Effect of ethanol on the binding of 35S-T-butylbicyclophosphorothionate to mouse brain membranes

The effect of in vitro addition of ethanol (0.02-1.0 M) on the binding of 35S-TBPS was examined in brain membranes from cerebellum and cortex of naive or chronically ethanol-treated C57B1 mice. In brain membranes of untreated animals, increasing concentrations of ethanol produced a dose-related inhibition of 35S-TBPS binding in the brain areas investigated. Additional studies showed that this effect of ethanol was due to a decreased affinity of 35S-TBPS for its binding sites. Chronic treatment of the animals with ethanol, which produced tolerance to and dependence on ethanol, did not alter ethanol's ability to inhibit the binding of 35S-TBPS. In naive animals, the in vitro addition of GABA or pentobarbital produced a pronounced inhibition of 35S-TBPS, both drugs being more potent in the cerebellum than in the cortex. Picrotoxin also produced a dose-dependent inhibition at 35S-TBPS, but was equally potent in the brain areas investigated. The inhibition by GABA or pentobarbital was not influenced by in vitro addition of a physiologically relevant concentration of ethanol (100 mM), whereas ethanol produced a significant increase in the IC50 values for picrotoxin both in the cortex and in the cerebellum. Furthermore, the inhibitory effects of GABA or pentobarbital on 35S-TBPS binding remained unchanged in animals chronically treated with ethanol for 7 days. Our data indicate that ethanol may affect the GABA receptor system through a rather specific interaction with the 35S-TBPS recognition site, but that this action of ethanol is not altered by the development of tolerance to and dependence on ethanol.     

Interaction of the Bacterial Ribonuclease Binase with the Polypeptide Inhibitor Barstar Based on Kinetic Data on Poly(U) Hydrolysis

The reaction of poly(U) hydrolysis catalyzed by binase while the latter is inhibited by barstar has been investigated. The inhibition constant for barstar and the apparent Michaelis constants for the inhibition by barstar in the presence of ethanol and NaCl have been determined. Both ethanol and NaCl enhance the inhibition by barstar. This suggests that the binding of barstar with binase is probably due to the interaction of hydrophobic sites rather than by electrostatic interaction between amino acid residues.     

Inhibition of succinate and NADH oxidases of submitochondrial particles by iron chelators and sulfhydryl reagents]

The inhibition of succinate- and NADH-oxidase activities of submitochondrial particles by 4,7-diphenyl-1,10-phenantroline was studied. The inhibition was shown to increase when the particles were pretreated with SH-reagents. The treatment of submitochondrial particles with ethanol in the presence of 1,10-phenantroline resulted in a complete inactivation of succinate oxidase and succinate: tetramethyl-n-phenyldiamine reductase; the succinate PMS reductase activity was only partially inhibited after such treatment. It is concluded that tetramethyl-n-phenyldiamine and phenazine metasulfate react with different sites of the succinate dehydrogenase complex. The changes in the properties of submitochondrial particles after ethanol--phenantroline treatment are apparently due to the effect of non-polar solvent rather than to the extraction of non-haem iron.     

Comparative study of the effects of short- and long-term ethanol treatment and alcohol withdrawal on phospholipid biosynthesis in rat hepatocytes

This study describes the effects of short- and long-term ethanol treatment and withdrawal on the biosynthesis of the phospholipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in hepatocytes isolated from rats, using isotopically labelled choline and ethanolamine as exogenous precursors. Our results demonstrate that short-term ethanol consumption increases the incorporation of exogenous polar bases into PC and PE, whereas long-term ethanol administration provokes a differential effect in both PC and PE biosynthesis via cytidine diphosphate derivatives (CDP-derivatives), decreasing PC synthesis and increasing the biosynthesis of PE. We suggest that the increased biosynthesis of PE after ethanol treatment results from changes in lipogenic substrates produced as a consequence of ethanol metabolism, whilst the specific inhibition of PC biosynthesis seems to be a consequence of alterations of enzymes involved in the CDP-choline pathway. With regard to the influence of ethanol on PE methylation to give PC, our results demonstrate that ethanol activates this pathway in short-term, as well as chronic ethanol treatment. Ethanol withdrawal returns the activity of the PC and PE pathways to control levels. The alterations in the biosynthesis of the main phospholipids, PC and PE, demonstrated in this study could be of a great physiological interest in determining the pathology of alcoholism.     

