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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The plasma
membrane of mammalian cells possesses rapid
Mg2+ transport mechanisms. The
identity of Mg2+ transporters is
unknown, and so are their properties. In this study,
Mg2+ 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 Mg2+
with negligible leak; 2) the
addition of Na+ or
Ca2+ induces a concentration- and
temperature-dependent efflux corresponding to 30-50% of the
intravesicular Mg2+;
3) the rate of flux is very rapid
(137.6 and 86.8 nmol total Mg2+ · µm2 · min
1
after Na+ and
Ca2+ addition, respectively);
4) coaddition of maximal
concentrations of Na+ and
Ca2+ induces an additive
Mg2+ efflux;
5) both
Na+- and
Ca2+-stimulated
Mg2+ effluxes are inhibited by
amiloride, imipramine, or quinidine but not by vanadate or
Ca2+ channel blockers;
6) extracellular
Na+ or
Ca2+ can stimulate
Mg2+ efflux in the absence of
Mg2+ gradients; and
7)
Mg2+ uptake occurs in LPM loaded
with Na+ but not with
Ca2+, thus indicating that
Na+/Mg2+
but not
Ca2+/Mg2+
exchange is reversible. These data are consistent with the operation of
two distinct Mg2+ transport
mechanisms and provide new information on rates of Mg2+ transport, specificity of the
cotransported ions, and reversibility of the transport.
sodium/magnesium antiporter; calcium/magnesium antiporter; hepatocytes
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INTRODUCTION |
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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
Mg2+ (4, 10, 24) following a
variety of metabolic interventions previously led to the assumption
that Mg2+ content remains
relatively constant within the cell and to the conclusion that cellular
enzymes cannot be regulated in vivo by changes in cytosolic
Mg2+. On the basis of the findings
that large amounts of "total"
Mg2+ 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 Mg2+ transport across the
plasma membrane occurs through at least two mechanisms. A
Na+/Mg2+
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 Mg2+ for 2 Na+ in chicken erythrocytes, and
possibly in other cell types as well (13). Recently, it was reported
that Mg2+ 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
Na+ (21, 29, 30). The presence of
a Na+-independent
Mg2+ 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
Mg2+ has not been defined. In
different experimental models or conditions, cellular
Mg2+ has been shown to exchange
for extracellular Mn2+ (6),
Ca2+ (29),
HCO3 (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
Na+,
Mg2+ accumulation is inhibited in
the absence of extracellular Ca2+
(29) or in the presence of the
Ca2+ channel blockers verapamil
(26) or nifedipine. Presently, it is also unclear whether the cellular
uptake and release of Mg2+ 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 Mg2+ 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 Mg2+ transport across the plasma membrane using whole cells is complicated by the fact that intracellular organelles are also active participants in cellular Mg2+ homeostasis (32). To overcome this complication and to minimize the possible buffering of Mg2+ 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 Mg2+ across the cell membrane.
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MATERIALS AND METHODS |
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Plasma Membrane Isolation
Liver plasma membrane (LPM) vesicles were isolated according to a procedure similar to that described by Prpic et 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 Mg2+ transport was noticed between freshly isolated LPM and vesicles quickly frozen and stored in liquid nitrogen for several days.Plasma Membrane Purity and Orientation
The purity of the LPM vesicles was assessed by using 5'-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 protein
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The orientation of 20 mM
Mg2+-loaded and unloaded vesicles
was assessed by measuring the
Na+-K+-ATPase
activity according to the procedure of Ismail-Beigi and Edelman (17),
by the 5'-nucleotidase assay (27), and by
[3H]ouabain binding
experiments. In loaded vesicles, the
Na+-K+-ATPase
activity (in µmol
Pi · mg
protein1 · min
1)
was 0.043 ± 0.006 in the absence of Triton and
increased to 0.35 ± 0.082 in the presence of Triton
(Table 1). When loaded vesicles were assessed for
5'-nucleotidase, the activity (in µmol Pi · mg
protein
1 · min
1)
was 0.55 ± 0.112 in the absence of Triton and 0.605 ± 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 ± 3 and 87 ± 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
Mg2+, 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
Mg2+ plus 5 mM ATP demonstrated an
inside-in orientation >80%.
