Action Of Ethanol Research Articles

INFLUENCE OF INOSINE GLYCYL-CYSTEINYL-GLUTAMATE DISODIUM ON THE CLINICAL COURSE AND OUTCOME OF THE EXPERIMENTAL ALCOHOL-INDUCED LIVER CIRRHOSIS

An evaluation of the inosine glycil-cysteinyl-glutamate disodium efficacy as a means for therapy of toxic alcoholinduced liver cirrhosis was performed in experiments on outbred white rats. Experimental modeling of liver cirrhosis was carried out by administrating intragastrically to rats two hepatotoxicants– 40% ethanol at a dose of 3 g/kg, every other day for three weeks and 1% dimethylnitrosamine at a dose of 5 mg/kg, intraperitoneally the first 4 days per week. Inosine glycyl-cysteinyl-glutamate disodium was administered subcutaneously at a dose of 30 mg/kg daily for three weeks; application of the drug was started after the administration of hepatotoxicants was ended and clinical and morphological indicators of toxic liver cirrhosis were proved. Evaluation of the efficiency of inosine glycyl-cysteinyl-glutamate disodium as a therapeutic means for alcohol-induced liver cirrhosis was performed by morphological, histological and biochemical methods. It was found out that a combined action of ethanol and dimethylnitrosamine led to the development of toxic liver cirrhosis, which manifested by an increase in the rats body weight , characteristic morphological and histological changes in liver tissues, increased activity of alanine and aspartate aminotransferase, gamma-glutamyltranspeptidase, alkaline phosphatase and total bilirubin level, and an increase in the blood plasma of concentration of interleukin-1β, interferon-γ and tumor necrosis factor-α. A course administration of inosine glycyl-cysteinyl-glutamate disodium contributed to a decrease in the volume of connective tissue in the liver of animals, decrease in the activity of hepatic enzymes, decrease in the level of total bilirubin and the concentration of pro-inflammatory cytokines in the blood plasma. Data obtained show the efficacy of inosine glycyl-cysteinyl-glutamate disodium as a means for treating alcohol-induced liver cirrhosis.

BK β1 subunit-dependent facilitation of ethanol inhibition of BK current and cerebral artery constriction is mediated by the β1 transmembrane domain 2.

Ethanol at concentrations obtained in the circulation during moderate-heavy episodic drinking (30-60mM) causes cerebral artery constriction in several species, including humans. In rodents, ethanol-induced cerebral artery constriction results from ethanol inhibition of large conductance voltage/Ca2+i -gated K+ (BK) channels in cerebral artery myocytes. Moreover, the smooth muscle-abundant BK β1 accessory subunit is required for ethanol to inhibit cerebral artery myocyte BK channels under physiological Ca2+i and voltages and thus constrict cerebral arteries. The molecular bases of these ethanol actions remain unknown. Here, we set to identify the BK β1 region(s) that mediates ethanol-induced inhibition of cerebral artery myocyte BK channels and eventual arterial constriction. We used protein biochemistry, patch-clamp on engineered channel subunits, reversible cDNA permeabilization of KCNMB1 K/O mouse arteries and artery in vitro pressurization. Ethanol inhibition of BK current was facilitated by β1 but not β4 subunits. Furthermore, only BK complexes containing β chimeras with β1 transmembrane (TM) domains on a β4 background or with a β1 TM2 domain on a β4 background displayed ethanol responses identical to those of BK complexes including wild-type β1. Moreover, β1 TM2 itself but not other β regions were necessary for ethanol-induced cerebral artery constriction. BK β1 TM2 is necessary for this subunit to enable ethanol-induced inhibition of myocyte BK channels and cerebral artery constriction at physiological Ca2+ and voltages. Thus, novel agents that target β1 TM2 may be considered to counteract ethanol-induced cerebral artery constriction and associated cerebrovascular conditions.

Ethanol binding sites on proteins

This study is on the analysis of ethanol binding sites on 3D structures of nonredundant proteins from the Protein Data Bank. The only one amino acid residue that is significantly overrepresented around ethanol molecules is Tyr. There are usually two or more Tyr residues in the same ethanol binding site, while residues of Thr, Asp and Gln are underrepresented around them. Residues of Ala and Pro are significantly underrepresented in ethanol binding surfaces. Several residues (Phe, Val, Pro, Ala, Arg, His, Ser, Asp) bind ethanol significantly more frequent if they are not included in beta strands. Residues of Ala, Ile and Arg preferably bind ethanol when they are included in an alpha helix. Ethanol molecules often make hydrogen bonds with oxygen and nitrogen atoms from the main chain of a protein. Because of this reason, the binding of ethanol may be associated with the decrease of the length of alpha helices and the disappearance of 3/10 helices. Obtained data should be useful for studies on new targets of the direct action of ethanol on enzymes, receptors, and transcription factors.

