Regulation of NAD- and NADP-linked isocitrate dehydrogenase by hydrocortisone in the brain and liver of male rats of various ages

The activity and hormonal regulation of NAD- and NADP-linked isocitrate dehydrogenase (EC 1.1.1.41 and 1.1.1.42, respectively) in the brain and liver of rats of various ages were investigated. The activity of NAD-linked isocitrate dehydrogenase of the brain was greater than cytoplasmic or mitochondrial NADP-linked isocitrate dehydrogenase. In contrast, the cytoplasmic NADP-isocitrate dehydrogenase of the liver predominates over both NAD- and mitochondrial NADP-isocitrate dehydrogenases at the three ages studied. The activity of NAD-isocitrate dehydrogenase increased in the brain (139%) and liver (17%) of rats upt o 33 weeks of age and decreased (57 and 39%, respectively) in old rats (85-week-old). The activity of cytoplasmic NADP-isocitrate dehydrogenase was maximum in immature (6-week-old) rat brain and decreased as the age of the rats increased; whereas, in liver, the activity of this enzyme was found to be maximum in adult rats (33-week-old). Brain mitochondrial NADP-isocitrate dehydrogenase activity increased (64%) in adult rats, but in liver it decreased (45 and 33% in 33- and 85-week-old rats, respectively). In both tissues, adrenalectomy and hydrocortisone treatment showed differential age-dependent response. Hydrocortisone-mediated induction of the level of enzymes was inhibited by actinomycin D.     

THE RELATIVE SIGNIFICANCE OF CO2-FIXING ENZYMES IN THE METABOLISM OF RAT BRAIN

To evaluate the relative significance of CO₂-fixing enzymes in the metabolism of rat brain, the subcellular distribution of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, NADP-isocitrate dehydrogenase and NADP-malate dehydrogenase, as well as the fixation of H¹⁴CO₃^? by the cytosol and the mitochondria was investigated. Pyruvate carboxylase and phosphoenol-pyruvate carboxykinase are mainly localized in the mitochondria whereas NADP-isocitrate dehydrogenase and NADP-malate dehydrogenase are present in both the cytosol and the mitochondria. In the presence of pyruvate rat brain mitochondria fixed H¹⁴CO₃^? at a rate of about 170 nmol/g of tissue/min whereas these organelles fixed negligible amounts of H¹⁴CO₃^? in the presence of α-ketoglutarate or phosphoenolpyruvate. Rat brain cortex slices fixed H¹⁴CO₃^? at a rate of about 7 nmol/g of tissue/min and it was increased by two-fold when pyruvate was added to the incubation medium. The carboxylation of α-ketoglutarate and pyruvate by the reversal of the cytosolic NADP-isocitrate dehydrogenase and NADP-malate dehydrogenase respectively was very low as compared to that by pyruvate carboxylase. The rate of carboxylation reaction of both NADP-isocitrate dehydrogenase and NADP-malate dehydrogenase was only about 1/10th of that of decarboxylation reaction of the same enzyme. It is suggested that under physiological conditions these two enzymes do not play a significant role in CO₂-fixation in the brain. In rat brain cytosol, citrate is largely metabolized to α-ketoglutarate by a sequential action of aconitate hydratase and NADP-isocitrate dehydrogenase. The operation of the citrate-cleavage pathway in rat brain cytosol is demonstrated. The data show that among four CO₂-fixing enzymes, pyruvate carboxylase, an anaplerotic enzyme, plays the major role in CO₂-fixation in the brain.     

Oxidative inactivation of reduced NADP-generating enzymes in E. coli: iron-dependent inactivation with affinity cleavage of NADP-isocitrate dehydrogenase

Treatment of E. coli extract with iron/ascorbate preferentially inactivated NADP-isocitrate dehydrogenase without affecting glucose-6-phosphate dehydrogenase. NADP-Isocitrate dehydrogenase required divalent metals such as Mg²⁺, Mn²⁺ or Fe²⁺ ion. Iron/ascorbate-dependent inactivation of the enzyme was accompanied with the protein fragmentation as judged by SDS-PAGE. Catalase protecting the enzyme from the inactivation suggests that hydroxyl radical is responsible for the inactivation with fragmentation. TOF-MS analysis showed that molecular masses of the enzyme fragments were 36 and 12, and 33 and 14 kDa as minor components. Based on the amino acid sequence analyses of the fragments, cleavage sites of the enzyme were identified as Asp307-Tyr308 and Ala282-Asp283, which are presumed to be the metal-binding sites. Ferrous ion bound to the metal-binding sites of the E. coli NADP-isocitrate dehydrogenase may generate superoxide radical that forms hydrogen peroxide and further hydroxyl radical, causing inactivation with peptide cleavage of the enzyme. Oxidative inactivation of NADP-isocitrate dehydrogenase without affecting glucose 6-phosphate dehydrogenase shows only a little influence on the antioxidant activity supplying NADPH for glutathione regeneration, but may facilitate flux through the glyoxylate bypass as the biosynthetic pathway with the inhibition of the citric acid cycle under aerobic growth conditions of E. coli.     

