Characterization of two Mg21 transporters in sealed
plasma membrane vesicles from rat liver
CHRISTIE CEFARATTI, ANDREA ROMANI, AND ANTONIO SCARPA
Department of Physiology and Biophysics, School of Medicine,
Case Western Reserve University, Cleveland, Ohio 44106-4970
sodium/magnesium antiporter; calcium/magnesium antiporter; hepatocytes
MAGNESIUM ION,
a major cation within the cell (8, 10), is
required for membrane structure stabilization (8, 10),
cell cycle regulation (22, 39), structural modification of
phosphometabolites (see Refs. 8 and 10 for review), and
the functioning of a large number of enzymes (10, 32).
The well-documented absence of significant changes in
cytosolic free Mg21 (4, 10, 24) following a variety of
metabolic interventions previously led to the assumption that Mg21 content remains relatively constant
within the cell and to the conclusion that cellular
enzymes cannot be regulated in vivo by changes in
cytosolic Mg21. On the basis of the findings that large
amounts of ‘‘total’’ Mg21 are accumulated or released by
cells within a few minutes in response to hormonal
stimulation (28–31), this assumption has recently been
challenged. Significant experimental evidence obtained
initially in nonmammalian cells (e.g., squid axon) and
more recently in several mammalian cell types suggests that Mg21 transport across the plasma membrane
occurs through at least two mechanisms. A Na1/Mg21
exchanger has been shown to operate in cardiac myocytes (29, 31, 38) as well as in hepatocytes (12, 28, 30),
thymocytes (15), and chicken and human erythrocytes
(12). This transport mechanism electroneutrally extrudes 1 Mg21 for 2 Na1 in chicken erythrocytes, and
possibly in other cell types as well (13). Recently, it was
reported that Mg21 extrusion from a variety of cell
types is stimulated by cAMP (15, 28, 31, 41), inhibited
by amiloride (13, 38), and not operative in the absence
of extracellular Na1 (21, 29, 30). The presence of a
Na1-independent Mg21 extrusion pathway has also
been proposed in human, chicken, and rat erythrocytes
(14). However, the requirement for a specific cation to
be accumulated in exchange for Mg21 has not been
defined. In different experimental models or conditions,
cellular Mg21 has been shown to exchange for extracellular Mn21 (6), Ca21 (29), HCO2
3 (12), or other cations or
anions (12), with various stoichiometries. Furthermore, recent experimental evidence indicates that in
hepatocytes (30) and cardiac myocytes (29), even in the
presence of extracellular Na1, Mg21 accumulation is
inhibited in the absence of extracellular Ca21 (29) or in
the presence of the Ca21 channel blockers verapamil
(26) or nifedipine. Presently, it is also unclear whether
the cellular uptake and release of Mg21 are performed
by the same transporter or by different transporters.
Collectively, these observations indicate the presence
in the plasma membrane of very active pathways for
Mg21 transport in and out of the cell. Yet the nature of
the transporters and the modality of operation are
unclear. With the exception of erythrocytes, the study of
Mg21 transport across the plasma membrane using
whole cells is complicated by the fact that intracellular
organelles are also active participants in cellular Mg21
homeostasis (32). To overcome this complication and to
minimize the possible buffering of Mg21 by intracellular compartments in this study, sealed plasma membrane vesicles, preloaded to contain a variety of trapped
ions and phosphonucleotides, were used to define the
operation and selectivity of the mechanism(s) by which
hepatocytes transport Mg21 across the cell membrane.
MATERIALS AND METHODS
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.
Plasma Membrane Isolation
Liver plasma membrane (LPM) vesicles were isolated
according to a procedure similar to that described by Prpic et
0363-6143/98 $5.00 Copyright r 1998 the American Physiological Society
C995
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Cefaratti, Christie, Andrea Romani, and Antonio
Scarpa. Characterization of two Mg21 transporters in sealed
plasma membrane vesicles from rat liver. Am. J. Physiol. 275
(Cell Physiol. 44): C995–C1008, 1998.—The plasma membrane of mammalian cells possesses rapid Mg21 transport
mechanisms. The identity of Mg21 transporters is unknown,
and so are their properties. In this study, Mg21 transporters
were characterized using a biochemically and morphologically standardized preparation of sealed rat liver plasma
membranes (LPM) whose intravesicular content could be set
and controlled. The system has the advantages that it is not
regulated by intracellular signaling machinery and that the
intravesicular ion milieu can be designed. The results indicate that 1) LPM retain trapped intravesicular total Mg21
with negligible leak; 2) the addition of Na1 or Ca21 induces a
concentration- and temperature-dependent efflux corresponding to 30–50% of the intravesicular Mg21; 3) the rate of flux is
very rapid (137.6 and 86.8 nmol total Mg21 · µm22 · min21 after
Na1 and Ca21 addition, respectively); 4) coaddition of maximal concentrations of Na1 and Ca21 induces an additive Mg21
efflux; 5) both Na1- and Ca21-stimulated Mg21 effluxes are
inhibited by amiloride, imipramine, or quinidine but not by
vanadate or Ca21 channel blockers; 6) extracellular Na1 or
Ca21 can stimulate Mg21 efflux in the absence of Mg21
gradients; and 7) Mg21 uptake occurs in LPM loaded with
Na1 but not with Ca21, thus indicating that Na1/Mg21 but not
Ca21/Mg21 exchange is reversible. These data are consistent
with the operation of two distinct Mg21 transport mechanisms and provide new information on rates of Mg21 transport, specificity of the cotransported ions, and reversibility of
the transport.
C996
MG21 TRANSPORT IN LIVER PLASMA MEMBRANE VESICLES
Plasma Membrane Purity and Orientation
The purity of the LPM vesicles was assessed by using
58-nucleotidase, cytochrome-c oxidase, and glucose-6-phosphatase activities as markers for plasma membrane, mitochondria, and endoplasmic reticulum (ER), respectively (27) (Table
1). Cytochrome-c oxidase and glucose-6-phosphatase activities were ,2 nmol cytochrome c oxidized · mg protein21 · min21
and 40 nmol Pi · mg protein21 · min21, respectively.
The orientation of 20 mM Mg21-loaded and unloaded
vesicles was assessed by measuring the Na1-K1-ATPase
activity according to the procedure of Ismail-Beigi and Edelman (17), by the 58-nucleotidase assay (27), and by [3H]ouabain binding experiments. In loaded vesicles, the Na1-K1ATPase activity (in µmol Pi · mg protein21 · min21 ) was 0.043 6
0.006 in the absence of Triton and increased to 0.35 6 0.082 in
the presence of Triton (Table 1). When loaded vesicles were
assessed for 58-nucleotidase, the activity (in µmol Pi · mg
protein21 · min21 ) was 0.55 6 0.112 in the absence of Triton
and 0.605 6 0.116 in the presence of Triton. Therefore, the
activities of these assays concurred in indicating that LPM
were mostly in the ‘‘inside-in’’ configuration (86 6 3 and 87 6
10%, respectively). Unloaded vesicles, instead, were mainly
in ‘‘inside-out’’ configuration, in agreement with the observation by Prpic et al. (25) (Table 1). The proper configuration in
loaded vesicles is primarily due to the presence of Mg21,
which has been shown to significantly contribute to membrane sidedness in similar systems (36, 37). The activity of
the enzymes in intact vesicles both in the presence and in the
absence of ouabain was correlated with that measured in
Triton X-100-treated vesicles (considered as 100%). The difference between the two activities indicates the percentage of
inside-out and inside-in vesicles. Also, LPM vesicles loaded
with 20 mM Mg21 plus 5 mM ATP demonstrated an inside-in
orientation .80%.
