Brian C. Shanley

Increased NMDA-induced excitability during ethanol withdrawal: a behavioural and histological study

Intrahippocampal injections of N-methyl-D-aspartic acid (NMDA) leads to neurodegeneration in a dose-dependent manner. Chronic administration of ethanol to animals leads to CNS tolerance and dependence. Hyperexcitability following ethanol withdrawal is thought to be related to increased sensitivity of the NMDA receptors. The purpose of this study was to investigate this predisposition to hyperexcitability by intrahippocampal injection of low dose of NMDA. Using control and ethanol-withdrawn male Wistar rats, behavioural indices were determined immediately after injection and morphological damage was assessed after a period of recovery. There was significantly increased hyperactivity in the ethanol-treated rats immediately after injection. Morphological damage resulting from 5 nmol of NMDA was significantly greater in the CA3 region of the hippocampus in these animals. These data support the hypothesis that ethanol dependence and subsequent withdrawal is associated with increased sensitivity to NMDA which may underlie ethanol withdrawal-associated brain damage.

Increased sensitivity of the hippocampus in ethanol-dependent rats to toxic effect of N-methyl-D-aspartic acid in vivo

Chronic administration of ethanol in animals leads to CNS tolerance and physical dependence. Subsequent withdrawal of ethanol causes hyperexcitability which is thought to be related to increased sensitivity of N-methyl-D-aspartic acid (NMDA) receptors. The purpose of this study was to investigate sensitivity to NMDA in ethanol-treated animals by detecting damage after intrahippocampal injection of NMDA. Choline acetyltransferase (ChAT) and glutamate decarboxylase (GAD) specific activity was used as markers of cholinergic and gamma-aminobutyric acid neurons, respectively. Ethanol-dependent animals were more liable to die following intrahippocampal injection of either 120 or 240 nmol of NMDA. There was a significantly greater decrease in hippocampal GAD but not ChAT specific activity in the surviving animals. These data support the hypothesis that ethanol dependence is associated with increased sensitivity to NMDA which may be responsible for excitotoxic brain damage and death.

Ethanol increases synaptosomal free calcium concentration.

The effect of ethanol on synaptosomal free calcium concentration [( Ca2+]i) and 45Ca2+ uptake was studied in vitro. Synaptosomes were prepared from forebrains of adult rats and incubated in the presence of ethanol 50-500 mM. [Ca2+]i determined by means of the Ca2+-sensitive dye, fura-2, was increased by ethanol in polarized and depolarized synaptosomes, whereas 45Ca2+ uptake was decreased. The results suggest that ethanol may influence neuronal calcium homeostasis by increasing rather than decreasing [Ca2+]i.

Immediate Early Gene Expression in the Rat Brain during Ethanol Withdrawal.

The temporal and spatial expression of three immediate early genes was investigated at the level of both mRNA and protein in the brains of rats undergoing ethanol withdrawal. Animals were made dependent by chronic vapor inhalation. All animals showed behavioral signs of withdrawal between 8 and 17 h after removal from ethanol vapor. A large, transient increase in the expression of whole brain c-fos, c-jun, and zif/268 mRNA was observed 12 h after withdrawal, and expression of their protein products was detected 15 to 24 h after withdrawal. Spatial variation in the expression of each protein was detected. All three proteins were present in the cerebral cortex, the olfactory bulb, the inferior colliculus, the granular cell layer of the cerebellum, and in the brain stem, but only C-JUN and ZIF/268 were detected in the hippocampus of animals undergoing withdrawal without overt seizures. C-FOS was detected in the hippocampus only in animals with overt seizures. These data reveal a complex pattern of immediate early gene expression during ethanol withdrawal, which may be associated with changes in neuronal plasticity and/or cell repair.

The formation and stability of imidazolidinone adducts from acetaldehyde and model peptides : A kinetic study with implications for protein modification in alcohol abuse

The kinetics of the reaction of acetaldehyde (AcH) with the alpha-amino group of several di- and tripeptides to form 2-methylimidazolidin-4-one adducts were determined at pH 7, 4, 37 degrees C, using reverse phase HPLC to separate peptides from adducts. The imidazolidin-4-one structure of the adducts was confirmed by 13C NMR spectroscopy. The reaction of val-gly-gly with AcH was shown to follow second-order kinetics over a wide range of concentrations of both reactants, with k2 = 0.734 +/- 0.032 M(-1) min(-1). Under conditions similar to those in the liver of an alcoholic during chronic ethanol oxidation ([Ach]o = 50-910 microm; [free peptide alpha-amino groups]o = 1.5 mM), the reaction proceeded until effectively all of the AcH had been consumed. The side chain of the N-terminal amino acid was shown not to have a marked effect on the rate of imidazolidinone formation. The decomposition of the imidazolidinone adduct of val-gly-gly and AcH was observed at 60-100 degrees C. Extrapolation of an Arrhenius plot to 37 degrees C provided an estimate of K(obs) of 0.002 h-1 (t1/2 approximately 14 days). Based on these kinetic studies, it is concluded that imidazolidinone adducts of AcH with proteins may be present in the liver and, possibly, in the blood of alcoholics.

