PHOSPHATIDYLETHANOL MIMICS ETHANOL MODULATION OF p42/44 MITOGEN-ACTIVATED PROTEIN KINASE SIGNALLING IN HEPATOCYTES
Annayya R. Aroor,
Geoffrey W. Custer,
Yu-I. Weng,
Youn Ju Lee and
Shivendra D. Shukla*
Department of Pharmacology, School of Medicine, University of MissouriColumbia, School of Medicine, Columbia, MO 65212, USA
Received 28 December 2001;
in revised form 14 March 2002;
accepted 4 April 2002
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ABSTRACT
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Aims: Although long-term exposure of hepatocytes to ethanol results in agonist-selective potentiation of p42/44 mitogen-activated protein kinase (MAPK) activation, mediators of this effect of ethanol are not known. Methods: We examined the role of phosphatidylethanol (PEth), a novel phospholipid formed exclusively in the presence of ethanol. Results: PEth accumulated in primary cultures of rat hepatocytes treated with ethanol. Exogenously added PEth potentiated angiotensin II-stimulated p42/44 MAPK similarly to that observed with ethanol treatment of cells for 24 h, a condition where PEth accumulates. PEth levels remained elevated 2 h after ethanol removal subsequent to a 24-h exposure, and the potentiating effects of ethanol were also present. PEth did not potentiate p42/44 MAPK activation by either epidermal growth factor or vasopressin, thus further mimicking the known agonist selectivity for this ethanol effect. Conclusions: These results offer a novel role for PEth as a mediator in the ethanol modulation of p42/44 MAPK cascade in hepatocytes.
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INTRODUCTION
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Acute and chronic exposure to ethanol leads to alterations in hepatic carbohydrate and lipid metabolism as well as protein and DNA synthesis (Bailey and Cunningham, 1998
; Yang et al., 1998
; Lieber, 2000
). However, the mechanisms and mediators of cellular adaptation to ethanol remain largely unknown. Ethanol modulates several signalling pathways (Hoek et al., 1987
, 1992
; Diehl et al., 1992
; Thurston and Shukla, 1992
; Shukla et al., 2001
), including agonist-stimulated p42/44 mitogen-activated protein kinase (MAPK) signalling in hepatocytes (Reddy and Shukla, 1996
; Chen et al., 1998
; Tomber et al., 1998
; Mckillop et al., 1999
; Reddy and Shukla, 2000
). Recently we reported agonist selectivity of ethanol potentiation of p42/44 MAPK signalling in hepatocytes (Weng and Shukla, 2000
). Although agonists acting at receptor tyrosine kinase [epidermal growth factor (EGF) and insulin] and G-protein coupled receptors (angiotensin II, adrenaline and vasopressin) caused p42/44 MAPK activation, ethanol selectively potentiated angiotensin II- and adrenaline-stimulated p42/44 MAPK activation. These effects of ethanol may be direct or mediated indirectly through metabolites derived from ethanol. Acetaldehyde alone was ineffective in mimicking the effects of ethanol on potentiation of agonist-stimulated p42/44 MAPK activity (Weng and Shukla, 2000
). Phosphatidylethanol (PEth) is a novel ethanol-derived bioactive lipid formed by a phospholipase D-catalysed reaction (Alling et al., 1983
). This abnormal phospholipid is formed exclusively in vivo during ethanol exposure in various animal tissues, including brain (Alling et al., 1984
). PEth accumulation in hepatocytes has been shown after alcohol administration in vivo and in vitro (Alling et al., 1983
; Gustavsson et al., 1990
). The accumulation of PEth may account for some of the pathophysiology associated with chronic ethanol use, as PEth is degraded slowly (Gustavsson et al., 1990
). PEth accumulates after phorbol ester stimulation of lymphocytes from chronic alcoholics, as compared to lymphocytes obtained from individuals who use ethanol moderately (Mueller et al., 1988
). The concentration of PEth in the blood from chronic alcoholics is in the range of 017.8 µmol/l (Varga et al., 2000
). PEth induces tolerance to fluidization by ethanol in artificial bilayers and inhibits ethanol activation of Na+-K+ ATPase in crude brain membranes (Lundqvist et al., 1993
), all of which have been attributed to chronic ethanol treatment. PEth has also been shown to stimulate phosphoinositide hydrolysis in neuronal cells, as does ethanol (Omodeo-Sale et al., 1991
). PEth activates protein kinase C (PKC) in vitro (Asaoka et al., 1988
) and in vivo (Aroor and Baker, 1996
) and phospholipase A2 in vitro (Chang et al., 2000
), thus suggesting a role for PEth in ethanol modulation of signal transduction. In the present study, we have investigated the effects of PEth on angiotensin II-stimulated p42/44 MAPK activation.
