Conjugated Dopamine in Superfusates of Slices of Rat Striatum

Abstract: An acid-hydrolyzable conjugate of 3,4-dihydroxyphenylethylamine (dopamine, DA) was detected in superfusates from slices from rat striatum. The concentrations of endogenous free and conjugated DA, and of the acid metabolites (3,4-dihydroxyphenylacetic acid [DOPAC] and homovanillic acid [HVA]) in superfusates were measured using HPLC with electrochemical detection. Conjugated DA in superfusates represented 10–20% of the free DA under basal conditions and during release evoked by p -tyramine (5 × 10⁻⁶ M to 5 × 10⁻⁴ M ); much smaller amounts of conjugated DA overflowed into superfusate when DA was released by equimolar concentrations of β-phenylethyl-amine. Surprisingly, inhibition of monoamine oxidase by the inhibitors N -methyl- N -propargyl-3-(2,4-dichlorophenoxy)propylamine hydrochlo-ride (clorgyline) or N -methyl- N -2-propynylbenylamine (pargyline) had little effect on the amounts of conjugated DA present in superfusate. Under basal conditions, the amounts of conjugated DA in superfusate were always less than the amounts of DOPAC but quite similar to the amounts of HVA. However, during release of DA evoked by p -tyramine the concentrations of conjugated DA in superfusate showed much more pronounced increases than those of the acidic metabolites.     

EFFECT OF COPPER STATUS ON BRAIN NEUROTRANSMITTER METABOLISM IN THE LAMB

Abstract— Ataxic and non-ataxic lambs reared under field conditions which gave rise to low copper status were treated with copper intravenously. Untreated ataxic animals served as controls. The neurotransmitter amines, dopamine, norepinephrine and serotonin, were determined in the anterior and posterior regions of the brain stem. Dopamine levels in the anterior region, including the corpus striatum, were significantly lower in the untreated animals than in those treated with copper. Norepinephrine levels were also lower but serotonin concentrations were not different. Plasma amine oxidase activity was markedly higher in the copper treated animals but monoamine oxidase activity in brain stem homogenates was not significantly affected. The monoamine oxidase activity in cortical and cerebellar homogenates was significantly lower in the treated animals than in the untreated animals.     

Pathological changes in platelet histamine oxidases in atopic eczema

Increased plasma histamine levels were associated with significantly lowered diamine and type B monoamine oxidase activities in platelet-rich plasma of atopic eczema (AE) patients. The diamine oxidase has almost normal cofactor levels (pyridoxal phosphate and Cu(2+)) but the cofactor levels for type B monoamine oxidase (flavin adenine dinucleotide and Fe(2+)) are lowered. The biogenic amines putrescine, cadaverine, spermidine, spermine, tyramine and serotonin in the sera, as well as dopamine and epinephrine in EDTA-plasma were found to be normal. It is unlikely, therefore, that these amines are responsible for the decreased activities of monoamine and diamine oxidase in these patients. The most likely causative factors for the inhibition of the diamine oxidase are nicotine, alcohol, food additives and other environmental chemicals, or perhaps a genetic defect of the diamine oxidase.     

Histochemical characterization of the monoamine-containing cells of the adenohypophysis in the chinese quail

Summary The demonstration of biogenic amine-containing cells is classically performed by means of the formaldehyde-induced fluorescence technique after administration of the monoamine precursors, L-dopa or 5-hydroxytryptophan (5-HTP). The injection of these two compounds rapidly leads to the formation of large amounts of dopamine or serotonin. The mechanism and site of decarboxylation are therefore of great importance.The biochemical pathway evoked above is studied in the avian adenohypophysis by two different methods involving enzyme inhibition. In the first method, a seryl-derivative of hydrazine (benserazide, Ro 4-4602) is used for the inhibition of the aromatic L-amino acid decarboxylase. This experiment supports the hypothesis that a variable part of L-dopa or 5-HTP can be decarboxylated out of the gland, although we agree with the possibility that, in most cases, biogenic amine precursors may be taken up by endocrine cells. Interference between benserazide and precursor may readily be excluded.The second way involves monoamine oxidase (MAO) inhibition by nialamide. In is based on the differential breakdown of L-dopa on one side, and dopamine and serotonin on the other side. While L-dopa is catabolized by O-methylation, the amines undergo an oxidative deamination by means of MAO. Inhibition of this enzyme leads to the accumulation of fluorescent material in the PAS-positive cells of the adenohypophysis.Observations made under various experimental conditions suggest that, in the Chinese quail in particular, a variable part of dopamine and/or serotonin is synthetized out of the adenohypophysis, and that this substances may be subsequently taken up by the PAS-positive cells of the gland where they possibly play a rôle in synthesis, storage and/or secretion of the polypeptide hormone. It is highly probable that these PAS-positive cells are to be regarded as corticotrophs, but it is possible that -gonadotrophs, melanotrophs and/or somatotrophs must also be regarded as aminergic endocrine cells.A comparison between the metabolism of biogenic amines in a peripheral organ such as hypophysis and the situation in the central nuclei of the brain, as regards the blood-brain barrier, is made.     

