Ethanol dissociates hormone-stimulated cAMP production from inhibition of TNF-alpha production in rat Kupffer cells

Andrew Aldred and Laura E. Nagy

Department of Nutrition, Case Western Reserve University, Cleveland, Ohio 44106


    ABSTRACT
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Abstract
Introduction
Materials and methods
Results
Discussion
References

Ethanol impairs hormone-stimulated cAMP production in a number of cell types, yet the effects of ethanol on downstream responses mediated by cAMP-dependent protein kinase (PKA) are not understood. Here we have investigated the effects of ethanol feeding on cAMP-mediated inhibition of tumor necrosis factor-alpha (TNF-alpha ) synthesis in rat Kupffer cells. Male Wistar rats were fed liquid diets containing 36% of calories as ethanol for 4 wk or were pair fed a control diet. Stimulation of cAMP production by the adenosine A2 receptor agonist 5'-(N-ethylcarboxamido)-adenosine (NECA), prostaglandin E2, or forskolin was decreased to 25% of control values in Kupffer cells isolated from ethanol-fed rats. This decrease was associated with a reduction in the quantity of immunoreactive Gsalpha protein in ethanol-fed rats, with no changes observed in Gialpha or Gbeta . TNF-alpha production was higher in ethanol-fed rats in response to stimulation with lipopolysaccharide or latex beads. Despite the profound reduction in the ability of hormone to increase cAMP production, NECA and prostaglandin E2 inhibited TNF-alpha production to an equivalent degree in Kupffer cells from ethanol- and pair-fed rats. Total activity and immuoreactive protein quantity of PKA did not differ between groups. Activation of PKA in response to a 15-min treatment with 1 µM NECA was reduced by 50% in ethanol-fed rats compared with control. Despite this reduction in activation, translocation of the catalytic subunit of PKA to the nucleus and phosphorylation of cAMP response element binding protein in response to activation were observed in Kupffer cells from both ethanol- and pair-fed rats. These data demonstrate that there is a dissociation between ethanol-induced desensitization of hormone-stimulated cAMP production in rat Kupffer cells and the downstream inhibition of TNF-alpha production mediated by cAMP.

G protein; tumor necrosis factor-alpha ; desensitization; lipopolysaccharide; macrophage; adenosine 3',5'-cyclic monophosphate


    INTRODUCTION
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Abstract
Introduction
Materials and methods
Results
Discussion
References

cAMP IS AN important regulator of cellular metabolism, gene transcription, differentiation, and proliferation. Ethanol disrupts hormone-stimulated cAMP production in almost all cell types examined. In many cells types, long-term exposure to ethanol both in vivo and in culture decreases cAMP production via receptors coupled to the stimulatory G protein, Gs, producing a heterologous desensitization (7). However, the interaction of ethanol with cAMP signalling pathways can vary among cell types. For example, long-term treatment of primary cultures of hepatocytes with ethanol increases receptor-stimulated cAMP production (29, 30). Similarly, long-term ethanol feeding in vivo increases cAMP production in liver membranes (3, 20) and adipocytes (41). Despite these clear effects of ethanol on cAMP production, very little is known about the consequences of these changes in cAMP production on downstream responses mediated by cAMP-dependent protein kinase (PKA). cAMP binds to the regulatory subunits (R) of PKA, inducing holoenzyme dissociation and the liberation of the catalytic subunit (PKA-C). Nuclear translocation of PKA-C is the rate-limiting step in the coupling of hormonal stimulation and cAMP-dependent gene transcription (16). Although activity of purified PKA is not affected by ethanol in vitro (24), chronic ethanol exposure decreases PKA activity in the embryonic chick brain (2) and rat striatum (34). Recent data also indicate that phosphorylation of the nuclear transcription factor cAMP response element binding protein (CREB) is increased after short-term, but not long-term, ethanol exposure in rat cerebellum (43). In contrast, treatment of NG108-15 neuroblastoma X glioma cell hybrids with ethanol results in the translocation of the C subunit to the nucleus, independent of hormonal activation (8). However, it is not known whether the C subunit is active under these conditions.

To investigate the relationship between ethanol-induced desensitization of cAMP production and downstream responses mediated by PKA, we have utilized a primary cell culture model of Kupffer cells. Kupffer cells, the resident macrophages in the liver, release fibrogenic and inflammatory cytokines, as well as superoxide anions, in response to bacterial lipopolysaccharide (LPS) or phagocytosis of cellular debris (42). Tumor necrosis factor-alpha (TNF-alpha ) is a major cytokine produced by Kupffer cells after exposure to LPS (33). cAMP mediates a feedback inhibitory pathway that decreases TNF-alpha production in Kupffer cells (33); elevation of cAMP in Kupffer cells by treatment with PGE2, beta -adrenergic agonists, adenosine A2 receptor agonists, and phosphodiesterase inhibitors decreases TNF-alpha and interleukin-1 production (4, 6, 13, 32, 35).

Activation of Kupffer cells is likely to play an important role in the development of localized inflammation and fibrosis in alcoholic liver disease, since inactivation of Kupffer cells by treatment with gadolinium chloride can prevent alcohol-induced liver injury in rats (1). Despite their recognized importance in the etiology of disease, very little is known about the effects of ethanol on Kupffer cell function. Therefore, we have investigated the effects of ethanol on hormone-stimulated cAMP production and subsequent inhibition of TNF-alpha production in Kupffer cells. By measuring the ability of hormones to inhibit TNF-alpha production, we were able to assess the impact of ethanol-induced desensitization of cAMP production on regulation of downstream responses mediated by PKA.


