Chronic EtOH administration alters liver Mg2+ homeostasis

Andrew Young, Christie Cefaratti, and Andrea Romani

Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4970


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Ethanol (EtOH) administration to rats for 4 wk markedly decreased Mg2+ content in several tissues, including liver. Total cellular Mg2+ accounted for 26.8 ± 2.4 vs. 36.0 ± 1.4 nmol Mg2+/mg protein in hepatocytes from EtOH-fed and control rats, respectively, and paralleled a 13% decrease in cellular ATP content. Stimulation of alpha 1- or beta -adrenergic receptor or acute EtOH administration did not elicit an extrusion of Mg2+ from liver cells of EtOH-fed rats while releasing 5% of total tissue Mg2+ content from hepatocytes of control rats. Despite the 25% decrease in Mg2+ content, hepatocytes from EtOH-fed rats did not accumulate Mg2+ following stimulation of protein kinase C signaling pathway, whereas control hepatocytes accumulated ~2 nmol Mg2+ · mg protein-1 · 4 min-1. Together, these data indicate that Mg2+ homeostasis and transport are markedly impaired in liver cells after prolonged exposure to alcohol. The inability of liver cells, and possibly other tissues, to accumulate Mg2+ can help explain the reduction in tissue Mg2+ content following chronic alcohol consumption.

hepatocytes; protein kinase C; adrenergic signaling; plasma membranes


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MAGNESIUM IS THE SECOND MOST abundant cation in mammalian cells after potassium (17, 18, 44, 45). Within the cell, Mg2+ is mainly localized inside mitochondria, endoplasmic reticulum, nucleus, and cytosol (8, 17, 45), in which it regulates numerous biological functions (reviewed in Ref. 44). The cellular abundance of Mg2+ and its physiological relevance for cell cycle, biochemical reactions, or enzyme and channel activity contrasts with the limited knowledge about the mechanism(s) regulating Mg2+ cellular homeostasis and transport. In recent years, several laboratories, including ours, have provided compelling evidence for the occurrence of large and fast fluxes of Mg2+ in and out of mammalian cells (see Ref. 45 for review). Although no mammalian Mg2+ transporter has been cloned to date, experimental and pharmacological evidence supports the operation of a Na+-dependent Mg2+ extrusion pathway, tentatively identifiable as a Na+/Mg2+ exchanger, in the majority of mammalian cells investigated (13, 18, 20). The operation of a Na+-independent Mg2+ extrusion pathway, which can use extracellular Ca2+, Mn2+, or anions to transport Mg2+, has also been reported (18, 19). These pathways can be activated via stimulation of alpha 1- and beta -adrenergic receptors in a variety of tissues, including heart (42, 49) and liver (10, 11, 43). We have recently observed that the stimulation of glucagon or beta -adrenoceptor in liver cells selectively activates the Na+-dependent Mg2+ extrusion pathway (10), whereas the administration of catecholamine activates both Na+-dependent and Na+-independent Mg2+ extrusion pathways (10). Current opinion is that the increase in cellular cAMP resulting from the activation of glucagon or beta -adrenergic receptor or from the use of cell-permeant cAMP analogs phosphorylates the putative Na+/Mg2+ exchanger and determines an extrusion of Mg2+ from the cell (20, 32, 37). In contrast, hormones or agents that decrease cellular cAMP level (39, 41, 42) or activate protein kinase C signaling (7, 38) prevent Mg2+ extrusion or induce an accumulation of Mg2+ within the cell. The nature of the Mg2+ entry pathway still remains undefined, because experimental data would support either the operation of a Mg2+ channel in cardiac myocytes (35) and kidney cells (34) or the reverse operation of the Na+/Mg2+ exchanger located in the basolateral portion of hepatocyte cell membrane (4). As for the physiological significance, changes in Mg2+ content appear to play a significant role in regulating specific mitochondrial dehydrogenases (33) and various cellular functions, including glycolysis (16).

Hypomagnesemia and a reduced Mg2+ content within several tissues, including heart and liver, have been observed following acute and chronic alcohol consumption in animal models and in chronic alcoholic patients (1, 14, 28, 52). Experimental evidence suggests that the decline in Mg2+ content is deleterious for the tissue in which it occurs (1, 28). Cardiac chrono- and inotropism (26), smooth muscle cell contractility (28), collagen deposition (36), and cell cycle (30) are all examples of functions that are affected to a varying extent by the decrease in cellular Mg2+ content. On the other hand, Mg2+ supplementation under these conditions ameliorates several metabolic functions compromised by ethanol (EtOH) administration (1, 52). Yet the mechanism(s) responsible for Mg2+ loss from the tissues, the relevance that a reduced Mg2+ content may have to the onset of alcohol-related pathologies such as cirrhosis and hepatitis, and the modality by which Mg2+ reintroduction exerts its beneficial effects are not fully investigated. Recently, we have reported that the acute infusion of EtOH in perfused liver results in a marked, dose-dependent extrusion of Mg2+ from the organ into the perfusate via a Na+-dependent mechanism (50). Mg2+ extrusion is associated with the transient reduction in cellular ATP content that occurs during the conversion of EtOH to acetaldehyde, because both processes can be largely prevented by pretreatment with the alcohol dehydrogenase inhibitor 4-methylpyrazole (50). On the basis of these results, we proposed that by impairing ATP homeostasis and enhancing its degradation (31) EtOH administration reduces the cellular buffering capacity for Mg2+, which results in an extrusion of Mg2+ from the cell (50).