Antidepressants and the monoamine masquerade

Neurotransmitter transporters have long been known to recognize related compounds as substrates, resulting in the accumulation and release of so-called "false transmitters." In this issue of Neuron, Zhou et al. show that when serotonin levels are elevated by inhibition of either serotonin reuptake or of monoamine oxidase, dopamine neurons accumulate serotonin. The results suggest that release of serotonin by dopamine neurons may contribute to the effects of multiple major classes of antidepressants.     

Different roles of dopamine and serotonin in conditioned passive avoidance response of rats

Deamination of dopamine and serotonin by monoamine oxidase was studied in the prefrontal cortex, striatum, hippocampus and amygdaloid complex of the brain of rats during retrieval of conditioned passive avoidance response. Changes in the dopamine and serotonin metabolism were observed in different brain structures. A decrease in dopamine-deaminating activity of monoamine oxidase was found in the hippocampus, striatum and prefrontal cortex. At the same time, serotonin-deaminating activity of the enzyme was decreased in the striatum and increased in the amygdaloid complex, whereas it did not change in the prefrontal cortex and hippocampus. The observed changes in dopamine metabolism in the prefrontal cortex and hippocampus and serotonin metabolism in the amygdaloid complex indicate that dopamine and serotonin are involved in the regulation of two different processes mediating the memory trace retrieval. Dopamine is involved in neuronal mechanisms of information processes providing the strategy of behavior, whereas serotonin is related to emotional mechanisms of memory.     

PRODUCTS OF BIOGENIC AMINE METABOLISM IN THE LOBSTER: SULFATE CONJUGATES

Octopamine, dopamine and serotonin, the three biogenic amines found in the lobster nervous system, are each converted by lobster tissues into two principal classes of products, A and B metabolites. In this paper, evidence is presented that the B metabolites are sulfate conjugates of the amines and their A metabolites. Two double-labelled conjugates were formed from each of the three amines during incubations of lobster nerves with tritiated amine and ³⁵SO₄. When the two octopamine conjugates were hydrolyzed by mild acid, one of the conjugates was converted to a mixture of ³⁵SO₄ and [³H]-octopamine, and the other to a mixture of ³⁵SO₄, [³H]octopamine, and [³H]metabolite A. [³H]Metabolite A was also converted to octopamine by acid hydrolysis. The results indicated that one of the double-labelled conjugates was octopamine-sulfate, and the other metabolite-A-sulfate. An enzyme fraction prepared from nerve homogenates catalyzed the synthesis of double-labelled sulfate conjugates from the tritiated amines and [³⁵S]3′-phosphoadenosine-5′-phospho-sulfate. Double-labelled conjugates formed in this way contained 1 mol of sulfate per mol of amine. Indirect evidence suggested that the sulfate was in ester linkage with the ring hydroxyls of the amines. Neither monoamine oxidase, nor catechol-O-methyl transferase is found in lobster tissues; therefore, in these animals, sulfation may be a major means of inactivation of the biogenic amines following their release from nerve endings.     

Neuromediator mechanisms of the effect of the antihistamine agent dimebone on the brain

Dimebone was shown to inhibit monoamine oxidase (MAO) deaminating dopamine and serotonin, decrease dopamine metabolism in the basal ganglia of the rat brain, increase noradrenaline level and depress dopamine deamination in the hypothalamus. Dimebone first increased and then diminished the release of dopamine in the cortex, with the concomitant MAO activation and the increase in dopamine and noradrenaline levels. The in vitro experiments have demonstrated that dimebone (10(-4)) preferentially inhibited MAO activity, type B and dopamine deamination in homogenates of different rat brain structures. The role of MAO inhibition in the mechanism of dimebone action on the catecholamine metabolism in the brain structures and its stimulating effect on CNS are discussed.     