For the [3H]ouabain binding procedure, after the loading
with Mg21, LPM vesicles were incubated in the presence of 1
mM ouabain to which 50 µCi/ml [3H]ouabain was added. After
5 min of incubation, the vesicles were rapidly sedimented in
microcentrifuge tubes, the supernatant was removed by
vacuum suction, and the radioactivity in the pellet was
measured by liquid scintillation counting. The binding occurring in intact LPM vesicles was compared with that observed
in LPM vesicles permeabilized by digitonin or Triton X-100, a
condition in which the maximal binding capacity of ouabain
to Na1-K1-ATPase was obtained. Similarly, in [3H]ouabain
binding experiments, the inside-in percentage was determined to be 72 6 5% for 20 mM Mg21-loaded vesicles. As
noted in Table 1, LPM vesicles used in these studies exhibited
17.5- and 15.1-fold enrichment compared with homogenate in
Na1-K1-ATPase and 58-nucleotidase activity, respectively.
Because the Na1-K1-ATPase is prevalent in basolateral
membranes (42) whereas the 58-nucleotidase is primarily
sorted to the apical membrane of hepatocytes (35), it appears
that our LPM preparation contains a similar enrichment in
both of these membrane portions.
To further ascertain LPM sidedness, 45Ca21 transport
experiments were performed in both unloaded and loaded
vesicles, as previously described by Prpic et al. (25). Unloaded
LPM, but not Mg21-loaded LPM, displayed a Ca21 uptake
similar to that reported by Prpic et al. (25) (data not shown).
In contrast, LPM loaded with 20 mM MgCl2 plus 45Ca21 and
10 mM ATP actively extruded 45Ca21 (data not shown). Under
both conditions, Ca21 transport was abolished in the presence
of A-23187. These data further demonstrate that loaded LPM
are primarily oriented inside-in.
Endogenous carryover of cations during preparation in
freshly isolated LPM was assessed for Na1, Mg21, Ca21, and
K1 by atomic absorbance spectroscopy (AAS), using a PerkinElmer 3100 spectrophotometer, and found to be minimal.
Negligible levels of intravesicular adenine phosphonucleotides were detected by HPLC (not shown).
Plasma Membrane Vesicle Volume and Surface Area
LPM volume and surface area were determined using two
different techniques. First, LPM radius and surface area were
Table 1. Enzyme activity of purified LPM
58-Nucleotidase
Fraction
No Triton
Triton
Mg21-ATPase
No Triton
Triton
Na1-K1-ATPase
No Triton
Triton
Glucose-6Phosphatase
Cytochrome-c
Oxidase
Homogenate
0.05 6 0.009 0.04 6 0.007 0.05 6 0.002 0.06 6 0.012 0.022 6 0.007 0.02 6 0.004 0.081 6 0.002 0.031 6 0.004
Unloaded LPM
0.17 6 0.02 0.324 6 0.014 0.63 6 0.27 0.65 6 0.23
0.37 6 0.18 0.202 6 0.17 0.027 6 0.0014 0.002 6 0.0004
Loaded LPM
(20 mM Mg21 ) 0.550 6 0.112 0.605 6 0.116 0.12 6 0.05 0.96 6 0.08 0.043 6 0.006 0.35 6 0.082
ND
ND
Enrichment
11.0
15.1
12.6
16.0
16.8
17.5
Values are means 6 SE; n 5 15, 8, 8, 4, and 6 for 58-nucleotidase, Mg21-ATPase, Na1-K1-ATPase, glucose-6-phosphatase, and cytochrome-c
oxidase, respectively. Enzyme activities are expressed as µmol Pi · mg protein21 · min21. Cytochrome-c oxidase activity is expressed as nmol
cytochrome c oxidized · mg protein21 · min21. All experimental protocols are described in MATERIALS AND METHODS. Enrichment was determined
by comparing activity in loaded liver plasma membrane (LPM) fraction to homogenate for 58-nucleotidase. For all other enzymatic parameters,
enrichment was determined by comparing fraction with highest activity (in presence of Triton) to homogenate. ND, not determined.
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al. (25). Male Sprague-Dawley rats (250–350 g) were anesthetized by intraperitoneal injection of 50 mg/kg body wt pentobarbital. The abdomen was opened, and the liver was perfused by the portal vein with 50 ml of isolation medium (in
mM: 250 sucrose, 5 potassium HEPES, and 1 EGTA; pH 7.4)
at 37°C. The liver was rapidly removed, finely minced,
washed twice in the isolation medium, and homogenized in 50
ml of medium using 10 passes with a loose-fitting pestle
followed by 3 passes with a tight-fitting pestle. The homogenate was diluted to 6% (wt/vol) with the same buffer and
sedimented at 1,400 g for 10 min. The pellets were recovered
and resuspended at 6% (final concentration) in the isolation
medium by four passes with the loose-fitting pestle. Percoll
(Pharmacia) was added to the resuspension in the proportions 1.4 ml Percoll to every 10.4 ml resuspension (25), and
the LPM were isolated by centrifugation at 34,500 g for 30
min. All operations after liver removal were carried out at
4°C. The top fluffy layer, containing the plasma membrane
vesicles, was collected, diluted 1:5 (vol/vol) with the incubation medium (250 mM sucrose and 50 mM Tris; pH 8.0), and
sedimented at 34,500 g for 30 min. The resulting pellets were
diluted to a final concentration of 5 mg protein/ml in incubation medium and stored in liquid nitrogen until used. No
difference in Mg21 transport was noticed between freshly
isolated LPM and vesicles quickly frozen and stored in liquid
nitrogen for several days.
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MG21 TRANSPORT IN LIVER PLASMA MEMBRANE VESICLES
Plasma Membrane Loading
Aliquots (5 ml) of LPM vesicles were resuspended in 25 ml
of incubation medium (1:5 vol/vol) in the presence of 20 mM
MgCl2 or, when specified, in medium containing other ions
such as Na1, Sr21, or Ca21, and loading was empirically
determined by four passes with a tight-fitting pestle at 4°C.
Effective and efficient loading can be obtained with use of this
Table 2. Characteristics of purified LPM
Characteristic
Value
Average diameter, µm
Total volume of membranes, µl/mg protein
Volume per vesicle, µl/vesicle or µm3/vesicle
Surface area, µm2
Number of vesicles per mg protein
Mg21 fluxes, nmol Mg21 · µm22 · min 21
Na1 induced
Ca21 induced
0.90 6 0.03
4.90 6 0.09
3.82 3 10210 or 0.382
2.50
1.30 3 1010
137.60
86.80
Morphological calculation of diameter was determined as described
in MATERIALS AND METHODS. Value represents average of 5,011 vesicles
fitting to a Gaussian function with a bin range of 0.1. Intravesicular
volume was determined as difference between 3H2O-accessible space
(intravesicular and extravesicular space) and [14C]polydextranaccessible space (extravesicular space); n 5 3. Details are described
in MATERIALS AND METHODS. Average volume and surface area per
individual vesicle were determined by assuming a spherical shape
and average diameter of 0.9 µm. Number of vesicles per mg protein
was calculated as ratio between average volume of vesicles per mg
protein (4.9 µl/mg protein) and average volume per individual vesicle
(3.82 3 10210 µl/vesicle). Flux (nmol Mg21 · µm22 · min 21 ) of ions
across LPM per unit of surface area was determined based on efflux of
Mg21 within 1 min after stimulation by 25 mM NaCl or 50 µM CaCl2 ;
20 mM magnesium gluconate-loaded vesicles incubated in presence
of 20 mM magnesium gluconate outside were demonstrated to have
largest Mg21 efflux within 1 min after stimulation by 25 mM NaCl
(344.1 6 158.8 nmol Mg21/mg protein) or 50 µM CaCl2 (217 6 18.2
nmol Mg21/mg protein). Fluxes were determined by dividing amplitude of efflux by surface area of 2.5 µm2. As discussed in text, fluxes
are underestimated because sedimentation technique used does not
permit initial rate measurements.
protocol. LPM vesicles were loaded with various concentrations of Mg21 (2, 5, 10, or 20 mM Mg21 ). Because total Mg21
concentration within the intact cell is 20 mM, this was the
concentration used throughout all experiments (unless stated
otherwise) to more closely approximate physiological conditions. In some experiments, the role of intravesicular phosphonucleotides or Pi was investigated by supplementing the
Mg21-containing loading medium with 5 mM ATP (either Tris
or disodium salt) or, alternatively, with adenosine 58-O(3-thiotriphosphate) (ATPgS), ADP (disodium salt), GTP
(lithium salt), GDP (lithium salt), pyrophosphate, Tris-Pi, or
NH4-Pi. In either case, the loaded vesicles were sedimented at
34,500 g for 10 min, resuspended, and filtered through Nitrex
nylon with a mesh opening of 475 µm. Mg21 fluxes were
determined as the amount of Mg21 released into the extravesicular space (supernatant experiments) or as the Mg21
content trapped within LPM (pellet experiments).