For the [3H]ouabain binding procedure, after the loading with Mg2+, 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 Na+-K+-ATPase was obtained. Similarly, in [3H]ouabain binding experiments, the inside-in percentage was determined to be 72 ± 5% for 20 mM Mg2+-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 Na+-K+-ATPase and 5'-nucleotidase activity, respectively. Because the Na+-K+-ATPase is prevalent in basolateral membranes (42) whereas the 5'-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, 45Ca2+ transport experiments were performed in both unloaded and loaded vesicles, as previously described by Prpic et al. (25). Unloaded LPM, but not Mg2+-loaded LPM, displayed a Ca2+ uptake similar to that reported by Prpic et al. (25) (data not shown). In contrast, LPM loaded with 20 mM MgCl2 plus 45Ca2+ and 10 mM ATP actively extruded 45Ca2+ (data not shown). Under both conditions, Ca2+ 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 Na+, Mg2+, Ca2+, and K+ by atomic absorbance spectroscopy (AAS), using a Perkin-Elmer 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 determined from an electron micrograph of a typical preparation of MgCl2-loaded LPM (×9,000 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.
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In the second technique, the intravesicular volume of LPM loaded with 20 mM Mg2+ 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 Mg2+ (2-4 mg protein/ml) were equilibrated for 1-10 min in incubation medium (pH 7.4, 37°C) labeled with [14C]polydextran and 3H2O (8 µCi/ml of each radionuclide). Aliquots (500 µl) were 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).
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 Na+, Sr2+, or Ca2+, 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 protocol. LPM vesicles were loaded with various concentrations of Mg2+ (2, 5, 10, or 20 mM Mg2+). Because total Mg2+ 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 Mg2+-containing loading medium with 5 mM ATP (either Tris or disodium salt) or, alternatively, with adenosine 5'-O(3-thiotriphosphate) (ATPLoading 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. Mg2+ (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.Mg2+ Measurement
Mg2+ fluxes or contents were measured by AAS. Hence, in all experiments in vesicle pellets or supernatants, total (rather than free) Mg2+ was measured. The Mg2+-loaded vesicles were incubated in the incubation medium at a final concentration of 250 µg protein/ml. For the supernatant measurements, the loaded LPM vesicles were resuspended in Mg2+-free incubation medium. This was necessary because large extravesicular Mg2+ concentrations interfere with the measurement of Mg2+ content in the supernatant by AAS. For the pellet measurement, to minimize the leakage of the entrapped Mg2+ 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 Mg2+ 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. Mg2+ content was measured in the supernatants by AAS. As for the Mg2+ 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 Mg2+. The supernatant and oil layer were removed by vacuum suction, and the pellet was digested overnight in 500 µl 10% HNO3. The Mg2+ content of the vesicles was measured by AAS in the acid extracts using Mg2+ standards in identical acid concentration. Similar procedures were also used for Na+- or Ca2+-loaded LPM.
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 ± 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 ![]() |
RESULTS |
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Electron Micrograph of Purified LPM
Figure 1 shows an electron micrograph of a typical preparation of LPM (×9,000 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 Mg2+-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 values are summarized in Table 2 and were used to calculate Mg2+ fluxes.
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Dose Response of Na+- and Ca2+-Induced Mg2+ 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 Na+ or Ca2+ release negligible Mg2+ over several minutes of incubation (control). The addition of NaCl (Fig. 2A) or CaCl2 (Fig. 2B) to the incubation medium prompts a sizable Mg2+ efflux from the vesicles in a dose-dependent fashion. Irrespective of the concentration of NaCl or CaCl2 used, Mg2+ efflux at 37°C is already complete within the first period of observation, 2 min after NaCl or CaCl2 addition. The amount of Mg2+ released at this point is not limited by an equilibration of Mg2+ gradients across the plasma membrane, as additional Mg2+ can be mobilized from the vesicles by the addition of a divalent cation ionophore (see Fig. 5).
