Modulation of rat striatal membrane fluidity: effects on age related differences in dopamine receptor concentrations

Rat striatal membrane fluidity and dopamine receptor concentrations were modulated in vitro by ethanol or cholesterol hemisuccinate treatment. In general, available receptor levels are directly proportional to membrane fluidity. However, receptor concentrations remained higher in mature membranes at all levels of fluidity attained. These results suggest that apparent loss of striatal dopamine receptors during aging is not due to membrane sequestration.     

Factors influencing the development of tolerance to ethanol

The influence of three parameters on the degree of acquired tolerance to ethanol was studied using goldfish as the animal model. These factors were: a) the rate of ethanol administration; b) the repeated performance of the behavioral test, and c) the prior presence of tolerance to a low dose of ethanol. Tolerance, as indicated by the increase in the ethanol brain levels associated with the loss of righting reflex (overturn test), was observed in fish exposed to 0.2, 0.4, or 0.6% (w/v) ethanol for 1 to 24 hours independently of the rate of ethanol administration. Fish exposed to 0.8% (w/v) ethanol developed tolerance only when this ethanol concentration was reached slowly (circa 3 hours). The increase in functional demand by the repeated testing of the fish and the presence of tolerance to a low dose of ethanol (0.4%) did not affect the degree of the observed tolerance in fish left to equilibrate with a 0.8% ethanol dose. The data support the suggestion that motor functions controlled by the central nervous system can adapt to a high concentration of ethanol if an adequate amount of time is allowed for this compensation to occur or if this concentration is reached slowly. Furthermore, in goldfish the degree of acquired tolerance is directly proportional to the concentration of the ethanol during the time of exposure and independent of the previous experience in alcohol solution.     

A new alcohol antagonist: phaclofen

The ability of the GABA(B) receptor antagonist, phaclofen to alter behavioral effects of ethanol was evaluated by loss of righting reflex (sleep time), motor incoordination (bar holding), spontaneous locomotion (open field activity) and hypothermia. Pretreatment with phaclofen significantly decreased the effects of ethanol on motor incoordination, locomotor activity and hypothermia. However, phaclofen had no effect on either pentobarbitalor diazepam-induced motor incoordination. Phaclofen slightly increased the ED50 for loss of the righting reflex but did not alter either the duration of reflex loss produced by ethanol or blood ethanol levels at awakening. Our results suggest phaclofen is rapidly inactivated resulting in difficulty in observing antagonism of long duration ethanol effects. These findings suggest that the GABA(B) system may play a role in mediating several important actions of ethanol.     

Interactive effects of ethanol and silver on sodium transport across toad skin

Both ethanol and silver ions have been shown to affect ion transport across various epithelia. This investigation was principally undertaken to further define mechanisms of silver ions and ethanol, and their possible interactions, on sodium transport across toad skin. Isolated toad skin, mounted between identical oxygenated amphibian bicarbonate Ringer solutions, maintained stable transepithelial potential differences (serosa positive) and short-circuit currents for several hours at 25 degrees C. It was observed that (1) ethanol inhibited the active transcellular component of sodium absorption and this effect was reversible; (2) inhibition of sodium transport by ethanol was directly proportional to the applied concentration; (3) pretreatment with silver ions prevented any ethanol effects; and (4) pretreatment with ethanol prevented any silver ion effects. It was concluded from these results that ethanol induced its inhibitory effects on membrane phospholipids thereby perturbing the function of a sulfhydryl ligand, while silver ion or silver chloride complex binding to this ligand would maintain its function in sodium transport despite the presence of ethanol.     

Chronic ethanol consumption in rats produces opioid antinociceptive tolerance through inhibition of mu opioid receptor endocytosis