Ethanol Stimulates Locomotion via a Gαs-Signaling Pathway in IL2 Neurons in Caenorhabditis elegans.

Alcohol is a potent pharmacological agent when consumed acutely at sufficient quantities and repeated overuse can lead to addiction and deleterious effects on health. Alcohol is thought to modulate neuronal function through low-affinity interactions with proteins, in particular with membrane channels and receptors. Paradoxically, alcohol acts as both a stimulant and a sedative. The exact molecular mechanisms for the acute effects of ethanol on neurons, as either a stimulant or a sedative, however remain unclear. We investigated the role that the heat shock transcription factor HSF-1 played in determining a stimulatory phenotype of Caenorhabditis elegans in response to physiologically relevant concentrations of ethanol (17 mM; 0.1% v/v). Using genetic techniques, we demonstrate that either RNA interference of hsf-1 or use of an hsf-1(sy441) mutant lacked the enhancement of locomotion in response to acute ethanol exposure evident in wild-type animals. We identify that the requirement for HSF-1 in this phenotype was IL2 neuron-specific and required the downstream expression of the α-crystallin ortholog HSP-16.48 Using a combination of pharmacology, optogenetics, and phenotypic analyses we determine that ethanol activates a Gαs-cAMP-protein kinase A signaling pathway in IL2 neurons to stimulate nematode locomotion. We further implicate the phosphorylation of a specific serine residue (Ser322) on the synaptic protein UNC-18 as an end point for the Gαs-dependent signaling pathway. These findings establish and characterize a distinct neurosensory cell signaling pathway that determines the stimulatory action of ethanol and identifies HSP-16.48 and HSF-1 as novel regulators of this pathway.

Ethanol differentially modulates P2X4 and P2X7 receptor activity and function in BV2 microglial cells

Neuroinflammation is one of the mechanisms leading to neurodegenerative brain damage induced by chronic alcohol (ethanol) exposure. Microglia play a major role in the development of innate immune responses to environmental injuries including ethanol. Adenosine 5″-triphosphate (ATP)-activated purinergic P2X receptor (P2XR) subtypes, P2X4Rs and P2X7Rs, are endogenously expressed in microglia and can modulate their activity. These 2 P2XR subtypes differ pharmacologically and functionally: 1) P2X4Rs are activated at lower (≤0.1 mM) whereas P2X7Rs – at higher (≥1.0 mM) ATP concentrations; 2) P2X4R activation contributes to the release of brain derived neurotrophic factor and its role in tactile allodynia and neuropathic pain is demonstrated; 3) Due to its role in the secretion of pro-inflammatory IL-1β, P2X7Rs have been implicated in the development of neurodegenerative pathologies, pain and morphine tolerance. To date, the roles of individual P2XR subtypes in ethanol effects on microglia and the functional consequences are not completely understood. Based on the existing knowledge on the pharmacological and functional differences between P2X4Rs and P2X7Rs, the present work tested the hypothesis that P2X4Rs and P2X7Rs play differential roles in ethanol action in microglia. Effects of ethanol on P2X4R and P2X7R activity, expression and functional consequences were determined using murine BV2 microglial cells. Ethanol (≥100 mM) inhibited P2X4Rs but was inactive on P2X7 channel activity. Ethanol (25, 100 mM) inhibited P2X4R-mediated microglia migration whereas it potentiated pore formation in P2X7Rs. Furthermore, ethanol (25, 100 mM) potentiated P2X7R-mediated IL-1β secretion from BV2 microglia. Ethanol also induced protein expression for both P2XR subtypes. Overall, the findings identify differential roles for P2X4Rs and P2X7Rs in regards to ethanol effects on microglia which may be linked to different stages of ethanol exposure.

