Enhanced sodium-dependent extrusion of magnesium in mutant cells established fro - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:04 |只看该作者 |倒序浏览 |打印

Department of Physiology, Tokyo Medical University, Tokyo, Japan, L* J: u( P9 q8 i7 Q  c

Department of Biochemistry II and Division of Nephrology and Hypertension, The Jikei University School of Medicine, Tokyo, Japan6 Y8 k5 F$ K# y/ e  y7 x' @
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ABSTRACT

To study the regulatory mechanisms of intracellular Mg2  concentration ([Mg2 ]i) in renal tubular cells as well as in other cell types, we established a mutant strain of mouse renal cortical tubular cells that can grow in culture media with very high extracellular Mg2  concentrations ([Mg2 ]o > 100 mM: 101Mg-tolerant cells). [Mg2 ]i was measured with a fluorescent indicator furaptra (mag-fura 2) in wild-type and 101Mg-tolerant cells. The average level of [Mg2 ]i in the 101Mg-tolerant cells was kept lower than that in the wild-type cells either at 51 mM or 1 mM [Mg2 ]o. When [Mg2 ]o was lowered from 51 to 1 mM, the decrease in [Mg2 ]i was significantly faster in the 101Mg-tolerant cells than in the wild-type cells. These differences between the 101Mg-tolerant cells and the wild-type cells were abolished in the absence of extracellular Na  or in the presence of imipramine, a known inhibitor of Na /Mg2  exchange. We conclude that Na -dependent Mg2  transport activity is enhanced in the 101Mg-tolerant cells. The enhanced Mg2  extrusion may prevent [Mg2 ]i increase to higher levels and may be responsible for the Mg2  tolerance.7 M& K6 u6 ~+ t
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membrane transport; sodium/magnesium exchange
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THE KIDNEYS ARE EXTREMELY important for control of body Mg2  balance through its urinary excretion, which is primarily regulated by tubular reabsorption. In the cortical thick ascending limb, Mg2  reabsorption is thought to occur by passive transport through the paracellular pathway. On the other hand, Mg2  reabsorption in distal tubules is transcellular and active; Mg2  enters into the tubular cells through the apical membrane by passive influx driven by the electrochemical gradient [possibly through some transient receptor potential (TRP) channels] and extruded from the cells through the basolateral membrane by active transport (for review, see Refs. 1 and 12). Extracellular Na -dependent Mg2  extrusion (the putative Na /Mg2  exchange) has been postulated as the active transport mechanism in renal tubular cells, as well as in other cell types (for reviews, see Refs. 5 and 15). However, the transporter molecules are not yet identified, hindering detailed characterization of the transport.) p' e! @9 E) X: p

A cultured cell line that highly expresses any target molecule, if established, could be a useful tool for molecular cloning. In the present study, as the first step toward molecular cloning of the Na /Mg2  exchanger, we established a mutant cell line from mouse renal cortical tubular (MCT) cells (9) that could grow in culture media with very high extracellular Mg2  concentration ([Mg2 ]o >100 mM: 101Mg-tolerant cells) by stepwise increases of Mg2  concentration in the culture media and selection of high-Mg2 -tolerant cells. Intracellular Mg2  concentration ([Mg2 ]i) was measured with a fluorescent indicator furaptra (mag-fura 2) in the 101Mg-tolerant and the wild-type cells, and the characteristics of their Mg2 -extruding activities were compared.
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Some of these results have been published previously in abstract form (22).9 f3 t7 U7 b& h+ o

MATERIALS AND METHODS+ q# V9 Z: s5 N2 l' w

Cell culture. A line of mouse cortical tubular epithelial cells, MCT cells (9), was kindly provided by Drs. I. Inoue and J. M. Lalouel of the University of Utah. The cells were maintained in DMEM (Invitrogen, Tokyo, Japan) supplemented with 5% FCS (Invitrogen) under 10% CO2-air at 37°C. For passage, cells were washed twice with PBS (137 mM NaCl, 2.7 mM KCl, 20 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.4) followed by dissociation with 0.25% trypsin (Invitrogen)." W; p: d! J3 G0 G