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MATERIALS AND METHODS
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Materials
Myelin basic protein (MBP) was purchased from Invitrogen (Carlsbad, CA, USA). [
-32P]ATP (specific activity 3000 Ci/mmol) was bought from Perkin Elmer Life Sciences (Boston, MA, USA). PEth (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanol) and p42/44 MAPK antibodies were purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA, USA) and Upstate Biotechnology (Lake Placid, NY, USA) or Cell Signaling Technology, Inc. (Beverly, MA, USA) respectively. Nitrocellulose and goat-anti (rabbit IgG) secondary antibody were purchased from Bio-Rad Laboratories (Hercules, CA, USA). All other chemicals were from Sigma Chemical Company (St Louis, MO, USA).
Isolation and culture of rat hepatocytes
Hepatocytes were isolated from SpragueDawley male rats weighing 200250 g by the two-step perfusion procedure using 0.035% collagenase as described earlier (Seglen, 1976
; Weng and Shukla, 2000
). Cell viability, assessed by exclusion of trypan blue, was 90 ± 5%. Isolated cells were resuspended in hepatocyte medium [Dulbeccos modified Eagles medium containing penicillin (100 U/ml) and streptomycin (100 µg/ml) supplemented with 10% fetal bovine serum]. Cells were plated onto rat collagen-coated culture dishes in the same medium at a density of 1 x 105 cells per cm2 in 60 or 100 mm dishes in 4 or 10 ml of hepatocyte medium, respectively. Two hours after plating, the medium was replaced with Dulbeccos modified medium containing penicillin (100 U/ml) and streptomycin (100 µg/ml). The dishes treated with ethanol were wrapped with parafilm to prevent evaporation of ethanol. The control dishes, as well as dishes used for treatment of hepatocytes with agonists and various lipids, were wrapped with parafilm to simulate identical treatment conditions.
Preparation of cell extracts
After treatment of hepatocytes, the cells were washed with ice-cold phosphate-buffered saline (PBS) and 0.5 ml of lysis buffer (10 mM HEPES, pH 7.5 containing 10 mM ß-glycerophosphate, 1 mM EDTA, 1 mM EGTA, 1 mM phenylmethylsulphonyl fluoride, 1 mM sodium orthovanadate, 1 mM dithiothreitol (DTT), 1 mM benzamidine, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 10 µg/ml pepstatin A. Cell lysates were prepared by scraping, followed by sonication and centrifugation for 30 min at 15 000 g. Protein concentration in the supernatant was determined by Bio-Rad DC protein assay kit and the samples were stored at 80°C.
Measurement of MAPK activity
A myelin basic protein in-gel kinase assay to measure p42/44 MAPK phosphotransferase activity was utilized as described previously (Weng and Shukla, 2000
). Briefly, equal amounts of extracts (20 µg) were fractionated on sodium dodecyl sulphate (SDS)polyacrylamide gels (10%) containing MBP (0.5 mg/ml), and SDS was removed by incubation in 50 mM Tris, pH 8.0, containing 20% isopropanol for 1 h at room temperature. The protein in the gel was denatured in 6 mol/l guanidium chloride and renatured overnight in 50 mM Tris, pH 8.0, containing 50 mM 2-mercaptoethanol and 0.1% Triton X-100 with several changes of buffer. The gel was incubated in kinase buffer containing 40 mM HEPES, pH 8.0, 1.5 mM EGTA, 40 µM ATP, 10 mM MgCl2, 2 mM DTT, and [
-32P]ATP (5 µCi/ml, 3000 Ci/mmol). The gel was washed with 5% trichloroacetic acid containing 1% sodium pyrophosphate (w/w), then dried and exposed to an X-ray film. Relative kinase activities were determined by scanning each band with a laser densitometer.
Immunoblotting
For immunoblots, proteins in extract supernatants (20 µg/lane) were separated by SDSpolyacrylamide gel electrophoresis (10% acrylamide) and transferred to nitrocellulose paper. The membrane was blocked for 2 h at room temperature in Tris-buffered saline containing 0.1% Tween 20 (TBST) and 5% non-fat dry milk. The blots were incubated overnight at 4°C with diluted primary antibody (1:1000) in TBST3% bovine serum albumin. After three washes in TBST, membranes were incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Proteins were detected by the Pierce ECL (enhanced chemiluminescence) method.