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At 2 min, the addition of 10 or 20 mM NaCl induces the extrusion of 12.7 ± 3.9 and 37.6 ± 6.4 nmol Mg2+/mg protein, respectively (n = 37). The maximum Mg2+ 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 Mg2+ efflux (Fig. 2A). In addition, Fig. 2A shows that the addition of 100 mM sucrose does not induce a Mg2+ efflux, suggesting that the mobilization of Mg2+ by 50 mM NaCl is not caused by a nonspecific rapid osmotic mismatch.
Figure 2B shows that the concentrations of CaCl2 able to induce Mg2+ extrusion are far lower than those of NaCl. In fact, Mg2+ extrusion is already detectable following the addition of 25 µM Ca2+ (44.9 ± 8.4 nmol Mg2+/mg protein) and is maximal when 500 µM CaCl2 is added to the system (160.5 ± 11.6 nmol Mg2+/mg protein) (n = 8). Under these conditions, the amount of Mg2+ released by Ca2+ is almost double that mobilized by maximal concentrations of Na+. Higher CaCl2 concentrations (in the millimolar range) do not result in larger Mg2+ efflux (not shown). Figure 2B also shows that the coaddition to LPM vesicles of 500 µM Ca2+ and 25 mM Na+, each individually producing maximal Mg2+ release, results in an additive amount of Mg2+ extruded. Specifically, the net Mg2+ extrusion induced by 25 mM Na+ plus 500 µM Ca2+ is 267.7 ± 17.0, compared with the Mg2+ efflux induced by Na+ or Ca2+ alone of 92.2 ± 13.8 and 160.5 ± 11.6 nmol Mg2+/mg protein, respectively.
The results reported in Fig. 2, A and B, reflect an increase in extravesicular Mg2+ content (supernatant experiments). However, a quantitatively similar decrease in Mg2+ content retained within the vesicles could be observed in pellet experiments (see Fig. 6). Table 3 shows that the Mg2+ extrusion activated by Na+ or Ca2+ is decreased by 50% in the presence of 100 µM amiloride, and Ca2+-induced Mg2+ extrusion is almost completely absent in the presence of 1 mM amiloride. Such a high concentration of amiloride has been shown to effectively inhibit Mg2+ extrusion via the putative Na+/Mg2+ 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 Mg2+ extrusion mechanism (7). However, the coaddition of amiloride and quinidine or imipramine failed to elicit additional inhibition of the Na+-dependent Mg2+ release (not shown). In contrast, the Ca2+ channel blockers nifedipine and verapamil, as well as inhibitors of the ER Ca2+ leak mechanism and the mitochondrial Ca2+ uniport, neomycin and ruthenium red, were ineffective at inhibiting Mg2+ extrusion from LPM (not shown).
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Mg2+ Efflux in Preloaded Vesicles
The operation of the Na+- and Ca2+-dependent Mg2+ extrusion mechanisms was also investigated in LPM vesicles preloaded with 10 mM Mg2+. The major difference in this preparation is that the LPM were loaded during isolation with a concentration of 10 mM Mg2+, 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 Mg2+ is even more evident. Figure 3 shows that in these vesicles 50 µM CaCl2 prompts a Mg2+ 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 Ca2+-mediated Mg2+ efflux is less (see Table 3).
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Resolution of Mg2+ Kinetics by Decreasing Temperature of Incubation Medium
Figure 4 shows the temperature dependence of Mg2+ 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 incubation temperature to 15 or 20°C, a progressive decrease in Mg2+ 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).
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Effects of the Ionophore A-23187
To determine the total amount of Mg2+ mobilizable from LPM vesicles, A-23187 (1 µg/ml) was used either in the absence of Na+ or after stimulation by Na+ (Fig. 5). As Fig. 5 shows, ~35% of the entrapped Mg2+ 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 Mg2+ after Na+ stimulation. The addition of A-23187 alone rapidly mobilizes ~70% of the intravesicular Mg2+ content. Thus ionophore by itself releases a total amount of Mg2+ comparable to that mobilized by Na+ plus the ionophore. Approximately 30% of the total Mg2+ is retained in LPM after addition of the ionophore. Based on the intravesicular water space, the total amount of Mg2+ releasable under those conditions is two- to threefold greater than the amount of "free" Mg2+ expected in the vesicles. This should be the result of dissociation between aspecifically bound Mg2+ and free Mg2+, as more free Mg2+ is released by the ionophore. Such a dissociation should, in principle, be facilitated by the intravesicular decrease in pH resulting from exchange of 1 Mg2+ out for 2 H+ in, facilitated by A-23187. Similar results were also obtained when Ca2+ was used instead of Na+ (not shown).