It is well known that the mu-opioid receptor (MOR) plays an important role in the rewarding properties of ethanol. However, it is less clear how chronic ethanol consumption affects MOR signaling. Here, we demonstrate that rats with prolonged voluntary ethanol consumption develop antinociceptive tolerance to opioids. Signaling through the MOR is controlled at many levels, including via the process of endocytosis. Importantly, agonists at the MOR that promote receptor endocytosis, such as the endogenous peptides enkephalin and β-endorphin, show a reduced propensity to promote antinociceptive tolerance than do agonists, like morphine, which do not promote receptor endocytosis. These observations led us to examine whether chronic ethanol consumption produced opioid tolerance by interfering with MOR endocytosis. Indeed, here we show that chronic ethanol consumption inhibits the endocytosis of MOR in response to opioid peptide. This loss of endocytosis was accompanied by a dramatic decrease in G protein coupled receptor kinase 2 (GRK2) protein levels after chronic drinking, suggesting that loss of this component of the trafficking machinery could be a mechanism by which endocytosis is lost. We also found that MOR coupling to G-protein was decreased in ethanol-drinking rats, providing a functional explanation for loss of opioid antinociception. Together, these results suggest that chronic ethanol drinking alters the ability of MOR to endocytose in response to opioid peptides, and consequently, promotes tolerance to the effects of opioids.     

Lack of cardiac alpha 1-adrenoceptor involvement in ethanol-induced cardiac hypertrophy

The development of cardiac hypertrophy was examined in rats given ethanol in a nutritionally adequate, liquid diet mixture, by intubation, in severely intoxicating doses at 8-h intervals for up to 96 h, alone or in combination with prazosin. Other groups of rats received isocalorically paired quantities of maltose-dextrin. Adrenal glands of rats receiving ethanol were larger than those from control animals. Prazosin did not affect this measure. In contrast, concurrent treatment with prazosin enhanced the loss of medullary catecholamines and noradrenaline from hearts of rats given ethanol, while it had no such effects in controls. Reflecting these changes, excreted quantities of catecholamines were markedly increased in rats given ethanol and prazosin. Hearts of animals given the combined treatment of ethanol and prazosin showed cardiomegaly at 24 h, when there was an increase of about 20% in proportional heart weight, an increase that persisted through the remaining 3 days of the study. At 48 h, hearts of animals given prazosin and ethanol were heavier than those given ethanol alone. A significant correlation between catecholamine excretion and the development of cardiac hypertrophy was identified. The results of the study show that prazosin can enhance effects of ethanol on the peripheral sympathetic nervous system. Moreover, the results suggest that postsynaptic alpha 1-adrenoceptor stimulation in the heart is not an important contributor to ethanol-induced cardiomegaly.     

Kinetics of apparent cell death in yeasts induced by ethanol

Summary Evidence is presented that adaptation of yeast cells to ethanol results in a reduced loss of cell viability induced by exposure to that agent. In line with earlier work, an exponential model is shown to apply when the concentration of ethanol exceeds a critical value, beyond which cell growth cannot occur. Such an exponential model is consistent with the absolute theory of reaction rates. Adaptation of yeast cells to 7% w/v ethanol lowers the specific rate of cell death at various ethanol concentrations by a factor of some 40 fold compared to a non-adapted culture.     

Physiological behaviour of Saccharomyces cerevisiae in aerated fed-batch fermentation for high level production of bioethanol

Saccharomyces cerevisiae was able to produce 20% (v/v) of ethanol in 45 h in a fully aerated fed-batch process recently developed in our laboratory. A notable feature of this process was a production phase uncoupled to growth, the extent of which was critical for high-level ethanol production. As the level of production was found to be highly variable, we investigated on this high variability by means of a detailed physiological analysis of yeast cells in two fed-batch fermentations showing the most extreme behaviour. We found a massive leakage of intracellular metabolites into the growth medium which correlated with the drop of cell viability. The loss of viability was also found to be proportional to the reduction of plasma membrane phospholipids. Finally, the fed-batch processes with the longest uncoupling phase were characterized by induction of storage carbohydrates at the onset of this phase, whereas this metabolic event was not seen in processes with a short uncoupling phase. Taken together, our results suggested that reproducible high-level bioethanol production in aerated fed-batch processes may be linked to the ability of yeast cells to impede ethanol toxicity by triggering a metabolic remodelling reminiscent to that of cells entering a quiescent GO/G1 state.     

