The characteristics of higher nervous activity and of the brain benzodiazepine system in rats subjected to ethanol exposure in the prenatal period

Exposure to ethanol during pregnancy results in the alternation of 3H-diazepam binding to synaptosomal neocortical membranes from the rat offspring. In male experimental rats, 14 days of age, binding level diminished to 11%. In two-month-old control rats Scatchard plot was biphasic. It has been shown that prenatal exposure to ethanol leads to changes in the nature of binding in two-month-age experimental animals, as compared with the control ones. 3H-diazepam binding changes went along with behavioural deviations. In experimental rats locomotor activity was increased in the "open field" test, passive avoidance conditioned reflex retention was decreased and elaboration parameters of active avoidance conditioned reflex were changed, as compared with the control ones. The data obtained show that higher integrative functions were disturbed by prenatal alcoholization. Correlations between benzodiazepine receptor state and behaviour were studied.     

Long-term ethanol alters the binding of 3H-opiates to brain membranes

In order to examine whether ethanol treatment has selective or differential effects on brain binding sites for opiates, male Sprague Dawley rats were fed for 15 or 21 days with a complete liquid diet containing 6.5% ethanol (v:v) or an isocaloric amount of sucrose. The binding of 3H-DADL-enkephalin, 3H-dihydromorphine and 3H-naloxone to the brain membranes from rats treated with ethanol was increased. However, addition of ethanol directly in the incubation medium decreased the binding of 3H-DADL enkephalin and increased the binding of 3H-dihydromorphine to brain membranes from both control and ethanol treated rats. Direct exposure of brain membranes to ethanol caused no significant change in the binding of 3H-naloxone. Thus chronic ethanol ingestion alters the binding of opiate ligands to brain membranes. Furthermore, the direct effect of ethanol appears to be different for the different classes of opiate binding sites.     

急、慢性乙醇处理后大鼠伏核内cAMP反应元件结合蛋白磷酸化的变化

采用免疫组织化学的方法,检测急性、慢性乙醇作用及戒断后大鼠伏核内cAMP反应元件结合蛋白(cAMP response element binding protein,CREB)磷酸化的变化。结果显示,急性腹腔注射乙醇后15min,伏核内磷酸化CREB(Phospho-CREB,p-CREB)蛋白明显增加,30min后达高峰,至1和6h后仍明显高于对照组。而慢性饮乙醇溶液显著降低大鼠伏核内P—CREB蛋白含量,在撤除乙醇后24、72h时,伏核内p—CREB蛋白含量仍明显较低,戒断后7d,恢复到正常水平。结果表明,急性乙醇处理增加伏核内CREB磷酸化作用,而慢性乙醇作用则降低伏核内CREB磷酸化作用,这可能是乙醇依赖的分子机制之一。     

Hepatic insulin binding following in utero exposure to ethanol

Insulin binding to liver membranes has been studied in term fetuses of rats fed ethanol-containing liquid diet during pregnancy . Pair-fed and ad libitum-fed controls received liquid diet in which maltose-dextrins were substituted isocalorically for ethanol. Food consumption and body weigh gain of ethanol- imbibing dams were 35% and 70% less than their ad libitum counterparts respectively. Ethanol-fed rats also exhibited less gain in body weight than pair-fed controls despite isocalorically equivalent food intake. The number of live pups was not different among the various groups; however, liver weight of fetuses exposed to ethanol in utero was 47% less than those of the pups of ad libitum control dams and 28% less than those of the offspring of pair-fed control rats. Insulin binding to liver membranes of fetuses exposed to ethanol in utero was lower than that of ad libitum controls but was not significantly different from that of the pair-fed control animals. Average affinity profiles showed a reduction in K at all levels of receptor occupancy in the fetuses of ethanol-fed rats. For fetuses of the pair-fed group, K was reduced only at fractional occupancy below 20% but not at higher fractional occupancy. Because of the similarity of insulin binding in the fetuses of the ethanol-fed rats and their pair-fed counterparts, effects of ethanol on insulin binding cannot account for the reduced hepatic glycogen stores previously reported in term fetuses.     

Peculiarities of H3-muscimol binding to neocortex membranes of rats exposed to the effects of ethanol in the prenatal period

White rats were subjected to ethanol exposure during 5-20 days of pregnancy. 3H-muscimol binding with synaptosomal neocortex membranes yielded from two month age offsprings was studied. 3H-muscimol binding level for experimental animals was 27% more than for control ones. Possible ways of GABAergic system malformation are discussed.     