PEth biosynthesis and degradation in cultured hepatocytes
Primary cultured rat hepatocytes were treated with 100 mM ethanol for 24 h, washed in ice-cold PBS and the lipids extracted (Omodeo-Sale et al., 1991
). PEth and phosphatidic acid standards were run on thin layer chromatography plates along with cell lipid extract by published methods (Nakamura and Handa, 1984
). Phospholipids were separated on Whatman silica gel 60 plates using an ethylacetate/isooctane/ acetic acid (61:28:11, v/v) water-saturated solvent system. After the chromatographic run, plates were dried and immersed in methanol/distilled water/glacial acetic acid (20:79:1) containing 0.03% brilliant blue R for 1 h. The plate was then immersed in 20% methanol in water for 10 min. After drying of the plates, the PEth was quantified by densitometry. The quantification was linear in the range of 0.5 to 10 nmol.
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RESULTS
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Accumulation and degradation of PEth in hepatocytes
The accumulation of PEth was assessed by treating hepatocytes with 100 mM ethanol for 24 h. Under these conditions, 189 ± 32.8 pmol of PEth/mg of cell protein were formed (Fig. 1
). In control (non-ethanol treated) cells, we were unable to detect any lipid band (by Coomassie blue staining) corresponding to the PEth region. The metabolic fate of endogenous PEth was followed by removing the ethanol from the medium and replacing it with ethanol-free medium. Compared to PEth accumulated at 24 h (100%), the levels of PEth at 2 and 20 h after ethanol removal were 73 and 26%, respectively. These studies show that significant accumulation of PEth occurred after ethanol treatment and that endogenously accumulated PEth remained elevated even after ethanol withdrawal.

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Fig. 1. Accumulation of phosphatidylethanol (PEth) in ethanol-treated hepatocytes analysed by thin layer chomatography. Hepatocytes were cultured for 24 h in the presence of 100 mM ethanol. After 24 h, the ethanol-containing medium was removed; the cells were then washed and further cultured in ethanol-free medium. Lipids were extracted and analysed by thin layer chomatography and Coomassie blue staining as described in Materials and methods. PEth together with phosphatidic acid (PA) standards were run in lanes 1 (8 nmol), 2 (4 nmol), 3 (2 nmol), 4 (1 nmol) and 5 (0.5 nmol). Lane 6, 24-h ethanol treatment; lane 7, 24-h ethanol treatment followed by no ethanol for 2 h. Thin layer chomatographic profile is from a typical experiment. There was no detectable PEth in control (non-ethanol treated) cells (not shown). The results are given as mean ± SEM (bars) of thee independent experiments.
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Potentiation of angiotensin II-stimulated p42/44 MAPK in hepatocytes treated with PEth
We have previously reported the ethanol potentiation of angiotensin II stimulation of p42/44 MAPK in hepatocytes after 24 h of exposure to ethanol (Weng and Shukla, 2000
). Under these conditions,
80% of the added ethanol remained in the dishes. Ethanol had no effect on angiotensin II-stimulated p42/44 MAPK activity when ethanol treatment of hepatocytes was for
12 h (Weng and Shukla, 2000
). The observed potentiation is therefore not a direct physical effect of ethanol. Ethanol did not show MAPK potentiation in hepatocytes exposed to lipopolysaccharide or cytokines. A role for acetaldehyde, the oxidatively derived metabolite of ethanol, has been ruled out in this potentiation (Weng and Shukla, 2000
). We therefore next examined the role of PEth in this phenomenon. PEth was prepared as a sonicated aqueous lipid emulsion in Dulbeccos medium. The cells were incubated with 20 µM PEth for 4 h and their extracts were analysed for p42/44 MAPK activity as determined by an in-gel kinase assay. Although PEth alone did not significantly activate p42/44 MAPK in hepatocytes, PEth treatment caused significant potentiation of angiotensin II-stimulated p42/44 MAPK activation (Fig. 2
). The increase in the p42/44 MAPK activity, as determined by the in-gel kinase assay, correlated with the increase in levels of activated forms of p42/44 MAPK, as determined by immunoblotting techniques to detect dually phosphorylated/activated p42/44 MAPK (Fig. 2
). The levels of p42/44 MAPK protein remained unaltered by PEth treatment thus excluding increased expression of p42/44 MAPK as the underlying cause for PEth potentiation of angiotensin II stimulated p42/44 MAPK activation (Fig. 2
).

