Neuroehemical Research, Vol. 21, No. 3, 1996, pp. 30~311
Ethanol Potentiates the Uptake of [I4C]Serine into Phosphatidylserine by Base-Exchange Reaction in NG 108-15 Cells F. D a v i d R o d r i g u e z , ~ C h i s t e r Ailing, z a n d L e n a G u s t a v s s o n z
(Accepted November 13, 1995)
Phospholipid base-exchange enzymes catalyze the incorporation of nitrogenous bases into phosphoglycerides by a calcium-dependent mechanism. In this study, we describe the effect of ethanol on the incorporation of radioactive serine, choline and ethanolamine into their respective phospholipids in a neuroblastoma x glioma hybrid cell line (NG 108- 15). Long term ethanol exposure induced a potentiation of the incorporation of [~4C]serine into phosphatidylserine. Moreover, the phosphorus content of PS was found to be increased after long-term ethanol exposure. No concomitant changes in the phosphorus content of other phospholipids were observed. The results indicate that in NG 108-15 cells, the incorporation of radiolabelled serine into PS is potentiated during chronic ethanol exposure. KEY WORDS: Ethanol; phospholipids; base-exchange enzymes; NG 108-15 cells; phosphatidylserine.
ethanolamine and choline into pre-existing phosphoglycerides. (5-10). Although there is not clear evidence of separate base-exchange molecular entities for the biosynthesis of PS, PC and PE, there are indications suggesting differential regulation of base-exchange activities by different factors such as divalent cations (5), unsaturated fatty acids (11), temperature (12), substrate concentration (13,14), substrate competition (7) or GTP and P2u purinergic agonists (15). PS is an anionic phospholipid playing an important modulatory role in the functioning of membrane-bound proteins (16,17). A representative example is the activation of protein kinase C by PS. (18,19). The participation of PS in several physiological events and its peculiar biosynthetic pathway, compared to other phospholipids, makes serine-exchange reaction an important site for the regulation and modification of the phospholipid composition of cell membranes. It has been reported that the amount of anionic phospholipids in rodent brain synaptic membranes increases after chronic ethanol administration. (20,21). On the other hand, Magruder and coworkers (22) observed
Different pathways are involved in the biosynthesis of phospholipids in animal tissues. The de novo biosynthesis of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) occurs mainly by incorporating choline and ethanolamine into the corresponding phospholipids, via a cytidyl diphosphoryl intermediate (1). PC and PE may also be synthesized by base-exchange reactions. Other alternatives for PE and PC biosynthesis exist, namely the decarboxylation pathway for PE (2) and the methylation pathway for PC (3,4). However, the base-exchange reaction is the only pathway for the biosynthesis of phosphatidylserine (PS) in mammalian cells. This feature makes PS a unique phospholipid. The base-exchange enzymes catalyze the calciumdependent non-energy requiting incorporation of serine, 1 Dept. of Biochemistry and Molecular Biology, University of Salamanca, 37007 Salamanca, Spain. 2 Dept. of Psychiatry and Neurochemistry, University of Lund, S-220 09 Lund, Sweden.
a d e c r e a s e in a c i d i c p h o s p h o l i p i d s a f t e r c h r o n i c e t h a n o l t r e a t m e n t . T h i s d i s c r e p a n c y m a y b e d u e to the use o f different model systems and protocols of alcohol exposure. T h e p a t t e r n o f e t h a n o l e x p o s u r e p l a y s a n i m p o r t a n t role o n the e t h a n o l - i n d u c e d c h a n g e s in n e g a t i v e l y c h a r g e d p h o s p h o l i p i d s (23,24). F u r t h e r m o r e , a n i o n i c p h o s p h o l i p i d s p l a y a n i m p o r t a n t role in the d e v e l o p m e n t o f m e m b r a n e t o l e r a n c e to e t h a n o l (25). F r o m t h e s e studies o n e c a n c o n c l u d e t h a t e t h a n o l s e e m s to h a v e a g r e a t e r i n f l u e n c e o n a n i o n i c p h o s p h o l i p i d s c o m p a r e d to the z w i t e r i o n s P E a n d PC. H o w e v e r , to w h a t e x t e n t t h e s e a l t e r a t i o n s in m e m b r a n e c o m p o s i t i o n reflect c h a n g e s in lipid m e t a b o l i s m h a s n o t p r e v i o u s l y b e e n e x p l o r e d in a cell m o d e l a n d its s u b c e l l u l a r c o m p a r t m e n t . O u r a i m in this s t u d y w a s to i n v e s t i g a t e to w h i c h e x t e n t the i n c o r p o r a t i o n o f n i t r o g e n o u s b a s e s into t h e i r r e s p e c t i v e p h o s p h o l i p i d s is a f f e c t e d b y e t h a n o l . W e s h o w that t h e u p t a k e o f r a d i o a c t i v e s e r i n e into PS is i n c r e a s e d in N G 108-15 cells c h r o n i c a l l y e x p o s e d to ethanol. T h e o b s e r v a t i o n w a s specific for serine b a s e exchange.
