the saturable system. Apparent Km values for iron uptake via the saturable system were higher in the presence of maltol and ethyl maltol than in the presence of NTA, presumably since the iron binds more avidly to the hydroxypyrones and so is less readily donated. Excess ligand, either pyrone or NTA, reduced the rate at which 59Fe was donated to the uptake system. The Vmax value for uptake from the pyrones was greater than from NTA. It is concluded that maltol, ethyl maltol and NTA can hold iron(III) in solution and donate it to an endogenous uptake system. But, the hydroxypyrones may be more suitable ligands for the oral administration of iron since, when complexed with iron, they lack the toxic effects associated with iron(III)-NTA and with iron(II) preparations.
Animals↗
Some in vitro effects of moclobemide and other MAO inhibitors on responses to sympathomimetic amines.
The use of inhibitors of monoamine oxidase (MAO) in the treatment of some forms of depression has been marred by the occurrence of unpleasant or even fatal side effects. Of these the most serious has been the risk of sudden hypertensive crises following the ingestion of food or drink containing indirectly-acting sympathomimetic amines such as tyramine (cheese effect). This effect, due to long lasting or irreversible inhibition of MAO in the gut and vasomotor neurones is much less obvious with the new generation of reversible inhibitors. Acceptance into clinical practice of these agents will depend upon the provision of clear and unequivocal evidence that there is little or no risk of unexpected cheese effects. The use of in vitro methods has provided a most sensitive way of revealing and measuring any such propensity in these new agents including moclobemide, cimoxatone and toloxatone. Use of isolated smooth muscles, such as the vas deferens and anococcygeus muscles of the rat together with vascular smooth muscle, typified by the perfused mesenteric arterial bed may be used as reliable preliminaries to in vivo and human volunteer studies in an attempt to introduce into clinical medicine an antidepressant that acts through the inhibition of MAO but carries little or no risk of the cheese effect.
Animals↗
Some properties of semicarbazide-sensitive amine oxidases.
The semicarbazide-sensitive amine oxidases (SSAOs) comprise a substantial but diffuse group of enzymes separable from classical monoamine oxidase in several respects. Differences in cofactor requirement, molecular weight and subcellular distribution are crucial for such a separation. Differential sensitivity to enzyme inhibitors, characterized by resistance to inhibition by acetylenic MAO inhibitors coupled with sensitivity to semicarbazide and some related compounds are characteristic of these enzymes. SSAO enzymes have been found in the plasma of man, ox, pig and horse, for example as well as in the solid tissues of many species. Extensive studies have so far failed to produce any conclusive evidence to indicate what the precise functions of many of these enzymes may be. Indeed in most cases there is no clear idea as to the nature of the preferred physiological substrate, although many amines with pharmacological activity have been shown to be substrates. The actions of these amines may be potentiated following inhibition of SSAO, but as yet little is known whether or not these actions can be important in vivo. An attempt is made in this review to bring together some of the evidence to see if there are indications for future endeavours.
Amine Oxidase (Copper-Containing)↗
Monoamine oxidase enzymes: are they important in the horse?
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Animals↗
Effects of the pyrones, maltol and ethyl maltol, on iron absorption from the rat small intestine.
The pyrones, 3-hydroxy-2-methyl-4-pyrone (maltol) and 3-hydroxy-2-ethyl-4-pyrone (ethyl maltol) chelate iron with a high affinity and selectivity. The resulting 1:3 (metal-ligand) complexes, being neutral, are able to partition readily across cell membranes and thus may facilitate iron transport across the intestinal wall. Absorption of radioactive iron (59Fe) in the presence of these pyrones was investigated in male rats 1, 2, 4 and 6 h after intraduodenal administration of a 7 micrograms dose and compared with that of 59Fe given as the sulphate, gluconate, fumarate or complexed to EDTA. Total body absorption and distribution were calculated from the 59Fe content of various tissue samples. With all the iron preparations used, blood levels of 59Fe were highest 1 h after injection whilst the 59Fe content at the major site of deposition, i.e. the bone marrow, increased up to 6 h. No 59Fe was found in the urine. Total body absorption of 59Fe was significantly higher from the pyrones than from the other four preparations. Over the dose range 0.7-700 micrograms, the proportion of 59Fe absorbed from both iron maltol and iron sulphate decreased with increasing dose. Enhanced 59Fe uptake from maltol was evident at 0.7-70 micrograms but not at 700 micrograms suggesting that use of these pyrones will not result in iron overload. Absorption of 59Fe given into the stomach was slower in onset but was sustained longer presumably via a steady delivery of iron to the duodenum from the gastric reservoir.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
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.
Amine Oxidase (Copper-Containing)↗