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
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ABSTRACT |
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
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INTRODUCTION |
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.
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MATERIALS AND METHODS |
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.
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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 |
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).
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.
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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
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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
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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 |
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
-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
-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.
 |
REFERENCES |
1.
Altura, B. M.,
A. Gebrewold,
B. T. Altura,
and
R. K. Gupta.
Role of brain [Mg2+]i in alcohol-induced hemorrhagic stroke in a rat model: a 31P-NMR in vivo study.
Alcohol
12:
131-136,
1995[Medline].
2.
Bradford, M. M.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem.
72:
248-254,
1976[Medline].
3.
Corkey, B. E.,
J. Duszynski,
T. L. Rich,
B. Matschinsky,
and
J. R. Williamson.
Regulation of free and bound magnesium in rat hepatocytes and isolated mitochondria.
J. Biol. Chem.
261:
2567-2574,
1986[Abstract/Free Full Text].
4.
De Young, M. B.,
G. Dubyak,
and
A. Scarpa.
Measurement of phosphometabolites in heart and muscle by high-perfomance liquid chromatography.
In: The Heart and Cardiovascular System: Scientific Foundations (2nd ed.), edited by H. A. Fozzard,
E. Haber,
R. B. Jennings,
A. M. Katz,
and H. E. Morgan. New York: Raven, 1990, vol. I, chapt. 24, p. 543-549.
5.
Domschke, S.,
W. Domschke,
and
C. S. Lieber.
Hepatic redox state: attenuation of the acute effects of ethanol induced by chronic ethanol consumption.
Life Sci.
15:
1327-1334,
1974[Medline].
6.
Feray, J. C.,
and
R. Garay.
Demonstration of a Na+:Mg2+ exchange in human red cells by its sensitivity to tricyclic antidepressant drugs.
Naunyn Schmiedebergs Arch. Pharmacol.
338:
332-337,
1988[Medline].
7.
Flatman, P. W.,
and
L. M. Smith.
Sodium-dependent magnesium uptake by ferret red cells.
J. Physiol. (Lond.)
443:
217-230,
1991[Abstract].
8.
Flink, E. B.
Magnesium deficiency in alcoholism.
Alcohol. Clin. Exp. Res.
10:
590-594,
1986[Medline].
9.
Galambos, J. T.
Natural history of alcoholic hepatitis. 3. Histological changes.
Gastroenterology
63:
1026-1035,
1972[Medline].
10.
Gandhi, C. R.,
and
C. H. Ross.
Influence of ethanol on calcium, inositol phospholipids and intracellular signalling mechanisms.
Experientia
45:
407-413,
1988.
11.
Gasbarrini, A.,
A. M. Borle,
P. Caraceni,
A. Colantoni,
H. Farghali,
F. Trevisani,
M. Bernardi,
and
D. H. van Thiel.
Effect of ethanol on adenosine trisphosphate, cytosolic free calcium, and cell injury in rat hepatocytes: time course and effect of nutritional status.
Dig. Dis. Sci.
41:
2204-2212,
1996[Medline].
12.
Gonzales-Calcin, J. L.,
J. B. Saunders,
and
R. Williams.
Effects of ethanol and acetaldehyde on hepatic plasma membrane ATPases.
Biochem. Pharmacol.
32:
1723-1728,
1983[Medline].
13.
Gunther, T.
Mechanisms and regulation of Mg2+ efflux and Mg2+ influx.
Miner. Electrolyte Metab.
19:
259-265,
1993[Medline].
14.
Gunther, T.,
and
J. Vormann.
Mg2+ efflux is accomplished by an amiloride sensitive Na+/Mg2+ antiport.
Biochem. Biophys. Res. Commun.
130:
540-545,
1985[Medline].
15.
Gunther, T.,
and
J. Vormann.
Activation of Na+/Mg2+ antiporter in thymocytes by cAMP.
FEBS Lett.
297:
132-134,
1992[Medline].
16.
Gunther, T.,
and
J. Vormann.
Na+-dependent Mg2+ efflux from isolated perfused rat livers.
Magnes. Bull.
14:
126-129,
1992.
17.
Gunzel, D.,
and
N. R. Schlue.
Sodium-magnesium antiport in Retzius neurones of the leech Hirudo medicinalis.
J. Physiol. (Lond.)
491:
595-608,
1996[Abstract].
18.
