Sodium gradient-dependent transport of magnesium in rat ventricular myocytes

Michiko Tashiro1 and Masato Konishi1,2

1 Department of Physiology, The Jikei University School of Medicine, Tokyo 105 - 8461, and 2 Department of Physiology, Tokyo Medical University, Tokyo 160-8402, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS.
DISCUSSION
REFERENCES

Cytoplasmic concentration of Mg2+ ([Mg2+]i) was measured with a fluorescent indicator furaptra in ventricular myocytes enzymatically dissociated from rat hearts (25°C). To study Mg2+ transport across the cell membrane, cells were treated with ionomycin in Ca2+-free (0.1 mM EGTA) and high-Mg2+ (10 mM) conditions to facilitate passive Mg2+ influx. Rate of rise of [Mg2+]i due to the net Mg2+ influx was significantly smaller in the presence of 130 mM extracellular Na+ than in its absence. We also tested the extracellular Na+ dependence of the net Mg2+ efflux from cells loaded with Mg2+. After [Mg2+]i was raised by ionomycin and high Mg2+ to the level 0.5-0.6 mM above the basal value (~0.7 mM), washout of ionomycin and lowering extracellular [Mg2+] to 1.2 mM caused rapid decline of [Mg2+]i in the presence of 140 mM Na+. This net efflux of Mg2+ was completely inhibited by withdrawal of extracellular Na+ and was largely attenuated by imipramine, a known inhibitor of Na+/Mg2+ exchange, with 50% inhibition at 79 µM. The relation between the rate of net Mg2+ efflux and extracellular Na+ concentration ([Na+]o) had a Hill coefficient of 2 and [Na+]o at half-maximal rate of 82 mM. These results demonstrate the presence of Na+ gradient-dependent Mg2+ transport, which is consistent with Na+/Mg2+ exchange, in cardiac myocytes.

Na+/Mg2+ exchange; cardiac muscle; antiport; magnesium; sodium


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS.
DISCUSSION
REFERENCES

CYTOPLASMIC FREE CONCENTRATION of Mg2+ ([Mg2+]i) in mammalian cardiac myocytes has been estimated with various methods and appears to be in the range of 0.5-1.0 mM, well below electrochemical equilibrium across the cell membrane (7, 19, 27, 31). Since it has been reported that submillimolar [Mg2+]i significantly influences many intracellular processes of cardiac muscles, including adenylate cyclase activity (2), K+ channels (see Refs. 1 and 25 for reviews), excitation-contraction coupling (36), Ca2+ sensitivity of myofilaments (8), and Ca2+ binding to intracellular sites (10, 20), [Mg2+]i must be tightly regulated by active extrusion from the cell to balance any passive leak influx of Mg2+. Na+/Mg2+ exchange may play an essential role as an active Mg2+ extrusion pathway in many types of cells (for review see Refs. 12 and 30), but experimental evidence so far obtained in cardiac muscle is very controversial. The concept of the existence of Na+/Mg2+ exchange has been supported by measurements of the net Mg2+ fluxes by atomic absorption spectroscopy (29) and also by measurements of [Mg2+]i either by ion-selective microelectrodes (14) or a fluorescent indicator (17). However, other studies with improved ion-selective microelectrodes (6, 7) and fluorescent indicators (6, 18, 26, 31) failed to provide any evidence for a Na+ gradient-dependent Mg2+ efflux in cardiac muscle.

This study describes [Mg2+]i measurements with a fluorescent indicator carried out to seek evidence for Na+/Mg2+ exchange in cardiac myocytes. The results show that Na+-dependent changes in [Mg2+]i are unmasked after facilitation of passive Mg2+ influx by an ionophore and strongly suggest the existence of a Na+ gradient-dependent Mg2+ efflux.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS.
DISCUSSION
REFERENCES

General. All experiments were carried out on single ventricular myocytes enzymatically isolated from male Wistar rats (250-300 g), as previously described (18, 20). After enzymatic digestion with 0.2 mg/ml collagenase (collagenase S-1; Nitta Zerachin, Tokyo, Japan) and 0.04 mg/ml protease (type XIV; Sigma, St. Louis, MO) in the presence of 0.6 mg/ml BSA (Sigma), cells were stored in 0.2 mM CaCl2-containing Tyrode solution at 6°C until used. Cells were placed in an experimental chamber on the stage of an inverted microscope (Diaphot; Nikon, Tokyo, Japan) and superfused with normal Tyrode solution containing (in mM) 138 NaCl, 5.9 KCl, 2.4 CaCl2, 1.2 MgCl2, 11.8 glucose, and 5 HEPES (pH 7.4). Only quiescent rod-shaped cells that gave an all-or-none response to a 5-ms field stimulation were used for the experiments. After the indicator was loaded by incubation of the cells with 4 µM furaptra-AM for 10 min at room temperature and washout of the AM ester for at least 10 min, fluorescence measurements were carried out under continuous flow of the perfusate at 25°C.