The subcellular location of isozymes of NADP-isocitrate dehydrogenase in tissues from pig, ox and rat

Antibodies against purified NADP-isocitrate dehydrogenase from pig liver cytosol and pig heart were raised in rabbits. The purified enzymes from these sources are different proteins, as demonstrated by differences in electrophoretic mobility and absence of crossreactivity by immunotitration and immunodiffusion. The NADP-isocitrate dehydrogenase in the soluble supernatant homogenate fraction from pig liver, kidney cortex, brain and erythrocyte hemolyzate was identical with the purified enzyme from pig liver cytosol, as determined by electrophoretic mobility and immunological techniques. The enzyme in extracts of mitochondria from pig heart, kidney, liver and brain was identical with the purified pig heart enzyme by the same criteria. However, the 'mitochondrial' isozyme was the major component also in the soluble supernatant fraction of pig heart homogenate. The 'cytosolic' isozyme accounted for only 1-2% of total NADP-isocitrate dehydrogenase in pig heart, as determined by separation of the isozymes with agarose gel electrophoresis and immunotitration. The mitochondrial isozyme was also the predominant NADP-isocitrate dehydrogenase in porcine skeletal muscle. The ratio of cytosolic/mitochondrial isozyme for porcine whole tissue extract, determined by immunotitration, was about 2 for liver and 1 for kidney cortex and brain. The distribution of isozymes in cell homogenate fractions from ox and rat tissues corresponded to that observed in organs of porcine origin. The mitochondrial and cytosolic isozymes from ox and rat tissues exhibited crossreactivity with the antibodies against the pig heart and pig liver cytosol enzyme, respectively, and the electrophoretic migration patterns were similar qualitatively to those found for the isozymes in porcine tissues. Nevertheless, there were species specific differences in the characteristics of each of the corresponding isozymes. NAD-isocitrate dehydrogenase was not inhibited by the antibodies, confirming that the protein is distinct from that of either isozyme of NADP-isocitrate dehydrogenase.     

Lipid metabolism by rat lung in vitro. Effect of starvation and re-feeding on utilization of [U-14C]glucose by lung slices

1. The incorporation of [U-(14)C]glucose into several lipid components of lung and liver slices, and the activities of glucose 6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), ;malic' enzyme (EC 1.1.1.40) and NADP-isocitrate dehydrogenase (EC 1.1.1.42) of the cell cytosol were examined in normal, starved and re-fed rats. 2. Lipogenesis and the activities of these enzymes in liver were decreased markedly in rats starved for 72h. Re-feeding starved rats on a fat-free diet for 72h resulted in the well documented hyperlipogenic response in liver, particularly in its ability to convert glucose into neutral lipid, and increased activities of glucose 6-phosphate dehydrogenase, ;malic' enzyme and 6-phosphogluconate dehydrogenase to values approx. 700, 470 and 250% of controls respectively. 3. Approx. 70% of the total label in lung lipids was present in the phospholipid fraction. Hydrolysis of lung phospholipids revealed that lipogenesis from glucose was considerable, with approx. 40% of the total phospholipid radioactivity present in the fatty acid fraction. 4. Incorporation of glucose into total lung lipids was decreased by approx. 40% in lung slices of starved rats and was returned to control values on re-feeding. Although phospholipid synthesis from glucose was decreased in lung slices of starved rats, the decrease proportionally was greater for the fatty acid fraction (approx. 50%) as compared with the glycerol fraction (approx. 25%). 5. The activities of lung glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and NADP-isocitrate dehydrogenase were not affected by the dietary alterations. ;Malic' enzyme activity was not detected in lung cytosol preparations. 6. The results are discussed in relation to the surface-active lining layer (surfactant) of the lung.     

