Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4970
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ABSTRACT |
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Ethanol
(EtOH) administration to rats for 4 wk markedly decreased
Mg2+ content in several tissues, including liver. Total
cellular Mg2+ accounted for 26.8 ± 2.4 vs. 36.0 ± 1.4 nmol Mg2+/mg protein in hepatocytes from EtOH-fed
and control rats, respectively, and paralleled a 13% decrease in
cellular ATP content. Stimulation of 1- or
-adrenergic receptor or acute EtOH administration did not elicit an
extrusion of Mg2+ from liver cells of EtOH-fed rats while
releasing 5% of total tissue Mg2+ content from hepatocytes
of control rats. Despite the 25% decrease in Mg2+ content,
hepatocytes from EtOH-fed rats did not accumulate Mg2+
following stimulation of protein kinase C signaling pathway, whereas
control hepatocytes accumulated ~2 nmol
Mg2+ · mg
protein
1 · 4 min
1.
Together, these data indicate that Mg2+ homeostasis and
transport are markedly impaired in liver cells after prolonged exposure
to alcohol. The inability of liver cells, and possibly other tissues,
to accumulate Mg2+ can help explain the reduction in tissue
Mg2+ content following chronic alcohol consumption.
hepatocytes; protein kinase C; adrenergic signaling; plasma membranes
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INTRODUCTION |
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MAGNESIUM IS THE
SECOND MOST abundant cation in mammalian cells after potassium
(17, 18, 44, 45). Within the cell, Mg2+ is mainly localized inside mitochondria, endoplasmic
reticulum, nucleus, and cytosol (8, 17, 45), in which it
regulates numerous biological functions (reviewed in Ref.
44). The cellular abundance of Mg2+ and its
physiological relevance for cell cycle, biochemical reactions, or
enzyme and channel activity contrasts with the limited knowledge about
the mechanism(s) regulating Mg2+ cellular homeostasis and
transport. In recent years, several laboratories, including ours, have
provided compelling evidence for the occurrence of large and fast
fluxes of Mg2+ in and out of mammalian cells (see Ref.
45 for review). Although no mammalian Mg2+
transporter has been cloned to date, experimental and pharmacological evidence supports the operation of a Na+-dependent
Mg2+ extrusion pathway, tentatively identifiable as a
Na+/Mg2+ exchanger, in the majority of
mammalian cells investigated (13, 18, 20). The operation
of a Na+-independent Mg2+ extrusion pathway,
which can use extracellular Ca2+, Mn2+, or
anions to transport Mg2+, has also been reported (18,
19). These pathways can be activated via stimulation of
1- and
-adrenergic receptors in a variety of tissues,
including heart (42, 49) and liver (10, 11, 43). We have recently observed that the stimulation of glucagon or
-adrenoceptor in liver cells selectively activates the
Na+-dependent Mg2+ extrusion pathway
(10), whereas the administration of catecholamine activates both Na+-dependent and
Na+-independent Mg2+ extrusion pathways
(10). Current opinion is that the increase in cellular
cAMP resulting from the activation of glucagon or
-adrenergic
receptor or from the use of cell-permeant cAMP analogs phosphorylates
the putative Na+/Mg2+ exchanger and determines
an extrusion of Mg2+ from the cell (20, 32,
37). In contrast, hormones or agents that decrease cellular cAMP
level (39, 41, 42) or activate protein kinase C signaling
(7, 38) prevent Mg2+ extrusion or induce an
accumulation of Mg2+ within the cell. The nature of the
Mg2+ entry pathway still remains undefined, because
experimental data would support either the operation of a
Mg2+ channel in cardiac myocytes (35) and
kidney cells (34) or the reverse operation of the
Na+/Mg2+ exchanger located in the basolateral
portion of hepatocyte cell membrane (4). As for the
physiological significance, changes in Mg2+ content appear
to play a significant role in regulating specific mitochondrial
dehydrogenases (33) and various cellular functions, including glycolysis (16).
Hypomagnesemia and a reduced Mg2+ content within several tissues, including heart and liver, have been observed following acute and chronic alcohol consumption in animal models and in chronic alcoholic patients (1, 14, 28, 52). Experimental evidence suggests that the decline in Mg2+ content is deleterious for the tissue in which it occurs (1, 28). Cardiac chrono- and inotropism (26), smooth muscle cell contractility (28), collagen deposition (36), and cell cycle (30) are all examples of functions that are affected to a varying extent by the decrease in cellular Mg2+ content. On the other hand, Mg2+ supplementation under these conditions ameliorates several metabolic functions compromised by ethanol (EtOH) administration (1, 52). Yet the mechanism(s) responsible for Mg2+ loss from the tissues, the relevance that a reduced Mg2+ content may have to the onset of alcohol-related pathologies such as cirrhosis and hepatitis, and the modality by which Mg2+ reintroduction exerts its beneficial effects are not fully investigated. Recently, we have reported that the acute infusion of EtOH in perfused liver results in a marked, dose-dependent extrusion of Mg2+ from the organ into the perfusate via a Na+-dependent mechanism (50). Mg2+ extrusion is associated with the transient reduction in cellular ATP content that occurs during the conversion of EtOH to acetaldehyde, because both processes can be largely prevented by pretreatment with the alcohol dehydrogenase inhibitor 4-methylpyrazole (50). On the basis of these results, we proposed that by impairing ATP homeostasis and enhancing its degradation (31) EtOH administration reduces the cellular buffering capacity for Mg2+, which results in an extrusion of Mg2+ from the cell (50).
