Effects of acetaldehyde on hepatocyte glycerol uptake and cell size: implication of aquaporin 9

Background: The effects of ethanol and acetaldehyde on uptake of glycerol and on cell size of hepatocytes and a role Aquaporin 9 (AQP9), a glycerol transport channel, were evaluated. Methods: The studies were done in primary rat and mouse hepatocytes. The uptake of [¹⁴C] glycerol was determined with hepatocytes in suspension. For determination of cell size, rat hepatocytes on coated dishes were incubated with a lipophilic fluorochrome that is incorporated into the cell membrane and examined by confocal microscopy. A three‐dimensional z scan of the cell was performed, and the middle slice of the z scan was used for area measurements. Results: Acute exposure to acetaldehyde, but not to ethanol, causes a rapid increase in the uptake of glycerol and an increase in hepatocyte size, which was inhibited by HgCl₂, an inhibitor of aquaporins. This was not observed in hepatocytes from AQP9 knockout mice, nor observed by direct application of acetaldehyde to AQP9 expressed in Xenopus Laevis oocytes. Prolonged 24‐hour exposure to either acetaldehyde or ethanol did not result in an increase in glycerol uptake by rat hepatocytes. Acetaldehyde decreased AQP9 mRNA and AQP9 protein, while ethanol decreased AQP9 mRNA but not AQP9 protein. Ethanol, but not acetaldehyde, increased the activities of glycerol kinase and phosphoenolpyruvate carboxykinase. Conclusions: The acute effects of acetaldehyde, while mediated by AQP9, are probably influenced by binding of acetaldehyde to hepatocyte membranes and changes in cell permeability. The effects of ethanol in enhancing glucose kinase, and phosphoenolpyruvate carboxykinase leading to increased formation of glycerol‐3‐phosphate most likely contribute to alcoholic fatty liver.     

Tolerance to disulfiram induced by chronic alcohol intake in the rat

Background: Disulfiram, an inhibitor of aldehyde dehydrogenase used in the treatment of alcoholism, is an effective medication when its intake is supervised by a third person. However, its therapeutic efficacy varies widely, in part due to the fact that disulfiram is a pro‐drug that requires its transformation into an active form and because it shows a wide range of secondary effects which often prevent the use of doses that ensure full therapeutic effectiveness. In this preclinical study in rats we report the development of tolerance to disulfiram induced by the chronic ingestion of ethanol, an additional source of variation for the actions of disulfiram with possible therapeutic significance, We also addresses the likely mechanism of this effect. Methods: Wistar‐derived rats bred for generations as high ethanol drinkers (UChB) were trained for either 3 days (Group A) or 30 days (Group B) to choose between ethanol (10% v/v) or water, which were freely available from 2 bottles on a 24‐hour basis. Subsequently, animals in both groups were administered disulfiram or cyanamide (another inhibitor of aldehyde dehydrogenase) and ethanol intake in this free choice paradigm was determined. Animals were also administered a standard dose of 1 g ethanol/kg (i.p) and arterial blood acetaldehyde was measured. Results: Disulfiram (12.5 and 25 mg/kg) and cyanamide (10 mg/kg) markedly inhibited ethanol intake (up to 60 to 70%) in animals that had ethanol access for only 3 days (Group A). However both drugs were inactive in inhibiting ethanol intake in animals that had consumed ethanol for 30 days (Group B). Following the injection of 1 g ethanol/kg, arterial blood acetaldehyde levels reached levels of 150 and 300 μM for disulfiram and cyanamide respectively, values which were virtually identical regardless of the length of prior ethanol intake of the animals. Conclusions: Chronic ethanol intake in high‐drinker rats leads to marked tolerance to the aversive effects of disulfiram and cyanamide on ethanol intake despite the presence of consistently high levels of blood acetaldehyde. These findings may have implications for the use of disulfiram for the treatment of alcoholism in humans.     

A Placebo-Controlled Double-Blind Comparative Clinical Study of the Disulfiram- and Calcium Carbimide-Acetaldehyde Mediated Ethanol Reactions in Social Drinkers

Treatment for 2 days with disulfiram (3.5 mg/kg once daily) and calcium carbimide (0.7 mg/kg twice daily) in social drinkers produced, as compared to controls, similar blood ethanol values, 2- to 3-fold increases in blood acetaldehyde, respectively, and increased heart rate, pulse pressure, skin temperature, and flushing following 0.15 g/kg of ethanol taken 12 hr after the last drug administration. Peak blood acetaldehyde concentration was greater for calcium carbimide compared to disulfiram (p less than 0.05) and subjects treated with calcium carbimide experienced greater discomfort compared to disulfiram due to palpitations and shortness of breath, and they reported less intention to drink during the reaction. However, neither drug produced sufficient aversion to curtail further drinking totally. With repeated drinks, there was an overall reduction of blood acetaldehyde concentration for calcium carbimide of 85% and for disulfiram of 35%. These data may provide a biochemical basis for the claims of certain alcoholics that they can drink to "burn off" the effects of these drugs.     