Compositions of media used for establishing Mg2 -tolerant cells are listed in Table 1. They were made up by mixing the vitamin mixture (Invitrogen) and other stock solutions prepared from individual chemicals. Osmolality of the culture media was measured by the freezing-point method using an osmometer (Osmotron-20; Orion, Tokyo, Japan).0 m9 b& E4 e8 O& k
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View this table:

Microscopy. Cells grown on a culture dish (Corning, Corning, NY) were observed and photographed with an inverted phase-contrast microscope (Olympus, Tokyo, Japan) equipped with a digital camera (PDMC Ie, Polaroid, Waltham, MA). For electron microscopy, cells on a culture dish were washed twice with PBS and fixed in 2% glutaraldehyde in 0.1 M Na -K  phosphate, pH 7.4 (Srensen's buffer) at 4°C for 18 h. The cells were postfixed in 1% osmium tetroxide in the same buffer for 2 h, dehydrated in a graded series of ethanol, and embedded in epoxy resin at the bottom of the culture dish. Ultrathin sections were cut with a diamond knife, mounted on a grid, and stained with uranylacetate and lead citrate. The specimens were examined and photographed with a transmission electron microscope (H7500, Hitachi, Tokyo, Japan).

Optical measurements. Either 101Mg-tolerant or wild-type cells were grown on glass-bottomed culture dishes (Matsunami Glass, Osaka, Japan) and placed on the stage of an inverted microscope (TE300, Nikon, Tokyo, Japan). Apparatus, methods for fluorescence measurements, and analyses have been described previously (19). Briefly, cell clusters in a 300-μm-diameter field were alternately illuminated with light beams of 350 nm (an isosbestic wavelength for Mg2 ) and 382 nm (Mg2 -sensitive wavelength) through a x40 objective (CFI S Fluor40, Nikon). A shutter for the excitation light beam was opened for 10 s, and emitted fluorescence at 500 nm [25 nm full width at half-maximum (FWHM)] at each excitation wavelength was low-pass filtered at 1.7 Hz, sampled at 20 Hz, and averaged over a 7-s period.* s8 p' t$ ^6 g; h1 X2 ^% m
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After measurement of the background fluorescence from the cell cluster within the optical field, cells were incubated with 5 μM AM ester of furaptra for 12 min at room temperature. The AM ester was then washed off for at least 10 min by continuous flow of the perfusate. The background fluorescence was subtracted from the total fluorescence measured after the indicator loading to calculate indicator fluorescence intensities with excitation at 350 nm (F350) and 382 nm (F382).
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Because the instability of optical components caused small drifts during the study, we occasionally measured the ratio of F382 and F350 (F382/F350) in a Ca2 - and Mg2 -free buffer solution (140 mM KCl, 10 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.05 mM furaptra, and 10 mM PIPES, pH 7.1) filled in thin-wall quartz capillaries (internal diameter of 50 μm) as a standard. All values of F382/F350 measured from cells were normalized to the standard F382/F350 value, and the normalized F382/F350 was converted to [Mg2 ]i with the equation
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where Rmin and Rmax are the F382/F360 values at zero [Mg2 ]i and saturating [Mg2 ]i, respectively. We used parameter values previously estimated in cardiac myocytes [Rmin of 0.969, Rmax of 0.223, and Kd of 5.30 mM (21)] because intracellular calibration of furaptra fluorescence signals was not successful in the MCT cells, owing to significant leakage of the indicator (20% in 20 min).

In some experiments, fura 2 was introduced by incubation with fura 2-AM for 20 min at room temperature. After background subtraction, F382/F360 of fura 2 was normalized to the standard F382/F360 measured in the Ca2 - and Mg2 -free solution (above) and used as the Ca2 -related signal. Because the present purpose was to compare relative changes in intracellular Ca2  concentration ([Ca2 ]i) in the 101Mg-tolerant and wild-type cells, no attempt was made to calibrate fura 2 F382/F360 in terms of [Ca2 ]i.