In the present study, the changes in cellular Mg2+ homeostasis and transport that occur in liver cells of rats exposed to EtOH in the diet for 4 wk were investigated as a first step in determining the short- and long-term implications that changes in cellular Mg2+ content may have for the proper functioning of specific processes within the cell (e.g., glycolysis) or cellular organelles (e.g., mitochondrial dehydrogenases) and the onset of EtOH-related liver pathologies. To this purpose, perfused organs, isolated cells, and purified plasma membrane vesicles were used as experimental models to determine to what extent chronic EtOH administration affects Mg2+ homeostasis and transport in liver cells. The obtained results indicate that prolonged EtOH administration decreases hepatic Mg2+ content by 1) affecting cytosolic ATP content and 2) impairing the ability of liver cells to accumulate Mg2+, thus preventing the hepatocyte from effectively restoring cellular Mg2+ homeostasis. In addition, the decrease in cellular Mg2+ content prevents the liver cells from extruding a significant amount of Mg2+ following adrenergic stimulation, de facto hampering the physiological significance of Mg2+ extrusion locally and systemically (25).
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MATERIALS AND METHODS |
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Materials. Collagenase (CLS2) was from Worthington. 125I-labeled Biotrak cAMP-RIA was from Amersham. Luciferin-luciferase and lactate dehydrogenase (LDH) enzymatic kits and all other chemicals and reagents were from Sigma. Lieber-De Carli diet for EtOH-treated and control animals was from Bio-Serve (Frenchtown, NJ).
Liquid diet. Male Sprague-Dawley rats (180-200 g body wt) were randomly divided into control and EtOH-treated groups and housed individually in metabolic cages. EtOH-treated rats were maintained for 4 wk with Lieber-De Carli diet containing 67 ml EtOH/l of liquid diet (6% EtOH, final concentration). Control rats received a daily amount of an isocaloric control diet comparable to that consumed by EtOH-treated rats (pair-fed protocol). Weight gain was recorded weekly for all experimental groups.
Determination of tissue cation content. Rats were killed by cranial dislocation. Aliquots from brain, skeletal muscles, heart, diaphragm, and splanchnic organs were collected, rinsed in 250 mM sucrose, blotted on absorbing paper, weighed, minced, and homogenized in 10% HNO3. After overnight extraction, the acid mixture was centrifuged at 2,000 g for 5 min to sediment denatured protein. The Na+, K+, Ca2+, and Mg2+ contents of the acid extracts were measured by atomic absorbance spectrophotometry (AAS) in a Perkin-Elmer 3100, calibrated with proper standards.
Perfused livers.
Fed male Sprague-Dawley rats (250-350 g body wt) were anesthetized
by intraperitoneal injection of sodium pentobarbital (50 mg/ml). The
abdomen was opened, and the liver was perfused via the portal vein with
a medium containing (in mM) 120 NaCl, 3 KCl, 1 CaCl2, 1.2 KH2PO4, 0.6 MgCl2, 12 NaHCO3, 15 glucose, and 10 HEPES, pH 7.2, at 37°C,
preequilibrated with O2/CO2 (95:5 vol/vol) at a
flow rate of 3.5-4
ml · g1 · min
1.
Following the cannulation, the liver was rapidly removed and placed on
a platform. After 5 min of equilibration, the perfusion medium was
replaced with another having similar composition but devoid of
Mg2+ (Mg2+-free medium). Contaminant
Mg2+ content of the medium was measured by AAS and found to
be ~8-10 µM. After 10 min of washout with
Mg2+-free medium (time 0), samples of the
effluent were collected at 30-s intervals and Mg2+ content
of the perfusate was measured by AAS. The samples collected during the
first 8 min provided a baseline for the subsequent infusion of EtOH or
adrenergic agonist. EtOH was infused in a range of concentrations
between 0.01% (1.6 mM) and 1% (160 mM) (50). A selective
1- (phenylephrine, 5 µM) or
- (isoproterenol, 10 µM) adrenergic agonist or the mixed adrenoceptor agonist
catecholamine (epinephrine, 5 µM) was infused at pharmacological
doses to exclude lack of an effect due to altered adrenoceptor
responsiveness. At the end of the perfusion, the liver was gently
blotted on adsorbing paper and weighed. Aliquots of 1-1.5 g were
homogenized in 5 volumes of 10% HNO3 and digested
overnight to measure total tissue Mg2+ content. The protein
content of the acid extract was sedimented in a refrigerated Beckman
J-6B centrifuge (2,000 g for 10 min), and the
Mg2+ content in the supernatant was measured by AAS.
Estimation of the total amount of
Mg2+ extruded.
To estimate the total amount of Mg2+ extruded from the
livers following EtOH or adrenergic agonist administration, the
Mg2+ content in the perfusate at the last five time points
before addition of the agent was averaged and subtracted from each of the time points under the curve of efflux. The net amount of
Mg2+ mobilized into the perfusate (nmol/ml) was then
calculated, taking into account the perfusion rate (3.5-4
ml · g1 · min
1)
and the time of collection (30 s), and expressed as micromoles. The
residual Mg2+ content of the perfused livers was also
calculated in the tissue homogenate as described above.
Collagenase-dispersed cells. Collagenase-dispersed rat hepatocytes were isolated according to the procedure of Seglen (47). After the isolation, hepatocytes were resuspended, at a final concentration of ~1 × 106 cells/ml, in a medium of the following composition (in mM): 120 NaCl, 3 KCl, 1.2 KH2PO4, 12 NaHCO3, 1.2 CaCl2, 1.2 MgCl2, 10 HEPES, and 10 glucose, pH 7.2, at 37°C, under O2/CO2 (95:5 vol/vol) flow, and kept at room temperature until used. Cell viability was 88 ± 3% for both EtOH-fed and control rats (n = 10/group), as assessed by trypan blue exclusion, and did not significantly change over 3-4 h (86 ± 2%). For the determination of Mg2+ transport, 1 ml of cell suspension was transferred into a Microfuge tube and the cells were rapidly sedimented at 600 g for 30 s. The supernatants were removed, and the cells were washed with 1 ml of a medium having the same composition as the one reported above but devoid of Mg2+ (incubation medium). The cells were then transferred in 10 ml of incubation medium, prewarmed at 37°C, and incubated therein under continuous stirring and O2/CO2 flow. After a few minutes of equilibration, 0.01, 0.1, or 1% EtOH or the reported concentrations of adrenergic agonists or protein kinase C-stimulating agents were added to the incubation system. At the time points reported in the figures, 700-µl aliquots of the incubation mixture were withdrawn in duplicate, and the cells were sedimented in Microfuge tubes. The Mg2+ content in the supernatant was measured by AAS.