Effect of rhodium (III) complex on experimental carcinogenesis in the livers of rats treated with thioacetamide

Enzyme activities and protein content were determined in the cytosolic and mitochondrial fractions of liver homogenates obtained from Rh(III) complex-, thioacetamide- and thioacetamide + Rh(III) complex-treated rats. The Rh(III) complex administered to nonthioacetamide-treated rats produced no significant changes either in the enzymatic activities assayed or in the protein concentration. The Rh(III) complex administered to thioacetamide-treated rats produced significant restoration of the following altered values: cytosolic and mitochondrial aspartate aminotransferase, glutamate dehydrogenase, NADP-isocitrate dehydrogenase, and protein concentration. However, a further increase was produced in the activities of glucose-6-phosphate dehydrogenase and malic enzyme. These increases can be interpreted in terms of an enhancement of the NADPH-dependent detoxifying processes and of nucleic acid synthesis and repair.     

DEVELOPMENT OF LIPOGENESIS IN RAT BRAIN CORTEX: THE DIFFERENTIAL INCORPORATION OF GLUCOSE AND ACETATE INTO BRAIN LIPIDS IN VITRO

—The oxidation to CO₂ and the incorporation of [U-¹⁴C]glucose and [U-¹⁴C]acetate into lipids by cortex slices from rat brain during the postnatal period were investigated. The oxidation of [U-¹⁴C]glucose was low in 2-day-old rat brain, and increased by about two-fold during the 2nd and 3rd postnatal weeks. The oxidation of [U-¹⁴C]acetate was increased markedly in the second postnatal week, but decreased to rates observed in 2-day-old rat brain at the time of weaning. Both labeled substrates were readily incorporated into non-saponifiable lipids and fatty acids by brain slices from 2-day-old rat. Their rates of incorporation and the days on which maximum rates occurred were different, however, maximum incorporation of [U-¹⁴C]glucose and [U-¹⁴]acetate into lipid fractions being observed on about the 7th and 12th postanatal days, respectively. The metabolic compartmentation in the utilization of these substrates for lipogenesis is suggested. The activities of glucose-6-phosphate dehydrogenase, cytosolic NADP-malate dehydrogenase, cytosolic NADP-isocitrate dehydrogenase, ATP-citrate lyase and acetyl CoA carboxylase were measured in rat brain during the postnatal period. All enzymes followed somewhat different courses of development; the activity of acetyl CoA carboxylase was, however, the lowest among other key enzymes in the biosynthetic pathway, and its developmental pattern paralleled closely the fatty acid synthesis from [U-¹⁴C]glucose. It is suggested that acetyl CoA carboxylase is a rate-limiting step in the synthesis de novo of fatty acids in developing rat brain.     

The dose-dependent effect of 3,5-dicarbomethoxyphenylbiguanide on activity of the glutathione antioxidant system in heart and blood serum of rats with experimental myocardial infarction

Administration of a synthetic compound with predicted anti-ischemic and cardioprotective activity, 3,5-dicarbomethoxyphenylbiguanide (3,5-DCMPBG) to rats with experimental myocardial infarction led to a decrease in the lipid peroxidation level, glutathione peroxidase activity, the level of reduced glutathione, activity of NADP-isocitrate dehydrogenase in the heart and blood serum, and activity of glucoso-6-phosphate dehydrogenase in heart in comparison with their levels in untreated animals with myocardial infarction. This may be attributed to a decrease of free radical processes and reduction of antioxidant system loading induced by the protective effect of the administered compound. At the same time the increase glutathione reductase activity observed under these conditions in the heart and blood serum is probably associated with specific influence of 3,5-DCMPBG on this enzyme.     

Regulation of mitochondrial NADP-isocitrate dehydrogenase in rat heart during ischemia

The changes in the regulation of at mitochondrial NADP-isocitrate dehydrogenase (NADP-ICDH) in a rat heart during have been analysed. Increase of enzyme activity in the cytosol and mitochondria of the heart ischemia was detected. Catalytic properties of the mitochondrial NADP-ICDH at norm and pathology have been compared on homogeneous enzyme preparations. Enzyme from the normoxic and ischemic heart showed the same electrophoretical mobility and molecular mass. Enzyme isolated from the ischemic heart mitochondria demonstrated higher activation energy and lower thermal stability. NADP-isocitrate dehydrogenase at the normoxic and ischemic conditions exhibited different K_m for substrates and regulatory behaviour in relation to ATP, ADP, 2-oxoglutarate, citrate, malate, reduced and oxidised glutathione. The inhibitory effect of the Fe²⁺ and H₂O₂ mixture associated with the generation of hydroxyl radicals was lower in the ischemic enzyme. We hypothesise that the specific features of regulation behaviour of NADP-ICDH from the ischemic tissues permits the enzyme to supply NADPH to the glutathione reductase/glutathione peroxidase system.     