Cell Culture and Ethanol Treatment. NG 108-15 cells (passage 20-40) were used. The cultivation of the cells was carried out in 35 mm diameter plastic dishes (NUNC S/A, Denmark) containing 1 ml of Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum, 2 mM 1-glutamine, 50 • HAT (final concentration: hypoxantine 0.1 mM, aminopterine 4 gM and thymidine 16 gM), 100 gg/ml streptomycin and 100 1E/ml penicillin (26). Antibiotics were from Astra Pharmaceuticals, Sweden. Other cell culture chemicals were from Flow Laboratories, UK. Medium was changed daily. The cells were maintained at 37~ in an incubator under a humidified atmosphere containing 10% COj90% air. Ethanol at different concentrations was added directly to the medium. Both control and ethanol-treated dishes were placed inside tightly capped plastic boxes together with an open dish containing an appropriate amount of ethanol. By using this method ethanol concentrations were kept constant over time (27). Incorporation of the Radiolabelled Bases into Their Respective Phospholipids in Intact Cells. Control and ethanol-treated cells were labeled with 0.5 gCi/ml of L-[~C(U)]serine (specific radioactivity 180.5 Ci/mmol), 2 gCi/ml of methyl [3H]choline (specific radioactivity 88.7 Ci/mmol) or 2 ~tCi/ml of 1,2 [t4C]ethanolamine (specific radioactivity 48 Ci/mmol) for different periods of time. The incorporation of label was studied after treating the cells for different periods of time in the presence of ethanol. In some experiments, the cells were labeled with [~4C]serine for 180 min. Afterwards, the label was withdrawn and the radioactivity in PS analyzed after different periods of incubation in [~4C]-serine-free medium. Once labeling was finished the cells were washed with Hank's balanced salt solution (without Mg2+ and Ca2§ and harvested in ice-cold 50 mM TRIS-HC1 buffer (pH 7.4) containing 2 mM EDTA. Preparation of Microsomes from NG 108-15 Cells. Microsomes from NG 108-15 cells were prepared by centrifugation in a buffer
containing 0.32 M sucrose, l mM EDTA and 2 mM HEPES (pH 7.4), as previously described (28,29). Briefly, the cells were homogenized and centrifuged at 20,000 g for 20 min. Thereafter the supematant was centrifuged at 100,000 g for 60 min. The microsomal fraction-containing pellet was then resuspended in a buffer containing 40 mM HEPES and 1 mM CaC12 (pH: 7.45), protein content measured, and stored at -80~ until use. Serine Base-Exchange Assay #z NG 108-15 Microsomes. An in vitro assay for serine base-exchange reaction was carried out according to Hu et al. (28) with minor modifications. Briefly, microsomal membranes (approximately 30 ~tg protein/tube) were incubated in a buffer containing 40 mM HEPES and 1 mM CaC12, at pH 7.45. After adding 4.5 p.Ci of [~4C]serine (50 [aM, final concentration), the samples were incubated at 37~ in a shaking bath for 20 minutes. The total volume of the samples was 100 gl. The reaction was terminated by adding 400 gl of chloroform: methanol (1:2, by volume). Thereafter, 100 gl of water and 150 p.1 chloroform were added, and lipids extracted and separated as indicated below. Lipid Extraction and Separation of Phospholipids. The extraction of lipids was carried out according to Bligh and Dyer (30). The lipidcontaining phase was evaporated under N2 and redissolved in 1 ml chloroform:methanol (2:1, by volume) and stored at -20~ until analysis. Separation of phospholipids was done by using a double onedimensional TLC separation method (31). Briefly, the lipid extracts were applied 8 cm from the bottom of 20 • 20 cm TLC plates (silica gel 60, Merk, Germany) previously dried in an oven (15 min at 100~ The solvent system used in the first direction was chloroform: methanol:28%aqueous ammonia (65:35:7.5, by volume). The outer lane containing phospholipid standards was removed and stained by exposure to iodine vapours to ascertain the location of the spot corresponding to PC. The plate was then cut horizontally just below the extrapolated PC spots. The upper