Effect of central adrenergic alpha receptor blockader IEM-611 on the activity of alcohol and aldehyde dehydrogenase in rat liver

The effect of IEM-611 (30 mg/kg) on alcohol consumption in rats under the conditions of voluntary choice between water and 15% ethanol was studied as that on alcohol dehydrogenase (ADH) in postmitochondrial supernatant and in NAD-dependent aldehyde dehydrogenases (A1DH) in liver mitochondria. Administration of IEM-611 during 6 or 12 days reduces ethanol consumption by 29 and 30%, respectively, activates ADH and appreciably decreases overall activity of NAD-dependent A1DH. At the same time the ADH/A1DH ratio increases. Activation of ADH and A1DH and the decreased ADH/A1DH ratio were disclosed in alcohol preferring rats as compared to water preferring animals. IEM-611 shifts enzymatic activity of ethanol metabolism towards the level characteristic for water preferring rats. It is suggested that variation of the ADH/A1DH ratio is one of the mechanisms responsible for the decreased ethanol consumption in rats.     

Loss of growth hormone-dependent characteristics of rat hepatocytes in culture

Summary The liver of rodents is sexually differentiated, i.e. the female liver differs from the male liver. This differentiation is largely controlled by the pattern of growth hormone (GH) secretion. We have attempted to maintain GH-dependent differentiation of cultured rat hepatocytes. We examined the level of alcohol dehydrogenase (ADH) activity, which responds to GH and is higher in female than in male liver, and the estrogen receptor, which is dependent on GH but is present in equal amounts in males and females. ADH activity was maintained in cells from male rats, but fell by 40% in cells from females in medium supplemented with insulin and dexamethasone. The estrogen receptor content of female cells fell dramatically to undetectable levels within 2 d of culture. Extensive supplementation of the medium failed to prevent the decrease in ADH activity in female cells; similarly, the addition of female sex steroids; rat serum; pituitary extracts; rat, human, or bovine GH; or ovine prolactin failed to maintain the enzyme activity. Insulin, dexamethasone, thyroid hormone plus GH or prolactin, or the combination of all five hormones also failed to prevent the loss of estrogen receptors. Short-term cultures of rat hepatocytes, although retaining the liver-specific expression of ADH at the male level, lose GH-dependent expression of the estrogen receptor and stimulation of ADH activity. Supported by grants AA 00081 and AA 06434 from the National Institute on Alcohol Abuse and Alcoholism, Bethesda, MD.     

Diabetes impairs the enzymatic disposal of 4-hydroxynonenal in rat liver

This study assesses whether the HNE accumulation we formerly observed in liver microsomes and mitochondria of BB/Wor diabetic rats depends on an increased rate of lipoperoxidation or on impairment of enzymatic removal. There are three main HNE metabolizing enzymes: glutathione-S-transferase (GST), aldehyde dehydrogenase (ALDH), and alcohol dehydrogenase (ADH). In this study we show that GST and ALDH activities are reduced in liver microsomes and mitochondria of diabetic rats; in contrast, ADH activity remains unchanged. The role of each enzyme in HNE removal was evaluated by using enzymatic inhibitors. The roles of both GST and ALDH were markedly reduced in diabetic rats, while ADH-mediated consumption was significantly increased. However, the higher level of lipohydroperoxides in diabetic liver indicated more marked lipoperoxidation. We therefore think that HNE accumulation in diabetic liver may depend on both mechanisms: increased lipoperoxidation and decreased enzymatic removal. We suggest that glycoxidation and/or hyperglycemic pseudohypoxia may be involved in the enzymatic impairment observed. Moreover, since HNE exerts toxic effects on enzymes, HNE accumulation, deficiency of HNE removal, and production of reactive oxygen species can generate vicious circles able to amplify the damage.     