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Fig. 2. Effects of phosphatidylethanol (PEth) on angiotensin II (Ang II)-stimulated p42/44 mitogen-activated protein kinase (MAPK) in hepatocytes. Primary cultures of hepatocytes were held in serum-free medium for 18 h, after which hepatocytes were treated with 20 µM PEth for 4 h. The cell extracts (20 µg of protein) were processed for p42/44 MAPK in-gel kinase assay and immunoblot analysis as described in Materials and methods. The p42/44 MAPK protein levels were the same, indicating that differences in the activation are not due to protein expression and that equal loading was also achieved in each lane. Results are representative of four independent experiments.
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The effects of PEth were dose dependent, except that a weaker inhibition of angiotensin II-stimulated p42/44 MAPK activation was observed at 50 µM concentrations of PEth (Fig. 3
). The incorporation of PEth into cells has been shown to require a few hours in cultured cells (Lundqvist et al., 1993
). When hepatocytes were treated with PEth for 10 min, PEth had no effect on angiotensin II-stimulated p42/44 MAPK (Fig. 3
). However, a 1 h incubation of hepatocytes with PEth caused potentiation of angiotensin II-stimulated p42/44 MAPK, although the magnitude of the effects at 1 h (control: 1.45/1.50-fold increase; PEth: 1.9/1.9 fold increase) was not as significant, as compared to treatment of hepatocytes with PEth for 4 h. In experiments where 20 µM PEth was added to cells for 4 h, we were able to detect, in hepatocytes, 760 ± 26 pmol PEth/mg protein. The specificity of PEth was investigated by comparing the effects of phosphatidylcholine (PC). Phosphatidylcholine (aqueous sonicated suspension) at a concentration of 20 µM had no significant effect on angiotensin II-stimulated p42/44 MAPK activation in control cells (data not shown).

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Fig. 3. Dose- and time-dependent effects of phosphatidylethanol (PEth) on p42/44 mitogen-activated protein kinase (MAPK) activation in hepatocytes. Primary cultures of hepatocytes were held in serum-free medium for 18 h, after which hepatocytes were treated with different concentrations of PEth for 4 h (upper panel) or treated with 20 µM PEth for 10 min and 1 h (lower panel). Cells were subsequently stimulated with 100 nM angiotensin II (Ang II) for 5 min and extracts were processed for phospho-p42/44 MAPK immunoblot analysis as described in Materials and methods. Results are representative of three independent experiments.
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Agonist-selective effects of PEth in potentiation of agonist-stimulated p42/44 MAPK
It has been demonstrated earlier that ethanol did not potentiate p42/44 MAPK activated by epidermal growth factor or vasopressin, but potentiated that by angiotensin II (Weng and Shukla, 2000
). We took advantage of this finding to determine whether the PEth effect was also agonist specific. As shown in Fig. 4
, similar to ethanol, PEth markedly potentiated angiotensin II-stimulated p42/44 MAPK, but had no potentiating effect on the EGF or vasopressin responses.

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Fig. 4. Agonist-selective effects of phosphatidylethanol (PEth) on p42/44 mitogen-activated protein kinase (MAPK) activation in hepatocytes. Primary cultures of hepatocytes were held in serum-free medium for 18 h after which hepatocytes were treated with 10 µM PEth for 4 h. This was followed by treatment of hepatocytes with epidermal growth factor (EGF) (10 ng/ml), angiotensin II (100 nM) or vasopressin (100 nM) for 5 min. The cell extracts were processed for p42/44 MAPK immunoblot analysis as described in Materials and methods. Results are representative of four independent experiments. Densitometric analyses of bands are shown as histograms in the lower panels and are presented as mean ± SEM (bars) for four experiments. Compared to control (no PEth) the PEth-treated samples were significantly different (*) where P-values were 0.0011 for p42 and 0.0019 for p44 MAPK.
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Persistent effects of ethanol on angiotensin II-stimulated p42/44 MAPK activation after ethanol withdrawal
The turnover rate of PEth is slow and accumulation of PEth has been shown after several hours of ethanol withdrawal in pancreatic islets, hepatocytes and NG-108 cells (Lundqvist et al., 1993
). In order to examine the potential role of PEth after removal of ethanol from the medium, hepatocytes were treated with ethanol for 24 h, and the medium was subsequently replaced by Dulbeccos modified Eagles medium containing 0.1% fetal bovine serum without ethanol. The cells were harvested at 2 and 20 h after ethanol withdrawal. As shown in Fig. 5
, potentiation of angiotensin II-stimulated p42/44 MAPK activation was observed at 2 h after ethanol withdrawal (control: 1.5/2.4-fold increase; 2 h ethanol withdrawal: 2.0/3.5-fold increase). However, the effects of ethanol were abolished at 20 h after ethanol withdrawal (Fig. 5
). PEth level did not decrease significantly at 2 h, but did at 20 h after ethanol withdrawal (Fig. 1
). Thus, a correlation between PEth level and the MAPK potentiation was observed. These results support a role for PEth as an endogenous modulator of angiotensin II-stimulated p42/44 MAPK activation.