A novel binding site for the native hepatic acute‐phase protein α1‐antitrypsin expressed on the human hepatoma cell line HepG 2 and intestinal cell line Caco 2

Abstract: Aims/Background:α1‐antitrypsin (α1‐AT) is a hepatic acute phase protein which predominantly inhibits neutrophil elastase. Besides this major function, we have also previously shown that α1‐AT markedly increased H‐ferritin mRNA expression and ferritin synthesis in the human hepatoma cell line HepG 2. These actions suggest that α1‐AT might interact with HepG 2 cells via a specific cell surface binding site. Methods and Results: Using radio‐labelled native α1‐AT, we observed saturable binding to HepG 2 cells with a dissociation constant (Kd) of 63.3±6.9 nM and a maximal density of binding sites (Bmax) of 0.34 ±0.05 pmol/10⁶ cells equivalent to 195800±29200 sites/cell. The binding of [¹²⁵I]α1‐AT was time dependent with a calculated association rate constant of 9.22±1.84×10⁴×M^?1×min^?1. Binding was highly specific since other acute phase proteins or protease inhibitors failed to block binding. Although α1‐AT‐trypsin, α1‐AT‐elastase and the pentapeptide FVYLI, the minimal binding sequence for the SEC receptor, increased [¹²⁵I]α1‐AT binding, in long term experiments these complexes failed to influence the number of α1‐AT binding sites. Specific, saturable binding of [¹²⁵I]α1‐AT was also found on the human intestinal epithelial Caco 2 cells, but not on fibroblast or leukaemic cell lines. Conclusion: These experiments demonstrate a specific, high affinity binding site for native α1‐AT on HepG 2 and Caco 2 cells, cell lines derived from tissues involved in the acute phase response.     

Effect of vasoactive intestinal peptide,somatostatin, neurotensin,cholecystokinin octapeptide,and secretin on intestinal absorption of amino acid in rat

The effects of vasoactive intestinal peptide (VIP), somatostatin (SRIF), neurotensin (NT), cholecystokinin octapeptide (CCK-8), and secretin (SEC) on the intestinal absorption of amino acid were investigated. Six groups of Wistar rats were studied: (1) controls; (2) VIP treated; (3) SRIF treated; (4) NT treated; (5) CCK-8 treated; (6) SEC treated. [³H] Leucine was given intraluminally through a cannula at the ligament of Treitz, a number of blood samples were obtained through a superior mesenteric vein catheter 1-60 min after administration of [³H]leucine, and the radioactivity of plasma was measured to evaluate the absorption of [³H]leucine. It was shown that VIP and SRIF significantly inhibited the absorption of [³H]leucine (by 59.1% and 38.7%, respectively), whereas NT, CCK-8, and SEC significantly enhanced absorption (by 44.2%, 49.6%, and 39.1%, respectively). Radioimmunoassays of VIP, SRIF, and NT showed that at least some of the hormones or peptides exerted their effects on absorption of leucine at or near their physiological concentrations.     

Alterations in Brain Glucose Utilization Accompanying Elevations in Blood Ethanol and Acetate Concentrations in the Rat

Background: Previous studies in humans have shown that alcohol consumption decreased the rate of brain glucose utilization. We investigated whether the major metabolite of ethanol, acetate, could account for this observation by providing an alternate to glucose as an energy substrate for brain and the metabolic consequences of that shift. Methods: Rats were infused with solutions of sodium acetate, ethanol, or saline containing ¹³C‐2‐glucose as a tracer elevating the blood ethanol (BEC) and blood acetate (BAcC) concentrations. After an hour, blood was sampled and the brains of animals were removed by freeze blowing. Tissue samples were analyzed for the intermediates of glucose metabolism, Krebs’ cycle, acyl‐coenzyme A (CoA) compounds, and amino acids. Results: Mean peak BEC and BAcC were approximately 25 and 0.8 mM, respectively, in ethanol‐infused animals. Peak blood BAcC increased to 12 mM in acetate‐infused animals. Both ethanol and acetate infused animals had a lower uptake of ¹³C‐glucose into the brain compared to controls and the concentration of brain ¹³C‐glucose‐6‐phosphate varied inversely with the BAcC. There were higher concentrations of brain malonyl‐CoA and somewhat lower levels of free Mg²⁺ in ethanol‐treated animals compared to saline controls. In acetate‐infused animals the concentrations of brain lactate, α‐ketoglutarate, and fumarate were higher. Moreover, the free cytosolic [NAD⁺]/[NADH] was lower, the free mitochondrial [NAD⁺]/[NADH] and [CoQ]/[CoQH₂] were oxidized and the ΔG′ of ATP lowered by acetate infusion from ?61.4 kJ to ?59.9 kJ/mol. Conclusions: Animals with elevated levels of blood ethanol or acetate had decreased ¹³C‐glucose uptake into the brain. In acetate‐infused animals elevated BAcC were associated with a decrease in ¹³C‐glucose phosphorylation. The co‐ordinate decrease in free cytosolic NAD, oxidation of mitochondrial NAD and Q couples and the decrease in ΔG′ of ATP was similar to administration of uncoupling agents indicating that the metabolism of acetate in brain caused the mitochondrial voltage dependent pore to form.     