Ethanol potentiates heat response in the carotid artery via TRPV1

AimEthanol is one of the most widely used recreational drugs in the world. At high concentrations, it can induce carotid artery vasoconstriction. Hyperthermia potentiates its effects resulting in carotid artery vasoconstriction at any concentration. The aim of this study is to investigate the interaction between ethanol and heating and to understand the underlying mechanisms leading to their synergistic effect. Materials and methodsIsometric tension of rabbit carotid artery ring segments suspended in organ baths filled with Krebs solution was recorded. Different concentrations of ethanol were examined at 37°C and during temperature elevation to39–43°C. Capsaicin and capsazepine were used to examine the mechanism of action of ethanol. Key findingsEthanol induced contraction at 37°C when the concentration reached 100mM. Contraction was observed at any concentration at higher temperatures. Ethanol potentiated heat-induced contraction. Capsaicin, the vanilloid receptor subtype1 (TRPV1) agonist, potentiated the vasoconstriction due to heating. While capsazepine, TRPV1 antagonist, abolished the effect of ethanol and its potentiation of heating-induced contraction, but it did not abolish the heating effect. SignificanceEthanol's mechanism of action and its effect on heating induced-vasoconstriction of the carotid artery is being mediated by TRPV1. The combination of ethanol and hyperthermia can lead to a synergistic effect on carotid vasoconstriction. This effect may induce brain damage and heat stroke. Development of new drugs act as TRPV1 antagonist can be used to prevent these fatal effects.

Mutation of the inhibitory ethanol site in GABAA ρ1 receptors promotes tolerance to ethanol-induced motor incoordination

Genes encoding the ρ1/2 subunits of GABAA receptors have been associated with alcohol (ethanol) dependence in humans, and ρ1 was also shown to regulate some of the behavioral effects of ethanol in animal models. Ethanol inhibits GABA-mediated responses in wild-type (WT) ρ1, but not ρ1(T6'Y) mutant receptors expressed in Xenopus laevis oocytes, indicating the presence of an inhibitory site for ethanol in the second transmembrane helix. In this study, we found that ρ1(T6'Y) receptors expressed in oocytes display overall normal responses to GABA, the endogenous GABA modulator (zinc), and partial agonists (β-alanine and taurine). We generated ρ1 (T6'Y) knockin (KI) mice using CRISPR/Cas9 to test the behavioral importance of the inhibitory actions of ethanol on this receptor. Both ρ1 KI and knockout (KO) mice showed faster recovery from acute ethanol-induced motor incoordination compared to WT mice. Both KI and KO mutant strains also showed increased tolerance to motor impairment produced by ethanol. The KI mice did not differ from WT mice in other behavioral actions, including ethanol intake and preference, conditioned taste aversion to ethanol, and duration of ethanol-induced loss of righting reflex. WT and KI mice did not differ in levels of ρ1 or ρ2 mRNA in cerebellum or in ethanol clearance. Our findings indicate that the inhibitory site for ethanol in GABAA ρ1 receptors regulates acute functional tolerance to moderate ethanol intoxication. We note that low sensitivity to alcohol intoxication has been linked to risk for development of alcohol dependence in humans.

On the Accuracy of In Vivo Ethanol and Acetaldehyde Monitoring, a Key Tile in the Puzzle of Acetaldehyde as a Neuroactive Agent.

Over the last 20 years researchers have explored the postulated role of acetaldehyde (ACD) as a mediator of some of the actions of ethanol (EtOH) in the central nervous system (CNS). However, efforts have been hampered mainly by the difficulty of directly measuring in vivo EtOH and ACD levels in the CNS and thus, our knowledge is based on indirect evidences. Although technically challenging, the development of reliable methods for in vivo measurement of ACD and EtOH is of paramount importance to solve the “puzzle of acetaldehyde as a neuroactive agent.” In this short review we discuss the recent advances on brain EtOH pharmacokinetic and state-of-the-art available techniques that could be used for in vivo detect EtOH and ACD both non-invasively (magnetic resonance spectroscopy), and invasively (microdialysis and biosensors). Among the different in vivo sampling techniques described, particular emphasis is paid to the field of enzyme-based amperometric biosensors. Biosensors have gained much attention in recent years for their ability to online monitor biological signals in vivo, and several micro- and nano-structured devices have been successfully used for in vivo studies. Owing to their high temporal and spatial resolution, biosensors could provide the adequate technology for studying in vivo EtOH pharmacokinetic.

