B.C. Shanley

Antibodies against acetaldehyde-modified epitopes: an elevated IgA response in alcoholics

Abstract. Several recent reports have shown that antibodies reactive with acetaldehyde (AcH)‐modified epitopes are present in alcoholics. However, similar antibodies have also been found in patients with nonalcoholic liver disease and control subjects. In each of these studies total immunoglobulin binding to the AcH‐modified proteins was measured, with no attempt being made to identify the classes of immunoglobulin involved. In the present study we employed an enzyme‐linked immunosorbent assay (ELISA) to assess the classes of immunoglobulin involved in this response, using plasma samples from 97 alcoholics with varying degrees of liver disease, 35 patients with non‐alcoholic liver disease and 33 control subjects. All three groups exhibited a large IgM response and a negligible IgG response. However, the alcoholics exhibited a significantly higher IgA response than either of the other groups. This suggests that the measurement of the IgA response to AcH‐modified epitopes may be a specific marker of ethanol abuse.

Acute administration of ethanol suppresses pentylenetetrazole-induced c-fos expression in rat brain

The effect of acute ethanol administration on pentylenetetrazole-induced c-fos expression in rat brain was studied. Pentylenetetrazole induced the rapid and transient expression of c-fos mRNA in rat brain. Maximal induction at a dose of 30 mg/kg was detected within 30 min and persisted for 60 min. Thereafter c-fos gene expression decreased to control levels by 180 min. No increase in c-fos mRNA was evident at doses of pentylenetetrazole < or = 20 mg/kg, whereas maximal elevation was seen at 30 or 40 mg/kg. This action was inhibited by acute ethanol treatment (blood alcohol level > or = 100 mg/dl). Acute ethanol treatment alone had no effect on c-fos gene expression.

Effects of chronic ethanol inhalation on the enhancement of benzodiazepine binding to mouse brain membranes by GABA.

Chronic ethanol inhalation produced no change in the number or affinity of [(3)H]flunitrazepam binding sites on well-washed synaptic membranes prepared from male Quackenbush mice, but produced a significant decrease in the capacity of GABA to enhance [(3)H]flunitrazepam binding. This decrease was characterised by a higher EC(50) (1.4 ? M compared to 0.6 ? M) and a lower maximal level of enhancement (162% compared to 172%) for tissue from the chronically treated animals compared to tissue from control animals. Acute ethanol treatment or ethanol incubated in vitro with the brain membranes did not produce changes in any of the [(3)H]flunitrazepam binding parameters. These results support other findings that chronic ethanol may affect the coupling of various sites on GABA-A receptor-ionophore complexes in brain.

Ethanol and protein kinase C in rat brain

The effect of chronic ethanol consumption on the catalytic activity of protein kinase C isolated from rat brain was studied in two different ways. Enzyme activity was first measured by phosphorylation of Histone IIIS in vitro. There was no change in the activity of the cytosolic enzyme. Membrane-associated enzyme activity was reduced in the ethanol-treated animal. This difference was not evident if the enzyme was stimulated by arachidonate. The reduction in enzyme activity was confirmed by analysis of the phosphorylation of endogenous substrates in intact synaptosomes. When the binding of the ligand [3H]phorbol dibutyrate was measured by quantitative autoradiography, increased binding to membrane-associated protein kinase C was observed in the CA1 region of the hippocampus but not in other brain regions. These results indicate that ethanol treatment results in a general reduction in membrane-associated protein kinase C activity as measured in vitro but the effect may not be consistent in all brain regions. The differential effect in the CA1 region of the hippocampus may be a reflection of a disruption in the normal regulation of protein kinase C activity in this area and may indicate that this region is a sensitive target for the action of ethanol.

Effects of acetaldehyde on polymerization of microtubule proteins

The in vitro effects of ethanol and acetaldehyde on polymerization of calf brain microtubular proteins (MTP) were examined. While ethanol up to 100 mM had no effect on the polymerization of MTP, acetaldehyde above 0.5 mM had an inhibitory effect. This effect was not dependent on the presence of microtubule-associated proteins (MAPs), since acetaldehyde had a similar effect on the polymerization of highly purified tubulin. Electron microscopy revealed that the number and the length of microtubules at equilibrium was reduced by the presence of acetaldehyde. Acetaldehyde raised the critical concentration for tubulin assembly and caused greater inhibition at lower tubulin concentrations. Acetaldehyde augmented the depolymerizing effects of Ca2+ on preassembled microtubules. In addition, acetaldehyde itself caused depolymerization of microtubules but only in the absence of MAPs. Long-term (19.5 h) incubation of MTP with acetaldehyde led to significant loss of polymerization ability which could not be reversed by removal of acetaldehyde. This loss of activity was apparently independent of the observed formation of reducible adducts between acetaldehyde and MTP.

Effects of chronic ethanol exposure on the gaba-benzodiazepine receptor complex in rat brain.