The Effects of Chronic Ethanol Consumption on the Formation of Phosphatidylethanolamine Molecular Species and Their Appearance at the Plasma Membrane

The purpose of our study was to determine whether chronic ethanol consumption affected membrane assembly by altering the formation of specific molecular species of phosphatidylethanolamine (PE) and their subsequent incorporation into the plasma membrane (PM). We investigated the effects on the PE species made by the two major pathways in hepatocytes: (1) from CDP-ethanolamine in the endo-plasmic reticulum, and (2) by the decarboxylation of phosphatidylserine (PS) in the mitochondria. Ethanol consumption exerted significant effects on the formation of ethanolamine-derived PE species and affected mainly two species, the 16:0/22:6 and 180/20:4 species. In cultured hepatocytes from ethanol-fed rats labeled with methanolamine for 0.25 to 4 hr, the amount of the [³H]16:0/22:6 PE species was decreased compared with that in control cells, whereas the amount of [₃H]1 80/20:4 species was increased. The amount of the [³H]1 6:0/22:6 PE species on the cell surface was also decreased in hepatocytes from ethanol-fed rats, whereas the amount of [³H]18:0/20:4 species was increased. In contrast, the profile of [³H]PE species formed in cells treated with [³H]serine exhibited minor alterations, and the profile of the serine-derived [³H]PE species on the cells surface was not altered after 4 hr of labeling. The changes in ethanolamine-derived species were apparently caused by time-dependent alterations in the metabolic processes, because the presence of 110 mM ethanol in the culture media did not affect the profiles of [³H]PE species in cells from control or ethanol-fed rats and was not required to sustain the altered profiles. The results indicate that the synthesis of specific PE molecular species and their appearance on the PM may occur by compartmentalized processes which are distinguishable by different sensitivities to ethanol consumption. The results indicate that ethanol consumption may contribute alcoholic hepatic injury by interfering with the metabolism of specific PE molecular species and their assembly into the PM.     

Effect of dehydroepiandrosterone on insulin sensitivity in Otsuka Long-Evans Tokushima-fatty rats

In order to clarify the effect of dehydroepiandrosterone (DHEA) on improvement of insulin resistance, we examined the effects of overexpression of wild-type protein kinase C-ζ (wt-PKCζ)/3-phosphoinositide-dependent protein kinase-1 (wt-PDK1) and kinase-inactive PKCζ/PDK1 (ΔPKCζ/ΔPDK1) on DHEA-induced [³H]2-deoxyglucose (DOG) uptake using the electroporation method in rat adipocytes. Overexpression of wt-PKCζ and wt-PDK1 significantly increased in DHEA-induced [³H]2-DOG uptake. Wortmannin completely suppressed DHEA-induced [³H]2-DOG uptake in wt-PKCζ- and wt-PDK1-transfected adipocytes. Overexpression of neither ΔPKCζ nor ΔPDK1 increased DHEA-induced [³H]2-DOG uptake. Otsuka Long-Evans fatty rats (OLETF), animal models of type 2 diabetes, and Long-Evans Tokushima rats (LETO) as control, were treated with 0.4% DHEA for 2 weeks. Insulin-induced [³H]2-DOG uptakes, activations of PI 3-kinase and PKCζ of adipocytes were significantly increased in DHEA-treated OLETF rats. Moreover, plasma glucose levels in OLETF rats after treatment with DHEA for 2 weeks were significantly lower than treatment without DHEA, but not in LETO rats. These results indicate that DHEA treatment may improve glucose tolerance through a PI 3-kinase-PKCζ pathway and downregulates adiposity in OLETF rats.     