Solutions and chemicals. Measurements of [Mg2 ]i were carried out in Ca2 -free solutions containing 0.5 mM EGTA to minimize any interference by Ca2 -related fluorescence change of furaptra (8, 18, 19). The normal-Mg2  solution contained (in mM) 150 NaCl, 4 KCl, 0.5 EGTA, 1 Mg(methanesulfonate)2, 10 glucose, and 5 HEPES, with pH adjusted with Tris ﹞ HCl to 7.40 at 25°C. The high-Mg2  solution contained 50 mM MgCl2 with NaCl concentration reduced to 75 mM to maintain the osmolality constant at 310 mosmol/kgH2O. In some experiments, Mg2  concentration was further increased to 101 mM, while Na  was eliminated. Low-Na  solutions were prepared by equimolar substitution of Na  with N-methyl-D-glucamine. For measurements of Ca2 -related signals of fura 2, Ca2  concentration of the solutions was raised by replacement of 0.5 mM EGTA with 2 mM CaCl2. Furaptra-AM (mag-fura 2-AM), furaptra (mag-fura 2, 4 K  salt), fura 2-AM, and fura 2 (5 K  salt) were purchased from Molecular Probes (Eugene, OR). Dextran (T-40, molecular weight of 36,000–43,000) was purchased from Amersham (Piscataway, NJ). All other chemicals were reagent grade.
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Data analysis.+ r. x6 X& k2 I$ n9 X; A7 i
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Nonlinear and linear least-square fittings were carried out with the program Kaleidagraph (version 3.501; Synergy Software, Reading, PA). The two-tailed Student's t-test was used for statistical comparison with the significance level set at P 6 r; L# s" z+ k
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RESULTS9 {+ `$ U# @% i6 u* K
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Establishment of Mg-tolerant MCT cells. The standard culture medium for MCT cell culture was replaced by media containing various concentrations of Mg2  (Table 1) and incubated replacing the medium with fresh media twice per week. In a medium containing 41 mM or a lower concentration of Mg2 , cells showed no apparent changes in their shape and growth rate. However, 80% of the cells in the 61 mM Mg2 -containing medium (Mg-61mM) and all of the cells in 81 mM or higher Mg2 -containing medium died within 7 days. In contrast, addition of 20% dextran (molecular weight of 40,000) to the 1 mM Mg medium (osmolality of 400 mosmol/kgH2O) did not affect the growth capability of the cells (unpublished data), indicating that high-Mg2  concentration rather than high osmolality (353 mosmol/kgH2O) of the 81 mM Mg medium caused cell extinction. The surviving cells in Mg-61mM medium started dividing thereafter, and the cells reached confluence 21 days after increasing Mg2  concentration. At this time point, the cells were dissociated with trypsin and diluted 10-fold into a fresh Mg-61mM medium. After passages in the Mg-61mM medium for 6 wk (i.e., dissociation and 10-fold dilution when cells reached confluence), the culture medium was changed to one containing 71 mM Mg. Thereafter, the Mg2  concentration of the medium was further increased every 10–12 wk in increments of 10 mM. Massive cell death was observed when Mg2  concentration was elevated from 71 to 81 mM and from 81 to 91 mM. The cells that had adapted to 81 and 101 mM Mg2  could be dislodged and stored in 10% DMSO and 90% FCS in liquid nitrogen, with 20–50% of viability at retrieval. MCT cells could be adapted in DMEM containing as much as 121 mM Mg2  without losing their growing capacity.

