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Am J Physiol Gastrointest Liver Physiol 275: G1106-G1116, 1998;
0193-1857/98 $5.00
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Vol. 275, Issue 5, G1106-G1116, November 1998

Acute effect of EtOH on Mg2+ homeostasis in liver cells: evidence for the activation of an Na+/Mg2+ exchanger

Patrick A. Tessman and Andrea Romani

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

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The acute administration of ethanol mobilizes a considerable amount of Mg2+ from perfused rat livers and isolated hepatocytes in a dose-dependent fashion in the absence of release of cellular K+ or lactate dehydrogenase (LDH) in the extracellular medium. Mg2+ extrusion becomes detectable within 2 min and reaches the maximum within 8 min after ethanol addition, declining toward the basal value thereafter irrespective of the persistence of alcohol in the perfusion system and the dose of ethanol administered. The effect is the result of a specific impairment of Mg2+ transport and/or regulatory mechanisms. In fact, Mg2+ extrusion does not occur under conditions in which 1) ethanol is replaced by an equivalent dose of DMSO, 2) amiloride or imipramine are used as inhibitors of the Na+/Mg2+ exchanger, 3) extracellular Na+ is replaced by an equimolar concentration of choline chloride, and 4) 4-methylpyrazole is used to specifically inhibit alcohol dehydrogenase and cytochrome P-4502E1. Finally, the observation that the cellular level of ATP is markedly reduced after acute ethanol administration would suggest that Mg2+ extrusion results from a decreased buffering capacity of cytosolic Mg-ATP complex.

sodium/magnesium exchanger; adenosine 5'-triphosphate; perfusion

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

ALCOHOL ABUSE IS ASSOCIATED with severe damage of several biological functions and activities within the cell (9, 18, 29, 49), including electrolyte homeostasis (10). These effects are exerted via a direct modification of biological membrane fluidity or via the production of reactive molecules (acetaldehyde, aldehyde derivatives, and free radical moieties) (30), which hamper the operation of signal transduction pathways and key enzymes located in the plasma membrane or in the membrane of intracellular organelles, in particular the endoplasmic reticulum and mitochondria (20).

As for ion homeostasis, severe impairment of cellular Ca2+ homeostasis (see Ref. 20 for a review), of Ca2+- and Mg2+-ATPase activity (12), and of Na+-K+-ATPase operation (47) has been observed after both acute and chronic ethanol (EtOH) administration. Changes in cellular and plasma Mg2+ homeostasis have also been observed. Acute EtOH ingestion is paralleled by an increase in the plasma Mg2+ level and in the urinary excretion of the cation (39), whereas chronic EtOH consumption is accompanied by a marked decrease in plasma and cellular Mg2+ content in humans (8) and animals (19) as well. Both these observations imply that Mg2+ can rapidly be extruded from organ(s) or tissue(s) into the bloodstream and that Mg2+ uptake and/or extrusion processes, especially in the kidney (40) and intestine (24), are affected to some extent by alcohol administration. Yet the modality of Mg2+ mobilization from the tissues and the subsequent changes in cellular Mg2+ homeostasis and redistribution are largely uncharacterized.

In recent years, an increasing number of reports indicated that cellular Mg2+ homeostasis is markedly affected by hormonal stimulation. Evidence provided by this (42-44) and other laboratories (15, 16, 23, 36, 50) suggests that major fluxes of Mg2+ can cross the plasma membrane of cardiac myocytes (44, 50), hepatocytes (16, 42, 43), and other mammalian cell types (15, 36) in either direction after a variety of hormonal stimuli. Mg2+ extrusion appears to occur through an increase in cytosolic cAMP level (15, 42, 44, 50), which activates a Na+/Mg2+ exchanger (16, 43, 50), likely the most represented Mg2+ transport mechanism in the plasma membrane of mammalian cells (see Refs. 13, 45 for reviews). The stoichiometry of this exchanger is still undefined because it appears to exchange 1 Na+ for 1 Mg2+ in human (33) and ferret red blood cells (7) and 2 Na+ for 1 Mg2+ in chicken erythrocytes (14). Irrespective of the exchange ratio, in the absence of more specific inhibitors, the transporter operation is blocked by submillimolar concentrations of amiloride (16, 50) or imipramine (6, 14) or by the removal of Na+ from the extracellular compartment (43). More potent amiloride derivatives such as 5-(N, N-hexamethylene)-amiloride and 5-(N, N-dimethyl)-amiloride are ineffective at inhibiting this Mg2+ transport mechanism in mammalian (51) and nonmammalian (17) cell types. The presence of a distinct Na+-independent Mg2+ transport mechanism in mammalian cell types has also been suggested (13, 45), but its modality of operation and inhibition are at present poorly characterized.

