Ethanol-induced liver injury: potential roles for egr-1.

Chronic ethanol-induced liver injury follows a typical progression from its earliest stage of steatosis to more advanced injury, characterized by the development of inflammation, hepatocyte necrosis/apoptosis, fibrosis and finally cirrhosis. Kupffer cells, the resident macrophage in the liver, play a critical role in the progression of liver injury. Increased exposure of Kupffer cells to lipopolysaccharide (LPS) during chronic ethanol exposure leads to the production of a number of inflammatory mediators, including tumor necrosis factor alpha (TNF-alpha). Recent evidence indicates that in addition to increased exposure to LPS, Kupffer cells also develop an enhanced sensitivity to LPS after chronic ethanol feeding. We have recently identified early growth response-1 (Egr-1), an immediate-early gene transcription factor, as an important contributor to increased LPS-stimulated TNF-alpha secretion by Kupffer cells after chronic ethanol exposure. In other models of tissue injury, such as ischemia-reperfusion in the lung, Egr-1 acts as a coordinator of the complex response to stress. Here we review the literature regarding the role of EGR-1 in regulation of a number of genes implicated in each of the stages of chronic ethanol-induced liver injury. In addition to the critical role of Egr-1 in generating maximal LPS-stimulated TNF-alpha expression, Egr-1 also controls the expression of a number of inflammatory mediators, including intercellular adhesion molecule (ICAM)-1, monocyte chemotactic protein (MCP)-1 and macrophage inflammatory protein (MIP)-2, as well as genes contributing to fibrosis, such as transforming growth factor (TFG)-beta1, platelet-derived growth factor PDGF-A chain and fibroblast growth factor (FGF). Understanding the contribution of Egr-1 to the expression of genes involved in the development of chronic ethanol-induced liver injury may lead to the development of improved therapies designed to prevent and/or reverse alcohol-induced liver injury.

Stabilization of tumor necrosis factor alpha mRNA by chronic ethanol: role of A + U-rich elements and p38 mitogen-activated protein kinase signaling pathway.

Increased expression of tumor necrosis factor alpha (TNFalpha) in response to chronic ethanol has been implicated in the pathogenesis of alcoholic liver disease. However, the molecular mechanisms by which ethanol increases the levels of TNFalpha are not well characterized. Utilizing Kupffer cells isolated from rats fed an ethanol containing diet and a murine macrophage cell line, RAW264.7, exposed to ethanol in culture, we have demonstrated that exposure to chronic ethanol results in an enhanced expression of lipopolysaccharide (LPS)-induced TNFalpha. While chronic ethanol had no effect on the rate of LPS-induced TNFalpha transcription as measured by nuclear run-on experiments, TNFalpha mRNA half-life was increased by chronic ethanol. Chronic ethanol also potentiated the activation of LPS-induced p38 mitogen-activated protein (MAP) kinase in Kupffer cells, as well as in RAW264.7 cells. Specific inhibition of p38 MAP kinase activation by SB203580 in Kupffer cells or by overexpression of dominant negative p38 MAP kinase in RAW264.7 cells blocked ethanol-mediated TNFalpha mRNA stabilization. Furthermore, using chimeric reporter constructs, we have shown that A + U-rich elements in the 3'-untranslated region of TNFalpha mRNA are not sufficient to impart ethanol-mediated stabilization on TNFalpha mRNA.

GLUT4 vesicle trafficking in rat adipocytes after ethanol feeding: regulation by heterotrimeric G-proteins.