2-Oxoglutarate: Oxidation and Role as a Potential Precursor of Cytosolic Acetyl-CoA for the Synthesis of Acetylcholine in Rat Brain Synaptosomes

The possibility that 2-oxoglutarate may supply acetyl units for the cytosolic synthesis of acetylcholine in rat brain synaptosomes was investigated. The contribution of [14C]2-oxoglutarate to the synaptosomal synthesis of [14C]acetylcholine was found to be negligible despite evidence for its uptake and oxidation. The activity of the enzymes NADP-isocitrate dehydrogenase (EC 1.1.1.42), aconitate hydratase (EC 4.2.1.3), and ATP citrate-lyase (EC 4.1.3.8) were measured in the synaptosol. NADP-isocitrate dehydrogenase and aconitate hydratase are present at three- to 1.5-fold higher activities than ATP citrate-lyase. It seems likely that these enzymes contribute to the metabolism of citrate and prevent detectable formation of cytosolic acetyl-CoA from exogenously added 2-oxoglutarate (or citrate). The data further suggest that ATP citrate-lyase may in part be associated with the mitochondrial fraction.     

beta-Sulfur substituted alpha-ketoglutarates as inhibitors and alternate substrates for isocitrate dehydrogenases and certain other enzymes

The RS-isomers of beta-mercapto-alpha-ketoglutarate, beta-methylmercapto-alpha-ketoglutarate and beta-methylmercapto-alpha-hydroxyglutarate have been synthesized. Beta-Mercapto-alpha-ketoglutarate was a potent inhibitor, competitive with isocitrate and noncompetitive with NADP+, of the mitochondrial NADP-specific isozyme from pig heart (Ki = 5 nM; Km (DL-isocitrate)/Ki(RS-beta-mercapto-alpha-ketoglutarate) = 650) and pig liver, the cytosolic isozyme from pig liver (I0.5 = 23 nM), and the NADP-linked enzymes from yeast (Ki = 58 nM) and Escherichia coli (Ki = 58 nM) at pH 7.4 and with Mg2+ as activator. beta-Mercapto-alpha-ketoglutarate was also an effective inhibitor of NADP-isocitrate-dehydrogenase activity in intact liver mitochondria. beta-Mercapto-alpha-ketoglutarate was a much less potent inhibitor for heart NAD-isocitrate dehydrogenase (Ki = 520 nM) than for the NADP-specific enzyme. beta-Methylmercapto-alpha-ketoglutarate (I0.5 = 10 microM) was a much less effective inhibitor than the beta-mercapto derivative for heart NADP-isocitrate dehydrogenase. The beta-sulfur substituted alpha-ketoglutarates were substrates for the oxidation of NADPH by heart NADP-isocitrate dehydrogenase without requiring CO2. beta-Methylmercapto-alpha-hydroxyglutarate, the expected product of reduction of beta-methylmercapto-alpha-ketoglutarate, did not cause reduction of NADP+ but it was an inhibitor competitive with isocitrate for NADP-isocitrate dehydrogenase. The beta-sulfur substituted alpha-ketoglutarate derivatives were alternate substrates for alpha-ketoglutarate dehydrogenase and the cytosolic and mitochondrial isozymes of heart aspartate aminotransferase but had no effect on glutamate dehydrogenase or alanine aminotransferase.     

Ethanol-induced Adaptation of Alcohol Dehydrogenase Activity in Rat Brain

ALTHOUGH the presence of alcohol dehydrogenase (ADH) in cerebral tissue has been established¹, a physiological role for such a brain ethanol-oxidizing system has been unclear. The brain may be more biochemically adaptive than was once thought²; thus, it seemed possible that brain ADH may be substrate-induced. We now report that significant elevations of brain ADH activity occur in alcohol-imbibing rats; no changes from control values were found in liver ADH, liver aldehyde dehydrogenase (AldDH), or brain AldDH activities.     