Characterizations of the Mg-tolerant cells. The 101Mg-tolerant cells grew at a significantly slower rate than the nontolerant (wild-type) cells. The approximate doubling time was 40 h for the 101Mg-tolerant cells and 13 h for the wild-type cells. The 101Mg-tolerant cells also showed a distinct morphology. Under a phase-contrast microscope, growing wild-type cells formed islet-like groups of monolayer cells, and each cell seemed to attach tightly. In contrast, the 101Mg-tolerant cells grew without forming groups, and cell-to-cell contact was very rough (Fig. 1). Transmission electron micrographs revealed that wild-type cells formed consecutive side-by-side membrane contact with adjacent cells, whereas the 101Mg-tolerant cells attached to each other only at the tips of processes. Desmosomes were often observed at the contact point of the 101Mg-tolerant cells, suggesting that nondesmosomal cellular attachment is impaired in these cells.* C+ W) [! m8 n9 }4 ]9 {& v% c# S
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Effect of extracellular Mg2  and Na  on [Mg2 ]i. Figure 2 summarizes the effects of [Mg2 ]o and extracellular Na  concentration ([Na ]o) on [Mg2 ]i in the 101Mg-tolerant and wild-type MCT cells. In the presence of 150 mM Na , [Mg2 ]i in the 101Mg-tolerant cells was significantly lower than that in the wild-type cells at 1 mM and also 51 mM [Mg2 ]o. In the absence of [Na ]o, however, there was no significant difference in [Mg2 ]i between the 101Mg-tolerant and wild-type cells. These results suggest that [Mg2 ]i of the 101Mg-tolerant cells is lowered by an extracellular Na -dependent mechanism.; o1 r, h# c4 j5 F1 e

Comparison of the Mg2  extrusion activity in the 101Mg-tolerant and the wild-type MCT cells. To evaluate the rate of Mg2  extrusion in the 101Mg-tolerant and wild-type cells, time-resolved measurements of [Mg2 ]i were carried out on reduction of [Mg2 ]o from 51 to 1 mM. In the presence of 150 mM Na , reduction of [Mg2 ]o to 1 mM caused a decrease in [Mg2 ]i, with the average rate much greater in the 101Mg-tolerant cells than in the wild-type cells (Fig. 3). The decrease in [Mg2 ]i was largely diminished in the absence of extracellular Na  in both cell types, indicating the Na -dependent Mg2  extrusion activity.
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For quantitative comparison of the rate of Mg2  extrusion in the 101Mg-tolerant and wild-type cells, we analyzed changes in [Mg2 ]i during the initial 7 min after reduction of [Mg2 ]o ([Mg2 ]i). The interval of 7 min was chosen because the greatest changes in [Mg2 ]i occurred during this period in the 101Mg-tolerant cells (Fig. 3). In the presence of 150 mM Na , [Mg2 ]i was found to be linearly related to initial [Mg2 ]i for both the 101Mg-tolerant cells and wild-type cells (Fig. 4A); values of [Mg2 ]i were more negative for the higher initial [Mg2 ]i. However, the slope of the relation was clearly steeper, and the decrease in the [Mg2 ]i was faster at any given initial [Mg2 ]i for the 101Mg-tolerant cells than for the wild-type cells (Fig. 4A). On the other hand, in the absence of extracellular Na , [Mg2 ]i values were close to zero independent of initial [Mg2 ]i in both cell types (Fig. 4B). These results suggest the existence of a Na -dependent Mg2  extrusion mechanism in the MCT cells and enhancement of its activity in the cells of acquired tolerance to very high [Mg2 ]o.
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The rate of Mg2  influx was also estimated in the 101Mg-tolerant and the wild-type cells by time-resolved measurements of [Mg2 ]i carried out after elevation of [Mg2 ]o from 1 to 51 mM to facilitate Mg2  influx. The solution of 51 mM [Mg2 ]o did not contain Na  to minimize Mg2  efflux. In 7 min, [Mg2 ]i slightly rose from 0.379 ± 0.161 to 0.472 ± 0.190 mM (n = 3) in the 101Mg-tolerant cells and from 0.752 ± 0.186 to 0.856 ± 0.260 mM (n = 3) in the wild-type cells; the [Mg2 ]i increments in the 101Mg-tolerant and the wild-type cells were not significantly different. Thus the rate of Mg2  influx appears to be similar in the 101Mg-tolerant cells and the wild-type cells.: ^. t/ c3 C% Q+ k# d

