Brain plasticity and cognitive functions after ethanol consumption in C57BL/6J mice.

Acute or chronic administrations of high doses of ethanol in mice are known to produce severe cognitive deficits linked to hippocampal damage. However, we recently reported that chronic and moderate ethanol intake in C57BL/6J mice induced chromatin remodeling within the Bdnf promoters, leading to both enhanced brain-derived neurotrophic factor (BDNF) expression and hippocampal neurogenesis under free-choice protocol. We performed here a series of cellular and behavioral studies to analyze the consequences of these modifications. We showed that a 3-week chronic free-choice ethanol consumption in C57BL/6J mice led to a decrease in DNA methylation of the Bdnf gene within the CA1 and CA3 subfields of the hippocampus, and upregulated hippocampal BDNF signaling pathways mediated by ERK, AKT and CREB. However, this activation did not affect long-term potentiation in the CA1. Conversely, ethanol intake impaired learning and memory capacities analyzed in the contextual fear conditioning test and the novel object recognition task. In addition, ethanol increased behavioral perseveration in the Barnes maze test but did not alter the mouse overall spatial capacities. These data suggested that in conditions of chronic and moderate ethanol intake, the chromatin remodeling leading to BDNF signaling upregulation is probably an adaptive process, engaged via epigenetic regulations, to counteract the cognitive deficits induced by ethanol.

Activation of brain histaminergic neurotransmission: a mechanism for cognitive effects of memantine in Alzheimer's disease.

We previously reported that some N-methyl-D-aspartate (NMDA)-receptor antagonists enhanced histamine neuron activity in rodents. Here, we have investigated the effects of memantine, an NMDA-receptor antagonist used for the treatment of Alzheimer's disease, on histaminergic neurotransmission. In vitro, memantine antagonized native NMDA receptors with a micromolar potency but had no effect at recombinant human histamine receptors. In vivo, a single administration of memantine increased histamine neuron activity, as shown by the 60% increase of tele-methylhistamine (t-MeHA) levels observed in the brain of mice. This increase occurred with an ED(50) of 0.3 ± 0.1 mg/kg, similar to that found on inhibition of ex vivo [(3)H]dizocilpine maleate (MK-801) binding (1.8 ± 1.3 mg/kg). Two days after pretreatment of mice with memantine at 5 mg/kg twice daily for 5 days, t-MeHA levels were enhanced by 50 ± 7% (p < 0.001), indicating a long-lasting activation of histamine neurons. Quantitative polymerase chain reaction analysis was used to explore genes involved in this persistent effect. H(3) receptor mRNAs were strongly increased, but the density of H(3) receptor binding sites was increased solely in hypothalamus (by 141 ± 24%). Up-regulations of brain-derived neurotrophic factor and NMDA-receptor 1 subunit mRNAs were also found but were restricted to hippocampus. mRNA expression of α7-nicotinic receptors remained unchanged in any region. Considering the well established cognitive effects of histamine neurons, the increase in brain t-MeHA levels after single or repeated administration of therapeutic doses of memantine suggests that the drug exerts its beneficial effects on cognitive deficits of Alzheimer's disease, at least partly, by activating histamine neurons.

Effects of betahistine at histamine H3 receptors: mixed inverse agonism/agonism in vitro and partial inverse agonism in vivo.

We previously suggested that therapeutic effects of betahistine in vestibular disorders result from its antagonist properties at histamine H(3) receptors (H(3)Rs). However, H(3)Rs exhibit constitutive activity, and most H(3)R antagonists act as inverse agonists. Here, we have investigated the effects of betahistine at recombinant H(3)R isoforms. On inhibition of cAMP formation and [(3)H]arachidonic acid release, betahistine behaved as a nanomolar inverse agonist and a micromolar agonist. Both effects were suppressed by pertussis toxin, were found at all isoforms tested, and were not detected in mock cells, confirming interactions at H(3)Rs. The inverse agonist potency of betahistine and its affinity on [(125)I]iodoproxyfan binding were similar in rat and human. We then investigated the effects of betahistine on histamine neuron activity by measuring tele-methylhistamine (t-MeHA) levels in the brains of mice. Its acute intraperitoneal administration increased t-MeHA levels with an ED(50) of 0.4 mg/kg, indicating inverse agonism. At higher doses, t-MeHA levels gradually returned to basal levels, a profile probably resulting from agonism. After acute oral administration, betahistine increased t-MeHA levels with an ED(50) of 2 mg/kg, a rightward shift probably caused by almost complete first-pass metabolism. In each case, the maximal effect of betahistine was lower than that of ciproxifan, indicating partial inverse agonism. After an oral 8-day treatment, the only effective dose of betahistine was 30 mg/kg, indicating that a tolerance had developed. These data strongly suggest that therapeutic effects of betahistine result from an enhancement of histamine neuron activity induced by inverse agonism at H(3) autoreceptors.

