1Department of Physiology, Tokyo Medical University, Tokyo, Japan; and 2Department of Biochemistry II and 3Division of Nephrology and Hypertension, The Jikei University School of Medicine, Tokyo, Japan
Submitted 7 March 2005 ; accepted in final form 7 May 2005
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ABSTRACT |
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membrane transport; sodium/magnesium exchange
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.
Some of these results have been published previously in abstract form (22).
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MATERIALS AND METHODS |
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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).
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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.
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).
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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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 36,00043,000) was purchased from Amersham (Piscataway, NJ). All other chemicals were reagent grade.
Data analysis.
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 < 0.05, unless otherwise noted. Statistical values were given as means ± SD.
RESULTS
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 1012 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 2050% 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.
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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).
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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.
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 04 mM Mg2+ (16).
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).
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).
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.
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).
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).
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.
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 (<500 µM; Ref. 6), and rat cardiac myocytes (80 µM; Ref. 17). Overall, the Na+-dependent Mg2+ extrusion activity observed in the present study can be attributed to the Na+/Mg2+ exchange, similar to reports in other cell types.
Conclusion.
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+.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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REFERENCES |
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