HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Cefaratti, C. Articles by Scarpa, A. Search for Related Content PubMed PubMed Citation Articles by Cefaratti, C. Articles by Scarpa, A. Social Bookmarking                      What's this?

J Biol Chem, Vol. 275, Issue 6, 3772-3780, February 11, 2000

Differential Localization and Operation of Distinct Mg²⁺ Transporters in Apical and Basolateral Sides of Rat Liver Plasma Membrane^*

Christie Cefaratti, Andrea Romani, and Antonio Scarpa

From the Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4970

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Upon activation of specific cell signaling, hepatocytes rapidly accumulate or release an amount of Mg²⁺ equivalent to 10% of their total Mg²⁺ content. Although it is widely accepted that Mg²⁺ efflux is Na⁺-dependent, little is known about transporter identity and the overall regulation. Even less is known about the mechanism of cellular Mg²⁺ uptake. Using sealed and right-sided rat liver plasma membrane vesicles representing either the basolateral (bLPM) or apical (aLPM) domain, it was possible to dissect three different Mg²⁺ transport mechanisms based upon specific inhibition, localization within the plasma membrane, and directionality. The bLPM possesses only one Mg²⁺ transporter, which is strictly Na⁺-dependent, bi-directional, and not inhibited by amiloride. The aLPM possesses two separate Mg²⁺ transporters. One, similar to that in the bLPM because it strictly depends on Na⁺ transport, and it can be differentiated from that of the bLPM because it is unidirectional and fully inhibited by amiloride. The second is a novel Ca²⁺/Mg²⁺ exchanger that is unidirectional and inhibited by amiloride and imipramine. Hence, the bLPM transporter may be responsible for the exchange of Mg²⁺ between hepatocytes and plasma, and vice versa, shown in livers upon specific metabolic stimulation, whereas the aLPM transporters can only extrude Mg²⁺ into the biliary tract. The dissection of these three distinct pathways and, therefore, the opportunity to study each individually will greatly facilitate further characterization of these transporters and a better understanding of Mg²⁺ homeostasis.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Magnesium, the second most abundant cation within mammalian cells, is necessary for a variety of metabolic and cellular functions (1-5). Under resting conditions, cellular-free Mg²⁺ concentration is held at 0.5-0.8 mM, well below its predicted electrochemical equilibrium, and approximately 5 mM Mg²⁺ is complexed with ATP and other metabolites (1, 5). In several tissues such as heart and liver, specific hormonal or metabolic stimulation causes, within a few minutes, a massive mobilization of cellular Mg²⁺ (6-9). Although cytosolic-free Mg²⁺ undergoes minimal changes, several intracellular signals leading to a cAMP increase induce a loss of 5-10% of total cellular Mg²⁺ (6-9). Conversely, signals activating protein kinase C result in an accumulation of Mg²⁺ and a consequent increase of total cellular Mg²⁺ by 5-10% (10).

These findings underscore the operation of a very powerful Mg²⁺ transport machinery within the plasma membranes of mammalian cells. Yet, the Mg²⁺ transporter(s) has not been isolated, and even the basic kinetic properties of Mg²⁺ cellular transport remain confusing and contradictory. For example, the dependence of Mg²⁺ release on extracellular Na⁺ has been established in invertebrate and mammalian cells (11-16) but Na⁺-independent pathways have also been reported (1, 8, 9, 17, 18). Equally contradictory are the findings of variable Na⁺/Mg²⁺ stoichiometries (1, 19), the role of trans-membrane potential, and the partial and variable effectiveness of inhibitors such as amiloride (for review see Refs. 1, 5, 18, and 20). In principle, these discrepancies could be accounted for by the co-existence of separate plasma membrane Mg²⁺ transporters each operating with different mechanisms and sensitivity to inhibitors. Hence, this work was designed to dissect putatively different Mg²⁺ transporters using an appropriate cellular model, the plasma membrane of rat hepatocytes.

In view of its size and secretory capacity, the liver should play a major role in Mg²⁺ homeostasis. Recent evidence in the literature demonstrates that the perfused liver or isolated hepatocytes can accumulate or release very large amounts of Mg²⁺ upon specific metabolic stimulation (7, 8). Recently, this laboratory was able to successfully obtain sealed liver plasma membranes (LPM)¹ to study Mg²⁺ transport across the hepatocyte cell membrane (21). This model provides well defined and controllable experimental conditions in which membrane transport can be quantitatively investigated in the absence of interfering intracellular signaling pathways or transport by organelles. Using sealed plasma membranes, we recently found that either extravesicular Na⁺ or Ca²⁺ could mobilize intravesicular Mg²⁺ (21).