Loading of Plasma Membrane Vesicles During Isolation
In some experiments (Fig. 3), LPM vesicles were loaded
during isolation, using a protocol similar to the one described
above, with the following modifications. Mg21 (10 mM) was
added to the buffer before the initial homogenization and was
maintained throughout the second resuspension, and the
Percoll ratio was increased to 3.5 ml Percoll for every 10.4 ml
resuspension. In this protocol, LPM were used immediately
after isolation. An advantage of this protocol is that LPM can
be simultaneously isolated and loaded.
Mg21 Measurement
Mg21 fluxes or contents were measured by AAS. Hence, in
all experiments in vesicle pellets or supernatants, total
(rather than free) Mg21 was measured. The Mg21-loaded
vesicles were incubated in the incubation medium at a final
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determined from an electron micrograph of a typical preparation of MgCl2-loaded LPM (39,000 magnification). The loaded
LPM were sedimented through an 11% Percoll gradient and
then processed for electron microscopy. The pellet was fixed
overnight in 0.1 M cacodylate buffer (pH 7.4), containing 2%
paraformaldehyde and 2.5% glutaraldehyde and then washed
three times in 0.1 M cacodylate buffer. Postfixation occurred
in 2% osmium tetraoxide for 1 h, and the pellet was washed
three times in double-distilled H2O. Next, the block was
stained in 1% aqueous solution of uranyl acetate for 1 h, and
dehydration took place in a series of graded ethanol treatments. The samples were infiltrated and embedded in Spurr
low-viscosity embedding medium (Ernest F. Fullman, Latham,
New York) and blocked out in Eppendorf microcentrifuge
tubes. The sample was polymerized for 48 h at 70°C. The
polymerized pellet block was sectioned on an ultramicrotome
(Research and Manufacturing, Tucson, AZ), specifically at the
center and parallel to the long axis of the Eppendorf tube,
with a section thickness of 75 nm. This permitted a view of
the density-separated LPM from the top to the bottom of the
pellet with relative size homogeneity (larger vesicles in the
bottom and smaller vesicles at the top of the pellet). Sections
were mounted on Formvar-supported copper slot grids and
contrasted with 2% uranyl acetate in 50% ethanol and 0.25%
lead citrate in 1 N NaOH (Electron Microscopy Sciences). The
section was examined and photographed in a transmission
electron microscope (model JEM 1200EX II, JEOL, Peabody,
MA) at 80 kV with an objective aperture of 50 µm.
A transparency marked with 1 line/in. and with the lines
perpendicular to the long axis of the microcentrifuge tube was
placed over the micrographs. The largest 3 diameters in a
field of ,25 vesicles along each line were averaged. Because
the section thickness is approximately one-tenth of the diameter of the vesicles, the largest diameter along the line most
closely represents the true size of the vesicles. The number of
vesicles along each grid line was counted, and all the vesicles
were assumed to be the size of the averaged largest diameters. A histogram of the diameters of all vesicles along the
line was generated, and the data were fitted to a Gaussian
function of the counted diameters. Based on these measurements and assumptions, an average diameter of 0.9 µm was
determined for the entire vesicle preparation. The surface
area of the LPM was calculated (Table 2) under the assumption that the vesicles were spherical in shape.
In the second technique, the intravesicular volume of LPM
loaded with 20 mM Mg21 was measured according to the
procedure of Johnson and Scarpa (18) and determined as the
difference in the pellet between the spaces accessible to 3H2O
and to [14C]polydextran. LPM loaded with 20 mM Mg21 (2–4
mg protein/ml) were equilibrated for 1–10 min in incubation
medium (pH 7.4, 37°C) labeled with [14C]polydextran and
3H O (8 µCi/ml of each radionuclide). Aliquots (500 µl) were
2
sedimented for 5 min at 7,000 g in a Fisher benchtop
microcentrifuge. The supernatant was removed, and the
pellet was dissolved overnight in 10% HNO3. The acid extract
was sedimented (45 s at 7,000 g), and the radioactivity in the
supernatant was measured by liquid scintillation counting
(Beckman LS3801).
C998
MG21 TRANSPORT IN LIVER PLASMA MEMBRANE VESICLES
Other Experimental Procedures
The intravesicular content of adenine phosphonucleotides
was measured by HPLC, as described previously (5). Protein
was measured according to the procedure of Bradford (1),
using BSA as a standard.
Chemicals
All chemicals were of the purest analytical grade (Sigma,
St. Louis, MO). [3H]ouabain was from ICN (Costa Mesa, CA).
All adenine and guanine nucleotides were from Calbiochem
(La Jolla, CA). 3H2O was obtained from Amersham, and
[14C]polydextran was from Dupont-NEN. Nitrex nylon mesh
was obtained from Tetko (Briarcliff Manor, NY).
Statistical Analysis
Data are presented as means 6 SE. Data were first
analyzed by one-way ANOVA. Multiple means were then
compared by Tukey’s multiple comparison test or by the
Student-Newman-Keuls method performed with the level of
statistical significance designated as P # 0.05.
RESULTS
Electron Micrograph of Purified LPM
Figure 1 shows an electron micrograph of a typical
preparation of LPM (39,000 magnification). The contamination by other intracellular organelles is small,
consistent with the biochemical determinations reported in Table 1. Based on a large body of data, the
preparation of Mg21-loaded LPM is between 75 and
85% ‘‘right configuration’’ (see Table 1 and MATERIALS
AND METHODS ). The average diameter, size, surface
area, and number of vesicles per milligram protein
were estimated in the whole population by a combination of radiochemical and morphological techniques as
described in MATERIALS AND METHODS and Table 2. These
Fig. 1. Electron micrograph of rat liver plasma membranes (LPM)
used for Mg21 transport. Details on preparation and fixation are
reported in MATERIALS AND METHODS. Magnification: 39,000.
values are summarized in Table 2 and were used to
calculate Mg21 fluxes.
Dose Response of Na1- and Ca21-Induced Mg21 Efflux
From LPM and Effects of Various Inhibitors
Figure 2, A and B, shows that LPM vesicles loaded
with 20 mM MgCl2 and suspended in a medium devoid
of Na1 or Ca21 release negligible Mg21 over several
minutes of incubation (control). The addition of NaCl
(Fig. 2A) or CaCl2 (Fig. 2B) to the incubation medium
prompts a sizable Mg21 efflux from the vesicles in a
dose-dependent fashion. Irrespective of the concentration of NaCl or CaCl2 used, Mg21 efflux at 37°C is
already complete within the first period of observation,
2 min after NaCl or CaCl2 addition. The amount of
Mg21 released at this point is not limited by an equilibration of Mg21 gradients across the plasma membrane, as additional Mg21 can be mobilized from the
vesicles by the addition of a divalent cation ionophore
(see Fig. 5).