Long-term ethanol consumption decreases insulin-stimulated glucose uptake in isolated rat adipocytes. Here we investigate the mechanisms for this decrease. Male Wistar rats were fed for 4 weeks with a liquid diet containing 35% of the calories from ethanol and compared with pair-fed controls. Stimulation of 3-O-methylglucose transport in isolated adipocytes by insulin was decreased by 70% after ethanol feeding. However, stimulation by insulin of the tyrosine phosphorylation of the p85 subunit of phosphoinositide 3-kinase and the phosphorylation of Akt were not affected by ethanol feeding. GLUT4 was mobilized from intracellular light microsomes in response to insulin in both pair-fed and ethanol-fed rats, resulting in 4.3-fold and 3.3-fold increases in GLUT4 associated with plasma membrane in pair-fed and ethanol-fed rats respectively. Surface-accessible GLUT4, assessed by a trypsin cleavage assay or cell-surface labelling with bis-mannose photolabel, was increased 2.3-fold and 1.6-fold respectively, in pair-fed rats after treatment with insulin. In contrast, insulin did not increase surface-accessible GLUT4 in ethanol-fed rats. Treatment of adipocytes with R-phenylisopropyladenosine, an adenosine A1 receptor agonist, increased the transport of 3-O-methylglucose and trypsin-accessible GLUT4, in adipocytes from both pair-fed and ethanol-fed rats. These results demonstrate that whereas the insulin-mediated signalling and translocation of GLUT4 to the plasma membrane is maintained after ethanol feeding, the final fusion of GLUT4 vesicles to the plasma membrane is disrupted, preventing the stimulation of glucose uptake by insulin. Fusion of GLUT4 with the plasma membrane can be stimulated by the activation of adenosine A1 receptors.

Ethanol stimulates glucose uptake and translocation of GLUT-4 in H9c2 myotubes via a Ca(2+)-dependent mechanism.

Short-term exposure to ethanol impairs glucose homeostasis, but the effects of ethanol on individual components of the glucose disposal pathway are not known. To understand the mechanisms by which ethanol disrupts glucose homeostasis, we have investigated the direct effects of ethanol on glucose uptake and translocation of GLUT-4 in H9c2 myotubes. Short-term treatment with 12.5-50 mM ethanol increased uptake of 2-deoxyglucose by 1.8-fold in differentiated myotubes. Pretreatment of H9c2 myotubes with 100 nM wortmannin, an inhibitor of phosphatidylinositol 3-kinase, had no effect on ethanol-induced increases in 2-deoxyglucose uptake. In contrast, preincubation with 25 microM dantrolene, an inhibitor of Ca(2+) release from the sarcoplasmic reticulum, blocked the stimulation of 2-deoxyglucose uptake by ethanol. Increased 2-deoxyglucose uptake after ethanol treatment was associated with a decrease in small intracellular GLUT-4 vesicles and an increase in GLUT-4 localized at the cell surface. In contrast, ethanol had no effect on the quantity of GLUT-1 and GLUT-3 at the plasma membrane. These data demonstrate that physiologically relevant concentrations of ethanol disrupt the trafficking of GLUT-4 in H9c2 myotubes resulting in translocation of GLUT-4 to the plasma membrane and increased glucose uptake.

Mobilization of GLUT-4 from intracellular vesicles by insulin and K(+) depolarization in cultured H9c2 myotubes.

The insulin-responsive glucose transporter, GLUT-4, moves from an intracellular compartment to the cell surface in response to insulin and/or muscle contraction. Treatment of H9c2 myotubes with insulin significantly increased uptake of 2-deoxyglucose. Depolarization of the myotubes by increasing extracellular [K(+)], which mimics the initial phases of excitation-contraction coupling, also increased 2-deoxyglucose uptake. The K(+)- but not insulin-evoked increase was blocked by dantrolene, an inhibitor of Ca(2+) release from the sarcoplasmic reticulum. In contrast, wortmannin, an inhibitor of phosphatidylinositol 3-kinase, blocked insulin- but not K(+)-stimulated 2-deoxyglucose uptake. Increased glucose uptake in response to insulin or K(+) depolarization was associated with increased GLUT-4 in plasma membranes and depletion of a population of small intracellular GLUT-4-containing vesicles. Similarly, in H9c2 cells transfected with c-myc-tagged GLUT-4, translocation of c-myc GLUT-4 to the cell surface was increased after stimulation with insulin or K(+) depolarization. Taken together, these data demonstrate that insulin and K(+) depolarization increase glucose uptake by recruiting GLUT-4 from intracellular vesicles to the plasma membrane of H9c2 myotubes via distinct signaling mechanisms.