Chronic alcohol feeding potentiates hormone-induced calcium signalling in hepatocytes.

Chronic alcohol consumption causes a spectrum of liver diseases, but the pathogenic mechanisms driving the onset and progression of disease are not clearly defined. We show that chronic alcohol feeding sensitizes rat hepatocytes to Ca2+ -mobilizing hormones resulting in a leftward shift in the concentration-response relationship and the transition from oscillatory to more sustained and prolonged Ca2+ increases. Our data demonstrate that alcohol-dependent adaptation in the Ca2+ signalling pathway occurs at the level of hormone-induced inositol 1,4,5 trisphosphate (IP3 ) production and does not involve changes in the sensitivity of the IP3 receptor or size of internal Ca2+ stores. We suggest that prolonged and aberrant hormone-evoked Ca2+ increases may stimulate the production of mitochondrial reactive oxygen species and contribute to alcohol-induced hepatocyte injury. ABSTRACT: 'Adaptive' responses of the liver to chronic alcohol consumption may underlie the development of cell and tissue injury. Alcohol administration can perturb multiple signalling pathways including phosphoinositide-dependent cytosolic calcium ([Ca2+ ]i ) increases, which can adversely affect mitochondrial Ca2+ levels, reactive oxygen species production and energy metabolism. Our data indicate that chronic alcohol feeding induces a leftward shift in the dose-response for Ca2+ -mobilizing hormones resulting in more sustained and prolonged [Ca2+ ]i increases in both cultured hepatocytes and hepatocytes within the intact perfused liver. Ca2+ increases were initiated at lower hormone concentrations, and intercellular calcium wave propagation rates were faster in alcoholics compared to controls. Acute alcohol treatment (25mm) completely inhibited hormone-induced calcium increases in control livers, but not after chronic alcohol-feeding, suggesting desensitization to the inhibitory actions of ethanol. Hormone-induced inositol 1,4,5 trisphosphate (IP3 ) accumulation and phospholipase C (PLC) activity were significantly potentiated in hepatocytes from alcohol-fed rats compared to controls. Removal of extracellular calcium, or chelation of intracellular calcium did not normalize the differences in hormone-stimulated PLC activity, indicating calcium-dependent PLCs are not upregulated by alcohol. We propose that the liver 'adapts' to chronic alcohol exposure by increasing hormone-dependent IP3 formation, leading to aberrant calcium increases, which may contribute to hepatocyte injury.

Mu-opioid receptor and delta-opioid receptor differentially regulate microglial inflammatory response to control proopiomelanocortin neuronal apoptosis in the hypothalamus: effects of neonatal alcohol

BackgroundOpioid receptors are known to control neurotransmission of various peptidergic neurons, but their potential role in regulation of microglia and neuronal cell communications is unknown. We investigated the role of mu-opioid receptors (MOR) and delta-opioid receptors (DOR) on microglia in the regulation of apoptosis in proopiomelanocortin (POMC) neurons induced by neonatal ethanol in the hypothalamus.MethodsNeonatal rat pups were fed a milk formula containing ethanol or control diets between postnatal days 2–6. Some of the alcohol-fed rats additionally received pretreatment of a microglia activation blocker minocycline. Two hours after the last feeding, some of the pups were sacrificed and processed for histochemical detection of microglial cell functions or confocal microscopy for detection of cellular physical interaction or used for gene and protein expression analysis. The rest of the pups were dissected for microglia separation by differential gradient centrifugation and characterization by measuring production of various activation markers and cytokines. In addition, primary cultures of microglial cells were prepared using hypothalamic tissues of neonatal rats and used for determination of cytokine production/secretion and apoptotic activity of neurons.ResultsIn the hypothalamus, neonatal alcohol feeding elevated cytokine receptor levels, increased the number of microglial cells with amoeboid-type circularity, enhanced POMC and microglial cell physical interaction, and decreased POMC cell numbers. Minocycline reversed these cellular effects of alcohol. Alcohol feeding also increased levels of microglia MOR protein and pro-inflammatory signaling molecules in the hypothalamus, and MOR receptor antagonist naltrexone prevented these effects of alcohol. In primary cultures of hypothalamic microglia, both MOR agonist [D-Ala 2, N-MePhe 4, Gly-ol]-enkephalin (DAMGO) and ethanol increased microglial cellular levels and secretion of pro-inflammatory cell signaling proteins. However, a DOR agonist [D-Pen2,5]enkephalin (DPDPE) increased microglial secretion of anti-inflammatory cytokines and suppressed ethanol’s ability to increase microglial production of inflammatory signaling proteins and secretion of pro-inflammatory cytokines. In addition, MOR-activated inflammation promoted while DOR-suppressed inflammation inhibited the apoptotic effect of ethanol on POMC neurons.ConclusionsThese results suggest that ethanol’s neurotoxic action on POMC neurons results from MOR-activated neuroinflammatory signaling. Additionally, these results identify a protective effect of a DOR agonist against the pro-inflammatory and neurotoxic action of ethanol.

