Serotonergic characteristics of rainbow trout divergent in stress responsiveness.

Juvenile rainbow trout divergent in their cortisol response to confinement stress (HR: high responsive or LR: low responsive fish) were exposed to either 1 or 3 h of confinement stress. Untreated fish served as control. After the exposure blood and brain samples were collected. From the blood samples, the levels of cortisol and catecholamines were determined, while the brain serotonergic and monoamineoxidase (MAO) activity was determined in four different brain areas (brain stem, hypothalamus, telencephalon and optic tectum). Our results show that the LR fish responds to handling stress with a higher increase in plasma epinephrine compared to HR fish. Our results also show that confinement stress leads to a larger increase in the serotonergic activity in the brain stem and telencephalon in LR fish compared to HR fish. These results support the hypothesis that stress coping strategies similar to those described in mammals also exists in fish. Further, our results have shown that the MAO activity increases in optic tectum and hypothalamus of rainbow trout during confinement stress, while it remains unchanged or decreases in brain stem and telencephalon. Moreover, the MAO activity does not differ between the two selection lines. This indicates that MAO participates actively in the stress response without contributing to the differences in stress coping strategies.

Cytoprotection by deprenyl and tolcapone in a cell culture model of cerebral ischaemia.

Foetal rat brain aggregation cultures were exposed to a single episode of anoxia and hypoglycaemia for 30 min. Lactate dehydrogenase specific activity was estimated in the culture medium after ischaemia as a marker of lost cell integrity. Release of lactate dehydrogenase was most prominent during the first 24 hr period after the ischaemic damage, then it gradually declined. Immediately after ischaemic exposure, the cultures were treated with different concentrations of L-deprenyl or tolcapone. Significantly lower amounts of lactate dehydrogenase leaked into the culture medium during the first 24 hr after the ischaemic episode in cultures treated with deprenyl or tolcapone (1-100 nM). These results suggest that deprenyl and tolcapone may reduce cell damage after ischaemia, at doses causing enzyme inhibition.

A cell culture model of cerebral ischemia as a convenient system to screen for neuroprotective drugs.

Aggregation cultures of rat brain were exposed to a combination of anoxia and hypoglycaemia for 30 minutes. Thereafter, the release of lactate dehydrogenase into the cell culture medium was monitored up to 4 days as a measure of cell damage after the ischemic insult. Some cultures were treated with different concentrations of deprenyl or tolcapone, selective inhibitors of monoamine oxidase B and catechol-O-methyltransferase, respectively. After 1 day in culture, the release of lactate dehydrogenase was significantly reduced in cultures treated with deprenyl (at 1 nM. 100 nM, and 10 microM), as well as in cultures treated with 1 nM or 100 nM tolcapone; 10 microM of tolcapone, on the other hand, resulted in a toxic effect on the cell aggregates. No differences in the release of lactate dehydrogenase into the medium was observed in the aggregates treated with drugs as compared with the control cultures after 2 or 4 days post-ischemia.

Oxidative stress decreases antioxidant enzyme activities in reaggregation cultures of rat brain cells.

The brain has been suggested to be especially sensitive to damage by reactive oxygen species. In this study, we examined the effects of hyperoxic conditions on the activities and mRNA levels of antioxidant enzymes in reaggregation cultures of rat forebrain cells. Cultures were exposed to 80% oxygen for 12-60 h starting on Days 17 and 33 in culture. Superoxide dismutase activities and mRNA levels were not affected by hyperoxia, whereas catalase activity was slightly decreased after 24 h in 80% oxygen at Day 17. Glutathione peroxidase activity was markedly decreased already after 12 h of hyperoxia, and decreased activities of glutathione reductase and glucose-6-phosphate dehydrogenase were also noted. The glutathione peroxidase mRNA levels were increased in hyperoxic cultures at Day 17 but not at Day 33. These results suggest that the enzymatic defense mechanisms against reactive oxygen species in the brain are rather weak and deteriorate during oxidative stress but that a potential for compensatory upregulation exists at least during the first postnatal weeks.

Ethanol-induced increase in catalase activity in reaggregation cultures of rat brain cells is due to increased oligodendrocyte differentiation.

Recent studies have shown that ethanol exposure of reaggregation cultures of fetal rat brain cells causes an increased activity and amount of catalase, and also an increased activity of the oligodendrocyte marker enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP). In the present study, reaggregation cultures were grown in the presence of 40 mM ethanol during 17 days, corresponding to a period in vivo from gestational day 17 to postnatal day 12. The activities of catalase, the peroxisomal marker enzyme acyl-coenzyme A oxidase and CNP were increased in ethanol-treated cultures. Immunofluorescence experiments confirmed that catalase mainly resided in oligodendrocytes. By immunoperoxidase staining for CNP followed by image analysis, it was observed that the proportion of CNP-positive cells, as well as the intensity of staining, was increased in ethanol-treated cultures. Thus, the previously reported increase in catalase protein and activity in ethanol-treated cultures might be due to a stimulation, or earlier onset, of oligodendrocyte differentiation.