About Mechanisms Responsible for the Depression of Alcohol Motivation Resulting from the Immunization of Albino Rats against Alcohol Dehydrogenase I: The Role of ADH Epitopes and ADH Activity in the Adrenals

Experimental results have demonstrated a significant decrease in the level of alcohol consumption by albino rats immunized with heterologous horse alcohol dehydrogenase. The role of ADH epitopes 9–14, 93–115, and 265–276 in this phenomenon was examined, and it was established that the latter sequence (265–276) plays the biggest role. The inhibition of ADH activity in the adrenals of immunized rats was much higher compared to the liver. We propose a hypothesis that the effect of alcohol dehydrogenase on alcohol consumption is connected with its role in catecholamine metabolism.     

Hepatic alcohol dehydrogenase activity in chick hepatocytes towards the major alcohols present in commercial alcoholic beverages: comparison with activities in rat and human liver

We have compared hepatic alcohol dehydrogenase activities in chick, rat and human liver with the major alcohols in commercial alcoholic beverages. 1. Chick and rat hepatic alcohol dehydrogenase was greater when assayed at a physiological pH in buffer containing chloride ions, as compared with the activity in pyrophosphate buffer at alkaline pH. 2. In contrast to reports of instability of ADH to freezing, we found the enzyme from all three species stable to freezing in 0.25 M sucrose. 3. Rat liver enzymatic activity was unstable in the presence of substrate, where as that of chick and human was not. 4. For all three species, the Km of hepatic ADH for substrate decreased with increasing chain length of alcohols. In both chick and human samples, the Vmax values for the higher chain alcohols were similar to that with ethanol, while in rat samples, ADH activity was dramatically lower with the higher chain alcohols compared to ethanol.     

In vivo contribution of Class III alcohol dehydrogenase (ADH3) to alcohol metabolism through activation by cytoplasmic solution hydrophobicity

Alcohol metabolism in vivo cannot be explained solely by the action of the classical alcohol dehydrogenase, Class I ADH (ADH1). Over the past three decades, attempts to identify the metabolizing enzymes responsible for the ADH1-independent pathway have focused on the microsomal ethanol oxidizing system (MEOS) and catalase, but have failed to clarify their roles in systemic alcohol metabolism. In this study, we used Adh3-null mutant mice to demonstrate that Class III ADH (ADH3), a ubiquitous enzyme of ancient origin, contributes to alcohol metabolism in vivo dose-dependently resulting in a diminution of acute alcohol intoxication. Although the ethanol oxidation activity of ADH3 in vitro is low due to its very high Km, it was found to exhibit a markedly enhanced catalytic efficiency (kcat/Km) toward ethanol when the solution hydrophobicity of the reaction medium was increased with a hydrophobic substance. Confocal laser scanning microscopy with Nile red as a hydrophobic probe revealed a cytoplasmic solution of mouse liver cells to be much more hydrophobic than the buffer solution used for in vitro experiments. So, the in vivo contribution of high-Km ADH3 to alcohol metabolism is likely to involve activation in a hydrophobic solution. Thus, the present study demonstrated that ADH3 plays an important role in systemic ethanol metabolism at higher levels of blood ethanol through activation by cytoplasmic solution hydrophobicity.     

Study on mesophilic and thermophilic alcohol dehydrogenases in gas-phase reaction

The initial reaction rate and the thermostability of the mesophilic alcohol dehydrogenase (ADH) from Lactobacillus brevis (LBADH), and the thermophilic ADH from Thermoanaerobacter sp. (ADH T) in gas-phase reaction were compared. The effects of water activity, cofactor-to-protein molar ratio, and reaction temperature on the reduction of acetophenone to 1-phenylethanol were studied. An optimal water activity of 0.55 in terms of productivity was found for both ADHs. The cofactor-to-protein molar ratio was chosen slightly higher than equimolar to increase both activity and thermostability. An excellent optimal productivity of 1,000 g x L(-1) x d(-1) for LBADH and 600 g x L(-1) x d(-1)for ADH T was found at 60 degrees C, while the highest total turnover numbers with respect to the enzyme were achieved at 30 degrees C and amounted to 4.2 million for LBADH and 1.7 million for ADH T, respectively. Interestingly, the ADH from the mesophilic L. brevisshowed the higher thermostability in the nonconventional medium gas phase.     