NMDA Receptor Binding in Adult Rat Brain after Several Chronic Ethanol Treatment Protocols

The amino acid L-glutamate is a major excitatory neurotransmitter that is involved in many CNS functions, including learning, memory, long-term potentiation, and synaptic plasticity. Acute exposures to ethanol (50 to 200 mM) have been shown to inhibit NMDA receptor responses, whereas chronic exposure to ethanol leads to adaptive supersensitivity thought to be involved in ethanol dependence and tolerance. To investigate the effects of chronic ethanol exposure on glutamate receptor density, we examined the binding of both NMDA and non-NMDA ligands in rat brain after several chronic ethanol treatment protocols using a number of different rat strains. No increases in the binding of [³H]MK-801, [³H]CGP 39653, or the polyamine specific competitive antagonist, [³H]ifenprodil, were seen after two well-used chronic ethanol treatments. These included the 2-week liquid diet developed by Frye et al. (J. Pharmacol. Exp. Ther. 216:306-314, 1981) and the 4-day binge treatment developed by Ma-jchrowicz (Psychopharmacologia 43:245-254, 1975). However, small increases in the binding of both the NMDA noncompetitive antagonist [³H]MK-801, as well as the competitive NMDA antagonist [³H]CGP 39653, were seen in select frontal brain regions after 3 weeks of the Walker-Freund chronic ethanol liquid diet When this chronic liquid diet treatment was extended to a period of 6 weeks, these increases in receptor binding were diminished to nonsignificant levels. The binding of the non-NMDA ligands [³H]AMPA and [³H]kainate were not significantly affected by either length of Walker-Freund liquid diet exposure. When rats were treated chronically with ethanol for 30 days using the paradigm developed by Tsukamoto et al. (Hepatology 5:224-232, 1985), small, but significant, increases in the binding of [³H]MK-801 were seen in the CA1 and dentate gyrus regions of the hippocampus. These studies indicate that robust increases in NMDA receptor binding do not occur with several chronic ethanol treatment protocols, and suggests that NMDA receptor supersensitivity during the development of tolerance and dependence to ethanol may not simply be due to changes in the density of NMDA receptors, but may involve other mechanisms.     

Effect of Bismuth and Nitecapone on Acetaldehyde Production by Helicobacter pylori

Salmela KS, Roine RP, Höök-Nikanne J, Kosunen TU, Salaspuro M. Effect of bismuth and nitecapone on acetaldehyde production by Helicobacter pylori. Scand J Gastroenterol 1994;29:528-531.

Background: We have recently shown that colloidal bismuth subcitrate inhibits cytosolic alcohol dehydrogenase of Helicobacter pylori as well as acetaldehyde production from excess ethanol. We now extend our studies to bismuth subsalicylate and nitecapone, a novel antiulcer agent.

Methods: Cytosol of H. pylori was incubated with 0.1% or 1% ethanol in the presence of different drug concentrations for 2 h, whereafter acetaldehyde formed was analyzed by head space gas chromatography. In addition, we incubated a culture solution containing intact bacteria with the drugs at 1% ethanol.

Results: Bismuth subsalicylate and nitecapone inhibit acetaldehyde formation from 0.1% ethanol by H. pylori cytosol at drug concentrations theoretically achievable in the stomach after intake of therapeutic doses of these drugs. Furthermore, colloidal bismuth subcitrate, bismuth subsalicylate, and nitecapone also inhibit acetaldehyde production by intact H. pylori, although rather high drug concentrations are required for this to occur.

Conclusions: Inhibition of H. pylori acetaldehyde formation may be one of the mechanisms by which bismuth and nitecapone exert their effect in the treatment of H. pylori-tented disorders.     

Alcohol metabolism in human cells causes DNA damage and activates the Fanconi anemia-breast cancer susceptibility (FA-BRCA) DNA damage response network

Background: We recently reported that exposure of human cells in vitro to acetaldehyde resulted in the activation of the Fanconi anemia–breast cancer susceptibility (FA‐BRCA) DNA damage response network. Methods: To determine whether intracellular generation of acetaldehyde from ethanol metabolism can cause DNA damage and activate the FA‐BRCA network, we engineered HeLa cells to metabolize alcohol by expression of human alcohol dehydrogenase (ADH) 1B. Results: Incubation of HeLa‐ADH1B cells with ethanol (20 mM) resulted in acetaldehyde accumulation in the media, which was prevented by co‐incubation with 4‐methyl pyrazole (4‐MP), a specific inhibitor of ADH. Ethanol treatment of HeLa‐ADH1B cells produced a 4‐fold increase in the acetaldehyde–DNA adduct and N²‐ethylidene‐dGuo and also resulted in the activation of the FA‐BRCA DNA damage response network, as indicated by a monoubiquitination of FANCD2 and phosphorylation of BRCA1. Ser 1524 was identified as 1 site of BRCA1 phosphorylation. The increased levels of DNA adducts, FANCD2 monoubiquitination, and BRCA1 phosphorylation were all blocked by 4‐MP, indicating that acetaldehyde, rather than ethanol itself, was responsible for all 3 responses. Importantly, the ethanol concentration we used is within the range that can be attained in the human body during social drinking. Conclusions: Our results indicate that intracellular metabolism of ethanol to acetaldehyde results in DNA damage, which activates the FA‐BRCA DNA damage response network.     