Pre-Clinical Studies with D-Penicillamine as a Novel Pharmacological Strategy to Treat Alcoholism: Updated Evidences.

Ethanol, as other drugs of abuse, is able to activate the ventral tegmental area dopamine (VTA-DA) neurons leading to positively motivational alcohol-seeking behavior and use, and, ultimately to ethanol addiction. In the last decades, the involvement of brain-derived acetaldehyde (ACD) in the ethanol actions in the mesolimbic pathway has been widely demonstrated. Consistent published results have provided a mechanistic support to the use of ACD inactivating agents to block the motivational and reinforcing properties of ethanol. Hence, in the last years, several pre-clinical studies have been performed in order to analyze the effects of the sequestering ACD agents in the prevention of ethanol relapse-like drinking behavior as well as in chronic alcohol consumption. In this sense, one of the most explored interventions has been the administration of D-Penicillamine (DP). These pre-clinical studies, that we critically summarize in this article, are considered a critical step for the potential development of a novel pharmacotherapeutic strategy for alcohol addiction treatment that could improve the outcomes of current ones. Thus, on one hand, several experimental findings provide the rationale for using DP as a novel therapeutic intervention alone and/or in combination to prevent relapse into alcohol seeking and consumption. On the other hand, its effectiveness in reducing voluntary ethanol consumption in long-term experienced animals still remains unclear. Finally, this drug offers the additional advantage that has already been approved for use in humans, hence it could be easily implemented as a new therapeutic intervention for relapse prevention in alcoholism.

Protective influence of hyaluronic acid on focal adhesion kinase activity in human skin fibroblasts exposed to ethanol.

AimThe aim of this study was to evaluate the effect of ethanol and hyaluronic acid (HA) on cell survival and apoptosis in cultured human skin fibroblasts. Regarding the mechanism of ethanol action on human skin fibroblasts, we investigated cell viability and apoptosis, expression of focal adhesion kinase (FAK), and the influence of HA on those processes.Materials and methodsStudies were conducted in confluent human skin fibroblast cultures that were treated with 25 mM, 50 mM, and 100 mM ethanol or with ethanol and 500 µg/mL HA. Cell viability was examined using methyl thiazolyl tetrazolium (MTT) assay and NC-300 Nucleo-Counter. Imaging of the cells using a fluorescence microscope Pathway 855 was performed to measure FAK expression.ResultsDepending on the dosage, ethanol decreased cell viability and activated the process of apoptosis in human skin fibroblasts. HA prevented the negative influence of ethanol on cell viability and prevented apoptosis. The analysis of fluorescence imaging using BD Pathway 855 High-Content Bioimager showed the inhibition of FAK migration to the cell nucleus, depending on the increasing concentration of ethanol.ConclusionThis study proves that downregulation of signaling pathway of FAK is involved in ethanol-induced apoptosis in human skin fibroblasts. The work also indicates a protective influence of HA on FAK activity in human skin fibroblasts exposed to ethanol.

Effects of Repeated Ethanol Exposures on NMDA Receptor Expression and Locomotor Sensitization in Mice Expressing Ethanol Resistant NMDA Receptors.