Determination of cytosolic cAMP levels.
cAMP level was determined in aliquots of cell extract by
125I-RIA as reported previously (50). Briefly,
collagenase-dispersed hepatocytes were incubated under the experimental
conditions described in Collagenase-dispersed cells. At
5-min intervals, aliquots of the incubation mixture (700 µl) were
withdrawn and the cells were sedimented in Microfuge tubes. The
supernatant was removed for determination of Mg2+ content
by AAS. The cell pellets were resuspended in 200 µl of assay buffer
(Biotrak; Amersham), boiled for 5 min, and stored at 20°C
until used.
Determination of ATP levels.
Cellular ATP level was also measured in isolated hepatocytes incubated
as reported previously (50). At 5-min intervals, 700-µl
aliquots of the incubation mixture were withdrawn and the cells were
sedimented in Microfuge tubes. The supernatant was removed, and the
cells were digested in perchloric acid (5% final concentration) for 5 min in ice. The acid mixture was then neutralized by the addition of
KHCO3, and the denatured protein was sedimented in a
refrigerated Beckman J-6B (2,000 g for 10 min). The
supernatants were removed and stored at 20°C until used. ATP
determination was carried out by luciferin-luciferase assay (detecting
sensitivity in the picomole to nanomole per milliliter range; Sigma),
using a LUMAT Berthold LB 9501 luminometer, or by HPLC, using a C18 RP
column (Millipore Waters) and 60 mM ammonium phosphate (pH 6.6 with
ammonium hydroxide) as the mobile phase (50). Adenine phosphonucleotide standards (1-20 nmol/ml) were injected for calibration.
Determination of glucose, LDH, and protein. Aliquots of the perfusate were collected every minute, and glucose content was determined by enzymatic kit (Sigma) as the variation in hexokinase-coupled NADH+ content at 340 nm. LDH activity in the perfusate or in the extracellular medium was measured by enzymatic kit (Sigma) sensitive to changes in the microunit-per-milliliter range and expressed as units per liter or as a percentage of the total amount of LDH releasable from digitonin-permeabilized hepatocytes. Cell viability was assessed by trypan blue exclusion test. Protein content was measured according to the procedure of Lowry et al. (29) using BSA as standard.
Cytosolic free Ca2+ measurement. Changes in cytosolic Ca2+ concentration ([Ca2+]) were measured in fura-2 AM-loaded hepatocytes as previously reported (39) by using a dual-wavelength excitation fluorometer (University of Pennsylvania Biomedical Instrumentation Group, Philadelphia, PA) equipped with a thermostated cuvet.
Plasma membrane isolation.
Total liver plasma membrane vesicles (tLPM) were isolated and stored as
described in detail by Cefaratti et al. (3). The purity of
plasma membrane vesicles (Table 1) was
assessed by using 5'-nucleotidase, cytochrome c oxidase, and
glucose 6-phosphatase activities as markers for plasma membrane,
mitochondria, and endoplasmic reticulum, respectively (3).
Negligible levels of cytochrome c oxidase and glucose
6-phosphatase activities were detected in tLPM preparations, whereas
both 5'-nucleotidase and Na+-K+-ATPase were
found to be enriched 12- and 8-fold in tLPM from control and EtOH-fed
livers, respectively (Table 1). The activity of the latter two enzymes
was also used to evaluate the orientation of tLPM vesicles
(3). The comparison of these activities to those of
detergent-disrupted vesicles (considered as 100%) confirmed our early
report (3) that 90% of EtOH-treated and control tLPM
were in the "inside-in" configuration following loading with Mg2+. The tLPM endogenous carryover of cations and adenine
phosphonucleotides was measured by AAS and HPLC, respectively, and
found to be negligible (not shown).
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Loading of tLPM and determination of Mg2+ fluxes. Five-milliliter aliquots of tLPM were resuspended in 25 ml of 250 mM sucrose and 25 mM HEPES (pH 7.4 with Tris) in the presence of 20 mM MgCl2 and were loaded by four passes in a Thomas C Potter with a tight-fitting pestle at 4°C (3). We have previously reported that this Mg2+ concentration is optimal to study Mg2+ transport in this experimental model (3). The mixture was sedimented at 34,500 g for 10 min in a Sorvall SS-34 rotor to remove excess extravesicular cation. The Mg2+-loaded vesicles were resuspended in 5 ml of the Mg2+-free medium and stored in ice until used. Loading efficiency was assessed by treating the vesicles with 2 µg/ml A23187 or 0.1% Triton X-100 and by measuring the amount of Mg2+ extruded in the extravesicular space or retained in the vesicle pellet by AAS, as reported previously (3).
Mg2+ fluxes were measured by AAS. An aliquot of Mg2+-loaded tLPM was incubated in the Mg2+-free medium at 37°C under continuous stirring at a final concentration of ~300 µg protein/ml. After 2 min of equilibration, aliquots of the incubation mixture were withdrawn in duplicate at 2-min intervals, and the vesicles were sedimented in Microfuge tubes at 7,000 g for 45 s (3). Total Mg2+ content in the supernatants was measured by AAS. The pellet was digested overnight in 500 µl of 10% HNO3. The denatured protein was sedimented in Microfuge tubes, and the Mg2+ content of the acid extract was measured by AAS. The first two time points after the equilibration period (time 0) were used to establish a baseline. Following the withdrawal of the second sample, 50 mM Na+ or 500 µM Ca2+ was added to the incubation mixture to elicit maximal Mg2+ extrusion from tLPM suspensions (3), and the incubation continued for six additional minutes. For simplicity, the data are reported as the net variation in extravesicular Mg2+ content, normalized per milligram of protein. To calculate net Mg2+ extrusion, Mg2+ content in the supernatant at the first two time points was calculated, averaged, and subtracted from the values of the subsequent time points of incubation.Statistical analysis. The data are reported as means ± SE. Data were first analyzed by one-way ANOVA. Multiple means were then compared by Tukey's multiple comparison test performed with a q value established for statistical significance of P < 0.05.