    MATERIALS AND METHODS
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Abstract
Introduction
Materials and methods
Results
Discussion
References

Materials. Adult male Wistar rats weighing 150 g were purchased from Harlan Sprague-Dawley (Indianapolis, IN). Liquid diet ingredients were as described (20). 125I-labeled cAMP was from ICN (St. Laurent, Quebec, Canada). Adenosine receptor agonists were purchased from Research Biochemicals (Natick, MA). [32P]ATP and the enhanced chemiluminescence detection kit were from Amersham (Oakville, Ontario, Canada). Antibodies to cAMP, Gsalpha , Gialpha , and the beta -subunit were from Gramsch Laboratories (Felsenfeldbruch, Germany) and New England Nuclear DuPont (Mississauga, Ontario, Canada). Adenosine deaminase, creatine kinase, human recombinant TNF-alpha , and the anti-mouse and anti-rabbit IgG-peroxidase antibodies were from Boehringer Mannheim (Dorval, Quebec, Canada). The PKA assay system was from GIBCO BRL (Burlington, Ontario, Canada). Monoclonal antibody to type I regulatory subunit of PKA (RI) was from Transduction Laboratories (Lexington, KY), and polyclonal antibodies to type II regulatory subunit of PKA (RII), phospho-CREB, and PKA-C were from Upstate Biotechnology (Lake Placid, NY). Ro 20-1724 was from Biomol Research Laboratories (Plymouth Meeting, PA). Texas red-conjugated anti-rabbit IgG and sytox green (nuclear stain) were from Molecular Probes (Eugene, OR). Antibodies to ED1 and ED2 were from Serotec (Toronto, Ontario, Canada). Other reagent grade chemicals were from Fisher Scientific (Missisaugua, Ontario, Canada) or Sigma (St. Louis, MO).

Animals and diets. Procedures using animals were approved by the Animal Care Committee at the University of Guelph or Case Western Reserve University. Adult male Wistar rats were housed in a temperature- and humidity-controlled environment with alternating 12:12-h light-dark cycles. After a 1-wk acclimatization period on standard rat chow, the animals were allowed free access to the LieberDeCarli liquid diet (23) containing 36% of calories from ethanol or were pair fed an identical isocaloric diet in which maltose dextrins were substituted for ethanol. After 4 wk, the animals were anesthetized with pentobarbital sodium, and Kupffer cells were isolated.

Livers were perfused via the portal vein first with modified Hanks' solution (free of Ca2+ and Mg2+) containing 1 mM EGTA and 10 mM HEPES and then with 0.05% collagenase (type I) in Williams E medium with 10 mM HEPES at a flow rate of 15 ml/min. A cell suspension was formed by gentle disruption of the collagenase-treated livers. The resulting suspension of cells was then treated further with 0.02% Pronase and 0.001% DNase for 15 min at 10°C (12). The cell suspension was then filtered over nylon (230- and 120-µm mesh) and washed two times by centrifugation at 500 g for 7 min in DMEM with 7.5% fetal bovine serum and 0.001% DNase. The final cell pellet was resuspended in Gey's balanced salt solution (GBSS) and 0.001% DNase, layered on a gradient containing 17.5 and 8.5% nycodenz, and centrifuged for 17 min at 1,500 g. In some control experiments, the collagenase-treated liver was not treated with Pronase. Instead, the liver cell suspension was centrifuged three times at 50 g for 5 min to pellet hepatocytes, and the supernatant was collected after each centrifugation. The pooled supernatant was then centrifuged at 500 g for 10 min to collect nonparenchymal cells. Kupffer cells were separated from this pellet on nycodenz as described above. The cells sedimenting between these two densities were collected by centrifugation at 500 g for 7 min and resuspended in 10 ml GBSS with 0.001% DNase. This sample was loaded on a centrifugal elutriator (2,500 rpm). Kupffer cells were collected at a flow rate of 45 ml/min. Isolated Kupffer cells were washed two times by centrifugation at 500 g for 7 min. Cells were either used immediately, plated for cell culture, or frozen at -70°C for future analysis. Cell viability was measured by trypan blue exclusion, and Kupffer cells were identified by endogenous peroxidase or naphthyl esterase activity (28) or the presence of the antigenic macrophage markers ED1 and ED2.

Cell culture. Isolated Kupffer cells were suspended in Iscove's modified Dulbecco's medium (IMDM) with 20% fetal bovine serum and penicillin-streptomycin at a concentration of 2 × 106 cells/ml and plated on tissue culture plates (96 well for TNF-alpha assay, 12 well for cAMP and PKA measurements, and LabTek chamber slides for immunohistochemistry). After 2 h, nonadherent cells were removed by aspiration, and fresh medium was supplied. Assays were carried out after 24 h in culture. Attached Kupffer cell number was constant over the 24-h culture period (data not shown).

Measurement of cAMP production. cAMP production in intact Kupffer cells was measured after 24 h in culture. Culture medium was aspirated, and cells were incubated in PBS with 25 mM HEPES, 10 mM glucose, 0.7 mM CaCl2, and 0.4 mM MgCl2, pH 7.4 (PBS), containing 1 µM Ro 20-1724, an inhibitor of phosphodiesterase, and 1 U/ml adenosine deaminase to remove any endogenously produced adenosine. Cells were then treated with and without agonists for 10 min as described in Figs. 1-6. Reactions were terminated by the addition of 0.1% Nonidet P-40 and 0.1 N HCl. Cells were incubated on ice for 10 min, and supernatants were removed and stored at -20°C for subsequent analysis for cAMP by RIA.

Western blot analysis. Kupffer cell homogenates or crude membrane preparations (29) were diluted to 1 mg protein/ml in SDS sample buffer. For the detection of Gialpha , Gsalpha , and Gbeta , 50 µg of crude membrane proteins were separated by SDS-PAGE using the Laemmli buffer system (22). Detection of PKA subunits was carried out in homogenates of cultured Kupffer cells. Proteins were then transferred to polyvinylidene difluoride membranes, and Western blotting was carried out as previously described (37). Immunoreactive protein quantity was assessed by scanning densitometry.