In the present study, the changes in cellular Mg2+ homeostasis and transport that occur in liver cells of rats exposed to EtOH in the diet for 4 wk were investigated as a first step in determining the short- and long-term implications that changes in cellular Mg2+ content may have for the proper functioning of specific processes within the cell (e.g., glycolysis) or cellular organelles (e.g., mitochondrial dehydrogenases) and the onset of EtOH-related liver pathologies. To this purpose, perfused organs, isolated cells, and purified plasma membrane vesicles were used as experimental models to determine to what extent chronic EtOH administration affects Mg2+ homeostasis and transport in liver cells. The obtained results indicate that prolonged EtOH administration decreases hepatic Mg2+ content by 1) affecting cytosolic ATP content and 2) impairing the ability of liver cells to accumulate Mg2+, thus preventing the hepatocyte from effectively restoring cellular Mg2+ homeostasis. In addition, the decrease in cellular Mg2+ content prevents the liver cells from extruding a significant amount of Mg2+ following adrenergic stimulation, de facto hampering the physiological significance of Mg2+ extrusion locally and systemically (25).


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Collagenase (CLS2) was from Worthington. 125I-labeled Biotrak cAMP-RIA was from Amersham. Luciferin-luciferase and lactate dehydrogenase (LDH) enzymatic kits and all other chemicals and reagents were from Sigma. Lieber-De Carli diet for EtOH-treated and control animals was from Bio-Serve (Frenchtown, NJ).

Liquid diet. Male Sprague-Dawley rats (180-200 g body wt) were randomly divided into control and EtOH-treated groups and housed individually in metabolic cages. EtOH-treated rats were maintained for 4 wk with Lieber-De Carli diet containing 67 ml EtOH/l of liquid diet (6% EtOH, final concentration). Control rats received a daily amount of an isocaloric control diet comparable to that consumed by EtOH-treated rats (pair-fed protocol). Weight gain was recorded weekly for all experimental groups.

Determination of tissue cation content. Rats were killed by cranial dislocation. Aliquots from brain, skeletal muscles, heart, diaphragm, and splanchnic organs were collected, rinsed in 250 mM sucrose, blotted on absorbing paper, weighed, minced, and homogenized in 10% HNO3. After overnight extraction, the acid mixture was centrifuged at 2,000 g for 5 min to sediment denatured protein. The Na+, K+, Ca2+, and Mg2+ contents of the acid extracts were measured by atomic absorbance spectrophotometry (AAS) in a Perkin-Elmer 3100, calibrated with proper standards.

Perfused livers. Fed male Sprague-Dawley rats (250-350 g body wt) were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/ml). The abdomen was opened, and the liver was perfused via the portal vein with a medium containing (in mM) 120 NaCl, 3 KCl, 1 CaCl2, 1.2 KH2PO4, 0.6 MgCl2, 12 NaHCO3, 15 glucose, and 10 HEPES, pH 7.2, at 37°C, preequilibrated with O2/CO2 (95:5 vol/vol) at a flow rate of 3.5-4 ml · g-1 · min-1. Following the cannulation, the liver was rapidly removed and placed on a platform. After 5 min of equilibration, the perfusion medium was replaced with another having similar composition but devoid of Mg2+ (Mg2+-free medium). Contaminant Mg2+ content of the medium was measured by AAS and found to be ~8-10 µM. After 10 min of washout with Mg2+-free medium (time 0), samples of the effluent were collected at 30-s intervals and Mg2+ content of the perfusate was measured by AAS. The samples collected during the first 8 min provided a baseline for the subsequent infusion of EtOH or adrenergic agonist. EtOH was infused in a range of concentrations between 0.01% (1.6 mM) and 1% (160 mM) (50). A selective alpha 1- (phenylephrine, 5 µM) or beta - (isoproterenol, 10 µM) adrenergic agonist or the mixed adrenoceptor agonist catecholamine (epinephrine, 5 µM) was infused at pharmacological doses to exclude lack of an effect due to altered adrenoceptor responsiveness. At the end of the perfusion, the liver was gently blotted on adsorbing paper and weighed. Aliquots of 1-1.5 g were homogenized in 5 volumes of 10% HNO3 and digested overnight to measure total tissue Mg2+ content. The protein content of the acid extract was sedimented in a refrigerated Beckman J-6B centrifuge (2,000 g for 10 min), and the Mg2+ content in the supernatant was measured by AAS.

Sedimentation of aliquots of the perfusate in Microfuge tubes (14,000 g for 10 min) and protein assay excluded the persistence of nonresident cellular and plasma proteins in the collected medium (50). The absence of cell damage was assessed by enzymatically measuring LDH activity in aliquots of the perfusate at 1-min intervals throughout the procedure. The release of K+ from damaged cells into the perfusate was measured by AAS in aliquots of the perfusate (50).

Estimation of the total amount of Mg2+ extruded. To estimate the total amount of Mg2+ extruded from the livers following EtOH or adrenergic agonist administration, the Mg2+ content in the perfusate at the last five time points before addition of the agent was averaged and subtracted from each of the time points under the curve of efflux. The net amount of Mg2+ mobilized into the perfusate (nmol/ml) was then calculated, taking into account the perfusion rate (3.5-4 ml · g-1 · min-1) and the time of collection (30 s), and expressed as micromoles. The residual Mg2+ content of the perfused livers was also calculated in the tissue homogenate as described above.