Proteasome activity and autophagosome content in liver are reciprocally regulated by ethanol treatment

The proteasome and autophagy are two major intracellular protein degradation pathways and the regulation of each by ethanol metabolism affects cellular integrity. Using acute and chronic ethanol feeding to mice in vivo, and precision-cut rat liver slices (PCLS) ex vivo, we examined whether ethanol treatment altered these proteolytic pathways. In acute studies, we gave C57Bl/6 mice either ethanol or phosphate-buffered saline (PBS) by gastric intubation and sacrificed them 12h later. PCLS were exposed to 0 or 50mM ethanol for 12 and 24h with or without 4-methylpyrazole (4MP). In chronic studies we pair-fed control and ethanol liquid diets for 4-6 weeks to transgenic mice, expressing the green fluorescent protein (GFP) fused to the autophagic marker, microtubule associated protein-1 light chain 3 (LC3). Acute ethanol administration elevated autophagosomes (AVs), as judged by a 1.5-fold increase in LC3II content over PBS-gavaged control mice. Hepatic proteasome activity was unaffected by this treatment. Compared with controls, ethanol exposure for 12 and 24h to PCLS inhibited proteasome activity by 1.5- to 3-fold and simultaneously enhanced AVs by 2- to 5-fold. The decrease in proteasome activity and the rise in AVs both depended on ethanol oxidation as its inhibition by 4-methylpyrazole (4MP) blocked both proteasome inhibition and AV induction. Hepatocytes from mice chronically consuming ethanol exhibited a 1.6-fold decline in proteasome activity, and a 4-fold rise in GFP-LC3 puncta compared with pair-fed control mice. When we exposed hepatocytes from these animals to MG262, a proteasome inhibitor, LC3II puncta per cell further increased 2- to 5-fold over untreated cells. Conclusion: Our findings demonstrate that ethanol metabolism generates oxidants, the levels of which differentially influence the activities of the proteasome and autophagy.     

Biomass production potential of grasslands in the oak savanna region of Minnesota, USA

Biomass harvested from grasslands formerly used for forage production or set aside for conservation has been identified as a potential source of bioenergy feedstocks. Our objective was to characterize yield and chemical composition of biomass harvested from existing grasslands in the oak savanna region of Minnesota and to determine whether aggregated soil properties and grassland type influence biomass yield and feedstock properties. The influence of soil type and dominant functional plant groups on biomass yield, theoretical ethanol yield, and mineral, ash, and lignocellulosic concentration was measured on biomass harvested from 32 grassland sites. Soils with high productivity ratings, as measured by the Minnesota Crop Productivity Index, produced 36 % more biomass than lower quality soils. Grasslands dominated by warm-season species produced 18 % more biomass than those dominated by cool-season species, when measured after senescence during the late-fall harvest time. Biomass harvested from sites dominated by cool-season grasses had higher N, Mg and Cl concentrations than those dominated by warm-season grasses, suggesting that such grasslands could have lower efficiency in thermochemical conversion processes and that repeated harvesting from such grasslands could remove nutrients from the systems. In addition, glucose and xylose concentrations were slightly higher in biomass from sites dominated by warm-season grasses, which resulted in an estimated additional 12 L?Mg^?1 of ethanol over those dominated by cool-season grasses. Combined with the greater yields, warm-season grasslands could produce an additional 376 L?ha^?1 year^?1.     

Lead acetate and arsenic trioxide induce instability of microsatellites at three different fragile sites (6q21, 9q32-9q33 and 15p14) within the genome of the rat

Human exposure to metals is of increasing concern due to the well-documented toxic and carcinogenic effects of metals and metal compounds, and the rising environmental levels due to industrial processes and pollution. It has been reported that metals can be genotoxic by several modes of action including generation of reactive oxygen species and inhibition of DNA repair. The aim of this study was to evaluate microsatellite instability (MSI) in three microsatellite loci (D6mit3, D9mit2 and D15Mgh1) located within three common fragile sites in the genome of the laboratory rat (6q21, 9q32-9q33 and 15p14) exposed to acute and chronic doses of a metal salt (lead acetate trihydrate) and a metalloid oxide (arsenic trioxide). In the acute and sub-chronic studies with the two compounds, MSI was observed in the three loci as deletions or additions of microsatellite repeats. The percentages of MSI were 36.4% and 42.1% for lead acetate and arsenic trioxide, respectively. Results of the present work indicate that the microsatellites located within fragile sites provide a convenient assay system to detect changes in DNA sequences resulting from exposure to genotoxic agents.     