A major difference in liver plasma membrane vesicles is that they release intravesicular Mg²⁺ in the presence of extravesicular Na⁺ or Ca²⁺ alone, without the need of metabolic stimulation (21) necessary in intact hepatocytes (7, 8). Under these conditions, cellular Mg²⁺ transport appears "uncoupled" and maximally operating in the absence of stimulatory cellular signals. One intriguing possibility is the presence in hepatocytes of a physiological "break" on Mg²⁺ transport, which could be transiently removed as the result of activation of cellular pathways. Such a break (i.e. protein phosphorylation and dephosphorylation) appears to be absent in LPM probably because they are devoid of ATP and signaling pathways.

Those studies also provided direct evidence that extracellular Na⁺ and Ca²⁺ elicited a larger Mg²⁺ efflux than either cation alone and suggested the presence of distinct Na⁺/Mg²⁺ and Ca²⁺/Mg²⁺ transporters (21). Unfortunately, both putative transporters seemed to be inhibited by the same inhibitor. Additionally, it was not clear whether the two transport mechanisms were localized within the same vesicle or in two distinct populations of vesicles.

The hepatocyte is a highly polarized cell with differential localization of enzymes or transporters in the two main membrane domains, the basolateral and apical plasma membrane. The present study has now taken advantage of the possibility of obtaining separate populations of sealed and properly oriented plasma membranes comprised mostly of either apical (aLPM) or basolateral (bLPM) domains.

The data presented here demonstrate that the Na⁺- dependent and Ca²⁺-dependent Mg²⁺ transporters are distinct in terms of localization within the plasma membrane, inhibition, and overall operation. The data indicate that two distinguishable Na⁺-dependent Mg²⁺ exchange mechanisms are localized to both the basolateral and apical domain, whereas a Ca²⁺/Mg²⁺ exchanger operates only in the apical domain. Such a finding should be of primary importance to kinetically characterize individual Mg²⁺ transporters and to better identify them.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Plasma Membrane Isolation-- Total LPM were isolated as described previously (21, 22). Briefly, male Harlan Sprague-Dawley rats (250-350 g) were anesthetized by intraperitoneal injection of 50 mg of pentobarbital/kg of body weight. The abdomen was open and the liver was perfused by the portal vein with 50 ml of isolation medium: 250 mM sucrose, 5 mM K-Hepes, 1 mM 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 three passes with a tight fitting pestle. The homogenate was diluted to 6% (w/v) with the same buffer and sedimented at 1400 × g for 10 min. The pellets were recovered and resuspended at 6% (final concentration) in the isolation medium by four passes with the tight fitting pestle. Percoll (Amersham Pharmacia Biotech) was added to the resuspension in the proportion of 1.4 ml of Percoll to every 10.4 ml of resuspension (21), and the LPM were isolated by centrifugation at 34,500 × g for 30 min. All operations following liver removal were carried out at 4 °C. The top fluffy layer, containing the plasma membrane vesicles, was collected, diluted 1:5 (v/v) with the incubation medium (250 mM sucrose, 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 of protein/ml in incubation medium and stored in liquid nitrogen until used. No difference in Mg²⁺ transport was noticed using freshly isolated LPM or vesicles quickly frozen and stored in liquid nitrogen for several days.

Isolation of aLPM and bLPM from Total LPM Population-- After isolation, LPM were washed with 5 volumes of 250 mM sucrose, 25 mM K-Hepes, pH 7.4 (washing and incubation buffer) and sedimented at 34,500 × g for 5 min in a SS-34 Sorvall rotor to remove residual Percoll carried over from isolation. LPM vesicles were resuspended in the same buffer to a concentration of 5 mg/ml and homogenized by 75 passes with a tight fitting pestle in a Thomas C Potter (21) to fully homogenize the vesicles. LPM were then layered on a discontinuous sucrose gradient (43%, 46%, and 52% w/v from top to bottom) and sedimented at 93,000 g_max for 60 min in a SW 28.1 Beckman rotor. The pellet (bLPM) and the bands at the top of the 43% and 43%/46% sucrose interface (aLPM) were carefully removed, diluted with 5 volumes of buffer, and sedimented at 34,500 × g for 10 min in a SS-34 Sorvall rotor. LPM were stored in liquid nitrogen until used (within one week). All procedures were carried out at 4 °C.

Plasma Membrane Purity and Orientation-- The purity of the LPM vesicles was assessed using 5'-nucleotidase, cytochrome c oxidase, and glucose 6-phosphatase activities as markers for plasma membrane, mitochondria, and endoplasmic reticulum, respectively (see Table I). Alkaline phosphatase, Na⁺/K⁺-ATPase, and 5'-nucleotidase were used to assess the purity of bLPM and aLPM. In addition, Na⁺/K⁺-ATPase and 5'-nucleotidase were used to assess LPM orientation (see Table I).