Microsomal Acetaldehyde Oxidation is Negligible in the Presence of Ethanol

The microsomal ethanol oxidizing system (MEOS), inducible by ethanol and acetone, oxidizes ethanol to acetaldehyde, which causes many toxic effects associated with excess ethanol. Recent studies reported that rat liver microsomes also oxidize acetaldehyde, thereby challenging the validity of the assessment of MEOS activity by measuring acetaldehyde production and suggesting that MEOS activity results in the accumulation not of acetaldehyde but, rather, of its less toxic metabolite, acetate. To address these issues, we compared both metabolic rates of ethanol and acetaldehyde and the effect of ethanol on the acetaldehyde metabolism. Liver microsomes were prepared from Sprague-Dawley rats induced either with acetone for 3 days or ethanol for 3 weeks. NADPH-dependent acetaldehyde (300 /μM) metabolism was measured in two ways: (1) by detection of acetaldehyde disappearance by headspace gas chromatography, and (2) by assessment of acetaldehyde oxidation by liquid scintillation counting of acetate formed from [1,2-¹⁴C]acetaldehyde. Ethanol (50 mM) oxidation was measured by gas chromatography. In acetone- and ethanol-induced rat liver microsomes, the acetaldehyde disappearance (p < 0.0001) and oxidation (p < 0.0001) rates were both significantly increased. The rates of acetaldehyde oxidation paralleled those of p-nitrophenol hydroxylation (r= 0.974, p < 0.0001), with a K_m of 82 ± 14 μM and a V_max of 4.8 ± 0.5 nmol/min/mg protein in acetone-induced microsomes. Acetaldehyde disappearance in acetone-induced microsomes and acetaldehyde oxidation in acetone-induced and ethanol-induced microsomes were significantly lower than the corresponding ethanol oxidation, with rates (nmol/min/mg protein) of 4.6 ± 0.6 versus 9.0 ± 0.8 (p < 0.005), 4.4 ± 0.3 versus 9.1 ± 0.5 (p < 0.0005), and 14.0 ± 0.9 versus 19.5 ± 1.8 (p < 0.05), respectively. The presence of 50 mM ethanol decreased this metabolism to 0.9 ± 0.3 (p < 0.005), 0.5 ± 0.1 (p < 0.001), and 1.8 ± 0.3 (p < 0.001), resulting in rates of acetaldehyde metabolism of only 9.8 ± 3.2%, 6.0 ± 0.5%, and 9.5 ± 1.2% (respectively) of those of ethanol oxidation. In conclusion, rat liver microsomes oxidize acetaldehyde at much lower rates than ethanol, and this acetaldehyde metabolism is strikingly inhibited by ethanol. Accordingly, acetaldehyde formation provides an accurate assessment of MEOS activity. Furthermore, because acetaldehyde production vastly exceeds its oxidation, the net result of MEOS activity is the accumulation of this toxic metabolite.     

Muscarinic Receptor Binding and Plasma Drug Concentration after the Oral Administration of Propiverine in Mice

Objectives: The current study was undertaken to characterize the binding of propiverine to muscarinic receptors in mouse tissues by measuring plasma concentrations of the drug and its metabolite. Methods: At 0.5–24 h after the oral administration of propiverine at pharmacologically relevant doses, muscarinic receptors in tissue homogenates were measured by a radioligand binding assay using [N‐methyl‐ ³H]scopolamine (NMS), along with the drug's concentration in plasma by the liquid chromatography‐tandem mass spectrometric method. Results: In the in vitro experiments, propiverine and its metabolite 1‐methy‐4‐piperidyl benzilate N‐oxide competed with [³H]NMS for binding sites in the bladder, submaxillary gland and heart of mice in a concentration‐dependent manner. After the oral administration of propiverine, dose‐ and time‐dependent increases in the dissociation constant for specific [³H]NMS binding were observed in the bladder and other tissues of mice, indicating that orally administered propiverine and/or its metabolite undergo significant binding to muscarinic receptors in mouse tissues. A longer‐lasting binding of muscarinic receptor was seen in the bladder than in the submaxillary gland at relatively low doses of propiverine. Furthermore, the decrease in maximal number of binding sites values for [³H]NMS binding was more remarkable in the bladder than submaxillary gland of propiverine treated mice. There was a dose‐dependent rise in the plasma concentrations of propiverine and 1‐methy‐4‐piperidyl benzilate N‐oxide in mice after the oral administration of propiverine. Conclusion: The oral administration of propiverine exerts a more prominent and longer‐lasting effect in the bladder than in the submaxillary gland of mice. The N‐oxide metabolite may contribute significantly to the blockade of muscarinic receptors caused by oral propiverine.     

