Neuronal differentiation modifies the effect of ethanol exposure on voltage-dependent calcium channels in NG 108-15 cells

The effect of prolonged (72 h) ethanol (200 mM) exposure on the labeling of L-type (using tritiated PN 200-110) and N-type (using iodinated ω-conotoxin) voltage-dependent calcium channels was investigated in cultured NG 108-15 cells. In undifferentiated cells ethanol produced an 80% increase in PN 200-110 B_max and no changes in ω-conotoxin binding. Differentiation had a profound effect on the response of cells to ethanol, which in differentiated neuron-like cells decreased w-conotoxin binding (−53.5%) leaving PN 200-110 labeling of L-type channels unaffected. The effect was time dependent and reversible upon ethanol withdrawal. The decreased ω-conotoxin binding was accompanied by a reduced ability of ω-conotoxin to inhibit K⁺-stimulated calcium uptake. The results demonstrate that in cultured NG 108-15 cells ethanol differentially affects DHP and ω-conotoxin-sensitive, voltage-dependent calcium channels and that the effect is also modulated by differentiation of the cell to a neuronal phenotype.     

姜黄素对氯化铝染毒的NG108 - 15细胞凋亡及PKC - NMDAR通路表达的影响

目的 探讨姜黄素对氯化铝染毒所致NG108 - 15细胞凋亡的影响及其机制,为铝致学习记忆损伤的治疗提供参考。方法 取对数生长期NG108 - 15细胞,随机分为空白对照组、DMSO组（溶剂对照）、姜黄素组（16 μmol/L姜黄素）、铝组（160 mg/L氯化铝染毒 ）、铝+姜黄素组（160 mg/L氯化铝染毒24 h后,给予16 μmol/L姜黄素处理24 h）。收集各组细胞,采用吖啶橙/嗅化乙锭（AO/EB）双荧光染色观察细胞凋亡形态,计数凋亡细胞数并计算凋亡率;流式细胞术检测细胞凋亡率, qRT - PCR检测细胞中PKC、NMDAR1、NMDAR2B 的mRNA表达水平,western blot检测细胞中凋亡相关蛋白（caspase3、Bax）和PKC、NMDAR1、NMDAR2B的蛋白表达水平。多组间均数的比较采用单因素方差分析（one - way ANOVA）,组间两样本均数的比较采用LSD法。结果 与空白对照组相比,铝染毒组的caspase3、Bax蛋白表达水平升高（t = - 5.547、 - 4.948,P＜0.001）,PKC、NMDAR1、NMDAR2B的mRNA（t = 4.926,P = 0.003;t = 6.330,P＜0.001;t = 4.224,P = 0.019）和蛋白（t = 20.638,P＜0.001;t = 4.509,P＜0.001;t = 17.388,P = 0.002）表达水平降低, AO/EB染色和流式细胞术结果均表明细胞凋亡率升高（t = - 5.153、 - 7.390,P＜0.001）;加入姜黄素处理后,与铝染毒组相比,铝+姜黄素组的caspase3、Bax蛋白表达水平降低（t = 2.930,P = 0.006;t = 4.907,P＜0.001）,PKC、NMDAR1、NMDAR2B 的mRNA（t = - 10.337、 - 6.621、 - 6.847,P＜0.001）和蛋白（t = - 30.551、 - 7.451、 - 26.294,P＜0.001）表达水平升高,AO/EB染色和流式细胞术结果均表明细胞凋亡率降低（t = 2.707,P = 0.01;t = 4.632,P＜0.001）。结论 上调PKC - NMDAR信号通路表达,可能是姜黄素减轻氯化铝染毒所致NG108 - 15细胞凋亡的机制之一。     

Alcohol-induced alterations in phosphoinositide hydrolysis in astrocytes

Cultured astrocytes exposed to ethanol were prelabeled with [3H]inositol and the accumulation of [3H]inositol phosphates was determined following stimulation with norepinephrine (NE). Acute doses of alcohol (25-200 mM) had little effect on phosphoinositide (PI) hydrolysis. However, chronic exposure for 7 days produced significant increases in hydrolysis with ethanol concentrations as low as 50 mM. The onset of alcohol-induced increases occurred within 4 hr of exposure, reaching maximum values by Days 3 to 5. Withdrawal resulted in the return of stimulated PI hydrolysis to pre-alcohol exposure levels after 2 days. The evidence provided suggests that chronic exposure of ethanol alters the alpha-adrenergic receptor characteristics of astrocytes and/or enhances the effector pathway leading to PI hydrolysis at a site distal to the receptor.     