Autoregulation of McA-RH7777 hepatoma cell proliferation by histamine H3 receptors.

Previous studies have suggested that histamine (HA) acts as an autocrine growth factor. We have explored the modulation of cell proliferation by HA using McA-RH7777 hepatoma cells. High L-histidine decarboxylase (HDC) expression and HA synthesis were found in McA-RH7777 cells. Whereas extracellular HA reached submicromolar concentrations, intracellular levels were very low, indicating that HA was secreted by the cells. McA-RH7777 cells also express H3-receptor (H3R) transcripts and proteins. Reverse transcriptase-polymerase chain reaction analysis detected only transcripts for the long isoform. Immunocytochemistry performed with a selective H3R antibody showed that most cells were immunoreactive. H3R binding sites (Bmax approximately 30 fmol/mg protein) were identified when [125I] iodoproxyfan binding was displaced by the agonist imetit. High-affinity binding also occurred at cytochrome P450 enzymes. This binding was not inhibited by HA, H3R agonists, or by a nonimidazole H3R antagonist but was displaced by imidazole H3R antagonists or by ketoconazole, a imidazole-containing cytochrome inhibitor. HA inhibited proliferation of McA-RH7777 hepatoma cells. The absence of uptake system, its much higher potency at H3Rs, and its low intracellular levels suggested that HA interacted with H3Rs rather than cytochromes. In agreement, both imidazole H3R antagonists, a nonimidazole H3R antagonist, and the HDC inhibitor alpha-monofluoromethyl histidine increased cell proliferation (up to approximately 60%), revealing a H3R-mediated inhibition by endogenous HA. Moreover, exogenous HA inhibited the increase induced by alpha-FMH or H3R antagonists with a nanomolar potency. In conclusion, our findings show that HA regulates proliferation of McA-RH7777 hepatoma cells by interacting with autoinhibitory H3Rs.

Modulation of stress proteins by Cd2+ in a human T cell line.

We previously showed in a human T cell line (CEM-C12 cells) that Cd2+ induced gene expression of stress proteins, metallothionein-IIA and heat shock protein 70 in a time- and dose-dependent manner. In the present study, CEM-C12 cells were pretreated for 24 h with 1 microM Cd2+ and then challenged with toxic concentrations of this metal. We found that maximal expression of the metallothionein-IIA and heat shock protein 70 genes was increased and this maximal level occurred at higher Cd2+ toxic concentrations. Actinomycin D chase experiments indicated that Cd2+ pretreatment did not modify metallothionein-IIA mRNA stability. The modulatory effect of Cd2+ pretreatment was dose-dependent from 100 pM to 1 microM. Such pretreatment also enhanced resistance to Cd2+ toxicity. Finally, verapamil, a calcium/potassium channel blocker displaced the dose-response curve for Cd2+ toxicity as well as metallothionein-IIA and heat shock protein 70 gene expression to higher Cd2+ concentrations.

Degranulacion inmunologica in vitro de basofilos humanos modulada por Pulmon histamina y Apis mellifica / In vitro immunologica degranulation of human basophils modulated by Pulmo histaminum and Apis mellifica