TNF-alpha assay. TNF-alpha secreted in the Kupffer cell culture medium was measured using a cytotoxicity assay. After culture for 20 h, cells were treated with LPS (Escherichia coli 026:B6) and/or receptor agonists as described in the Figs. 1-6. After a further 4 h in culture, cell culture medium was removed and stored at -20°C. Serial dilutions of the conditioned cell culture medium were added at 100-µl volumes to 96-well culture dishes. WEHI 164 cells (100 µl), which are sensitive to TNF-alpha cytotoxicity, were added to each well at 5 × 105 cells/ml in IMDM containing 10% FBS and 0.5 µg/ml actinomycin D. Standard curves were generated in each plate using human recombinant TNF-alpha . The plates were cultured for 20 h, and then 20 µl of 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide were added to each well. After 4 h, 150 µl of medium were removed from each well, and 100 µl of isopropanol-0.04 N HCl were added to develop metabolized crystals. The plates were then stored in the dark at room temperature overnight, and absorbance of each well at 550 nm was read in an ELISA plate reader. The concentration of TNF-alpha in the Kupffer cell medium was calculated based on the standard curve and was corrected for cell concentration in the original Kupffer cell culture.

Determination of PKA activity. Activity of PKA, as well as stimulation in response to 10-6 M 5'-(N-ethylcarboxamido)-adenosine (NECA), was measured by incubating cells with and without 10-6 NECA for 15 min in PBS. Reactions were terminated by the addition of ice-cold 10 mM potassium phosphate, pH 6.8, 10 mM EDTA, 0.5 mM isobutyl methylxanthine, 150 mM KCl, and 1% Triton X-100. Cells were extracted on ice for 30 min, and activity of PKA was assayed immediately. PKA activity was measured using the kemptide assay over a 5-min period in the presence (for total activity) or absence (for basal or hormone-activated activity) of 40 µM dibutyryl-cAMP. Nonspecific activity was measured by the addition of 4 µM protein kinase inhibitor peptide.

Immunohistochemistry. Kupffer cells cultured on LabTek chamber slides were washed two times in PBS and stimulated with or without 10-6 M NECA or 10 nM forskolin for 30 min. After stimulation, cells were fixed for 30 min in 4% freshly prepared paraformaldehyde. Some cells were preincubated with the fluorescent nuclear stain sytox green for 30 min before fixation to visualize the nucleus. Slides were then quenched by two 1-min washes in 25 mM glycine and one 5-min wash in PBS and were blocked in PBS with 2% BSA, 5% fish gelatin, and 0.02% saponin for 2 h at room temperature. Slides were then incubated with antibody to the PKA-C (1:200) or phospho-CREB (1:200) in blocking buffer overnight at 4°C. Slides were washed three times for 15 min in fresh blocking buffer and then incubated for 1 h with Texas red-conjugated anti-rabbit IgG (1:200) for 1 h in blocking buffer. Slides were finally washed three times for 15 min in blocking buffer and two times for 5 min in PBS. Slides were then mounted and examined by laser scanning confocal microscopy under a ×60 lens. Nonspecific binding was determined by incubation with secondary antibody alone.

Statistical analysis. Because of the limited number of Kupffer cells available from each animal, data from several feeding trials are presented in this study; each trial consisted of five rats per feeding group. Values reported are means ± SE. Data were analyzed by Student's t-test or the general linear models procedure blocking for trial effects if data from more than one trial were utilized. Differences between diet groups were assessed by Tukey's test or least-square means. Statistical analyses were performed on the SAS statistical package for the personal computer.


    RESULTS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

Kupffer cell isolation procedure. To understand the effects of ethanol on receptor-stimulated cAMP production in Kupffer cells, we first optimized the Kupffer cell isolation procedures to ensure that receptor-mediated cAMP production was not impaired. The standard procedure for Kupffer cell isolation involves digestion with Pronase, a relatively nonselective protease, to destroy hepatocytes contaminating the preparation. Because hormone/cytokine receptors are exposed at the cell surface, Pronase digestion may impair their function. Therefore, we measured receptor-stimulated cAMP production in Kupffer cells isolated under differing Pronase concentrations for increasing periods of incubation compared with Kupffer cells isolated in the absence of Pronase (collagenase only; Table 1). At low Pronase concentrations, short digestion times, and lower digestion temperatures (data not shown), receptor-stimulated cAMP signal transduction and plating efficiency in Pronase-treated Kupffer cells were similar to Kupffer cells isolated without Pronase, with a fivefold increase in Kupffer cell yield (Table 1). cAMP production after 24 h in culture increased relative to freshly isolated cells and did not differ between cells isolated in the absence of Pronase and 0.02% 15-min Pronase exposure (data not shown).

                              
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Table 1.   Kupffer cell isolation procedures

Receptor-stimulated cAMP production in Kupffer cells. Rats were allowed free access to the Lieber-DeCarli liquid diet containing 36% of calories from ethanol or were pair fed a control diet that isocalorically substituted maltose dextrins for ethanol. Growth of the rats on these diets was equivalent (Table 2). Kupffer cell yields, viability, plating efficiency, and cell numbers after 24 h in culture were also equivalent between the two treatment groups (Table 2). Gsalpha and Gialpha protein quantity was measured by Western blotting in freshly isolated Kupffer cells from ethanol and pair-fed rats. Kupffer cells from both feeding groups expressed the long and short forms of Gsalpha , but the total quantity of Gsalpha immunoreactivity (long and short forms combined) was 41% less than in ethanol-fed rats compared with pair-fed rats (Table 3). The quantity of Gialpha was not affected by ethanol feeding. The decrease in quantity of Gsalpha in Kupffer cells from ethanol-fed rats was maintained during 24 h of culture in the absence of ethanol (Fig. 1 and Table 3). After culture for 24 h, the quantity of immunoreactive Gialpha and Gbeta did not differ between the groups (Table 3). To test the functional significance of this decrease in Gsalpha , receptor-stimulated cAMP production was measured in intact Kupffer cells. Basal cAMP production did not differ between groups (Fig. 2). However, stimulation of cAMP production by agents that activate adenylyl cyclase independently of receptors (forskolin), as well as via adenosine A2 receptor and PGE2 receptor (Fig. 2), was lower in Kupffer cells from ethanol-fed rats. These data indicate that chronic ethanol feeding decreases receptor-stimulated cAMP production and that this heterologous desensitization can be observed in Kupffer cells for at least 24 h in culture.