In the present study, the possibility that acute administration of EtOH can mobilize Mg2+ from liver cells was investigated. The reported results indicate that EtOH mobilizes Mg2+ in a dose-dependent manner from perfused livers and from collagenase-dispersed hepatocytes in the absence of K+ and lactate dehydrogenase (LDH) release and significant changes in cellular cAMP level. The extrusion of Mg2+ correlates well with a concomitant decrease in cytosolic ATP level. The dependence of EtOH-mediated Mg2+ extrusion on the presence of a physiological concentration of Na+ in the extracellular compartment and its inhibition by amiloride or imipramine suggest that Mg2+ extrusion occurs via the putative Na+/Mg2+ exchanger reported to operate in liver plasma membrane. Finally, because the EtOH-induced Mg2+ extrusion is prevented by the infusion of 4-methylpyrazole (4-MP), a specific inhibitor of cytosolic alcohol dehydrogenase and endoplasmic reticulum cytochrome P-4502E1, but not by the aldehyde dehydrogenase inhibitor cyanamide (CyN), indication is there that the biotransformation of EtOH to acetaldehyde plays an important role in impairing cellular Mg2+ homeostasis and transport.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Perfused livers. Fed male Sprague-Dawley rats (250-350 g body wt) were anesthetized with an intraperitoneal injection of pentobarbital sodium. The abdomen was opened, and the liver was perfused via the portal vein with a Krebs-Henseleit medium containing 139 mM Na+, 114 mM Cl-, 4.7 mM K+, 1 mM Ca2+, 1.2 mM H2PO-4, 0.6 mM Mg2+, 12 mM HCO-3, 15 mM glucose, and 10 mM HEPES, pH 7.2, at 37°C, preequilibrated with O2-CO2 (95:5 vol/vol) at a flow of 3.5-4 ml · g-1 · min-1. After cannulation, the liver was rapidly removed and placed on a platform. After a 5-min equilibration, the perfusion medium was replaced with another having a similar composition but devoid of Mg2+ (Mg2+-free medium). The Mg2+ contaminant in the medium was ~10-15 µM as measured by atomic absorbance spectrophotometry (AAS) in a Perkin-Elmer 3100. After an 8-min washout with the Mg2+-free medium (time 0), effluent samples were collected at 30-s intervals, and the Mg2+ content in the perfusate was measured by AAS. The first 10 min provided a baseline for the subsequent EtOH infusion. 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. Two different protocols were used for EtOH administration. In the first protocol, EtOH was infused at a dose of 0.01 (1.6 mM), 0.1 (16 mM), or 1% (160 mM) starting at the end of the washout period and maintained throughout the experimental procedure (time = 45 min). In the second protocol, the reported doses of EtOH were introduced in the perfusion system at the end of the washout period and infused for only 8 min. After EtOH withdrawal, the perfusion was continued for an additional 25-27 min. At the end of the experiment, the liver was gently dried with 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 (1,000 g for 10 min), and the Mg2+ content in the supernatant was measured by AAS.

To ascertain the dependence of Mg2+ movement on the presence of extracellular Na+, in a separate set of experiments, the livers were perfused with a medium devoid of Na+ (NaCl replaced with an equimolar concentration of choline chloride).

The absence of cell damage was assessed by enzymatically measuring LDH activity in aliquots of the perfusate at 3-min intervals throughout the procedure. The release of K+ from damaged cells into the perfusate was assessed by AAS.

Estimation of the total amount of Mg2+ extruded. To estimate the total amount of Mg2+ extruded from the liver, Mg2+ content in the perfusate of the last five time points before EtOH administration was averaged and subtracted from each of the following time points under the curve of efflux. The net amount of Mg2+ mobilized into the perfusate (in 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 is expressed as micromoles. For comparison, the total residual Mg2+ content of the perfused livers in the homogenate was calculated as described in Perfused livers.

Collagenase-dispersed cells. Collagenase-dispersed rat hepatocytes were isolated according to the procedure of Seglen (46). After isolation, the hepatocytes were resuspendend at a final concentration of ~1 × 106 cells/ml in a medium having 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% (n = 8) as assessed by trypan blue exclusion test and did not significantly change over 3-4 h (85 ± 2%). For the determination of Mg2+ movement, 1 ml of cell suspension was transferred in a Microfuge tube, and the cells were rapidly sedimented at 600 g for 30 s. The supernatant was 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 few minutes of equilibration, 0.01, 0.1, or 1% EtOH was added to the incubation system. At the time points reported in Figs. 1-6, 700-µl aliquots of the incubation mixture were withdrawn in duplicate, and the cells were sedimented in Microfuge tubes. Mg2+ content in the supernatant was measured by AAS. After achievement of maximal Mg2+ extrusion, digitonin (final concentration 50 µg/ml) was added to the incubation system, and the residual Mg2+ content in the cytosol was measured in aliquots of the incubation mixture as described above.

Determination of cytosolic cAMP levels. Cytosolic cAMP level was determined in perfused livers and in suspensions of isolated hepatocytes by 125I RIA (Biotrak, Amersham). For determination of cellular cAMP level in the perfused liver, 0.5 g of the organ was removed before, during, and after EtOH administration, homogenated (20% wt/vol) in assay buffer (Biotrak-Amersham), boiled for 5 min, and stored at -20°C until used. cAMP levels were also determined in aliquots of cell extract. Collagenase-dispersed hepatocytes were incubated under the experimental conditions described in Collagenase-dispersed cells. 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 Mg2+ content was measured by AAS. The cell pellets were resuspended in 200 µl of assay buffer (Biotrak, Amersham) and processed as reported above for the cell pellets.