Disorders in memory and learning in offspring of alcoholized female rats,and a possibility for correction of these changes

We studied the effect of alcohol intoxication of albino female rats on the process of learning and memory of their adult (two-month-old) offspring and also a possibility for correction of the observed changes using dolivin. During pregnancy and lactation, female rat of the experimental group obtained 15% solution of ethanol instead of water. To estimate the successfulness of spatial learning of their offspring, we used a multiway elevated labyrinth; the level of consolidation of memory traces was estimated using a passive avoidance test in a chamber with dark and illuminated sections. In the tested offspring rats, prenatal exposure to ethanol induced dramatic drops in the indices of both the above tests. The use of dolivin simultaneously with ethanol exposure allowed us to demonstrate the clear protective properties of this complex preparation containing such antioxidants as hypoxen and vitamin E: disorders in behavior of the offspring of alcoholized females were smoothed significantly. Neirofiziologiya/Neurophysiology, Vol. 40, No. 2, pp. 130–136, March–April, 2008.     

Physical activity in osteopenia treatment improved the mass of bones directly and indirectly submitted to mechanical impact

The effect of physical activity in the treatment of osteopenia induced by ovariectomy was studied in 34 two-month-old Wistar female rats. Animals were divided into three groups in which two were formed by ovariectomized (OVX) animals and the other one had sham-operated animals. Group 1, active OVX'd rats; group 2, sedentary OVX'd rats and group 3, sham-operated ones (control). After three months of daily physical activity in a motor-driven treadmill all rats were sacrificed. In order to perform a histomorphometric analysis, long bones, vertebrae, and nasal bone were selected at necropsy. Ovariectomized rats which exercised showed an increased trabecular bone volume, cortical thickness in the long bones and vertebrae and also an increased nasal bone thickness. Physical activity also increased the connection of osteocytes. It was concluded that physical activity in osteopenia treatment increases and restores the mass of bones directly and indirectly submitted to physical impact.     

Change of dopamine, serotonin and opioid neuromediator systems in the brain of rats adapted to prolonged effect of ethanol

AIM OF THE STUDY: To investigate the effect of in vivo short-term ethanol administration (i.p., 1.5 g/kg, 6 h) on binding characteristics of opioid receptor agonists in rat midbrain, as well as the contents of dopamine, serotonin and their precursors and metabolites in midbrain, striatum and hypothalamus of rats after long-term alcohol consumption. The methods of receptor binding assay and high performance liquid chromatography with electrochemical detection were used. The data obtained suggest that the response of neurotransmitter systems to short-term ethanol administration in different regions of rats brain is not identical. Our findings demonstrate that short-term ethanol administration may modulate dopaminergic transmission in the rat hypothalamus and striatum but this effect may be attenuated by down-regulation of OP, in the midbrain after long-term alcohol consumption. Serotonin system in hypothalamus becomes more sensitive to short-term ethanol administration after the long-term ethanol-containing liquid diet in comparison with control rats. Our results suggest that reinforcing properties of ethanol may be partially mediated by mechanisms involving the ethanol-induced disturbing of dopaminergic metabolism in the midbrain and hypothalamus and serotoninergic metabolism in hypothalamus.     

Evaluation of the state of the progeny of mice with damaged kidneys]

It was established that increased mortality was characteristic of newborn mice from females with dystrophic lesions of the renal tissue. The kidneys of newborns from these females had a less relative weight as compared with newborns from healthy mice. Other organs of experimental and control newborns did not differ in their relative weight. Hypertrophy of the tubule cells with the signs of picnosis of nuclei was noted in the kidneys of experimental newborn animals. In two-month-old mice of the experimental and control groups the kidneys and other organs (liver, heart, lungs, spleen) had no substantial distinctions in the relative weight. The concentration of urea in the blood of two-month-old mice from females with injured kidneys under protein load was higher than in control two-month-old mice from females treated with 0,85% solution of NaCl which speaks of decreased resistance of kidneys in the animals of experimental group against pathogenic factors.     