part of the plate contained the PC and PE spots. The lower part of the plate was turned 180~ and chromatographed in the solvent system chloroform:acetone:methanol:acetic acid:water (50:15:10:10:5, by volume). With this method acidic phospholipids such as PS, phosphatidylinositol (PI) and phosphatidic acid (PA) remained at the origin in the first solvent system but migrated in the second solvent system. The areas in the silica gel corresponding to PC, PE, PI, and PS were identified with authentic standards and scraped off after visualization with iodine. The radioactivity in the spots was measured by scintillation counting. The total radioactivity in the lipids was determined in aliquots taken from the organic phase. Determination of Phosphorus and Protein Content. Phosphorus content in different phospholipids was measured by using a colorimetric method (32) after separation of lipids on the thin layer chromatography plates as described above. Proteins were quantified according to Bradford (33). Statistical Analysis. Results are presented as means -+ S.E.M. Statistical significances were evaluated by Student's t-test.
Incorporation o f f4C]Serine, pH]Choline, and [a4C]Ethanolamine into Phospholipids in NG 108-15 Cells. N G 108-15 cells w e r e i n c u b a t e d in the p r e s e n c e o f r a d i o l a b e l l e d serine for 30 m i n to 2 4 hours. Fig. 1 s h o w s the u p t a k e o f r a d i o a c t i v i t y o v e r t i m e in d i f f e r e n t p h o s p h o l i p i d s . T h e i n c o r p o r a t i o n o f [14C]serine into PS
Ethanol and Base-Exchange enzymes
Radioactivity in PS (cpm / mg protein) 8000
Time of labelling (hours)
Time of labelling (hours)
Fig. 1. Time-course of incorporation of [~4C]serineinto phospholipids. Intact cells were labeled in the presence of radioactive serine as indicated in Experimental procedure, and radioactivity determined in different phospholipids after separation on TLC plates. PS (open circles), PE (open squares) and PC (open triangles). All values are the mean _+ SEM of four experiments done in triplicate.
Table I. Effect of Acute Ethanol Exposure on the Uptake of [~4C]Serine, [3H]Choline and [14C]Ethanolamineinto PS, PC, and PE Respectively Radioactivity (cpm/mg protein) PS
Control 4,040 _+ 790 165,000 _+ 33,000 101,000 _+ 5,900 Ethanol (100 mM) 3,380 _+ 900 181,000 _+ 18,000 120,000 _+ 8,100 All values are expressed as mean + SEM of three experiments done in triplicate. Cells were labeled in the presence of [~4C]serine, [~4C]ethanolamine, or [3H]choline for three hours in the presence or absence of 100 mM ethanol, and the radioactivity in the respective phospholipid determined.
was linear up to approximately six hours o f labelling, after which the uptake curve reached a plateau. Three hours after addition o f label, radioactivity was detected in PE. This observation m a y reflect the time lag necessary for the newly synthesized PS to be transported to the mitochondria and decarboxytated to PE (9). After 6 hours o f labeling and onwards some radioactivity was detected in PC. This probably reflects the conversion o f radiolabeled PE into PC by stepwise methylation. When cells were labeled with [3H]choline the radioactivity in PC reached a steady-state between 6 and 12 hours o f labeling (data not shown). Similarly, the radioactivity in PE reached a plateau after 6 hours when cells were incubated in the presence o f [14C]ethanolamine. A significant amount o f radioactivity was detected in PC after
Fig. 2. Influence of chronic ethanol exposure on the time-course of uptake of [~4C]serine into phosphatidylserine. The radioactivity was analyzed in control (open squares) and NG 108-15 cells exposed to 100 mM ethanol for 2 days (filled squares). Results are mean _+ SEM of four experiments done in triplicate. Asterisk denotes statistical significance p < 0.05 (Student's t-test).
labelling the cells with [~4C]ethanolamine, indicating the conversion o f radioactive PE to PC through the methylation pathway (data not shown).