Determination of ATP levels. For determination of ATP level in the perfused liver, 0.5 g of the organ was removed before, during, and after EtOH administration, homogenated (20% wt/vol) in 5% perchloric acid, and digested for 5-10 min in ice. The acid mixture was then neutralized by the addition of KHCO3, and the denaturated protein was sedimented in a refrigerated Beckman J-6B (1,500 g for 10 min). The supernatants were removed and stored at -20°C until used. Determination of ATP level was carried out by a luciferin-luciferase assay (detecting sensitivity in the pmol-nmol/ml range; Sigma) with a LUMAT Berthold LB 9501 luminometer or by HPLC with a C18 RP column (Millipore Waters) and 60 mM ammonium phosphate (pH 6.6 with ammonium hydroxyde) as the mobile phase (4). No significant changes in flow rate and Mg2+ baseline were observed as a consequence of tissue removal (Figs. 1 and 2). Cellular ATP level was also measured in isolated hepatocytes incubated as reported in Collagenase-dispersed cells. 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 and processed as reported for the tissue homogenate. Adenine nucleotides standards (1-20 nmol/ml) were injected for calibration.


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  		Fig. 1.   Extrusion of Mg2+ from rat livers perfused with ethanol (EtOH) for 35 min. Data points were determined every 30 s but are reported at 90-s intervals for simplicity. Data are means ± SE of 5 different preparations for each dose of EtOH tested. Data were first analyzed by 1-way ANOVA. Multiple means were then compared by Tukey's multiple comparison test performed with a q value established for significance at P < 0.05. * Significant difference from control. ** Significant difference from 0.01% EtOH. # Significant difference from all samples.


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  		Fig. 2.   Extrusion of Mg2+ from rat livers perfused with EtOH (A) or DMSO (B) for 8 min. Data points were determined every 30 s but are reported at 90-s intervals for simplicity. Data are means ± SE of 6 different experiments in A and 4 different experiments in B. Data were first analyzed by 1-way ANOVA. Multiple means were then compared by Tukey's multiple comparison test performed with a q value established for significance at P < 0.05. * Significant difference from control. ** Significant difference from 0.01% EtOH.

Protein determination, LDH measurements, and statistical analysis. Protein content was measured according to the procedure of Bradford (2), with BSA as a standard. LDH activity in the perfusate or the extracellular medium was measured with an enzymatic kit (Sigma) sensitive to detect changes in the microunit per milliliter range and is expressed as units per liter or as a percentage of the total amount of LDH released from digitonin-permeabilized hepatocytes. Cell viability was also assessed by trypan blue exclusion test.

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 significance of P < 0.05.

Chemicals. Collagenase (CLS2) was from Worthington. 125I Biotrak cAMP RIA was from Amersham. Luciferin-luciferase and LDH enzymatic kits and all the other chemicals and reagents were from Sigma.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Acute EtOH administration induces Mg2+ extrusion from liver cells in a dose-dependent manner. The acute administration of EtOH to perfused rat livers induced a release of cellular Mg2+ into the perfusate. As Fig. 1 shows, the infusion of 0.01, 0.1 or 1% EtOH to isolated rat livers induced a marked extrusion of Mg2+ from the organ. Irrespective of the dose of EtOH administered, the release of Mg2+ became evident within 2 min, reached the maximum within 8 min from the introduction of EtOH into the perfusion system, and slowly declined toward the basal level thereafter despite the persistence of EtOH in the perfusion medium. By comparison, no appreciable amount of Mg2+ was released from control livers into the perfusate over 45 min of perfusion (Figs. 1 and 2A). Mg2+ extrusion in the perfusate was already evident in livers perfused with 0.01% (1.6 mM) EtOH and markedly increased in livers perfused with 0.1 (16 mM) or 1% EtOH (160 mM; Fig. 1). The total amount of Mg2+ released in the perfusate, calculated as the area under the curve of efflux (see MATERIALS AND METHODS) accounts for 1.35, 1.64, and 3.20 µmol Mg2+ for livers treated with 0.01, 0.1, and 1% EtOH, respectively.

The maximal Mg2+ extrusion was achieved within 8 min from the addition of EtOH to the perfusate irrespective of the dose of EtOH infused and the persistence of alcohol in the perfusion system. Thus, in the results shown thereafter, EtOH was infused only for this period of time. As Fig. 2A shows, the amounts of Mg2+ extruded from rat livers perfused with 0.01, 0.1, or 1% EtOH for 8 min are almost superimposable to those reported in Fig. 1 (1.21, 1.70, and 3.84 µmol Mg2+ for the three doses of EtOH, respectively).