1. El efecto de las altas diluciones de dos medicamentos homeopaticos; Pulmon histamina (Pulm. hist.) y Apis mellifica (Apis m.) usadas para el tratamiento de enfermedades alergicas, han sido evaluados in vitro por la degranulacion de los basofilos humanos (tipo de leucocitos con anticuerpos de inmunoglobulina E -IgE- sobre su superficie, que cuando son expuestos a anticuerpos anti -IgE liberan histamina de sus granulos intracelulares). Los experimentos fueron llevados a cabo en estudio ciego. 2. La degranulacion de los basofilos, inducidos por 1.66x10 potencia -9 m de anticuerpos anti-IgE resulto significativamente inhibida en la presencia de Pulm. hist. 5C (5A dilucion centesimal de Pulm. hist.) y Pulm. hist. 15C.; en un 28.8//y 28.6//respectivamente, y por el 65.8//en la presencia de Apis m. 9C la degranulacion de los basofilos inducida por 1.66x10 potencia -16 a 1.66x10 potencia -18 m de anticuerpos anti -IgE resulto tambien inhibida por una alta dilucion de Pulm. hist. y Apis m. con una inhibicion de cerca del 100//con Pulmon hist. 18C y Apis m. 10C. Una alternancia de inhibicion, inactividad y estimulacion se observo cuando los basofilos fueron incubados en la presencia de una serie de diluciones de Pulm. hist. y Apis m. 3. La investigacion de la eficacia clinica de altas diluciones de Pulmon hist. y Apis m. deberia ser estudiada en enfermedades alergicas, en forma paralela con estudios in vitro y ex vivo en ensayos biologicos

Degranulacao de basofilo humano ativada por anti-soro anti-IgE diluido em alto grau / Human basophil degranulation triggeded by dilute antiserum against IgE

Quando basofilos polimorfonucleares humanos, um tipo decelula branca do sangue com anticorpos do tipo da imunoglobulina E (IgE) em sua superficie, sao expostos a anticorpos anti-IgE, eles liberam histamina de seus granulos intracelulares e modificam suas propriedades corantes. Isto pode ser demonstrado em diluicoes de anti-IgE que variam de 1x10 (10 elevado a segunda potencia) a 1x10 (10 elevado a centesima vigesima potencia); alem desse limite, ha sucessivos picos de degranulacao de 40 a 60 porcento dos basofilos, a despeito da suposta ausencia de quaisquer moleculas anti-IgE nas diluicoes mais elevadas. Visto que as diluicoes precisam ser acompanhadas por vigorosas agitacoes para que os efeitos sejam observados, a transmissao de informacao biologica poderia estar relacionada a organizacao molecular da agua

Human basophil degranulation triggered by very dilute antiserum against IgE.

When human polymorphonuclear basophils, a type of white blood cell with antibodies of the immunoglobulin E (IgE) type on its surface, are exposed to anti-IgE antibodies, they release histamine from their intracellular granules and change their staining properties. The latter can be demonstrated at dilutions of anti-IgE that range from 1 x 10(2) to 1 x 10(120); over that range, there are successive peaks of degranulation from 40 to 60% of the basophils, despite the calculated absence of any anti-IgE molecules at the highest dilutions. Since dilutions need to be accompanied by vigorous shaking for the effects to be observed, transmission of the biological information could be related to the molecular organization of water.

In vitro immunological degranulation of human basophils is modulated by lung histamine and Apis mellifica.

1. The effect of high dilutions of two homeopathic drugs Lung histamine (Lung his) and Apis mellifica (Apis mel) used for the treatment of allergic diseases has been assessed on in vitro human basophil degranulation. Experiments were conducted blind. 2. Basophil degranulation induced by 1.66 X 10(-9) M anti-IgE antibody was significantly inhibited in the presence of 5 Lung his (5th centesimal dilution of Lung his) and 15 Lung his (15th centesimal dilution of Lung his) by 28.8% and 28.6% respectively and by 65.8% in the presence of 9 Apis mel (9th centesimal dilution of Apis mel). Basophil degranulation induced by 1.66 X 10(-16) to 1.66 X 10(-18) M anti-IgE antibody was also inhibited by high dilutions of Lung his and Apis mel with an inhibition of nearly 100% with 18 Lung his (18th centesimal dilution of Lung his) and 10 Apis mel (10th centesimal dilution of Apis mel). An alternance of inhibition, inactivity and stimulation was observed when basophils were incubated in the presence of serial dilutions of Lung his and Apis mel. 3. The investigation of the clinical efficacy of high dilutions of Lung his and Apis mel should be envisaged in allergic diseases in parallel with in vitro and ex vivo biological assays.