Harper, C. G.,
and
J. J. Krill.
Neuropathology of alcoholism.
Alcohol Alcohol.
25:
207-213,
1990[Medline].
19.
Hemmingsen, R.,
and
P. Kramp.
Effect of acute ethanol intoxication, chronic ethanol intoxication, and ethanol withdrawal on magnesium and calcium metabolism in the rat.
Psychopharmacology (Berl.)
67:
255-259,
1980[Medline].
20.
Hoek, J. B.,
A. P. Thomas,
T. A. Rooney,
K. Higashi,
and
E. Rubin.
Ethanol and signal transduction in the liver.
FASEB J.
6:
2386-2396,
1992[Abstract/Free Full Text].
21.
Iles, K. E.,
and
L. E. Nagy.
Chronic ethanol feeding increases the quantity of G
s-protein in rat liver plasma membrane.
Hepatology
21:
1154-1160,
1995[Medline].
22.
Ismail-Beigi, F.,
and
I. S. Edelman.
The mechanism of the calorigenic action of thyroid hormone.
J. Gen. Physiol.
57:
710-722,
1971[Abstract/Free Full Text].
23.
Jakob, A.,
J. Becker,
G. Schottli,
and
G. Fritzsch.
1-Adrenergic stimulation causes Mg2+ release from perfused rat livers.
FEBS Lett.
246:
127-130,
1989[Medline].
24.
Kayne, L. H.,
and
D. B. N. Lee.
Intestinal magnesium absorption.
Miner. Electrolyte Metab.
19:
210-217,
1993[Medline].
25.
Koss, K. L.,
and
R. D. Grubbs.
Elevated extracellular Mg2+ increases Mg2+ buffering through a Ca-dependent mechanism in cardiomyocytes.
Am. J. Physiol.
267 (Cell Physiol. 36):
C633-C641,
1994[Abstract/Free Full Text].
26.
Kunitoh, S.,
S. Imaoka,
T. Hiroi,
Y. Yabusaki,
T. Monna,
and
Y. Funae.
Acetaldehyde as well as ethanol is metabolized by human CYP2E1.
J. Pharmacol. Exp. Ther.
280:
527-532,
1997[Abstract/Free Full Text].
27.
Leffert, H. L.,
K. S. Koch,
M. Fehlmann,
W. Heiser,
P. J. Lad,
and
H. Skelly.
Amiloride blocks cell-free protein synthesis at levels attained inside cultured rat hepatocytes.
Biochem. Biophys. Res. Commun.
108:
738-745,
1982[Medline].
28.
Leffert, H. L.,
K. S. Koch,
P. Shapiro,
H. Kelly,
J. Hubert,
C. Monken,
P. J. Lad,
J. Sala-Trepat,
and
F. V. Chisari.
Primary cultures, monoclonal antibodies and nucleic acid probes as tools for studies of hepatic structure and function.
In: Cirrhosis of the Liver: Methods and Field of Research, edited by N. Tygstrup,
and F. Orlandi. Amsterdam: Elsevier Science, 1987, p. 121-140.
29.
Lieber, C. S.
Alcohol, liver, and nutrition.
J. Am. Coll. Nutr.
10:
602-632,
1991[Abstract].
30.
Lieber, C. S.
Ethanol metabolism, cirrhosis and alcoholism.
Clin. Chim. Acta
257:
59-84,
1997[Medline].
31.
Lin, R. C.,
R. A. Sidner,
M. J. Fillenwarth,
and
L. Lumeng.
Localization of protein-acetaldehyde adducts on cell surface of hepatocytes by flow cytometry.
Alcohol. Clin. Exp. Res.
16:
1125-1129,
1992[Medline].
32.
Loomis, C. W.,
and
J. F. Brien.
Specificity of hepatic aldehyde dehydrogenase inhibition by calcium carbimide (calcium cyanamide) in the rat.
Can. J. Physiol. Pharmacol.
61:
431-435,
1983[Medline].
33.
Ludi, H.,
and
H. J. Schatzmann.
Some properties of a system for sodium-dependent outward movement of magnesium from metabolizing human red blood cells.
J. Physiol. (Lond.)
390:
367-382,
1987[Abstract].
34.
Masson, S.,
F. Desmoulin,
M. Sciaky,
and
P. J. Cozzone.
Catabolism of adenine nucleotides and its relation with intracellular phosphorylated metabolite concentration during ethanol oxidation in perfused rat liver.