                              
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Table 2.   Kupffer cell isolation from ethanoland pair-fed rats


                              
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Table 3.   Immunoreactive quantity of guanine nucleotide regulatory protein subunits in Kupffer cells isolated from ethanol- and pair-fed rats



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Fig. 1.   Long-term ethanol feeding decreases in Gsalpha protein but not Gialpha in rat Kupffer cells. Kupffer cells were isolated from ethanol (EF)- and pair (PF)-fed rats and maintained in culture for 24 h. Crude membrane fractions were then prepared from the cultured cells, and Gsalpha protein quantity was determined by Western blot. Blot is representative of data presented in Table 3 for 24-h cultures.


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Fig. 2.   Ethanol feeding decreases receptor-stimulated cAMP production by cultured Kupffer cells. Kupffer cells were isolated from ethanol (EtOH)- and pair-fed rats and cultured for 24 h in Iscove's modified Dulbecco's medium with 20% FBS. Cell culture medium was aspirated, and cells were incubated for 10 min in PBS with 1 µM Ro 20-1724 and 1 U/ml adenosine deaminase for 10 min. cAMP production was then stimulated with agonist for a further 15 min at 37°C. Reactions were terminated by the addition of 0.1 N HCl-0.2% Nonidet P-40. cAMP was measured by RIA. Values represent means ± SE; n = 4-6 rats. * P < 0.05 compared with pair-fed rats. NECA, 5'-(N-ethylcarboxamido)-adenosine.

Regulation of TNF-alpha production. The concentration of TNF-alpha was increased in Kupffer cells from both ethanol- and pair-fed rats by treatment with LPS or latex beads for 4 h (Fig. 3A). TNF-alpha quantity was greater in nonstimulated (basal) Kupffer cells and in cells exposed to latex beads from ethanol-fed rats compared with control (Fig. 3A). Similarly, at 400 ng/ml LPS, TNF-alpha release was greater in ethanol-fed compared with pair-fed rats, but, after treatment with 4,000 ng/ml LPS, there was no difference between groups (Fig. 3A). A second dose response was conducted to evaluate responses to lower LPS concentrations; TNF-alpha release was greater in Kupffer cells from ethanol-fed rats after treatment with 0.4-40 ng/ml LPS compared with pair-fed rats (Fig. 3B).


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Fig. 3.   Stimulation of tumor necrosis factor-alpha (TNF-alpha ) production by latex beads or lipopolysaccharide (LPS) is increased after chronic ethanol feeding. Kupffer cells from pair-fed and ethanol-fed rats were isolated and cultured for 24 h. Cells were treated with and without LPS or latex beads for a further 4 h. Medium was collected, and TNF-alpha concentration was determined in a cytotoxicity assay. Values represent means ± SE. A: = 9-11 rats for all conditions except for 4,000 ng/ml LPS where n = 4 rats. B: n = 4 rats. * P < 0.05 compared with pair-fed rats.

Because the production of cAMP in response to a broad range of concentrations of both NECA and PGE2 was reduced in ethanol-fed rats (Fig. 2), we asked whether these agents could modulate TNF-alpha production in cells stimulated with LPS or latex beads. Both 10-4 M NECA and 10-6 M PGE2 decreased the production of TNF-alpha in Kupffer cells from pair-fed rats (Fig. 4). Surprisingly, in ethanol-fed rats, a similar degree of inhibition was observed when cells were treated with 10-4 M NECA and 10-6 M PGE2, as well as 1 mM dibutyryl-cAMP (Fig. 4). Inhibition of TNF-alpha production was also equivalent between pair-fed and ethanol-fed animals when Kupffer cells were pretreated with 10-6 M NECA. TNF-alpha production in response to 400 ng/ml LPS was reduced to 63 ± 28% of control in pair-fed rats and 64 ± 14% of control in ethanol-fed rats (n = 3-4).


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Fig. 4.   Activation of Gsalpha -coupled hormone receptors decreases the concentration of TNF-alpha in Kupffer cells cultured from ethanol (hatched bars)- and pair (open bars)-fed rats. Kupffer cells were isolated and cultured for 24 h. Cells were treated with and without hormone for 1 h and then were stimulated with LPS or latex beads for a further 4 h. Medium was collected, and TNF-alpha concentration was determined in a cytotoxicity assay. Basal TNF-alpha concentrations were 52 ± 18 and 81 ± 18 ng/105 cells after exposure to beads (A), 52 ± 15 and 117 ± 19 after treatment with 400 ng/ml LPS (B), and 176 ± 56 and 128 ± 66 after treatment with 4,000 ng/ml LPS (C) for pair-fed and ethanol-fed rats, respectively. Values represent means ± SE; n = 6-10 rats. There was no significant effect of diet. * P < 0.05 compared with basal. db cAMP, dibutyryl-cAMP.

The measurements of cAMP production reported in Fig. 2 were conducted under conditions designed to assess cAMP synthesis; assays were carried out over short time periods in the presence of an inhibitor of type IV phosphodiesterase (Ro 20-1724), the predominant phosphodiesterase isoform in monocytes (36). In contrast, inhibition of TNF-alpha production by hormone was measured over longer periods of hormone treatment and in the absence of phosphodiesterase inhibitor. If ethanol feeding decreased the activity of phosphodiesterase, cAMP might accumulate over longer time periods in Kupffer cells from ethanol-fed rats despite decreased rates of cAMP synthesis. cAMP content was therefore measured in cultured Kupffer cells after longer periods of treatment with hormone in the absence of Ro 20-1724. cAMP content was 0.33 ± 0.14 and 0.07 ± 0.01 pmol/106 cells after 15 min and 1.1 ± 0.35 and 0.09 ± 0.05 after 60 min of stimulation with 10-6 M NECA in pair-fed and ethanol-fed rats, respectively (n = 3-4, P < 0.05 for ethanol-fed compared with pair-fed rats), indicating that the cAMP content of Kupffer cells from ethanol-fed rats reflected the decreased rate of cAMP synthesis.