Effect of Acute Ethanol Treatment on the Uptake of Radiolabeled Nitrogenous Bases. The uptake o f [~4C]serine, [3H]choline and [~4C]ethanolamine was analyzed in cells in the presence o f 100 m M ethanol and radiolabel for 3 hours. No alteration in the incorporation o f either radiolabel was observed (Table I) when ethanol was present in the medium, thus indicating that acute ethanol exposure did not interfere with the incorporation o f either base into its corresponding phospholipid.
Effect of Long-Term Ethanol Treatment on the Incorporation of Radioactive Serine, Choline, and Ethanolamine into Phospholipids. The effect o f long-term ethanol exposure on the in vivo incorporation o f radioactive serine into PS was studied after different times o f labeling. Cells were treated with ethanol (100 raM) for 2 days before the addition o f label. No effect on the protein content o f the cells was detected. After 3 hours o f labeling, a significant increase in [~4C]PS was observed in cells exposed to ethanol. The increase was maintained up to 12 hours after addition o f [14C]serine (Fig. 2). The effect was already observed when the concentration o f ethanol was 25 m M (Fig. 3). A time-course study indicated that the increase o f radioactivity in PS was apparent after 24 hours o f ethanol exposure (Fig. 4). To ascertain whether this increased incorporation o f serine into PS was due to a slower disappearance o f label from PS, an experiment was performed where the cells
Rodrlguez, Ailing, and Gnstavsson Radioactivity In PS (cpm/mg protein)
Radioactivity In PS (cpm/mg protein)
o conlro| acute ethanol
50 100 200
. . . . . . . . . 1
Time after withdrawal of label (hours) Fig. 3, Effect of different ethanol concentrations on the accumulation of radioactive serine into phosphatidylserine. NG 108-15 cells were incubated in the presence of different concentrations of ethanol for 2 days. Afterwards, the cells were incubated with radioactive serine (0.5 gCi/ml), in the presence of the corresponding ethanol concentration, for 180 rain and the radioactivity measured in PS. The acute group was exposed to 100 mM ethanol only during the labelling period (180 min) Results are the mean _+ SEM of three experiments done in triplicate. Asterisk indicates statistical significance compared to both control and acute ethanol groups (p < 0.05, Student's t-test).
Radioactivity in PS (cpm/mg protein) 7000-
6000' 5000' 4OOO30002000 1000 0
/// /// /// ///
/// /// /// /.,-/ /// ///
/// //J /// /// /// //"/ /// //# /// ///
Fig. 4. Influence of time of ethanol exposure on the uptake of radioactive serine into PS. Control (open bars) and ethanol-exposed cells (100 mM for the times indicated) (hatched bars) were incubated with [14Clserine (0.5 lttCi/ml) for 180 rain, harvested, and radioactivity in PS measured. Results are expressed as mean + SEM of three experiments done in triplicate. All ethanol values are statistically significant when compared with the corresponding control values (p < 0.05, Student's t-test).
were labeled with radioactive serine for 3 hours9 Afterwards, the radiolabel was washed out and the radioactivity determined in PS after different periods of time in cells maintained in fresh medium with no radiolabel present. Fig. 5 shows a parallel initial rate o f decay o f
Fig. 5. Effect of chronic ethanol exposure on the time-dependent disappearance of radioactivity from radiolabelled PS9 Control (open squares) and ethanol-treated cells (100 mM, 2 days) (filled squares) were incubated with 0.5 l.tCi/ml radioactive serine for 180 rain. At time zero, radiolabel was withdrawn and fresh medium added to the cells. Results are the mean ___ SEM of four experiments done in triplicate. Statistical significance (*) was assessed by Student's t-test (p < 0.05).