Collagenase-dispersed cells. Collagenase-dispersed rat hepatocytes were isolated according to the procedure of Seglen (47). After the isolation, hepatocytes were resuspended, at a final concentration of ~1 × 106 cells/ml, in a medium of the following composition (in mM): 120 NaCl, 3 KCl, 1.2 KH2PO4, 12 NaHCO3, 1.2 CaCl2, 1.2 MgCl2, 10 HEPES, and 10 glucose, pH 7.2, at 37°C, under O2/CO2 (95:5 vol/vol) flow, and kept at room temperature until used. Cell viability was 88 ± 3% for both EtOH-fed and control rats (n = 10/group), as assessed by trypan blue exclusion, and did not significantly change over 3-4 h (86 ± 2%). For the determination of Mg2+ transport, 1 ml of cell suspension was transferred into a Microfuge tube and the cells were rapidly sedimented at 600 g for 30 s. The supernatants were removed, and the cells were washed with 1 ml of a medium having the same composition as the one reported above but devoid of Mg2+ (incubation medium). The cells were then transferred in 10 ml of incubation medium, prewarmed at 37°C, and incubated therein under continuous stirring and O2/CO2 flow. After a few minutes of equilibration, 0.01, 0.1, or 1% EtOH or the reported concentrations of adrenergic agonists or protein kinase C-stimulating agents were added to the incubation system. At the time points reported in the figures, 700-µl aliquots of the incubation mixture were withdrawn in duplicate, and the cells were sedimented in Microfuge tubes. The Mg2+ content in the supernatant was measured by AAS.

Determination of cytosolic cAMP levels. cAMP level was determined in aliquots of cell extract by 125I-RIA as reported previously (50). Briefly, collagenase-dispersed hepatocytes were incubated under the experimental conditions described in Collagenase-dispersed cells. At 5-min intervals, aliquots of the incubation mixture (700 µl) were withdrawn and the cells were sedimented in Microfuge tubes. The supernatant was removed for determination of Mg2+ content by AAS. The cell pellets were resuspended in 200 µl of assay buffer (Biotrak; Amersham), boiled for 5 min, and stored at -20°C until used.

Determination of ATP levels. Cellular ATP level was also measured in isolated hepatocytes incubated as reported previously (50). At 5-min intervals, 700-µl aliquots of the incubation mixture were withdrawn and the cells were sedimented in Microfuge tubes. The supernatant was removed, and the cells were digested in perchloric acid (5% final concentration) for 5 min in ice. The acid mixture was then neutralized by the addition of KHCO3, and the denatured protein was sedimented in a refrigerated Beckman J-6B (2,000 g for 10 min). The supernatants were removed and stored at -20°C until used. ATP determination was carried out by luciferin-luciferase assay (detecting sensitivity in the picomole to nanomole per milliliter range; Sigma), using a LUMAT Berthold LB 9501 luminometer, or by HPLC, using a C18 RP column (Millipore Waters) and 60 mM ammonium phosphate (pH 6.6 with ammonium hydroxide) as the mobile phase (50). Adenine phosphonucleotide standards (1-20 nmol/ml) were injected for calibration.

Determination of glucose, LDH, and protein. Aliquots of the perfusate were collected every minute, and glucose content was determined by enzymatic kit (Sigma) as the variation in hexokinase-coupled NADH+ content at 340 nm. LDH activity in the perfusate or in the extracellular medium was measured by enzymatic kit (Sigma) sensitive to changes in the microunit-per-milliliter range and expressed as units per liter or as a percentage of the total amount of LDH releasable from digitonin-permeabilized hepatocytes. Cell viability was assessed by trypan blue exclusion test. Protein content was measured according to the procedure of Lowry et al. (29) using BSA as standard.

Cytosolic free Ca2+ measurement. Changes in cytosolic Ca2+ concentration ([Ca2+]) were measured in fura-2 AM-loaded hepatocytes as previously reported (39) by using a dual-wavelength excitation fluorometer (University of Pennsylvania Biomedical Instrumentation Group, Philadelphia, PA) equipped with a thermostated cuvet.

Plasma membrane isolation. Total liver plasma membrane vesicles (tLPM) were isolated and stored as described in detail by Cefaratti et al. (3). The purity of plasma membrane vesicles (Table 1) was assessed by using 5'-nucleotidase, cytochrome c oxidase, and glucose 6-phosphatase activities as markers for plasma membrane, mitochondria, and endoplasmic reticulum, respectively (3). Negligible levels of cytochrome c oxidase and glucose 6-phosphatase activities were detected in tLPM preparations, whereas both 5'-nucleotidase and Na+-K+-ATPase were found to be enriched 12- and 8-fold in tLPM from control and EtOH-fed livers, respectively (Table 1). The activity of the latter two enzymes was also used to evaluate the orientation of tLPM vesicles (3). The comparison of these activities to those of detergent-disrupted vesicles (considered as 100%) confirmed our early report (3) that >= 90% of EtOH-treated and control tLPM were in the "inside-in" configuration following loading with Mg2+. The tLPM endogenous carryover of cations and adenine phosphonucleotides was measured by AAS and HPLC, respectively, and found to be negligible (not shown).