The quantitative histochemistry of a chemically induced ependymoblastoma. I. Enzymes

Enzymes from several metabolic pathways were studied quantitatively in homogenates and in homogeneous areas of frozen-dried cryostat sections of an experimental, mouse ependymoblastoma, mouse mammary carcinoma, and mouse melanoma, growing as transplants in mouse brain. Micro analyses were performed in fiveto sixfold replicates on portions of tumour with a dry weight of 0·03-0·2 μg. A close resemblance of the enzyme spectrum of the ependymoblastoma to that of immature brain was noted. Hexokinase and malate dehydrogenase were lower and lactate, glucose-6-phosphate, and NADP⁺-linked isocitrate dehydrogenases, and β-glucuronidase higher in the ependymoblastoma than in whole, adult mouse brain. Mouse mammary carcinoma had a higher level of hexokinase and lower levels of lactate and glucose-6-phosphate dehydrogenases and β-glucuronidase than ependymoblastoma. The concentration of malate dehydrogenase was lower and that of lactate, glucosed-6-phosphate, and NADP⁺-linked isocitric dehydrogenases was higher in the melanoma than in the ependymoblastoma. β-Glucuronidase levels were similar in these two neoplasms. It is suggested that the relatively high levels of several NADP⁺-linked enzymes in the ependymoblastoma may be related to increased capacity for lipid synthesis.     

Changes in NADP-isocitrate dehydrogenase isoenzyme levels in Acinetobacter calcoaceticus in response to acetate

Abstract During adaptation of Acinetobacter calcoaceticus to growth on acetate the specific activity of NADP-isocitrate dehydrogenase increased. This response is unique, as in other bacteria grown under the same conditions the activity of the enzyme decreases as a result of covalent phosphorylation. Moreover, A. calcoaceticus is also unusual in containing two distinct isoenzymes of NADP-isocitrate dehydrogenase. It has here been shown that the adaptation of A. calcoaceticus to acetate is accompanied by an increase in the relative proportion of the larger, allosteric, isoenzyme with a concomitant decrease in the level of the smaller, non-allosteric, isoenzyme.     

Glutathione system adaptation to acute stress in the heart of rats during different regimes of hypoxia training

The intensity of lipid peroxidation (LPO), reduced and oxidized glutathione (GSH and GSSG) contents, glutathione reductase, glutathione peroxidase, glutathione-S-transferase, glucose-6-phosphate dehydrogenase (G-6-PDH), and NADP-isocitrate dehydrogenase (NADP-IDH) activities were studied in the heart of male rats exposed to two modes of intermittent hypoxic training (IHT): I-breathing in normobaric chamber with 7% O2 gas mixture for 5 min with 15 min normoxic intervals 4 times daily during 3 weeks; II-breathing by 12% O2 gas mixture in the same manner). After adaptation to hypoxia, the rats were subjected to 6h-immobilization stress. It has been shown that stress action after IHT (regime I) caused the increase in LPO and the shift of GSH/GSSG to disulfides. A disbalance in antioxidative defense system was determined by the decrease in glutatione peroxidase, G-6-PDH activities, and GSH content. The support of glutathione reductase activity under stress in this group with simultaneous decrease of enzyme activity in the pentose phosphate pathway was realized through the participation of NADP-IDH. Hypoxic training in regime II induced LPO decrease in the heart tissue after stress. The increase in the heart GSH content, optimal balance of glutathione-related enzymes in this group evidences for the dependence of adaptation effects on the vigor of hypoxic exposition. Our results suggest the active participation of glutathione system in the formation of adaptation reactions under the extreme factor influences through the action on intracellular red/ox potential as well as effectiveness of antioxidant defense.     

Aluminum decreases the glutathione regeneration by the inhibition of NADP-isocitrate dehydrogenase in mitochondria

Effect of aluminum on the NADPH supply and glutathione regeneration in mitochondria was analyzed. Reduced glutathione acted as a principal scavenger of reactive oxygen species in mitochondria. Aluminum inhibited the regeneration of glutathione from the oxidized form, and the effect was due to the inhibition of NADP-isocitrate dehydrogenase the only enzyme supplying NADPH in mitochondria. In cytosol, aluminum inhibited the glutathione regeneration dependent on NADPH supply by malic enzyme and NADP-isocitrate dehydrogenase, but did not affect the glucose 6-phosphate dehydrogenase dependent glutathione formation. Aluminum can cause oxidative damage on cellular biological processes by inhibiting glutathione regeneration through the inhibition of NADPH supply in mitochondria, but only a little inhibitory effect on the glutathione generation in cytosol.     