The intra-acinar distribution patterns of alcohol-dehydrogenase activity in the liver of juvenile, castrated and testosterone-treated rats

Rat liver alcohol dehydrogenase shows characteristic sex-differences with respect to activity and heterotopy. For the recognition of gonadal influences on the intra-acinar distribution patterns luminometric determinations of ADH activity were carried out on 50-150 ng lyophilized liver tissue samples which had been microdissected along the sinusoidal length. Juvenile rats of both sexes showed equally high alcohol dehydrogenase activity, which surpassed the adult values by a factor of 2 in males and 1.3 in females. The distribution pattern was rather flat, with a weak maximum at the beginning of the last third of the sinusoid. Castration of adult male and female rats resulted in an increase of alcohol dehydrogenase activity to around the prepubertal values. The intra-acinar profiles showed a gradual increase in activity from low periportal values to a peak near the perivenous zone. Only the hepatocytes directly adjacent to the efferent venule showed an even lower activity. Administration of testosterone to castrated animals had no effect on the ADH activity in males and resulted in only a slight decrease of enzyme activity in females. The intra-acinar distribution patterns showed an intermediary peak at the end of the second third of the sinusoidal length in males and a gradual increase of activity, beginning periportally, in the direction of the perivenous zone in females. The present findings on total activity of ADH and its distribution patterns in the liver are considered to be the result of complex hormonal alterations rather than a specific effect of testosterone.     

Sexual dimorphic expression of ADH in rat liver: importance of the hypothalamic-pituitary-liver axis

Hepatic alcohol dehydrogenase (ADH) activity is higher in female than in male rats. Although sex steroids, thyroid, and growth hormone (GH) have been shown to regulate hepatic ADH, the mechanism(s) for sexual dimorphic expression is unclear. We tested the possibility that the GH secretory pattern determined differential expression of ADH. Gonadectomized and hypophysectomized male and female rats were examined. Hepatic ADH activity was 2.1-fold greater in females. Because protein and mRNA content were also 1.7- and 2.4-fold greater, results indicated that activity differences were due to pretranslational mechanisms. Estradiol increased ADH selectively in males, and testosterone selectively decreased activity and mRNA levels in females. Effect of sex steroids on ADH was lost after hypophysectomy; infusion of GH in males increased ADH to basal female levels, supporting a role of the pituitary-liver axis. However, GH and L-thyroxine (T4) replacements alone in hypophysectomized rats did not restore dimorphic differences for either ADH activity or mRNA levels. On the other hand, T4 in combination with intermittent administration of GH reduced ADH activity and mRNA to basal male values, whereas T4 plus GH infusion replicated female levels. These results indicate that the intermittent male pattern of GH secretion combined with T4 is the principal determinant of low ADH activity in male liver.     

Aldehyde dehydrogenase activity and level of dopamine in certain sections of the brain of rats preferring and refusing ethanol

Aldehyde dehydrogenase activity (KF 1.2.1.3) of cytosol fractions of brain structures (hypothalamus, midbrain and new cortex) as well as dophamine content in these structures were studied in comparative aspect in rats preferring and rejection ethanol. It has been shown that there were two isoforms of aldehyde dehydrogenases (aldehyde dehydrogenase 1 and aldehyde dehydrogenase 2) in cytosol fractions of all investigated brain structures of animals preferring ethanol while only aldehyde dehydrogenase 2 has been found in the new cotex of rats rejecting ethanol. Thus, aldehyde-dehydrogenase activity is higher in the animals preferring ethanol than in those ones rejecting ethanol. Content of dophamine in the rats preferring ethanol is higher than in those ones rejecting ethanol both in the hypothalamus and new cortex. Differences between the studied groups of animals can underlie the pathologic attraction to alcohol.     