Coupling of Dopamine Oxidation (Monoamine Oxidase Activity) to Glutathione Oxidation Via the Generation of Hydrogen Peroxide in Rat Brain Homogenates

Abstract: Homogenates of perfused rat brain generated oxidized glutathione from reduced glutathione during incubation with dopamine or serotonin. This activity was blocked by pargyline. a monoamine oxidase inhibitor, or by catalase, a scavenger of hydrogen peroxide. These results demonstrate formation of hydrogen peroxide by monoamine oxidase and the coupling of the peroxide to glutathione peroxidase activity. Oxidized glutathione was measured fluorometrically via the oxidation of NADPH by glutathione reductase. In the absence of added dopamine or serotonin, a much smaller amount of reduced glutathione was oxidized: this activity was blocked by catalase, but not by pargyline. Therefore, endogenous production of hydrogen peroxide, not linked to monoamine oxidase activity, was present. These results indicate that glutathione peroxidase (linked to hexose monophosphate shunt activity) can function to eliminate hydrogen peroxide generated by monoamine oxidase and other endogenous sources in aminergic neurons.     

I. Determination of the endogenous and evoked release of catecholamines from the hypothalamus and caudate nucleus of the conscious and unrestrained rat

The action of cathecholamines within the CNS is important for the expression of numerous vegetative and behavioral functions. To understand the role these amines play, it is necessary to measure changes in the levels of these transmitter substances by utilizing new developments and methodology in the behaving animal. Utilizing new developments in methodology, it is possible to measure the release of amines into perfusates obtained from specific sites in the brain of the rat under basal and evoked conditions without prior purification or concentration.Using the push-pull perfusion technique, perfusates were obtained from the hypothalamus and caudate nucleus and analyzed by liquid chromatography with electrochemical detection. It is possible to readily determine basal release of dopamine from the caudate nucleus. Detection of both dopamine and noradrenaline is possible under ephedrine stimulated conditions from both the caudate nucleus and the hypothalamus. Although levels of serotonin (5-HT) were detected in brain perfusates, it may not be of neuronal origin. It may be possible to use these techniques to delineate the roles these amines play in various physiological functions.     

DISTRIBUTION OF BIOGENIC AMINES AND RELATED ENZYMES IN THE RAT PITUITARY GLAND

Abstract— Biogenic amines and related enzymes were quantitatively measured in the pituitary gland of the rat. The sensitivity of the assays used allows the determinations to be performed in single pituitary lobes. Relatively high values of histamine and serotonin were found in all three lobes, with higher amounts in the posterior and intermediate lobes. Highest catecholamine concentrations were detected in the posterior lobe, and only very low amounts of dopamine were measured in the anterior lobe. Throughout the gland, norepinephrine concentrations were low, about one-tenth that of dopamine. Tryptamine could not be detected. High levels of A and B monoamine oxidase were found in all three pituitary lobes. Tyrosine hydroxylase activity was measured in the posterior and intermediate lobes, but was not detected in the anterior lobe. Tryptophan hydroxylase was present in all three pituitary lobes. A relatively low catechol- O -methyltransferase activity was found in the anterior lobe, and none was detected in the intermediate and posterior lobe. Choline acetyltransferase, dopamine-β-hydroxylase and phenylethanolamine- N -methyltransferase activities could not be detected.     

Biogenic Aldehydes in Brain: On Their Preparation and Reactions with Rat Brain Tissue

When 1 mM serotonin, dopamine, or norepinephrine was incubated with a monoamine oxidase preparation (mitochondrial membranes) in the presence of 4 mM sodium bisulfite, 85-95% of the amines were oxidized to the corresponding aldehydes. In the absence of bisulfite, the recoveries were only approximately 30%, and dark colored products were formed during the incubations. The aldehydes derived from tyramine, octopamine, methoxytyramine, and normetanephrine were also prepared by the use of this method. The bisulfite-aldehyde compounds were stable during storage at -20 degrees C. Bisulfite-free aldehyde solutions were made by diethylether extraction. When the aldehydes derived from dopamine or serotonin were incubated with rat brain homogenates, they were found to disappear in an aldehyde dehydrogenase- and aldehyde reductase-independent manner. The disappearance of the latter aldehyde was more pronounced, and the results indicated that this aldehyde may react with both proteins and phospholipids.     

Effect of L-tryptophan and restraint stress on hypothalmic and brain serotonin turnover, and pituitary TSH and prolactin release in rats.