To exclude that the effect of EtOH was due to a modification of plasma membrane fluidity, livers were perfused with 1% (~128 mM) DMSO, an organic solvent that affects the integrity of biological membranes (38). Under these experimental conditions, no Mg2+ was released into the perfusate during an 8- (Fig. 2B) or 30-min (data not shown) perfusion. Furthermore, EtOH-induced Mg2+ extrusion was not accompanied by release of cellular K+ (data not shown) or LDH in the perfusate (Table 1). Because no release of LDH was observed after addition of 0.1 or 1% EtOH (Table 1), this parameter was not assessed for the lowest dose of EtOH used (0.01%). By contrast, when the livers were perfused with 10% EtOH, a massive release of cellular Mg2+ (~9 µmol Mg2+) into the perfusate was observed, which was accompanied by a net release of ~100 and ~200 U LDH/l within 3 and 8 min, respectively, from the addition of EtOH to the system. RIA analysis of cAMP level and HPLC determination of ATP content were performed on portions of the organs removed before EtOH introduction into the system, after 8 min of EtOH administration, and at a later time point (40 min) after EtOH withdrawal. Although cytosolic cAMP level did not significantly change during alcohol infusion (data not shown), a transient and significant decrease in ATP level (40%) at the peak of EtOH administration was observed (Table 1).

                              
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  		Table 1.   ATP and ADP content and LDH release in perfused livers treated in vitro with varying doses of EtOH

Mg2+ efflux occurs via a Na+/Mg2+ exchanger. A Na+/Mg2+ exchanger is considered to be the main Mg2+ extrusion mechanism in mammalian cell types (reviewed in Refs. 13, 45) including hepatocytes (16, 43). To determine whether the Mg2+ extrusion induced by EtOH occurs through the operation of this exchanger, rat livers were perfused with a Krebs-Henseleit medium in which Na+ was replaced by an equimolar concentration of choline chloride (43). As seen in Fig. 3A, the absence of Na+ in the perfusion medium drastically hampered the ability of EtOH to mobilize Mg2+ from liver cells. The operation of a Na+/Mg2+ exchanger is further supported by the results reported in Fig. 3, B and C, which show that the presence of 1 mM amiloride or 200 µM imipramine, respectively, in the perfusion medium reduced the amplitude of Mg2+ extrusion induced by 1% EtOH by ~70% (1.28 and 1.71 µmol are the total amounts of Mg2+ released during the 8-min perfusion with 1% EtOH in the presence of amiloride and imipramine, respectively, vs. 3.84 µmol of Mg2+ released in the absence of inhibitors). The administration of any of the two inhibitors in the absence of EtOH infusion did not induce any appreciable change in Mg2+ baseline in the perfusate compared with the control value (data not shown) nor affected the cellular ATP level (Table 1). Also, the coaddition of amiloride and imipramine to the perfusion medium failed to elicit additional inhibition of the EtOH-induced Mg2+ extrusion (data not shown). As Fig. 3, B and C, show, the withdrawal of amiloride or imipramine, respectively, restored, to a lesser extent, the EtOH-induced Mg2+ extrusion in the absence of a detectable release of LDH in the perfusate (Table 1). The reduced amount of Mg2+ extruded appears to correlate well with the reduction in cellular ATP content induced by EtOH in the presence of amiloride (Table 1).


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  		Fig. 3.   Extrusion of Mg2+ from rat liver perfused with EtOH in absence of extracellular Na+ (A) or in presence of extracellular Na+ and amiloride (B) or imipramine (C). Livers were perfused with EtOH dissolved in a medium in which extracellular Na+ was replaced with an equimolar concentration of choline chloride (A). Amiloride or imipramine was introduced in perfusion medium 3 min before EtOH administration and removed 5 min after EtOH withdrawal (B and C, respectively). Data points were determined every 30 s but are reported at 90-s intervals for simplicity. Data are means ± SE of 5 different experiments. Data were first analyzed by 1-way ANOVA. Multiple means were then compared by Tukey's multiple comparison test performed with a q value established for significance at P < 0.05. * Significant difference from control.

