Department of Psychiatry, University of Mainz, Untere Zahlbacher Straße 8, 55131 Mainz, Germany
Received 6 July 1999; in revised form 11 October 1999; accepted 7 November 1999
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ABSTRACT |
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INTRODUCTION |
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Our study aimed to evaluate the correlation of age and basal and stimulated AC activity of lymphocytes from alcohol-dependent patients after detoxification in comparison to healthy control persons.
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MATERIALS AND METHODS |
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Preparation of lymphocytes
Lymphocytes were isolated by centrifugation through a Ficoll gradient (Boyum, 1968). Peripheral blood was collected in 9-ml EDTA tubes (1.6 mg/ml blood; Sarstedt, Germany) and diluted 1:3 with 1 x PBS buffer (phosphate-buffered saline). Ten ml of Ficoll (density = 1.077 g/cm3; Biochrom KG, Berlin, Germany) was layered under 40 ml of diluted blood. After 15 min of centrifugation at 1200 g at room temperature (RT), the lymphocytes were removed from the Ficoll/plasma interphase. The cells were washed with 30 ml 1 x PBS buffer and centrifuged for 15 min at 300 g and RT. The cell pellet was resuspended in 1 ml 1 x PBS buffer. An aliquot was taken for assessing cell number and viability by trypan-blue exclusion. Cells were pelleted by 30 min centrifugation at 500 g and 4°C and frozen at 80°C for a minimum of 12 h before membrane preparation.
Preparation of lymphocyte membranes
As described previously (Pauly et al., 1999), lymphocytes were thawed and resuspended to a concentration of 107 cells/ml in buffer I, 10 mM HEPES (ICN Biochemicals, Meckenheim, Germany), 2 mM EGTA (ethylene glycerol-bis(ß-aminoethyl ether)N,N,N,N-tetraacetic acid; ICN Biochemicals), 4 mM MgCl2 (Fluka, Neu-Ulm, Germany), pH 6.8. Ninety-five to 98% of the lymphocytes were disrupted by 60 strokes (glass/glass; type S) in a precooled Dounce homogenizer (Braun, Melsungen, Germany). Nuclei were separated by 92-min centrifugation at 500 g and 4°C. Membranes were pelleted by a 30-min centrifugation at 44 000 g and 4°C. The membrane pellet was resuspended in 200 µl of buffer II, containing 10 mM Tris/HCl pH 7.4 (Roth, Karlsruhe, Germany), 1 mM EDTA (Sigma, Munich, Germany), 0.5 mM DTT (dithiothreitol; Sigma), 0.5 mM PMSF (phenylmethanesulphonylfluoride; Fluka), 1 mM benzamidine (Sigma), 0.1 mM benzethonium-chloride (Sigma), 0.5 µM sodium-orthovanadate (Na3VO4; Sigma), 64 ng STI (soybean trypsin inhibitor; Sigma), 1.76 µg TPCK (l-1-chlor-3-(4-tosylamido)-4-phenyl-2 butanon; Boehringer Mannheim, Germany), 440 ng TLCK (N
-tosyl-l-lysine-chloromethyl ketone hydrochloride; Boehringer Mannheim), 56 ng aprotinin (from bovine lung; Trasylol; Fluka), 12 mM Tris, 2 mM MgCl2, 10% glycerol (87%; Fluka). An aliquot was taken for determination of membrane protein content (Bradford, 1976
) using bovine serum albumin as standard (Protein-assay; Bio-Rad, Munich, Germany). The membrane fraction was frozen at 80°C for a minimum of 12 h until further use. Storage of more than 6 months had no measurable influence on enzyme activity.