At 2 min, the addition of 10 or 20 mM NaCl induces
the extrusion of 12.7 6 3.9 and 37.6 6 6.4 nmol
Mg21/mg protein, respectively (n 5 37). The maximum
Mg21 extrusion is observed following the addition of 25
mM NaCl (,90 nmol/mg protein), whereas larger concentrations of NaCl do not result in a significantly
greater Mg21 efflux (Fig. 2A). In addition, Fig. 2A shows
that the addition of 100 mM sucrose does not induce a
Mg21 efflux, suggesting that the mobilization of Mg21
by 50 mM NaCl is not caused by a nonspecific rapid
osmotic mismatch.
Figure 2B shows that the concentrations of CaCl2
able to induce Mg21 extrusion are far lower than those
of NaCl. In fact, Mg21 extrusion is already detectable
following the addition of 25 µM Ca21 (44.9 6 8.4 nmol
Mg21/mg protein) and is maximal when 500 µM CaCl2
is added to the system (160.5 6 11.6 nmol Mg21/mg
protein) (n 5 8). Under these conditions, the amount of
Mg21 released by Ca21 is almost double that mobilized
by maximal concentrations of Na1. Higher CaCl2 concentrations (in the millimolar range) do not result in larger
Mg21 efflux (not shown). Figure 2B also shows that the
coaddition to LPM vesicles of 500 µM Ca21 and 25 mM
Na1, each individually producing maximal Mg21 re-
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concentration of 250 µg protein/ml. For the supernatant
measurements, the loaded LPM vesicles were resuspended in
Mg21-free incubation medium. This was necessary because
large extravesicular Mg21 concentrations interfere with the
measurement of Mg21 content in the supernatant by AAS. For
the pellet measurement, to minimize the leakage of the
entrapped Mg21 down its concentration gradient, the loaded
LPM vesicles were resuspended in the presence (20 mM) of
the cation used for the loading. In the majority of pellet
experiments, the external ion concentration was 2 mM after
1:10 dilution in the incubation mixture, unless otherwise
stated. In some experiments, the concentrations of intra- and
extravesicular Mg21 were kept identical at 20 mM.
After 1 min of equilibration, aliquots of the incubation
mixture were withdrawn at 1- to 2-min intervals, and the
vesicles were sedimented by rapid centrifugation (7,000 g for
1 min) in microcentrifuge tubes. Mg21 content was measured
in the supernatants by AAS. As for the Mg21 content in LPM
vesicles (pellet measurement), aliquots of the incubation
medium were sedimented in microcentrifuge tubes through
an oil layer (dimethyl phthalate, dibutyl phthalate, and
dioctyl phthalate 2:3:4) to remove extravesicular Mg21. The
supernatant and oil layer were removed by vacuum suction,
and the pellet was digested overnight in 500 µl 10% HNO3.
The Mg21 content of the vesicles was measured by AAS in the
acid extracts using Mg21 standards in identical acid concentration. Similar procedures were also used for Na1- or Ca21loaded LPM.
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MG21 TRANSPORT IN LIVER PLASMA MEMBRANE VESICLES
lease, results in an additive amount of Mg21 extruded.
Specifically, the net Mg21 extrusion induced by 25 mM
Na1 plus 500 µM Ca21 is 267.7 6 17.0, compared with
the Mg21 efflux induced by Na1 or Ca21 alone of 92.2 6
Table 3. Percent inhibition of Mg21 efflux in
LPM by amiloride
Inhibition, %
Amiloride
Concentration, µM
500 µM CaCl2
stimulation
25 mM NaCl
stimulation
0
25
100
500
1,000
0
39.2 6 6.8
58.0 6 6.0
78.3 6 3.4
99.9 6 0.2
0
3.1 6 1.1
47.2 6 14.6
ND
58.6 6 0.7
Values are means 6 SE of 4 independent experiments. Aliquots of
incubation mixture were withdrawn and centrifuged, and Mg21
content in supernatant was measured by atomic absorbance spectroscopy as described for Fig. 2.
Fig. 2. Net Mg21 efflux as a function of extravesicular Na1 and/or
Ca21 concentration. Dose-response for NaCl-induced (A) or for CaCl2induced (B) Mg21 efflux from 20 mM MgCl2-loaded LPM vesicles,
incubated in absence of extravesicular Mg21. Experiments were
performed as described in MATERIALS AND METHODS. Vesicles (250
µg/ml) were incubated at 37°C in a reaction medium containing 250
mM sucrose and 50 mM Tris-HEPES (pH 7.4). After 2 min of
equilibration, an aliquot corresponding to 500 µl of incubation
medium was withdrawn and rapidly sedimented in a microcentrifuge
tube. Supernatant was assayed for Mg21 content by atomic absorbance spectrophotometry (AAS). After second withdrawal, concentration of NaCl or CaCl2 indicated was added, and aliquots of sample
were withdrawn in duplicate at indicated time points and processed
as above. Shown is net change in Mg21 content in supernatant with
respect to that before ion addition. Data are means 6 SE of 37 and 8
preparations for Na1 and Ca21, respectively. ANOVA and Tukey’s test
for multiple comparisons or Student-Newman-Keuls method were
performed at all time points. * P , 0.05 vs. control and/or 100 mM
sucrose for Tukey’s test; ** P , 0.05 vs. control for Student-NewmanKeuls method. Additionally, in B, asterisks were omitted for clarity
for 500 µM CaCl2 alone vs. 500 µM CaCl2 1 25 mM NaCl (P , 0.05).
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13.8 and 160.5 6 11.6 nmol Mg21/mg protein, respectively.
The results reported in Fig. 2, A and B, reflect an
increase in extravesicular Mg21 content (supernatant
experiments). However, a quantitatively similar decrease in Mg21 content retained within the vesicles
could be observed in pellet experiments (see Fig. 6).
Table 3 shows that the Mg21 extrusion activated by Na1
or Ca21 is decreased by 50% in the presence of 100 µM
amiloride, and Ca21-induced Mg21 extrusion is almost
completely absent in the presence of 1 mM amiloride.
Such a high concentration of amiloride has been shown
to effectively inhibit Mg21 extrusion via the putative
Na1/Mg21 exchanger in several mammalian cell types,
including hepatocytes (13, 38). A quantitatively similar
inhibition (not shown) was also observed using 200 µM
quinidine or 200 µM imipramine, two other nonspecific
inhibitors of the Mg21 extrusion mechanism (7). However, the coaddition of amiloride and quinidine or
imipramine failed to elicit additional inhibition of the
Na1-dependent Mg21 release (not shown). In contrast,
the Ca21 channel blockers nifedipine and verapamil, as
well as inhibitors of the ER Ca21 leak mechanism and
the mitochondrial Ca21 uniport, neomycin and ruthenium red, were ineffective at inhibiting Mg21 extrusion
from LPM (not shown).
C1000
MG21 TRANSPORT IN LIVER PLASMA MEMBRANE VESICLES
Mg21 Efflux in Preloaded Vesicles
Resolution of Mg21 Kinetics by Decreasing
Temperature of Incubation Medium
Figure 4 shows the temperature dependence of Mg21
extrusion in supernatant experiments from 20 mM
MgCl2-loaded LPM vesicles incubated in a thermostated vessel at 15, 20, or 37°C following stimulation
with 25 mM NaCl. Through a decrease in the incuba-
Fig. 4. Temperature dependence of Mg21 efflux from loaded LPM
vesicles. LPM vesicles were loaded with 20 mM MgCl2 in absence of
extravesicular Mg21, incubated at 15, 20, or 37°C, and stimulated
with 25 mM NaCl. Efflux of Mg21 from LPM into supernatant is
expressed as percent of value before cation addition. Experimental
conditions were similar to those for Fig. 2. Data are means 6 SE of 5
preparations. ANOVA and Tukey’s test for multiple comparisons were
performed at all time points. * P , 0.05 vs. control.
tion temperature to 15 or 20°C, a progressive decrease
in Mg21 extrusion rate is observed, and the process
starts to be kinetically resolved. A qualitatively similar
temperature dependence was also observed in LPM
stimulated by CaCl2 (not shown).