We also investigated the effects of inhibition of phosphodiesterase activity on TNF-alpha production in Kupffer cells from ethanol- and pair-fed rats. If a decrease in phosphodiesterase activity after ethanol feeding contributed to the inhibition of TNF-alpha production despite desensitization of cAMP synthesis, measurement of TNF-alpha production in the presence of Ro 20-1724 should then reflect the decreased cAMP production in Kupffer cells from the ethanol-fed rats. TNF-alpha production was inhibited when cells from both pair-fed and ethanol-fed rats were preincubated with 0.1-10 µM Ro 20-1724 for 1 h before LPS stimulation (Fig. 5). Treatment with 0.1-10 µM Ro 20-1724 in the absence of LPS had no effect on TNF-alpha release (data not shown). TNF-alpha production did not differ between ethanol- and pair-fed rats, even in the presence of the phosphodiesterase inhibitor.


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Fig. 5.   Inhibition of LPS-stimulated TNF-alpha production by Ro 20-1724. Kupffer cells were isolated and cultured for 24 h, and cells were treated with 0-10 µM Ro 20-1724 for 1 before addition of 400 ng/ml LPS for a further 4 h. Medium was collected, and TNF-alpha concentration was measured. Values represent means ± SE; n = 7 rats. There was no effect of diet. * P < 0.05 compared with cells treated with LPS alone.

Activity and quantity of PKA. The inhibitory effect of cAMP on TNF-alpha production is associated with a decrease in mRNA for TNF-alpha in macrophages and Kupffer cells (14, 45). cAMP-dependent regulation of transcription requires the activation of PKA and translocation of the catalytic subunit to the nucleus (16). We therefore compared the activity and quantity of PKA in Kupffer cells from ethanol and pair-fed rats. Basal and total activity of PKA, measured in cell homogenates in the absence or presence of 40 µM dibutyryl-cAMP, did not differ between groups, as measured by the kemptide assay (Table 4). Similarly, there was no difference in the quantity of immunoreactive RI, RII, or C subunits of PKA in homogenates of cultured Kupffer cells from pair-fed and ethanol-fed rats (Table 4). However, activation of PKA in response to hormonal stimulation (activity ratio) in intact Kupffer cells was decreased after ethanol feeding. Treatment of Kupffer cells from pair-fed rats with 10-6 M NECA for 15 min resulted in the activation of 42% of total PKA (Table 4). In cells from ethanol-fed rats, the extent of activation was reduced by 50% compared with the pair-fed group (Table 4), reflecting the decreased ability of NECA to activate cAMP production after ethanol feeding.

                              
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Table 4.   Activity and quantity of cAMP-dependent protein kinase in Kupffer cells from ethanol- and pair-fed rats

As additional measures of hormonal stimulation of PKA activity in Kupffer cells, the translocation of PKA-C to the nucleus and phosphorylation of CREB were assessed by immunohistochemistry. In nonstimulated cells, PKA-C was predominantly localized to extranuclear regions of Kupffer cells isolated from both ethanol- and pair-fed rats (Fig. 6A). Translocation of the catalytic subunit was observed after 30 min of stimulation with 10-6 M NECA in both groups (Fig. 6A). Similarly, the appearance of phospho-CREB in the nucleus was observed after 30 min of treatment with 10 nM forskolin in cells from both ethanol- and pair-fed animals (Fig. 6B). Although immunohistochemistry does not allow for a quantitative comparison between ethanol- and pair-fed animals, Kupffer cells from both groups responded to hormonal activation, despite the reduced production of cAMP and percent activation of PKA after ethanol feeding.


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Fig. 6.   Localization of the catalytic subunit of cAMP-dependent protein kinase (PKA-C) and phospho-CREB (P-CREB) in response to activation of cAMP production. Kupffer cells isolated from ethanol- and pair-fed rats were cultured for 24 h. Cells were treated with and without 10-6 M NECA (A) or 10 nM forskolin (B) for 30 min. Cells were then fixed in paraformaldehyde, and localization of the catalytic subunit and phospho-CREB was assessed by immunohistochemistry. The nucleus was labeled with sytox green in A. Images are representative of cells from at least 4 pairs of animals.


    DISCUSSION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

Chronic ethanol feeding to rats caused a desensitization of hormone-stimulated cAMP production in isolated and cultured Kupffer cells. This ethanol-induced desensitization was characterized by a decrease in the quantity of Gsalpha protein and decreased receptor-stimulated cAMP production. Surprisingly, this desensitization of the cAMP signaling cascade did not interfere with the ability of hormones coupled to Gsalpha to inhibit TNF-alpha production after treatment with LPS or latex beads. Although total PKA activity was not affected by ethanol feeding, activation of PKA in response to hormonal stimulation was reduced in Kupffer cells from ethanol-fed rats compared with controls. However, translocation of PKA-C to the nucleus and phosphorylation of CREB in response to hormonal stimulation were observed after ethanol feeding, despite the profound reduction in cAMP production in response to hormones acting via Gsalpha . These data suggest that ethanol exposure resulted in a dissociation between cAMP production and the downstream inhibition of TNF-alpha production mediated by PKA.

The cAMP signal transduction cascade is under complex regulatory control; mechanisms for enhancing or dampening responses to hormonal activation can occur via regulation of cAMP concentration per se or the activity of PKA. Concentration of cAMP in response to activation can be controlled by receptor function and quantity, G protein function and quantity, and activity of adenylyl cyclase and phosphodiesterases. Regulation of activity of PKA can be mediated by changes in subunit expression, activity, and localization (19). Activity of PKA can also be regulated depending on the quantity and localization of the heat-stable inhibitor of PKA (PKI). PKI associates with PKA-C in the nucleus, inhibiting its activity and mediating its export from the nucleus (39, 40). Finally, there is also regulation of the quantity of cAMP-responsive transcription factors, CREB and cAMP response element modulator (CREM) (15).