Serotonin-stimulated phosphoinositide hydrolysis in platelets from post-withdrawal alcoholics.

It has previously been reported that serotonin-stimulated second messenger formation is inhibited in platelets from alcoholics undergoing detoxification. Serotonin-stimulated signal transduction was therefore analysed in platelets from young post-withdrawal alcoholics to elucidate whether the previously reported inhibition is a state or trait marker of alcoholism. No difference between post-withdrawal alcoholics and controls was found with regard to serotonin-stimulated [32P]-phosphatidylinositol 4,5-bisphosphate hydrolysis or [32P]-phosphatidic acid formation. The results indicate that the inhibited serotonin2 receptor function seen in alcoholics undergoing detoxification is a state-dependent rather than a trait-dependent marker.     

Chronic ethanol inhibits receptor-stimulated phosphoinositide hydrolysis in rat liver slices.

The effects of chronic ethanol feeding on norepinephrine (NE)- and arginine-vasopressin (AVP)-stimulated phosphoinositide (PI) hydrolysis in rat liver slices was determined. The maximum NE-stimulated PI response was significantly reduced by 40% in liver slices from 8-month-old rats which had been treated for 5 months with a liquid diet containing ethanol compared to pair-fed controls. The maximum AVP-stimulated PI response was decreased by 39% in liver slices from the ethanol-fed rats compared to control. EC50 values for NE- and AVP-stimulated PI hydrolysis in liver slices were not affected by the chronic ethanol treatment. Similar reductions in the maximal NE- and AVP-stimulated PI hydrolysis (28% and 27%, respectively) were found in 22-month-old rats which had been maintained on an ethanol containing diet for 5 months compared to pair-fed controls. The binding of [3H]prazosin and [3H]AVP to liver plasma membranes from 8-month-old ethanol-fed rats was not significantly different from binding to liver membranes from sucrose-fed controls. Our data suggest that chronic ethanol ingestion may lead to a reduction in PI-linked signal transduction in liver.     

Control of endometrial phosphoinositide hydrolysis and prostaglandin F2alpha secretion in pigs

This study examined the control of endometrial phosphoinositide (PI) hydrolysis and prostaglandin (PG) F2alpha release by oxytocin (OT) and vasopressin in cyclic pigs on Day 15 post oestrus. In Expt 1, OT and arginine-vasopressin (AVP) increased endometrial PI hydrolysis (P<0.01), but only OT stimulated (P<0.01) PGF2alpha secretion. In Expt 2, OT and lysine-vasopressin (LVP) increased PI hydrolysis (P<0.01). No difference was detected between the 100 nM and 200 nM concentrations of OT or between the 100 nM and 200 nM concentrations of LVP. The increase in PI hydrolysis was greater (P<0.05) for 100 nM OT plus 100 nM LVP than for the 100 or 200 nM concentrations of OT or LVP alone. In Expt 3, OT (P<0.01) and LVP increased (P<0.01) PI hydrolysis. An OT antagonist abolished (P<0.01) the OT-induced increase in PI hydrolysis, but did not significantly alter the LVP-induced increase. A type 2 VP antagonist completely inhibited (P<0.01) the LVP-induced increase in PI hydrolysis, but only partially antagonized the stimulatory effect of OT (P<0.01). These results are consistent with the proposal that OT acts through specific receptors to promote endometrial PGF2alpha secretion in pigs.     

Ethanol promotes hydrolysis of 3H-labeled sialoconjugates from brain of mice in vitro

Acute administration of ethanol reportedly decreases total sialic acid in brain. Here, we tested the hypothesis in brain and liver that the decrement is due to increased hydrolysis of sialoglycoconjugates. Mouse tissue slices were pulse-labeled with N-[³H]acetyl-D-mannosamine, the precursor of sialic acid. Incorporation was linear for up to 4 hr of incubation. When the labeled slices were incubated with three concentrations of ethanol (0.1, 0.5, and 1 M) for 5 hr, labeled liver sialoconjugates were significantly affected only at 0.5 and 1 M ethanol, whereas labeled brain sialoconjugates were markedly decreased even at 100 mM ethanol. Sialidase activity decreased steadily with increasing concentration of ethanol, indicating that the increased hydrolysis was not attributable to an enhanced sialidase activity. n-Propanol and t-butanol had the same degradative effect as ethanol on sialocompounds; and 3 mM pyrazole, an inhibitor of alcohol dehydrogenase (ADH), had no effect on ethanol-induced degradation of sialocompounds. The protein/DNA ratio in liver showed a steady decrease with increasing ethanol. The data thus confirm the in vivo reports of ethanol-enhanced cleavage and rule out any increase in sialidase activity as a major cause.     