Effect of mouse peritoneal macrophages of orally administered very high dilutions of silica.

The activity of very high dilutions of silica, a substance cytotoxic for macrophages, was tested on the synthesis by mouse peritoneal macrophages of the inflammatory ether-lipid paf-acether and its precursor lyso paf-acether. C57Bl6 female mice received for 25 days either 1.66 X 10(-11) M silica (11 sil) or 1.66 X 10(-19) M (19 sil) (final concentration) in the tap-water they were given to drink while control mice remained untreated. Isolated macrophages from mice treated with 11 sil produced 44.2 and 30.8% more paf-acether than cells from untreated mice in the presence of 50 and 200 micrograms zymosan (Z)/ml respectively. When 19 sil was given to the mice, the respective increases were 67.5 and 38%. In an experiment with a blind design, the mice were either untreated or received 19 sil or saline submitted to the same dilution procedure (19 sal). After administration of 19 sil, paf-acether synthesis was 55.5 and 33.5% higher upon stimulation with 50 and 200 micrograms Z/ml, respectively, than in the 19 sal group. In a third blind experiment, macrophages from mice that received 19 sil formed 61.3 and 28.6% more paf-acether upon stimulation with 50 and 200 micrograms Z/ml respectively, as compared to mice receiving 19 sal or lactose submitted to the same dilution procedure (19 lac). There was no difference between the 19 sal and the 19 lac groups. The differences between control and silica-treated mice were highly statistically significant in all experiments. There was no effect on the synthesis of lyso paf-acether. These results demonstrate clear ex vivo cellular effect of high dilutions of silica, that cannot be explained in our present state of knowledge.

[Oral contraceptives and blood platelets]. / Contraceptifs oraux et plaquettes sanguines.

PIP: This work describes a study of the effects of combined oral contraceptives (OCs) on lipid biosynthesis in platelets of female rats and women. A highly significant hypercoagulability due solely to increased activity of platelet factor 3 can be observed in women using combined OCs. The phospholipidic nature of factor 3 has been demonstrated. Phospholipids are implicated in the aggregation of platelets because they are the essential constituents of the platelet membranes and the precursors of prostaglandins. Platelets actively synthesize their own lipids, and combined OCs modify serum lipid metabolism. In each experiment, a control group of rats weighing 180-200 g received .5 ml/g body weight of olive oil once daily for 4 days. 3 groups of experimental rats received .5 ml of olive oil containing 10 mcg of ethinyl estradiol (EE) and 250 mcg of lynestrenol or 10 mcg of EE alone or 250 mcg of lynestrenol alone per 100 g of body weight. The doses were the equivalent of 1/2 that required to block ovulation in adult female rats. Platelets were studied on the 5th day. In another experiment a group of rats was given a triple dose of EE and lynestrenol on the 1st study day. Platelets were studied on days 1, 3, 5, and 8. Lipid biosynthesis was studied by incorporation of carbon 14 labelled acetate and mevalonate precursors. Radioactivity was measured for the lipids as a whole and for different lipid fractions separated by chromatography. Incorporation of carbon 14 labelled acetate was augmented by 44.6% in animals receiving EE and lynestrenol and by 43% in animals receiving EE alone, but was not modified in animals receiving lynestrenol alone. In animals receiving a triple dose of hormones, incorporation was maximal on the 3rd day, diminished on the 5th day, and normal after 8 days. The EE component thus appears to be responsible for modifications in platelet lipid metabolism during OC use. The response appears after a latency period and seems to be irreversible, since the duration of life of platelets is 4-5 days. The increased synthesis occurs mainly in cholesterol and its precursors lanosterol and dihydrolanosterol. Supplemental in vitro experiments suggested that lanosterol was responsible for the increased platelet activity. 17 nonsmoking women aged 32 years on average who took no medications were compared to 18 women aged 30 years on average who took OCs with estrogen doses of 30-40 mcg for at least 6 months. As in the rat studies, lipid biosynthesis was analyzed by incorporation of carbon 14 labelled acetate or mevalonate in the platelets. Compared to control women, the women on OCs showed an augmentation of 37% in incorporation of mevalonate and 28% of acetate. The labelled acetate showed a higher incorporation at the level of each of the lipid fractions. Mevalonate showed the highest augmentation in the lanosterol fraction. 43% of the women taking OCs showed an increased platelet sensitivity to thrombine. The increased sensitivity was correlated with increased lanosterol synthesis, but the relation was only observed in women taking OCs. The phenomenon is of interest because of its possible relationship to the increased risk of thromboembolic accidents in women taking OCs.^ieng

Effect of dietary (n--3) fatty acids on platelet function and lipid metabolism in rats.