Chronic ethanol exposure desensitizes G protein-stimulated cAMP production in a number of cell types (7), similar to the decrease in hormone-dependent cAMP production found in freshly isolated and cultured Kupffer cells after chronic ethanol feeding (Figs. 1-2). Degradation of cAMP by phosphodiesterases does not appear to be a target for ethanol action; we found no effect of ethanol on activity of phosphodiesterase in hepatocytes (30), and inhibition of type IV phosphodiesterase decreased TNF-alpha production to an equivalent extent in Kupffer cells from ethanol- and pair-fed rats (Fig. 5). Ethanol feeding had no effect on the total in vitro activity of PKA measured in Kupffer cell extracts (Table 4). However, activation of PKA in the intact cell by the adenosine receptor agonist NECA was 50% lower in Kupffer cells after ethanol feeding compared with controls (Table 4). cAMP production in response to 10-6 M NECA was only 27% of control values in Kupffer cells after ethanol feeding (Fig. 2), indicating that there was amplification of the hormone-dependent signal in ethanol-fed rats at this point in the cAMP signalling cascade. Moreover, despite the lower degree of activation of PKA in response to 10-6 M NECA, activation was sufficient in the Kupffer cells from ethanol-fed rats to detect translocation of PKA-C to the nucleus within 30 min after stimulation.

In some cell types, ethanol may also regulate the localization/translocation of PKA-C. In cultured neuroblastoma X glioma hybrid cells (NG108-15), chronic exposure to ethanol during culture results in the movement of PKA-C to the nucleus independent of the application of exogenous agonist (8). Because PKA-C was not sequestered to the nucleus in nonstimulated Kupffer cells from ethanol-fed animals, agonist-independent movement of PKA-C to the nucleus is not likely to account for the dissociation of cAMP production from inhibition of TNF-alpha by hormones in our system. Moreover, if PKA-C was localized to the nucleus in the absence of agonist, we would expect lower TNF-alpha release in ethanol-fed rats, even in the absence of agonist. In contrast to this prediction, TNF-alpha release is higher in ethanol-fed rats in the absence of agonist. Because the mislocalization of the catalytic subunit is restored in NG108-15 cells within 9 h of ethanol removal (8), it is possible that any ethanol-induced changes in localization of PKA-C have recovered in Kupffer cells from ethanol-fed rats after 24-h culture in the absence of ethanol.

Another step in the cAMP signal transduction cascade that may be targeted by long-term ethanol feeding is the regulation of transcriptional control by the nuclear transcription factor CREB. However, very little information is available on the potential effects of ethanol on CREB phosphorylation/dephosphorylation, interaction with other nuclear transcription factors, or cAMP response elements. In rat cerebellum, short-term ethanol exposure increases the phosphorylation of CREB, whereas chronic ethanol has no effect (43). In Kupffer cells, ethanol feeding did not impact on the ability of forskolin to increase the level of immunoreactive phospho-CREB in Kupffer cells.

Taken together, these data suggest that ethanol feeding potentiates responses to cAMP. Despite very low rates of cAMP accumulation after ethanol feeding, localization of the catalytic subunit to the nucleus and its ability to phosphorylate CREB in response to stimulation of cAMP production were maintained. The mechanism for this potentiation is not known. It may be that chronic ethanol exposure specifically enhances the amplification of the cAMP signal at one or more points in the signalling cascade. Activation of PKA by cAMP may be one site of amplification, since endogenous activation of PKA by 10-6 M NECA was reduced by only 50%, whereas cAMP production at this concentration was reduced by 75%. Alternatively, it is possible that no unique amplification mechanism is required after ethanol feeding, i.e., the normal mechanisms of signal amplification from cAMP to the downstream response may be sufficient to amplify the signal from the small amount of second messenger generated in response to hormonal activation after ethanol feeding.

Kupffer cells, the resident macrophage in the liver, have been implicated in the development of alcoholic liver injury (1, 26). Long-term alcohol consumption is associated with an increase in circulating TNF-alpha (21, 25, 44) and an increased response to endotoxin treatment (18). Increased TNF-alpha observed after alcohol consumption (21, 25, 44) is likely due both to increased exposure to circulating endotoxins (21, 26) and enhanced Kupffer cell responses to this activation. When rats fed ethanol for 6 wk are injected with LPS, the concentration of circulating TNF-alpha was fivefold greater than in LPS-treated controls (17). We have found that basal release of TNF-alpha and the response to low concentrations of LPS were higher in Kupffer cells isolated from ethanol-fed rats compared with pair-fed controls (Fig. 3). The mechanism for this enhanced production of TNF-alpha is not known. However, our data indicate that increased TNF-alpha after ethanol feeding is not due to a loss in the activity of the feedback inhibitory pathway mediated by cAMP. We have been able to isolate the effects of ethanol on cAMP-mediated inhibition of TNF-alpha release and have found that Kupffer cells from ethanol-fed rats retain the ability to inhibit TNF-alpha production via cAMP-dependent hormonal activation. Thus the increased susceptibility to endotoxin-induced liver injury observed after ethanol feeding (18) is likely the result of an enhanced stimulatory response of Kupffer cells to activation by endotoxin rather than an impairment in the inhibitory feedback loop mediated by cAMP.


    ACKNOWLEDGEMENTS

This work was supported in part by grants from the Alcoholic Beverage Medical Research Foundation and National Sciences and Engineering Research Council, Canada.


    FOOTNOTES

Present address of A. Aldred: Dept. of Health Studies and Gerontology, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1.

Address for reprint requests: L. E. Nagy, Dept. of Nutrition, Case Western Reserve Univ., Cleveland, OH 44106-4906.

Received 1 October 1997; accepted in final form 10 September 1998.