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Mg2+ Transport Is Not Due to Displacement of Bound Mg2+
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 Mg2+ concentrations were equal). Under these conditions, the Mg2+ content remaining in the pellet was measured (see MATERIALS AND METHODS). Therefore, Fig. 6 represents the amount of Mg2+ released from LPM as decreasing values. In vesicles loaded with magnesium gluconate, the addition of 25 mM sodium isethionate mobilizes 344.1 ± 158.8 nmol Mg2+/mg protein, whereas 50 µM calcium gluconate induces an efflux of 217.1 ± 18.2 nmol Mg2+/mg protein (Fig. 6) within 1 min after addition. Also, in this system, 1 mM amiloride inhibits Na+- (Fig. 6) and Ca2+-dependent (not shown) Mg2+ efflux. It is noteworthy that in the presence of 20 mM extravesicular Mg2+ the amount of Mg2+ 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 Mg2+ (Fig. 2). Consistent with this interpretation, the amount of Mg2+ trapped within the vesicles resuspended in the absence of extracellular Mg2+ (Fig. 2) is approximately twofold less than LPM resuspended in the presence of 20 mM Mg2+.
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Mg2+ Efflux Is Specific for the Monovalent Cation Na+
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 Na+/Mg2+ antiporter for Na+. Neither Li+ nor K+ is able to elicit a Mg2+ efflux under these conditions.
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Similarities Between Mg2+ and Sr2+ Release From Isolated LPM
Sr2+ has been shown to act as a substitute for Mg2+ in a variety of cell transport systems (29). Therefore, LPM vesicles were loaded with 20 mM Sr2+ (Fig. 8), and intravesicular Sr2+ content was measured using AAS. As Fig. 8 shows, the net loss of Sr2+ from the vesicles is quantitatively comparable to the net Mg2+ increase in the extravesicular medium (Fig. 2). For example, 25 mM NaCl and 50 µM Ca2+ induce an efflux of 95.9 ± 7.8 and 74.0 ± 31.3 nmol Sr2+/mg protein, respectively. Figure 8 also shows the inhibitory effect of 200 µM imipramine on both the Na+- and Ca2+-dependent Mg2+ effluxes. Quantitatively similar data were obtained using SrCl2 labeled with 85Sr2+ (data not shown).
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Bidirectionality of Mg2+ Transport Mechanism(s)
Na+/Mg2+ 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 Mg2+ was added at the concentrations of MgCl2 indicated in Fig. 9. Under these experimental conditions, a marked extrusion of Na+ from the vesicles is also observed (data not shown). Figure 9B shows that, under the same conditions, Sr2+ is also accumulated in a quantitatively and kinetically similar fashion, indicating that Sr2+ could replace Mg2+ for Na+/Mg2+ exchange in either direction. The addition of Mg2+ or Sr2+ induces a large uptake that is complete within 1 min after addition.
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Figure 10 shows the result of an experiment in which the effect of Mg2+ or Ca2+ was measured in Na+-loaded vesicles resuspended in the absence of Na+. The addition of 5 mM Mg2+ to the extravesicular compartment prompts a net Na+ efflux of 235.3 ± 8.7 nmol Na+/mg protein (Fig. 10), an amount approximately double that of Mg2+ released from Mg2+-loaded vesicles stimulated by 25 mM NaCl (Fig. 2A; 92.8 ± 13.8 nmol Mg2+/mg protein). In contrast, under identical conditions, 5 mM CaCl2 fails to induce Na+ extrusion. The small uptake of Na+ observable under control and Ca2+ conditions is likely due to the carryover of Na+ bound to the external face of the vesicle.
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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 Mg2+ can induce the release of Na+ but not of Ca2+ from the vesicles, suggesting that the Na+/Mg2+ antiporter but not the Ca2+/Mg2+ mechanism can operate in either direction.