Evidence from a large number of preclinical studies suggests that chronic exposure to drugs of abuse, such as psychostimulants or ethanol induces changes in glutamatergic transmission in key brain areas associated with reward and control of behavior. These changes include alterations in the expression of ionotropic glutamate receptors including N-methyl-D-aspartate receptors (NMDAR) that are important for regulating neuronal activity and synaptic plasticity. NMDA receptors are inhibited by ethanol and reductions in NMDA-mediated signaling are thought to trigger homestatic responses that limit ethanol's effects on glutamatergic transmission. Following repeated exposures to ethanol, these homeostatic responses may become unstable leading to an altered glutamatergic state that contributes to the escalations in drinking and cognitive deficits observed in alcohol-dependent subjects. An important unanswered question is whether ethanol-induced changes in NMDAR expression are modulated by the intrinsic sensitivity of the receptor to ethanol. In this study, we examined the effects of ethanol on NMDAR subunit expression in cortical (orbitofrontal, medial prefrontal), striatal (dorsal and ventral striatum) and limbic (dorsal hippocampus, basolateral amygdala) areas in mice genetically modified to express ethanol-resistant receptors (F639A mice). These mice have been previously shown to drink more ethanol than their wild-type counterparts and have altered behavioral responses to certain actions of ethanol. Following long-term voluntary drinking, F639A mice showed elevations in GluN2A but not GluN1 or GluN2B expression as compared to wild-type mice. Mice treated with repeated injections with ethanol (2–3.5 g/kg; i.p.) showed changes in NMDAR expression that varied in a complex manner with genotype, brain region, subunit type and exposure protocol all contributing to the observed response. F639A mice, but not wild-type mice, showed enhanced motor activity following repeated ethanol injections and this was associated with differences in NMDAR subunit expression across brain regions thought to be involved in drug sensitization. Overall, while the results of the study suggest that NMDARs with reduced sensitivity to ethanol favor the development of locomotor sensitization, they also show that intrinsic ethanol sensitivity is not the sole determinant underlying changes in NMDAR expression following repeated exposures to ethanol.

High-Throughput Screening of Potential Skin Penetration-Enhancers Using Stratum Corneum Lipid Liposomes: Preliminary Evaluation for Different Concentrations of Ethanol.

In this study, we developed a technique for high-throughput screening (HTS) of skin penetration-enhancers using stratum corneum lipid liposomes (SCLLs). A fluorescent marker, sodium fluorescein (FL), entrapped in SCLLs was prepared to provide a preliminary evaluation of the effect of different concentrations of ethanol on the disruption effect of SCLLs, which is an alternative for skin penetration-enhancing effects. In addition, SCLLs containing a fluorescent probe (DPH, TMA-DPH, or ANS) were also prepared and utilized to investigate SCLL fluidity. The results using SCLL-based techniques were compared with conventional skin permeation and skin impedance test using hairless rat skin. The obtained correlations were validated between FL leakage, SCLL fluidity with various probes, or skin impedance and increases in the skin permeation enhancement ratio (ER) of caffeine as a model penetrant. As a result, FL leakage and SCLL fluidity using ANS were considered to be good indices for the skin penetration-enhancing effect, suggesting that the action of ethanol on the SC lipid and penetration-enhancing is mainly on the polar head group of intercellular lipids. In addition, this screening method using SCLL could be utilized as an alternative HTS technique for conventional animal tests. Simultaneously, the method was found to be time-saving and sensitive compared with a direct assay using human and animal skins.

Thiadiazole ring opening in the derivatives of 2-substituted 5-(1,2,3-thiadiazol-4-yl)-3-furoic acid under the action of bases

Transformations of the derivatives of 2-substituted 5-(1,2,3-thiadiazol-4-yl)-3-furoic acid under the action of bases has been studied. In the presence of potassium tert-butylate in THF, the studied compounds decompose with the cleavage of the thiadiazole ring, liberation of nitrogen, and formation of labile acetylene thiolates. In the presence of methyl iodide, these salts form stable 2-methylthioethynylfurans. Under the action of sodium ethylate in ethanol, thiadiazole ring of ethyl [2-methyl-5-(1,2,3-thiadiazol-4-yl)]-3-furoate is split to form the corresponding sodium acetylene thiolate. Under the action of ethanol, two molecules of this salt give bis(furyl)dithiafulvene. In the DMF–potassium carbonate system, acetylene thiolates react with primary and secondary amines giving thioamides of (4-ethoxycarbonyl-5-methylfur-2-yl)acetic acid. Treating of ethyl 2-methyland 2-N-morpholinomethyl-5-(1,2,3-thiadiazol-4-yl)-3-furoates with hydrazine hydrate leads to hydrazinolysis of the ester group and cleavage of thiadiazole ring resulting in the formation of hydrazides of 4-hydrazinocarbonylfur-2-ylacetic acid. In the case of ethyl 2-acetoxymethyl- and 2-(4-nitrophenoxy)methyl-5-(1,2,3-thiadiazol-4-yl)-3-furoates, thiadiazole ring is retained and exclusively hydrazinolysis of the ester groups is observed.

