Dopamine D2-receptor gene polymorphisms in Scandinavian chronic alcoholics.

Alterations in the dopamine system have been hypothesized as a predisposing factor in alcoholism. The presence of the TaqI A1 and B1 alleles adjacent to the dopamine D 2-receptor gene (DRD2) was studied in Scandinavian alcoholic inpatients (n = 74), alcoholics autopsied at a forensic clinic (n = 19) and controls (n = 81). There were no significant differences between controls and the alcoholics, but a tendency of increased DRD2 TaqI A1 or B1 allele frequencies in alcoholic groups selected for severity (i.e. severity according to DSM-III-R criteria, early onset or severe medical complications due to alcohol abuse) and decreased frequencies in the corresponding less severe alcoholic group. The present study does not yield evidence for the hypothesis of an association between the DRD2 TaqI A1 or B1 alleles and alcoholism.

Norepinephrine metabolite in CSF correlates with ethanol consumption and heredity in humans.

Previous studies showed that in cerebrospinal fluid (CSF) the concentration of 3-methoxy-4-hydroxy-phenylglycol (MHPG), the main metabolite of norepinephrine (NE), was positively correlated with blood ethanol concentrations in both healthy volunteers and in alcoholics. In this preliminary study we have extended those results by correlating MHPG concentrations in CSF with reported ethanol consumption and other indices of alcohol problems before and after consumption of 60-120 g of ethanol. MHPG in CSF correlates negatively with reported ethanol consumption, presence of first-degree relatives with alcohol problems, and presence of memory lapses, and correlates positively with age and the amount of ethanol consumed in the experiment. These results suggest that MHPG may indicate not only a high alcohol consumption but also a familial or genetic predisposition for alcoholism.

Brain regional specificity and time-course of changes in the NMDA receptor-ionophore complex during ethanol withdrawal.

Previous work, using membrane receptor binding techniques, demonstrated an increase in hippocampal MK-801 binding sites in mice after chronic ethanol ingestion. The current studies, using quantitative autoradiography, demonstrate that chronic ethanol ingestion also produces increases in MK-801 binding in cerebral cortex, striatum and thalamus, as well as in hippocampus. The persistence of changes in MK-801 binding paralleled the time-course for ethanol withdrawal seizure susceptibility. These results support the hypothesis that an increase in the number of NMDA receptor/channel complexes in hippocampus, and possibly other brain regions, plays a role in the generation or expression of ethanol withdrawal seizures.

NMDA receptors in mice bred to be prone or resistant to ethanol withdrawal seizures.

Selective breeding has produced replicate lines of mice that are prone (WSP) or resistant (WSR) to ethanol withdrawal seizures. Ethanol-naive WSP mice inherently have a greater number of hippocampal binding sites for the NMDA receptor-gated ion channel blocker, MK-801, than ethanol-naive WSR mice. After chronic ethanol ingestion, hippocampal (but not cerebral cortical) MK-801 binding sites increase in both lines of mice. However, the number of MK-801 binding sites in the ethanol-treated WSR mice does not exceed the number of MK-801 binding sites in untreated WSP mice. At the time of ethanol withdrawal, the number of hippocampal MK-801 binding sites in each line of WSP mice is 50-70% higher than the number of such sites in WSR mice. Given the past evidence for a role of the NMDA receptor in seizures, the results implicate hippocampal NMDA receptor-gated channels in the generation of ethanol withdrawal seizures.

Ethanol and the NMDA receptor.

The actions of glutamate, the major excitatory amino acid in the CNS, are mediated by three receptor subtypes: kainate, quisqualate and N-methyl-D-aspartate (NMDA) receptors. Ethanol, in vitro, is a potent and selective inhibitor of the actions of agonists at the NMDA receptor. Following chronic ethanol ingestion, the number of NMDA receptor-ion channel complexes in certain brain areas is increased. This increase may contribute to the generation of ethanol withdrawal seizures, since administration of an NMDA receptor antagonist can reduce these seizures. The results suggest that certain acute behavioral effects of ethanol, such as effects on memory, as well as certain aspects of ethanol withdrawal, may involve the NMDA receptor.

Ethanol withdrawal seizures and the NMDA receptor complex.

Prior biochemical and electrophysiological studies have shown that low doses of ethanol inhibited calcium influx through the N-methyl-D-aspartate (NMDA) receptor/ionophore. The present data show that chronic ethanol treatment results in an increase in the number of NMDA receptor/ionophore complexes in the hippocampus, a brain area known to be associated with ethanol withdrawal seizure activity. Treatment during withdrawal with NMDA-exacerbated handling induced withdrawal seizures in the ethanol-dependent mice, while administration of the NMDA receptor-associated calcium channel antagonist MK-801 decreased the occurrence and severity of the withdrawal seizures in a dose-dependent manner. The results are consistent with the hypothesis that the up-regulation of the NMDA receptor systems following chronic ethanol treatment may mediate the seizures associated with ethanol withdrawal in dependent animals.