Origins of the high catalytic activity of human alcohol dehydrogenase 4 studied with horse liver A317C alcohol dehydrogenase

The turnover numbers and other kinetic constants for human alcohol dehydrogenase (ADH) 4 ("stomach" isoenzyme) are substantially larger (10-100-fold) than those for human class I and horse liver alcohol dehydrogenases. Comparison of the primary amino acid sequences (69% identity) and tertiary structures of these enzymes led to the suggestion that residue 317, which makes a hydrogen bond with the nicotinamide amide nitrogen of the coenzyme, may account for these differences. Ala-317 in the class I enzymes is substituted with Cys in human ADH4, and locally different conformations of the peptide backbones could affect coenzyme binding. This hypothesis was tested by making the A317C substitution in horse liver ADH1E and comparisons to the wild-type ADH1E. The steady-state kinetic constants for the oxidation of benzyl alcohol and the reduction of benzaldehyde catalyzed by the A317C enzyme were very similar (up to about 2-fold differences) to those for the wild-type enzyme. Transient kinetics showed that the rate constants for binding of NAD(+) and NADH were also similar. Transient reaction data were fitted to the full Ordered Bi Bi mechanism and showed that the rate constants for hydride transfer decreased by about 2.8-fold with the A317C substitution. The structure of A317C ADH1E complexed with NAD(+) and 2,3,4,5,6-pentafluorobenzyl alcohol at 1.2 ? resolution is essentially identical to the structure of the wild-type enzyme, except near residue 317 where the additional sulfhydryl group displaces a water molecule that is present in the wild-type enzyme. ADH is adaptable and can tolerate internal substitutions, but the protein dynamics apparently are affected, as reflected in rates of hydride transfer. The A317C substitution is not solely responsible for the larger kinetic constants in human ADH4; thus, the differences in catalytic activity must arise from one or more of the other hundred substitutions in the enzyme.     

Purification and Properties of Rape Alcohol Dehydrogenase

Germinating seeds with the highest specific activity (24 hour germination) were used for isolation of alcohol dehydrogenase, ADH, from rape (Brassica napus L. cv. T?ebi?ská). The rape ADH was purified by fractionation with ammonium sulphate, desalting on Sephadex G 25, chromatography on DEAE cellulose and gel filtration on Sephadex G 150. Using this isolation procedure, enzyme with a specific activity 85.6 times higher than that of the crude extract was obtained. The molecular weight of the enzyme obtained is 66.000. The enzyme is a metallo-enzyme containing sulfhydryl groups as evidence by the inhibitory effect of chelating compounds and thiol reagents. The optimum pH for the oxidation of ethanol is 8.5 and for reduction of acetaldehyde 7.0. The enzyme exhibits a relatively wide substrate specificity towards alcohols. Dimethyl-sulphoxide (DMSO), some amides and oximes and some intermediates of the carbohydrate metabolism act as ADH inhibitors, ATP as analogue of NAD also exhibits an inhibitory effect. The inhibitory effect of heterocyclic substances (pyrazol, imidazol, pyridine) is similar to the effect on liver alcohol dehydrogenase.     

Circadian rhythms of alcohol dehydrogenase and MEOS in the rat

The circadian peak in alcohol dehydrogenase (ADH) activity fell near the time of maximal blood ethanol clearance rates both in groups of rats injected with a single ethanol dose (acute group) and in rats continuously exposed to ethanol for 22 weeks (chronic group). However, at all timepoints investigated ADH activity levels were lower and fluctuated less in the chronic group than in either the acute or control (ethanol naive) groups. In contrast, activity levels of the microsomal ethanol oxidizing system (MEOS) revealed a prominent rhythm that was 180 degrees out of phase with the ADH rhythm in the chronic group, while MEOS activity showed very low levels in the acute and control groups and did not vary over the circadian span.     