Role of the slo1 CRAC4 Motif in BK Channel's Ethanol Sensitivity

Cholesterol (CLR) has been recognized for its ability to interact with ion channels. Among ion channels that sense CLR presence are large conductance, voltage/Ca2+-gated K+ (BK) channels. Functional BK channels result from tetrameric association of pore-forming slo1 subunits. CLR inhibition of slo1 current may result from CLR-sensing by the slo1 cytosolic tail domain (CTD). CTD contains seven CLR-recognition amino acid consensus (CRAC) motifs that contribute to the overall CLR sensitivity of the channel, with the contribution by CRAC4 being studied (Singh et al., 2012). Alcohol (ethanol) also modifies BK channel function, this effect resulting from an orchestration of several factors that include [Ca2+]i. In native membranes, ethanol increases slo1 channel open probability (Po) at [Ca2+]i<20-30 µM whereas reduces Po at [Ca2+]i<30 µM. Both CLR-enrichment and CLR-depletion of native membranes modify ethanol action on BK channels. Thus, we set to determine the role of CLR-sensing by the CRAC4 motif in the slo1 channel's ethanol sensitivity. Slo1 cloned from rat cerebral artery myocytes (cbv1) and the cbv1CRAC4T450F were incorporated into POPE:POPS (3:1 w/w) bilayers in 300/30 mM K+i/K+o gradient. At 0.3 μM Ca2+i, ethanol increased cbv1 Po by ≈3-fold. At 300 μM Ca2+i, ethanol failed to modify cbv1 Po in CLR-free bilayers but increased cbv1 Po in CLR-containing (33 mol%) bilayers. The discrepancy in ethanol effect between native membranes and planar bilayers may likely arise from their difference in proteo-lipid, including CLR, composition. Cbv1CRAC4T450F did not respond to ethanol in CLR-free bilayers, but was activated by ethanol in presence of 33 mol% CLR. Collectively, the similarity of ethanol modulation of BK channel activity studied with cbv1 vs. cbv1CRAC4T450F suggests that CRAC4 does not contribute to CLR modulation of ethanol action on slo1 at high [Ca2+]i. Support: R01AA023764 (AB), R37AA11560 (AD).

The forensic medical evaluation of the changes in the cerebral tissue in the case of sudden death from alcoholic cardiomyopathy

This article presents the results of the retrospective analysis of the protocols of forensic medical autopsies and histological studies of the cerebral tissues together with the data obtained by their statistical treatment. The objective of present work was to study and evaluate the structural changes in the cerebral tissues associated with sudden cardiac death from alcoholic cardiomyopathy. It was shown that the morphological changes in the endothelial lining of the microcirculatory bed of the cerebral tissue can be a consequence of both the direct cytotoxic action of ethanol or its metabolites and the influence of cellular modulators liberation of which results in enhanced vascular permeability associated with trophic disturbances in the tissue. These changes provide a substrate for the development of the dystrophic and necrobiotic processes in the main structural components of the organ in question. The clustering of glial cells around atrophic neutrons or instead of the dead ones in the brain as well as around the microcysts of the cerebral tissue as a compensatory response to the lesion can be in its turn considered as a pathognomonic sign of chronic alcoholic intoxication. The severe dystrophic and destructive processes proceeding in the main components of the histohematological barrier revealed in the cerebral tissue as a result of the present analysis are believed to reflect the toxic action of ethanol and/or its metabolites that play an important role in the development of dyscirculatory disorders responsibly for cerebral tissue hypoxia.

Presynaptic Ethanol Actions: Potential Roles in Ethanol Seeking.