Temperature and enzyme activity in poikilotherms. Isocitrate dehydrogenases in rainbow-trout liver

1. The kinetics of the thermally induced enzyme variants of the supernatant NADP-isocitrate dehydrogenase from rainbow-trout liver are investigated. 2. Fish acclimatized to 2 degrees C (cold-adapted enzyme) and 17 degrees C (warm-adapted enzyme) show different relative distributions of the three NADP-isocitrate dehydrogenase isoenzymes; this has been demonstrated with electrophoresis and electrofocusing techniques. 3. Plots of K(m) versus temperature for the cold-adapted and warm-adapted enzyme variants are complex in nature with apparent maximal enzyme-substrate affinity corresponding to the temperature at which the trout is acclimatized. Both substrates, dl-isocitrate and NADP(+), give similar curves although the magnitude of the K(m) change with temperature is much decreased in the case of NADP(+). 4. E(a) values of approx. 18kcal/mol were determined for both the cold-adapted and warm-adapted enzyme variants. 5. In an attempt to determine how velocities can be increased at low temperatures, cation, pH requirements, metabolite and enzyme concentrations were examined. 6. NAD-isocitrate dehydrogenase could not be detected in trout tissues.     

Regulation of liver and brain hexose monophosphate dehydrogenases by insulin and dietary intake in the female rat

Summary Liver glucose 6-phosphate dehydrogenase and phosphogluconate dehydrogenase activities were significantly decreased in both diabetic and fasted rats. Treatment of diabetic rats with insulin resulted in liver glucose 6-phosphate dehydrogenase and phosphogluconate dehydrogenase activities that were significantly greater than controls. Insulin promoted an increase in food consumption that was blocked by adrenaline. Insulin, when administered together with adrenaline, restored hepatic glucose 6-phosphate dehydrogenase and phosphogluconate dehydrogenas activities of diabetic animals to control values, without altering food consumption. Brain glucose 6-phosphate dehydrogenase and phosphogluconate dehydrogenase activities were not significantly altered by either dietary restriction, diabetes or insulin treatment. These results demonstrate a dissociation between the action of insulin on hepatic glucose 6-phosphate dehydrogenase activity and its action to increase food intake.Abbreviations NADP⁺ oxidoreductase, EC 1.1.1.49 Glucose 6-P dehydrogenase, GPD, D-glucose-6-phosphate - NADP⁺ 2-oxidoreductase (decarboxylating), EC 1.1.1.44 phosphogluconate dehydrogenase, PGD, 6-phospho-D-gluconate     

Enhanced 5-HT<Subscript>2A</Subscript> Receptors in Brain Stem and ALDH Activity in Brain Stem and Liver: 5-HT<Subscript>2A</Subscript> Regulation on ALDH in Primary Hepatocytes Cultures In Vitro

Brain serotonin (5-HT) modulates the neural effects of ethanol. In the present study, we investigated the changes in 5-HT level, 5-HT_2A receptor binding and aldehyde dehydrogenase (ALDH) activity in brain stem and liver of ethanol treated rats and 5-HT_2A regulation on ALDH in hepatocyte cultures in vitro. The 5-HT content in the brain stem and liver significantly decreased with an increased 5-HIAA/5-HT ratio in the ethanol treated rats compared to control. Scatchard analysis of [³H] (±)2,3-dimethoxyphenyl-1-[2-(-4-piperidine)-methanol] [³H] MDL 100907 against ketanserin in brain stem of ethanol treated rats showed a significant increase in B _max without any change in K _d compared to control. The competition curve for [³H] MDL 100907 against ketanserin fitted one-site model in both control and ethanol treated rats with unity as Hill slope value. A significant increase in V _max of ALDH activity in liver and a significant decrease in K _m in liver and brain stem of ethanol treated rats compared to control was observed. In 24 h culture studies, an increase in enzyme activity was observed in cells in medium with 10% ethanol. The elevated ALDH activity in ethanol treated cells was reversed to control level in presence of 10⁻⁵ and 10⁻⁷ M 5-HT. Ketanserin, an antagonist of 5-HT_2A, reversed the effect of 5HT on 10% ethanol induced ALDH activity in hepatocytes. Our results showed that there was a decreased 5-HT content with an enhanced 5-HT_2A receptor and aldehyde dehydrogenase activity in the brain stem of alcohol treated rats and in vitro hepatocyte cultures. The enhanced ALDH activity in ethanol supplemented hepatocytes was reversed to control level in presence of 10⁻⁵ and 10⁻⁷ M 5-HT.