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RESULTS |
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Male Sprague-Dawley rats fed for 4 wk with a 6% EtOH-enriched liquid diet weighed 7% less than rats receiving an isocaloric control liquid diet (392.6 ± 5.5 vs. 421.6 ± 4.6 g body wt, respectively; n = 11 for both experimental groups).
The determination of tissue cation content indicated a marked decrease
in Mg2+ content in several tissues of EtOH-fed animals
compared with control rats (Table 2). All
of the muscles examined (abdomen, hind limb, soleus, quadriceps, heart,
and diaphragm) presented a decrease in Mg2+ content varying
between 12 and 19% (Table 2). The liver was also affected. Despite a
20% increase in liver mass compared with control animals (13.52 ± 0.50 vs. 11.21 ± 0.41 g of tissue, respectively; n = 11; P < 0.05), EtOH-fed rats
presented a 14% decrease in liver Mg2+ content (57.9 ± 4.1 vs. 67.1 ± 5.0 nmol/mg protein). A marked decrease in
Mg2+ content was also observed in lung, spleen, and blood
cells, whereas brain, testicles, adipose tissue, and pancreatic tissue
from EtOH-fed rats presented little or no change in Mg2+
content compared with control animals (Table 2). Mg2+
content was markedly increased in the urine of EtOH-fed rats (128%;
Table 2). K+ content was also decreased in skeletal muscles
and liver, whereas Na+ and Ca2+ content were
increased (Table 2).
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Our laboratory has extensively utilized perfused livers and collagenase-dispersed hepatocytes to elucidate the hormonal regulation of Mg2+ transport and homeostasis (10, 11, 37-39, 43). Hence we used these two experimental models to investigate to what extent the observed decrease in Mg2+ content altered the ability of liver cells to extrude Mg2+ following hormonal stimulation and whether Mg2+ could be accumulated into the cells to restore Mg2+ homeostasis.
Figure 1 shows that, in the absence of a
stimulatory agent, livers of control rats retained their cellular
Mg2+ content over 45 min of perfusion. The administration
of epinephrine (Fig. 1A) or norepinephrine (not shown; Ref.
43) elicited the extrusion of a sizable amount of
Mg2+ from the organs into the perfusate in a time-dependent
fashion. Selective 1- (phenylephrine) or
-
(isoproterenol) adrenergic agonists also elicited Mg2+
extrusion (Fig. 1A) from the livers, although the amount of
Mg2+ mobilized by each agonist was quantitatively smaller
than that mobilized by the mixed agonist catecholamine (~ 0.9, 1.8, and 3.3 µmol Mg2+ following isoproterenol, phenylephrine,
and epinephrine administration, respectively). In contrast, the
infusion of these agents in livers explanted from EtOH-fed animals did
not induce a detectable extrusion of Mg2+ into the
perfusate, albeit pharmacological doses of the agonists were infused
(Fig. 1B). To determine whether the nonresponsiveness to
adrenergic agonists depended on altered adrenoceptor sensitivity, 250 µM of the cell-permeant cAMP analog 8-Cl-cAMP (Fig.
2) was infused to increase cellular level
of cAMP and induce Mg2+ extrusion bypassing the
-adrenoceptor (10, 37, 43). Consistent with previous
reports from this laboratory (10), 8-Cl-cAMP elicited an
extrusion of Mg2+ from livers of control rats that was
quantitatively similar to that elicited by isoproterenol under similar
experimental conditions (~1.8 µmol under both conditions). In
contrast, this agent failed to elicit a Mg2+ extrusion in
livers from EtOH-fed rats (Fig. 2). In addition, livers from EtOH-fed
rats exhibited a reduced isoproterenol- or epinephrine-induced glucose
extrusion (1.9 and 9.8 mmol/l, respectively) compared with control
animals (~50 mmol/l) (Fig. 3). The
infusion of varying EtOH concentrations (Fig.
4B) also failed to induce a
Mg2+ extrusion from livers of EtOH-fed rats, whereas a
time- and dose-dependent Mg2+ mobilization was observed in
control livers under similar experimental conditions (Fig.
4A; see also Ref. 50).
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To better quantify the decrease in tissue Mg2+ content, hepatocytes were isolated by collagenase digestion. Total Mg2+ content in hepatocytes from EtOH-treated rats accounted for 26.03 ± 1.24 nmol/mg protein (n = 14), which represented a 25% decrease in Mg2+ content compared with the value determined in hepatocytes from control rats (36.18 ± 0.52 nmol/mg protein; n = 10; P < 0.05). The lower hepatic Mg2+ content paralleled a 13% decrease in cellular ATP content (12.15 ± 0.13 vs. 14.03 ± 0.20 nmol ATP/mg protein for EtOH-fed and control livers, respectively; n = 14; P < 0.05).
A typical incubation time course for Mg2+ extrusion in
hepatocytes is reported in Fig.
5A. As the figure shows, no
Mg2+ is extruded from liver cells until an agonist is added
to the incubation system. Following agonist addition, Mg2+
is extruded from the cells into the extracellular compartment in a
time-dependent fashion, reaching the maximum at 4-6 min after the
addition. Prolonged incubations did not result in a larger Mg2+ extrusion (not shown; Refs. 37 and 39).