Modulation of prolactin binding sites in vitro by membrane fluidizers. Effects on male prostatic and female hepatic membranes in alcohol-fed rats

The objectives of this study were (i) to determine if in vivo administration of ethanol to rats produced changes in apparent lipid fluidity and prolactin binding capacity of male prostatic and female hepatic membranes and (ii) to compare the effects of membrane fluidizers (aliphatic alcohols) in vitro on prolactin binding of prostatic and hepatic membranes in control and alcohol-fed animals. In vitro ethanol has been shown by us previously to increase prolactin receptor levels presumably by unmasking cryptic prolactin receptors. The degree of fluidization was monitored by a fluorescence polarization method using 1,6-diphenylhexatriene. Adult male and female rats were given either water or 4% ethanol as the sole source of drinking fluid for a period of 6 weeks. No significant changes in plasma prolactin were observed between control and ethanol-treated groups of either sex. However, the microviscosity parameter, inversely related to lipid fluidity, was increased approx. 34% and 40%, respectively, in male prostatic and female rat hepatic membranes after ethanol feeding. Furthermore, 125I-prolactin binding capacity was decreased approx. 30% and 26%, respectively, in prostatic and hepatic membranes of alcohol fed animals. In vitro treatment with aliphatic alcohols had no effect on either microviscosity or prolactin binding in hepatic or prostatic membranes from ethanol-fed rats, but both fluidized and increased prolactin binding in the same membrane preparations from control rats. Our observations are consistent with the direct relationship between membrane fluidity and prolactin receptor levels. The changes in prostatic and hepatic membranes after alcohol feeding, namely decreased prolactin receptor levels, decreased fluidity and increased resistance to the fluidizing effects of in vitro aliphatic alcohols may reflect a fundamental membrane defect.     

乳酸杆菌培养上清抑制慢性酒精大鼠小肠上皮TLR4-TBK1通路

目的:探讨保加利亚乳酸杆菌培养上清(supernatant recovered from lactobacillus bulgaricus culture MRS broth,LBG-S)对慢性酒精摄入大鼠小肠上皮细胞Toll样受体4(Toll-like receptor 4,TLR4)-TANK结合激酶-1(TANK binding kinase-1,TBK1)信号通路的作用。方法:雄性健康Wistar大鼠30只(2月龄,体重250~300 g)分为三组:2月龄对照组(即基线对照组),顺应1周后即处死;9月龄对照组,自由取食标准鼠粮及饮用双蒸水7月;慢性酒精组,9月龄,饮用含25%乙醇的双蒸水连续6月。处死大鼠后,分离培养并计数各组大鼠小肠黏膜中的大肠杆菌和乳酸杆菌;分离并培养各组大鼠的小肠上皮细胞,在有或无LBG-S(10μg/m L)预处理的情况下,给予脂多糖(lipopolysaccharide,LPS,10 EU/m L)刺激后,Western blot检测各组大鼠小肠分离上皮细胞中的TLR4及TBK1水平,酶联免疫吸附法检测各组分离小肠上皮细胞培养上清中的肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)和干扰素-γ(interferon-γ,IFN-γ)。结果:慢性饮酒后,大鼠肠腔内乳酸杆菌数量明显低于相应对照组(P0.05);大肠杆菌数量无明显增加。LPS能明显升高各组分离大鼠小肠上皮细胞TLR4、TBK1以及生成TNF-α和IFN-γ的水平(P0.05),且慢性酒精组升高幅度明显大于2月龄及9月龄对照组(P0.05)。LBG-S的预处理能明显抑制LPS对各组分离大鼠小肠上皮细胞TLR4、TBK1以及生成TNF-α和IFN-γ水平的上调作用(P0.05)。结论:慢性酒精摄入导致大鼠小肠上皮TLR4-TBK1通路对LPS的高敏感性,LBG-S能明显抑制这一高敏感性。     

State of the opioid system in rats with different sensitivity to ethanol

In this work we investigated the content of opioids in plasma blood and the changes caused by ethanol (1.5 g/kg, i.p.) in midbrain opioid receptors of rats with different sensitivity to ethanol, as determined according to the duration of ethanol-induced sleep (DEIS). A receptor binding technique with selective delta-DADLE and mu-DAGO radioligands were used. Ethanol even at low dose produced changes in the midbrain opioid receptors which appeared after 6 hours. The response of the receptors and the content of opioids in plasma blood of rats with different DEIS were not identical.     

Correlation of glutamate binding activity with glutamate-binding protein immunoreactivity in the brain of control and alcohol-treated rats

Specific antibodies raised against a glutamate binding protein purified from bovine brain were used to trace the immunoreactivity of this protein in rat brain subcellular fractions. In the subcellular fractions obtained from whole brain homogenates, the synaptic membranes had the highest immunochemical reactivity towards the anti-glutamate-binding protein antibodies. The combination of measurements of glutamate binding activity and glutamate-binding protein immunoreactivity indicated that in brain synaptic membranes from control animals the highest activity in these two measures was associated with a synaptic plasma membrane subfraction that was enriched with synaptic junctions. In animals treated with ethanol for 14 days, there was a significant increase in the density of synaptic membrane glutamate binding sites. This increase in glutamate binding capacity was correlated with a greater than two-fold increase in the glutamate binding activity and binding protein immunoreactivity of the light synaptic membrane subfraction, a subfraction which does not contain many recognizable synaptic junctions. Acute administration of ethanol to rats produced a moderate but non-significant decrease in glutamate binding capacity of synaptic membranes. The increase in the number of glutamate binding protein subunits in brain plasma membranes may be an adaptive response of central nervous system neurons to the acute effects of ethanol on glutamate synaptic transmission.     