                              
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Table 1.   Enzymatic activity in liver plasma membrane vesicles from control and EtOH-fed rats

Loading of tLPM and determination of Mg2+ fluxes. Five-milliliter aliquots of tLPM were resuspended in 25 ml of 250 mM sucrose and 25 mM HEPES (pH 7.4 with Tris) in the presence of 20 mM MgCl2 and were loaded by four passes in a Thomas C Potter with a tight-fitting pestle at 4°C (3). We have previously reported that this Mg2+ concentration is optimal to study Mg2+ transport in this experimental model (3). The mixture was sedimented at 34,500 g for 10 min in a Sorvall SS-34 rotor to remove excess extravesicular cation. The Mg2+-loaded vesicles were resuspended in 5 ml of the Mg2+-free medium and stored in ice until used. Loading efficiency was assessed by treating the vesicles with 2 µg/ml A23187 or 0.1% Triton X-100 and by measuring the amount of Mg2+ extruded in the extravesicular space or retained in the vesicle pellet by AAS, as reported previously (3).

Mg2+ fluxes were measured by AAS. An aliquot of Mg2+-loaded tLPM was incubated in the Mg2+-free medium at 37°C under continuous stirring at a final concentration of ~300 µg protein/ml. After 2 min of equilibration, aliquots of the incubation mixture were withdrawn in duplicate at 2-min intervals, and the vesicles were sedimented in Microfuge tubes at 7,000 g for 45 s (3). Total Mg2+ content in the supernatants was measured by AAS. The pellet was digested overnight in 500 µl of 10% HNO3. The denatured protein was sedimented in Microfuge tubes, and the Mg2+ content of the acid extract was measured by AAS. The first two time points after the equilibration period (time 0) were used to establish a baseline. Following the withdrawal of the second sample, 50 mM Na+ or 500 µM Ca2+ was added to the incubation mixture to elicit maximal Mg2+ extrusion from tLPM suspensions (3), and the incubation continued for six additional minutes. For simplicity, the data are reported as the net variation in extravesicular Mg2+ content, normalized per milligram of protein. To calculate net Mg2+ extrusion, Mg2+ content in the supernatant at the first two time points was calculated, averaged, and subtracted from the values of the subsequent time points of incubation.

Statistical analysis. The data are reported as means ± SE. Data were first analyzed by one-way ANOVA. Multiple means were then compared by Tukey's multiple comparison test performed with a q value established for statistical significance of P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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REFERENCES

Male Sprague-Dawley rats fed for 4 wk with a 6% EtOH-enriched liquid diet weighed 7% less than rats receiving an isocaloric control liquid diet (392.6 ± 5.5 vs. 421.6 ± 4.6 g body wt, respectively; n = 11 for both experimental groups).

The determination of tissue cation content indicated a marked decrease in Mg2+ content in several tissues of EtOH-fed animals compared with control rats (Table 2). All of the muscles examined (abdomen, hind limb, soleus, quadriceps, heart, and diaphragm) presented a decrease in Mg2+ content varying between 12 and 19% (Table 2). The liver was also affected. Despite a 20% increase in liver mass compared with control animals (13.52 ± 0.50 vs. 11.21 ± 0.41 g of tissue, respectively; n = 11; P < 0.05), EtOH-fed rats presented a 14% decrease in liver Mg2+ content (57.9 ± 4.1 vs. 67.1 ± 5.0 nmol/mg protein). A marked decrease in Mg2+ content was also observed in lung, spleen, and blood cells, whereas brain, testicles, adipose tissue, and pancreatic tissue from EtOH-fed rats presented little or no change in Mg2+ content compared with control animals (Table 2). Mg2+ content was markedly increased in the urine of EtOH-fed rats (128%; Table 2). K+ content was also decreased in skeletal muscles and liver, whereas Na+ and Ca2+ content were increased (Table 2).

                              
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Table 2.   Tissue cation content in EtOH-fed and control rats

Our laboratory has extensively utilized perfused livers and collagenase-dispersed hepatocytes to elucidate the hormonal regulation of Mg2+ transport and homeostasis (10, 11, 37-39, 43). Hence we used these two experimental models to investigate to what extent the observed decrease in Mg2+ content altered the ability of liver cells to extrude Mg2+ following hormonal stimulation and whether Mg2+ could be accumulated into the cells to restore Mg2+ homeostasis.

Figure 1 shows that, in the absence of a stimulatory agent, livers of control rats retained their cellular Mg2+ content over 45 min of perfusion. The administration of epinephrine (Fig. 1A) or norepinephrine (not shown; Ref. 43) elicited the extrusion of a sizable amount of Mg2+ from the organs into the perfusate in a time-dependent fashion. Selective alpha 1- (phenylephrine) or beta - (isoproterenol) adrenergic agonists also elicited Mg2+ extrusion (Fig. 1A) from the livers, although the amount of Mg2+ mobilized by each agonist was quantitatively smaller than that mobilized by the mixed agonist catecholamine (~ 0.9, 1.8, and 3.3 µmol Mg2+ following isoproterenol, phenylephrine, and epinephrine administration, respectively). In contrast, the infusion of these agents in livers explanted from EtOH-fed animals did not induce a detectable extrusion of Mg2+ into the perfusate, albeit pharmacological doses of the agonists were infused (Fig. 1B). To determine whether the nonresponsiveness to adrenergic agonists depended on altered adrenoceptor sensitivity, 250 µM of the cell-permeant cAMP analog 8-Cl-cAMP (Fig. 2) was infused to increase cellular level of cAMP and induce Mg2+ extrusion bypassing the beta -adrenoceptor (10, 37, 43). Consistent with previous reports from this laboratory (10), 8-Cl-cAMP elicited an extrusion of Mg2+ from livers of control rats that was quantitatively similar to that elicited by isoproterenol under similar experimental conditions (~1.8 µmol under both conditions). In contrast, this agent failed to elicit a Mg2+ extrusion in livers from EtOH-fed rats (Fig. 2). In addition, livers from EtOH-fed rats exhibited a reduced isoproterenol- or epinephrine-induced glucose extrusion (1.9 and 9.8 mmol/l, respectively) compared with control animals (~50 mmol/l) (Fig. 3). The infusion of varying EtOH concentrations (Fig. 4B) also failed to induce a Mg2+ extrusion from livers of EtOH-fed rats, whereas a time- and dose-dependent Mg2+ mobilization was observed in control livers under similar experimental conditions (Fig. 4A; see also Ref. 50).