EtOH-induced Mg2+ extrusion correlates with a decrease in cellular ATP level and not with adenylyl cyclase activation. Previous reports from this (42, 44) and other groups (15, 16, 36) indicate that the Na+/Mg2+ exchanger can be activated by an increase in cytosolic cAMP, most likely through a phosphorylation process (15). Experimental evidence also suggests that chronic EtOH administration can increase cellular cAMP level by affecting the redistribution of heterotrimeric G proteins in the plasma membrane of several cell types including hepatocytes (21). To ascertain whether EtOH activates the Na+/Mg2+ exchanger via an increase in cytosolic cAMP, collagenase-dispersed hepatocytes resuspended in a Krebs-Henseleit medium devoid of Mg2+ (see MATERIALS AND METHODS) were used to measure intracellular levels of cAMP after the stimulation by varying doses of EtOH in a more accurate manner than in perfused organs. Isolated hepatocytes responded to stimulation with EtOH by extruding Mg2+ in a dose- and time-dependent fashion qualitatively similar to that observed in perfused livers, provided that Na+ was present in the extracellular medium (Fig. 4). In the absence of extracellular Na+, the amplitude of Mg2+ extrusion induced by 1% EtOH is markedly reduced (~60%) and almost superimposable to that observed in control cells (Fig. 4). Confirming the results obtained in perfused livers (Table 1), isolated hepatocytes treated in vitro with 0.1 or 1% EtOH did not release cytosolic LDH in the extracellular compartment over a 15-min incubation (Table 2), and the trypan blue exclusion test did not show evidence of significant changes in cell viability on addition of 0.01, 0.1, or 1% EtOH vs. control cells over a 15-min treatment (from 88 ± 2% at the start to 87 ± 1, 86 ± 2, and 87 ± 1% vs. 86 ± 2%, respectively). Similarly, Na+ removal did not elicit a detectable increase in LDH release (Table 2) or in the number of trypan blue-permeant cells (from 87 ± 2% at the start to 86 ± 1% after 15 min). Also, RIA determinations indicated that the cellular level of cAMP was not modified after the acute administration of 0.1 or 1% EtOH (Table 2). Because no significant release in cytosolic LDH or changes in cellular cAMP level were observed after the administration of the two highest doses of EtOH, these biochemical parameters were not assessed for the lowest dose of EtOH (0.01%). By contrast, the increasing concentrations of EtOH induced a progressive decrease in cellular ATP content (Table 2). This decrease was markedly attenuated in liver cells treated with 1% EtOH in the presence of amiloride (Table 2). Interestingly, under conditions in which the hepatocytes were stimulated by 1% EtOH in the absence of extracellular Na+, cellular ATP level was not significantly different from the control value (P > 0.05; Table 2).


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  		Fig. 4.   Extrusion of Mg2+ from collagenase-dispersed hepatocytes treated in vitro with varying concentrations of EtOH. Isolated hepatocytes (~100,000 cells/ml) were stimulated in vitro by addition of EtOH in presence or absence of extracellular Na+. pt, Protein. Mg2+ value at time = 0 was subtracted from all the following time points. EtOH was added to incubation mixture at time = 0 after withdrawal of the 1st sample. Data are means ± SE of 5 different preparations. Data were first analyzed by 1-way ANOVA. Multiple means were then compared by Tukey's multiple comparison test performed with a q value established for significance at P < 0.05. * Significant difference from control. # Significant difference from sample incubated in absence of extracellular Na+.

                              
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  		Table 2.   LDH release and cAMP and ATP content in collagenase-dispersed hepatocytes treated in vitro with varying doses of EtOH

Mg2+ extrusion is not accompanied by intracellular Mg2+ redistribution. To ascertain whether EtOH mobilizes Mg2+ from the cytosol or whether a redistribution of the cation occurs between the cytosol and intracellular organelles, digitonin (50 µg/ml) was added to hepatocyte suspensions after the maximal EtOH-induced Mg2+ extrusion was attained. The amount of residual cytosolic Mg2+ mobilized by the addition of digitonin was reduced in cells pretreated with 0.01, 0.1, and 1% EtOH (41.03, 41.10, and 36.73 nmol Mg2+/mg protein, respectively) compared with untreated cells (46.45 nmol Mg2+/mg protein; Table 3). The difference between these values and the control value accounts for the quantity of Mg2+ previously released from the cells (~3.7, ~3.8, and ~5.3 nmol Mg2+/mg protein for 0.01, 0.1, and 1% EtOH-treated cells, respectively, vs. ~2.3 nmol Mg2+/mg protein for control cells; Fig. 4, Table 2).

                              
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  		Table 3.   Net Mg2+ release in supernatant from collagenase-dispersed hepatocytes treated in vitro with varying doses of EtOH

Metabolic conversion of EtOH to acetaldehyde is involved in the extrusion of Mg2+. Liver cells metabolize EtOH to acetaldehyde via cytosolic alcohol dehydrogenase (EC 1.1.1.1) (29) and cytochrome P-4502E1 (EC 1.14.15.6), a specific isoform of the cytochrome P-450 superfamily located within the endoplasmic reticulum (29). To a lesser extent, EtOH is also oxidized by the peroxisomal catalase (EC 1.11.1.6) (30). Acetaldehyde is then converted to acetic acid via cytosolic and mitochondrial aldehyde dehydrogenase (EC 1.2.1.3) (29) and also via cytochrome P-4502E1 activity (26), although the presence of an alcohol dehydrogenase isoform within the nucleus (28) may suggest additional sites of metabolic modifications.

