Platelet Adenylyl Cyclase Activity in Alcoholics and Subtypes of Alcoholics

Adenylyl cyclase (AC) activity was measured in membrane preparations of platelets from control and alcoholic subjects. The sample consisted of 51 alcoholics who were categorized as type I or type II using the criteria of Gilligan et al. ( Genet. Epidemiol. 4:395–414, 1987) and 54 normal controls. Alcoholic males exhibited significantly lower values than controls in basal and fluoride-stimulated platelet AC activity. When male alcoholics were segregated into type I and type II categories, the platelet AC activity did not differ between subtypes, and both subtypes had AC activity that was below control values. Western blot analysis of the quantity of G_sα and G_iα proteins in a subset of male controls and alcoholic subjects demonstrated no significant relationship between quantity of G proteins and AC activity. The results confirm lower platelet AC activity in male alcoholics, compared with controls. Given the lack of quantitative relations between G_sα and G_iα proteins and AC activity, the results support the contention that individual differences in platelet AC activity in the alcoholic subjects may reflect quantitative or qualitative differences in the AC catalytic units.     

Effects of Chronic Ethanol Consumption on G Proteins in Brain Areas Associated with the Nigrostriatal and Mesolimbic Dopamine Systems

This study examined the effects of chronic ethanol consumption on the content of G proteins in brain areas associated with the nigrostriatal and mesolimbic dopamine systems of male Fischer 344 rats, aged 3, 5, or 13 months at the time of killing. In addition, G protein mRNA was assessed in 3-month-old rats. G proteins were examined in ethanol-fed rats because a number of studies have implicated these proteins with both the acute and chronic effects of ethanol. Brain areas associated with the nigrostriatal and mesolimbic dopamine systems were examined because of the evidence that these systems are sensitive to ethanol. The brain areas examined include the substantia nigra (SN), striatum (ST), globus pallidas (GP), frontal cortex (FCX), nucleus accumbens (NA), ventral tegmental area (VTA), and ventral pallidum (VP).
These experiments demonstrated that the 3-month-old rats that consumed a 6.6% (v/v) ethanol-containing liquid diet for 4 weeks had a significant (∽30–40%) increase in the mRNA content of G_13α, in the FCX, VTA, and VP, and a significant (∽20%) decrease of that for G_0α in the SN. Nonetheless, the content of the G_0α protein subunit was not altered. In addition, there were no significant differences in the content of the proteins detected by antibodies to G_6α, G_0α, G_11α/G_12α, and G_0α/G_13α in the FCX, NA, and ST of similarly treated older rats (5 and 13 months). The content of mRNA for the other G proteins examined in the seven brain areas of 3-month-old rats was unaffected by chronic ethanol exposure.     

Chlorzoxazone pharmacokinetics as a marker of hepatic cytochrome P4502E1 in humans

Objective: Previous in vitro studies have demonstrated that hepatic P4502E1 metabolizes chlorzoxazone (CZX, a commonly used muscle relaxant) to 6-hydroxychlorzoxazone (6-OH-CZX). We thus assessed whether measurement of the plasma 6-OH-CZX/CZX ratio after a CZX challenge could serve as a marker of hepatic P4502E1 content.
Methods: Three subject groups were included: recently drinking alcoholics (  N = 6  ), abstinent alcoholics (  N = 5  ), and nonalcoholic subjects with liver disease (  N = 5  ) undergoing liver biopsy. Excess tissue was procured for immunochemical determination of hepatic P4502E1 content. Within an hour of the biopsy, 750 mg CZX was administered orally and serial plasma samples were collected for 6 h.
Results: Recently drinking alcoholic subjects had a higher area under the curve for plasma 6-OH-CZX (1.354 ± 0.258 μg · min · ml⁻¹) then abstinent alcoholic subjects (0.296 ± 0.080 μg · min · ml⁻¹, p < 0.005) and subjects with nonalcoholic liver disease (0.428 ± 0.061 μg · min · ml⁻¹,   p < 0.005  ). The use of the plasma 6-OH-CZX/CZX ratio at 90, 120, and 180 min discriminated between recently drinking alcoholic and nondrinking subjects. Hepatic P4502E1 content significantly correlated with the maximal 6-OH-CZX concentration (  r = 0.76  , p = 0.001) and other pharmacokinetic parameters. In the recently drinking group, the area under the curve for plasma 6-OH-CZX significantly decreased after 8 days of abstinence.
Conclusions: Measurement of plasma 6-OH-CZX after administration of a CZX challenge can serve as a marker of hepatic P4502E1 activity and thus help avoid adverse drug reactions secondary to P4502E1 induction, particularly in heavy drinkers.     