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Fig. 1.   Adrenergic agonist-induced Mg2+ extrusion from perfused livers of ethanol (EtOH)-fed and control-fed rats. Livers from control (A) and EtOH-fed (B) rats were perfused as reported in MATERIALS AND METHODS. Epinephrine (Epi; 5 µM), phenylephrine (Phe; 5 µM), or isoproterenol (Iso; 10 µM) was infused for times shown. Data are means ± SE of 4 livers for each experimental condition for both control and EtOH-fed animals. * Statistically significant vs. basal values. Labeling is omitted for phenylephrine- and epinephrine-treated livers for simplicity. All of the values under the curve of efflux in A are statistically significant vs. values reported in B. Labeling is omitted for simplicity.



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Fig. 2.   cAMP-induced Mg2+ extrusion from perfused livers of EtOH-fed and control-fed rats. Livers from control were perfused with 250 µM 8-Cl-cAMP (a cell-permeant cAMP analog) for times shown. Data are means ± SE of 4 livers for both control and EtOH-fed animals. All values under the curve of efflux for control animals are statistically significant vs. basal values (see Fig. 1A) and vs. cAMP-stimulated livers from EtOH-fed rats. Labeling is omitted for simplicity.



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Fig. 3.   Adrenergic agonist-induced glucose extrusion from perfused livers of EtOH-fed and control-fed rats. Glucose extrusion was measured as reported in MATERIALS AND METHODS in aliquots of the perfusate. Data are means ± SE of 4 livers for each experimental condition for both control and EtOH-fed animals. * Statistically significant vs. control-fed rats.



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Fig. 4.   Alcohol-induced Mg2+ extrusion from perfused livers of EtOH-fed and control-fed rats. Livers from control (A) and EtOH-fed (B) rats were perfused with 0.01%, 0.1%, or 1% EtOH. Alcohol doses were infused for times shown. Data are means ± SE of 5 livers for each experimental condition for both control and EtOH-fed animals. * Statistically significant vs. basal values.

To better quantify the decrease in tissue Mg2+ content, hepatocytes were isolated by collagenase digestion. Total Mg2+ content in hepatocytes from EtOH-treated rats accounted for 26.03 ± 1.24 nmol/mg protein (n = 14), which represented a 25% decrease in Mg2+ content compared with the value determined in hepatocytes from control rats (36.18 ± 0.52 nmol/mg protein; n = 10; P < 0.05). The lower hepatic Mg2+ content paralleled a 13% decrease in cellular ATP content (12.15 ± 0.13 vs. 14.03 ± 0.20 nmol ATP/mg protein for EtOH-fed and control livers, respectively; n = 14; P < 0.05).

A typical incubation time course for Mg2+ extrusion in hepatocytes is reported in Fig. 5A. As the figure shows, no Mg2+ is extruded from liver cells until an agonist is added to the incubation system. Following agonist addition, Mg2+ is extruded from the cells into the extracellular compartment in a time-dependent fashion, reaching the maximum at 4-6 min after the addition. Prolonged incubations did not result in a larger Mg2+ extrusion (not shown; Refs. 37 and 39). For simplicity, the net amount of Mg2+ extruded at 6 min (i.e., 4 min after the agonist addition) was calculated as indicated in MATERIALS AND METHODS and was reported for the subsequent figures. Liver cells from control animals extruded ~2-2.5 nmol Mg2+/mg protein within 4 min from the addition of 0.01% EtOH, adrenergic agonist, cell-permeant cAMP, or thapsigargin (Fig. 5B). In contrast, hepatocytes isolated from EtOH-fed rats (Fig. 5B) mobilized a smaller amount of Mg2+ (0.4-0.6 nmol · mg protein-1 · 4 min-1) under similar experimental conditions. The determination of cAMP level in isolated hepatocytes from EtOH-fed rats stimulated by epinephrine or isoproterenol indicated a limited increase in second messenger level from basal level (+23% and +17%, respectively) following stimulation by beta -adrenergic agonist compared with controls (+65% and +45% for epinephrine- and isoproterenol-stimulated cells, respectively; Table 3).