To ascertain whether EtOH affects Mg2+ homeostasis per se or via its metabolic conversion to acetaldehyde and acetic acid, livers were perfused with 1% EtOH in the presence of 4-MP, a specific inhibitor of alcohol dehydrogenase (48) and cytochrome P-4502E1 (35), or in the presence of CyN, a selective inhibitor of aldehyde dehydrogenase (32). These agents directly inhibit their target enzymes and do not affect EtOH accumulation within the cells (32, 35, 48). The results, reported in Fig. 5, indicate that the presence of 50 µM 4-MP in the perfusion medium (Fig. 5A) was sufficient to completely prevent the Mg2+ extrusion induced by 0.01 or 0.1% EtOH (Fig. 5A) and to markedly decrease the extrusion induced by 1% EtOH (Fig. 5B). When the dose of 4-MP was increased to 200 µM, a slightly larger inhibition of the Mg2+ extrusion induced by 1% EtOH was observed (Fig. 5B). The total amount of Mg2+ released from livers perfused with 1% EtOH and pretreated with 50 or 200 µM 4-MP accounted for ~0.73 and ~0.38 µmol, respectively, vs. ~3.8 µmol in livers perfused with 1% EtOH in the absence of the inhibitor (Fig. 2A). The cellular ATP level of isolated hepatocytes pretreated with 50 or 200 µM 4-MP and stimulated by 0.1 or 1% EtOH were not significantly different from the control values (data not shown). By contrast, pretreatment with 500 µM (data not shown) or 1 mM CyN (Fig. 6) did not affect the Mg2+ mobilization induced by 0.01, 0.1, or 1% EtOH (Fig. 6, A and B).


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  		Fig. 5.   Extrusion of Mg2+ from rat livers perfused with varying doses of EtOH in presence of different doses of 4-methylpyrazole (4-MP). Inhibitor 4-MP was introduced in perfusion medium at time = 0 and maintained throughout experimental procedure. Livers were perfused with 0.01 or 0.1% EtOH in presence of 50 µM 4-MP (A) or with 1% EtOH in presence of 50 or 200 µM 4-MP (B). Data points were determined every 30 s but are reported at 90-s intervals for simplicity. Data are means ± SE of 4 different preparations for each experimental condition. Data were first analyzed by 1-way ANOVA. Multiple means were then compared by Tukey's multiple comparison test performed with a q value established for significance at P < 0.05. * Significant difference from control.


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  		Fig. 6.   Extrusion of Mg2+ from rat livers perfused with varying doses of EtOH in presence of cyanamide. Inhibitor cyanamide was introduced into perfusion medium at time = 0 and maintained throughout experimental procedure. Livers were perfused with 0.01 or 0.1% EtOH (A) or 1% EtOH (B). Data points were determined every 30 s but are reported at 90-s intervals for simplicity. Data are means ± SE of 4 different preparations for each experimental condition. Data were first analyzed by 1-way ANOVA. Multiple means were then compared by Tukey's multiple comparison test performed with a q value established for significance at P < 0.05.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Chronic EtOH consumption induces severe alteration in structure, function, and ion distribution within the cell (10,29). Modifications in Ca2+, Na+, or K+ homeostasis have been attributed to a direct effect of EtOH on Ca2+ regulatory mechanisms (20, 41) and Na+-K+-ATPase (47), respectively. A marked and persistent decrease in plasma and cellular Mg2+ content in chronic alcoholics has also been observed (8). Although the implications of Mg2+ depletion for cell functioning have not been completely elucidated, the administration of Mg2+ to humans (29) or animals (52) appears to prevent, attenuate, and eventually revert several functions in liver (29), cardiac (52), and smooth muscle cells (1) compromised by chronic EtOH consumption. The observation that the plasma Mg2+ level increases after acute EtOH administration (39), whereas both plasma and cellular Mg2+ content decrease in humans (8) and animals (19) after chronic EtOH consumption, suggests that the mechanisms that control cellular Mg2+ homeostasis and/or transport across cell plasma membrane are directly or indirectly modified by EtOH administration.

Involvement of the putative Na+/Mg2+ exchanger in the EtOH-induced Mg2+ extrusion from liver cells. The effect of acute administration of EtOH on cellular Mg2+ homeostasis was investigated in perfused rat livers and in suspensions of collagenase-dispersed hepatocytes. In both experimental models, the addition of varying doses of EtOH results in the net extrusion of 15-20% of total Mg2+ content per gram of liver tissue in the extracellular compartment in a dose- and time-dependent fashion. The absence of K+ and LDH release in the perfusate or the extracellular compartment, together with the inability of 1% DMSO to reproduce the effect of EtOH, suggests that the release of Mg2+ induced by EtOH does not result from a nonselective increase in membrane permeability but rather occurs through the operation of a specific transport process. In fact, all the concentrations of EtOH tested (including 10% EtOH; data not shown) induce Mg2+ extrusion only in the presence of a physiological concentration of Na+ in the extracellular medium, the process being inhibited under conditions in which extracellular Na+ is replaced with an equimolar concentration of choline chloride or in which amiloride or imipramine is present in the perfusion medium. Although nonspecific, amiloride and imipramine are the only known inhibitors of the putative Na+/Mg2+ exchanger (6, 14). When added to the perfusion system before EtOH administration, both agents inhibit ~70% of the Mg2+ extrusion elicited by EtOH. Because they inhibit, to a similar extent, the beta -adrenergic-induced cAMP-mediated Mg2+ extrusion from cardiac (50) or liver (16) cells, it can be excluded that the residual Mg2+ extrusion observed under our experimental conditions depends on a direct effect of EtOH on plasma membrane fluidity. The possibility that amiloride may block Mg2+ extrusion by inhibiting protein synthesis (27), although likely, is not fully consistent with the observation that 1) the inhibitory effect of amiloride is quantitatively and qualitatively similar to that exerted by imipramine, an agent that to our knowledge does not affect protein synthesis; 2) it disappears, as in imipramine-treated livers, within 1 min from the withdrawal of the agent from the perfusion medium, a lag time that is consistent with a direct effect on the transport mechanism at the plasma membrane level rather than with the removal of protein synthesis inhibition; and 3) the addition of the protein synthesis inhibitor cycloheximide (10 µM or higher dose) to the perfusion medium is ineffective at preventing the EtOH-induced Mg2+ extrusion (data not shown).