The dose response and time course effects of L-tryptophan and restraint stress on the metabolism of serotonin and release of thyroid stimulating hormone (TSH) and prolactin (PRL) were tested in male rats. Both treatments increased serotonin turnover in the hypothalamus (H) and remaining brain tissue minus the cerebellum (brain) as determined by enhanced accumulation of serotonin following monoamine oxidase (MAO) inhibition. L-tryptophan but not restraint stress elevated levels of tryptophan in the cerebellum. Both L-tryptophan and restraint stress inhibited TSH release and stimulated PRL release. These findings indicate that enhanced rates of serotonin turnover produced by L-tryptophan and physical restraint are associated with inhibition of TSH and stimulation of PRL release from the anterior pituitary.     

Development of monoamine oxidase activity and monoamine effects on glutamate release in cerebellar neurons and astrocytes

Activities of monoamine oxidase (MAO) A and B were measured during the first month of postnatal development in mouse cerebellum and in primary cultures of either cerebellar granule cells or cerebellar astrocytes, derived from 7-day-old cerebella. In addition, effects of the two monoamines, serotonin (a MAO A substrate) and phenylethylamine (a MAO B substrate) on the release of glutamate under resting conditions and in a transmitter related fashion (i.e., potassium-induced, calcium-dependent glutamate release) were studied during the same period. Both MAO A and MAO B activities increased during in vivo development (beginning around postnatal day 14) and in cultured astrocytes (during a comparable time period and to a similar extent), but remained constant at a low level in granule cells. In 4-day-old cerebellar granule cell cultures there was no potassium-induced glutamate release but serotonin as well as phenylethylamine reduced the release in both the presence and absence of excess potassium. In 8- and 12-day-old granule cell cultures and in 8- and 18-day old astrocyte cultures there was a pronounced glutamate release during superfusion with 50 mM K⁺. In both neurons and astrocytes this response was inhibited by 1 nM of either serotonin or phenylethylamine. In the astrocytes the inhibition was followed by an increased release of glutamate in both the presence and absence of the high potassium concentration, whereas the 8-day-old neurons showed only a slight increase in glutamate release after the with-drawal of the monoamine and only in the absence of excess potassium. The response was almost identical in 8-and 18-day-old astrocytes in spite of the marked difference in MAO activities.Special issue dedicated to Dr. Paola S. Timiras.     

Effect of co-administration of varenicline and antidepressants on extracellular monoamine concentrations in rat prefrontal cortex

Since a substantial proportion of smokers have comorbid mood disorders, the smoking cessation aid varenicline might occasionally be prescribed to patients who are simultaneously treated with antidepressants. Given that varenicline is a selective nicotinic acetylcholine receptor partial agonist and not a substrate or inhibitor of drug metabolizing enzymes, pharmacokinetic interactions with various classes of antidepressants are highly unlikely. It is, however, conceivable that varenicline may have a pharmacodynamic effect on antidepressant-evoked increases in central monoamine release. Interactions resulting in excessive transmitter release could cause adverse events such as serotonin syndrome, while attenuation of monoamine release could impact the clinical efficacy of antidepressants. To investigate this we examined whether varenicline administration modulates the effects of the selective serotonin reuptake inhibitor sertraline and the monoamine oxidase inhibitor clorgyline, given alone and combined, on extracellular concentrations of the monoamines serotonin, dopamine, and norepinephrine in rat brain by microdialysis. Given the important role attributed to cortical monoamine release in serotonin syndrome as well as antidepressant activity, the effects on extracellular monoamine concentrations were measured in the medial prefrontal cortex. Responses to maximally effective doses of sertraline or clorgyline and of sertraline plus clorgyline were the same in the absence as in the presence of a relatively high dose of varenicline, which by itself had no significant effect on cortical monoamine release. This is consistent with the binding profile of varenicline that has insufficient affinity for receptors, enzymes, or transporters to inhibit or potentiate the pharmacologic effects of antidepressants. Since varenicline neither diminished nor potentiated sertraline- or clorgyline-induced increases in neurotransmitter levels, combining varenicline with serotonergic antidepressants is unlikely to cause excessive serotonin release or to attenuate antidepressant efficacy via effects on cortical serotonin, dopamine or norepinephrine release.     

Monoamine oxidase activity measurements using radioactive substrates

The use of Amberlite CG-50, Dowex 50 and solvent extraction for separation of the oxidation products of the biogenic amines are compared, and measurements of monoamine oxidase activity using ¹⁴C-labeled biogenic amines are described. K_m data for tyramine, dopamine, tryptamine, and serotonin for monoamine oxidase activity of rabbit brain mitochondria are reported. Rates of product formation from [¹⁴C]tyramine are compared with polarographic measurements of oxygen utilization using purified MAO and intact mitochondria from rabbit liver and brain. Difficulties in comparative measurements of monoamine oxidase activity and some reasons for wide variations in published data are discussed.     