Ethanol Disrupts Carbamylcholine-Stimulated Release of Arachidonic Acid from Chinese Hamster Ovary Cells Expressing Different Subtypes of Human Muscarinic Receptor

Ethanol disrupts signal transduction mediated by a variety of G-protein coupled receptors. We examined the effects of ethanol on arachidonic acid release mediated by muscarinic acetylcholine receptors. Chinese hamster ovary (CHO) cells transfected with the different subtypes of human muscarinic receptors (M1 to M5) were incubated with [³H]arachidonic acid ([³H]AA) for 18 hr, washed, and exposed to the cholinergic agonist carbamylcholine for 15 min. Carbamylcholine induced [³H]AA release from CHO cells expressing M1, M3, or M5, but not M2 or M4, muscarinic receptors. Dose response curves revealed that carbamylcholine stimulated [³H]AA release by up to 12-fold with an EC₅₀ of ～0.4 μM; maximal responses were obtained with 10 μM carbamylcholine. Exposure of M1-, M3-, or M5-expressing cells to ethanol for 5 min before stimulating with carbamylcholine reduced [³H]AA release by 40 to 65%; 50% of the maximal inhibition was obtained with an ethanol concentration of 30 to 50 mM. Ethanol did not affect basal [³H]AA release measured in the absence of carbamylcholine. Dose response curves suggest that ethanol acts as a noncompetitive inhibitor of muscarinic receptor-induced [³H]AA release insofar as maximal [³H]AA release was depressed in the presence of ethanol with no apparent change in the EC₅₀ for stimulation by carbamylcholine. Exposure of CHO cells to 38 mM ethanol for 48 hr increased [³H]AA release induced by carbamylcholine without affecting basal [³H]AA release or altering the EC₅₀ for carbamylcholine. These results indicate that ethanol acutely inhibits muscarinic receptor signaling through the arachidonic acid pathway in a noncompetitive manner, but chronically enhances muscarinic signaling through the same pathway.     

Ethanol metabolism in the generation of new antigenic determinants on liver cells.

Antibodies directed against ethanol altered liver cell components have been detected in the serum of nearly 50% of patients with alcoholic liver disease although the pathogenetic mechanisms are unclear. The importance of ethanol metabolism in the generation of new antigenic determinants on liver cells was investigated by in vivo inhibition of alcohol or acetaldehyde dehydrogenase and an induced cytotoxicity assay. There was a significant reduction in cytotoxicity to hepatocytes isolated from rabbits treated with ethanol 1 g/kg when the metabolism of ethanol to acetaldehyde by alcohol dehydrogenase was inhibited. In contrast when the oxidation of acetaldehyde was inhibited by disulfiram cytotoxicity was significantly enhanced. These results show that ethanol metabolism is integral to the expression of the ethanol related determinant and suggest that an impaired ability to metabolism acetaldehyde could lead to the development of immunological reactions to alcohol altered liver membrane antigens.     

Locomotor activity induced by noncompetitive NMDA receptor antagonists versus dopamine transporter inhibitors: opposite strain differences in inbred long-sleep and short-sleep mice

Background: The actions of ethanol in the brain involve multiple neuroreceptor systems, including glutamatergic N‐methyl‐D‐aspartate receptor (NMDAR) channels. In a novel environment, both ethanol and the noncompetitive NMDAR antagonist MK‐801 stimulate locomotor activity to a lesser extent in inbred long‐sleep (ILS) mice compared with inbred short‐sleep (ISS) mice. The behaviorally activating effects of noncompetitive NMDAR antagonists are thought to involve increased monoamine neurotransmission. Thus, in this study, we sought to determine whether: (1) habituation to the behavioral environment alters the differential locomotor‐stimulant effects of noncompetitive NMDAR antagonists in ILS and ISS mice and (2) the differential behavioral sensitivity of ILS and ISS mice to noncompetitive NMDAR antagonists is mediated through direct inhibition of the dopamine transporter (DAT). Methods: Open field locomotor activity was measured following acute systemic injection of saline or drug. [³H]DA uptake parameters were determined in striatal synaptosomes prepared from drug‐naïve mice. Results: Habituation to the testing environment abolished the strain differences in saline‐induced locomotor activity. However, ethanol‐ as well as MK‐801‐treated ILS mice still exhibited reduced locomotor activity compared with ISS mice, suggesting that a drug‐environment interaction is not the primary explanation for the strain differences. The noncompetitive NMDAR antagonists phencyclidine and ketamine also induced significantly lower locomotor activity in ILS than in ISS mice. In contrast, the DAT inhibitors cocaine and GBR 12909 and the DA releaser amphetamine induced greater locomotor activity in ILS than in ISS mice, a strain difference opposite that of the noncompetitive NMDAR antagonists. Furthermore, the differential behavioral effect found with DAT inhibitors was not mediated by differences in the affinity nor number of striatal DATs between ILS and ISS mice. Conclusions: Our results support the conclusion that the differential locomotor‐stimulant effects of ethanol and noncompetitive NMDAR antagonists in ILS and ISS mice are not mediated through direct inhibition of DAT.     