Human GABA, Receptor αl and α3 Subunits Genes and Alcoholism

γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain. GABA effects are largely mediated by binding to the postsynaptic GABA_A receptor, causing the opening of an integral chloride-ion channel. The GABA_A antagonists picrotoxin and bicuculline reduce some ethanol-induced behaviors, such as motor impairment, sedation, and hypnosis. The role of this receptor in alcoholism is further supported by effective alleviation of alcohol withdrawal symptoms by GABA_A agonists. To determine the role of the GABA, receptor (GABR) genes in the development of alcoholism, we have used α₁ and α ₃ simple sequence repeat polymorphisms in a sample of unrelated alcoholics, alcoholic probands with both parents, and psychiatrically normal controls. For the GABRα₁ gene, the differences between allele frequencies, when all alleles were compared together, were not significant between total alcoholics, subtypes of alcoholics, and normal controls. However, for GABRα₃, the differences between total alcoholics and normal controls were significant when all alleles were compared together. The differences between subtypes of alcoholics and normal controls were not significant. The results of haplotype relative risk analysis for both genes, GABRα₁ and GABRα₃, were also negative. It is possible that the sample size in the haplotype relative risk is too small to have power to detect the differences in transmitted versus nontransmitted alleles. There is a need for a replication study in a large family sample, that will allow haplotype relative risk or affected sib-pair analysis.     

Activated Lymphocytes (CD25+ CD69+ Cells) and Decreased CD19+ Cells in Well-Nourished Chronic Alcoholics without Ethanol-Related Diseases

To assess lymphocyte subsets and expression of activation antigens in peripheral blood lymphocytes (PBLs) in chronic alcoholism, a cross-sectional study with 30 well-nourished chronic alcoholics and 30 controls was performed. Studies included detailed clinical and laboratory evaluation, nutritional status assessment, and determination of lymphocyte subpopulations, as well as activation antigens. A significant decrease of B cells (CD19⁺) was observed in chronic alcoholics, compared with controls ( p < 0.001). A significant increase of PBLs expressing CD69 and CD25 ( p < 0.01, both) in chronic alcoholics was also detected, whereas CD71 expression was unaffected. In addition, T lymphocytes expressing HLA-DR were significantly higher in chronic alcoholics than controls ( p < 0.05). The serum level of soluble interleukin-2 receptor was also significantly higher in the alcoholic group, compared with controls ( p = 0.04). Moreover, the estimated total lifetime dose of ethanol consumed correlated positively with the percentage of PBLs expressing CD25 ( r = 0.48; p = 0.01) and negatively with PBLs expressing CD71 ( r = -0.39; p = 0.04). By contrast, the changes were not related to age, nutritional status, or the presence of other ethanol-related diseases. In conclusion, chronic alcoholics present a significant decrease of B cells and an "incomplete activation state" of PBLs that depends on the dose of ethanol consumed.     

Compartmentalization regulates the interaction between the platelet integrin αIIbβ3 and ICln

The volume-regulating protein, ICln, interacts with the conserved KxGFFKR α-integrin signature motif. ICln is an abundant protein (4455 ± 650 molecules/platelet) found exclusively in the soluble cytosolic fraction of unactivated platelets. In contrast, its binding partner, the platelet integrin α_IIbβ₃, is present in detergent-insoluble fractions associated with membrane and cytoskeleton subcellular localizations. This study investigated factors that regulate the interaction of ICln with α_IIbβ₃ during platelet activation. His-tagged recombinant ICln bound equally to purified α_IIbβ₃ and to integrin from resting or activated platelets. Binding was not affected by direct integrin activation with Mn⁺⁺ or by inhibitors of integrin occupancy (abciximab, RGD). However, the capacity for interaction between integrin and recombinant ICln was slowly downregulated following prolonged platelet activation for >300 s. In parallel, ICln redistributed to membrane and cytoskeletal platelet subcellular fractions. The time-course of this redistribution preceded the downregulation of integrin binding capacity and suggests that only a short window of opportunity exists for ICln interaction with α_IIbβ₃ to occur. Thus, although ICln has the inherent capacity to bind to α_IIbβ₃ regardless of its activation state, it can only do so following platelet activation. Activation-dependent subcellular redistribution of ICln represents a novel, temporally-regulated mechanism for control of integrin function in platelets.     