Biochemistry
32:
1025-1031,
1993[Medline].
35.
Matsumoto, H., K. Matsubayashi, and Y. Fukui. Evidence that
cytochrome P-4502E1 contributes to ethanol elimination at low doses:
effects of diallyl sulfide and 4-methyl pyrazole on ethanol elimination
in the perfused rat liver. Alcohol. Clin. Exp.
Res. 20, Suppl.:
12A-16A, 1996.
36.
Matsuura, T.,
Y. Kanayama,
T. Inoue,
T. Takeda,
and
I. Morishima.
cAMP-induced changes in intracellular free Mg2+ levels in human erythrocytes.
Biochim. Biophys. Acta
1220:
31-36,
1993[Medline].
37.
Moreno, A.,
and
X. Pares.
Purification and characterization of a new alcohol dehydrogenase from human stomach.
J. Biol. Chem.
266:
1128-1133,
1991[Abstract/Free Full Text].
38.
Mrak, R. E.
Opposite effects of dimethyl sulfoxide and ethanol on synaptic membrane fluidity.
Alcohol
9:
513-517,
1992[Medline].
39.
Peng, T.,
and
H. J. Gitelman.
Ethanol-induced hypocalcemia, hypermagnesemia and inhibition of the serum calcium-raising effect of parathyroid hormone in rats.
Endocrinology
94:
608-611,
1974[Medline].
40.
Quamme, G. A.
Magnesium homeostasis and renal magnesium handling.
Miner. Electrolyte Metab.
19:
218-225,
1993[Medline].
41.
Roche, E.,
and
M. Prentki.
Calcium regulation of immediate-early response genes.
Cell Calcium
16:
331-338,
1994[Medline].
42.
Romani, A.,
E. Dowell,
and
A. Scarpa.
Cyclic AMP induced Mg2+ release from rat liver hepatocytes, permeabilized hepatocytes and isolated mitochondria.
J. Biol. Chem.
266:
24376-24384,
1991[Abstract/Free Full Text].
43.
Romani, A.,
C. Marfella,
and
A. Scarpa.
Hormonal stimulation of Mg2+ uptake in hepatocytes: regulation by plasma membrane and intracellular organelles.
J. Biol. Chem.
268:
15489-15495,
1993[Abstract/Free Full Text].
44.
Romani, A.,
and
A. Scarpa.
Hormonal control of magnesium transport in the heart.
Nature
346:
841-844,
1990[Medline].
45.
Romani, A.,
and
A. Scarpa.
Regulation of cell magnesium.
Arch. Biochem. Biophys.
298:
1-12,
1992[Medline].
46.
Seglen, P. O.
Preparation of isolated rat liver cells.
Methods Cell Biol.
13:
29-83,
1976[Medline].
47.
Swann, A. C.
Ethanol and (Na+,K+)-ATPase: alteration of Na+-K+ selectivity.
Alcohol. Clin. Exp. Res.
14:
922-927,
1990[Medline].
48.
Theorell, H.,
B. Chance,
T. Yonetani,
and
N. Oshino.
The combustion of alcohol and its inhibition by 4-methylpyrazole in perfused rat livers.
Arch. Biochem. Biophys.
15:
434-444,
1972.
49.
Urbano-Marques, A.,
R. Estruch,
F. Navarro-Lopez,
J. M. Grau,
L. Mont,
and
E. Rubin.
The effects of alcoholism on skeletal and cardiac muscle.
N. Engl. J. Med.
320:
409-415,
1989[Abstract].
50.
Vormann, J.,
and
T. Gunther.
Amiloride-sensitive net Mg2+ efflux from isolated perfused rat hearts.
Magnesium
6:
220-224,
1987[Medline].
51.
Wolf, F.,
A. Di Francesco,
V. Covacci,
and
A. Cittadini.
cAMP activates magnesium efflux via the Na/Mg antiporter in ascites cells.
Biochem. Biophys. Res. Commun.
202:
1209-1214,
1994[Medline].
52.
Zou, L. Y.,
F. Wu,
B. T. Altura,
R. L. Barbour,
and
B. M. Altura.
Beneficial effects of high magnesium on alcohol-induced cardiac failure.
Magnesium Trace Elem.
10:
409-419,
1991-1992[Medline].
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