Attenuation of fatty acid-induced apoptosis by low-dose alcohol in neonatal rat cardiomyocytes

Moderate alcohol consumption has been shown to reduce the morbidity and mortality from coronary heart disease. Ethanol elicits its protective effects via mechanisms that include activation of protein kinases linked to growth and survival. Our results in isolated neonatal rat cardiomyocytes demonstrate that repeated short-term, low-dose exposure to ethanol is sufficient to activate the growth and/or survival pathways that involve PKC-epsilon, Akt, and AMP-activated kinase. In addition, we are able to induce apoptosis in these cardiomyocytes using the saturated fatty acid palmitate. Pretreatment with multiple low-dose ethanol exposures attenuates the apoptotic response to palmitate. This protection is manifested by a reduction in caspase-3-like activity, decreased mitochondrial loss of cytochrome c, and decreased loss of the mitochondrial lipid cardiolipin. We previously reported that incubation of cardiomyocytes with palmitate results in decreased production of reactive oxygen species compared with cells incubated with the nonapoptotic fatty acid oleate. In the present study, we observed an increase in the production of superoxide and the rates of fatty acid oxidation in cardiomyocytes pretreated with ethanol and then exposed to fatty acids. The level of superoxide production in palmitate-treated cells returns to the levels observed in oleate-treated cells after ethanol exposure. Taken together with our observed increase in AMP-activated kinase activity, we propose that ethanol pretreatments stimulate oxidative metabolism and electron transport within cardiomyocytes. We postulate that stimulation of palmitate metabolism may protect cardiomyocytes by preventing accumulation of unsaturated precursor molecules of cardiolipin synthesis. Maintaining cardiolipin levels may be sufficient to prevent the mitochondrial loss of cytochrome c and the downstream activation of caspases.     

Metoprolol suppresses the development of ethanol-induced cardiac hypertrophy in the rat

Subacute, severe intoxication with ethanol stimulates the peripheral sympathetic nervous system in the rat and enhances the excretion of adrenaline and noradrenaline. In association with this effect there is a rapid development of cardiac hypertrophy, with proportional heart weight increasing by 12% within 48 h. At this time adrenal medullary adrenaline content was depressed by more than 35%, whereas nonadrenaline content of the adrenal and heart were not affected. Metoprolol (20 mg/kg, t.i.d.) was without effect when used alone and had little if any impact on the ethanol-induced changes. Metoprolol (100 mg/kg, t.i.d.) reduced adrenal catecholamine content, but not cardiac noradrenaline content, and diminished cardiac weight in control animals. The combination of ethanol with the high dose of methoprolol enhanced the loss of medullary catecholamine and reduced cardiac noradrenaline content, whereas cardiac weight was the same as in control animals. A correlation between sympathetic activation and increasing cardiac mass and its antagonism by metoprolol implies a beta-adrenoceptor mediated link in the cardiac hypertrophy induced by ethanol.     

Ethanol-induced changes in the fatty acid composition of Lactobacillus hilgardii, its effects on plasma membrane fluidity and relationship with ethanol tolerance

The effect of environmental ethanol concentration on the fatty acid composition of strains of Lactobacillus hilgardii, differing in their tolerance to ethanol, was determined. A marked increase in the proportion of lactobacillic acid (a cyclopropane fatty acid) and a decrease in oleic and vaccenic acids with increasing ethanol concentration was observed. The amount of lactobacillic acid determined at standard conditions (25°C, 0% ethanol) was found to be proportional to the ethanol tolerance of the strains studied. The effect of this alcohol on plasma membrane fluidity was studied by differential scanning calorimetry. The adaptive response to growth in the presence of high concentrations of ethanol produced membranes which, within the limits of ethanol tolerance, maintained the fluidity and integrity in an environment which tends to increase membrane rigidity. When pre-adapted cells are analysed in the absence of environmental ethanol there is a measurabie increase in fluidity. It is proposed that this phenomenon may be correlated with the increase in the proportion of lactobacillic acid. The existence of a relationship between membrane fluidity and ethanol tolerance is discussed.     

Ethanol self‐administration in serotonin transporter knockout mice: unconstrained demand and elasticity

Low serotonin function is associated with alcoholism, leading to speculation that increasing serotonin function could decrease ethanol consumption. Mice with one or two deletions of the serotonin transporter (SERT) gene have increased extracellular serotonin. To examine the relationship between SERT genotype and motivation for alcohol, we compared ethanol self‐administration in mice with zero (knockout, KO), one (HET) or two copies (WT) of the SERT gene. All three genotypes learned to self‐administer ethanol. The SSRI, fluvoxamine, decreased responding for ethanol in the HET and WT, but not the KO mice. When tested under a progressive ratio schedule, KO mice had lower breakpoints than HET or WT. As work requirements were increased across sessions, behavioral economic analysis of ethanol self‐administration indicated that the decreased breakpoint in KO as compared to HET or WT mice was a result of lower levels of unconstrained demand, rather than differences in elasticity, i.e. the proportional decreases in ethanol earned with increasing work requirements were similar across genotypes. The difference in unconstrained demand was unlikely to result from motor or general motivational factors, as both WT and KO mice responded at high levels for a 50% condensed milk solution. As elasticity is hypothesized to measure essential value, these results indicate that KO value ethanol similarly to WT or HET mice despite having lower break points for ethanol .     