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Fig. 5.   Mg2+ extrusion in hepatocytes from EtOH-fed and control-fed rats. Hepatocytes from both experimental groups were incubated as reported in MATERIALS AND METHODS. Epinephrine (5 µM), phenylephrine (5 µM), isoproterenol (10 µM), 8-Cl-cAMP (cAMP; 250 µM), or thapsigargin (Thaps; 2 µM) was added after removal of samples at 2 min. A typical experiment is shown in A. Net Mg2+ extrusion at 6 min (i.e., 4 min after the agonist addition) is shown in B. Data in B are means ± SE of 5 different preparations, each performed in quadruplicate, for all of the experimental conditions reported. * Statistically significant vs. net Mg2+ extrusion in liver cells from EtOH-fed rats.


                              
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Table 3.   cAMP content in liver from EtOH-fed vs. control rats

Whereas activation of cAMP/protein kinase A pathway elicits Mg2+ extrusion, stimulation of protein kinase C signaling pathway results in an accumulation of Mg2+ within liver cells (38, 39). Figure 6A shows that the addition of vasopressin, carbachol, phorbol myristate acetate derivatives, or the diacylglycerol analogs oleoylacetyl glycerol and stearoylarachidonolyl glycerol resulted in a net accumulation of ~2 nmol Mg2+ · mg protein-1 · 4 min-1 in hepatocytes from control animals. Liver cells from EtOH-fed rats were, in contrast, unable to accumulate Mg2+ irrespective of the agent (or dose; not shown) administered to stimulate protein kinase C signaling pathway and the extracellular Mg2+ concentration utilized (i.e., contaminant or 1 mM; Fig. 6B). Basal cytosolic free [Ca2+], although slightly higher in hepatocytes from EtOH-fed rats compared with control cells (108 ± 12 vs. 90 ± 7 nM, respectively; n = 5 for both experimental conditions) did not approach statistical significance. In line with data reported by others (51), mobilization of Ca2+ by 10 nM vasopressin was ~30% lower in hepatocytes from EtOH-fed vs. control rats (215 ± 21 vs. 305 ± 35 nM, respectively; n = 5 for both experimental conditions; P < 0.05).


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Fig. 6.   Mg2+ uptake in hepatocytes from EtOH-fed and control-fed rats. Hepatocytes from both experimental groups were stimulated by the addition of 20 nM vasopressin (VP or Vasopres), 100 µM carbachol, 20 nM phorbol 12,13-dibutyrate (PDBU), or 20 nM oleoylacetyl glycerol (OAG) to the incubation mixture. A typical experiment is shown in A. Net Mg2+ extrusion at 6 min (i.e., 4 min after agonist addition) is shown in B. Data are means ± SE of 5 different preparations, each performed in quadruplicate, for all of the experimental conditions reported. * Statistically significant vs. basal and vs. net Mg2+ accumulation in liver cells from EtOH-fed rats.

The operation of distinct Mg2+ transport mechanisms has been evidenced in purified liver plasma membrane vesicles (3, 4), a model in which intravesicular Mg2+ content is artificially rendered similar to the intracellular concentration (20 mM) and the transport from the vesicle into the extravesicular space is virtually similar to the extrusion from the cell into the extracellular space. Hence we used this experimental model to ascertain whether the lack of Mg2+ transport observed in perfused livers and isolated hepatocytes could be ascribed to an intrinsic alteration of Mg2+ transporters. As Fig. 7 indicates, plasma membrane vesicles isolated from control rats extruded ~120 and ~150 nmol Mg2+/mg protein when stimulated by 50 mM Na+ and 500 µM Ca2+, respectively (see also Ref. 3). In contrast, Mg2+-loaded plasma membranes from EtOH-fed rats did not extrude Mg2+ efficiently when stimulated with Na+ or Ca2+ (-70% for both conditions). Concentrations of Na+ and Ca2+ larger than those reported in the figure did not elicit a larger Mg2+ mobilization from plasma membrane vesicles (not shown).


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Fig. 7.   Mg2+ extrusion in plasma membrane vesicles from EtOH-fed and control-fed rats. Total liver plasma membrane vesicles (tLPM) were isolated from livers of EtOH-fed and control rats and incubated in ion-free medium as reported in MATERIALS AND METHODS. After a few minutes of equilibration, 50 mM NaCl or 500 µM CaCl2 was added to the incubation mixture after the withdrawal of samples at 2 min. Additional samples were withdrawn at 2-min intervals. A typical experiment is shown in A. Net Mg2+ extrusion was determined as described in MATERIALS AND METHODS and is shown in B. Data are means ± SE of 5 different preparations, each performed in quadruplicate, for all of the experimental conditions reported. * Statistically significant vs. Mg2+ extrusion in tLPM from control-fed rats.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Extensive investigation in various experimental models and alcoholic patients (1, 14, 28, 52) indicates that chronic EtOH consumption alters physiological functions and ion pattern within several tissues. Tissue Na+ and Ca2+ content have been reported to increase as a result of an altered operation of transport mechanisms (15, 48) and cell signaling (2, 21, 22). A decrease in tissue Mg2+ content has also been reported to occur in brain, heart, and other tissues, but the causes for this decrease still remain largely unclear. Although experimental evidence supports a role of Mg2+ in regulating collagen deposition (36), glycogen phosphorylase activation (16), and mitochondrial alpha -ketoglutarate dehydrogenase activity (33), it is not fully determined to what extent these cellular processes are impaired by the decrease in Mg2+ content within the tissues nor the long-term implications that changes in the activity of these functions may have for the development of EtOH-related hepatic pathologies.