Six groups of rats were fed diets containing 40% (by weight) lipids, mostly as saturated fatty acids (from 78 to 90%), with a basic amount of linoleic acid (18:2) (1.9%). In four groups, 5% of the saturated fats were substituted with an oil (vegetable: corn, rapeseed; or fish: cod liver, maxepa) and in one group 0.6% of the saturated fats was replaced by eicosapentaenoic acid ethyl ester. The diets supplied different amounts of 18:2 (1.9 to 10%), 18:3 (0.2 to 1.2%), 20:5 (n--3) (eicosapentaenoic acid) (0 to 1.3%) and 22:6(n--3) (0 to 1.6%). After 3-8 months on diets, platelet aggregation, plasma and platelet cholesterol, fatty acids and incorporation of [14C]acetate and [14C]arachidonate into platelet lipids were investigated. The three diets supplying eicosapentaenoic acid (1.3%) induced an approximately 80-fold increase in this fatty acid in plasma and platelet phospholipids, mainly at the expense of arachidonic acid. This was associated with an increase of the incorporation of arachidonic acid into platelet PE and PS. The incorporation of acetate in the (n--3) fatty-acid-fed animals was markedly increased into 22:5 and 22:6(n--3) at the expense of 22:4(n--6). Platelet aggregation induced by ADP was similar in the six groups. The response to thrombin was lower in animals fed corn and maxepa oils. Collagen aggregation tended to be lower in the fish-oil groups. Platelet aggregation to collagen was significantly negatively correlated to the level of eicosapentaenoic acid in platelet phospholipids, while the aggregation to thrombin was related to the level of 20:3(n--9). In the present study in rat, the (n--3) fatty acids added in small amounts to a saturated fat diet over a period of several months induced drastic changes in platelet lipid metabolism and composition without comparable effects on platelet behaviour.

Vitamin E prevents the platelet abnormalities induced by estrogen in rat.

Platelet lipid biosynthesis in relation to aggregation has been studied in female rats treated with ethynylestradiol and fed laboratory chow or a vitamin E-deficient diet. In both normal and vitamin E-deficient rats, administration of ethynylestradiol highly significantly (p less than .001) increased the biosynthesis of total lipids but mostly of lanosterol (+ dihydrolanosterol) by thirteen-fold in normal rats and by nine-fold in vitamin E-deficient rats. The increased lipid synthesis was associated with a higher response of platelets to thrombin-induced aggregation. Concomitant administration of alpha-tocopherol acetate in both normal and vitamin E-deficient rats depressed markedly the enhanced lipid synthesis and aggregation induced by estrogen. Administration of ethynylestradiol lowered considerably the level of vitamin E in plasma but not in platelets. Treatment by tocopherol partly corrected the low plasma level of vitamin E resulting from estrogen administration. In vitro addition of lanosterol to platelets highly significantly increased the response of platelets to thrombin- and ADP-induced aggregation. This hyperaggregability was almost entirely inhibited by preincubation of platelets with tocopherol acetate. In the present in vivo and in vitro studies, alpha-tocopherol was able to neutralize most of the adverse effects of estrogen on blood platelets.

Effects of dietary lipids on behaviour, lipid biosynthesis and lipid composition, in rat platelets.