Gender differences in response to sertraline pharmacotherapy in Type A alcohol dependence

We previously established that Babor Type A "lower-risk/severity" alcoholics (n = 55) had better treatment response to fourteen weeks of sertraline (200 mg/day) than placebo, a finding not present for Type B "higher-risk/severity" alcoholics (n = 45). This exploratory study extended these results by examining the original sample for gender differences in response to sertraline pharmacotherapy. Type A alcoholic men, but not Type A alcoholic women, had consistently better outcomes with sertraline compared to placebo on several common drinking measures: time to relapse, days drinking, days drinking heavily, drinks per drinking day, and number of those continually abstinent. There were no significant differences in drinking with sertraline compared to placebo in Type B alcoholic men or women.     

Haemoglobin F Texas I (α2γ25Glu→Lys): a Variant of Haemoglobin F

A survey of 3000 cord bloods in Great Britain yielded one electrophoretically abnormal variant of Haemoglobin F in an infant of West Indian parentage. This haemoglobin resembled Haemoglobin F Texas. Haemoglobin F Texas might either be α₂γ₂^5Glu→^Lys or α₂γ₂^6Glu→^Lys. The present variant F has its substitution in the fifth residue of the γ-chain, and is designated Haemoglobin F Texas I.     

Effect of Alcohol Drinking on Gene Expression of Hepatic O6-Methylguanine DNA Methyltransferase in Chronic Liver Diseases

O⁶-methylguanine DNA methyltransferase (MGMT) is a repair protein that transfers methyl groups from O⁶-methylguanine to a cysteine acceptor in its own molecule, and restores DNA to its undamaged state. If left unrepaired, O⁶-methylguanine can pair with either a thymine or a cytosine, causing a C-G to T-A transition, which is considered to be one of the molecular mechanisms of both mutagenesis and carcinogenesis. The expression of MGMT mRNA in liver tissue was quantitatively assessed by the competitive reverse transcrip-tion-polymerase chain reaction method in patients with chronic liver diseases with or without alcohol drinking. MGMT mRNA expression was 1.4 ± 0.9 pg/μg RNA in control livers. Its expression in chronic hepatitis was 3.8 ± 0.7 in alcoholics and 2.7 ± 0.8 in nonalcoholics, which were not statistically different. MGMT mRNA expression in liver cirrhosis was significantly low, compared with that in chronic hepatitis, and 0.8 ± 0.3 in alcoholics and 0.5 ± 0.1 in nonalcoholics, which also were not significantly different. The present study shows that MGMT mRNA was not decreased in patients with chronic liver diseases with alcohol drinking, compared with those without alcohol drinking.     

Deficiency of endogenous prostanoids in gastric and duodenal ulcer diseases

The levels of prostaglandin E₂ (PGE₂), 6-keto-prostaglandin F₁α (PGF₁α) and thromboxane B₂ (TXB₂) in endoscopic biopsy specimens from the gastric and duodenal mucosa of healthy volunteers and ulcer patients were measured by radio-immunoassay. The PGE₂ and PGF₁α levels in the mucosa of the corpus of the stomach were lower and the TXB₂ level was higher in 10 patients with gastric ulcer in the corpus than in the 16 healthy subjects. The PGE₂ level in the antral mucosa of 14 patients with gastric ulcer in the antrum was lower than in the controls. In 18 patients with duodenal ulcer, PGE₂ deficiency was more widespread in the entire gastric and duodenal mucosa while the reduced PGF₁α level was limited in the gastric corpus. Lower levels of PGE₂ in patients with antral or duodenal ulcer and of PGE₂ and PGF₁α in patients with corpus ulcer in the anatomical mucosal area including the ulcer site may predispose the mucosa to ulceration.     