The effects of starving and refeeding on the intra-acinar distribution pattern of alcohol-dehydrogenase activity in rat liver

Microquantitative determinations of ADH activity were carried out on the livers of male and female rats. The animals were either starved for 84 h, or starved and then refed with a carbohydrate-rich diet for 6 nights. When the enzyme activity is expressed in mumoles/min/g dry weight, fasting does not appear to alter liver ADH activity, while in starved and subsequently refed rats it is diminished by 20%. Microquantitative measurements of ADH activity in 50-150 ng lyophilized tissue samples, microdissected the whole way along the sinusoidal length, made the computer-aided plotting of intra-acinar distribution patterns possible. The results showed that, under the feeding conditions selected, only minor changes in the ADH activity profiles occur in the liver acinus. These are within the range of the standard deviations of the normal mean values. From these results it can be deduced that fasting and refeeding do not lead to specific inhibition or induction of liver ADH activity. - The decrease of ADH activity of total liver (mumol/min) per total body weight in starved rats is obviously the result of a loss of protein which affects the liver cells of all acinar zones almost equally.     

Cloning and sequencing of the gene coding for alcohol dehydrogenase of Bacillus stearothermophilus and rational shift of the optimum pH.

Using Bacillus subtilis as a host and pTB524 as a vector plasmid, we cloned the thermostable alcohol dehydrogenase (ADH-T) gene (adhT) from Bacillus stearothermophilus NCA1503 and determined its nucleotide sequence. The deduced amino acid sequence (337 amino acids) was compared with the sequences of ADHs from four different origins. The amino acid residues responsible for the catalytic activity of horse liver ADH had been clarified on the basis of three-dimensional structure. Since those catalytic amino acid residues were fairly conserved in ADH-T and other ADHs, ADH-T was inferred to have basically the same proton release system as horse liver ADH. The putative proton release system of ADH-T was elucidated by introducing point mutations at the catalytic amino acid residues, Cys-38 (cysteine at position 38), Thr-40, and His-43, with site-directed mutagenesis. The mutant enzyme Thr-40-Ser (Thr-40 was replaced by serine) showed a little lower level of activity than wild-type ADH-T did. The result indicates that the OH group of serine instead of threonine can also be used for the catalytic activity. To change the pKa value of the putative system, His-43 was replaced by the more basic amino acid arginine. As a result, the optimum pH of the mutant enzyme His-43-Arg was shifted from 7.8 (wild-type enzyme) to 9.0. His-43-Arg exhibited a higher level of activity than wild-type enzyme at the optimum pH.     

Liver alcohol dehydrogenase activity in the female rat: Effects of ovariectomy and estradiol administration

The effects of ovariectomy and administration of estradiol on the activity of liver alcohol dehydrogenase and on the rate of ethanol elimination were determined in female Sprague-Dawley rats. The activity of the enzyme and the rates of ethanol elimination in the female sham-operated animals were higher than obtained previously in male rats of the same age. Ovariectomy had no effect on liver alcohol dehydrogenase and on rates of ethanol elimination. Estradiol administration resulted in an increase in liver weight and in total liver alcohol dehydrogenase activity per animal in sham-operated but not in ovariectomized animals. The increase in enzyme activity after estradiol administration in sham-operated animals was not associated with a significant increase in the rate of ethanol elimination, suggesting that the enzyme activity in female rats is not rate-limiting in in vivo ethanol oxidation.     

Alcohol dehydrogenase (ADH). Influence of homo- and heterologous ADH administration on albino rats craving for alcohol and on ADH isozyme activity in the liver.

The effects of homo- and heterologous alcohol dehydrogenase (ADH) administration into albino rats were investigated. It was found that homologous ADH increases and heterologous ADH decreases the craving for ethanol. The latter effect was accompanied by the appearance of anti-ADH-3 antibodies and by a decrease in ADH-3 activity in the liver. Craving for alcohol decreased after both active and passive immunization against ADH.