Effect of Acute N-Nitro-l-Arginine Methyl Ester (L-NAME) Hypertension on Glucose Tolerance,Insulin Levels,and [3H]-Deoxyglucose Muscle Uptake

This study was conducted to test the hypothesis that acute, widespread N-nitro-l-arginine methyl ester (L-NAME) induced vasoconstriction and hypertension may affect glucose tolerance and insulin sensitivity in normal rats. Comparisons were made of blood pressure, intravenous glucose tolerance, and insulin response and [³H]-deoxyglucose tissue uptake between L-NAME and control treated rats.Chronically instrumented, awake rats were administered L-NAME (30 mg/kg) (n = 8) or saline (0.3 mL) (n = 8) intravenously. After blood pressure stabilized, a bolus injection containing glucose (300 mg/kg) and trace [³H]-deoxyglucose was administered. Arterial blood was sampled for evaluation of glucose tolerance, insulin response, and [³H]-deoxyglucose muscle uptake.L-NAME treated rats had a persistent 54 ± 4 mm Hg blood pressure rise while fasting, and postload plasma glucose and insulin responses did not differ, nor did heart and striated muscle [³H]-deoxyglucose uptake differ.In conclusion, acute L-NAME induced hypertension does not result in glucose intolerance, hyperinsulinemia, or decreased [³H]-deoxyglucose muscle uptake.     

Influence of Serum in Hemodialysis Patients on the Expression of Intestinal and Hepatic Transporters for the Excretion of Pravastatin

It is known that the lipid‐lowering agent pravastatin, which is not metabolized by cytochrome P450, is eliminated as an unchanged drug in bile and urine. It is interesting to note that the non‐renal clearance of pravastatin in end‐stage renal failure patients is decreased compared with that of healthy volunteers. This study investigated the influence of uremic serum and toxins on the transport mechanisms of pravastatin to elucidate the cause of decreased non‐renal clearance in end‐stage renal failure patients. Caco‐2 and Hep3B cells were used as models of intestinal epithelial cells and hepatocytes respectively. Normal and uremic serum were deproteinized by treatment with methanol. 3‐Carboxy‐4‐methyl‐5propyl‐2‐furanpropanoic acid (CMPF), hippuric acid, indole‐3‐acetic acid, 3‐indoxyl sulfate, and p‐cresol were chosen as uremic toxins. Uremic serum‐treated Caco‐2 cells exhibited significantly increased accumulation of pravastatin and significantly decreased expression of MRP2 mRNA compared with normal serum‐treated Caco‐2 cells. In addition, the expression of MRP2 mRNA tended to decrease in cells treated with CMPF, indole‐3‐acetic acid, or 3‐indoxyl sulfate. Uremic serum‐treated Hep3B cells showed a significantly decreased initial uptake rate of pravastatin; furthermore, the expressions of OATP1B1 and OATP2B1 mRNA were decreased compared to normal serum‐treated Hep3B cells. These results suggest that the decrease in the non‐renal clearance of pravastatin in end‐stage renal failure patients is partly induced by the downregulation of intestinal MRP2 and hepatic OATP1B1 and/or OATP2B1 by various uremic toxins in end‐stage renal failure patients.     