Ethanol exposure alters K+- but not bradykinin-induced dopamine release in PC12 cells

The effects of ethanol on [3H]dopamine release were investigated in cultured PC12 cells using two methods to stimulate dopamine release: exposure to depolarizing concentrations of extracellular K+ and incubation with the highly active secretagogue, bradykinin. Both K+ and bradykinin dose-dependently increased [3H]dopamine release. The mean +/- S.E.M. EC50 for K+ was 35.8 +/- 1.2 mM; for bradykinin it was 1.07 +/- 0.23 x 10(-7) M. The characteristics of the bradykinin-stimulated dopamine release showed it to be dependent on extracellular Ca2+ and was attenuated by 1 mM Co2+ or 1 mM Ni2+. However, release was unaffected by either the voltage-dependent Ca2+ channel antagonist, verapamil, or the dihydropyridine (DHP) Ca2+ channel agonist, BAY K 8644. In contrast, 1 mM Co2+ completely blocked, verapamil inhibited and BAY K 8644 augmented K+-stimulated [3H]dopamine release. PC12 cells acutely exposed to ethanol (100 and 200 mM) showed diminished K+-stimulated [3H]dopamine release but an unaltered bradykinin-stimulated response. Cells exposed to 200 mM ethanol for 6 days showed significantly enhanced [3H]dopamine release in response to high concentrations of K+ but no changes were observed in their response to bradykinin. These data provide evidence that ethanol, within the same cell, can differentially affect neurotransmitter release, dependent upon the secretagogue used.     

Ethanol and temperature effects on five membrane bound enzymes

The effects of ethanol on the activities of five membrane bound enzymes were determined using a crude membrane preparation obtained from cortex of long-sleep (LS) and short-sleep (SS) mice. These two mouse lines were selectively bred for differences in duration of ethanol-induced sleep time. The enzymes studied were two forms of NaK-ATPase, Mg-ATPase, 5′nucleotidase, and acetylcholinesterase. Arrhenius plots of the ethanol-temperature-enzyme activity studies indicate specificity in ethanol's actions. NaK-ATPase activity consists of two enzymes which were distinguished by sensitivity to ouabain. The Arrhenius plot of the high ouabain sensitivity enzyme (low K_i) exhibited a transition temperature which was reduced twice as much by ethanol in LS membranes as in SS membranes. Ethanol did not affect the transition temperature of the high K_i NaK-ATPase but the control (no ethanol) transition temperature was 2.7° higher in SS membranes. Arrhenius plots of Mg-ATPase activity did not exhibit a transition temperature and ethanol did not alter enzyme activity. Ethanol did not alter the transition temperatures of 5′nucleotidase or acetylcholinesterase but the control transition temperature for acetylcholinesterase was 2.3° higher in SS membranes. These results indicate specificity in ethanol's actions on membranes and that inhibition of the lipid-enzyme interactions for the low K_i NaK-ATPase is correlated with the difference in sensitivity to ethanol seen between the LS and SS mice.     

Ro15-4513 enhances and attenuates motor stimulant effects of ethanol in rats

The actions of the imidazobenzodiazepine, Ro15-4513 (2.5 mg/kg IP), ethanol (0.50 g/kg, 0.75 g/kg, 1.25 g/kg), and Ro15-4513 in combination with ethanol were assessed using low activity male Charles River (CD) rats. Horizontal (ambulatory) and nonhorizontal (rearing, grooming, etc.) activity were measured in the open field with a Digiscan activity system. When Ro15-4513 was given alone, animals responded in a manner similar to that of the control animals. Ethanol alone, however, resulted in a significant enhancement of activity, with the effect being most pronounced at 0.50 g/kg for horizontal activity and 1.25 g/kg for nonhorizontal activity. A potentiation of the ethanol-induced stimulant effect was observed following pretreatment with Ro15-4513 on the horizontal activity measure for the 1.25 g/kg ethanol group. An attenuation of activity was observed for this group on the nonhorizontal activity measure. Ro15-4513, however, did not affect the stimulation produced by the other doses of ethanol on the nonhorizontal measure. The results suggest that possibility of distinct neurochemical mechanisms in the mediation of horizontal and nonhorizontal activity and that, perhaps, GABA-benzodiazepine mechanisms may play different roles in mediating their ETOH-induced stimulant effect.     