Effects of nerve growth factor on X-irradiated reaggregation cultures of rat brain cells.

The effects of exogenously added nerve growth factor (NGF) on reaggregation cultures of foetal rat brain cells after X-irradiation with 2 Gy were studied. Irradiation caused decreased protein and DNA levels, which was not prevented by NGF. The activities of the cholinergic marker enzymes choline acetyl transferase and acetylcholine esterase were increased in irradiated cultures. However, no difference in the activities of these enzymes was found between irradiated and unirradiated NGF-treated cultures. Irradiation did not affect the activity of the marker enzyme for oligodendrocytes (2',3'-cyclic nucleotide 3'-phosphodiesterase), but caused an increase in the astrocyte marker (glutamine synthetase) activity. This effect on astrocytes was prevented by NGF.

Inhibition of aldehyde dehydrogenases by methylene blue.

The effect of the redox dye methylene blue on the stability of NADH and on the activity of the enzyme aldehyde dehydrogenase (ALDH; EC 1.2.1.3) was examined. NADH was measured by HPLC with fluorometric or spectrophotometric detection. The ALDH activity assays were carried out by following the formation of 3,4-dihydroxyphenylacetic acid (DOPAC) from 3,4-dihydroxyphenylacetaldehyde (DOPAL) using HPLC and electrochemical detection. Incubation of NADH solutions in the presence of methylene blue resulted in a time-dependent direct oxidation of NADH. Methylene blue inhibited the human erythrocyte and leukocyte ALDHs and the rat liver mitochondrial low-Km ALDH in a concentration-dependent manner. The inactivation was reversible by dilution, and kinetic analysis indicated that methylene blue inhibits the rat liver mitochondrial low-Km and human erythrocyte ALDHs competitively with respect to DOPAL, while no effect of the NAD+ concentration was apparent. For the rat liver low-Km ALDH, a Ki of 8.4 +/- 2.8 microM (mean +/- SD; N = 5) was calculated. The inhibition of ALDH and the resulting decrease in the redox effect on the NAD system bound to alcohol dehydrogenase (EC 1.1.1.1) could explain the protective effect of methylene blue against metabolic redox effects of ethanol.

Increase in catalase activity in developing rat brain cell reaggregation cultures in the presence of ethanol.

The aim of this investigation was to study the effects of ethanol on antioxidant enzymes in the developing brain, using reaggregation cultures of fetal rat brain cells as a model. The cultures were grown in the presence of 20 and 40 mM ethanol from day 2 until day 44 of the culture period, corresponding to a period in vivo from gestational day 17 to postnatal day 37. The catalase (EC 1.11.1.6) activity was consistently increased at all observation periods, from culture day 11 to day 44, by both doses of ethanol, and an immunoblot showed that the amount of catalase protein was markedly increased. The activities of manganese and copper-zinc superoxide dismutase (EC 1.15.1.1) and glutathione peroxidase (EC 1.11.1.9) were largely unaffected.

Development of antioxidant enzymes in rat brain and in reaggregation culture of fetal brain cells.

The development of antioxidant enzymes in rat brain and reaggregation cultures of fetal brain cells was studied from embryonic day 15 to postnatal day 45. Both in vivo and in culture, the copper-zinc superoxide dismutase activity first increased and then decreased with age, whereas the manganese superoxide dismutase activity increased throughout the period. Catalase showed a maximum activity at day 5 after birth, thereafter decreasing to adult level around day 30, both in vivo and in culture. The glutathione peroxidase activity increased from the first week after birth and reached adult level at day 45. In culture, the activity of this enzyme was slightly lower. The good correlation between the development of the antioxidant enzymes in vivo and in culture suggests that reaggregation cultures might be a valuable system for studying defense mechanisms against free radicals in the brain.

Effects of low-dose X-irradiation on mouse-brain aggregation cultures.