    REFERENCES
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

1.   Adachi, Y., B. U. Bradford, W. Gao, H. K. Bojes, and R. G. Thurman. Inactivation of Kupffer cells prevents early alcohol-induced liver injury (Abstract). Hepatology 20: 453, 1994[Medline].

2.   Beeker, K., D. Deane, C. Elton, and S. Pennington. Ethanol-induced growth inhibition in embryonic chick brain is associated with changes in cytoplasmic cyclic AMP-dependent protein kinase regulatory subunit. Alcohol Alcohol. 23: 477-482, 1988[Medline].

3.   Blumenthal, R. S., I. W. Flinn, O. Proske, D. G. Jackson, R. G. Tena, M. C. Mitchell, and A. M. Feldman. Effects of chronic ethanol exposure on cardiac receptor-adenylyl cyclase coupling: studies in cultured embryonic chick myocytes and ethanol fed rats. Alcohol. Clin. Exp. Res. 15: 1077-1083, 1991[Medline].

4.   Bulut, V., A. Severn, and F. Y. Liew. Nitric oxide production by murine macrophages is inhibited by prolonged elevation of cyclic AMP. Biochem. Biophys. Res. Commun. 195: 1134-1138, 1993[Medline].

5.   Corbin, J. D. Determination of the cAMP-dependent protein kinase activity ratio in intact tissues. Enzymes 99: 227-232, 1983.

6.   Currin, R. T., L. J. Reinstein, S. N. Lichtman, R. G. Thurman, and J. J. Lemasters. Inhibition of tumor necrosis factor release from cultured rat Kupffer cells by agents that reduce graft failure from storage injury. Transplant. Proc. 25: 1631-1632, 1993[Medline].

7.   Diamond, I., and A. S. Gordon. Cellular and molecular neuroscience of alcoholism. Physiol. Rev. 77: 1-20, 1997[Abstract/Free Full Text].

8.   Dohrman, D., I. Diamond, and A. S. Gordon. Ethanol causes translocation of cAMP-dependent protein kinase catalytic subunit to the nucleus. Proc. Natl. Acad. Sci. USA 93: 10217-10221, 1996[Abstract/Free Full Text].

9.   D'Souza, N. B., G. J. Bagby, S. Nelson, C. H. Lang, and J. J. Spitzer. Acute alcohol infusion suppresses endotoxin-induced serum tumor necrosis factor. Alcohol. Clin. Exp. Res. 13: 295-297, 1989[Medline].

10.   Earnest, D. L., E. R. Abril, C. S. Jolley, and F. Martinez. Ethanol and diet-induced alterations in Kupffer cell function. Alcohol Alcohol. 28: 73-83, 1993[Abstract].

11.   Fox, E. S., C. H. Cantrell, and K. A. Leingang. Inhibition of the Kupffer cell inflammatory response by acute ethanol: NF-kappa B activation and subsequent cytokine production. Biochem. Biophys. Res. Commun. 225: 134-140, 1996[Medline].

12.   Friedman, S. L., and F. J. Roll. Isolation and culture of hepatic lipocytes, Kupffer cells and sinusoidal endothelial cells by density gradient centrifugation with Stractan. Anal. Biochem. 161: 1233-1247, 1987.

13.   Funaki, N., S. Arii, Y. Adachi, H. Higashituji, S. Fujita, M. Furutani, M. Mise, S. Ishiguro, T. Kitao, J. Tanaka, and T. Tobe. Effect of PGE2 on interleukin-1 and superoxide release from primary-cultured human hepatic macrophages. Life Sci. 51: 1339-1346, 1992[Medline].

14.   Grewe, M., R. Gausling, K. Gyufko, R. Hoffmann, and K. Decker. Regulation of the mRNA expression for tumor necrosis factor alpha in rat liver macrophages. J. Hepatol. 20: 811-818, 1994[Medline].

15.   Habener, J. F., C. P. Miller, and M. Vallejo. cAMP dependent regulation of gene transcription by cAMP response element-binding protein and cAMP response element modulator. Vitam. Horm. 51: 1-52, 1995[Medline].

16.   Hagiwara, M., P. Brindle, R. Harootunian, J. Armstrong, W. Rivier, R. Vale, R. Tsien, and M. Montminy. Coupling of hormonal stimulation and transcription via cyclic AMP-responsive factor CREB is rate limited by nuclear entry of protein kinase A. Mol. Cell. Biol. 13: 4852-4859, 1993[Abstract].

17.   Hansen, J., D. L. Cherwitz, and J. I. Allen. The role of tumor necrosis factor alpha in acute endotoxin-induced hepatotoxicity in ethanol-fed rats. Hepatology 20: 461-474, 1994[Medline].

18.   Honchel, R., L. Marsono, D. Cohen, S. Shedlofsky, and C. McClain. A role for tumor necrosis factor in alcohol enhanced endotoxin liver injury. In: The Physiological and Pathological Effects of Cytokines, edited by C. A. Dinarello, M. J. Kluger, M. C. Powanda, and J. J. Oppenheim. New York: Wiley-Liss, 1990, vol. 10B, p. 171-176.

19.   Houslay, M. D., and G. Milligan. Tailoring cAMP-signalling responses through isoform specificity. Trends Biochem. Sci. 22: 217-224, 1997[Medline].

20.   Iles, K., and L. E. Nagy. Chronic ethanol feeding increases the quantity of Gas-protein in rat liver plasma membranes. Hepatology 21: 1154-1160, 1995[Medline].

21.   Khoruts, A., L. Stahnke, C. J. McClain, G. Logan, and J. I. Allen. Circulating tumor necrosis factor, interleukin-1 and interleukin-6 concentrations in chronic alcoholic patients. Hepatology 13: 267-276, 1991[Medline].

22.   Laemmli, U. K. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227: 680-685, 1970[Medline].

23.   Lieber, C. S., and L. M. DeCarli. The feeding of alcohol in liquid diets: two decades of application and 1982 update. Alcohol. Clin. Exp. Res. 6: 523-531, 1982[Medline].