Extracellular Na  dependence. To analyze Na  dependence of the Mg2  extrusion, we measured [Mg2 ]i in the 101Mg-tolerant cells at various [Na ]o. For this purpose, we selected cell clusters with a similar initial [Mg2 ]i of 0.9 mM (0.91 ± 0.13 mM, n = 24). The results of a series of experiments carried out with the same subculture on the same day clearly showed that [Na ]o accelerated, in a concentration-dependent manner, the decrease in [Mg2 ]i induced by reduction of [Mg2 ]o from 51 to 1 mM (Fig. 5A). The data thus obtained from a total of 24 cell clusters were explained by the Hill-type curve with a Hill coefficient of 2 and half activation at 25 mM [Na ]o (Fig. 5B).

Effect of imipramine. We also examined the effects of imipramine, a known inhibitor of the Na /Mg2  exchange in erythrocytes (4, 6) and cardiac myocytes (8, 17), on the rate of decrease in [Mg2 ]i at 150 mM [Na ]o. In the selected clusters of the 101Mg-tolerant cells that had similar initial [Mg2 ]i of 0.8 mM (0.813 ± 0.106 mM, n = 20), imipramine slowed decreased [Mg2 ]i in a concentration-dependent manner (Fig. 6); [Mg2 ]i in the presence of 200 μM imipramine was not significantly different from that in the absence of extracellular Na  (Scheffé's post hoc analysis; P > 0.05). Thus imipramine appears to inhibit most, if not all, of the extracellular Na -dependent Mg2  extrusion activity with a half-inhibitory concentration between 50 and 200 μM.6 Q; C! K0 }. I7 {! B
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Comparison of Na /Ca2  exchange activity in the Mg2 -tolerant and the wild-type MCT cells. Because it has been reported that the Na /Ca2  exchanger can transport Mg2  and may play a role in the extrusion of Mg2  from cells (18), we compared Na /Ca2  exchange activity in the 101Mg-tolerant cells with that in the wild-type cells by monitoring fura 2 fluorescence signals. In the presence of extracellular 2 mM Ca2 , withdrawal of extracellular Na  induced a slight decrease in fura 2 F382/F360, as expected for the small elevation of [Ca2 ]i, and F382/F360 rapidly returned to the base level after reintroduction of Na  (Fig. 7). In the 101Mg-tolerant and wild-type cells, Na  withdrawal caused changes of fura 2 F382/F360 in 2 min by –0.050 ± 0.0015 (n = 4) and –0.058 ± 0.022 (n = 5), respectively, from the initial levels of 0.815 ± 0.023 and 0.725 ± 0.058, respectively. These Na -free-induced changes in F382/F360 in the 101Mg-tolerant cells and the wild-type cells were not significantly different, suggesting similarly low activity of the Na /Ca2  exchanger in both cell types. Low activity of the Na /Ca2  exchanger was consistent with low expression levels of mRNA; DNA microarray analysis indicated that mRNA levels for NCX1 (a major isoform known to be expressed in kidney; e.g., 13) were under the detection threshold in both 101Mg-tolerant cells and wild-type cells (data not shown).

DISCUSSION

General. We successfully established high-Mg2 -tolerant MCT cells, probably through genetic change(s) in the cellular genome due to selection under high-Mg2  conditions. The implications of the morphological changes in the high-Mg2 -tolerant cells (Fig. 1) are not known at this point. In the present study, we focused on the functional differences in Mg2  homeostasis of the 101Mg-tolerant and wild-type cells.
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Quantitative measurements of [Mg2 ]i require calibration of furaptra F382/F360 in the cell interior (intracellular calibration), since properties of furaptra are likely altered in the cytoplasm (16, 19) probably as a result of the indicator binding to cellular proteins. Although we used parameter values previously estimated in cardiac myocytes (Rmin of 0.969, Rmax of 0.223, Kd of 5.30 mM), similar parameter values were also obtained for furaptra in tenia cecum: Rmin of 0.986, Rmax of 0.199, and Kd of 5.43 mM (16). Thus it appears that intracellular properties of the indicator are similar in different cell types and may cover the potential differences in the calibrations for the MCT cells vs. cardiac myocytes. Note, however, that values of Rmax and Rmin are instrument dependent and must be determined in each system. It has been reported that neither addition of imipramine (up to 200 μM) nor equimolar substitution of K  by Na  (up to 20 mM) markedly affects furaptra F382/F360 in the solutions containing 0–4 mM Mg2  (16).' _# X6 a* S" q) \! Z/ `