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

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 microM 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.

Moderate dietary protein and energy restriction modulate cAMP-dependent protein kinase activity in rat liver.

Very low protein diets result in a desensitization of hepatic cAMP signaling in rats, which is characterized by a loss of cAMP-dependent protein kinase (PKA) activity and type I regulatory subunit (RI). Here we have tested whether more moderate protein restriction (Trial 1) or energy restriction (Trial 2) also modulates hepatic PKA quantity and activity. In trial 1, weanling rats were allowed free access to diets containing normal protein (15%, AL-NP), moderately restricted protein (12.5%, AL-MP) and low protein (7.5%, AL-LP); in trial 2, rats were allowed free access to diet containing 15% (AL-NP) or 0.5% protein (very low protein, AL-VLP) or were energy restricted by pair-feeding a diet isonitrogenous to AL-NP but at 65% of the energy intake (ER-IN) for 14 d. Body weights were lower (P < 0.05) by d 14 in all restricted groups compared with the AL-NP group. The quantity of cytosolic RI was lower (P < 0.05) in AL-LP and AL-VLP, but not in AL-MP or ER-IN, compared with AL-NP. In contrast, there was no effect of diet on RI in the particulate fraction. RII was not changed by moderate and low protein diets in either the cytosol or particulate fraction. However, type II regulatory subunit (RII) was greater in the cytosol of ER-IN and in the particulate fraction of AL-VLP (P < 0.05) compared with AL-NP. Specific activity of PKA was lower in the cytosol and particulate fraction (P < 0.05) in the AL-VLP and ER-IN groups compared with the AL-NP group. In contrast, specific activity of PKA was maintained in cytosol from AL-LP, but lower in the particulate fraction (P < 0.05) compared with AL-NP. In summary, protein restricted-diets lower RI subunit in the cytosol; however, only in rats fed very low protein diets is this loss of RI associated with lower cytosolic PKA activity. In contrast, energy restriction lowers PKA activity in the cytosol and particulate fractions, independent of signficant reduction in RI or RII subunits. Taken together, these data indicate that moderate protein and energy restrictions have differential effects on activity and quantity of PKA.

Deposition of cellular fibronectin increases before stellate cell activation in rat liver during ethanol feeding.

Cellular fibronectin (cFN)-a structural extracellular matrix protein-facilitates cell adhesion, migration, and differentiation during organ development; wound healing; tissue regeneration; and fibrogenic processes. cFN is deposited early in various fibrotic diseases and seems to function as a template for deposition of other extracellular matrix proteins, such as collagen type I and laminin, in the injured area. We have compared the relative changes in cFN levels with other pathogenic markers of alcoholic liver injury over time of ethanol feeding in the rat. Male Wistar rats were allowed free access to a liquid diet containing 36% of total energy as ethanol or pair-fed an isocaloric control diet for 4, 8, and 12 weeks. Serum alanine aminotransferase activity and total liver lipid were increased in ethanol-fed animals, compared with pair-fed controls after 4, 8, and 12 weeks of feeding. Liver lipid content was higher in ethanol-fed rats as early as 4 weeks and was further increased by 12 weeks of feeding. Total fibronectin and cFN protein quantity was greater in liver from ethanol-fed rats after 8 and 12 weeks (fibronectin: 2.3-fold and 2.6-fold; cFN: 4.3-fold and 2.6-fold higher than pair-fed at 8 and 12 weeks, respectively). alpha-Smooth muscle actin, an indicator of hepatic stellate cell activation, was increased in the liver of ethanol-fed rats after 12 weeks of feeding (344% higher compared with pair-fed), with no differences observed at any earlier time points. In summary, increases in hepatic immunoreactive cFN content were observed subsequent to increased liver lipid concentration, but before hepatic stellate cell activation in rats fed the ethanol-based diet. These data suggest that deposition of cFN in the liver during long-term ethanol consumption may represent an early response to injury similar to that observed in other models of liver injury and wound healing.