The increased rate of ethanol removal from blood of clofibrate-treated rats (Short Communication)

After ethanol injection similar metabolite changes were found in livers of clofibrate-treated and non-treated rats. The rate of ethanol removal from blood of clofibrate-treated rats was increased by 50%, but this was directly proportional to the increased liver size caused by clofibrate. No other difference needs to be postulated to explain the increased rate of ethanol removal from blood.     

A comparison of the effects of alcohol on mice selectively bred for differences in ethanol sleep-time

The activities of liver alcohol dehydrogenase and aldehyde dehydrogenase were virtually identical in two lines of mice selectively bred for differences in “sleep-time” to ethanol. Similarly, the rates of blood ethanol disappearance were identical for these mouse lines. The ED50 values for loss of righting reflex with ethanol were approximately two-fold greater in the short-sleep than in the long-sleep mice. These results suggest that the two lines of mice differ in their central nervous system sensitivity to ethanol.     

Rhizomorph Formation in Fungi III. The Effect of Ethanol on the Synthesis of DNA and RNA and Uptake of Asparagine and Phosphate in Armillaria mellea

Growth and rhizomorph formation in Armillaria mellea (Vahl ex Fr.) Quél. can be stimulated by ethanol when grown on a synthetic glucose medium. The content of DNA (deoxy-ribonucleic acid) and RNA (ribonucleic acid) in cultures of A. mellea have been followed during a growth period in relation to growth (dry weight) and ethanol uptake with 3 different initial ethanol concentrations. The continuous increase in dry weight as well as DNA and RNA contents during growth showed similar exponential rates as long as ethanol was present in the medium. The final amounts were proportional to the initial ethanol concentration. A further supply of ethanol caused a similar proportional increase in dry weight and also in the DNA and RNA contents, which indicates that the growth stimulated by ethanol is caused by cell divisions rather than an accumulation of lipids or polysaccharides. Uptake of asparagine and phosphate were also stimulated by ethanol.     

Impacts of main factors on bioethanol fermentation from stalk juice of sweet sorghum by immobilized Saccharomyces cerevisiae (CICC 1308)

In order to attain a higher ethanol yield and faster ethanol fermentation rate, orthogonal experiments of ethanol fermentation with immobilized yeast from stalk juice of sweet sorghum were carried out in the shaking flasks to investigate the effect of main factors, namely, fermentation temperature, agitation rate, particles stuffing rate and pH on ethanol yield and CO(2) weight loss rate. The range analysis and analysis of variance (ANOVA) were applied for the results of orthogonal experiments. Results showed that the optimal condition for bioethanol fermentation should be A(4)B(3)C(3)D(4), namely, fermentation temperature, agitation rate, particles stuffing rate and pH were 37 degrees C, 200rpm, 25% and 5.0, respectively. The verification experiments were carried out in shaking flasks and 5L bioreactor at the corresponding parameters. The results of verification experiments in the shaking flasks showed that ethanol yield and CO(2) weight loss rate were 98.07% and 1.020gh(-1), respectively. The results of ethanol fermentation in the 5L bioreactor showed that ethanol yield and fermentation time were 93.24% and 11h, respectively. As a result, it could be concluded that the determined optimal condition A(4)B(3)C(3)D(4) was suitable and reasonable for the ethanol fermentation by immobilized Saccharomyces cerevisiae. The conclusion in the research would be beneficial for application of ethanol fermentation by immobilized S. cerevisiae from stalk juice of sweet sorghum.     