Effects of the Ionophore A-23187
Fig. 3. Mg21 efflux from vesicles preloaded with 10 mM Mg21. Shown
is effect of extravesicular cations on vesicles preloaded with 10 mM
Mg21 in absence of extravesicular Mg21 (see MATERIALS AND METHODS ).
Experimental conditions were similar to those for Fig. 2. CaCl2 (50
µM) induces a net efflux of 124 6 40.4 nmol Mg21/mg protein vs. a net
efflux of 22.7 6 4.2 nmol Mg21/mg protein induced by NaCl. Data are
means 6 SE of 4 preparations. ANOVA and Tukey’s test for multiple
comparisons were performed at all time points. * P , 0.05 vs. control,
50 mM NaCl, and 50 µM CaCl2 1 1 mM amiloride.
To determine the total amount of Mg21 mobilizable
from LPM vesicles, A-23187 (1 µg/ml) was used either
in the absence of Na1 or after stimulation by Na1 (Fig.
5). As Fig. 5 shows, ,35% of the entrapped Mg21 is
mobilized by the addition of 50 mM NaCl (Fig. 5) to the
extravesicular medium. The subsequent addition of
ionophore mobilizes an additional 50% of the residual
intravesicular Mg21 after Na1 stimulation. The addition of A-23187 alone rapidly mobilizes ,70% of the
intravesicular Mg21 content. Thus ionophore by itself
releases a total amount of Mg21 comparable to that
mobilized by Na1 plus the ionophore. Approximately
30% of the total Mg21 is retained in LPM after addition
of the ionophore. Based on the intravesicular water
space, the total amount of Mg21 releasable under those
conditions is two- to threefold greater than the amount
of ‘‘free’’ Mg21 expected in the vesicles. This should be
the result of dissociation between aspecifically bound
Mg21 and free Mg21, as more free Mg21 is released by
the ionophore. Such a dissociation should, in principle,
be facilitated by the intravesicular decrease in pH
resulting from exchange of 1 Mg21 out for 2 H1 in,
facilitated by A-23187. Similar results were also obtained when Ca21 was used instead of Na1 (not shown).
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.1 on June 15, 2017
The operation of the Na1- and Ca21-dependent Mg21
extrusion mechanisms was also investigated in LPM
vesicles preloaded with 10 mM Mg21. The major difference in this preparation is that the LPM were loaded
during isolation with a concentration of 10 mM Mg21, in
contrast to the previous protocol in which vesicles were
loaded with 20 mM MgCl2 after isolation. With this
preparation, the effectiveness of CaCl2 with respect to
NaCl in mobilizing Mg21 is even more evident. Figure 3
shows that in these vesicles 50 µM CaCl2 prompts a
Mg21 extrusion similar to that reported in Fig. 2B,
whereas the extrusion induced by NaCl is markedly
smaller (compare Fig. 3 with Fig. 2A). Furthermore,
the inhibitory effect of amiloride on the Ca21-mediated
Mg21 efflux is less (see Table 3).
MG21 TRANSPORT IN LIVER PLASMA MEMBRANE VESICLES
C1001
Mg21 Efflux Is Specific for the Monovalent Cation Na1
The experiment shown in Fig. 7 was also performed
under conditions of identical MgCl2 concentration inside and outside the vesicles. Figure 7 demonstrates
the stringent specificity of the Na1/Mg21 antiporter for
Na1. Neither Li1 nor K1 is able to elicit a Mg21 efflux
under these conditions.
Similarities Between Mg21 and Sr21 Release From
Isolated LPM
Fig. 5. Mg21 mobilization by A-23187. Percent movement of Mg21
from 20 mM Mg21-loaded LPM vesicles with 2 mM MgCl2 in
extravesicular solution. After 1 min of equilibration, 500-µl aliquots
were withdrawn and rapidly sedimented through an oil layer (see
MATERIALS AND METHODS ). Supernatant and oil layer were removed,
and pellets were digested overnight in 500 µl 10% HNO3. Mg21
content in acid extract was assayed by AAS. After second withdrawal,
25 mM NaCl or 1 µg/ml A-23187 was added. Aliquots of incubation
medium were withdrawn at indicated time points and processed as
above. Mg21 content retained in vesicles following addition of NaCl or
A-23187 is expressed as a percent of Mg21 content at time 0. Initial
Mg21 content before ion addition was 345.3 6 9.8 nmol Mg21/mg
protein. Data are means 6 SE of 4 preparations. ANOVA and Tukey’s
test for multiple comparisons were performed at all time points. * P ,
0.05 vs. control.
Mg21 Transport Is Not Due to Displacement
of Bound Mg21
The experiment shown in Fig. 6 was performed under
conditions in which the concentrations of MgCl2 or
magnesium gluconate were identical inside and outside
the vesicles (i.e., inside and outside Mg21 concentrations were equal). Under these conditions, the Mg21
content remaining in the pellet was measured (see
MATERIALS AND METHODS ). Therefore, Fig. 6 represents
the amount of Mg21 released from LPM as decreasing
values. In vesicles loaded with magnesium gluconate,
the addition of 25 mM sodium isethionate mobilizes
344.1 6 158.8 nmol Mg21/mg protein, whereas 50 µM
calcium gluconate induces an efflux of 217.1 6 18.2
nmol Mg21/mg protein (Fig. 6) within 1 min after
addition. Also, in this system, 1 mM amiloride inhibits
Na1- (Fig. 6) and Ca21-dependent (not shown) Mg21
efflux. It is noteworthy that in the presence of 20 mM
extravesicular Mg21 the amount of Mg21 released is
considerably larger than that reported in Fig. 2. Most
likely, this is a consequence of a passive leakage of the
cation during the loading procedure and resuspension
in 0 mM Mg21 (Fig. 2). Consistent with this interpretation, the amount of Mg21 trapped within the vesicles
resuspended in the absence of extracellular Mg21 (Fig.
2) is approximately twofold less than LPM resuspended
in the presence of 20 mM Mg21.
Fig. 6. Vesicles loaded with 20 mM magnesium gluconate were
incubated in 20 mM magnesium gluconate. Shown is Mg21 efflux in
vesicles loaded and resuspended in presence of 20 mM magnesium
gluconate. Mg21 efflux is expressed as net change in Mg21 content
retained within LPM vs. content at time 0. After 1 min of equilibration, 25 mM sodium isethionate or 50 µM calcium gluconate was
added to incubation system. Mg21 content retained in vesicles was
assayed as described for Fig. 5. Initial Mg21 content before ion
addition was 1,275 6 131.1 nmol Mg21/mg protein. This value is
,3-fold higher than that of Fig. 5 and is consistent with a lesser Mg21
diffusion outside vesicles prepared and stored with 20 rather than 2
mM Mg21. Data are means 6 SE of 3 preparations. ANOVA and
Student-Newman-Keuls method for multiple comparisons were performed at all time points. * P , 0.05 vs. control. Additionally, asterisks
for 25 mM sodium isethionate vs. 25 mM sodium isethionate 1 1 mM
amiloride were omitted for clarity (P , 0.05).
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Sr21 has been shown to act as a substitute for Mg21 in
a variety of cell transport systems (29). Therefore, LPM
vesicles were loaded with 20 mM Sr21 (Fig. 8), and
intravesicular Sr21 content was measured using AAS.
As Fig. 8 shows, the net loss of Sr21 from the vesicles is
quantitatively comparable to the net Mg21 increase in
the extravesicular medium (Fig. 2). For example, 25
mM NaCl and 50 µM Ca21 induce an efflux of 95.9 6 7.8
and 74.0 6 31.3 nmol Sr21/mg protein, respectively.