Plasma Membrane Loading-- 5-ml aliquots of aLPM or bLPM vesicles were resuspended in 25 ml of incubation medium (1:5 v/v) in the presence of 20 mM MgCl₂ or, when specified, Na⁺ or Ca²⁺ and loading was empirically determined by four passes in a Thomas C Potter with a tight fitting pestle at 4 °C as described previously (21).

Mg²⁺ Measurement-- Mg²⁺ fluxes were measured by atomic absorption spectophotometry (AAS) in a Perkin-Elmer model 3100 as the total content of Mg²⁺ in the supernatant after fast sedimentation of the vesicles. The Mg²⁺-loaded LPM vesicles were resuspended in the Mg²⁺-free incubation medium previously mentioned. An aliquot of LPM vesicles was incubated in the Mg²⁺-free medium, at a final concentration of 250-350 µg of protein/ml. After a 1-min equilibration, aliquots of the incubation mixture were withdrawn in duplicate at 1-2 min intervals, and the vesicles sedimented by rapid centrifugation (7000 × g for 1 min) in microfuge tubes. Mg²⁺ content was measured in the supernatants by AAS. Similar procedures were also used for Na⁺- or Ca²⁺-loaded LPM.

As for the Mg²⁺ content in LPM vesicles (pellet measurement), aliquots of the incubation medium were sedimented in microfuge tubes through an oil layer (dimethyl-phthalate:dibutyl-phthalate: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 of 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.

Protein Assay-- Protein was measured according to the procedure of Bradford (23), using bovine serum albumin as a standard.

Chemicals-- All chemicals and assays were of the purest analytical grade (Sigma). Nitrex nylon mesh was obtained from Tetko, Inc. (Briarcliff Manor, NY). Percoll was from Amersham Pharmacia Biotech.

Statistical Analysis-- Data are presented as mean ± S.E. Data were first analyzed by one-way ANOVA (analysis of variance). Multiple means were then compared by the Student-Newman-Keuls method multiple comparison test performed with statistical significance designated at a p value of 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Purity and Orientation of bLPM and aLPM after Isolation-- The purity and enrichment of bLPM and aLPM were assessed using 5'-nucleotidase and alkaline phosphatase as apical markers and Na⁺/K⁺-ATPase as a basolateral marker (24-26). The maximal activity (considered as 100%) was obtained by using 0.06% Triton X-100 (21). Table I shows the activities and the enrichment of bLPM and aLPM as compared with homogenate and total LPM prior to separation. As can be noted, bLPM were enriched 19- and 14-fold over homogenate and aLPM, respectively, as measured by Na⁺/K⁺-ATPase activity. By contrast, aLPM were enriched 4- and 15-fold over bLPM as measured by 5'-nucleotidase and alkaline phosphatase, respectively. The protein recovery of bLPM and aLPM from the total LPM population was 34.6% ± 12% and 38.2% ± 7%, respectively (or 0.67 and 0.71 mg of protein/g of liver). As described previously, correct (inside-in) orientation of LPM is greatly affected by the presence of Mg²⁺, which has been shown to induce correct membrane sidedness (27, 28). The sidedness of LPM was determined as previously reported (21). Both the Mg²⁺-loaded aLPM and bLPM were approximately 80% inside-in.

View this table: [in this window] [in a new window]   Table I Enzyme activity of purified basolateral and apical liver plasma membranes Alkaline phosphatase is expressed as µmol of p-nitrophenyl hydrolyzed/mg of protein1/min1, whereas all the other data are expressed as µmol Pi/mg of protein1/min1. All experimental protocols were performed as previously described (21). All data are reported as mean ± S.E.; n = 15, 8, 8, and 10 for 5'-nucleotidase, MgATPase, Na+/K+-ATPase, and alkaline phosphatase in homogenate and total LPM, respectively. n = 10, 6, 6, and 10 for 5'-nucleotidase, MgATPase, Na+/K+-ATPase, and alkaline phosphatase in aLPM and bLPM, respectively.