Chronic ethanol feeding impairs glucose tolerance but does not produce skeletal muscle insulin resistance in rat epitrochlearis muscle.

Herein, we have investigated whether male Wistar rats develop impaired glucose tolerance after ethanol feeding. Rats were fed a liquid diet providing 35% calories from ethanol (EF) or a control diet that isocalorically replaced ethanol with maltose-dextrins for 4 weeks. Intravenous glucose tolerance was impaired in EF rats compared with pair-fed (PF), but not ad libitum (AL) controls. Areas under the intravenous glucose tolerance test curve were 5476 +/- 516 mm2, 3056 +/- 421 mm2, and 4199 +/- 613 mm2 (p < 0.05) for AL, PF, and EF rats, respectively. Initial plasma insulin concentrations in EF rats were comparable with PF rats; however, 15 min after a dextrose challenge, plasma insulin levels in EF rats were 39% lower than PF rats. Because skeletal muscle is the primary sink for insulin-mediated glucose disposal, the development of skeletal muscle insulin resistance after ethanol feeding could contribute to impaired glucose tolerance. Total GLUT1 was not affected by diet in either red or white muscle. No difference in the total quantity of insulin-responsive glucose transporter, GLUT4, was observed in red muscle. In contrast, GLUT4 was 20% lower in white muscle from EF rats, compared with PF and AL rats. However, insulin-stimulated glucose transport into the epitrochlearis, a white muscle group, was not impaired with ethanol feeding. These data demonstrate that chronic ethanol feeding impairs glucose tolerance; impaired glucose tolerance was associated with an inability to maintain plasma insulin levels, rather than the development of skeletal muscle insulin resistance.

Chronic ethanol feeding in a high-fat diet decreases insulin-stimulated glucose transport in rat adipocytes.

Ethanol consumption has been associated with glucose intolerance and insulin resistance and is suggested to be an independent risk factor in the development of non-insulin-dependent diabetes mellitus. We have investigated the long-term effects of ethanol consumption on insulin-regulated glucose transport in rat adipocytes. Male Wistar rats were fed a high-fat liquid diet containing 35% ethanol (ethanol fed) or a control diet that isocalorically substituted maltose dextrin for ethanol (ad libitum). A third group was pair fed the control diet. Basal rates of 2-deoxyglucose uptake were similar in adipocytes from all three groups. Treatment with insulin increased 2-deoxyglucose uptake in ad libitum- and pair-fed rats but did not stimulate uptake in ethanol-fed rats. Similarly, although okadaic acid increased 2-deoxyglucose uptake in pair-fed rats, it had no effect in ethanol-fed rats. GLUT-1 quantity was greater in pair-fed and ethanol-fed rats compared with ad libitum controls. GLUT-4 was decreased in ethanol-fed compared with pair-fed rats but was not different from ad libitum controls. In ad libitum- and pair-fed rats, insulin increased the translocation of GLUT-4 to the cell surface by 2.0-fold. In contrast, translocation of GLUT-4 was not observed after insulin stimulation of ethanol-fed rats, paralleling the loss of insulin-stimulated glucose uptake. In ethanol-fed rats, GLUT-4 protein quantity was negatively associated with increased Gs alpha protein and isoproterenol-stimulated adenosine 3',5'-cyclic monophosphate production. These data suggest that loss of insulin-stimulated glucose uptake in rat adipocytes after chronic ethanol feeding is at least partially due to decreased movement of GLUT-4 to the cell surface after insulin stimulation.

Very low protein diets induce a rapid decrease in hepatic cAMP-dependent protein kinase followed by a lower increase in adenylyl cyclase activity in rats.