Effect of an antiserum to brain gamma gamma-enolase (protein 14-3-2) on ethanol consumption by rats

The influence of intracisternal administration of antiserum to the neurospecific brain gamma gamma-enolase (aS-gamma gamma) on the consumption of 7.5% ethanol solution by rats was studied. Injection of aS-gamma gamma decreased the ethanol intake by the rats which had been drinking 15% solution of ethanol for 7 months as a single source of liquid. In vitro aS-gamma gamma caused 4-fold inhibition of the gamma gamma-enolase activity while it did not influence the alpha alpha-enolase activity. Intracisternal administration of aS-gamma gamma shifted enolase isoenzyme spectra in the direction of the decrease of gamma gamma-enolase content. It is suggested that the effect of aS-gamma gamma on ethanol consumption is due to inhibition of the activity of gamma gamma-enolase which participates in energy metabolism in neurons.     

Enhanced catabolism to acetaldehyde in rostral ventrolateral medullary neurons accounts for the pressor effect of ethanol in spontaneously hypertensive rats

We have previously shown that ethanol microinjection into the rostral ventrolateral medulla (RVLM) elicits sympathoexcitation and hypertension in conscious spontaneously hypertensive rats (SHRs) but not in Wistar-Kyoto (WKY) rats. In this study, evidence was sought to implicate the oxidative breakdown of ethanol in this strain-dependent hypertensive action of ethanol. Biochemical experiments revealed significantly higher catalase activity and similar aldehyde dehydrogenase (ALDH) activity in the RVLM of SHRs compared with WKY rats. We also investigated the influence of pharmacological inhibition of catalase (3-aminotriazole) or ALDH (cyanamide) on the cardiovascular effects of intra-RVLM ethanol or its metabolic product acetaldehyde in conscious rats. Compared with vehicle, ethanol (10 μg/rat) elicited a significant increase in blood pressure in SHRs that lasted for the 60-min observation period but had no effect on blood pressure in WKY rats. The first oxidation product, acetaldehyde, played a critical role in ethanol-evoked hypertension because 1) catalase inhibition (3-aminotriazole treatment) virtually abolished the ethanol-evoked pressor response in SHRs, 2) intra-RVLM acetaldehyde (2 μg/rat) reproduced the strain-dependent hypertensive effect of intra-RVLM ethanol, and 3) ALDH inhibition (cyanamide treatment) uncovered a pressor response to intra-RVLM acetaldehyde in WKY rats similar to the response observed in SHRs. These findings support the hypothesis that local production of acetaldehyde, due to enhanced catalase activity, in the RVLM mediates the ethanol-evoked pressor response in SHRs.     

Involvement of oxygen and mitochondrial function in the metabolism of D-xylulose by Saccharomyces cerevisiae

Mitochondrial function associated with oxygen was required for growth of Saccharomyces cerevisiae on D-xylulose. The requirement was shown by (i) the inhibition of growth of a wild-type strain under anaerobic conditions, (ii) the inhibition of aerobic growth after treatment with inhibitors of mitochondrial function, and (iii) the lack of aerobic and anaerobic growth of nuclear and cytoplasmic petites. The mitochondrial function was associated with the channeling of catabolites of D-xylulose to growth processes, since ethanol was formed even when growth was inhibited. Mitochondrial function was implicated as well in determining the extent of growth and the concentration of ethanol in aerobic cultures of the wild-type. In such cultures, the concentration of ethanol decreased and growth increased concomitantly as aeration rate increased. A factor in this relation was considered to be the relatively poor ability of D-xylulose to inhibit the oxidative utilization of ethanol.     

Ethanol-induced changes in lipid composition of Escherichia coli: Inhibition of saturated fatty acid synthesis in vitro

Growth of Escherichia coli in the presence of ethanol results in the synthesis of lipids containing elevated proportions of unsaturated fatty acids. Previous in vivo experiments indicated that the ethanol-induced changes in fatty acid composition result from a preferential inhibition of saturated fatty acid synthesis. In this study, the inhibition of saturated fatty acid synthesis by ethanol was confirmed in vitro. This inhibition was not membrane mediated and resulted from a direct action of ethanol on the soluble enzymes of fatty acid synthesis. The addition of ethanol resulted in a decrease in chain length of both saturated and unsaturated acyl products in vitro. Experiments with enzymes prepared from several fatty acid synthesis mutants of E. coli indicate that β-hydroxydecanoyl-acyl carrier protein dehydrase is not the site of the ethanol inhibition of saturated fatty acid synthesis. The two condensing enzymes are the probable sites for inhibition by ethanol.     

