A continuous spectrophotometric assay for monoamine oxidase and related enzymes in tissue homogenates.

A continuous, peroxidase-linked spectrophotometric assay is described which is suitable for measuring monoamine and diamine oxidase and semicarbazide-sensitive amine oxidase activities in tissue homogenates. In the assay, 4-aminoantipyrine is oxidized and then condenses with vanillic acid to give a red quinoneimine dye. The absorbance at 498 nm is proportional to the amount of hydrogen peroxide released in the amine oxidase reaction. The molar absorption coefficient of the dye at pH 7.6 was 4654 M-1 cm-1. The method is suitable for use with any amine oxidase substrate which has a higher oxidation-reduction potential than does 4-aminoantipyrine. Following preincubation of rat liver homogenates with selective monoamine oxidase (MAO)-A and -B inhibitors, kinetic constants were obtained for metabolism of the mixed substrate, p-tyramine. Inhibition of MAO in rat liver homogenates was also measured following administration of the antidepressant, phenelzine. This inexpensive assay which employs reagents with low toxicity can thus be used to determine the degree of inhibition of MAO elicited by potential antidepressant and anti-parkinsonian agents. Drugs, their metabolites, and environmental toxins can also be screened as possible amine oxidase substrates or inhibitors, and kinetic constants for turnover of novel substrates can be determined.

Inhibition of chick embryo lysyl oxidase by various lathyrogens and the antagonistic effect of pyridoxal.

Lysyl oxidase, which cross-links collagen and elastin, was obtained from chick embryo bone and cartilage and its substrate, elastin, from aorta. The enzyme was studied using an improved assay which enabled the stability of the substrate to be monitored. The enzyme was fully inhibited in vivo by beta-aminopropionitrile, semicarbazide, thiosemicarbazide and isoniazid and in vitro by beta-aminopropionitrile and semicarbazide but only partially by thiosemicarbazide and isoniazid. Penicillamine, which solubilizes collagen by labilizing Schiff base cross-links in vivo and which prevents stable cross-link formation in vitro indirectly by binding to aldehyde groups on collagen, was shown to have no direct inhibitory effect on lysyl oxidase in vivo or in vitro. Homocysteine, which also solubilizes collagen by a mechanism similar to penicillamine does not inhibit lysyl oxidase either in vivo or in vitro. Pyridoxal reversed the inhibition of lysyl oxidase by semicarbazide and isoniazid in vivo but was unable to reverse that produced by either beta-aminopropionitrile or thiosemicarbazide. These results can be explained by the presence of a sulphydryl group near the active site of lysyl oxidase, which can form a complex with the nitrile group on beta-aminopropionitrile or with the thiol group on thiosemicarbazide leading to irreversible inhibition.

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.

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.

Amine oxidase enzymes of sheep blood vessels and blood plasma: a comparison of their properties.

1. A comparison between the biochemical properties of semicarbazide-sensitive amine oxidase (SSAO) activities has been made in sheep blood plasma and arterial wall. 2. The metabolism of benzylamine (BZ) by blood plasma was resolved into high affinity (Km 2.76 +/- 0.24 microM) and low affinity (Km 743 +/- 49 microM) activities. Spermidine metabolism was by a single component (Km 174 +/- 22 microM) and this amine reduced the metabolism of high concentrations of BZ. 3. A single component metabolised BZ in arterial homogenates (Km 11.3 +/- 1.3 microM) and which metabolised spermidine at a very slow rate. 4. Dopamine was deaminated by plasma SSAO and competed with both low and high concentrations of BZ. Dopamine also interfered with arterial metabolism of BZ. 5. These results suggest that there are two SSAO enzymes in sheep blood plasma, with one having similar properties to SSAO in the arterial wall.

Enzymic oxidation of capsaicin.

The oxidation of capsaicin (8-methyl-N-vanillyl-6-nonenamide) has been investigated by means of electrochemical, enzymic and chemical procedures. Capsaicin appears to form a fluorescent dimer comparable with those known to be formed from some other compounds bearing the vanillyl(4-hydroxy-3-methoxybenzyl-) group. If such a dimer of capsaicin were to be formed in tissues, it would bind tightly to lipid structures and its formation would prove difficult to follow. Tests on other substances bearing the vanillyl group that might be used to investigate the dimerization reaction in tissues and tissue extracts showed that 4-hydroxy-3-methoxyphenylacetic acid is a poor second substrate for peroxidase reactions. It was found that 2-methoxy-4-methylphenol (creosol) was more suitable. These results support the suggestion that the oxidation of capsaicin may be involved in some of its biological actions.