Recent evidence indicates that cAMP-mediated responses are desensitized in liver during malnutrition. While receptor-stimulated production of cAMP is increased in hepatocytes from rats fed very low protein diets for 14 d, activity of cAMP-dependent protein kinase (PKA) is decreased in liver cytosol. The present study investigated the time course for this desensitization. Weanling rats were fed either a 0.5 (malnourished) or 15% protein (control) diet for 1, 3, 7 or 14 d. Total PKA activity decreased after only 3 d of feeding the low protein diet. This decrease was confined to the cytosolic compartment and was associated with a lower quantity of immunoreactive RI regulatory subunit of PKA, with no difference in the quantity of immunoreactive RII regulatory subunit. In contrast, basal-, MnCl2- and guanine nucleotide regulatory protein-stimulated adenylyl cyclase activities were not greater in liver membranes of malnourished rats than in those of the control rats until the 2nd wk of feeding. Greater activity was paralleled by an increase in the quantity of the stimulatory guanine nucleotide regulatory protein at d 14. The inhibitory guanine nucleotide regulatory protein quantity did not differ between dietary groups. Greater cAMP production was not mediated by changes in PKA phosphorylation of adenylyl cyclase because preincubation of membranes with purified PKA catalytic subunit decreased MnCl2-stimulated cAMP production equally in liver membranes of both control and malnourished rats. Similarly, treatment with alkaline phosphatase decreased adenylyl cyclase activity but did not eliminate the difference in adenylyl cyclase activity between control and malnourished rats. These data demonstrate that loss of PKA activity is an early response to a low protein diet and that, subsequently, a number of molecular adaptations occur which increase cAMP production. These changes may be adaptive responses to malnutrition that maintain essential cAMP-dependent functions.

Equilibrative adenosine transport in rat hepatocytes after chronic ethanol feeding.

Acute treatment of cells with ethanol in vitro inhibits adenosine uptake via equilibrative nucleoside transporters. After longer periods of exposure to ethanol in culture, rechallenge with ethanol no longer inhibits adenosine uptake. Herein, we have investigated the long-term effects of ethanol consumption in vivo on equilibrative nucleoside transport. Rats were fed a liquid diet containing 35% of calories as ethanol (ethanol-fed). Control rats were pair-fed a liquid diet that isocalorically substituted maltose dextrins for ethanol. After 4 weeks of ethanol consumption, nucleoside transport was measured in isolated hepatocytes. Uptake of [3H]adenosine was lower in ethanol-fed rats compared with control. Influx of the nonmetabolizable nucleoside analog, [3H]formycin B, was also decreased after ethanol feeding. However, neither the number of nitrobenzylthioinosine (NBMPR) binding sites or inhibition of adenosine uptake by NBMPR were affected by ethanol feeding. In controls, acute treatment of isolated hepatocytes with 100 mM ethanol inhibited [3H]adenosine uptake by 30-40%. However, in ethanol-fed rats, acute challenge with ethanol did not inhibit [3H]adenosine uptake. These data demonstrate that long-term ethanol feeding decreases equilibrative nucleoside transport in hepatocytes independent of a change in the number of nucleoside transporters and renders adenosine uptake insensitive to inhibition by ethanol.

Chronic ethanol feeding increases the quantity of G alpha s-protein in rat liver plasma membranes.

The liver is a primary target for both acute and chronic effects of ethanol. Because ethanol is known to alter the function of guanine nucleotide regulatory proteins (G-proteins), changes in hepatic G-proteins could contribute to the adverse effects of ethanol on liver function. Male Wistar rats were fed a liquid diet containing 36% of calories as ethanol for 4 weeks. Control rats were pair-fed or allowed free access to a diet that isocalorically substituted maltose dextrins for ethanol. Liver plasma membranes were isolated and separated into basolateral and canalicular fractions by sucrose-density gradients. Enrichment of marker enzymes (5'-nucleotidase for canalicular membranes and forskolin-stimulated adenylyl cyclase activity for basolateral membranes) was not affected by ethanol feeding. Quantity of G alpha s and G alpha i proteins in membrane fractions was determined by immunoblot. After ethanol feeding, immunoreactive G alpha s protein was increased in liver plasma membranes compared with pair-fed controls. G alpha i and G alpha s were present in both the basolateral and canalicular fractions of the plasma membrane in control and ethanol-fed rats. G alpha s quantity in the basolateral membrane was greater in ethanol-fed rats compared with controls, with no differences in G alpha s observed in canalicular membranes. The quantity of G alpha i did not change in response to ethanol feeding in any of the membrane fractions. Treatment of isolated plasma and basolateral membranes with 10 mumol/L 5'-guanylimidophosphate, a nonhydrolyzable guanosine triphosphate analogue that activates G-proteins, increased cAMP production to a greater extent in ethanol-fed rats compared with controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Activity of cAMP-dependent protein kinase is reduced in protein-energy malnourished rats.