The influence of adrenal medullectomy on the development of ethanol-induced cardiac hypertrophy

Cardiac hypertrophy was assessed in adrenal medullectomized and sham control rats given ethanol in a liquid diet mixture, intragastrically, in severely intoxicating doses at 8-h intervals for 48 and 96 h. The ethanol treatments produced increases of some 20% in wet and dry proportional heart weights in the control animals, but this rapid development of hypertrophy was less apparent in the medullectomized rats. Hearts of the medullectomized animals given ethanol were heavier than those of pair-matched controls given isocaloric maltose-dextrin in the diet mix. These increases were not statistically significant. The ethanol treatments produced, in addition, equivalent increases in the weight of the adrenal glands of both medullectomized and control animals. Medullectomy was evaluated by analysis of adrenal glands and urine samples for catecholamines. The majority of adrenals from the demedullated group had nondetectable amounts of catecholamines and minimal quantities were found in the remainder. Adrenaline was not detected generally in urine samples from this group. The adrenaline contents of adrenals from sham controls given ethanol were markedly reduced by the ethanol treatments, whereas the quantities in urine were many times greater than those from rats given maltose-dextrin. Urinary noradrenaline levels were increased in both control and medullectomized rats given ethanol. The results of this study identify that adrenal medullary catecholamines participate in the rapid development of cardiac hypertrophy that results from severe, continuing ethanol intoxication in the rat.     

Alcohol stimulates Na+/Ca2+ exchange in brain mitochondria

Ethanol, at low concentrations, specifically stimulates the Na(+)-dependent Ca2(+)-efflux in brain mitochondria. In addition, at higher concentrations, ethanol inhibits the Na(+)-independent Ca2(+)-efflux. The electrogenic Ca(+)-uptake system is not affected by ethanol. The specific stimulation of Na+/Ca2+ exchange reaches a maximum of 60% stimulation, with half-maximal stimulation at 130 mM ethanol. The inhibition of the Na(+)-independent efflux is proportional to the ethanol concentration, becoming significant only above 200 mM, with 50% inhibition at 0.5 M. The inhibition of the Na(+)-independent efflux is, in large part, due to an inhibition of the activation of the Cyclosporin-sensitive pore. Long-term ethanol-feeding had no effect on the Ca2+ transport systems and their sensitivity to acute ethanol treatment. It is suggested that the stimulation of the Na(+)-dependent Ca2(+)-efflux, which is the dominant Ca2+ efflux pathway in brain mitochondria, contributes to the intoxicating effects of ethanol.     

Volatile transport in frozen aqueous solutions. II. Influence of system parameters

Frozen aqueous butanol solutions are evaluated for the influence of sample preparation and equilibration conditions on the loss of butanol.It is shown that an increase of equilibration temperature results in increased butanol loss, both water independent and water dependent. The freezing rate will influence the equilibration retention level, with faster freezing resulting in smaller butanol loss. An increase in butanol concentration gave the same percentage of butanol loss, i.e., the amount of butanol lost was directly proportional to the initial concentration.     

Effect of bax deletion on ethanol sensitivity in the neonatal rat cerebellum

The developing cerebellum is highly sensitive to ethanol during discrete neonatal periods. This sensitivity has been linked to ethanol-induced alterations in molecules of the Bcl-2 survival-regulatory gene family. Ethanol exposure during peak periods of cerebellar sensitivity, for example, results in increased expression of proapoptotic proteins of this family, while overexpression of the antiapoptotic Bcl-2 protein in the nervous system protects against ethanol neurotoxicity. For the present study, neonatal mice with a targeted deletion of the proapoptotic bax gene were used to determine whether elimination of this protein would mitigate ethanol toxicity. bax knock-out and wild-type mice pups were exposed to ethanol via vapor inhalation during the maximal period of neonatal cerebellar ethanol sensitivity and cerebellar tissue was subsequently assessed for Purkinje and granule cell number and ethanol-mediated generation of reactive oxygen species (ROS). The results revealed that: (1) ethanol exposure during the peak period of cerebellar vulnerability resulted in substantial loss of Purkinje cells in wild-type animals, but not in bax knock-outs; (2) granule cells in the bax gene-deleted animals were not similarly protected from ethanol effects; and (3) levels of ROS following acute ethanol exposure were appreciably enhanced in the wild-type animals but not in the bax knock-outs. These results imply that Bax is important to ethanol-induced Purkinje cell death during critical neonatal periods, but that ethanol effects on granule cells may function at least partially independent of this apoptosis agonist. Amelioration of ethanol-mediated increases in ROS production in the knock-outs may contribute to the observed effects.