AC assay
As a measure of AC activity, the formation of 32P-cAMP from -32P-ATP was quantified. Frozen membranes were thawed and repelleted for 30 min at 41 000 g and 4°C. Membranes were diluted with buffer III, pH 6.8, containing 20 mM HEPES, 3 mM EGTA, 0.53 mM CaCl2 (Fluka), 3 mM MgCl2, 110 mM KCl (Sigma), 10 mM LiCl (Sigma), 0.1 mM ATP (ICN Biochemicals) to a final concentration of 1 µg protein/µl. Aliquots of 20 µl were then incubated for 10 min at 30°C with 100 µl of reaction mixture containing 30 mM MOPS (Roth), 15 mM creatine phosphate (ICN Biochemicals), 4.5 mM theophylline (Sigma), 16 mM ATP, 0.5 mM DTT, 5 mM MgCl2, 50 µg creatine phosphokinase (Sigma), 0.450.5 µCi
-32P-ATP (specific activity: 3000 Ci/mmol; NEN-Dupont, Wilmington, DE, USA). For stimulation, the reaction mixture contained additionally either 20 µM guanosine-5-o-(3-triphosphate) (GTP
S; Sigma) or 100 µM forskolin (Sigma). The reaction was stopped by adding 500 µl of Stop-mix consisting of 2% SDS (Roth), 0.1 mM cAMP (Fluka), and 3H-cAMP (specific activity: 250 mCi; NEN-Dupont) which served as internal standard.
Separation of substrate and reaction product was achieved by column chromatography on Al2O3 material (aluminium oxide 90 active, neutral; Merck, Darmstadt, Germany). Columns (internal diameter:1 cm) were filled with a slurry of 2.5 ml Al2O3 in 1:1.5 w/v 0.1 M imidazole pH 7.4 (Sigma) and rinsed with 2 ml of 0.1 M imidazole pH 7.4 prior to use. The volume of the reaction mixture was adjusted to 1 ml with 0.1 M imidazole pH 7.4 (Sigma) and applied to the column.
The first elution was carried out by applying 1 additional ml of 0.1 M imidazole, pH 7.4, to the column and collecting the eluent (2 ml) in a 6-ml scintillation vial (Beckman Instruments, Munich, Germany). Thereafter, the elution was repeated three times, each time applying 2 ml of 0.1 M imidazole, pH 7.4, to the column and collecting the eluent in separate scintillation vials. Eluent samples were mixed with 2 ml of a scintillation cocktail (Ready-Gel, Beckman Instruments) and measured by liquid scintillation spectroscopy using the LS 6000 series liquid scintillation system (Beckman Instruments). The counts per minute value of the first elution containing background radiation was subtracted from the counts per minute values of the eluent samples containing the 3H-labelled internal standard and thus the 32P-cAMP product.
Statistical analysis
For evaluation of correlations between age and AC activity, the correlation coefficient was calculated using the Spearman rank correlation coefficient. Correlations were considered as significant for P < 0.05. The two-tailed t-test was used in the evaluation of group differences. Differences were considered as significant for P < 0.05. The Levene test for the equality of variances was used.
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RESULTS |
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When patients' mean AC activity (n = 68) was compared with those of controls (n = 44), patients showed a trend towards increased AC activity under all conditions tested, which, however, did not consistently reach significance levels (Table 1). In particular, mean AC activity in patients under 40 years of age was higher than that of the corresponding controls. The respective P-values were 0.02, 0.01, and 0.02 for basal, GTP
S-stimulated, and forskolin-stimulated AC activity. The AC activity of patients in the over 55-year age group was significantly lower than those of the controls with respective P-values of 0.020, 0.004, and 0.01.
To find out whether the observed age-related reductions of enzyme activity in the alcohol-dependent patients were also related to the duration of regular alcohol intake, we additionally correlated basal and stimulated AC activity with the duration of regular alcohol intake as indicated by the respective item (years of ethanol consumption on three or more occasions per week, item 1 b of the drug and ethanol use section) of the Addiction Severity Index (ASI, 5th edn, McLellan et al., 1992) that was available for 44 patients. Under these conditions, no significant effect of the duration of ethanol intake on AC activity could be demonstrated (Fig. 2). Nevertheless, the duration of regular ethanol intake was correlated with the age of the patients (correlation coefficient 0.43; P = 0.002). No systematic effects on AC activity were found, when smokers and non-smokers were compared within each group (data not shown).