Dose Response of Na⁺ and Ca²⁺ Induced Mg²⁺ Efflux from bLPM and aLPM and the Effect of Various Inhibitors-- Fig. 1A shows that bLPM loaded with 20 mM MgCl₂ and suspended in the absence of Na⁺ and Ca²⁺ released negligible Mg²⁺ over several min of incubation (control). The addition of increasing concentrations of extravesicular NaCl induced a dose-dependent efflux of Mg²⁺, which was maximal at 10 mM NaCl (138.1 ± 15.1 nmol of Mg²⁺/mg of protein, n = 4). Concentrations of NaCl larger than those reported in the figure (e.g. 25 or 50 mM) did not induce a statistically significant increase in Mg²⁺ efflux (142.9 ± 12.5 nmol of Mg²⁺/mg of protein, n = 5, for 25 mM NaCl versus 138.1 ± 15.1 nmol of Mg²⁺/mg of protein, n = 4, for 10 mM NaCl). Irrespective of the concentration of NaCl used, Mg²⁺ efflux at 37 °C was maximal within the first min after the Na⁺ addition (Fig. 1). In contrast to what was previously observed in the total LPM population (21), the addition of 500 µM CaCl₂ to bLPM elicited a negligible release of Mg²⁺ (18.4 ± 6.0 nmol of Mg²⁺/mg of protein as compared with 267.7 ± 17.0 in the total LPM population (21).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Na+, but not Ca2+, induces Mg2+ efflux from 20 mM MgCl2-loaded basolateral LPM (A). Imipramine but not amiloride inhibits Mg2+ efflux (B). Mg2+ efflux is reported as the net change in Mg2+ content in the supernatant with respect to that before the addition of extravesicular Na+ or Ca2+ (Fig. 1A). Dose response for NaCl induced Mg2+ efflux from 20 mM MgCl2-loaded bLPM vesicles incubated in the absence of extravesicular Mg2+. All experiments were performed as described under "Experimental Procedures." Vesicles (250 µg/ml) were incubated at 37 °C in a reaction medium containing 250 mM sucrose, 25 mM K-Hepes, pH 7.4. After a 2-min equilibration, an aliquot corresponding to 500 µl of incubation medium was withdrawn in duplicate and rapidly sedimented in microfuge tubes. Mg2+ content in the supernatant was measured by AAS. After withdrawal, the concentrations of NaCl or CaCl2 indicated in the figure were added, and an aliquot of the incubation mixture was withdrawn in duplicate at the reported time points and processed as above. The figure represents the net change in Mg2+ content in the supernatant with respect to that present before ion addition. In Fig. 1B, 1 mM amiloride or 200 µM imipramine was added 2 min before Na+ addition. Data are mean ± S.E. of six and eight different preparations for Na+ for Ca2+, respectively. ANOVA and or the Student-Newman-Keuls method was performed at all time points. *, p < 0.05 versus control and imipramine.

Several agents have been reported to inhibit the Na⁺/Mg²⁺ transporter. Amiloride has been shown to effectively inhibit Mg²⁺ extrusion via the putative Na⁺/Mg²⁺ exchanger in several mammalian cell types, including hepatocytes (5, 8, 14, and 18). Amiloride also has been shown to inhibit the Na⁺/H⁺ exchanger as well as Na⁺ entry pathways in epithelia (29, 30). In addition, Feray and Garay (13) demonstrated that tricyclic antidepressant drugs, such as imipramine, are nonspecific inhibitors of Mg²⁺ transport, i.e. Na⁺/Mg²⁺ exchange, in human red cells. Interestingly, the addition of 1 mM amiloride to bLPM did not inhibit Na⁺-induced Mg²⁺ efflux (Fig. 1B) as compared with a 50% inhibition observed in the total LPM population (21). In fact, 1 mM amiloride enhances the Na⁺-induced Mg²⁺ extrusion 3 min after addition of NaCl (Fig. 1B), a phenomenon for which we have no explanation. By contrast, the addition of 200 µM imipramine effectively inhibited Na⁺-induced Mg²⁺ mobilization up to approximately 85% (Fig. 1B).

Fig. 2, A and B, illustrate the Na⁺ and Ca²⁺ dose-dependent effect, respectively, on Mg²⁺ efflux in aLPM. In Fig. 2A maximal Mg²⁺ efflux is induced by much greater concentrations of NaCl than in bLPM (e.g. 25 mM induces an efflux of 184.7 ± 36.7 nmol of Mg²⁺/mg of protein). At variance from bLPM, Ca²⁺ could also induce Mg²⁺ efflux in these vesicles. The maximal Mg²⁺ efflux induced by 500 µM CaCl₂ was 227.2 ± 50.2 nmol of Mg²⁺/mg of protein, n = 4 (Fig. 2B). The concentrations of Na⁺ or Ca²⁺ able to elicit maximal Mg²⁺ efflux from aLPM are very similar to those effective in the total LPM population (21). Also, in contrast to bLPM, 1 mM amiloride inhibited both Na⁺- and Ca²⁺-induced Mg²⁺ efflux in aLPM to a comparable extent (Fig. 2C). 200 µM imipramine was equally effective at inhibiting both Mg²⁺ extrusion mechanisms (not shown).