CD151对脐静脉内皮细胞PI3K表达及细胞增殖的影响

目的研究CD151对脐静脉内皮细胞(HUVEC)增殖及PI3K表达的影响,以进一步研究其生物学作用及其在血管生成中的作用机制。方法通过构建p-AAV-CD151质粒并转染HUVEC,采用MTT法测定HUVEC增殖的变化,W estern B lot检测CD151及PI3K表达。结果p-AAV-CD151转染组CD151表达明显增加,p-AAV-CD151转染组与正常对照组、p-AAV-GFP转染组比较表达差异有统计学意义(P<0.05)。MTT法检测正常对照组、p-AAV-GFP转染组、p-AAV-CD151转染组的OD值分别为0.1663±0.0095、0.169±0.0085、0.2457±0.0044,p-AAV-CD151转染组较p-AAV-GFP转染组和正常对照组细胞增殖能力增强,差异有统计学意义(P<0.05)。p-AAV-CD151转染组PI3K表达明显增加,p-AAV-CD151转染组与正常对照组、p-AAV-GFP转染组比较表达差异有统计学意义(P<0.05)。结论CD151具有促进内皮细胞增殖的作用,CD151可能通过上调PI3K表达,促进内皮细胞增殖达到促进血管新生的作用。     

乙醇对小鼠胚胎脑发育毒性的体外实验研究

目的　探讨乙醇的脑发育毒性及其可能的作用机制。　方法　采用植入后全胚胎培养(whole embryo culture,WEC)和微团培养技术。　结果　全胚胎培养结果显示 ,随着乙醇浓度的增加(1.0 0 ,2 .0 0 ,4.0 0 g/ L) ,胚胎脑畸形率明显上升 ,呈剂量反应关系 ,最常见的脑畸形为脑神经管未闭。中脑细胞微团培养结果表明 ,随着染毒剂量的增加 ,不同浓度乙醇 (1.0 0～ 16.0 0 g/ L )对中脑细胞的增殖和分化有明显的抑制作用 ,呈剂量反应关系 ,ID5 0 (50 %细胞分化抑制浓度 )、IP5 0 (50 %细胞增殖抑制浓度 )分别为 8.89g/ L和 12 .2 1g/ L。　结论　乙醇抑制胚胎脑细胞的增殖和分化可能是器官形成期乙醇脑发育毒作用的重要机制之一     

Ethanol effects in an anxiety/defense test battery

Two tests, components of an Anxiety/Defense Test Battery, have been designed to measure risk assessment, inhibition of nondefensive behaviors, and movement arrest, all of which occur in the natural defense patterns of rats to threatening stimuli. In these tests, which used a nonpainful threat stimulus (a cat), ethanol (0.6 and 1.2 g/kg) increased two risk assessment behaviors on an initial test day, and produced a wider pattern of changes in all three patterns on a retest (no cat) day, 5 days later. The pattern of results obtained is compatible with a view that defensive behaviors occur in a fixed sequence with the decreasing intensity of threat an important factor in the transition from one defensive behavior to the next, and with ethanol at these doses producing a mild and relatively nonspecific anxiolytic effect. Comparison of male and female subjects on these tasks also suggested that females are more defensive than males, a finding which agrees with a variety of human anxiety studies but is at variance with previous rodent literature.     

Ethanol induces locomotor activating effects in preweanling Sprague-Dawley rats

Abuse of drugs exerts biphasic motor activity effects, which seem to be associated with their motivational effects. In the case of ethanol, heterogenous rat strains appear to be particularly sensitive to the sedative effects of the drug. In contrast, ethanol's activating effects have been consistently reported in rats genetically selected for ethanol affinity. Heightened ethanol affinity and sensitivity to ethanol's reinforcement are also observed in nonselected rats during early ontogeny. In the present study, we examined psychomotor effects of ethanol (1.25 and 2.5 g/kg) in 8-, 12-, and 15-day-old pups. Motor activity in a novel environment was assessed 5-10 or 15-20 min following drug treatment. Rectal temperatures and latency to exhibit the righting reflex were recorded immediately after locomotor activity assessment. Ethanol exerted clear activating effects at 8 and 12 days of age (Experiments 1a and 1b) and to a lesser extent at 15 days. At this age, ethanol enhanced locomotor activity in the first testing interval (Experiment 1b) and suppressed locomotion at 15-20 min (Experiment 1a). Ethanol-mediated motor impairment was more pronounced in the youngest group (postnatal day 8) than in the older ones. Blood ethanol concentrations were equivalent in all age groups. The present study indicates that preweanling rats are sensitive to ethanol's stimulating effects during the second postnatal week, and suggest that specific periods during early ontogeny of the rat can provide a valuable framework for the study of mechanisms underlying ethanol's stimulation and reinforcement effects.     