24.   Machu, T. K., R. W. Olsen, and M. D. Browning. Ethanol has no effect on cAMP-dependent protein kinase, protein kinase C or Ca2+-calmodulin-dependent protein kinase II-stimualated phosphorylation of highly purified substrates in vitro. Alcohol. Clin. Exp. Res. 15: 1040-1046, 1991[Medline].

25.   McClain, C. J., and D. A. Cohen. Increased tumor necrosis factor production by monocytes in alcoholic hepatitis. Hepatology 9: 349-351, 1989[Medline].

26.   McClain, C., D. Hill, J. Schmidt, and A. M. Diehl. Cytokines and alcoholic liver disease. Semin. Liver Dis. 13: 170-182, 1993[Medline].

27.   Meinkoth, J. L., Y. Ji, S. S. Taylor, and J. R. Feramisco. Dynamics of the distribution of cAMP-dependent protein kinase in living cells. Proc. Natl. Acad. Sci. USA 87: 9595-9599, 1990[Abstract].

28.   Munthe-Kaas, A. C., T. Berg, P. O. Seglen, and R. Seljelid. Mass isolation and culture of rat Kupffer cells. J. Exp. Med. 141: 1-10, 1975[Abstract].

29.   Nagy, L. E., and S. E. F. deSilva. Ethanol increases receptor-dependent cAMP production in cultured hepatocytes by decreasing Gi-mediated inhibition. Biochem. J. 286: 681-686, 1992[Medline].

30.   Nagy, L. E., and S. E. F. deSilva. Adenosine A1 receptors mediate chronic ethanol-induced increases in receptor-stimulated cAMP in cultured hepatocytes. Biochem. J. 304: 205-210, 1994[Medline].

31.   Nolan, J. P. Intestinal endotoxins as mediators of hepatic injury: an idea whose time has come again. Hepatology 10: 887-891, 1989[Medline].

32.   Parmely, M. J., W. Zhou, C. K. I. Edwards, D. R. Borcherding, R. Silverstein, and D. C. Morrison. Adenosine and a related carbocyclic nucleoside analogue selectively inhibit tumor necrosis factor-alpha production and protect mice against endotoxin challenge. J. Immunol. 151: 389-396, 1993[Abstract/Free Full Text].

33.   Peters, T., U. Karck, and K. Decker. Interdependence of tumor necrosis factor, prostaglandin E2 and protein synthesis in LPS-exposed rat Kupffer cells. Eur. J. Biochem. 191: 583-589, 1990[Abstract].

34.   Rius, R. A., S. Govoni, F. Battaini, and M. Trabucchi. Cyclic AMP-dependent protein phosphorylation is reduced in rat striatum after chronic ethanol treatment. Brain Res. 365: 355-359, 1986[Medline].

35.   Shimozato, T., M. Iwata, H. Kawada, and N. Tamura. Human immunoglobulin preparation for intravenous use induces elevation of cellular cyclic adenosine 3':5'-monophosphate levels, resulting in suppression of tumour necrosis factor alpha and interleukin-1 production. Immunology 72: 497-501, 1991[Medline].

36.   Souness, J. E., M. Griffin, M. Christopher, K. Ebsworth, L. C. Scott, K. Pollock, M. N. Palfreyman, and J. A. Karlsson. Evidence that cyclic AMP phosphodiesterase inhibitors suppress TNF alpha generation from human monocytes by interacting with a "low affinity" phosphodiesterase 4 conformer. Br. J. Pharmacol. 118: 649-658, 1996[Abstract].

37.   Stephen, L. L., and L. E. Nagy. Very low protein diets induce a rapid decrease in hepatic cAMP-dependent protein kinase followed by a slower increase in adenylyl cyclase activity in rats. J. Nutr. 126: 1799-1807, 1996[Medline].

38.   Victorov, A. V., and J. B. Hoek. Secretion of prostaglandins elicited by lipopolysaccaride and ethanol in cultured rat Kupffer cells. Biochem. Biophys. Res. Commun. 215: 691-607, 1995[Medline].

39.   Wen, W., J. L. Meinkoth, R. Y. Tsien, and S. S. Taylor. Identification of a signal for rapid export of proteins from the nucleus. Cell 82: 463-473, 1995[Medline].

40.   Wen, W., S. S. Taylor, and J. L. Meinkoth. The expression and intracellular distribution of the heat-stable protein kinase inhibitor is cell cycle regulated. J. Biol. Chem. 270: 2041-2046, 1995[Abstract/Free Full Text].

41.   Wilkes, J., L. DeForrest, and L. E. Nagy. Chronic ethanol feeding in a high-fat diet decreases insulin-stimulated glucose transport in rat adipocytes. Am. J. Physiol. 271 (Endocrinol. Metab. 34): E477-E484, 1996[Abstract/Free Full Text].

42.   Winwood, P. J., and M. J. P. Arthur. Kupffer cells: their activation and role in animal models of liver injury and human liver disease. Semin. Liver Dis. 13: 50-59, 1993[Medline].

43.   Yang, X., A. M. Diehl, and G. S. Wand. Ethanol exposure alters the phosphorylation of cyclic AMP responsive element binding protein and cyclic AMP responsive element binding activity in rat cerebellum. J. Pharmacol. Exp. Ther. 278: 338-346, 1996[Abstract].

44.   Yoshioka, K., S. Kakumu, M. Arao, Y. Tsutum, and M. Inoue. Tumor necrosis factor alpha production in peripheral blood mononuclear cells of patients with chronic liver disease. Hepatology 10: 769-773, 1989[Medline].

45.   Zhong, W. W., P. A. Burke, M. E. Drotar, S. R. Chavali, and R. A. Forse. Effects of prostaglandin E2, cholera toxin and 8-bromo-cAMP on lipopolysaccharide induced gene expression of cytokines in human macrophages. Immunology 84: 446-452, 1995[Medline].


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