Glucagon decreases glutathione synthesis in hepatocytes from well-nourished rats. However, in hepatocytes from malnourished rats, glucagon does not inhibit glutathione synthesis, suggesting a desensitization of cAMP-mediated signal transduction. We investigated the mechanism for this desensitization of cAMP-mediated responsiveness in malnourished rats by comparing the signal transduction pathways in rats fed very low protein diets (0.5 g protein/100 g diet) with those of rats fed diets adequate in protein (15 g protein/100 g diet) for 2 wk. Glucagon receptor and forskolin-stimulated cAMP production were greater in hepatocytes from malnourished rats. Stimulation of adenylyl cyclase with forskolin, guanine nucleotides or manganese in hepatic membranes was also enhanced after malnutrition. Moreover, quantity of the stimulatory guanine nucleotide regulatory protein was 70-80% greater in hepatocytes from malnourished rats but the inhibitory guanine nucleotide regulatory protein was not different. These results suggested that desensitization of cAMP-mediated signal transduction after malnutrition occurred at a site distal to cAMP production. Maximal activity of cAMP-dependent protein kinase was 60% lower in liver homogenates from malnourished rats compared with controls. This difference in activity was confined to the cytosolic compartment, with no difference in activity observed in the particulate fraction. Lower activity of cAMP-dependent protein kinase in the cytosol of malnourished rats was associated with a 43% reduction in the quantity of regulatory subunit type I, with no difference in the regulatory subunit type II. These data indicate that desensitization of cAMP signal transduction in rat liver after malnutrition is due to a decrease in the quantity and activity of cAMP-dependent protein kinase.

Adenosine A1 receptors mediate chronic ethanol-induced increases in receptor-stimulated cyclic AMP in cultured hepatocytes.

Cellular responses to adenosine depend on the distribution of the two adenosine receptor subclasses. In primary cultures of rat hepatocytes, adenosine receptors were coupled to adenylate cyclase via A1 and A2 receptors which inhibit and stimulate cyclic AMP production respectively. R-(-)-N6-(2-phenylisopropyl)-adenosine (R-PIA), the adenosine A1 receptor-selective agonist, inhibited glucagon-stimulated cyclic AMP production with an IC50 of 19 nM. This inhibition was blocked by the A1-specific antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPDX). 5'-N- Ethylcarboxamidoadenosine (NECA), an agonist which stimulates A2 receptors, increased cyclic AMP production with an EC50 of 0.6 microM. Treatment of primary cultures of rat hepatocytes with 100 mM ethanol for 48 h decreases the quantity and function of the inhibitory guanine-nucleotide regulatory protein (G(i)), resulting in a sensitization of receptor-stimulated cyclic AMP production [Nagy and deSilva (1992) Biochem. J. 286, 681-686]. When cells were cultured with 2 units/ml adenosine deaminase, to degrade extracellular adenosine, ethanol-induced increases in cyclic AMP production were completely prevented. Moreover, the specific A1-receptor antagonist, CPDX, also blocked the chronic effects of ethanol on receptor-stimulated cyclic AMP production. Treatment with adenosine deaminase or CPDX also prevented the decrease in quantity of the alpha subunit protein of G(i) observed in hepatocytes after chronic treatment with ethanol. Taken together, these results suggest that activation of adenosine A1 receptors on primary cultures of hepatocytes is involved in the development of chronic ethanol-induced sensitization of receptor-stimulated cyclic AMP production.