Biochemical and morphological differentiation in reaggregating mouse-brain cell cultures after low-dose radiation (0.5 Gy) in vitro was studied. Cells were irradiated on culture day 2, corresponding to embryonic day 15-16, and different glial and neuronal markers were followed through development to postnatal day 40. The shape and size of irradiated aggregates were more irregular and smaller compared with controls. Total amounts of DNA and protein were significantly lower in irradiated aggregates than in controls between days 8 and 20. After 30 days in culture activities of the glial markers glutamine synthetase (GS) and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) were lower in X-irradiated aggregates than in controls. However, after 40 days the CNP activity in irradiated aggregates increased to levels above those of the controls. Irradiated and control aggregates did not differ significantly in neuronal marker enzyme activities, i.e. choline acetyltransferase (ChAT), acetylcholine esterase (AChE) and glutamic acid decarboxylase (GAD) measured on a per mg protein basis. On days 20 and 30 the amount of nerve growth factor (NGF) was two-fold higher in irradiated aggregates compared with non-irradiated ones, suggesting that, after irradiation, surviving cells in culture were induced to produce more NGF. After 40 days the amount of NGF in irradiated aggregates had decreased to the level found in the control aggregates.

Effects of disulfiram and coprine on rat brain tryptophan hydroxylation in vivo.

The effects of disulfiram and coprine on brain tryptophan hydroxylation, and on the brain-levels of serotonin and 5-hydroxyindole-3-acetic acid, were studied in 45 and 235 days old rats. Both drugs were found to affect the parameters measured. Disulfiram increased the rate of tryptophan hydroxylation and the serotonin level in young rats, while these parameters appeared to be unaffected in old disulfiram-treated rats. In contrast, coprine increased the rate of tryptophan hydroxylation and possibly also the serotonin level in old rats while no significant effects were seen in young coprine-treated rats. Regarding the 5-hydroxyindole-3-acetic acid concentration, this appeared to be increased by disulfiram in both age-groups, while no significant effects were found with coprine. The lack of similarity in the action of disulfiram and coprine, which are both potent aldehyde dehydrogenase inhibitors, suggests that the effects found were not caused by an impaired metabolism of monoamine-derived biogenic aldehydes.

Reactions of biogenic aldehydes with hemoglobin.

The aldehyde derivatives of dopamine and serotonin, 3,4-dihydroxyphenylacetaldehyde (DOPAL) and 5-hydroxyindole-3-acetaldehyde (5-HIAL), respectively, were incubated with human hemoglobin under physiological conditions. Both DOPAL and 5-HIAL, as well as dopamine, showed a time-dependent disappearance during the incubations, whereas this was not observed with serotonin. The amounts of free aldehydes recovered after incubation with hemoglobin, as analysed by high-performance liquid chromatography with electrochemical detection, corresponded to the amounts of acid metabolites formed in enzymatic assays, when the samples instead were incubated with aldehyde dehydrogenase. Incubations with DOPAL, 5-HIAL, or dopamine, and hemoglobin also resulted in distinct increases in the absorption spectra between 250-350 nm, whereas no similar increase was observed with serotonin. Addition of sodium borohydride to the incubates, which is used to stabilize Schiff base adducts between aldehydes and proteins, resulted in reduction of DOPAL and 5-HIAL to their corresponding alcohol metabolites. However, a rapid initial disappearance of the aldehydes, as analysed from the recoveries of the alcohol metabolites, was observed, followed by a more slow disappearance rate throughout the incubation period.

Neurons in primary cultures from five defined rat brain regions--cellular composition and morphological appearance.

Primary cultures from 15-17 days old fetal rat cerebral cortex, striatum, hippocampus, substantia nigra and brain stem were grown for ten days. Cell aggregates were formed one to two days after seeding. The cell bodies migrated peripherally from the clusters during development and networks of processes were formed. The cultures from the different brain regions contained predominantly neurons, stained by an antiserum against the neuron-specific enolase (NSE). There were differences in morphological appearance of the aggregates and also of the single neuronal cells cultivated from the various brain regions. On the bottom of the culture dishes a monolayer was formed of predominantly undifferentiated (mesenchymal-like) cells. Some cells of the monolayer stained for the astrocyte markers glial fibrillary acidic protein (GFAp) or S-100. The majority of the cells were, however, unstained to these markers. Very few endothelial cells and macrophages were observed.

Effect of acute ethanol administration on human blood aldehyde dehydrogenase activity.

The aldehyde dehydrogenase (EC 1.2.1.3) activity in human blood was measured after acute oral administration of two different doses of ethanol to healthy subjects. No changes in aldehyde dehydrogenase activity were observed in either of the two dose groups, as compared to control subjects receiving no ethanol. Likewise, incubation of human whole blood samples with 20 mM ethanol for 2 hr caused no change in aldehyde dehydrogenase activity. The results show that human blood aldehyde dehydrogenase activity is not affected by acute ethanol administration.

Effects of disulfiram, cyanamide and 1-aminocyclopropanol on the aldehyde dehydrogenase activity in human erythrocytes and leukocytes.