C1002
MG21 TRANSPORT IN LIVER PLASMA MEMBRANE VESICLES
by 25 mM NaCl (Fig. 2A; 92.8 6 13.8 nmol Mg21/mg
protein). In contrast, under identical conditions, 5 mM
CaCl2 fails to induce Na1 extrusion. The small uptake
of Na1 observable under control and Ca21 conditions is
likely due to the carryover of Na1 bound to the external
face of the vesicle.
Figure 11 shows the result of experiments in which
LPM vesicles were loaded with 20 mM NaCl (Fig. 11A)
or with 10 mM CaCl2 (Fig. 11B) and resuspended in a
medium containing the same concentration of either
cation. The addition of 5 mM Mg21 can induce the
release of Na1 but not of Ca21 from the vesicles,
suggesting that the Na1/Mg21 antiporter but not the
Ca21/Mg21 mechanism can operate in either direction.
Several laboratories have reported the presence of an
ATP-dependent Mg21 transport in both mammalian (9,
21) and nonmammalian (2) cell types. HPLC determination showed that unloaded LPM vesicles contained no
detectable ATP or other phosphonucleotides (not shown).
Fig. 7. Stimulation of LPM by monovalent cations. Shown is data for
LPM loaded with 20 mM MgCl2, incubated in presence of 20 mM
MgCl2, and stimulated by 25 mM LiCl, KCl, or NaCl. Mg21 movement
is expressed as net decrease in Mg21 content retained in pellet. LPM
Mg21 content was assayed as described for Fig. 5. Initial Mg21
content before ion addition was 1,252 6 170.8 nmol Mg21/mg protein.
Data are means 6 SE of 3 preparations. ANOVA and Tukey’s test for
multiple comparisons were performed at all time points. * P , 0.05 vs.
control, 25 mM LiCl, and 25 mM KCl.
Figure 8 also shows the inhibitory effect of 200 µM
imipramine on both the Na1- and Ca21-dependent Mg21
effluxes. Quantitatively similar data were obtained
using SrCl2 labeled with 85Sr21 (data not shown).
Bidirectionality of Mg21 Transport Mechanism(s)
Na1/Mg21 transport can operate in either direction.
Figure 9A shows the result of an experiment in which
isolated LPM vesicles were loaded with 20 mM NaCl, as
described in MATERIALS AND METHODS, and extravesicular Mg21 was added at the concentrations of MgCl2
indicated in Fig. 9. Under these experimental conditions, a marked extrusion of Na1 from the vesicles is
also observed (data not shown). Figure 9B shows that,
under the same conditions, Sr21 is also accumulated in
a quantitatively and kinetically similar fashion, indicating that Sr21 could replace Mg21 for Na1/Mg21 exchange in either direction. The addition of Mg21 or Sr21
induces a large uptake that is complete within 1 min
after addition.
Figure 10 shows the result of an experiment in which
the effect of Mg21 or Ca21 was measured in Na1-loaded
vesicles resuspended in the absence of Na1. The addition of 5 mM Mg21 to the extravesicular compartment
prompts a net Na1 efflux of 235.3 6 8.7 nmol Na1/mg
protein (Fig. 10), an amount approximately double that
of Mg21 released from Mg21-loaded vesicles stimulated
Fig. 8. LPM vesicles loaded with 20 mM Sr21. Efflux of Sr21 from
LPM vesicles loaded with 20 mM SrCl2 and incubated in presence of 2
mM extravesicular SrCl2. Aliquots of incubation mixture were withdrawn at indicated time points and processed as described for Fig. 5.
Sr21 content in LPM was measured by AAS. Initial Sr21 content
before ion addition was 120.3 6 19.6 nmol Sr21/mg protein. This
value is ,3 times smaller than that of Fig. 5 under similar conditions.
This difference could be accounted for by lack of endogenous Sr21, as
opposed to endogenous Mg21 bound to vesicles. Data are means 6 SE
of 3 preparations. ANOVA and Tukey’s test for multiple comparisons
were performed at all time points. * P , 0.05 vs. control and both
imipramine-treated samples at all time points.
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Contribution of Phosphonucleotides or Phosphate
Groups to Mg21 Fluxes
MG21 TRANSPORT IN LIVER PLASMA MEMBRANE VESICLES
C1003
Therefore, LPM were loaded with 20 mM Mg21 in the
absence or presence of 5 mM concentrations of different
phosphonucleotides or phosphate. The results in Fig.
12 show the effects of Na1 or Ca21 stimulation on Mg21
extrusion within 2 min after addition under a variety of
phosphate loading conditions. Under all loading condi-
tions in the absence of cation stimulation (control),
LPM tightly retain trapped intravesicular Mg21 (Fig.
12). Both in the absence and in the presence of intravesicular ATP, the addition of Na1 or Ca21 induces
quantitatively similar Mg21 effluxes from the vesicles
(Fig. 12). LPM loaded with Mg21 in the presence of 5
mM Tris-Pi mobilize an amount of Mg21 comparable to
that of vesicles loaded solely with Mg21 after the
addition of Na1 but mobilize a much larger amount of
Mg21 after Ca21 addition. Interestingly, both the Na1and Ca21-induced Mg21 releases are completely inhibited in vesicles loaded with 5 mM ATPgS. Also, in the
presence of ATP or Pi, amiloride (1 mM), quinidine, or
imipramine (200 µM) inhibits both the Na1- and Ca21mediated Mg21 effluxes (not shown). Lastly, qualita-
Fig. 9. Influx of Mg21 (A) or Sr21 (B) into 20 mM NaCl-loaded LPM
vesicles. Accumulation of Mg21 (A) or Sr21 (B) into LPM vesicles
loaded with 20 mM NaCl and resuspended in presence of 2 mM NaCl
in extravesicular solution. Experimental conditions were similar to
those for Fig. 5. At indicated time points after addition of indicated
concentrations of Mg21 or Sr21, aliquots of incubation mixture were
withdrawn and sedimented through an oil layer as described in
MATERIALS AND METHODS. Acid extract of pellets was assayed for Mg21
or Sr21 content by AAS. Initial Na1 content before ion addition was
1,995.1 nmol Na1/mg protein. One experiment typical of four similar
experiments for both experimental conditions (Mg21 and Sr21 ) is
shown.
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Fig. 10. Na1 efflux from 20 mM NaCl-loaded vesicles. Efflux of Na1
from LPM loaded with 20 mM NaCl, incubated in absence of external
NaCl, and stimulated by 5 mM Mg21 or 5 mM Ca21. Experimental
conditions and Mg21 determinations were as for Fig. 2. Initial Na1
content before ion addition was 2,625.4 6 114.2 nmol Na1/mg
protein. Data are means 6 SE of 3 preparations. ANOVA and Tukey’s
test for multiple comparisons were performed at all time points. * P ,
0.05 vs. control and 5 mM CaCl2.
C1004
MG21 TRANSPORT IN LIVER PLASMA MEMBRANE VESICLES
DISCUSSION
Fig. 11. Bidirectionality of Na1, but not Ca21, transport mechanism.
Net Na1 or Ca21 efflux was measured in vesicles loaded with 20 mM
NaCl and resuspended in presence of 20 mM NaCl outside (A) or
vesicles loaded with 10 mM CaCl2 and resuspended in presence of 10
mM CaCl2 outside (B). After 1 min of equilibration, 5 mM MgCl2 was
added to incubation system. Na1 or Ca21 content retained in vesicles
was assayed by AAS as for Fig. 5. Initial ion contents before ion
addition were 1,671.3 6 67.5 and 585.7 6 68.1 nmol Na1 or Ca21/mg
protein, respectively. Data are means 6 SE of 3 preparations. ANOVA
and Tukey’s test for multiple comparisons were performed at all time
points. * P , 0.05 vs. control.
tively similar Mg21 fluxes are observed in vesicles in
which ATP is replaced with an equivalent concentration of ADP (disodium or Tris salt), GTP (lithium salt),
or GDP (lithium salt) (data not shown).