Brain forskolin binding in mice dependent on and tolerant to ethanol.

Chronic ethanol ingestion by mice was previously shown to result in decreased activation of adenylate cyclase by guanine nucleotides and beta-adrenergic agonists, and in the loss of the high affinity beta-adrenergic agonist binding site in frontal cortex and hippocampus but not in cerebellum. These results indicate a regional specificity of ethanol's actions on beta-adrenergic receptors, the guanine nucleotide binding protein (Gs) and/or adenylate cyclase. To further detail the anatomical specificity of the effects of ethanol ingestion on receptor-coupled adenylate cyclase (AC) systems we have quantified the binding of [3H]forskolin to brain sections of control and ethanol-fed mice. High-affinity forskolin binding, thought to represent the complex of the alpha-subunit of Gs (as) and AC, was decreased in several brain areas including frontal cortex and hippocampus, but not in cerebellum, nucleus accumbens and certain other brain areas of ethanol-fed mice. Guanine nucleotides, such as Gpp(NH)p, generally enhanced forskolin binding in control animals. In ethanol-fed mice, however, Gpp(NH)p failed to enhance forskolin binding in most brain regions. These findings suggest that chronic ethanol ingestion may decrease the amount or function of as-AC in certain brain regions. Moreover, the regulation of the formation of this complex in different brain regions may affect responses to ethanol ingestion in mice.

An analysis of postmortem brain samples from 32 alcoholic and nonalcoholic individuals for protein III, a neuronal phosphoprotein.

Protein phosphorylation is a primary mechanism of intracellular signal transduction, and abnormalities in protein phosphorylation have been implicated in the pathogenesis of several specific diseases. Protein III is a neuronal phosphoprotein that is associated with synaptic vesicles and is probably involved in the regulation of neurotransmitter release. Analysis of 32 postmortem brains has confirmed our previous report that variant forms of protein III with higher apparent molecular weights are found frequently in the brains of alcoholic individuals but rarely in the brains of nonalcoholic individuals who did not suffer from any other medical or neuropsychiatric disorders. Eight of 14 (57%) brain samples from alcoholic individuals and four of eight (50%) brain samples from suspected alcoholic individuals had variant forms, while none of 10 samples from nonalcoholic individuals had variant forms. Previous data indicate that variant forms of protein III are also associated with other neurodegenerative conditions, including various dementias, and, possibly, normal aging.

Beta-adrenergic receptor binding in brain of alcoholics.

The binding of agonists and antagonists to beta-adrenergic receptors in brain tissue obtained postmortem in nonalcoholic controls and matched intoxicated and sober alcoholics was measured to assess the state of the receptors and their coupling to adenylate cyclase. Binding of antagonist, iodocyanopindolol, to cerebral cortical and cerebellar membrane preparations was not different in alcoholics compared to that in controls, suggesting that the number of beta-adrenergic receptors was not affected by chronic ethanol ingestion. Agonist binding data, however, indicated the loss of the high-affinity agonist binding state of the beta-adrenergic receptor, representing the receptor-guanine nucleotide binding protein (Gs) complex. Such changes were observed in cerebral cortex but not in cerebellum of intoxicated alcoholics. These data suggest that cerebral cortical beta-adrenergic receptors are uncoupled from adenylate cyclase in these subjects. In cerebral cortical and cerebellar membranes of sober alcoholics both the high- and low-affinity agonist binding sites were observed. These findings are similar to those seen in animal studies and suggest that the effect of chronic ethanol ingestion on beta-adrenergic receptor-adenylate cyclase coupling is brain region specific and reversible with abstinence. Ethanol-induced changes in the coupling of receptors to adenylate cyclase may contribute to the physiological and behavioral manifestations of alcohol abuse.

Hippocampal and cerebellar beta-adrenergic receptors and adenylate cyclase are differentially altered by chronic ethanol ingestion.

Chronic ethanol ingestion by mice resulted in the loss of high-affinity beta-adrenergic agonist binding sites and a significant decrease in activation of adenylate cyclase by guanine nucleotides and beta-adrenergic agonists in the hippocampus, although no significant change was noted in the total number of beta-adrenergic receptors, as defined by the binding of the antagonist [125]iodocyanopindolol. In cerebellum, chronic ethanol ingestion resulted in a 16% decrease in the total concentration of beta-adrenergic receptors and in a decrease in the affinity for agonist of the high-affinity beta-adrenergic agonist binding sites. However, neither the amount of the high-affinity agonist binding sites nor the activation of adenylate cyclase by agonist was affected. The different responses to ethanol in hippocampus and cerebellum may result from quantitative differences in distribution of beta 1- and beta 2-adrenergic receptors in the tested brain areas and/or differential effects of ethanol on stimulatory guanine nucleotide binding protein in these brain areas.