Role of adenosine A1 receptors in inhibition of receptor-stimulated cyclic AMP production by ethanol in hepatocytes.

Brief exposure of primary cultures of hepatocytes to ethanol had a biphasic effect on glucagon receptor-dependent cyclic AMP (cAMP) production: 25-50 mM ethanol decreased cAMP levels, whereas treatment with 100-200 mM ethanol increased cAMP. This biphasic effect was also observed after pretreatment with 10 microM 4-methylpyrazole, an inhibitor of alcohol dehydrogenase. Adenosine A1 and A2 receptors in primary cultures of rat hepatocytes are coupled to inhibition and stimulation of adenylyl cyclase, respectively. Since primary cultures of hepatocytes release adenosine into their extracellular media, we tested whether the acute effects of ethanol on cAMP were mediated by extracellular adenosine. Co-incubation with 2 U/mL adenosine deaminase prevented inhibition of cAMP production by 25-50 mM ethanol, but had no effect on stimulation by 100-200 mM ethanol. Pretreatment of hepatocytes with 110 nM 8-cyclopentyl-1,3-dimethylxanthine, an adenosine A1 receptor antagonist, also completely blocked the inhibitory effects of ethanol on cAMP production. Low concentrations of ethanol enhanced the inhibitory effects of R(-)N6-(2-phenylisopropyl)adenosine, an A1 receptor agonist, on cAMP production in cells pretreated with adenosine deaminase to remove endogenous adenosine. These data suggest that endogenously produced adenosine can be an important modulator of the effects of ethanol on receptor-stimulated cAMP production in primary cultures of rat hepatocytes.

Regulation of hepatocyte glutathione by amino acid precursors and cAMP in protein-energy malnourished rats.

Hepatic glutathione concentration is decreased in protein-energy malnutrition. Malnourished rats are able to replenish hepatic glutathione after oral supplementation with L-2-oxothiazolidine-4-carboxylate, a cysteine pro-drug, to levels that are higher than in control rats. These results suggest that, even if a normal amount of amino acids for glutathione synthesis is provided, homeostatic control of glutathione concentration after protein-energy malnutrition is abnormal. The rate limiting enzyme for glutathione synthesis, gamma-glutamylcysteine synthetase, is subject to both short and long term hormonal control. Therefore, we used hepatocytes isolated from weanling rats fed a very low protein diet (0.5 g protein/100 g diet) or a diet adequate in protein for 2 wk to investigate whether a loss of hormonal control could contribute to abnormal regulation of hepatic glutathione. Glutathione concentration in hepatocytes isolated from protein-energy malnourished rats was 82% lower than in controls. In vitro supplementation of isolated hepatocytes with oxothiazolidine-4-carboxylate or methionine increased glutathione concentration in hepatocytes from malnourished rats to concentrations equivalent to control cells. However, when hepatocytes were incubated with cysteine, total glutathione in malnourished rats exceeded that of controls. Treatment of cells from control rats with 50 nmol/L glucagon or 1 mmol/L db-cAMP decreased glutathione concentration by 25-43%. In contrast, the glutathione concentration in hepatocytes of rats fed the low protein diet did not respond to treatment with glucagon or db-cAMP. These data indicate that glutathione synthesis is insensitive to regulation by cAMP in rats with protein-energy malnutrition.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of ethanol-induced changes in adenosine transport on cAMP production in rat hepatocytes.

Ethanol-induced increases in extracellular adenosine in primary cultures of rat hepatocytes are due to inhibition of adenosine uptake via the nucleoside transporter, as well as increased adenosine production resulting from ethanol metabolism. Chronic treatment of hepatocytes with ethanol during culture increases receptor-stimulated cAMP production. This increase is due, at least in part, to a decrease in Gi mediated inhibition of cAMP production and can be prevented by co-culture with the adenosine A1 receptor antagonist, cyclopentyltheophylline. These data suggest that increased extracellular adenosine, acting via adenosine A1 receptors, mediates the chronic effects of ethanol on cAMP in hepatocytes.