Physiological and pathological influences on sheep blood plasma amine oxidase: effect of pregnancy and experimental alloxan-induced diabetes mellitus.

A copper-containing amine oxidase is present in sheep blood plasma and has a high capacity to deaminate spermine and spermidine. The physiological function of this enzyme remains to be determined. Sheep blood plasma amine oxidase (SPAO) was measured by its ability to deaminate spermidine (700 microM) using a peroxidase-linked colorimetric assay developed for microtitre plates. SPAO activity has been studied in a group of Welsh Mountain sheep with experimental alloxan-induced diabetes. This resulted in an increase in SPAO activity which reached a peak of 70 days after alloxan treatment (60 per cent increase). This change could be seen in both pregnant and non-pregnant diabetic sheep. In normal pregnant ewes, SPAO activity remained stable for the first 100 days of pregnancy but declined by 50 per cent in the last month of pregnancy. Together, these findings suggest that SPAO activity is controlled by hormonal influences. This sensitive and convenient assay method could provide clues as to the physiological significance of SPAO and may be a useful clinical chemical indicator in the sheep.

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.

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.

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.

GABAA and GABAB receptor-mediated inhibition of release of 5-hydroxytryptamine in the intermediate lobe of the rat pituitary gland.

Isolated neurointermediate lobes or neural lobes of the rat pituitary gland attached to the pituitary stalk were incubated in vitro and the spontaneous or electrically (pituitary stalk stimulation, 5 Hz, 1,500 pulses) evoked release of 5-hydroxytryptamine was determined. The evoked release of 5-hydroxytryptamine from the neurointermediate lobe was increased fivefold in the presence of the dopamine receptor antagonist (-)-sulpiride (1 microM). The evoked release of 5-hydroxytryptamine from the isolated neural lobe was not altered by (-)-sulpiride. The evoked release of 5-hydroxytryptamine from the isolated neural lobe in the presence of (-)-sulpiride was less than 5% of that from the combined neurointermediate lobe showing that most of the 5-hydroxytryptamine released from the combined neurointermediate lobe originated in the intermediate lobe. The evoked release of 5-hydroxytryptamine from the neurointermediate lobe in the presence of (-)-sulpiride showed a diurnal variation. It was three to five times higher between 9.30 and 14.00 h than between 8.30 and 9.30 h or between 14.00 and 16.00 h. The 5-hydroxytryptamine tissue content at the end of the incubation experiments also showed similar variations which were, however, less pronounced. The evoked release of 5-hydroxytryptamine from the neurointermediate lobe, in the presence of (-)-sulpiride, was reduced by the preferential GABAA receptor agonist muscimol or the selective GABAB receptor agonist (-)-baclofen in a concentration-dependent manner. Bicuculline, a selective GABAA receptor antagonist inhibited the effect of muscimol, but not that of (-)-baclofen. Bicuculline alone did not affect the release of 5-hydroxytryptamine from the gland. It is concluded that the release of 5-hydroxytryptamine in the intermediate lobe is influenced by both dopaminergic and GABAergic mechanisms.

Acetylcholinesterase in the rat neurohypophysis is decreased after dehydration and released by stimulation of the pituitary stalk.

Specific acetylcholinesterase and non-specific cholinesterases are present in all three lobes of the rat pituitary gland. This paper describes two new observations on hypophyseal acetylcholinesterase. Firstly, a prolonged increase of neurohormone secretion evoked by dehydration and sodium loading was accompanied by a decrease in the acetylcholinesterase activity localized to the neural lobe, where acetylcholinesterase has previously been demonstrated in fine nerve fibres. Secondly, electrical stimulation of the pituitary stalk in vitro elicited the release of acetylcholinesterase and non-specific cholinesterases from the combined neural and intermediate lobe indicating that the enzyme can be released from nerve endings in the hypophysis by action potentials. The observed loss of enzyme activity during dehydration may be the consequence of a prolonged activation of cholinergic nerves in the gland, leading to an increased release of acetylcholinesterase, which is not immediately replaced by fresh enzyme. The decrease in acetylcholinesterase in the neural lobe during dehydration may also be connected with its peptidase function and thus with the previously observed loss of substance P from the neural lobe during dehydration [Holzbauer et al. (1984) Neurosci. Lett. 47, 23-28].

Intracerebroventricular dopamine attenuates sodium-induced but not angiotensin-induced drinking in minipigs.