Because the extracellular Na  dependence and imipramine sensitivity found in the present study are similar to those of the Na /Mg2  exchange reported in other cell types with different techniques (see below), most of the changes in [Mg2 ]i observed in the present experimental conditions likely reflect Mg2  transport across the cell membrane, rather than alterations in intracellular Mg2  buffering and sequestration by organelles. In Ca2 -free conditions where [Ca2 ]i does not change significantly, competition of binding sites between Ca2  and Mg2  is probably minimized, and Mg2  fluxes between the cytoplasm and mitochondria are also suppressed (2). Although we used N-methyl-D-glucamine to replace Na  for low-Na  solutions in the present study, we previously reported that the rates of extracellular Na -dependent Mg2  efflux were essentially unaffected by use of tetramethylammonium to replace Na  in cardiac myocytes (19).8 t( R, y9 w2 V9 v9 u. X
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Acquisition of Mg2  tolerance. The increase in Mg2  concentration accompanied the reduction of Na  concentration in the culture media (Table 1). It is unlikely, however, that low-Na  concentration plays a principal role in the prevention of cell growth because massive cell death was observed when Mg2  concentration was elevated from 71 to 81 mM and from 81 to 91 mM (see above), whereas Na  concentration was kept constant at 30 mM (Table 1). It is possible, however, that low Na  concentration may facilitate Mg2  overloading of the cells by partial inhibition of the Na -dependent Mg2  extrusion (Fig. 5).; N" j5 Q! }: _# O

The mechanism responsible for low [Mg2 ]i in the Mg2 -tolerant cells could be attributed to 1) decreased Mg2  influx, 2) increased Mg2  extrusion, and 3) other intracellular changes. The subsequent kinetic study of [Mg2 ]i by the fluorescent indicator suggests that point 2, above, is the most likely mechanism. Mg2  influx is likely mediated by Mg2 -permeable TRP channels, such as TRP-M6 and TRP-M7, which can permeate Mg2  either in the presence or in the absence of extracellular Na  (11). Suppression of this Na -independent Mg2  influx pathway could lead to Mg2  tolerance. However, the rate of Mg2  influx (measured at 51 mM [Mg2 ]o, above) appears to be similar in the 101Mg-tolerant cells and the wild-type cells and is probably much slower at 1 mM [Mg2 ]o than that of Mg2  efflux. Alternatively, facilitation of the Na -independent Mg2  efflux through the TRP channels, if it occurs under condition of a reversed electrochemical gradient of Mg2 , could play a role in the acquisition of Mg2  tolerance. It should be noted, however, that Mg2  extrusion in the absence of extracellular Na  (possibly via the Na -independent passive pathway) was similar in the 101Mg-tolerant and wild-type cells and was very slow even in the absence of extracellular Ca2 , in which Mg2  permeation through the channels was enhanced (Fig. 4B). Thus suppression of passive Mg2  influx or enhancement of passive Mg2  extrusion, if any, does not seem to explain the difference between the 101Mg-tolerant cells and the wild-type cells.& |+ [5 @& ^3 P, B$ _( w