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Contribution of Phosphonucleotides or Phosphate Groups to Mg2+ Fluxes
Several laboratories have reported the presence of an ATP-dependent Mg2+ 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). Therefore, LPM were loaded with 20 mM Mg2+ in the absence or presence of 5 mM concentrations of different phosphonucleotides or phosphate. The results in Fig. 12 show the effects of Na+ or Ca2+ stimulation on Mg2+ extrusion within 2 min after addition under a variety of phosphate loading conditions. Under all loading conditions in the absence of cation stimulation (control), LPM tightly retain trapped intravesicular Mg2+ (Fig. 12). Both in the absence and in the presence of intravesicular ATP, the addition of Na+ or Ca2+ induces quantitatively similar Mg2+ effluxes from the vesicles (Fig. 12). LPM loaded with Mg2+ in the presence of 5 mM Tris-Pi mobilize an amount of Mg2+ comparable to that of vesicles loaded solely with Mg2+ after the addition of Na+ but mobilize a much larger amount of Mg2+ after Ca2+ addition. Interestingly, both the Na+- and Ca2+-induced Mg2+ releases are completely inhibited in vesicles loaded with 5 mM ATP
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DISCUSSION |
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After hormonal stimulation, large fluxes of Mg2+ 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 Mg2+ equivalent to 5-10% of their total cellular Mg2+, whereas cytosolic free Mg2+ undergoes relatively little change (28, 30). Based on the total Mg2+ content (15-20 mM, depending on the cell type; see Ref. 32 for a review), the amount of Mg2+ 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
Mg2+ fluxes require a
physiological concentration of extracellular Na+ and are inhibitable by
amiloride (13, 38, 40) or by other agents that affect
Na+ transport, such as imipramine
or quinidine (7). Although these studies strongly support the operation
of a
Na+/Mg2+
antiporter, the requirement of additional ions, including
Ca2+,
HCO3, or
Cl
, has been observed in
several cell types (14, 26, 30). These findings raise the question of
the number and identity of Mg2+
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 Mg2+ transport system(s) in plasma membranes from rat hepatocytes, where Mg2+ transport is particularly active, and to determine whether Mg2+ 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 Mg2+ 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 well-characterized rat LPM was used. This system has the advantage of providing single compartments in which the intravesicular milieu can be designed, modified, and controlled.
Basic Properties of Mg2+ Transport
Mg2+ extrusion and entry. Tightly sealed LPM suspended in the absence of extravesicular Na+ or Ca2+ retained intravesicular Mg2+ over a 10-min incubation period. Based on the reported values of intravesicular water space, the amount of total Mg2+ (or Na+) 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 Mg2+ or Na+ to aspecific binding sites (33). The addition of Na+ or Ca2+ induces a rapid total Mg2+ efflux from the vesicles. The amount of Mg2+ releasable within 2 min by micromolar concentrations of Ca2+ ranges between 30 and 50% of the total trapped Mg2+, whereas the amount of Mg2+ releasable by Na+, at a concentration of 25 mM Na+ or higher, is ~35%. These results differ from those obtained in isolated hepatocytes, in which no Mg2+ is released in the presence of a physiological concentration of Na+ or Ca2+ in the extracellular space. In the intact hepatocyte, a smaller percentage of the total Mg2+ is released under conditions in which cellular cAMP increases (28), whereas a Mg2+ uptake of similar magnitude is induced by activation of protein kinase C (30). The difference in the amount of Mg2+ mobilized from these two experimental systems does not account for differences in Mg2+ gradients across the plasma membrane of dispersed cells or LPM and may be related to regulatory mechanisms that physiologically modulate Mg2+ efflux in intact cells. Hence, LPM provide a system in which basic kinetic properties of Mg2+ transport can be better defined, since plasma membrane Mg2+ transport is effectively "uncoupled" from cellular regulatory mechanisms. Consistent with this interpretation, it is possible that the reduced effectiveness of Na+ in mobilizing Mg2+ from preloaded vesicles (Fig. 3) in comparison with LPM loaded with Mg2+ 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 metabolites in the LPM, or to a different distribution of vesicle orientation. Preloaded vesicles provide a tool in which the Ca2+-dependent mechanism can be observed independently of the Na+-induced Mg2+ 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 Mg2+ fluxes. Na+ can induce Mg2+ release from Mg2+-loaded vesicles. Under appropriate conditions, this transport is fully reversible, as extravesicular Mg2+ can be accumulated in the vesicle in exchange for trapped Na+. Ca2+ can also induce Mg2+ extrusion from Mg2+-loaded vesicles in the absence of Na+, but Mg2+ cannot elicit an extrusion of Ca2+ from Ca2+-loaded vesicles (Fig. 10B). Hence, under these conditions, the operation of the Na+/Mg2+ antiporter appears to be bidirectional, whereas that of the Ca2+/Mg2+ exchanger is unidirectional.