Drinking in response to dopamine (100 micrograms) injected into the lateral cerebral ventricles (i.c.v.), alone, or together with angiotensin II (AII, 300 ng) or hypertonic NaCl (1.4 Osm) was studied in water-replete minipigs trained to obtain their water under operant conditions. Dopamine itself was ineffective at producing drinking and it did not affect AII-induced drinking. Drinking in response to hypertonic NaCl was significantly attenuated by dopamine. Therefore in minipigs angiotensin-induced drinking does not appear to operate through a dopaminergic mechanism whereas sodium-induced drinking does.

Variations in the monoamine oxidase-inhibitory activity ("tribulin?") in pig's urine.

Sandler and his colleagues (see Sandler, 1982) have demonstrated the presence of an endogenous inhibitor of the enzyme monoamine oxidase (MAO) and of benzodiazepine receptor binding in the urine and blood plasma of man and rat. The concentrations of this material increased under stress conditions and it has been named "tribulin". In the present experiments MAO-inhibitory activity was found in extracts of urine and plasma samples of domestic pigs. Evidence was obtained that the inhibitory activity was higher when pigs experienced slight discomfort. Thus it appears that pigs produce a substance similar to tribulin. It may become possible to use such MAO-inhibitory activity as an indicator in the assessment of interaction with the environment in pig husbandry.

The metabolism of dopamine in the blood of ruminant animals: formation of 3,4-dihydroxyphenylacetaldehyde.

A stable derivative of 3,4-dihydroxyphenylacetaldehyde (DOPAL) has been prepared that yields a solution of the parent aldehyde when dissolved in 1 M-hydrochloric acid. Dopamine, when injected into the bloodstream of a sheep, is metabolized to DOPAL which is then converted to 3,4-dihydroxyphenylethanol by aldehyde reductase activity associated with the cellular components of the blood, most probably the erythrocytes. In vitro, dopamine is metabolized by ruminant blood plasma to DOPAL, not to DOPET as previously reported. Dopamine is as good a substrate for sheep plasma amine oxidase as benzylamine. The plasma amine oxidase of ruminant animals could be a protection against the effects of dopamine released from the mast cells in these species.

Uric acid is released in the zona incerta of the subthalamic region of the sheep during rumination and in response to feeding and drinking stimuli.

The technique of intracranial dialysis was used to sample changes in monoamine metabolite and uric acid concentrations in the zona incerta (ZI) of the subthalamic region of conscious sheep during rumination and in response to the presentation of feeding and drinking stimuli. Sequential 10-min dialysis samples were taken over a 5-h period with stimuli presented at hourly intervals. For each animal, sampling was carried out weekly for 2-3 months. Results from HPLC analysis showed that concentrations of uric acid rose significantly in the ZI, but not in the cortex or peripheral plasma, during rumination, eating, drinking or exposure to the sight or smell of food. No response to control stimuli--loud noise, smell of amyl acetate or sight of a syringe--was found. Concentrations of 5-hydroxyindol-3-yl-acetic acid in the ZI were unaffected.

Release of endogenous 5-hydroxytryptamine from the intermediate lobe of the rat pituitary gland is inhibited by endogenous dopamine.

The release of endogenous 5-hydroxytryptamine (5-HT) from the in vitro incubated neurointermediate lobe (NIL) of the rat hypophysis was measured by HPLC with electrochemical detection. 5-HT release evoked by electrical stimulation of the pituitary stalk from the NIL, but not from the isolated neural lobe (NL) was enhanced in the presence of the dopamine receptor antagonist sulpiride (1 microM). The opiate receptor antagonist naloxone (1 microM) had no effect on the evoked release of 5-HT from the NIL or NL. In conclusion, the release of endogenous 5-HT from the intermediate lobe is under inhibitory control of endogenous dopamine.

A semi-automated fluorimetric method for measuring acetylcholinesterase activity in small volumes of cerebrospinal fluid and tissue extracts using acetylcholine as substrate.

A fluorimetric method for the assay of acetylcholinesterase (AChE; EC 3.1.1.7) in tissue extracts and cerebrospinal fluid, using acetylcholine as the substrate, is described. The method is based on the measurement of hydrogen peroxide, formed by the oxidation of choline resulting from the action of AChE on acetylcholine, by means of horseradish peroxidase and 4-hydroxy-3-methoxyphenylacetic acid (HVA) to yield a fluorescent derivative. Choline, if present in any sample to be analysed, is first removed by a modification of the peroxidase reaction during preincubation.