View larger version (29K): [in this window] [in a new window]   Fig. 2.   NaCl-induced (A) or CaCl2-induced (B) Mg2+ efflux from 20 mM MgCl2-loaded aLPM and inhibition by amiloride (C). Amiloride (1 mM) was added 2 min before the Ca2+ or Na+ addition. All experiments were performed as described in the legend to Fig. 1. Data are mean ± S.E. of three preparations. ANOVA and the Student-Newman-Keuls method for multiple comparisons were performed at all time points. *, p < 0.05 versus control and amiloride.

Partial Kinetics of Mg²⁺ Efflux-- The Mg²⁺ efflux at 37 °C shown in the previous experiments was already maximal at the first time observation point, after 2 min. Hence, it was difficult to distinguish whether the Mg²⁺ appearing in the supernatant was the result of net efflux and/or the release of Mg²⁺ from surface binding sites. Some kinetic resolution can be achieved by lowering the temperature of incubation. Fig. 3, A and B, show the rates of Na⁺- or Ca²⁺-dependent Mg²⁺ extrusion in the supernatant from 20 mM MgCl₂-loaded bLPM (Fig. 3A) or aLPM (Fig. 3B) incubated in a thermostated vessel at 10 °C. Under these conditions, the efflux of Mg²⁺ is comparable, but the rates of release are much slower and kinetically distinguishable.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Temperature dependence of Mg2+ efflux from loaded bLPM (A) or aLPM (B). bLPM (A) or aLPM (B) vesicles were loaded with 20 mM MgCl2 in the absence of extravesicular Mg2+, incubated at 10 °C, and stimulated with NaCl or CaCl2. Efflux of Mg2+ from LPM into the supernatant is expressed as a percentage of the value before cation addition. Experimental conditions were similar to those described in Fig. 1. Data are mean ± S.E. of three and five different preparations for Control and 10 mM NaCl, respectively, for bLPM, and three, six, and four different preparations for Control, 25 mM NaCl, and 500 µM CaCl2 for aLPM. ANOVA and the Student-Newman-Keuls method for multiple comparisons were performed at all time points. *, p < 0.05 versus control.

Specificity of Transport-- Na⁺-dependent Mg²⁺ extrusion in bLPM is specific for Na⁺. Addition of 25 mM KCl did not elicit a significant Mg²⁺ extrusion, and the addition of 25 mM LiCl elicited an efflux of 55.7 ± 29.1 nmol of Mg²⁺/mg of protein. The efflux induced by Li⁺ was not statistically significant from the control but was statistically significant from 10 mM NaCl (Fig. 4A). Similarly, the addition of 25 mM LiCl or 25 mM KCl to aLPM also elicited negligible Mg²⁺ efflux (not shown), indicating that the Mg²⁺ extrusion observed in aLPM strictly depends on extracellular Na⁺. Compared with Na⁺, the Ca²⁺ requirement for Mg²⁺ release from aLPM is far less specific, because Mg²⁺ efflux could also be induced by the addition of equimolar concentrations of other divalent cations (Fig. 4B).

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Cation specificity in promoting Mg2+ efflux. Mg2+ efflux from bLPM following stimulation by 10 mM NaCl, 25 mM KCl, or 25 mM LiCl (A). In B, Mg2+ efflux from loaded aLPM can be stimulated by Ca2+ or various other divalent cations. All experiments were performed as described in the legend to Fig. 1. Data are mean ± S.E. of four preparations. ANOVA and the Student-Newman-Keuls method for multiple comparisons were performed at all time points. *, p < 0.05 versus control for A. In B all time points were significant (p < 0.05) versus control. Asterisks were omitted for clarity.

Bidirectionality of the Mg²⁺ Exchange-- Fig. 5A shows the result of a set of experiments in which bLPM loaded with 20 mM NaCl were stimulated by the addition of varying concentrations of extravesicular Mg²⁺. The observation that extravesicular Mg²⁺ can induce a Na⁺ efflux from Na⁺-loaded bLPM demonstrates that the Na⁺/Mg²⁺ extrusion mechanism is bidirectional. On the other hand, when aLPM were loaded with 20 mM Na⁺ and stimulated with Mg²⁺, no Na⁺ efflux or Mg²⁺ influx was observed, indicating that the Na⁺/Mg²⁺ transport mechanism in these vesicles is unidirectional (Fig. 5B). When similar experiments were performed in aLPM loaded with CaCl₂, the addition of external MgCl₂ did not induce Ca²⁺ extrusion or, consequently, Mg²⁺ uptake. This indicates that the Ca²⁺/Mg²⁺ transporter in aLPM is also unidirectional, i.e. Mg²⁺ extrusion for Ca²⁺ uptake (Fig. 5C).