The rate of
Mg2+ flux is
very rapid.
Within the first observation point (1 min), 344.1 ± 158.8 and 217.1 ± 18.2 nmol Mg2+/mg protein are released following
stimulation with 25 mM Na+ and 50 µM Ca2+,
respectively. Under the assumption of a surface area of 2.5 µm2/mg protein (Table 2), fluxes
of 137.6 and 86.8 nmol
Mg2+ · µm2 · min
1
in exchange for Na+ and
Ca2+, 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.
Mg2+ efflux is the result of true transport. Mg2+ efflux is not the result of a passive leakage from Mg2+-loaded vesicles, for the following reasons: 1) unstimulated LPM do not release entrapped Mg2+ over 10 min of incubation even in the presence of a gradient across the plasma membrane (Fig. 2); 2) Na+ or Ca2+ can mobilize Mg2+ from vesicles under conditions in which the concentrations of Mg2+ inside and outside the vesicles are similar; and 3) both Na+- and Ca2+-induced Mg2+ effluxes are inhibited by various agents. Mg2+ efflux is not the result of a sudden change in osmolarity. A change in osmolarity may be significant upon addition of large Na+ concentrations but is negligible upon addition of micromolar concentrations of Ca2+. Furthermore, the addition of 100 mM sucrose (Fig. 2A), 50 mM LiCl, or 50 mM KCl (Fig. 7) fails to replace Na+ or Ca2+ in mobilizing Mg2+.
Mg2+ efflux is not the result of displacement of Mg2+ loosely bound to extravesicular structures, for the following reasons: 1) the rate of Mg2+ efflux is better time resolved by decreasing the temperature of incubation; 2) both Na+- and Ca2+-stimulated Mg2+ effluxes can still be measured in vesicles when the extra- and intravesicular Mg2+ concentrations are equal (Fig. 6); 3) the cation ionophore A-23187 (Fig. 5) equilibrates Mg2+ from the same pool mobilizable by Na+ or Ca2+; and 4) Mg2+ extrusion does not occur when Na+ is replaced by KCl or LiCl (Fig. 7) and is markedly inhibited by amiloride, quinidine, or imipramine (Table 3 and Fig. 8).Sr2+ can effectively replace Mg2+ as the transported cation. When Sr2+-loaded vesicles are stimulated by Na+ or Ca2+, the net Sr2+ efflux is quantitatively similar to the efflux of Mg2+ observed under similar experimental conditions. Moreover, Sr2+ efflux is inhibited by the same agents that inhibit Mg2+ fluxes. Because 28Mg2+ is difficult to obtain and use, whereas 85Sr2+ is a readily available isotope, the ability of Sr2+ to substitute for Mg2+ will provide a more useful radioactive tracer than 28Mg2+ to monitor the movement of Mg2+ in different tissues, cells, or organelles.
Mg2+ efflux
from vesicles loaded with Pi or
phosphonucleotides.
The relevance of the intravesicular content of ATP for the
Na+- and/or
Ca2+-dependent
Mg2+ 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 Mg2+ plus 5 mM ATP did not
significantly modify the amplitude of
Mg2+ extrusion induced by
Na+ or
Ca2+. The ineffectiveness of ATP
to modify Mg2+ movement and the
inability of 100 µM or 1 mM vanadate to affect Mg2+ transport (not shown) suggest
that a phosphorylation event or the modulation of
Mg2+ transport mechanism(s) via
ATPases is not an absolute requirement for
Mg2+ transport. Interestingly, the
presence of ATPS fully inhibits Mg2+ release by
Ca2+ or
Na+, whereas
Tris-Pi enhances only the
Ca2+-activated
Mg2+ extrusion by 50%. Taken
together, these data suggest an exchanger whose operation
is enhanced by the presence of Pi
only when Mg2+ is exchanged for
Ca2+.