Ethanol and synaptosomal calcium homeostasis

The effect of ethanol on synaptosomal calcium homeostasis was studied in the rat using the fluorescent dye, fura-2, and 45Ca uptake. The mitochondrial poison, cyanide, caused a substantial rise in intracellular free Ca2+ concentration, [Ca2+]i, over that of control synaptosomes. This rise was enhanced by ethanol. Ethanol also augmented the rise in [Ca2+]i induced by ouabain, indicating that modulation of Na(+)-Ca2+ exchange is probably not the underlying mechanism. The Ca2+ channel blockers, verapamil and La3+, also failed to inhibit the rise in [Ca2+]i caused by ethanol. Preincubation of synaptosomes with caffeine, however, caused a significant decrease in the rise of [Ca2+]i due to ethanol, suggesting that ethanol exerts effects on Ca2+ homeostasis at the level of the endoplasmic reticulum.

The reaction of acetaldehyde with brain microtubular proteins: formation of stable adducts and inhibition of polymerization

Stable adducts were formed by treatment of bovine brain microtubular proteins (MTP) with acetaldehyde, followed by gel filtration to remove excess acetaldehyde. The extent of stable adduct formation was determined using [14C]acetaldehyde and was correlated with acetaldehyde concentration and reaction time. Significant inhibition of MTP polymerization was observed at adduct concentrations of 0.6 mol acetaldehyde/mol tubulin dimer. The data suggest that repeated exposure of MTP to low concentrations of acetaldehyde, as would occur in the brain and other tissues of alcoholics, may inhibit MTP polymerization with neurological consequences.

Studies on regenerating liver and hepatoma plasma membranes—i. lipid and protein composition

1. Plasma membranes were isolated from normal liver, Morris hepatoma 7288C and regenerating liver, 6, 15, 24, and 48 hr after partial hepatectomy. 2. The cholesterol/phospholipid ratio was lower in regenerating liver 6 hr after partial hepatectomy (0.51) compared to the sham control (0.68), returning to normal after 15 hr. This was accompanied by a small increase in palmitic acid (16:0). There were no other changes in the lipid composition in regenerating hepatocytes in the first 48 hr after partial hepatectomy. 3. Analysis of lipid composition showed a higher cholesterol/phospholipid ratio in the hepatoma plasma membrane compared to normal liver accompanied by an increase in saturation of the fatty acyl groups of the phospholipids. There were also significant changes in the phospholipid classes. 4. There was no change in the two-dimensional electrophoretic profile of membrane proteins in the early stages of liver regeneration, however hepatoma membranes showed significant differences in protein profile. 5. These changes in the lipid composition of the hepatoma plasma membrane would have the effect of decreasing the average fluidity of the membrane and together with the changes in protein composition may be significant in the altered growth of the hepatoma. Changes in the lipid composition of the hepatocyte plasma membrane early in liver regeneration may reflect the onset of renewed cell division.

Studies on regenerating liver and hepatoma plasma membranes—ii. membrane fluidity and enzyme activity

1. Rat hepatocyte plasma membranes isolated from Morris hepatoma 7288C, normal and regenerating liver were labelled with the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene. 2. Steady-state fluorescence polarisation measurements indicated an increased fluidity of the membranes in the early stages of regeneration, returning to normal levels after 48 hr. 3. There was a decrease in hepatoma plasma membrane fluidity compared to normal hepatocytes. Changes in fluorescence polarisation with temperature (Arrhenius studies) indicate an increase in the lower critical temperature for the membrane lipid thermotropic transition of hepatoma compared to normal liver plasma membranes. 4. These changes in membrane lipid fluidity alter the activation of some intrinsic and extrinsic membrane bound enzymes.

Effect of ethanol on cholesterol and phospholipid composition of HeLa cells.

Chronic exposure of animals to ethanol leads to changes in membrane lipid composition which may be related to the development of tolerance and physical dependence. The object of the present study was to investigate this phenomenon at a cellular level. HeLa cells were grown in the presence of ethanol (86 mM) for periods of up to 9 days. Both the cholesterol and phospholipid concentration of these cells increased during this period but the cholesterol:phospholipid ratio remained unchanged. Among the phospholipid classes phosphatidic acid decreased while phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine increased rapidly, returning toward control values by 9 days. Significant decreases were observed in saturated (14:0, 16:0) and monoenoic (16:1, 18:1) fatty acids while the major polyenoic fatty acid (20:4) increased. It is concluded that cultured mammalian cells represent a useful model for investigation of the direct effects of ethanol on membrane lipid metabolism.