Chronic treatment of male Wistar rats with ethanol by inhalation did not affect the binding of [(3)H]flunitrazepam, [(3)H]GABA or [(3)H]muscimol to extensively washed synaptic membranes. Neither the affinity (K(d)) nor the number of binding sites (Bmax) for these ligands was changed. However, GABA enhancement of [(3)H]flunitrazepam binding was significantly decreased by approx. 40% in ethanol-treated animals (172% compared to 215%). Acute treatment with ethanol did not produce changes in the binding of [(3)H]flunitrazepam or [(3)H]muscimol. These findings suggest that chronic ethanol treatment leads to uncoupling of the various receptor sites on the GABA-benzodiazepine receptor ionophore-complex in the brain.

FOS and JUN as markers for ethanol-sensitive pathways in the rat brain

The expression of proteins coded by the immediate early genes of the fos family and c-jun was used to study the effect of acute ethanol administration on convulsant-induced neuronal activity in rat brain. Immunoreactivity for both types of protein was induced by either SC injection of pentylenetetrazole or by IP injection of N-methyl-D-aspartic acid. Both agents elicited distinct patterns of behaviour and a high level of FOS-immunoreactivity in the cerebral cortex and hippocampus. Acute IP doses of ethanol (1.0-3.0 g/kg) significantly reduced the behaviours and FOS-immunoreactivity induced in the cerebral cortex by both pentylenetetrazole and N-methyl-D-aspartic acid. Pentylenetetrazole-induced FOS-immunoreactivity in the hippocampus was also inhibited by ethanol. In contrast, N-methyl-D-aspartic acid-induced FOS-immunoreactivity in the hippocampus was not inhibited by any dose of ethanol. c-JUN immunoreactivity showed a distinct pattern of induction in the hippocampus after injection of N-methyl-D-aspartic acid. Ethanol (3.0 g/kg) inhibited N-methyl-D-aspartic acid-induced c-JUN-immunoreactivity in the hippocampus and cerebral cortex. The differences in inhibition of immunoreactivity suggest that the sensitivity of the NMDA- and GABAA-related neuronal pathways to ethanol varies among different anatomical structures.

The effect of chronic ethanol consumption on muscarinic receptors in rat brain

Ethanol (15% v/v) was administered in the drinking water to male Wistar rats over period of 3 months. Binding properties of muscarinic receptors were studied in synaptosomes from selected brain areas using [(3)H]quinuclidinyl benzilate and its displacement by the selective antagonist, pirenzepine and the agonist, carbachol. Dissociation constants (K(d)) of all three ligands in the cerebral cortex, hippocampus and striatum of ethanol-treated groups did not differ from those in controls. Density of [(3)H]quinuclidinyl benzilate binding sites in the cortex of ethanol-treated animals was approx. 50% higher than in controls (2.06 +/- 0.2 and 1.32 +/- 0.2 pmol/mg of protein respectively, mean +/- SD, n = 6, P < 0.001). This was largely attributable to an increase in M(1) binding sites as shown by pirenzepine displacement studies. In the hippocampus and striatum binding capacity of muscarinic receptors was not affected by ethanol treatment. Synthesis of acetylcholine in cerebral cortex prisms from ethanol-treated animals was not inhibited under resting conditions, but stimulation of synthesis by high K(+) concentration was significantly altenuated by comparison with controls. These results suggest that chronic ethanol consumption induces changes in cholinergic neurotransmission in selected brain areas.

Chronic ethanol administration alters binding of [35S]t-butylbicyclophosphorothionate to the GABA-benzodiazepine receptor complex in rat brain

Chronic treatment of male Wistar rats with ethanol (15% v/v) in drinking fluid for a period of 3 months affected the binding of the chloride channel antagonist, [(35)S]TBPS, to well-washed synaptic membranes and slide-mounted brain slices, the affinity for [(35)S]TBPS in brains of ethanol-treated animals was significantly decreased in comparison to controls while receptor density was increased (P < 0.001). However, other well described treatments, viz. an ethanol-containing liquid diet and chronic inhalation of ethanol failed to demonstrate changes in the binding of [(35)S]TBPS in brain preparations. Our findings suggest that long-term administration of ethanol can induce alterations in the characteristics of the ionophore component of the GABA-benzodiazepine receptor complex. This may have relevance to ethanol-induced neuronal damage.

Autoradiographic studies on the effect of ethanol on muscarinic receptors in rat brain

The effect of ethanol vapour inhalation on the binding characteristics of muscarinic receptors in rat brain has been studied using quantitative autoradiography. After 14 days in ethanol vapour there was increased binding of [(3)H]QNB to the cortex of 4- and 6-week-old animals, but not 11-week-old animals, and to the hippocampus of the 6-week-old animals. Displacement studies indicated that the effect of ethanol was due to an increase in the density of low-affinity carbachol binding sites. There was no effect on the binding of [(3)H]QNB in the presence of pirenzipine. The data strongly suggest that the increase in binding after ethanol exposure is due to an increase in M(1) sites in the younger animals possibly linked to age-related plasticity of the nervous system.