For simplicity, the net amount of Mg2+ extruded at 6 min
(i.e., 4 min after the agonist addition) was calculated as indicated in
MATERIALS AND METHODS and was reported for the subsequent
figures. Liver cells from control animals extruded ~2-2.5 nmol
Mg2+/mg protein within 4 min from the addition of 0.01%
EtOH, adrenergic agonist, cell-permeant cAMP, or thapsigargin (Fig.
5B). In contrast, hepatocytes isolated from EtOH-fed rats
(Fig. 5B) mobilized a smaller amount of Mg2+
(0.4-0.6 nmol · mg
protein1 · 4 min
1)
under similar experimental conditions. The determination of cAMP level
in isolated hepatocytes from EtOH-fed rats stimulated by epinephrine or
isoproterenol indicated a limited increase in second messenger level
from basal level (+23% and +17%, respectively) following stimulation
by
-adrenergic agonist compared with controls (+65% and +45% for
epinephrine- and isoproterenol-stimulated cells, respectively; Table
3).
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Whereas activation of cAMP/protein kinase A pathway elicits
Mg2+ extrusion, stimulation of protein kinase C signaling
pathway results in an accumulation of Mg2+ within liver
cells (38, 39). Figure
6A shows that the addition of
vasopressin, carbachol, phorbol myristate acetate derivatives, or the
diacylglycerol analogs oleoylacetyl glycerol and stearoylarachidonolyl glycerol resulted in a net accumulation of ~2 nmol
Mg2+ · mg
protein1 · 4 min
1 in
hepatocytes from control animals. Liver cells from EtOH-fed rats were,
in contrast, unable to accumulate Mg2+ irrespective of the
agent (or dose; not shown) administered to stimulate protein kinase C
signaling pathway and the extracellular Mg2+ concentration
utilized (i.e., contaminant or 1 mM; Fig. 6B). Basal
cytosolic free [Ca2+], although slightly higher in
hepatocytes from EtOH-fed rats compared with control cells (108 ± 12 vs. 90 ± 7 nM, respectively; n = 5 for both
experimental conditions) did not approach statistical significance. In
line with data reported by others (51), mobilization of
Ca2+ by 10 nM vasopressin was ~30% lower in hepatocytes
from EtOH-fed vs. control rats (215 ± 21 vs. 305 ± 35 nM,
respectively; n = 5 for both experimental conditions;
P < 0.05).
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The operation of distinct Mg2+ transport mechanisms has
been evidenced in purified liver plasma membrane vesicles (3,
4), a model in which intravesicular Mg2+ content is
artificially rendered similar to the intracellular concentration (20 mM) and the transport from the vesicle into the extravesicular space is
virtually similar to the extrusion from the cell into the extracellular
space. Hence we used this experimental model to ascertain whether the
lack of Mg2+ transport observed in perfused livers and
isolated hepatocytes could be ascribed to an intrinsic alteration of
Mg2+ transporters. As Fig. 7
indicates, plasma membrane vesicles isolated from control rats extruded
~120 and ~150 nmol Mg2+/mg protein when stimulated by
50 mM Na+ and 500 µM Ca2+, respectively (see
also Ref. 3). In contrast, Mg2+-loaded plasma
membranes from EtOH-fed rats did not extrude Mg2+
efficiently when stimulated with Na+ or Ca2+
(70% for both conditions). Concentrations of Na+ and
Ca2+ larger than those reported in the figure did not
elicit a larger Mg2+ mobilization from plasma membrane
vesicles (not shown).
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DISCUSSION |
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Extensive investigation in various experimental models and
alcoholic patients (1, 14, 28, 52) indicates that chronic EtOH consumption alters physiological functions and ion pattern within
several tissues. Tissue Na+ and Ca2+ content
have been reported to increase as a result of an altered operation of
transport mechanisms (15, 48) and cell signaling (2,
21, 22). A decrease in tissue Mg2+ content has also
been reported to occur in brain, heart, and other tissues, but the
causes for this decrease still remain largely unclear. Although
experimental evidence supports a role of Mg2+ in regulating
collagen deposition (36), glycogen phosphorylase activation (16), and mitochondrial -ketoglutarate
dehydrogenase activity (33), it is not fully determined to
what extent these cellular processes are impaired by the decrease in
Mg2+ content within the tissues nor the long-term
implications that changes in the activity of these functions may have
for the development of EtOH-related hepatic pathologies.
During the last decade, our laboratory has extensively contributed to
the characterization of the hormone-controlled mechanisms that regulate
cellular and whole body Mg2+ homeostasis (10, 11, 24,
37-39, 43). We have reported that the increase in cellular
cAMP elicited via -adrenoceptor stimulation induces a marked
extrusion of cellular Mg2+ from cardiac (42)
and liver cells (37, 43). The process is likely to be more
general, in that infusion of
-adrenoceptor agonist raises plasma
Mg2+ level by 15-20% in anesthetized rats
(24). In contrast, agents or hormones that decrease cAMP
level (38-43) or stimulate protein kinase C signaling
pathway (38) prevent Mg2+ extrusion from the
cell and/or revert Mg2+ extrusion into a Mg2+ accumulation.
Recently, we started to investigate the mechanisms responsible for the decrease in liver Mg2+ content following EtOH administration. We have reported that acute EtOH administration induces a marked extrusion of Mg2+ from liver cells in a time- and dose-dependent manner, decreasing liver Mg2+ content by 5-10% (50). The time course of Mg2+ extrusion matches the transient decrease in cellular ATP content observed under these experimental conditions (31, 50), thus suggesting that Mg2+ is essentially mobilized from the cytosolic MgATP pool.
The objective of the present study was to investigate the effect that chronic EtOH administration exerts on Mg2+ homeostasis and on hormone-mediated Mg2+ extrusion and uptake in liver cells as a first step toward elucidating how changes in cellular Mg2+ content affect specific hepatocyte functions.