Effects of chronic ethanol ingestion on mouse brain beta-adrenergic receptors (BAR) and adenylate cyclase.

Previous work showed that chronic ethanol ingestion by C57BL mice resulted in reduced stimulation of cerebral cortical adenylate cyclase (AC) activity by isoproterenol (ISO) and guanine nucleotides (GN). To investigate the mechanism of this change we have assessed the effect of chronic ethanol ingestion on agonist and antagonist binding to BAR in cerebral cortex (mainly beta 1-AR) and cerebellum (mainly beta 2-AR). C57BL mice were fed ethanol in a liquid diet for seven days and were withdrawn for various intervals. Agonist (ISO) binding data were best fit by a two-site model (high and low affinity states) in cortical membranes of control mice. GN induced conversion to a one site model (low affinity state). At the time of withdrawal, ISO binding data in cortical membranes were best fit by a one-site model even in the absence of GN. Antagonist binding was not affected. These results resemble those seen after heterologous desensitization, indicating "uncoupling" of receptor and AC. Control cerebellar ISO binding data were similar to cortical data. Chronic ethanol ingestion, however, did not produce data fit by a one site model in cerebellum. The affinity for ISO of the high affinity state of the BAR was significantly decreased at the time of withdrawal. ISO-stimulated AC-activity in cerebellar membranes was not affected by chronic ethanol ingestion, indicating that, in contrast to cerebral cortex, the cerebellar BAR was not uncoupled from AC.

Effect of ethanol on mouse cerebral cortical beta-adrenergic receptors.

Low concentrations of ethanol (10-100 mM), added to assays in vitro, altered agonist (isoproterenol) binding to mouse cerebral cortical beta-adrenergic receptors in a reversible manner. Ethanol decreased the affinity of the high affinity form of the receptor for isoproterenol but had no effect on the affinity of the low affinity form of the receptor, the proportion of high and low affinity forms of the receptor, the total number of agonist-binding sites, or antagonist binding. The selective effect of ethanol on the properties of the high affinity agonist-binding site suggested that ethanol alters the characteristics of the complex of the receptor and Gs, the guanine nucleotide-binding protein. In cerebral cortical membranes of mice that had ingested ethanol chronically, isoproterenol binding data were best fit by a one-site model, even in the absence of guanine nucleotides. This change, when considered together with previously reported changes in adenylate cyclase activity, is reminiscent of heterologous desensitization of the beta-adrenergic receptor. Thus, both acute and chronic ethanol administration may produce changes in adrenergic function in brain.

Comparison of the effects of ethanol on beta-adrenergic receptors in heart and brain.

Low, physiologically-attainable concentrations of ethanol affect agonist binding to cerebral cortical and cardiac beta-adrenergic receptors. In cerebral cortex, ethanol decreases the affinity of the high-affinity state of the receptor for isoproterenol. This may reflect a direct action of ethanol on the receptor. Ethanol also potentiates the action of guanine nucleotides on agonist binding, suggesting a second site of action at Ns. In heart, ethanol increases the proportion of low-affinity binding sites, an effect which is similar to that of guanine nucleotides, and may also indicate an action of ethanol at Ns. After chronic ethanol ingestion, the total number of cardiac beta-adrenergic receptors is decreased, but the proportion of high-affinity sites is increased. This change could reflect an increased sensitivity to catecholamines. In cerebral cortex, chronic ethanol results in a single, low-affinity binding site for agonist, compatible with an "uncoupled" receptor. Such a change also occurs during homologous desensitization, and may result from increased norepinephrine turnover during chronic ethanol ingestion. The differential responses to ethanol of similar receptors in heart and brain exemplify the specificity of ethanol's actions on various organ systems.

Clinical conditions and central dopamine metabolism in alcoholics during acute withdrawal under treatment with different pharmacological agents.

A group of 45 male alcoholics were studied during acute withdrawal. Patients were kept in hospital and treated with amobarbital (15 patients), oxazepam (15 patients), and melperone (15 patients) respectively in a double-blind design. Clinical symptoms were rated with a modified version of the Comprehensive Psychopathological Rating Scale after 1, 4 and 7 days. Blood pressure, body temperature and pulse rate were also recorded. Lumbar cerebrospinal fluid was collected after 1 and 7 days. A group of healthy males served as controls. The three treatment groups showed only small differences with regard to the investigated clinical items, except for a higher incidence of epileptic fits being evidenced in the melperone group. Levels of HVA in the cerebrospinal fluid did not differ between the treatment groups and the controls and did not change during treatment. Statistically significant correlations were noted between levels of HVA and auditory and visual hallucinations as well as concentration difficulties. Assuming that HVA levels reflect the activity of the central nervous dopamine system, the findings indicate a connection between central dopamine metabolism, psychotic symptoms and possibly other symptoms during acute alcohol withdrawal in man.