During the last decade, our laboratory has extensively contributed to the characterization of the hormone-controlled mechanisms that regulate cellular and whole body Mg2+ homeostasis (10, 11, 24, 37-39, 43). We have reported that the increase in cellular cAMP elicited via beta -adrenoceptor stimulation induces a marked extrusion of cellular Mg2+ from cardiac (42) and liver cells (37, 43). The process is likely to be more general, in that infusion of beta -adrenoceptor agonist raises plasma Mg2+ level by 15-20% in anesthetized rats (24). In contrast, agents or hormones that decrease cAMP level (38-43) or stimulate protein kinase C signaling pathway (38) prevent Mg2+ extrusion from the cell and/or revert Mg2+ extrusion into a Mg2+ accumulation.

Recently, we started to investigate the mechanisms responsible for the decrease in liver Mg2+ content following EtOH administration. We have reported that acute EtOH administration induces a marked extrusion of Mg2+ from liver cells in a time- and dose-dependent manner, decreasing liver Mg2+ content by 5-10% (50). The time course of Mg2+ extrusion matches the transient decrease in cellular ATP content observed under these experimental conditions (31, 50), thus suggesting that Mg2+ is essentially mobilized from the cytosolic MgATP pool.

The objective of the present study was to investigate the effect that chronic EtOH administration exerts on Mg2+ homeostasis and on hormone-mediated Mg2+ extrusion and uptake in liver cells as a first step toward elucidating how changes in cellular Mg2+ content affect specific hepatocyte functions.

Altered Mg2+ homeostasis in EtOH-fed animals. The administration of EtOH (6%) in the diet for 4 wk results in a marked decrease in Mg2+ content within several tissues, including liver. Compared with control animals, the decrease in hepatic Mg2+ content in EtOH-fed rats accounts for ~14% in the whole tissue and ~25% in isolated hepatocytes. Most likely, the discrepancy in the percentage of decrease between two values can be ascribed to technical manipulations or the heterogeneity inherent to whole tissue processing vs. cell isolation by collagenase before Mg2+ content determination. As already observed after acute EtOH administration (50), the decrease in Mg2+ content elicited by prolonged EtOH consumption is associated with a decrease in cellular ATP level (-13%). This observation indicates that common mechanisms are activated by acute and chronic EtOH administration, which result in a decrease of Mg2+ buffering capacity within the cell and in a loss of Mg2+ from the cytosolic MgATP pool. In the acute model of EtOH administration, the cellular conversion of EtOH to acetaldehyde inverts the NAD+/NADH ratio and favors the formation of glyceraldehyde-3-phosphate, which acts as a Pi trap and removes the inhibition by phosphate on AMP deaminase, allowing the degradation of ATP to proceed to uric acid and allantoin (31). Whereas the decrease in ATP level is transient (~8 min) during acute EtOH administration (50), in the chronic EtOH model reported here, no recovery in cellular ATP level is observed, most likely because of the continuous administration of alcohol with the diet. The amplitude of ATP decrease under acute and chronic experimental conditions is comparable, thus pointing to the lack of recovery in ATP content as the possible main cause for the persistent low level of cellular ATP and, consequently, Mg2+ content. Assuming an MgATP concentration of ~3 mM in the hepatocyte cytosol (6) and the differing dissociation constants for MgADP (~250 µM) and MgAMP (~8.13 mM) vs. MgATP (~80 µM), the lower cellular ATP content results in a major change in buffering capacity for Mg2+. Consequently, a larger amount of Mg2+ will dissociate from the degrading phosphonucleotides and be lost from the cell, most likely via a Na+-dependent mechanism (50). This scenario can explain the increase in Na+ content observed in the tissues in which Mg2+ was decreased, including liver (Table 1), as well as the lack of an effect of acute EtOH administration on these hepatocytes, since an acute infusion of EtOH will target the cytosolic Mg2+ pool, which is already depleted by the prolonged administration of EtOH with the diet. Because total Mg2+ concentration within the hepatocyte accounts for 18-20 mM (44), even a 14% decrease in total tissue Mg2+ content (the most conservative of our determinations) represents a major change in terms of cellular content (~2.5 mM). This value is about threefold larger than cytosolic free Mg2+ (estimated to be ~0.7-1 mM; Ref. 6), and about twofold larger than the combined amounts of free Mg2+ in the cytosol and Mg2+ no longer bound to ATP (a 13% decrease in total ATP content would account for ~650 µM Mg2+; Ref. 50). Hence we have to assume that a considerable portion of Mg2+ lost from the cell is coming from intracellular organelles such as mitochondria, endoplasmic reticulum, and nucleus, which all contain ~16-18 mM total Mg2+ under physiological conditions (8). The loss of Mg2+ from any, or more than one, of these compartments will then affect the Mg2+ content of the organelle, with obvious consequences on the activity rate of enzymes therein located (e.g., alpha -ketoglutarate dehydrogenase in the mitochondria; Ref. 33).