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Fig. 5. Residual effects of ethanol on angiotensin II (Ang II)-induced p42/44 mitogen-activated protein kinase (MAPK) activation in hepatocytes. Primary cultures of hepatocytes were treated with 100 mM ethanol for 24 h followed by replacement with ethanol-free medium. Cells were harvested 2 and 20 h after ethanol withdrawal and were then stimulated with Ang II (100 nM) for 5 min. The cell extracts were processed for p42/44 MAPK immunoblot analysis as described in Materials and methods. Results are representative of two independent experiments.
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DISCUSSION
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This is the first report establishing the ethanol-mimetic effects of PEth in ethanol modulation of MAPK signal transduction in hepatocytes. The role of PEth as an important mediator in ethanol potentiation of angiotensin II-stimulated p42/44 MAPK is provided by three lines of evidence. First, potentiation of angiotensin II-stimulated p42/44 MAPK by PEth addition was observed with pathophysiologically relevant concentrations of PEth. Second, there was similar agonist selectivity between PEth and ethanol. Third, after ethanol removal, the potentiating effects persisted as long as PEth levels remained elevated.
PEth is a unique phospholipid produced by the phospholipase D-catalysed transphosphatidylation reaction in the tissues exposed to ethanol. The synthesis of PEth has the potential to disrupt cellular function both by inhibition of normal phospholipase D products involved in intracellular signalling, and through the direct effects of PEth. The present study provides evidence for the direct effects of PEth in mimicking ethanol potentiation of angiotensin II-stimulated p42/44 MAPK signalling in hepatocytes. Furthermore, phosphatidylcholine was not effective in potentiating angiotensin II-stimulated p42/44 MAPK, thus indicating that the effects of PEth were not a non-specific lipidic effect. This raises the intriguing possibility that PEth specifically modulates agonist-stimulated signal transduction.
The mechanisms underlying the agonist-selective effects of ethanol and PEth are presently unknown and therefore require investigation. Whereas ethanol and PEth potentiated angiotensin II-stimulated MAPK, EGF- or vasopressin-stimulated MAPK was not affected by ethanol or PEth. Recent studies demonstrated PKC-dependent stimulation of MAPK by angiotensin II and PKC-independent stimulation by EGF in primary cultures of hepatocytes (Dajani et al., 1999
). Since ethanol and PEth have been shown to activate PKC, therefore PKC dependency may account for the agonist selectivity. However, vasopressin-stimulated MAPK has also been shown to be PKC-dependent, yet is not affected by ethanol. Therefore, other factors may also contribute to this selectivity. Interestingly, such complexity was also noted with angiotensin II effects on c-fos expression, which was PKC dependent, but vasopressin was not effective in stimulating c-fos expression in primary culture of hepatocytes (Gonzalez-Espinosa and Garcia-Sainz, 1992
).
Ample evidence exists that ethanol influences the concentration of key intracellular signal molecules, such as cyclic adenosine 3'5'-monophosphate, stimulatory and inhibitory G-proteins, and the activities of various protein kinases (Hoek et al., 1987
, 1992
; Diehl et al., 1992
; Thurston and Shukla, 1992
; Mckillop et al., 1999
). Direct effects of PEth on membrane structure, which have been reported, include a decrease in membrane fluidity, and fusion of membranes (Omodeo-Sale et al., 1991
). PEth activates protein kinase C in vitro (Asaoka et al., 1988
) and in vivo (Aroor and Baker, 1996
) and phospholipase A2 in vitro (Chang et al., 2000
). It is therefore tempting to speculate that many of the signal modulatory effects of ethanol may be attributable to PEth. The effect of PEth is also mimicked after a few hours of ethanol withdrawal, suggesting that this may underlie the mechanism involved in the persistent effects of ethanol during intermittent consumptions and in cellular adaptation to ethanol in individuals who misuse alcohol.
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ACKNOWLEDGEMENTS
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We thank Dr Steven Halenda for helpful discussions and Ms Pam Burgess for typing the manuscript. This work was supported by NIAAA grant AA11962.
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FOOTNOTES
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* Author to whom correspondence should be addressed. 
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