The appraisal of the level of photo- and peroxide resistance of human erythrocytic and lymphocytic cells in presence of biogenic amines

The changes of photo- and peroxide resistance of erythrocytic and lymphocytic cells of human under influence of UV and some active oxygen forms in presence of biogenic amines such as serotonin, dopamine, adrenalin, histamine had been investigated. While investigating the degree of photohemolysis of erythrocytes it was discovered that biogenic compounds raise UV stability of erythrocytic membranes. By using the method of chemiluminescence it was established that biogenic amines increased the degree of peroxide resistance of human erythrocytic and lymphocytic cells. The decrease of the level of erythrocytic diene conjugates under influence of UV by serotonin and histamine was also discovered.     

Monoamine oxidase activity of rat brain under cold exposure

Cold stress and cold adaptation were studied for their effect on the activity and substrate specificity of the monoamine oxidase A and B and on the Km of serotonin deamination in the rat brain mitochondria and supernatant. Mitochondrial monoamine oxidase Km with serotonin is established to increase more than twice under cold stress and decrease considerably in cold adapted rats. The lowering of the mitochondrial monoamine oxidase A activity is accompanied by the appearance of serotonin and the glucosamine deaminating activity in supernatant. The data suggest that decrease in the monoamine oxidase activity under cold stress may be caused by both release of the enzyme from mitochondrial membrane and changes in its catalytic property alteration.     

Lack of effect of corticotropin releasing factor on hypothalamic dopamine and serotonin synthesis turnover rates in rats

Catecholamine and serotonin neurons in the hypothalamus regulate the secretion of corticotropin releasing factor (CRF). We considered the possibility that CRF might in turn affect the activity of these aminergic neurons. We examined the effect of intracisternal administration of synthetic CRF on the synthesis turnover rates of dopamine and serotonin in the hypothalamus of adult male rats using two different methods to assess turnover. In one study, we measured the accumulation of L-dihydroxyphenylalanine (L-DOPA) or 5-hydroxytryptophan (5-HTP) in mediobasal hypothalamus after L-aromatic amino acid decarboxylase inhibition with m-hydroxybenzylhydrazine 20 min before sacrifice, and in the second study we measured the accumulation of dopamine, norepinephrine, epinephrine and serotonin after monoamine oxidase inhibition with pargyline 20 min before sacrifice. The commercial CRF which we administered intraarterially increased plasma ACTH and corticosterone concentrations. Intracerebral CRF 5 to 20 micrograms 20 min before sacrifice or 20 micrograms 110 min before sacrifice did not alter the m-hydroxybenzylhydrazine-induced accumulation of L-DOPA or 5-HTP when compared with saline vehicle-injected controls. CRF 20 micrograms did not alter basal concentration or pargyline-induced accumulation of the catecholamines or serotonin in whole hypothalamus when compared with saline vehicle-injected controls. Thus, intracisternal administration of CRF did not alter hypothalamic dopamine or serotonin synthesis rates as assessed by two nonsteady state turnover methods. The data suggest that the release of CRF from neurons in hypothalamus does not alter the activity of catecholamine or serotonin neurons in the hypothalamus of normal adult male rats.     

Changes in tissue concentrations of sulfhydryl groups in relation to the metabolism of biogenic amines in vitro.

Quantitative changes in nonprotein sulfhydryl (NP-SH) and protein sulfhydryl (P-SH) contents of rat tissue homogenates after incubation in the presence and absence of serotonin and dopamine were investigated. According to the enzymic determination of NP-SH using partially purified rat liver formaldehyde dehydrogenase, 81.4% of NP-SH in rat brain could be considered as reduced glutathione (GSH). The decrease in GSH was significantly less with added NAD in liver homogenate, but not in brain. On the other hand, addition of pargyline to the incubation mixture resulted in a smaller decrease in GSH-lost. These facts revealed that the decrease in GSH in the presence and absence of serotonin or dopamine is dependent on both the activities of monoamine oxidase and aldehyde dehydrogenase of tissues, which are involved in formation of aldehyde derivatives of these amines. In addition, decrease of P-SH in the presence of serotonin or dopamine may be protected by addition of GSH as a scavenger of the aldehyde formed.