Fig. 12. Loading with phosphonucleotides and phosphate moieties.
Net change in Mg21 content of LPM loaded with MgCl2 (20 mM) and
indicated phosphonucleotides (5 mM), incubated in presence of 2 mM
Mg21 in extravesicular medium. Comparison of stimulatory effect on
differently loaded LPM of indicated concentrations of Na1 or Ca21 at
2 min. Amount of Mg21 retained in vesicles was assayed as for Fig. 5.
Initial Mg21 content before ion addition was 292.5 6 26.2 nmol
Mg21/mg protein. Data are means 6 SE of 10, 16, 3, and 3 preparations for control (i.e., only Mg21 ), ATP, Tris-Pi, and adenosine
58-O-(3-thiotriphosphate) (ATPgS) loading conditions, respectively.
ANOVA and Tukey’s test for multiple comparisons were performed at
all time points. Stimulation of Mg21 efflux by Na1 or Ca21 is
significantly different under loading with 20 mM Mg21 1 5 mM
ATPgS (* P , 0.05). In presence of 20 mM Mg21 1 5 mM TRIS-Pi there
is a significant difference upon stimulation by Ca21 compared with
other loading conditions (** P , 0.05).
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After hormonal stimulation, large fluxes of Mg21
across the cell have been observed in hepatocytes (28)
and other cell types (15, 23, 29, 31, 38). Depending on
the signal transduction pathway activated, within 5
min of stimulation hepatocytes release or accumulate
an amount of Mg21 equivalent to 5–10% of their total
cellular Mg21, whereas cytosolic free Mg21 undergoes
relatively little change (28, 30). Based on the total Mg21
content (15–20 mM, depending on the cell type; see Ref.
32 for a review), the amount of Mg21 translocated
across the plasma membrane is very large, implying
the presence of transport mechanisms that are very
abundant and/or operate with a rapid turnover.
During the last two decades, a large body of evidence
has been obtained in molluscan (2) and in mammalian
(21, 40) cells that indicates that Mg21 fluxes require a
physiological concentration of extracellular Na1 and
are inhibitable by amiloride (13, 38, 40) or by other
agents that affect Na1 transport, such as imipramine or
quinidine (7). Although these studies strongly support
the operation of a Na1/Mg21 antiporter, the require2
ment of additional ions, including Ca21, HCO2
3 , or Cl ,
has been observed in several cell types (14, 26, 30).
MG21 TRANSPORT IN LIVER PLASMA MEMBRANE VESICLES
Basic Properties of Mg21 Transport
Mg21 extrusion and entry. Tightly sealed LPM suspended in the absence of extravesicular Na1 or Ca21
retained intravesicular Mg21 over a 10-min incubation
period. Based on the reported values of intravesicular
water space, the amount of total Mg21 (or Na1 ) trapped
within the vesicles is three to four times larger than
that expected based on the value of the concentration of
cation used for loading. This difference can be accounted for by aspecific binding of Mg21 or Na1 to
aspecific binding sites (33). The addition of Na1 or Ca21
induces a rapid total Mg21 efflux from the vesicles. The
amount of Mg21 releasable within 2 min by micromolar
concentrations of Ca21 ranges between 30 and 50% of
the total trapped Mg21, whereas the amount of Mg21
releasable by Na1, at a concentration of 25 mM Na1 or
higher, is ,35%. These results differ from those obtained in isolated hepatocytes, in which no Mg21 is
released in the presence of a physiological concentration of Na1 or Ca21 in the extracellular space. In the
intact hepatocyte, a smaller percentage of the total
Mg21 is released under conditions in which cellular
cAMP increases (28), whereas a Mg21 uptake of similar
magnitude is induced by activation of protein kinase C
(30). The difference in the amount of Mg21 mobilized
from these two experimental systems does not account
for differences in Mg21 gradients across the plasma
membrane of dispersed cells or LPM and may be
related to regulatory mechanisms that physiologically
modulate Mg21 efflux in intact cells. Hence, LPM
provide a system in which basic kinetic properties of
Mg21 transport can be better defined, since plasma
membrane Mg21 transport is effectively ‘‘uncoupled’’
from cellular regulatory mechanisms. Consistent with
this interpretation, it is possible that the reduced
effectiveness of Na1 in mobilizing Mg21 from preloaded
vesicles (Fig. 3) in comparison with LPM loaded with
Mg21 after isolation (Fig. 2A) can be attributed to a
partial retention of regulatory mechanism(s) within the
vesicles, to the residual presence of cations or metabo-
lites in the LPM, or to a different distribution of vesicle
orientation. Preloaded vesicles provide a tool in which
the Ca21-dependent mechanism can be observed independently of the Na1-induced Mg21 extrusion. Last,
because both basolateral and apical membranes are
present in the LPM population (see MATERIALS AND
METHODS for details), the possibilities arise that there
are two distinct membrane populations rendering the
distinct transport mechanisms or that the transporters
are ubiquitously distributed.
Direction of Mg21 fluxes. Na1 can induce Mg21 release
from Mg21-loaded vesicles. Under appropriate conditions, this transport is fully reversible, as extravesicular Mg21 can be accumulated in the vesicle in exchange
for trapped Na1. Ca21 can also induce Mg21 extrusion
from Mg21-loaded vesicles in the absence of Na1, but
Mg21 cannot elicit an extrusion of Ca21 from Ca21loaded vesicles (Fig. 10B). Hence, under these conditions, the operation of the Na1/Mg21 antiporter appears
to be bidirectional, whereas that of the Ca21/Mg21
exchanger is unidirectional.
The rate of Mg21 flux is very rapid. Within the first
observation point (1 min), 344.1 6 158.8 and 217.1 6
18.2 nmol Mg21/mg protein are released following
stimulation with 25 mM Na1 and 50 µM Ca21, respectively. Under the assumption of a surface area of 2.5
µm2/mg protein (Table 2), fluxes of 137.6 and 86.8 nmol
Mg21 ·µm22 ·min21 in exchange for Na1 and Ca21, respectively, can be calculated. These fluxes are two to four
orders of magnitude greater than those reported in
intact hepatocytes for other ions (3, 34). Although very
high, these fluxes are underestimated severalfold due
to the inability to time resolve the initial rate.
Mg21 efflux is the result of true transport. Mg21 efflux
is not the result of a passive leakage from Mg21-loaded
vesicles, for the following reasons: 1) unstimulated
LPM do not release entrapped Mg21 over 10 min of
incubation even in the presence of a gradient across the
plasma membrane (Fig. 2); 2) Na1 or Ca21 can mobilize
Mg21 from vesicles under conditions in which the
concentrations of Mg21 inside and outside the vesicles
are similar; and 3) both Na1- and Ca21-induced Mg21
effluxes are inhibited by various agents. Mg21 efflux is
not the result of a sudden change in osmolarity. A
change in osmolarity may be significant upon addition
of large Na1 concentrations but is negligible upon
addition of micromolar concentrations of Ca21. Furthermore, the addition of 100 mM sucrose (Fig. 2A), 50 mM
LiCl, or 50 mM KCl (Fig. 7) fails to replace Na1 or Ca21
in mobilizing Mg21.
Mg21 efflux is not the result of displacement of Mg21
loosely bound to extravesicular structures, for the
following reasons: 1) the rate of Mg21 efflux is better
time resolved by decreasing the temperature of incubation; 2) both Na1- and Ca21-stimulated Mg21 effluxes
can still be measured in vesicles when the extra- and
intravesicular Mg21 concentrations are equal (Fig. 6);
3) the cation ionophore A-23187 (Fig. 5) equilibrates
Mg21 from the same pool mobilizable by Na1 or Ca21;
and 4) Mg21 extrusion does not occur when Na1 is
replaced by KCl or LiCl (Fig. 7) and is markedly
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These findings raise the question of the number and
identity of Mg21 transport mechanisms in the cell
plasma membrane. Furthermore, fundamental questions about the molecular identity of these transporters, their kinetics and operative parameters, and their
overall regulation still await answers.