Ethanol produces intoxication through actions on numerous molecular and cellular targets. Adaptations involving these and other targets contribute to chronic drug actions that underlie continued and problematic drinking. Among the mechanisms involved in these ethanol actions are alterations in presynaptic mechanisms of synaptic transmission, including presynaptic protein function and excitation-secretion coupling. At synapses in the central nervous system (CNS), excitation-secretion coupling involves ion channel activation followed by vesicle fusion and neurotransmitter release. These mechanisms are altered by presynaptic neurotransmitter receptors and prominently by G protein-coupled receptors (GPCRs). Studies over the last 20-25years have revealed that acute ethanol exposure alters neurotransmitter secretion, with especially robust effects on synapses that use the neurotransmitter gamma-aminobutyric acid (GABA). Intracellular signaling pathways involving second messengers such as cyclic AMP and calcium are implicated in these acute ethanol actions. Ethanol-induced release of neuropeptides and small molecule neurotransmitters that act on presynaptic GPCRs also contribute to presynaptic potentiation at synapses in the amygdala and hippocampus and inhibition of GABA release in the striatum. Prolonged exposure to ethanol alters neurotransmitter release at many CNS GABAergic and glutamatergic synapses, and changes in GPCR function are implicated in many of these neuroadaptations. These presynaptic neuroadaptations appear to involve compensation for acute drug effects at some synapses, but "allostatic" effects that result in long-term resetting of synaptic efficacy occur at others. Current investigations are determining how presynaptic neuroadaptations contribute to behavioral changes at different stages of alcohol drinking, with increasing focus on circuit adaptations underlying these behaviors. This chapter will discuss the acute and chronic presynaptic effects of ethanol in the CNS, as well as some of the consequences of these effects in amygdala and corticostriatal circuits that are related to excessive seeking/drinking and ethanol abuse.

Pharmacological characterization of Cirsium ligulare Boiss. (Asteraceae) herb decoction

Background/Aim. Data on phytochemical and pharmacological investigations of genus Cirsium Mill. (Asteraceae) are scarce. Some data suggest that decoctions or infusions prepared from these plants are used in folk medicine as tonics, particularly in inflammatory, liver and stomach diseases. So far there have been no pharmacological investigations related to Cirsium ligulare (C. ligulare) Boiss. Accordingly, the aim of this study was to estimate antioxidative, anti-inflammatory and gastroprotective activities of aqueous extracts of C. ligulare herb prepared as 5% and 10% decoctions. Methods. Antioxidative activity was determined using the method of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging. Investigations of anti-inflammatory (a model of systemic inflammatory response induced by endotoxin of Escherichia coli and carrageenan-induced rat paw oedema model for local inflammatory response), as well as gastroprotective effects (a model of stress-ulcer induced by absolute ethanol), were conducted in adult female Wistar rats that were given the aqueous extracts of C. ligulare herb per os. Indomethacin and ranitidine were used as reference drugs for evaluation of local anti-inflammatory and gastroprotective effects, respectively. Results. The results demonstrated that aqueous extracts of C. ligulare herb produced strong antioxidative activity, diminished body weight loss induced by endotoxin, significantly reduced carrageenan-induced paw oedema, and prevented the ulcerogenic action of absolute ethanol. Both anti-inflammatory and gastroprotective activities of the extract tested were comparable to those of the reference drugs. Conclusion. Presented results justify the traditional use of C. ligulare herb decoctions and further phytochemical and pharmacological investigations are warranted.

Lithium ascorbate as a protector of human blood biomolecules under ethanol impact

Background: Alcoholism is an acute social problem worldwide for negative impact on health. This problem is closely associated with social stress and drinking traditions. Previously, it was established exhaustion of blood antioxidant in alcoholism patients. It requires development and investigation of substances with protective properties under ethanol impact, on both cells and biomolecules. Aims and Objectives: Lithium salts are widely used in medicine as mood stabilizers for mental pathologies, including alcoholism. In this work, we investigate lithium ascorbate for the protection of human blood plasma lipids and proteins under ethanol impact. Materials and Methods: Well-known antioxidants - carnosine and ascorbic acid - were used as reference drugs. Heparinized venous blood was used as an experimental substrate. The ethanol and tested substances in the form of water (physiological)solutions were added to blood in vitro. Protection effects were measured and estimated as concentrations of carbonyls proteins and products of lipid peroxidation in blood plasma. Results: It was shown similar protective action of lithium ascorbate and carnosine on human plasma biomolecules against damaging action of ethanol. No protective effect revealed for ascorbic acid. Conclusion: Lithium ascorbate and carnosine were proved to possess the protection effects for blood lipids and proteins under ethanol impact. Normothymic and bloodprotective properties are desirable for psychotropic drugs, and thus, lithium salts could be considered as prospective agents in the treatment of addictions and other pathologies associated with oxidative stress.