Rats of either sex were fed for 18 and 34 weeks respectively diets containing 40% (by weight) lipids with polyunsaturated fatty acids representing 1.34% or 13.2% of total calories. Platelet reactivity to thrombin, platelet fatty acid composition and incorporation of [14C]acetate into platelet lipids were investigated. Diets rich in saturated fatty acids markedly increased platelet sensitivity to thrombin. The concentration of 20:3 and 22:3 of the (n - 9) series and of 20:3 and 22:5 of the (n - 6) series were increased at the expense of 18:2 and 22:4 of the (n - 6) family in platelet lipids. 20:4 (n - 6) was unchanged. The fatty acid changes were more pronounced in male rats and after 34 weeks. [14C]Acetate incorporation into total platelet lipids and particularly into choline phosphoglycerides and ceramides was lower in animals fed saturated fats. This diet reduced the synthesis of 16:0 and of 22:4(n - 6) in platelet total fatty acids, while that of 22:3(n - 9) was markedly enhanced. This study showed that long-term feeding of high-saturated-low-polyunsaturated fat diets in rats induced marked changes in platelet lipid synthesis and composition, in both sexes. The lipid synthesis modification appears to be more pronounced in males than in females. The changes in the fatty acids 20:3(n - 9), 22:3(n - 9) and 22:4(n - 6) appeared to be closely related to platelet behaviour. The balance between the content and synthesis of these last fatty acids might be of significance for the effect of diet on thrombogenesis.

Influence of sex and dietary fats on platelet lipid biosynthesis in rat.

The platelet biosynthesis of total lipids, lipid fractions and fatty acids was determined by incorporation of [14C]acetate in normal and castrated rats of both sexes. Comparison was made between animals fed laboratory chow alone, and animals receiving, in addition, for 4 days by stomach tube a saturated (cream) or polyunsaturated (sunflower seed oil) fat. In male rats, the polyunsaturated fat increased slightly the total platelet lipid biosynthesis. The saturated fat drastically reduced it by 43% in comparison to the polyunsaturated fat-fed animals. Normal female rats did not exhibit a similar difference in the platelet lipid synthesis. However, the inhibitory effect of saturated fat on lipid synthesis could be observed in castrated females, although it was less pronounced (27% reduction) than in castrated or normal males (43%). Administration to castrated males of estradiol for 1 month almost completely inhibited the difference induced by the feeding of the different fats in the lipid platelet synthesis of male rats. This difference in the platelet lipid biosynthesis between male and female rats, normal and castrated, was observed mostly in the phospholipid (especially phosphatidylcholine), monoacylglycerol and triacylglycerol fractions and affected primarily the synthesis of the three main saturated fatty acids, 14:0, 16:0 and 18:0. Thus, it seems that, in rat, the short-term administration of a saturated fat induces drastic changes in the platelet lipid biosynthesis, but only in males. The protection observed in females appears to be essentially dependent upon estrogens.

Biosynthesis of platelet lipids in relation to aggregation in women using oral contraceptives.

The platelet lipid biosynthesis in relation to platelet aggregation and lipemia was studied by 14C-acetate and mevalonate incorporation into platelets of seventeen women without medication and of eighteen women using a low estrogen oral contraceptive. The lipid biosynthesis was significantly increased by 59% (mevalonate) and 38% (acetate) in women on oral contraceptives. From mevalonate, lipid synthesis was increased mostly in the lanosterol-dihydrolanosterol fraction (p less than .01). From acetate, lipid synthesis was significantly enhanced in all the lipid classes. In the oral contraceptive group, the response of platelets to thrombin aggregation was only slightly higher, but HDL-cholesterol was significantly lower. However, in the women using oral contraceptives, the percentage of abnormal values in HDL-cholesterol, thrombin-aggregation and acetate incorporation into lanosterol was similar. Thus, more than 40% of the women studied here, using low estrogen oral contraceptives, presented an increase in platelet lipid biosynthesis, especially in the lanosterol-dihydrolanosterol fraction, which was significantly correlated (p less than .05) with the response of their platelets to thrombin-induced aggregation.

Oral contraceptive and Platelet lipid biosynthesis in female rats: dose-response relationship.

Female rats were treated with different doses of an oral contraceptive (ethinyl estradiol + lynestrenol) and lipid biosynthesis was studied in blood platelets by acetate incorporation into different fractions separated by thin layer chromatography. A marked increase in lipid biosynthesis was observed, especially in the sterol fractions (cholesterol and lanosterol-dihydrolanosterol). It was dose-dependent, observed after a lag-phase, maximal in 3 days and normalized in 8 days. Thus, the oral contraceptive studied here appears to modify platelet lipid biosynthesis for the entire life of the platelets.