Biological effects and metabolic interactions after chronic and acute administration of 4-methylpyrazole and ethanol to rats

4-Methylpyrazole in a dose producing an inhibition of alcohol dehydrogenase of about 60% was given alone or in combination with ethanol (10%) as sole drinking fluid to growing rats in periods up to 38 weeks. No effects were observed on the weight curves. Hematologic analyses showed normal values for blood and bone marrow. Studies of liver function with transaminase, bilirubin and albumin did not reveal any functional changes. Kidney function was normal as judged by creatinine and normal electrolytes. Electronmicroscopy of liver, kidney, and heart did not reveal any changes related to treatment. Combined treatment of ethanol and 4-methylpyrazole caused an increase of the microsomal drug-metabolizing activity. Chronic administration of ethanol and 4-methylpyrazole indicated that there is a mutual interaction in the metabolism of ethanol and 4-methylpyrazole, leading to a higher concentration of both ethanol and 4-methylpyrazole in the blood. Acute experiments, where alcohol dehydrogenase is saturated with ethanol, indicated a much slower elimination of 4-methylpyrazole. Administration of ethanol and 4-methylpyrazole in acute experiments showed a lower concentration of 4-hydroxymethylpyrazole in the blood indicating that ethanol interferes with the 4-methylpyrazole- and/or 4-hydroxymethyl-pyrazole-metabolizing enzymes. The present investigation has shown that the acute and chronic toxicity of 4-methylpyrazole alone or in combination with ethanol is minimal at doses that are effective in blocking ethanol metabolism in the rat. Because of its low toxicity and powerful inhibitory capacity, 4-methylpyrazole should be a potential tool for experimental clinical investigation of alcohol metabolism and its effects. 4-Methylpyrazole is also a potential therapeutic agent in methanol or ethylene glycol poisoning.     

Acute inhibition by ethanol of intestinal absorption of glucose and hepatic glycogen synthesis on glucose refeeding after starvation in the rat.

1. Intragastric administration of ethanol (75 mmol/kg body wt.) at 1 h before glucose refeeding of 24 h-starved rats inhibited hepatic glycogen deposition (by 69%) and synthesis (by approx. 70%), but was without significant effect on muscle glycogen deposition and synthesis. 2. Treatment of ethanol-administered rats with methylpyrazole (an inhibitor of alcohol dehydrogenase) did not significantly diminish the inhibitory effect of ethanol on hepatic glycogen deposition after glucose refeeding, suggesting that the inhibition was not dependent on ethanol metabolism. 3. Ethanol delayed and diminished intestinal glucose absorption, at least in part by delaying gastric emptying. 4. At a lower dose (10 mmol/kg body wt.), ethanol inhibited hepatic glycogen repletion and synthesis without compromising intestinal glucose absorption. Ethanol inhibited glycogen deposition (by 40%) in hepatocytes from starved rats provided with glucose + lactate + pyruvate as substrates, consistent with it having a direct effect to diminish hepatic glycogen synthesis by inhibition of gluconeogenic flux at a site(s) between phosphoenolpyruvate and triose phosphate in the pathway. 5. It is concluded that ethanol acutely impairs hepatic glycogen repletion by inhibition at at least two distinct sites, namely (a) intestinal glucose absorption and (b) hepatic gluconeogenic flux.     

Design and optimization of quinazoline derivatives as melanin concentrating hormone receptor 1 (MCHR1) antagonists: part 2

Melanin concentrating hormone receptor 1 (MCHR1) antagonists have potential for the treatment of obesity and several CNS disorders. In the preceding article, we have described a novel series of quinazolines as MCHR1 antagonists and demonstrated in vivo proof of principle with an early lead. Herein we describe the detailed SAR and SPR studies to identify an optimized lead candidate having good efficacy in a sub-chronic DIO model with a good cardiovascular safety window.     

Interaction of ethanol with opiate receptors

Addition of ethanol to rat brain homogenate containing opiate receptors inhibits at a concentration of 50 mM the stereospecific binding of 3H-naloxone at 37 degrees C but not at 0 degree C, with the ID50 being 462 mM under these conditions. The temperature-dependent inhibition of the ligand binding suggests that ethanol does not compete with naloxone for specific binding sites of opiate receptors and changes the structure of lipids in biological membranes. Scatchard's analysis has demonstrated that apart from a decrease in the number of highly affinity binding sites of 3H-naloxone, the total amount of the binding sites remains unchanged both in the presence and absence of ethanol and constitutes 453 and 549 fmol/mg protein. It is assumed that ethanol might interconvert highly and low-affinity binding sites. Analysis of the effect of ethanol on 3H-naloxone binding with opiate receptors contained by synaptic membranes obtained from animals with varying predisposition to voluntary alcoholization has shown that ethanol inhibits to a greater degree ligand binding with membranes obtained from rats predisposed to alcoholization. The possibility of the involvement of receptors in the biochemical mechanisms by which the initial alcoholic motivation is effected is under discussion.     