Tolerance to ethanol-induced contractions of vascular smooth muscle: Role of endothelium

Ethanol, at high concentrations, produced a dose-dependent contraction of male rat aortic rings, $\text{in vitro}$. Mechanical removal of endothelial cells from aortic rings of control rats resulted in a small, but significant, shift of the ethanol dose-response curve to the right without a change in the maximal contraction. Removing the endothelial cells of aortic rings obtained from rats intoxicated with ethanol for two days significantly shifted the ethanol dose-response curve to the left and significantly increased the maximal contraction induced by ethanol. A comparison of the ethanol dose-response curves in aortic rings with endothelium obtained from control rats with those obtained from intoxicated rats indicated a significant shift to the right with no change in maximal response. No significant changes were observed when the responses of aortic rings without endothelium obtained from control and intoxicated rats were compared. These observations confirm that tolerance to ethanol can be demonstrated in vascular smooth muscle. In addition, they demonstrate that the endothelium is required for the development of tolerance to ethanol in the aorta.     

Hypersensitivity to the tremorogenic action of nicotine in rats withdrawn from ethanol

The tremorogenic effect of nicotine was studied in control rats and in rats withdrawn for 16-48 h from chronic ethanol administration. The intensity of tremor was measured electronically. Rats withdrawn from ethanol showed a marked hypersensitivity to the tremorogenic action of nicotine. Propranolol abolished the nicotine-induced tremor in the control rats. Propranolol did not, however, reduce the intensity of nicotine-induced tremor in rats withdrawn from ethanol. Thus, the observed hypersensitivity does not seem to be mediated by a sympathetic beta-adrenergic mechanism.     

Chronic ethanol exposure increases the binding of HuR to the TNFalpha 3'-untranslated region in macrophages

Tumor necrosis factor alpha (TNFalpha) expression is a key mediator of ethanol-induced liver disease. Increased lipopolysaccharide (LPS)-stimulated TNFalpha expression in macrophages after chronic ethanol feeding is associated with a stabilization of TNFalpha mRNA (Kishore, R., McMullen, M. R., and Nagy, L. E. (2001) J. Biol. Chem. 276, 41930-41937). Here we show that the 3'-UTR of murine TNFalpha mRNA was sufficient to mediate increased LPS-stimulated expression of a luciferase reporter in RAW 264.7 macrophages after chronic ethanol exposure. Further, we show that HuR, a nuclear/cytoplasmic shuttling protein, which binds to TNFalpha mRNA, is required for increased expression of TNFalpha after chronic ethanol. In Kupffer cells, HuR was primarily localized to the nucleus and then translocated to the cytosol in response to LPS in both pair- and ethanol-fed rats. After chronic ethanol feeding, HuR quantity in the cytosol was greater, both at baseline and in response to LPS, compared with pair-fed controls. Using RNA gel shift assays, we found that LPS treatment increased HuR binding to the 65-nucleotide A + U-rich element of the TNFalpha 3'-UTR by 2-fold over baseline in Kupffer cells from pair-fed rats. After chronic ethanol feeding, HuR binding to the TNFalpha A + U-rich element was increased by more than 5-fold at baseline and in response to LPS, compared with pair-fed controls. Down-regulation of HuR expression by RNA interference prevented the chronic ethanol-induced increase in expression of luciferase reporters containing the TNFalpha 3'-UTR. Taken together, these data demonstrate that increased binding of HuR to the TNFalpha 3'-UTR contributes to increased LPS-stimulated TNFalpha expression in macrophages after chronic ethanol exposure.     