radioactivity in PS in control and ethanol-treated cells. This indicates that ethanol did not interfere with the degradation o f the newly synthesized radioactive PS. The possibility that ethanol would affect the transport of radioactive serine into the intracellular compartment was examined by incubating the cells with 0.5 gCi/ml of radiolabelled serine. After 3 hours o f incubation the label was withdrawn and the cells were washed three times with 50 mM ice-cold TRIS-HCI buffer (pH 7.4), harvested, and the radioactivity measured. The radioactivity accumulated in the control cells (344,000 _+ 16,000 cpm/mg protein) was not significantly different from the radioactivity accumulated in cells treated with 100 mM ethanol for 2 days (291,000 _+ 33,000 cprn/mg protein) (means + SEM o f nine separate determinations). These results indicate that ethanol did not interfere with the process of uptake of serine to the intracellular space. The incorporation o f ['4C]ethanolamine and [3H]choline into PE and PC respectively was also studied in NG 108-15 cells incubated with ethanol (100 raM) for 2 days. Control and ethanol-treated cells were incubated in the presence of either [3H]choline (2 !aCi/ml) or ['4C]ethanolamine (2 gCi/ml) for 180 rain, harvested and the radioactivity incorporated in PC and PE determined. Ethanol had no effect on the incorporation o f [~4C]ethanolamine into PE (Radioactivity in PE: 190,000 _+ 12,000 cpm/mg protein in ethanol-treated cells corn-
Ethanol and Base-Exchange enzymes
Table II. Incorporation of ['aC]Serine into PS in NG 108-15 Microsomes
nmol of PS/mg protein Time of Labeling (min)
Ethanol Added In Vitro
Ethanol (2 days)
1.4 + 0.2 2.5 _+ 0.2
1.3 + 0.1 2,4 • 0.2
1.8 _+ 0.1" 3.3 +_ 0.2*
All values are the mean _+ S.E.M. of three experiments done in triplicate. Cells were labeled for 5 or 20 minutes in the presence of approximately 50 BM [L4C]serine as indicated in Experimental procedure section and radioactivity determined in PS after separation on TLC plates. In the ethanol added in vitro group, 100 mM ethanol was added to the tube during the assay. The ethanol (2 days) group represents the microsomes isolated from cells which were treated with 100 mM ethanol for 2 days. Asterisk indicates statistical significance (p<0.05, Student's t-test)
pared to 166,000 + 33,000 cpm/mg protein in controls. The results are the mean _+ SEM of three experiments done in triplicate) but significantly reduced the incorporation of [3H]choline into PC (Radioactivity in PC: 79,000 _+ 3,000 cpm/mg protein in ethanol-treated cells versus 103,000 + 5,000 cpm/mg protein in control cells. The results are the mean + SEM of three experiments done in triplicate. Statistical significance: p < 0.05, Student's t-test). Effect of Ethanol on the in Vitro Incorporation of [14C]Serine into PS in NG 108-15 Microsomes. To further investigate if the effect of long-term ethanol exposure on serine uptake could also be observed in an in vitro assay of base-exchange enzyme, the uptake of radioactive serine was studied in microsomal fractions. The incorporation of [~4C]serine into PS was increased in microsomes isolated from cells treated with 100 mM ethanol for two days, whereas no alteration was observed when the same concentration of ethanol was added to control microsomes during the assay (Table II). Effect of Chronic Ethanol Treatment on the Mass ofPhospholipids. NG 108-15 cells were treated with t 00 mM ethanol for 2 days and the amount of phosphorus in different phospholipids was determined after separation by TLC. The following results shown are the mean + SEM of three separate experiments done in triplicate. Ethanol induced a significant increase in the amount of phosphatidylserine (15.4 _+ 1.07 nmol/mg protein in ethanol-treated cells compared to 12.6 + 0.6 nmol/mg protein in control cells, p < 0.05, Student's t-test). No significant changes were observed among the other phospholipids analyzed: PC (120 _+ 8 nmol/mg protein in ethanol-treated cells compared to 107 _+ 4 nmol/mg protein in control cells), PE (36 + 2 nmol/mg protein in ethanol-treated cells compared to 34 _+ 2 nmol/mg
protein in control cells) and Phosphatidylinositol, PI (16.9 + 1.6 nmol/mg protein in ethanol-treated cells compared to 14.5 _+ 1.1 in control cells) or in the total amount of phospholipids (Data not shown).