Na+-Dependent Mechanism
Because intracellular Mg2+ is well below its electrochemical equilibrium in vivo (8), there is general consensus that Mg2+ transport across the plasma membrane could utilize the driving force of other cations, in particular Na+ (8). The operation of Na+-dependent Mg2+ 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 Na+/Mg2+ 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 Na+ in for 1 Mg2+ 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 Na+/Mg2+ antiporter is inhibited in intact cells by amiloride (13, 21, 38), imipramine, or quinidine (7) but not by ouabain (7), furosemide (7), bumetanide (7), 4,4'-dinitrostilbene-2,2'-disulfonic acid, or DIDS (7). Consistent with these observations, the data reported here for LPM vesicles indicate the presence of a Na+/Mg2+ 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 Na+ 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 Na+- and the Ca2+-dependent mechanisms by 50% or more (Table 3). Because amiloride is a nonspecific inhibitor of Na+ transport mechanisms, its effect on Mg2+ fluxes may possibly be indirect, i.e., through inhibition of other transport mechanisms such as the Na+/H+ 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 Na+ or Ca2+ mobilizes quantitatively similar amounts of entrapped cation from Mg2+- or Sr2+-loaded vesicles. In contrast, the addition of 5 mM Ca2+ results in virtually no extrusion of Na+ from 20 mM Na+-loaded LPM. This result indicates that the Na+/Ca2+ exchanger, which has negligible activity in hepatocytes (19), is not involved in the observed Mg2+ transport. Furthermore, the requirement for Na+ is very stringent because equimolar concentrations of Li+ or K+ do not induce a significant Mg2+ efflux from LPM (Fig. 7).
Ca2+-Dependent Mechanism
Several experimental reports from this (29) and other (8) laboratories indicate that Mg2+ transport across the plasma membranes of intact cells requires a physiological concentration of extracellular Ca2+. The present study suggests the presence of a Ca2+/Mg2+ exchange mechanism that operates at very low external Ca2+ concentrations (µM range). Compared with the Na+ requirement, the Ca2+ requirement is far less specific, since a Mg2+ efflux comparable to that prompted by Ca2+ could also be induced by addition of equimolar concentrations of other divalent cations (Ca2+At variance with what has been observed in intact cells (29), the Ca2+ channel inhibitors nifedipine and verapamil are ineffective at inhibiting the extrusion of Ca2+-dependent Mg2+ efflux, whereas amiloride, imipramine, and quinidine all inhibit the process to various extents. Although the range of Ca2+ concentrations tested (25-500 µM) is below the physiological extracellular Ca2+ concentration, the Mg2+/Ca2+ exchanger appears to operate effectively at these very low concentrations. Thus it can be hypothesized that in intact hepatocytes this system represents a constitutively active background exchanger that is very tightly controlled and that a critical controlling component(s) is lost during isolation of LPM.
At present, we have not yet identified an inhibitor able to selectively block the Ca2+- or Na+-induced Mg2+ efflux. The inhibitory effect of amiloride, imipramine, and quinidine on both the Na+- and the Ca2+-activated effluxes could be consistent with an activation by Ca2+ and Na+ on the same Mg2+ transport mechanism. However, this possibility is not supported by several lines of evidence. First, the coaddition of Na+ and Ca2+ elicits an additive efflux of Mg2+ that is greater than that prompted by any concentration of either ion alone. Second, in preloaded vesicles, the Mg2+ movement elicited by Na+ is almost completely abolished, whereas the movement by Ca2+ remains quantitatively unaffected. Third, the Na+/Mg2+ exchanger operates in either direction, whereas the Ca2+/Mg2+ mechanism does not. These observations point to the presence and operation of two distinct mechanisms that favor the exchange of Mg2+ for Na+ and divalent cations, respectively.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-18708.
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. §1734 solely to indicate this fact.
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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