Chronic ethanol administration impairs the binding and endocytosis of asialo-orosomucoid in isolated hepatocytes

We have examined the effect of ethanol administration on receptor-mediated endocytosis of asialo-orosomucoid by isolated hepatocytes. Significantly less ligand was bound, internalized, and degraded by hepatocytes isolated from rats fed an ethanol diet for 5-7 weeks than by cells isolated from chow-fed or pair-fed controls. Reduced binding was shown to be primarily due to a decreased number of cell surface receptors rather than to a lowered affinity of the receptor for its ligand. This reduction in cell surface receptors resulted in a marked inhibition of internalization and degradation of ligand by hepatocytes from the ethanol-fed rats. In addition, a defect in the initial stages of receptor-ligand internalization was also indicated, since less surface-bound ligand was internalized and subsequently degraded in cells from the ethanol-treated animals as compared to controls. Rates of internalization and degradation of internalized ligand were, however, similar for all three groups, suggesting that neither degradation per se nor rate of delivery of internalized ligand to the lysosomes was affected by ethanol feeding. Receptor recycling was impaired in ethanol-fed rats, as indicated by a decrease in the binding site number after stimulation of endocytosis for 120 min when compared to initial binding capacity. Receptor recycling was not impaired in hepatocytes from control animals. These results indicate that chronic ethanol feeding impairs the process of receptor-mediated endocytosis by the liver; the major cause of this impairment appears to be due to a decreased number of cell surface asialoglycoprotein receptors in the ethanol-fed animals, along with a decreased ability of these cells to internalize all of the surface-bound ligand.     

Study of the possibility of hyperparathyroidism development in rats exposed to internal irradiation by iodine radioisotope

The possibility of hyperparathyroidism development secondary to earlier internal irradiation with radioactive iodine was studied experimentally in Wistar rats. This report describes the parathyroid morphology and biochemical findings for animals irradiated with 131I at the doses of 4.5, 40, or 80 Gy. The interval between the radiation exposure of two-month-old rats and their examination for thyroid and parathyroid pathology was 14 months. Neither hypercalcemia nor hypophosphatemia was found. Moreover, the level of calcium in serum slightly decreased following 40 and 80 Gy irradiation. The increased incidence of parathyroid fibrosis and hypofunctional structure transformation were revealed.     

The response of circulating parameters of bone mineral metabolism to ethanol- and EDTA-induced hypocalcemia in the rat

The mechanism of the acute hypocalcemia that follows acute ethanol administration has not been established. Measurements of parathyroid hormone (PTH) performed during this hypocalcemia reveal conflicting results. We compared the response of ionized calcium (Ca²⁺), immunoreactive PTH and bone Gla protein (BGP) after ethanol- and EDTA-induced hypocalcemia. 103 male Sprague Dawley rats each weighing approximately 300 g received ethanol and 100 rats of similar weight received EDTA. In each of these studies the animals were divided into experimental and control groups. The ethanol-treated rats received ethanol, 2 g/kg body weight, by ip injection and the EDTA-treated rats received 100 mg EDTA/kg body weight by im injection. Controls received normal saline by the corresponding route of administration. Rats were sacrificed at 0, 30, 60, 90, 180 and 360 min for the measurement of the above parameters. In both experimental groups Ca²⁺ levels were significantly reduced to the same degree by 30 min with return to control values by 360 min. There was no significant difference in immunoreactive PTH, and BGP between control and ethanol-treated groups. In the EDTA-treated rats, however, PTH values were significantly increased at 30 (P < 0.005) and BGP at 60 and 90 minutes (P < 0.005) vs. control. Therefore acute ethanol administration appears to blunt the PTH response to hypocalcemia. A direct inhibitory effect of ethanol on osteoblast function ie BGP production cannot be excluded. In addition, PTH may stimulate BGP.     

Relationship between myeloperoxidase activity and the ontogenic response of rat gastric mucosa to ethanol.

We have examined the gastric luminal content of Na+, K+, and protein and mucosal levels of myeloperoxidase in rats between the ages of 10 and 60 days in response to luminal instillation of ethanol (20 and 50% w/v). In control animals the appearances of ions and protein and myeloperoxidase activities were low and similar in all age groups. Luminal content of cations and protein increased in response to both 20 and 50% ethanol and were greater in animals older than 20 days when compared with younger rats. However, ethanol treatment resulted in a significant degree of mucosal cellular disruption and erosions in both young and mature rats. Myeloperoxidase activities in response to ethanol were not greater than control until animals were older than 20 days. Treatment of rats aged 10-60 days with intraperitoneal glycogen (1%) resulted in peritoneal granulocyte infiltration. The concentration of peritoneal cells increased as animals aged. With the exception of day 15, the myeloperoxidase content of the peritoneal leukocytes did not vary significantly at other ages examined. These data suggest that (1) mucosal efflux of Na+, K+, and protein in response to luminal ethanol increase as rats age from 10 to 60 days; (2) the ontogenic development of ethanol-induced cation and protein appearance parallel the increase in myeloperoxidase activity in the gastric mucosa; and (3) the increase in mucosal myeloperoxidase activity in response to ethanol likely reflects increased granulocyte infiltration as rats age.