Altered Mg2+ homeostasis in EtOH-fed
animals.
The administration of EtOH (6%) in the diet for 4 wk results in a
marked decrease in Mg2+ content within several tissues,
including liver. Compared with control animals, the decrease in hepatic
Mg2+ content in EtOH-fed rats accounts for ~14% in the
whole tissue and ~25% in isolated hepatocytes. Most likely, the
discrepancy in the percentage of decrease between two values can be
ascribed to technical manipulations or the heterogeneity inherent to
whole tissue processing vs. cell isolation by collagenase before
Mg2+ content determination. As already observed after acute
EtOH administration (50), the decrease in Mg2+
content elicited by prolonged EtOH consumption is associated with a
decrease in cellular ATP level (13%). This observation indicates
that common mechanisms are activated by acute and chronic EtOH
administration, which result in a decrease of Mg2+
buffering capacity within the cell and in a loss of Mg2+
from the cytosolic MgATP pool. In the acute model of EtOH
administration, the cellular conversion of EtOH to acetaldehyde inverts
the NAD+/NADH ratio and favors the formation of
glyceraldehyde-3-phosphate, which acts as a Pi trap and
removes the inhibition by phosphate on AMP deaminase, allowing the
degradation of ATP to proceed to uric acid and allantoin
(31). Whereas the decrease in ATP level is transient (~8
min) during acute EtOH administration (50), in the chronic
EtOH model reported here, no recovery in cellular ATP level is
observed, most likely because of the continuous administration of
alcohol with the diet. The amplitude of ATP decrease under acute and
chronic experimental conditions is comparable, thus pointing to the
lack of recovery in ATP content as the possible main cause for the
persistent low level of cellular ATP and, consequently, Mg2+ content. Assuming an MgATP concentration of ~3 mM in
the hepatocyte cytosol (6) and the differing dissociation
constants for MgADP (~250 µM) and MgAMP (~8.13 mM) vs. MgATP
(~80 µM), the lower cellular ATP content results in a major change
in buffering capacity for Mg2+. Consequently, a larger
amount of Mg2+ will dissociate from the degrading
phosphonucleotides and be lost from the cell, most likely via a
Na+-dependent mechanism (50). This scenario
can explain the increase in Na+ content observed in the
tissues in which Mg2+ was decreased, including liver (Table
1), as well as the lack of an effect of acute EtOH administration on
these hepatocytes, since an acute infusion of EtOH will target the
cytosolic Mg2+ pool, which is already depleted by the
prolonged administration of EtOH with the diet. Because total
Mg2+ concentration within the hepatocyte accounts for
18-20 mM (44), even a 14% decrease in total tissue
Mg2+ content (the most conservative of our determinations)
represents a major change in terms of cellular content (~2.5 mM).
This value is about threefold larger than cytosolic free
Mg2+ (estimated to be ~0.7-1 mM; Ref.
6), and about twofold larger than the combined amounts of
free Mg2+ in the cytosol and Mg2+ no longer
bound to ATP (a 13% decrease in total ATP content would account for
~650 µM Mg2+; Ref. 50). Hence we have to
assume that a considerable portion of Mg2+ lost from the
cell is coming from intracellular organelles such as mitochondria,
endoplasmic reticulum, and nucleus, which all contain ~16-18 mM
total Mg2+ under physiological conditions (8).
The loss of Mg2+ from any, or more than one, of these
compartments will then affect the Mg2+ content of the
organelle, with obvious consequences on the activity rate of enzymes
therein located (e.g.,
-ketoglutarate dehydrogenase in the
mitochondria; Ref. 33).
Transport in EtOH-fed animals.
Hormone-modulated Mg2+ transport mechanisms are also
markedly affected by a prolonged exposure to EtOH in the diet. Although the decrease in cellular Mg2+ content is sufficient to
explain the lack of Mg2+ extrusion following -adrenergic
agonist or cell-permeant cAMP addition, the determination of basal
cellular cAMP level, which in hepatocytes from EtOH-fed rats is not
dissimilar from that of control cells (3.6 ± 0.4 vs. 3.8 ± 0.2 pmol/mg protein, respectively) and increases only marginally in
response to isoproterenol or epinephrine administration
(+17-23%), and the observation that glucose extrusion from
EtOH-fed livers is also markedly reduced compared with control organs
indicate that EtOH exerts an inhibitory effect on various elements of
the adrenergic signaling cascade, including G proteins and
phosphodiesterase (21, 22). Furthermore, the marginal
increase in cAMP level and the reduced output of glucose observed
following adrenergic stimulation indicate that hormonal activated
glycolysis is markedly hampered in EtOH-fed rats and cannot effectively
contribute to maintaining a physiological level of ATP within liver
cells. In addition, the reduced Mg2+ extrusion observed in
purified tLPM (Fig. 7) suggests that EtOH administration affects the
Mg2+ transport mechanisms in the plasma membrane, either
directly [EtOH-related modification of the phospholipid environment
(25) or EtOH-protein interaction (26)] or
indirectly (via the decrease in cellular ATP content). Cefaratti et al.
(5), in fact, have proposed that the Mg2+
transport mechanisms operating in purified plasma membrane vesicles become phosphorylated and active during the isolation procedure. Hence
it is conceivable that the decrease in ATP content and the defects in
cAMP signaling elicited by EtOH alter the modus operandi or the
activity rate of the Mg2+ transport mechanisms in the cell membrane.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute on Alcohol Abuse and Alcoholism Grant R9-AA-11593-A2
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FOOTNOTES |
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Address for reprint requests and other correspondence: A. Romani, Dept. of Physiology and Biophysics, Case Western Reserve Univ., 10900 Euclid Ave., Cleveland, OH 44106-4970 (E-mail: amr5{at}po.cwru.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpgi.00153.2002
Received 22 April 2002; accepted in final form 23 September 2002.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Altura, MB,
Gebrewold A,
Altura BT,
and
Gupta RK.