It has to be noticed that control cells incubated both in the absence or in the presence of external Na+ release 1-2 nmol Mg2+/mg protein over time (Fig. 4). Because cell viability does not change during the incubation period, this Mg2+ extrusion likely occurs via the basal operation of the not-better-characterized Na+-independent transport mechanism present in the plasma membrane of several mammalian cell types including hepatocytes (13, 45). This background extrusion of Mg2+ is not enhanced in hepatocytes treated with EtOH in the absence of external Na+ (Fig. 4). Taken together, these observations strongly support the hypothesis that, after EtOH administration, liver cells extrude Mg2+ from an intracellular pool, most likely the cytosol, via a transport mechanism that specifically requires extracellular Na+ and can be tentatively identified with the Na+/Mg2+ exchanger reported to operate in the plasma membrane of mammalian cells (13, 45). Interestingly, hepatocytes incubated in the absence of extracellular Na+ appear to release less cytosolic Mg2+ after the administration of digitonin (Table 3). Because the hepatocytes have not mobilized more Mg2+ at previous time points or during the transfer to the Na+-free medium, the possibility is there that the inability to extrude Mg2+ across the plasma membrane may result in a redistribution of the cation among intracellular organelles.

Role of cytosolic ATP on Mg2+ extrusion. The absence of detectable changes in cellular cAMP level excludes a role of the second messenger in the activation of the Na+/Mg2+ exchanger, at variance to what has been proposed to occur after beta -adrenergic stimulation (5, 15, 36, 42-44). Thus it is possible that the exchanger is directly activated by an increase in cytosolic free Mg2+ concentration ([Mg2+]). Such an increase could be consequent to a redistribution of Mg2+ between the cytosol and intracellular organelles and/or to a significant decrease in cytosolic buffering capacity. The decrease in cellular ATP level and the observation that digitonin mobilizes a reduced amount of Mg2+ from EtOH-treated hepatocytes compared with control cells would indicate that a decreased buffering capacity and not cellular Mg2+ redistribution is responsible for the increase in cytosolic free [Mg2+] and, consequently, Mg2+ extrusion.

The decrease in cellular ATP content measured in livers perfused with 1% EtOH (40%; Table 1) and in hepatocyte suspensions (30, 40, and 50% decreases for doses of 0.01, 0.1, and 1% EtOH, respectively, vs. a 10-15% decrease in control cells; Table 2) is comparable to that measured by other authors in perfused livers (34) and in isolated hepatocytes (11) treated in vitro with varying concentrations of EtOH. Figure 7 depicts the possible mechanism by which EtOH administration would result in ATP depletion and Mg2+ extrusion. The intracellular conversion of EtOH to acetaldehyde via alcohol dehydrogenase and cytochrome P-4502E1 results in an inversion of the redox state of the NAD+-NADH couple (5, 34). The increase in NADH content would favor the formation of glyceraldehyde 3-phosphate (GAP) (29, 34), which, by acting as a trap for Pi, would decrease the cellular Pi content and remove the inhibitory effect of this moiety on AMP deaminase (34). Consequently, ATP is degraded to ADP, AMP, and finally uric acid and allantoin (34). It is noteworthy that the time course of Mg2+ extrusion into the perfusate (~8 min; Figs. 1 and 2) parallels those of the decrease in cellular Pi content and the increase in GAP concentration measured by Masson et al. (34). The return of Mg2+ extrusion to the basal level, therefore, would correspond to the restoration of a normal cellular Pi concentration and ATP level (Table 1) through the activation of the penthose cycle (34). The decreased buffering capacity of ATP for Mg2+ would result in an increase in cytosolic free [Mg2+] that could directly activate the Na+/Mg2+ exchanger, thereby resulting in Mg2+ extrusion. Unfortunately, an accurate measurement of the increase in cytosolic free [Mg2+] by Mag-Fura 2 is hampered by the concomitant changes in pyridine nucleotide fluorescence induced by EtOH administration. Thus we can only estimate the increase in cytosolic free Mg2+ based on the decrease in ATP content reported in Tables 1 and 2. Assuming a concentration of 2.5-3 mM for the Mg-ATP complex in the cytosol of liver cells (3) and not considering Mg2+ binding to other cytosolic proteins or metabolites, the 50% decrease in ATP level observed in cells treated with 1% EtOH would result in an increase in cytosolic free [Mg2+] of ~1.2 mM. Because ADP and AMP can partially complex Mg2+, although with a lower affinity [the dissociation constant for the Mg-ADP complex is ~3 times lower than that for MgATP (25)], the net increase in cytosolic free [Mg2+] can be estimated to be ~800 µM. Similarly, net increases in free [Mg2+] of ~650 and ~500 µM can be calculated to occur, although more gradually (Table 2), in hepatocytes treated in vitro with 0.1 and 0.01% EtOH, respectively. A further support to the hypothesis that Mg2+ is mobilized from a cytosolic Mg-ATP complex as a consequence of the decrease in cytosolic buffering capacity is provided by the comparison of the decrease in cytosolic ATP level with the amount of Mg2+ extruded across the plasma membrane of hepatocytes treated with 1% EtOH (net change ~6-7 nmol/mg protein for both parameters at time = 15 min; Table 2, Fig. 4).