Haemoglobin F Texas II (α2γ26 Glu→Lys), the Second of the Haemoglobin F Texas Variants

S ummary A survey of 13 ,000 cord bloods in Great Britain yielded an electrophoretically abnormal variant of Haemoglobin F which resembled Haemoglobin F Texas. Haemoglobin F Texas might be either α₂γ₂^5G1u→Lys or α₂γ₂^6Glu→Lys. The first has been described and was named Haemoglobin F Texas I. The present variant has the same substitution in the sixth residue of the γ chain and is designated as Haenioglobin F Texas II.     

Defective Calcium Increase and Inositol Phosphate Production in Anti-CD3-Stimulated Lymphocytes of Alcoholics without Progressive Liver Disease

Intracellular free calcium concentration, phosphoinositide turnover, and inositol phosphate production were analyzed in peripheral blood lymphocytes from seven well-nourished alcoholic patients without severe acute or chronic liver disease, before and after stimulation with anti-CD₃ antibody. Seven comparable nondrinkers were studied as controls. A lower increase in intracellular free calcium concentration was detected in alcoholics, after anti-CD₃ stimulation of lymphocytes, than in control subjects. Lymphocyte activation generated inositol phosphates in both controls and alcoholics, but inositol phosphate production was significantly lower in alcoholics. The agreement between these findings indicates that the reduction in inositol phosphates is one of the most important events in the early phases of lymphocyte activation in alcoholics.     

The β+ IVS, I-NT no. 6 (T→C) thalassaemia in heterozygotes with an associated Hb Valletta or Hb S heterozygosity in homozygotes from Malta

Summary. In vitro DNA amplification and dot blot analysis with synthetic allele specific oligonucleotides (ASO) identified the β IVS, I-6 (T→C) thalassaemia in 78% of 32 chromosomes from 16 β-thalassaemia homozygotes in Malta. The preponderance of a single thalassaemia mutation in one population is unusual. The β⁺ IVS, I-6C thalassaemia mutation was also found in three carriers who had an associated β globin heterozygosity, i.e. Hb Valletta (or α₂β₂^87PRO) or Hb S (or α₂β₂^6VAL). The proportion of Hb A in these cases (av. = 29.7%) provided objective documentation of the relatively mild effect of this mutation on in vivo globin gene expression. However, the expression of homozygous disease was more severe in developing children compared to adults. The β⁺ IVS, I-6C mutation complicates population testing because heterozygotes can have Hb A₂ levels below those classically associated with β thalassaemia.     

Biochemical Studies of a New Class of Alcohol Dehydrogenase Inhibitors from Radix puerariae

Two potent, reversible inhibitors of human alcohol dehydrogenase; (ADH) isozymes were isolated from Radix puerariae (RP, commonly, known as kudzu root) and identified as the isoflavones daidzein and genistein. The 4-methoxy derivatives of daidzein (trivial name, for-mononetin) and genistein (biochanin A), minor constituents of RP, were also shown to be ADH inhibitors. All of these isoflavones inhibit 1 the human -γ₁γ₂-ADH isozyme competitively with respect to ethanol] and uncompetitively with respect to NAD⁺. A survey of more than 40 structurally related compounds revealed one more isoflavone (prunetin) and four flavones (7-hydroxyflavone, apigenin, galangin, and kaempferol) that inhibit ADH. The isoflavone inhibitors, however, are far more potent than the flavone inhibitors. Among the isoflavones studied, genistein is the most potent with K_i , = 0.1 μM toward γ₂γ₂- ADH. Human ADH isozymes differ in their sensitivity to these inhibitors in the order γ₂γ₂- , γ₁γ₁ > α₁α₁⁻,>⁻ > xx-ADH. These inhibitors, do not affect the β₁β₁ ,⁻ and β₂β₂ -ADH isozymes at concentrations as high as 20 μM. Rat and rabbit class I ADHs are also inhibited by these. isoflavone inhibitors. The 7-O-glucosyl derivatives of daidzein, genistein, formononetin, and biochanin A do not inhibit ADH, but are potent aldehyde dehydrogenase inhibitors.     