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Reversibility of the Na+-induced, but not the Ca2+-induced, Mg2+ transport. Net Na+ or Ca2+ efflux in bLPM (A) or aLPM (B) loaded with 20 mM NaCl and resuspended in the absence of extravesicular NaCl or aLPM loaded with 20 mM CaCl2 and resuspended in the absence of external CaCl2 (C). The Na+ or Ca2+ content in the supernatant was assayed by AAS as described for Mg2+ in the legend to Fig. 1. Data presented are mean ± S.E. of four different preparations. ANOVA and the Student-Newman-Keuls method for multiple comparisons were performed at all time points. *, p < 0.05 versus control.

Table II compares the initial loading content under each experimental loading condition as well as the percentage of cation releasable upon thr addition of Triton X-100. Table II also reconfirms that when no movement is observed such as in Fig. 5, B or C, the absence of efflux is not due to mismatched loading conditions.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Properties of Plasma Membrane Mg²⁺ Transport-- As indicated in the Introduction, the regulation of Mg²⁺ homeostasis in liver and other tissues is poorly understood. In response to activation of specific cell signaling, it has been shown that massive amounts of total Mg²⁺ can be translocated within minutes in or out of hepatocytes (7, 8, 10). This indicated a very powerful and rapid transport machinery of Mg²⁺ transport within the plasma membrane.

Several groups have demonstrated that the plasma membrane of different cell types possesses very active Mg²⁺ transport mechanisms (6-14). Data in the literature support the operation of at least two distinct Mg²⁺ transport mechanisms in mammalian cells tentatively identified as a Na⁺-dependent and a Na⁺-independent exchange pathway, respectively (8, 9, 12-17). Experiments performed using a variety of perfused organs or isolated cells indicate that Mg²⁺ transporters operate with varying stoichiometric ratios and transport properties (for review see Ref. 1, 3, 5, 18, 19, or 20) under different experimental conditions. This information makes it difficult to reconcile the available data in a comprehensive scheme.

These conflicting results could be consistent with the operation of several Mg²⁺ transporters within the same cell, each operating under different conditions and sensitivity to inhibitors. In this work, we were successful in obtaining effective separation of LPM into two fractions enriched in aLPM or bLPM, which maintained low permeability to cations and proper sidedness. Using these preparations to study Mg²⁺ fluxes, it was possible to observe that the plasma membrane of rat liver possesses three kinetically distinct Mg²⁺ transport pathways. These transporters are distinguishable in terms of localization within the plasma membrane, sensitivity to inhibitors, requirement of cation for co-transport, and reversibility.

The overall conclusion is that the apical LPM possesses two distinct Mg²⁺ transporters, one Ca²⁺- and one Na⁺-dependent. In contrast, the basolateral LPM contains only one Mg²⁺ transporter dependent upon Na⁺. Partial evidence indicates that the Mg²⁺ transporters activated by Na⁺ in the apical and basolateral membranes may be distinct. The kinetic properties of these transporters are outlined in Table III.

We conducted several controls as previously shown (21) to conclusively demonstrate that the Mg²⁺ extruded in the supernatant is the result of net translocation across the membrane and that Mg²⁺ efflux is not the result of a passive leakage from Mg²⁺-loaded vesicles or a release from surface binding sites after the addition of a cation or osmotic mismatch. Briefly, these experiments include the following: (a) unstimulated LPM do not release entrapped Mg²⁺ over several min of incubation even in the presence of a gradient across the plasma membrane (Figs. 1 and 2); (b) both Na⁺- and Ca²⁺-induced Mg²⁺ effluxes are completely prevented in the presence of inhibitors; (c) 25 mM LiCl or 25 mM KCl (Fig. 4A) fail to replace Na⁺ in mobilizing Mg^{2 +}; (d) at lower temperatures Mg²⁺ release induced by Ca²⁺ or Na⁺ is slow and kinetically resolvable; and (e) most of the Mg²⁺ releasable by Ca²⁺ or Na⁺ can be the same Mg²⁺ pool released by detergent or cation ionophore (21).

Properties of Mg²⁺ Transport in the Basolateral Membrane-- Only Na⁺ can induce the release of intravesicular Mg²⁺ from bLPM. The Na⁺ requirement is specific, because no other monovalent or divalent cation can induce Mg²⁺ efflux. The process is fully reversible, as extravesicular Mg²⁺ effectively mobilizes intravesicular Na⁺. It is noteworthy that imipramine, but not amiloride, is effective at inhibiting Na⁺-induced Mg²⁺ extrusion in bLPM (Fig. 1B). On the contrary, amiloride inhibits the Na⁺/Mg²⁺ mechanism in aLPM by >85% (Fig. 2C). As the ratio of aLPM to bLPM is ~1 on a protein basis, the differential effectiveness of amiloride to inhibit Mg²⁺ transport in bLPM versus aLPM could explain our previous observation that amiloride inhibits Mg²⁺ transport in total LPM by approximately 50% (21).