2,5-己二酮对SK-N-SH细胞中神经生长因子表达水平的研究

目的分析2,5-己二酮对人SK-N-SH细胞中神经生长因子表达水平变化的研究,探讨正己烷中毒的分子机制。方法分别以0、10、20、40 mM的2,5-己二酮持续染毒12 h后,以Q-PCR及Western Blot方法检测各组人SKN-SH细胞中神经生长因子的变化趋势。结果 2,5-己二酮染毒后,人SK-N-SH细胞神经生长因子mRNA及其蛋白表达水平均明显下调,可见染毒后SK-N-SH后细胞分泌的神经生长因子是降低的。结论 2,5-己二酮可以引起人SK-N-SH细胞NGF表达水平降低,说明NGF表达降低在2,5-己二酮对人SK-N-SH细胞的早期毒性及慢性毒作用机制中起着重要作用。     

Ethanol signals for apoptosis in cultured skin cells.

Ethanol is commonly used in cosmetic and pharmaceutical preparations. To test whether ethanol may cause apoptosis in skin cells, we treated A431 epidermoid skin cells and neonatal human primary skin cells with different concentrations of ethanol, for different time periods. Ethanol was toxic to cells in both a dose- and time-dependent manner and increased the percentage of cells undergoing apoptosis. Treatment of cells with 40 and 100 mM ethanol increased release of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) into culture medium and increased its expression in cells. The TNF-alpha was toxic to A431 epidermoid skin cells at concentrations similar to those released by cells on exposure to ethanol. Ethanol-treated cells examined by electron microscopy showed organelle damage, condensed chromatin, and apoptotic bodies. Therefore, even at low concentrations, ethanol may induce apoptosis in skin cells by enhancing the effects of TNF-alpha.     

Ethanol effects on local cerebral glucose utilization in high-alcohol-drinking and low-alcohol-drinking rats.

Divergent ethanol-drinking behavior in rats selectively bred for high- or low-ethanol-drinking behavior could be related to differences in the sensitivity of the CNS to ethanol. In the current study, we examined the effects of acute (i.e., single injection) ethanol administration on local cerebral glucose utilization (LCGU) within selected brain regions of high-alcohol-drinking (HAD) and low-alcohol-drinking (LAD) rats. Adult, male, HAD and LAD rats from replicate line 2 were injected intraperitoneally with saline, or ethanol, at doses of 0.25 g/kg or 1.0 g/kg, during their dark cycle; 10 min later, [14C]-2-deoxyglucose ([14C]-2-DG; 125 microCi/kg) was injected into the femoral vein. Timed arterial blood samples were collected over 45 min and assayed for plasma glucose, ethanol, and [14C]-2-DG levels. Rats were then decapitated, and their brains were quickly extracted and frozen in isopentane at -50 degrees C. Coronal brain sections were prepared and apposed to x-ray film for 2 days, and image densities were determined by using quantitative autoradiography. Data were collected from several key limbic (nucleus accumbens, ventral tegmental area, olfactory tubercle, amygdala, hippocampus, ventral pallidum, and septum), basal ganglia, cortical (medial prefrontal, frontal, parietal, temporal, occipital, entorhinal, piriform, and cingulate), and subcortical (thalamus, habenula, and superior colliculus) structures. After administration of both low (0.25 g/kg) and moderate (1.0 g/kg) doses of ethanol, LCGU values were lower, relative to those for saline controls, in several CNS regions (lateral septum; posterior cingulate, frontal, parietal, and temporal cortices; dorsomedial striatum; and dorsomedial thalamus) of LAD but not HAD rats. These findings may indicate that certain CNS regions of LAD-2 rats are more sensitive than regions of HAD-2 rats to the effects of low-to-intermediate doses of ethanol.     

The effects of ethanol and Ro 15-4513 on elevated plus-maze and rotarod performance in long-sleep and short-sleep mice

The effects of ethanol and diazepam were examined in long-sleep (LS) and short-sleep (SS) mice using the elevated plus-maze. Ethanol had more pronounced effects in SS mice than in LS mice. In contrast, LS mice were more sensitive to the effects of diazepam on the elevated plus-maze. The ataxic effects of ethanol were measured by rotarod performance. SS mice were more resistant to the ataxic effects of a 2.0 g/kg dose of ethanol than LS mice. Ro 15-4513 reversed ethanol's ataxic effects when administered after ethanol in both LS mice and SS mice. Pentobarbital-induced ataxia was unaffected by treatment with Ro 15-4513. Studies of competition of Ro 15-4513 on 3H-flunitrazepam binding indicated that LS and SS mice did not differ in this measure in cortex, cerebellum or hippocampus.