The effects of the aldehyde dehydrogenase (ALDH; EC 1.2.1.3) inhibitors disulfiram, cyanamide and 1-aminocyclopropanol (ACP) on the ALDH activities in human erythrocytes and leukocytes were studied. Assays were performed by incubating intact or sonicated blood cells in the presence of different concentrations of the inhibitors, using 3,4-dihydroxyphenylacetaldehyde, the aldehyde derived from dopamine oxidation, as the substrate. The amount of acid metabolite formed was measured using high-performance liquid chromatography with electrochemical detection. The erythrocyte ALDH was extremely sensitive to disulfiram, and only about 0.5 microM was needed to cause a 50% inhibition of the activity. The leukocyte activity was less sensitive, and showed a similar degree of inhibition at an 100-fold higher concentration of disulfiram. Cyanamide and ACP were both potent inactivators of the leukocyte ALDH activity, giving a 50% inhibition at concentrations of 10 and 50 microM, respectively, whereas the erythrocyte activity was much less affected. Diethyldithiocarbamate, the reduced metabolite of disulfiram, and coprine, from which ACP is derived, were much less effective inhibitors of the erythrocyte and leukocyte ALDH activities than were disulfiram and ACP.

Effects of ethanol, acetaldehyde and disulfiram on the metabolism of biogenic aldehydes in isolated human blood cells and platelets.

The effects of ethanol, acetaldehyde and disulfiram on the metabolism of biogenic aldehydes were measured in different human blood fractions. Intact erythrocytes, leukocytes and platelets were incubated in phosphate-buffered saline with 3,4-dihydroxyphenylacetaldehyde (DOPAL) or 5-hydroxy-indole-3-acetaldehyde (5-HIAL), the aldehydes derived from dopamine and serotonin, respectively. The disappearance of the aldehyde and the formation of acid and alcohol metabolites were analysed in the presence of different concentrations of ethanol, acetaldehyde or disulfiram using high-performance liquid chromatography with electrochemical detection. Ethanol at a concentration of 20 mM did not affect the biogenic aldehyde metabolism. High concentrations of acetaldehyde caused a dose-dependent inhibition of the disappearance rate of the biogenic aldehydes and of the formation rate of acid metabolites. In incubations with leukocytes or platelets, the inhibition of the acid formation was associated with a slight increase in the formation of the alcohol metabolites. Disulfiram at a concentration of 50 microM totally inhibited the metabolism of DOPAL and 5-HIAL in incubations with erythrocytes or platelets, whereas much less inhibition was observed in incubations with leukocytes.

Aldehyde dehydrogenase activity and vascular disease in type II diabetes--a comparison between 2 different assays for activity.

We have recently reported that type II diabetic subjects with macroangiopathy have a higher activity of aldehyde dehydrogenase (ALDH) in blood than those without clinical vascular disease. ALDH activity was measured as the elimination of acetaldehyde added to a blood homogenate in vitro. We have re-examined our clinical material with another assay of ALDH which uses indole-3-acetaldehyde as substrate and measures the formation of indole-3-acetic acid. A negative correlation between the half-life of acetaldehyde and the formation of indole-3-acetic acid was found in the group of subjects free from vascular disease (r = -0.55, p less than 0.01). Thus, a rapid elimination of acetaldehyde corresponded to a rapid formation of indole-3-acetic acid. No such correlation was found in subjects with macroangiopathy. These results suggest that the 2 groups, with and without clinical vascular disease, have differences in isoenzyme composition, in the kinetic properties of the enzyme, or in the non-enzymatic binding of acetaldehyde.

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.

Effects of aldehyde dehydrogenase inhibitors on hexobarbital sensitivity and neuroamine metabolism in rat brain.

Several authors have found that the aldehyde dehydrogenase (ALDH) inhibitor, disulfiram, prolongs hexobarbital-induced anaesthesia. It was suggested that this effect was caused by an alteration of the serotonergic system in brain, mediated by elevated levels of biogenic aldehydes. In the present study, disulfiram (300 mg/kg) was found to cause a 4-fold prolongation of hexobarbital-induced anaesthesia, while coprine (another potent ALDH-inhibitor) had no effect. This strongly suggested that biogenic aldehydes were not involved in this effect of disulfiram. The hexobarbital concentration in brain, at the electroencephalogram (EEG) criteria used for measuring hexobarbital sensitivity, was unaffected in rats given 75-300 mg/kg disulfiram, indicating that factors other than an increased brain hexobarbital sensitivity were responsible for the prolonged anaesthesia. Also 10-100 mg/kg coprine did not affect hexobarbital sensitivity measured this way. No alteration of the dopamine level in brain was found in rats given disulfiram, and in both disulfiram- and coprine-treated rats, similar changes in the serotonergic system were found. However, the level of norepinephrine was decreased in brains of disulfiram-treated rats, but it was unaffected by coprine. Thus, norepinephrine may have been involved in the prolongation of hexobarbital-induced anaesthesia caused by disulfiram.