Properties of Mg²⁺ Transport in the Apical Membrane-- The data from aLPM demonstrate that the apical domain of hepatocytes has both a Na⁺/Mg²⁺ and a Ca²⁺/Mg²⁺ transport mechanism. The Na⁺/Mg²⁺ exchange in aLPM operates in a manner similar to the Na⁺/Mg²⁺ exchanger described in bLPM in terms of specificity for Na⁺. However, three main differences are evident: (a) the dose response for extravesicular Na⁺ in aLPM is different than in bLPM in that the concentration for maximal activity is left-shifted in bLPM (Fig. 1A), (b) the transport is almost completely inhibited by amiloride (Fig. 2C), and (c) the process is not reversible because extravesicular Mg²⁺ cannot elicit efflux of intravesicular Na⁺ (Fig. 5B). These differences can be accounted for by the presence of two separate Na⁺/Mg²⁺ antiporters in the apical and basolateral sides of hepatocytes or by an identical Mg²⁺ transporter differently regulating Na⁺ exchange. In the latter alternative, the difference in Na⁺ affinity, amiloride sensitivity, and reversibility will be confined to the different mechanism of Na⁺ influx in either plasma membrane domain.

The Mg²⁺ release mechanism dependent upon extracellular Ca²⁺ and found in the apical membrane is a novel observation identified for the first time in mammalian cells. The data from aLPM demonstrate that the Ca²⁺/Mg²⁺ transporter is strictly localized in this domain and that the transporter is nonselective as it can be activated by several divalent cations (Fig. 4B). The fact that this transporter is unidirectional suggests that in vivo it may operate only in terms of Mg²⁺ efflux and Ca²⁺ uptake.

Physiological Significance of Hepatocyte Mg²⁺ Transporters-- The distribution of the three Mg²⁺ transport mechanisms within the hepatocyte is diagrammatically illustrated in Fig. 6. Based upon the limited share of knowledge of intra- and extracellular Mg²⁺ homeostasis in liver, or for that matter, in any other tissue, any discussion of the physiological role of these different transporters in hepatocytes remains speculative. Owing to its large mass, blood flow, and active secretory capacity, the liver is positioned to play a key role in the homeostasis of metabolites and ions such as Mg²⁺. Hepatocytes are polarized cells in which transport between hepatocytes and plasma is regulated by the basolateral membrane, whereas that between hepatocyte and biliary tract is regulated by the apical membrane.

View larger version (116K): [in this window] [in a new window]   Fig. 6.   Multiple Mg2+ transport in the hepatocyte. See "Discussion" for details.

The basolateral membrane accounts for approximately 90% of the surface area of the hepatocyte plasma membrane. Hence, the basolateral Na⁺-dependent Mg²⁺ transport, described by several groups, should play the primary role in hepatocyte Mg²⁺ homeostasis as well as in plasma Mg²⁺ homeostasis. The novel finding of this work is that the basolateral Na⁺/Mg²⁺ overall exchange is reversible, whereas the Na⁺-dependent and Ca²⁺-dependent Mg²⁺ transport mechanisms in the apical membrane are not. This implies that the basolateral Na⁺-dependent Mg²⁺ transporter is most likely the effector for the observed large and rapid changes in plasma (31) and tissue (6-10) Mg²⁺ observed in either direction after the addition of - or -adrenergic agonists (7-9, 32), protein kinase C activation (10), insulin (33), etc.

In contrast, the apical membrane Mg²⁺ transporter is unidirectional and can only transport Mg²⁺ from the hepatocytes to the bile in exchange for Ca²⁺ or Na⁺. Data in the literature indicate that the concentration of total Ca²⁺ in the bile ranges from 2 to 16 mM (34). The unidirectional Ca²⁺/Mg²⁺ exchange could be responsible for decreasing biliary Ca²⁺ thereby preventing the formation of bile stones or be an additional mechanism contributing to the overall reabsorption of Ca²⁺ (34-36). The physiological significance of the unidirectional Na⁺-dependent Mg²⁺ efflux from hepatocytes through the apical membrane is equally conjectural and may operate in tandem with the described Na⁺/bile acid uptake or for direct Mg²⁺ extrusion (37). It will be interesting to observe whether these transporters are up-regulated in conditions of chronic alcoholism or diabetes when the tissue concentrations of Mg²⁺ in liver and other tissues dramatically decrease (38, 39).

Although the present data may provide more questions than answers as to the physiological and metabolic implications of liver Mg²⁺ transport, they dissect an overall complex Mg²⁺ cellular transport in individual components. Identifying three individual transporters on the basis of localization, inhibition, reversibility, and cation specificity will permit the design of experimental approaches to further investigate the distribution of these Mg²⁺ transporters in other tissues, the more detailed mechanism of transport and the physiological significance in terms of Mg²⁺, and other ion and metabolic homeostasis.