Role of brain [Mg2+]i in alcohol-induced hemorrhagic stroke in a rat model. A 31P-NMR in vivo study.
Alcohol
12:
131-136,
1985.
2.
Bokkala, S,
Rubin E,
and
Joseph SK.
Effect of chronic ethanol exposure on inositol tris-phosphate receptors in WB rat liver epithelial cells.
Alcohol Clin Exp Res
23:
1875-1883,
1999[ISI][Medline].
3.
Cefaratti, C,
Romani A,
and
Scarpa A.
Characterization of two Mg2+ transporters in sealed plasma membrane vesicles from rat liver.
Am J Physiol Cell Physiol
275:
C995-C1008,
1998
4.
Cefaratti, C,
Romani A,
and
Scarpa A.
Differential localization and operation of distinct Mg2+ transporters in apical and basolateral sides of rat liver plasma membrane.
J Biol Chem
275:
3772-3780,
2000
5.
Cefaratti, C,
Zakhari YD,
Bond M,
Ruse C,
and
Scarpa A.
Protein kinase A dependent phosphorylation activates Mg2+ efflux from hepatocytes (Abstract).
Biophys J
80:
1035A,
2001.
6.
Corkey, BE,
Duszinski J,
Rich TL,
Matschinsky B,
and
Williamson JR.
Regulation of free and bound magnesium in rat hepatocytes and isolated mitochondria.
J Biol Chem
261:
2567-2574,
1986
7.
Csermely, P,
Fodor P,
and
Somogyi J.
The tumor promoter tetradecanoylphorbol-13 acetate elicits the redistribution of heavy metal in subcellular fractions of rabbit thymocytes as measured by plasma emission spectroscopy.
Carcinogenesis
8:
1663-1666,
1987[Abstract].
8.
Dalal, P,
Romani A,
and
Scarpa A.
Redistribution of Mg2+ in isolated hepatocytes upon chemical hypoxia (Abstract).
Biophys J
74:
A193,
1998.
9.
Domenicotti, C,
Paola D,
Lamedica A,
Ricciarelli R,
Chiarpotto E,
Marinari UM,
Poli G,
Melloni E,
and
Pronzato MA.
Effects of ethanol metabolism on PKC activity in isolated rat hepatocytes.
Chem Biol Interact
100:
155-163,
1996[ISI][Medline].
10.
Fagan, TE,
and
Romani A.
Activation of Na+- and Ca2+-dependent Mg2+ extrusion by 1- and
-adrenergic agonists in rat liver cells.
Am J Physiol Gastrointest Liver Physiol
279:
G943-G950,
2000
11.
Fagan, TE,
and
Romani A.
1-Adrenoceptor-induced Mg2+ extrusion from rat hepatocytes occurs via Na+-dependent transport mechanism.
Am J Physiol Gastrointest Liver Physiol
280:
G1145-G1156,
2001
12.
Fagan, TE,
and
Romani A.
Altered Mg2+ homeostasis in liver cells from streptozotocin treated rats (Abstract).
Endocrinology
222:
PI-345,
2001.
13.
Flatman, PW.
Mechanisms of magnesium transport.
Annu Rev Physiol
53:
259-271,
1991[ISI][Medline].
14.
Flink, EB.
Magnesium deficiency in alcoholism.
Alcohol Clin Exp Res
10:
590-594,
1986[ISI][Medline].
15.
Gandhi, CR,
and
Ross DH.
Influence of ethanol on calcium, inositol phospholipids and intracellular signaling mechanisms.
Experentia
45:
407-413,
1989[Medline].
16.
Gaussin, V,
Gailly P,
Gillis JM,
and
Hue L.
Fructose-induced increase in intracellular free Mg2+ ion concentration in rat hepatocytes: relation with the enzymes of glycogen metabolism.
Biochem J
326:
823-827,
1997[ISI][Medline].
17.
Gunther, T.
Functional compartmentation of intracellular magnesium.
Magnesium
5:
53-59,
1986[ISI][Medline].
18.
Gunther, T.
Mechanisms and regulation of Mg2+ efflux and Mg2+ influx.
Miner Electrolyte Metab
19:
250-265,
1993[ISI][Medline].
19.
Gunther, T,
and
Hollriegl V.
Na+- and anion-dependent Mg2+ influx in isolated hepatocytes.
Biochim Biophys Acta
1149:
49-54,
1993[ISI][Medline].
20.
Gunther, T,
and
Vormann J.
Activation of Na+/Mg2+ antiport in thymocytes by cAMP.
FEBS Lett
297:
132-143,
1992[ISI][Medline].
21.
Hoek, JB,
Thomas AP,
Rooney TA,
Higashi K,
and
Rubin E.
Ethanol and signal transduction in the liver.
FASEB J
6:
2386-2396,
1992
22.
Hoek, JB,
Thomas AP,
Rubin R,
and
Rubin E.
Ethanol-induced mobilization of calcium by activation of phosphoinositide-specific phospholipase C in intact hepatocytes.
J Biol Chem
262:
682-691,
1987
23.
Jakob, A,
Becker J,
Schottli G,
and
Fritzsch G.
1-Adrenergic stimulation causes Mg2+ release from perfused rat livers.
FEBS Lett
246:
127-130,
1989[ISI][Medline].
24.
Keenan, D,
Romani A,
and
Scarpa A.
Differential regulation of circulating Mg2+ in the rat by 1- and
2-adrenergic receptor stimulation.
Circ Res
77:
973-983,
1995
25.
Kelly-Murphy, S,
Waring AJ,
Roteenberg H,
and
Rubin E.
Effects of chronic ethanol consumption on the partition of lipophilic compounds into erythrocytes membranes.
Lab Invest
50:
174-183,
1984[ISI][Medline].
26.
Korpi, ER,
Makela R,
and
Uusi-Oukari M.