Reliability of the [13C]‐acetate breath test in the measurement of gastric emptying of ethanol solutions: a methodological study

Background: The [ ¹³ C]‐acetate breath test is a clinically well‐established test for measuring gastric emptying of liquids and correlates significantly with scintigraphy. However, no studies have been undertaken to examine the relationship between gastric emptying measured by the [ ¹³ C]‐acetate breath test and ultrasonography. Furthermore, it is not known how ethanol may affect gastric emptying as measured by the [ ¹³ C]‐acetate breath test. This is particularly important because of the additional steps of absorption, metabolism and exhalation of the tracer involved in the [ ¹³ C]‐acetate breath test compared to the simple measurement of gastric emptying by ultrasonography. The aim of the present study was to examine the relationship between the gastric half emptying times measured by the [ ¹³ C]‐acetate breath test and by ultrasonography and to determine the effect of ethanol on the comparability between both methods. Methods: On separate days, 10 healthy, fasted randomly selected subjects received the following solutions by gastric tube: 500?mL water, 4% and 10% (v/v) ethanol, 5.5% and 11.4 % (w/v) glucose. Gastric half emptying times of the test solutions were simultaneously determined using the [ ¹³ C]‐acetate breath test and ultrasonography of the fundus and the antrum. Results: The gastric emptying rates determined by ultrasonography of the fundus correlated significantly with the results obtained using the [ ¹³ C]‐acetate breath test for all test solutions (r?=?0.64–0.92, P?r?=?0.94, P?=?0.001) was used. The difference between the mean half‐emptying times obtained using the [ ¹³ C]‐acetate breath test and ultrasonography of the fundus was 55?±?1.8?min. This difference was significantly longer when 4% and 10% (v/v) ethanol solutions were given (61.2?±?3.3 and 71.2?±?2.9?min) compared to water (P?Conclusions: A good correlation was found between gastric emptying as measured by the [ ¹³ C]‐acetate breath test and ultrasonography of the fundus. Measurements that were obtained using the [ ¹³ C]‐acetate breath test demonstrate a longer gastric half emptying time compared with those obtained using ultrasonography. This difference is even more marked when ethanol solutions are used. Presumably, this is because ethanol affects the absorption and/or the hepatic metabolism of the tracer. The [ ¹³ C]‐acetate breath test is therefore not a reliable gastric emptying test for comparison of different solutions, especially when ethanol‐containing liquids are used.     

Characterization of the High Affinity [H]Nociceptin Binding Site in Membrane Preparations of Rat Heart: Correlations with the Non-opioid Dynorphin Binding Site

The binding parameters of [³H]nociceptin were examined in membrane preparations of rat heart and compared with those of [³H]dynorphin A-(1-13) ([³H]Dyn A-(1-13)). Scatchard analysis of [³H]nociceptin binding revealed the presence of two distinct sites: a high affinity (K_d: 583 nm) low capacity (B_max: 132 pmol/mg protein) site and a low affinity (K_d: 10 316 nm) high capacity (1552 pmol/mg protein) site. Dyn A and related peptides were potent competitors of the binding to the high affinity site with the following rank order of potencyα-neo-endorphin>Dyn A-(2-13)=Dyn A-(3-13)>Dyn A-(5-13)>Dyn A-(1-13)>Dyn A>Dyn B>Dyn A-(6-10)>>Dyn A-(1-8). Nociceptin was 6.7 times less potent than Dyn A with a K_iof 4.8μmas compared with 0.72μmfor Dyn A. The order of potency of the various peptides in inhibiting [³H]nociceptin binding correlated well (r=0.93) with their ability to compete with the binding of [³H]Dyn A-(1-13) (Dumont and Lemaire, 1993). In addition, the high affinity [³H]nociceptin and non-opioid [³H]Dyn A-(1-13) sites were both sensitive to NaCl (120 mm) and the phospholipase C (PLC) inhibitors, U-73122 and neomycin (100μm). The binding activities were less affected by the weak PLC inhibitor, U-73343, and no effect was observed with the non-hydrolysable GTP analogs, Gpp(NH)p and GTP-γ-S. Nociceptin (1–50μm) was also shown to inhibit the uptake of [³H]noradrenaline ( [³H]NA) by cardiac synaptosomal preparations. In spontaneously hypertensive rats (SHR), the potency of nociceptin in inhibiting [³H]NA uptake was increased by 1.6-fold as compared with Wistar Kyoto (WKY) control rats and such effect was accompanied by comparable increased levels of cardiac ORL₁mRNA and [³H]nociceptin high affinity sites. These changes correlated well with the previously observed increased levels of non-opioid cardiac [³H]Dyn A-(1-13) sites in SHR (1.3 times as compared with WKY) and increased potency of Dyn A-(1-13) in inhibiting [³H]NA uptake by cardiac synaptosomes in SHR (2.2-fold as compared with WKY) (Dumont and Lemaire, 1995). The results demonstrate that in rat heart the characteristics of the high affinity, low capacity [³H]nociceptin binding site are similar to those of the non-opioid Dyn binding site. The stimulation of this site by nociceptin, Dyn A or related peptides is more likely to produce a modulation of PLC activity and [³H]NA uptake and may participate to the pathophysiology of hypertension.