	ACKNOWLEDGEMENTS

We thank Drs. Meredith Bond and Ulrich Hopfer in this department for valuable scientific discussion.

	FOOTNOTES

^* This work was supported by National Institutes of Health 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. Section 1734 solely to indicate this fact.

To whom correspondence and reprint requests should be addressed: Dept. Physiology and Biophysics, School of Medicine, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106-4970. Tel.: (216) 368-3400; Fax: (216) 368-3952; E-mail: axs15@po.cwru.edu.

	ABBREVIATIONS

The abbreviations used are: LPM, liver plasma membranes; bLPM, basolateral liver plasma membranes; aLPM, apical liver plasma membranes; AAS, atomic absorption spectophotometry.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				G. Wiesenberger, K. Steinleitner, R. Malli, W. F. Graier, J. Vormann, R. J. Schweyen, and J. A. Stadler Mg2+ Deprivation Elicits Rapid Ca2+ Uptake and Activates Ca2+/Calcineurin Signaling in Saccharomyces cerevisiae Eukaryot. Cell, April 1, 2007; 6(4): 592 - 599. [Abstract] [Full Text] [PDF]

				H. A. Almulla, P. G. Bush, M. G. Steele, D. Ellis, and P. W. Flatman Loading rat heart myocytes with Mg2+ using low-[Na+] solutions J. Physiol., September 1, 2006; 575(2): 443 - 454. [Abstract] [Full Text] [PDF]

				M. Schweigel, H.-S. Park, B. Etschmann, and H. Martens Characterization of the Na+-dependent Mg2+ transport in sheep ruminal epithelial cells Am J Physiol Gastrointest Liver Physiol, January 1, 2006; 290(1): G56 - G65. [Abstract] [Full Text] [PDF]

				L. M. Torres, J. Youngner, and A. Romani Role of glucose in modulating Mg2+ homeostasis in liver cells from starved rats Am J Physiol Gastrointest Liver Physiol, February 1, 2005; 288(2): G195 - G206. [Abstract] [Full Text] [PDF]

				T. E. Fagan, C. Cefaratti, and A. Romani Streptozotocin-induced diabetes impairs Mg2+ homeostasis and uptake in rat liver cells Am J Physiol Endocrinol Metab, February 1, 2004; 286(2): E184 - E193. [Abstract] [Full Text] [PDF]

				J. T. Wright Jr, M. Rahman, A. Scarpa, M. Fatholahi, V. Griffin, R. Jean-Baptiste, M. Islam, M. Eissa, S. White, and J. G. Douglas Determinants of Salt Sensitivity in Black and White Normotensive and Hypertensive Women Hypertension, December 1, 2003; 42(6): 1087 - 1092. [Abstract] [Full Text] [PDF]

				A. Young, C. Cefaratti, and A. Romani Chronic EtOH administration alters liver Mg2+ homeostasis Am J Physiol Gastrointest Liver Physiol, January 1, 2003; 284(1): G57 - G67. [Abstract] [Full Text] [PDF]

				W. J. Haynes, C. Kung, Y. Saimi, and R. R. Preston An exchanger-like protein underlies the large Mg2+ current in Paramecium PNAS, November 26, 2002; 99(24): 15717 - 15722. [Abstract] [Full Text] [PDF]

				T. E. Fagan and A. Romani {alpha}1-Adrenoceptor-induced Mg2+ extrusion from rat hepatocytes occurs via Na+-dependent transport mechanism Am J Physiol Gastrointest Liver Physiol, June 1, 2001; 280(6): G1145 - G1156. [Abstract] [Full Text] [PDF]

				R. M. Touyz, C. Mercure, and T. L. Reudelhuber Angiotensin II Type I Receptor Modulates Intracellular Free Mg2+ in Renally Derived Cells via Na+-dependent Ca2+-independent Mechanisms J. Biol. Chem., April 20, 2001; 276(17): 13657 - 13663. [Abstract] [Full Text] [PDF]

				A. Graschopf, J. A. Stadler, M. K. Hoellerer, S. Eder, M. Sieghardt, S. D. Kohlwein, and R. J. Schweyen The Yeast Plasma Membrane Protein Alr1 Controls Mg2+ Homeostasis and Is Subject to Mg2+-dependent Control of Its Synthesis and Degradation J. Biol. Chem., May 4, 2001; 276(19): 16216 - 16222. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Cefaratti, C. Articles by Scarpa, A. Search for Related Content PubMed PubMed Citation Articles by Cefaratti, C. Articles by Scarpa, A. Social Bookmarking                      What's this?