Ethanol: novel actions on nerve cell physiology explain impaired functions.
News Physiol Sci
13:
164-170,
1998
27.
Lester, DS,
and
Baumann D.
Action of organic solvents on protein kinase C.
Eur J Pharmacol
206:
301-308,
1991[Medline].
28.
Lieber, CS.
Mechanism of ethanol induced hepatic injury.
Pharmacol Ther
46:
1-41,
1990[ISI][Medline].
29.
Lowry, OH,
Rosenbrough NJ,
Farr AL,
and
Randall RJ.
Protein measurement with the folin phenol reagent.
J Biol Chem
193:
265-275,
1951
30.
Maguire, ME.
Magnesium and cell proliferation.
Ann NY Acad Sci
551:
201-215,
1988[Abstract].
31.
Masson, S,
Desmoulin F,
Sciaky M,
and
Cozzone PJ.
Catabolism of adenine nucleotides and its relation with intracellular phosphorylated metabolite concentration during ethanol oxidation in perfused rat liver.
Biochemistry
32:
1025-1031,
1993[ISI][Medline].
32.
Matsuura, T,
Kanayama Y,
Inoue T,
Takeda T,
and
Morishima I.
cAMP-induced changes in intracellular Mg2+ levels in human erythrocytes.
Biochim Biophys Acta
1220:
31-36,
1993[ISI][Medline].
33.
Panov, A,
and
Scarpa A.
Independent modulation of the activity of -ketoglutarate dehydrogenase complex by Ca2+ and Mg2+.
Biochemistry
35:
427-432,
1996[ISI][Medline].
34.
Quamme, GA,
and
Dai LS.
Presence of a novel influx pathway for Mg2+ in MDCK cells.
Am J Physiol Cell Physiol
259:
C521-C525,
1990
35.
Quamme, GA,
and
Rabkin SW.
Cytosolic free magnesium in cardiac myocytes: identification of a Mg2+ influx pathway.
Biochem Biophys Res Commun
167:
1406-1412,
1990[ISI][Medline].
36.
Rayssiguier, Y,
Chevalier F,
Bonnet M,
Kopp J,
and
Durlach J.
Influence of magnesium deficiency on liver collagen after carbon tetrachloride or ethanol administration to rats.
J Nutr
115:
1656-1662,
1985[ISI][Medline].
37.
Romani, A,
Dowell E,
and
Scarpa A.
Cyclic AMP-induced Mg2+ release from rat liver hepatocytes, permeabilized hepatocytes, and isolated mitochondria.
J Biol Chem
266:
24376-24384,
1991
38.
Romani, A,
Marfella C,
and
Scarpa A.
Regulation of Mg2+ uptake in isolated rat myocytes and hepatocytes by protein kinase C.
FEBS Lett
296:
135-140,
1992[ISI][Medline].
39.
Romani, A,
Marfella C,
and
Scarpa A.
Hormonal stimulation of Mg2+ uptake in hepatocytes: regulation by plasma membrane and intracellular organelles.
J Biol Chem
268:
15489-15495,
1993
40.
Romani, A,
Marfella C,
and
Scarpa A.
Regulation of magnesium uptake and release in the heart and in isolated ventricular myocytes.
Circ Res
72:
1139-1148,
1993[Abstract].
41.
Romani, A,
Matthews V,
and
Scarpa A.
Parallel stimulation of glucose and Mg2+ accumulation by insulin in rat hearts and cardiac ventricular myocytes.
Circ Res
86:
326-333,
2000
42.
Romani, A,
and
Scarpa A.
Hormonal control of Mg2+ in the heart.
Nature
346:
841-844,
1990[ISI][Medline].
43.
Romani, A,
and
Scarpa A.
Norepinephrine evokes a marked Mg2+ efflux from liver cells.
FEBS Lett
269:
37-40,
1990[ISI][Medline].
44.
Romani, A,
and
Scarpa A.
Regulation of cell magnesium.
Arch Biochem Biophys
298:
1-12,
1992[ISI][Medline].
45.
Romani, A,
and
Scarpa A.
Regulation of cellular magnesium.
Front Biosci
5:
D720-D734,
2000[ISI][Medline].
46.
Ron, D,
Vagts AJ,
Dohrman DP,
Yaka R,
Jiang Z,
Yao L,
Crabbe J,
Grisel JE,
and
Diamond I.
Uncoupling of IIPKC from its targeting protein RACK1 in response to ethanol in cultured cells and mouse brain.
FASEB J
14:
2303-2314,
2000
47.
Seglen, PO.
Preparation of isolated rat liver cells.
Methods Cell Biol
13:
29-83,
1976[Medline].
48.
Swann, AC.
Ethanol and (Na+,K+)-ATPase: alteration of Na+-K+ selectivity.
Alcohol Clin Exp Res
14:
922-927,
1990[ISI][Medline].
49.
Vormann, J,
and
Gunther T.
Amiloride sensitive net Mg2+ efflux from isolated perfused rat hearts.
Magnesium
6:
220-224,
1987[ISI][Medline].
50.
Tessman, PA,
and
Romani A.
Acute effect of EtOH on Mg2+ homeostasis in liver cells: evidence for the activation of an Na+/Mg2+ exchanger.
Am J Physiol Gastrointest Liver Physiol
275:
G1106-G1116,
1998
51.
Zhang, BH,
Horsfield BP,
and
Farrell GC.
Chronic ethanol administration to rats decreases receptor-operated mobilization of intracellular ionic calcium in cultured hepatocytes and inhibits 1,4,5-inositol trisphosphate production: relevance to impaired liver regeneration.
J Clin Invest
98:
1237-1244,
1996
52.
Zou, LY,
Wu L,
Altura BT,
Barbour RL,
and
Altura BM.
Beneficial effect of high magnesium on alcohol-induced cardiac failure.
Magnes Trace Elem
10:
409-419,
1991-1992[Medline].