Changes in buffering and sequestration of intracellular Mg2  do not seem to be reconcilable with the observed effects of extracellular Na  and imipramine, unless these mechanisms are highly dependent on Na  and imipramine; [Mg2 ]i of the 101Mg-tolerant cells was markedly reduced in the absence of extracellular Na  or in the presence of imipramine (200 μM) to the levels similar to those observed in the wild-type cells (Fig. 6).
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The average levels of basal [Mg2 ]i estimated at normal [Mg2 ]o of 1 mM were in the submillimolar range in both the 101Mg-tolerant and wild-type cells, as reported with various methods in a number of different cell types (for review, see Ref. 14). In the high [Mg2 ]o conditions (51 mM), the average [Mg2 ]i of the wild-type MCT cells was increased above 1.0 mM, whereas that of the 101Mg-tolerant cells remained lower than 1.0 mM in the presence of extracellular Na . The lower [Mg2 ]i found in the 101Mg-tolerant cells probably accounts for, at least in part, their acquisition of high-Mg2  tolerance. However, it is also possible that there are other changes in intracellular Mg2  metabolism (i.e., intracellular binding and sequestration) in the 101Mg-tolerant cells that makes the cells resistant to high [Mg2 ]o. It should also be noted that some of genetic changes in the 101Mg-tolerant cells could result from, rather than cause, the Mg2  tolerance. Further studies are required to determine the precise mechanisms of the Mg2  tolerance.

Enhanced Mg2  extrusion in the 101Mg-tolerant cells. The present results clearly indicate that the Na -dependent net Mg2  efflux can significantly lower [Mg2 ]i of the 101Mg-tolerant cells within several minutes. Because influx of Mg2  appears to be rate limited by low permeability of the cell membrane for Mg2 , the enhanced net Mg2  efflux in the 101Mg-tolerant cells observed in the present study likely reflects, for the most part, the active Mg2  extrusion activity. The contribution of the Na /Ca2  exchanger on the enhanced Mg2  extrusion is probably, if any, minor, because the extracellular Na -dependent changes in [Ca2 ]i (as judged from fura 2 fluorescence) appear to be similar in the 101Mg-tolerant and the wild-type cells (Fig. 7).! _, R7 F- b# Y5 c% b

The Mg2  extrusion from the 101Mg-tolerant cells had a K1/2 value for Na  of 25 mM; i.e., the transport was half-activated at 25 mM [Na ]o (Fig. 5). This K1/2 value is similar to those reported in chicken erythrocytes (25 mM; Ref. 7), membrane vesicles from rabbit ileum (16 mM; Ref. 10), and smooth muscle of guinea pig tenia cecum (27 mM; Ref. 16). Thus the extracellular Na  dependence of Mg2  extrusion observed in the present study is consistent with those previously reported, although different estimates for K1/2 have also been reported in different cell types (for review, see Refs. 5 and 15). The Hill coefficient of 2 for the relation between [Na ]o and the rate of Mg2  extrusion (Fig. 5) could be explained by Na /Mg2  exchange with a stoichiometry of 2:1. However, further studies are necessary to establish the stoichiometry of the transport.
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Imipramine inhibited the Na -dependent Mg2  extrusion with half inhibition occurring between 50 and 200 μM (Fig. 6), the range roughly compatible to reported IC50 values of the agent for the putative Na /Mg2  exchange in human red blood cells (25 μM; Ref. 4), ferret red blood cells (
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Conclusion.
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We have successfully established a mutant strain of MCT cells that can grow in the culture media containing very high Mg2  concentrations (>100 mM). Optical measurements of [Mg2 ]i revealed enhanced Mg2  extrusion activity from the 101Mg-tolerant cells that was dependent on [Na ]o and was inhibited by imipramine. These properties of the Mg2  transport are consistent with those reported for the Na /Mg2  exchange in various cell types. We conclude that the 101Mg-tolerant cells established in the present study may be useful to identify Mg2  transporter molecules and to understand molecular mechanisms of the Na -dependent transport of Mg2 .

GRANTS
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This study was supported by grants from The Salt Science Research Foundation (0039), the Uehara Memorial Foundation, a Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science (14370016), and "High-Tech Research Center" Project for Private Universities, with matching fund subsidy from Ministry of Education, Culture, Sports, Science, and Technology, 2003–2007.; F6 L- U5 S% P
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ACKNOWLEDGMENTS

The authors are indebted to Prof. J. Patrick Barron of the International Medical Communications Center of Tokyo Medical University for review of this manuscript.

FOOTNOTES

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.

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