In Vivo Effect of Chronic Ethanol Consumption on the Fatty Acid Composition of Phosphatidylinositols in Resting and Anti-CD3-Activated Lymphocytes

Fatty acid composition of phosphatidylinositols was analyzed in peripheral blood lymphocytes from nine alcoholic patients who were well nourished and without severe acute and chronic liver disease, before and after stimulation with anti-CDs antibody. Six comparable nondrinkers were studied as controls. A reduction in unsaturated fatty acid (mainly arachidonic) and an increase in palmitic and stearic acid molar content were observed in phosphatidylinositol (PI), phos-phatidylinositol-4-phosphate (PIP), and phosphatidylinositol-4,5-bis-phosphate (PIP₂) in unstimulated samples from alcoholic patients in comparison with normal subjects, leading to a significant decrease in the saturated/unsaturated ratio. In controls, anti-CD₃ stimulation caused a marked decrease in arachidonic acid relative molar content counterbalanced by an increase in other polyunsaturated fatty acid relative molar content in PI, PIP, and PIP₂ fractions. Interestingly, after anti-CD₃ stimulation, alcoholic patients show the same trend of modification in the fatty acid composition resulting in a sharp reduction of arachidonic acid relative molar content. These results support the hypothesis of an alteration in nutrients being responsible for immune derangement in alcoholics.     

Respiratory Responsiveness in Male Alcoholics after Withdrawal

Peripheral and central chemosensitivity were assessed in eight abstinent male alcoholic patients and in seven healthy normal male subjects. Hypoxic rebreathing (15% O₂, 7% CO₂) was used to stimulate the peripheral and central chemoreceptors together. Hyperoxic rebreathing (93% O₂, 7% CO₂) was used to examine the central chemoreceptor response independently of the peripheral chemoreceptors. Minute ventilation was examined in relation to end-tidal PACO₂ under the two conditions. Neither peripheral nor central chemoreceptor responses were signficantly altered in the alcoholic as compared to the control subjects. Abnormal chemoreceptor function is therefore unlikely to be an important factor in the development of abnormal respiratory events during sleep in abstinent alcoholic subjects.     

Association of Restriction Fragment-Length Polymorphisms in the Alcohol Dehydrogenase 2 Gene with Alcoholic Brain Atrophy

Alcohol abuse can induce brain atrophy, but it only occurs in some alcoholics. To investigate whether genetic polymorphism of alcoholmetabolizing enzymes [including alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH)] was related to alcoholic brain atrophy, we determined restriction fragment-length polymorphisms of the ADH2 and ALDH2 genes in 77 male alcoholics. Computed tomography was used to determine the severity of brain atrophy. Digestion with Mae III and Mbo II after polymerase chain reaction amplification showed that the ADH2¹ gene frequency was significantly higher in patients with brain atrophy than in those without brain atrophy ( x ²= 9.274, p < 0.01), whereas no significant association was observed between brain atrophy and the ALDH2 gene. Multivariate analysis (including age, total alcohol intake, liver cirrhosis, and ADH2 genotype) showed that the ADH2¹/ADH2¹ genotype was associated with alcoholic brain atrophy. These findings suggest that the ADH2¹ allele may be associated with alcoholic brain atrophy.     

Ethanol Increases GABAA Responses in Cells Stably Transfected with Receptor Subunits

Ethanol enhancement of GABA_A receptor function has been found in some, but not all, studies. These results suggest the existence of ethanol-sensitive and -resistant receptors that may differ in subunit composition, although methodological differences (e.g., ³⁸Cl^- flux versus membrane currents) could also contribute to the different results. To examine these possibilities, we used mouse L(tk^-) cells stably transfected with α₁+β or α+β₁+γ_2L GABA_A receptor subunit DNAs and compared ³⁸Cl flux with whole-cell, patch-clamp measurements of GABA_A receptor function. Both techniques detected a similar modulation of the GABA receptor by ethanol, flunitrazepam, and pentobarbital. The potentiating action of ethanol required the -γ-subunit and was maximal at a concentration of 10 m m . Similar ethanol potentiation was obtained with brief (20 msec) or long (2 sec) applications of GABA. Analysis of data obtained from individual cells expressing α₁β_1-γ_2L subunits showed considerable variability in sensitivity to ethanol, particularly with concentrations of 30 and 100 m m . Ethanol potentiated GABA action if the cells were grown on coverslips coated with polylysine, but had no effect on GABA_A receptors of cells grown on uncoated coverslips. Thus, ethanol action was influenced by the growth matrix. Taken together, these data indicate that a y-subunit is necessary, but not sufficient, for ethanol sensitivity in this cell system. We suggest that posttranslational processing, particularly receptor phosphorylation, may also be important and that stably transfected cells will be useful in elucidating these events.