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  		Fig. 7.   Schematic depiction of cellular modifications occurring after EtOH administration in liver cells (see text for details). CytP450IIE1, cytochrome P-4502E1; E.R., endoplasmic reticulum; MITO, mitochondria.

Presently, we do not have an explanation why, in the absence of extracellular Na+, cellular ATP level decreased only by 10% over 15 min (i.e., a decrease comparable to that observed in control cells; Table 2) despite the presence of 1% EtOH in the incubation system. A tentative explanation might be that, in the absence of extracellular Na+, Na+-K+-ATPase, which accounts for ~38% of the energy consumption in liver cells (22), does not operate at a very high rate, and no major hydrolysis of cellular ATP occurs. Consequently, only a minimal amount of hydrolyzed Pi will be routed toward GAP formation (34) after EtOH administration, and the buffering capacity of cytosolic ATP for Mg2+ will remain largely unaffected. This interpretation may also explain why amiloride, which per se does not modify the cellular level of ATP, partially prevents the decrease in ATP content induced by 1% EtOH (Table 2). Most likely, the effect is attained by limiting the amount of Na+ that enters the cell, further supporting the hypothesis that at least part of the inhibitory effect of the drug on Mg2+ efflux is exerted at the level of the Na+/Mg2+ exchanger.

Role of EtOH metabolism. The extrusion of Mg2+ strictly depends on EtOH metabolism within the cell. The different effectiveness of 4-MP and CyN at preventing the EtOH-induced Mg2+ efflux from perfused livers (Figs. 5 and 6) suggests that Mg2+ extrusion from liver cells is consequent to the conversion of EtOH to acetaldehyde, a process that would lead to an increased formation of GAP discussed previously (Fig. 7) and not to the oxidation of acetaldehyde to acetic acid. Because cytosolic alcohol dehydrogenase should already be saturated at the highest doses of EtOH used in some of experimental protocols (37), the production of acetaldehyde after the administration of 1% EtOH should be mostly attributed to the operation of cytochrome P-4502E1 in the endoplasmic reticulum and to other metabolic enzymes located elsewhere within the cell (28, 30). Consistent with the mechanism illustrated in Fig. 7, by blocking the conversion of EtOH to acetaldehyde, 4-MP would prevent the inversion of the redox state of pyridine nucleotides, the increase in glycerol 3-phosphate, and the subsequent decrease in cytosolic ATP.

The formation of acetaldehyde-protein adducts in the cytosol of liver cells and their subsequent migration to the plasma membrane has been observed (31). If acetaldehyde has a role in modulating the activity of the Na+/Mg2+ exchanger, the extrusion of Mg2+ from EtOH-treated livers should be potentiated when CyN is present to inhibit the metabolic conversion of (acet)aldehyde to acetic acid. Because the potentiation is not observed (Fig. 6), it can be concluded that either the Na+/Mg2+ exchanger is already fully active after the initial conversion of EtOH to acetaldehyde, so that the excess of acetaldehyde produced cannot further activate the transporter, or acetaldehyde is not involved in Mg2+ extrusion. The perfusion of isolated livers with 30-50 µM acetaldehyde (concentrations measured within liver cells treated with EtOH) failed to induce a detectable mobilization of Mg2+ from the organs (data not shown). This result and the absence of potentiation are thus consistent with the hypothesis that the increase in cytosolic free [Mg2+] subsequent to the decrease in ATP content induced by EtOH administration is sufficient to determine Mg2+ mobilization, provided that Na+ is present in the extracellular compartment to favor the extrusion.

    ACKNOWLEDGEMENTS

The constructive comments and criticisms of Dr. A. Scarpa and Dr. J. Hoek during the preparation of the manuscript are gratefully acknowledged.

    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grant HL-18708.

Address for reprint requests: A. Romani, Dept. of Physiology and Biophysics, Case Western Reserve Univ., 10900 Euclid Ave., Cleveland, OH 44106-4970.

Received 6 October 1997; accepted in final form 1 July 1998.

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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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