Characteristics of receptors and enzymes in brains of human alcoholics.

Acute and chronic ethanol administration to animals has been shown to produce changes in the turnover of numerous neurotransmitters, as well as to change the characteristics of certain neurotransmitter receptors. In the present study, brains obtained from human alcoholics and matched control subjects were examined for similar changes. In frontal cortex, the affinity of opiate receptors for dihydromorphine was significantly reduced in brains of alcoholics. Judging from animal studies, this change may reflect alterations in opiate receptor-effector coupling processes. No changes were observed in muscarinic cholinergic or beta-adrenergic receptors in humans, in contrast to animals, possibly because of the protracted abstention from alcohol among the alcoholic patients prior to death. Similarly, no changes in the activities of choline acetyltransferase, tyrosine hydroxylase or MAO-B were observed in brains of alcoholics as compared to the control population. Our studies suggest that many of the alterations witnessed during alcohol intoxication and withdrawal in animals may be more subtle in humans, or may be reversible with abstinence.

5-Hydroxyindoleacetic acid in cerebrospinal fluid in alcoholic patients under different clinical conditions.

Levels of 5-hydroxyindoleacetic acid, 5-HIAA, the main serotoninergic metabolite in the central nervous system, were investigated in the cerebrospinal fluid, CSF, of male alcoholics and healthy controls. During intoxication, 5-HIAA correlated positively to blood ethanol concentration and levels gradually decreased by length of abstinence. In patients investigated after three months of alcohol abstinence, subnormal levels were noted. Assuming that the level of 5-HIAA in the CSF reflects central serotoninergic activity, the results support the assumption of subnormal activity in abstinent alcoholics and an activation during abuse.

Measurement of 5-hydroxytryptophol and 5-hydroxyindoleacetic acid in human and rat brain and plasma.

The levels of 5-hydroxyindoleacetic acid (5-HIAA) and free and total 5-hydroxytryptophol (5-HTOL) in human and rat brain regions and plasma were determined by a specific capillary column gas chromatographic--mass spectrometric method. The human brains were obtained 2-3 hours post mortem, and the levels of 5-HIAA were in the range of 0.48-31.3 nmoles/g in the regions investigated. The levels of free and total 5-HTOL were 10.9-387 pmoles/g and 14.5-821 pmoles/g, respectively. The ratio of total 5-HTOL to 5-HIAA was in the range of 0.6-5.5%. In human plasma the levels of free and total 5-HTOL were 0.9 +/- 0.3 and 2.9 +/- 0.8 pmoles/ml +/- S.E.M., respectively. In regions of rat brain, the 5-HIAA levels ranged from 0.37-2.84 nmoles/g. Free and total 5-HTOL were in the range of 11.4-56.1 and 16.2-77.1 pmoles/g, respectively. The ratio of total 5-HTOL and 5-HIAA ranged from 2.3-5.1%. Higher levels of 5-HIAA and 5-HTOL occurred in the rat pineal gland. In rat plasma the levels of free and total 5-HTOL were 1.34 +/- 0.06 and 21.6 +/- 1.6 pmoles/ml +/- S.E.M., respectively.

Central nervous system noradrenaline metabolism and alcohol consumption in man.

The levels of 3-methoxy-4-hydroxy ethyleneglycol, MOPEG, the main metabolite of noradrenaline in the CNS, were determined in lumbar cerebrospinal fluid of healthy male volunteers. The subjects had been alcohol-free for at least one week and were investigated after intake of 60, 80 or 120 g of ethanol. One week before or after the experiment control samples were obtained. All subjects showed increased MOPEG levels during intoxication, and the MOPEG elevation was significantly correlated to blood ethanol concentration. During control conditions MOPEG levels were fairly constant over time but there were higher levels at night than in the morning. The baseline levels of MOPEG were negatively correlated to the reported habitual alcohol intake of the subjects, and also to the occurrence of dependency traits in their drinking pattern. Subjects with first-degree relatives with alcohol problems had lower MOPEG levels than the rest. Assuming that changes of MOPEG levels in cerebrospinal fluid are dependent on changes of central nervous system noradrenaline turnover, the results indicate that alcohol intoxication is related to a central noradrenergic stimulation, and that the activity of central noradrenaline systems is of importance for longterm alcohol habits in man.