Interactions among Positions in the Third and Fourth Membrane-associated Domains at the Intersubunit Interface of the N-Methyl-D-aspartate Receptor Forming Sites of Alcohol Action

The N-methyl-d-aspartate (NMDA) glutamate receptor is a major target of ethanol in the brain. Previous studies have identified positions in the third and fourth membrane-associated (M) domains of the NMDA receptor GluN1 and GluN2A subunits that influence alcohol sensitivity. The predicted structure of the NMDA receptor, based on that of the related GluA2 subunit, indicates a close apposition of the alcohol-sensitive positions in M3 and M4 between the two subunit types. We tested the hypothesis that these positions interact to regulate receptor kinetics and ethanol sensitivity by using dual substitution mutants. In single-substitution mutants, we found that a position in both subunits adjacent to one previously identified, GluN1(Gly-638) and GluN2A(Phe-636), can strongly regulate ethanol sensitivity. Significant interactions affecting ethanol inhibition and receptor deactivation were observed at four pairs of positions in GluN1/GluN2A: Gly-638/Met-823, Phe-639/Leu-824, Met-818/Phe-636, and Leu-819/Phe-637; the latter pair also interacted with respect to desensitization. Two interactions involved a position in M4 of both subunits, GluN1(Met-818) and GluN2A(Leu-824), that does not by itself alter ethanol sensitivity, whereas a previously identified ethanol-sensitive position, GluN2A(Ala-825), did not unequivocally interact with any other position tested. These results also indicate a shift by one position of the predicted alignment of the GluN1 M4 domain. These findings have allowed for the refinement of the NMDA receptor M domain structure, demonstrate that this region can influence apparent agonist affinity, and support the existence of four sites of alcohol action on the NMDA receptor, each consisting of five amino acids at the M3-M4 domain intersubunit interfaces.     

Iron overload prevents oxidative damage to rat brain after chlorpromazine administration

The hypothesis tested is that Fe administration leads to a response in rat brain modulating the effects of later oxidative challenges such as chlorpromazine (CPZ) administration. Either a single dose (acute Fe overload) or 6 doses every second day (sub-chronic Fe overload) of 500 or 50 mg Fe-dextran/kg, respectively, were injected intraperitoneally (ip) to rats. A single dose of 10 mg CPZ/kg was injected ip 8 h after Fe treatment. DNA integrity was evaluated by quantitative PCR, lipid radical (LR^·) generation rate by electron paramagnetic resonance (EPR), and catalase (CAT) activity by UV spectrophotometry in isolated brains. The maximum increase in total Fe brain was detected after 6 or 2 h in the acute and sub-chronic Fe overload model, respectively. Mitochondrial and nuclear DNA integrity decreased after acute Fe overload at the time of maximal Fe content; the decrease in DNA integrity was lower after sub-chronic than after acute Fe overload. CPZ administration increased LR^· generation rate in control rat brain after 1 and 2 h; however, CPZ administration after acute or sub-chronic Fe overload did not affect LR^· generation rate. CPZ treatment did not affect CAT activity after 1–4 h neither in control rats nor in acute Fe-overloaded rats. However, CPZ administration to rats treated sub-chronically with Fe showed increased brain CAT activity after 2 or 4 h, as compared to control values. Fe supplementation prevented brain damage in both acute and sub-chronic models of Fe overload by selectively activating antioxidant pathways.     

Characterization of the inhibitory effects of hyperprolactinaemia on the mechanism controlling LH secretion in chronically ovariectomized rats

The time-course of the inhibitory effect of hyperprolactinaemia on LH secretion was delineated. Hyperprolactinaemia was induced in ovariectomized rats with injections of domperidone or ovine prolactin and circulating LH levels were measured from 1 h to 9 days after the treatment. Inhibition of LH secretion occurred within 2-4 h after treatment, and was maintained (provided that serum prolactin remained elevated) for a period of 6 days only. Thereafter LH levels increased to become insignificantly different from control levels on Day 9. A reduction in pituitary responsiveness was not associated with the acute or sub-chronic inhibition of LH secretion, although a significant fall in responsiveness was observed simultaneously with the return of serum LH levels to control values. No changes in hypothalamic LH-RH content was found. It is concluded that an impairment of pituitary function is not responsible for the inhibitory action of prolactin on LH secretion.