DISCUSSION The present study demonstrates that the in vivo and the in vitro incorporation of radiolabelled serine into PS was augmented by ethanol in NG 108-15 cells. This effect was only apparent when ethanol was present for at least one day of exposure. Moreover, the effect was observed at ethanol concentrations as low as 25 mM, indicating that this phenomenon can occur at ethanol concentrations which are obtainable in vivo, in humans. The uptake of [t4C]serine into PS was increased after chronic ethanol treatment. There was an increase in the initial uptake rate. After washing the label away, the initial rate of disappearance of radioactivity was not affected. This observation indicates that ethanol is able to induce an increased PS biosynthesis, via the baseexchange reaction. Notably, the augmented incorporation of radioactive serine into PS occurred both in intact cells and in microsomes. Since the only known mechanism for PS synthesis in mammalian cells is the baseexchange reaction which is predominantly present in the endoplasmic reticulum (5,41,42), our results from microsomes further support the implication of the baseexchange reaction in the observed effects of ethanol. In addition, as a consequence of an increased synthesis an augmentation in the amount of PS occurred. It has been reported that the incorporation of radioactive serine into PS through the base-exchange reaction was increased in synaptosomes obtained from ethanol-dependent rats (34). On the other hand, a recent report (28) has shown that the biosynthesis of PS, measured by analyzing the incorporation of radiolabelled serine into PS, was decreased in newborn rat pup cerebrum after in utero exposure to ethanol. The differences observed in rats may be due to different sensitivities of base-exchange enzymes depending on the stage of development or cell populations (35). Serine base-exchange reaction serves as the only established pathway for the synthesis of PS in mammalian cells, whereas choline and ethanolamine baseexchange reactions represent one in several alternatives for the biosynthesis of PC and PE respectively, and basically would participate in the remodelling of pre-existing phospholipids (8,9). An important finding in this study is that ethanol selectively induced an increased serine uptake and affected the incorporation of other ba-
310 ses in a different manner. Ethanol had no effect on the incorporation o f r a d i o l a b e l l e d ethanolamine into PE. The incorporation o f radioactive choline into PC was slightly inhibited by ethanol, but no significant quantitative changes in PC were detected. The biosynthesis o f PC occurs through several metabolic pathways and we can not exclude that ethanol m a y affect them in different manners. It m a y be possible that ethanol only affects the incorporation o f radiolabel into a pool o f PC (for instance trough base-exchange reaction), which is very small compared to the total amount o f PC and accordingly those changes would be difficult to detect when measuring the total amount o f PC. Several possibilities should be considered for the mechanism by which ethanol increased serine baseexchange activity. Such a mechanism could be caused b y a direct interaction between alcohol and the enzyme(s) or by altering some enzymatic regulatory factors such as the lipid environment or the concentration o f Ca 2+ (34,36-38). Since the effect appeared after chronic ethanol exposure, a possible effect on the expression o f the protein should not be ruled out. Several studies have indicated the regulation o f base-exchange reactions by factors such as temperature (12), divalent cations (5), substrate concentration (13), substrate competition (7) or GTP (15). Ethanol also effects serine base-exchange reaction. Ethanol treatment has been reported to increase anionic phospholipids in rodents (20,21). In another study a decrease in the content o f acidic phospholipids after chronic ethanol exposure has been shown (22). In our laboratory we have found that ethanol influences the content o f acidic phospholipids depending on the pattern o f alcohol exposure (23,24). Regardless o f differences encountered in the literature, (possibly due to the use o f different protocols o f treatment, biological systems, ethanol concentration, etc.) ethanol seems to have some influence on negatively charged phospholipids. Consequently, these changes in the composition o f specific phospholipids m a y influence the function o f membranebound proteins, b y altering the composition o f specific lipid domains. PS participates in the modulation and regulation o f different proteins, such as receptors and enzymes. Therefore, the control o f its metabolism m a y play a crucial role in cell function. It is known that PS is involved in the functioning o f membrane-bound enzymes such as Na+-K+-dependent ATPase (39) or Ca2+-dependent ATPase (40). Changes in the content o f PS could have an important influence in synaptic neurotransmission by altering for instance the release o f neurotransmitter to the synaptic space (34). The increase o f PS biosynthesis as
Rodrlguez, Ailing, and Gustavsson a result o f ethanol exposure, observed in this study, could therefore affect cell function. In conclusion, we have reported in this study that ethanol raises the incorporation o f serine into PS. This effect induces a net increase in the content o f PS in the cell. The consequences o f this finding need further analysis and consideration.
ACKNOWLEDGMENTS This work was supported by the Swedish Alcohol Research Fund, The Albert P~hlson Foundation and the Swedish Medical Research Council (projects no 03P-08895, 21X-05249 and 04X-10837) and the University of Salamanca (Spain). The technical assistance of Mrs Befit F/irjh is gratefully acknowledged.
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