Transport in EtOH-fed animals. Hormone-modulated Mg2+ transport mechanisms are also markedly affected by a prolonged exposure to EtOH in the diet. Although the decrease in cellular Mg2+ content is sufficient to explain the lack of Mg2+ extrusion following beta -adrenergic agonist or cell-permeant cAMP addition, the determination of basal cellular cAMP level, which in hepatocytes from EtOH-fed rats is not dissimilar from that of control cells (3.6 ± 0.4 vs. 3.8 ± 0.2 pmol/mg protein, respectively) and increases only marginally in response to isoproterenol or epinephrine administration (+17-23%), and the observation that glucose extrusion from EtOH-fed livers is also markedly reduced compared with control organs indicate that EtOH exerts an inhibitory effect on various elements of the adrenergic signaling cascade, including G proteins and phosphodiesterase (21, 22). Furthermore, the marginal increase in cAMP level and the reduced output of glucose observed following adrenergic stimulation indicate that hormonal activated glycolysis is markedly hampered in EtOH-fed rats and cannot effectively contribute to maintaining a physiological level of ATP within liver cells. In addition, the reduced Mg2+ extrusion observed in purified tLPM (Fig. 7) suggests that EtOH administration affects the Mg2+ transport mechanisms in the plasma membrane, either directly [EtOH-related modification of the phospholipid environment (25) or EtOH-protein interaction (26)] or indirectly (via the decrease in cellular ATP content). Cefaratti et al. (5), in fact, have proposed that the Mg2+ transport mechanisms operating in purified plasma membrane vesicles become phosphorylated and active during the isolation procedure. Hence it is conceivable that the decrease in ATP content and the defects in cAMP signaling elicited by EtOH alter the modus operandi or the activity rate of the Mg2+ transport mechanisms in the cell membrane.

The decrease in amplitude of Mg2+ transport in tLPM from EtOH-fed rats vs. that of control tLPM (~70%) is essentially similar to the decrease in amplitude of Mg2+ extrusion observed in the corresponding hepatocytes (~75%). On the other hand, the lack of detectable Mg2+ extrusion in perfused liver might depend on the dilution of the minimal amount of extruded Mg2+ in the high volume of perfusate (>= 40 ml/min) or in an altered delivery and/or interaction of the agonist to the specific receptor.

Lastly, hepatocytes from EtOH-fed rats are unable to accumulate Mg2+ from the extracellular compartment following stimulation of protein kinase C signaling pathway, irrespective of the stimulatory agent and the extracellular Mg2+ concentration utilized. We have previously reported that Mg2+ uptake in liver cells is inhibited under conditions in which protein kinase C is downregulated (38) or cytosolic Ca2+ is increased (39), whereas the absence of extracellular Ca2+ does not affect the amplitude of Mg2+ uptake. Both protein kinase C and Ca2+ signaling pathways are altered in various ways by chronic EtOH administration (9, 27, 46). Consistent with reports from other laboratories, EtOH treatment does not appear to significantly alter basal cytosolic free [Ca2+], although the responsiveness to vasopressin is decreased (see also Ref. 51). Yet a direct effect of EtOH on the transport mechanism responsible for Mg2+ uptake at the plasma membrane level cannot be excluded altogether. In this respect, we have recently observed (12) that in hepatocytes from diabetic rats, which also present reduced Mg2+ content and inability to accumulate the cation following hormonal stimulation, the bidirectional Na+/Mg2+ exchanger located in the basolateral domain of the plasma membrane is unable to reverse its modus operandi and elicit Mg2+ uptake. Alternatively, it is possible that Mg2+ uptake within the hepatocyte is an energy-dependent process, which is thus affected by the reduced level of ATP detected within the cell. Further studies are required to determine which of these possibilities is the correct one and whether this is a persistent or a transient phenomenon.

To our knowledge, this is the first study investigating the effect of chronic EtOH administration on liver Mg2+ homeostasis and transport. The reported results confirm the data obtained in liver cells acutely stimulated by EtOH (unpublished observations) and indicate that prolonged alcohol utilization results in a major loss of cellular Mg2+ within hepatocytes and affects the ability of liver cells to transport and accumulate Mg2+ effectively, thus providing an explanation for the decrease in tissue Mg2+ content observed in chronic alcoholics. Most importantly, our results indicate that hepatocytes are unable to accumulate Mg2+ following hormonal stimulation despite the marked loss (-20-25%) in total cation content. The loss in Mg2+ appears to affect to a similar extent all of the cellular Mg2+ pools, including that associated with ATP in the cytoplasm, therefore strengthening the hypothesis that the decrease in cellular ATP content, most likely for metabolic reasons (28, 31), is partially responsible for the Mg2+ loss. However, the pharmacological approach we used here to evaluate Mg2+ content within mitochondria and endoplasmic reticulum does not rule out the possibility that Mg2+ redistributes between these organelles and the cytoplasm during the time course of our experimental protocol. Presently, we have no information as to whether Mg2+ and ATP content decrease further with a longer EtOH administration or whether switching back to a pellet diet can restore Mg2+ and ATP content within liver cells and other tissues. However, the decrease in ATP and Mg2+ content measured in our chronic model is quantitatively similar to the decrease measured in liver cells acutely infused with EtOH (50), thus suggesting that a new cellular set point has already been attained. The implications of the decrease in Mg2+ and ATP content on liver metabolism and collagen deposition are presently under investigation.


    ACKNOWLEDGEMENTS

This work was supported by National Institute on Alcohol Abuse and Alcoholism Grant R9-AA-11593-A2


    FOOTNOTES

Address for reprint requests and other correspondence: A. Romani, Dept. of Physiology and Biophysics, Case Western Reserve Univ., 10900 Euclid Ave., Cleveland, OH 44106-4970 (E-mail: amr5{at}po.cwru.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpgi.00153.2002

Received 22 April 2002; accepted in final form 23 September 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Gastrointest Liver Physiol 284(1):G57-G67
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