The present study was undertaken to characterize
kinetically the operation of Mg21 transport system(s) in
plasma membranes from rat hepatocytes, where Mg21
transport is particularly active, and to determine
whether Mg21 uptake and/or release operates through
one or several mechanisms. Such a study in intact cells
is complicated by the fact that intracellular organelles
play a major role in cellular Mg21 homeostasis (11, 32)
and by the presence of intracellular ions, metabolites,
and signaling or controlling machinery. Hence, a simpler system consisting of sealed, purified, and wellcharacterized rat LPM was used. This system has the
advantage of providing single compartments in which
the intravesicular milieu can be designed, modified,
and controlled.
C1005
C1006
MG21 TRANSPORT IN LIVER PLASMA MEMBRANE VESICLES
Na1-Dependent Mechanism
Because intracellular Mg21 is well below its electrochemical equilibrium in vivo (8), there is general consensus that Mg21 transport across the plasma membrane
could utilize the driving force of other cations, in
particular Na1 (8). The operation of Na1-dependent
Mg21 transport mechanisms has been reported by
several laboratories (13, 21, 41) with kinetic characteristics and parameters that vary considerably among
different cell types. For example, the operation of a Na1/
Mg21 antiporter has been observed in chicken erythrocytes (13) as well as in human red blood cells (7, 21) and
in rat hepatocytes (30) and cardiac myocytes (29). Yet
the stoichiometry of operation of this antiporter appears to vary in different cell types. In fact, an electroneutral exchange of 2 Na1 in for 1 Mg21 out has been
reported in chicken and turkey erythrocytes (13) but
not in human or other mammalian erythrocytes (21). It
is also controversial whether this antiporter requires
ATP to operate.
Irrespective of the exchange ratio, the Na1/Mg21
antiporter is inhibited in intact cells by amiloride (13,
21, 38), imipramine, or quinidine (7) but not by ouabain
(7), furosemide (7), bumetanide (7), 4,48-dinitrostilbene2,28-disulfonic acid, or DIDS (7). Consistent with these
observations, the data reported here for LPM vesicles
indicate the presence of a Na1/Mg21 exchanger that is
inhibited by amiloride, imipramine, and quinidine to
approximately the same degree, irrespective of different loading or stimulatory conditions. The amiloride
derivatives 5-(N,N-hexamethylene)-amiloride and
5-(N,N-dimethyl)-amiloride (41), which are more specific and more effective at inhibiting other Na1 transport mechanisms at lower concentrations than amiloride, proved to be ineffective on LPM up to 200 µM, a
finding consistent with reports by Wolf et al. (41) and by
Gunzel and Schlue (16). Under our experimental conditions, 100 µM amiloride inhibits both the Na1- and the
Ca21-dependent mechanisms by 50% or more (Table 3).
Because amiloride is a nonspecific inhibitor of Na1
transport mechanisms, its effect on Mg21 fluxes may
possibly be indirect, i.e., through inhibition of other
transport mechanisms such as the Na1/H1 exchanger.
This may explain why the degree of inhibition varies
amply (50–90%) in our experiments under conditions
in which the ionic composition of the extra- and intravesicular milieu differs significantly. Comparing different
sets of experimental data results in a variety of stoichiometric exchange ratios, suggesting the involvement of
additional ions in compensating charge differential
directly or indirectly.
As for the specificity of the exchanger, the addition of
Na1 or Ca21 mobilizes quantitatively similar amounts
of entrapped cation from Mg21- or Sr21-loaded vesicles.
In contrast, the addition of 5 mM Ca21 results in
virtually no extrusion of Na1 from 20 mM Na1-loaded
LPM. This result indicates that the Na1/Ca21 exchanger, which has negligible activity in hepatocytes
(19), is not involved in the observed Mg21 transport.
Furthermore, the requirement for Na1 is very stringent
because equimolar concentrations of Li1 or K1 do not
induce a significant Mg21 efflux from LPM (Fig. 7).
Ca21-Dependent Mechanism
Several experimental reports from this (29) and
other (8) laboratories indicate that Mg21 transport
across the plasma membranes of intact cells requires a
physiological concentration of extracellular Ca21. The
present study suggests the presence of a Ca21/Mg21
exchange mechanism that operates at very low external Ca21 concentrations (µM range). Compared with
the Na1 requirement, the Ca21 requirement is far less
specific, since a Mg21 efflux comparable to that prompted
by Ca21 could also be induced by addition of equimolar
concentrations of other divalent cations (Ca21 : Co21 5
Mn21 . Sr21 . Ba21 . Cu21 : Cd21 ), as preliminary
experiments indicate (data not shown).
At variance with what has been observed in intact
cells (29), the Ca21 channel inhibitors nifedipine and
verapamil are ineffective at inhibiting the extrusion of
Ca21-dependent Mg21 efflux, whereas amiloride, imipramine, and quinidine all inhibit the process to various
extents. Although the range of Ca21 concentrations
tested (25–500 µM) is below the physiological extracellular Ca21 concentration, the Mg21/Ca21 exchanger
appears to operate effectively at these very low concentrations. Thus it can be hypothesized that in intact
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inhibited by amiloride, quinidine, or imipramine (Table
3 and Fig. 8).
Sr21 can effectively replace Mg21 as the transported
cation. When Sr21-loaded vesicles are stimulated by
Na1 or Ca21, the net Sr21 efflux is quantitatively
similar to the efflux of Mg21 observed under similar
experimental conditions. Moreover, Sr21 efflux is inhibited by the same agents that inhibit Mg21 fluxes.
Because 28Mg21 is difficult to obtain and use, whereas
85Sr21 is a readily available isotope, the ability of Sr21 to
substitute for Mg21 will provide a more useful radioactive tracer than 28Mg21 to monitor the movement of
Mg21 in different tissues, cells, or organelles.
Mg21 efflux from vesicles loaded with Pi or phosphonucleotides. The relevance of the intravesicular content
of ATP for the Na1- and/or Ca21-dependent Mg21 efflux
mechanisms was investigated by adding a ‘‘cytosol-like’’
concentration of ATP to LPM vesicles at the time of
loading. The loading of LPM vesicles with 20 mM Mg21
plus 5 mM ATP did not significantly modify the amplitude of Mg21 extrusion induced by Na1 or Ca21. The
ineffectiveness of ATP to modify Mg21 movement and
the inability of 100 µM or 1 mM vanadate to affect Mg21
transport (not shown) suggest that a phosphorylation
event or the modulation of Mg21 transport mechanism(s) via ATPases is not an absolute requirement for
Mg21 transport. Interestingly, the presence of ATPgS
fully inhibits Mg21 release by Ca21 or Na1, whereas
Tris-Pi enhances only the Ca21-activated Mg21 extrusion by 50%. Taken together, these data suggest an
exchanger whose operation is enhanced by the presence
of Pi only when Mg21 is exchanged for Ca21.
MG21 TRANSPORT IN LIVER PLASMA MEMBRANE VESICLES
We thank Dr. Meredith Bond and John Gabrovsek (Electron
Microscopy Core Facility, Cleveland Clinic Foundation, Cleveland,
OH) for assistance with the electron micrographs and Dr. Carlos
Obejero-Paz for assistance in statistically evaluating the data obtained from the electron micrographs.
This work was supported by National Heart, Lung, and Blood
Institute Grant HL-18708.
Address for reprint requests: A. Scarpa, Dept. of Physiology and
Biophysics, School of Medicine, Case Western Reserve University,
10900 Euclid Ave., Cleveland, OH 44106-4970.
Received 10 February 1998; accepted in final form 18 June 1998.
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