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DISCUSSION |
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Alcoholic patients differ in many respects from healthy controls, so that group differences cannot simply be attributed to the ethanol consumption nor to dependence, but may also be influenced by different life styles, or dietary and other factors. In particular, cigarette smoking is more frequent among alcoholic patients, than healthy controls. Because lymphocytes express nicotinic acetylcholine receptor channels (Hiemke et al., 1996), varying smoking habits might account for some of the differences. However, these channels are not directly coupled through the AC system and no systematic effects on AC activity were found when smokers and non-smokers were compared within each group. In addition, published experimental data do not support the hypothesis that nicotine has a major impact on AC activity in peripheral lymphocytes (Geng et al., 1996
).
When mean AC activity of the patients and controls were compared, the patients showed a trend towards increased AC activity that was largely due to the large variance in younger and middle-aged alcoholics. The increased variance of AC activity in the alcoholic patients deserves attention, because it appears to reflect a distortion of the AC signalling system in these patients that may be related to alcoholism. So far, we cannot offer a conclusive explanation why some patients showed AC activity far different from the mean activity. As we have pointed out in previous studies (Szegedi et al., 1998; Pauly et al., 1999
), actual blood-ethanol levels of actively drinking patients as well as acute withdrawal effects may modulate basal and stimulated AC activity in peripheral blood cells. Because we measured activity only days after the cessation of withdrawal symptoms, state-dependent factors may still play a role.
Several studies have reported a difference in AC activity in blood cells between alcoholic patients and controls, but the influence of age has not previously been systematically studied. Diamond et al. (1987) showed that lymphocytes of 10 actively drinking patients (mean age 48.6 ± 3.6 years) exhibited reduced basal and adenosine receptor-stimulated AC activity, compared to non-alcoholic controls. Parsian et al. (1996) described significantly reduced basal and caesium fluoride-stimulated AC activity in platelets of 51 male alcoholic patients, compared to 54 normal controls. However, the mean age of the patients was only 31.7 ± 10.9 years. Furthermore, blood-ethanol levels, which were not specified in the aforementioned studies, might also be responsible for the reported changes in the AC system. This point is also made in the study by Waltman et al. (1993), in which AC activity in lymphocytes of 22 actively drinking alcoholic patients was compared with that of 41 abstinent alcoholics and 54 controls. The AC activity was reduced in the (abstinent) alcoholics, but non-significantly increased in the actively drinking alcoholic patients.
The finding that age did not affect AC activity of lymphocytes in healthy controls might be perceived as surprising, since a decline in the responsiveness of the AC system is part of the ageing process in many tissues. However, the age dependency of AC activity of human lymphocytes has not as yet been studied conclusively, and conflicting data have been reported in the literature. Mader et al. (1988), in agreement with our data on healthy controls, were unable to demonstrate any age-related differences in basal, isoprenaline- or forskolin-stimulated AC activity. In contrast, Krall et al. (1981) reported an age-related reduction in lymphocyte AC activity. Likewise, relatively little is known about the effects of ageing on related components of the signal transduction machinery. Barki-Harrington et al. (1996) found the G-proteins Gs, G
i, and G
q in lymphocytes did not change with age. Total lymphocyte activity of protein kinase C, a kinase only indirectly affected by cAMP by cross talk mechanisms, has been described as inversely related to age (De Petrillo, 1994
).
Since the age-dependent decrease was found for both basal and stimulated AC activity, we conclude that the observed decline was most probably attributable to the AC protein, rather than to other parts of the second messenger system. It remains to be shown how far the observed changes were also relevant for receptor-mediated stimulation of AC. Based on our findings, it seems likely that stimulation of a G-protein coupled receptor would similarly decrease with age in alcoholic patients.
In summary, this study provides evidence that the AC activity of lymphocytes of detoxified alcoholic patients is inversely correlated with the age of the patients, but not with the duration of regular alcohol intake. Decreased AC activity was present in patients older than 55 years, whereas younger patients showed a greater variance than controls.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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