The apparatus, methods for fluorescence measurements, and analysis have been described previously (22, 34, 35). Briefly, excitation light beams of 350 and 382 nm were switched at 100 Hz and focused with a ×40 objective (CF Fluor 40, Nikon), and the emitted fluorescence at 500 nm (40 nm full width at half-maximum) at each excitation wavelength was measured from single myocytes. The background fluorescence was estimated from the measurement before the indicator loading (see below) and was subtracted from the total fluorescence measured after the indicator loading to calculate indicator fluorescence intensity at each excitation wavelength and the ratio of the indicator fluorescence intensities (R). Basal [Mg2+]i was calculated from the basal R of furaptra measured at the beginning of each experiment.

Calibration of furaptra signals. The ratio of furaptra fluorescence intensities measured with excitation at 382 and 350 nm [R = F(382)/F(350)] was used as a Mg2+-related signal. Slow drift of the optical instruments (e.g., aging of the lamp) was corrected by occasional measurement of R in a Ca2+-Mg2+-free buffer solution (in mM: 140 KCl, 10 NaCl, 1 EDTA, 1 EGTA, 0.025 furaptra, and 10 PIPES, pH 7.1) as a standard. All values of the measured R were normalized to the standard R value taken with identical optics and were converted to [Mg2+]i with the standard equation
[Mg<SUP>2+</SUP>]<SUB>i</SUB> = <IT>K</IT><SUB>D</SUB>[(R − R<SUB>min</SUB>)&cjs0823;  (R<SUB>max</SUB> − R)]
where Rmin and Rmax are the R values at zero [Mg2+]i and saturating [Mg2+]i, respectively, and KD is the dissociation constant. We used parameter values previously estimated in smooth muscle cells of tenia at 25°C: Rmin = 0.986, Rmax= 0.199 and KD = 5.43 mM (34). The calibration procedure also included a correction for the small offset of R (+0.035) found in cardiac myocytes in a previous study (34). This offset was observed shortly after application of an ionophore cocktail containing Br-A-23187 and other ionophores, and was attributed to an artifact unrelated to [Mg2+]i. Direct effects of imipramine (200 µM) and ionomycin (10 µM) on the furaptra R were tested at 0-4 mM [Mg2+] in vitro and were found to be negligible. It has been reported that equimolar substitution of Na+ by N-methyl-D-glucamine (NMDG+) up to 40 mM has little influence on the furaptra R in solutions containing 0-4 mM [Mg2+] (35).

We found that ionomycin caused a slow decrease in the cell autofluorescence (Fig. 1). Cell autofluorescence excited at either 350 or 380 nm decreased exponentially with time of ionomycin treatment (time constant for the decay: 12.4 min at 350 nm; 21.8 min at 380 nm) and partially recovered after ionomycin washout (time constant for the recovery: 15.0 min at 350 nm; 4.5 min at 380 nm). The analysis of [Mg2+]i measurements from the cells treated with ionomycin for 30-60 min (see Figs. 3-7) therefore included correction of the cell autofluorescence; we calculated, at each wavelength, the autofluorescence at any time during the [Mg2+]i measurement run using the fitted parameters of the exponential function for the decay and the recovery (see legend to Fig. 1).


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Fig. 1.   Long-term observation of cell autofluorescence, cell F(lambda ), excited at 350 nm (A) and 382 nm (B). Intensities of autofluorescence relative to values measured just before ionomycin application (the first data points in A and B) were plotted as a function of time during application and washout of ionomycin. Ionomycin (10 µM) was added to the high-Mg2+ solution (10 mM) containing either 130 mM Na+ (5 cells) or 130 mM N-methyl-D-glucamine (NMDG+; 5 cells) for the periods indicated by the horizontal bars. Cells were then perfused with Ca2+-free Tyrode solution containing 130 mM Na+. Values of F(350) were 0.551±0.042 (Na+) and 0.544±0.052 (NMDG+) at time = 30 min, and were 0.645±0.047 (Na+) and 0.598±0.032 (NMDG+) at time = 50 min. Values of F(382) were 0.721±0.057 (Na+) and 0.763±0.073 (NMDG+) at time = 30 min, and were 0.780±0.090 (Na+) and 0.773±0.052 (NMDG+) at time = 50 min. Because the ionomycin effect was not significantly different in the presence and in the absence of extracellular Na+, the data from the 10 cells were treated as a single data set; each symbol represents a mean (±SE) value measured from the total of 10 cells. Solid lines are the least-squares fit of the mean values by an exponential (plus constant) function of the form F(lambda ) = c + a × exp(-t/tau ), where t is time after the first data point in the fitted range, and tau  is a time constant. Constants c and a give, respectively, an offset and a scaling factor. At each wavelength (lambda ), four data points between 0 and 30 min () were fitted for the decay during the ionomycin treatment, and three data points between 30 and 50 min (the last  and two open circle ) were fitted for the recovery after washout.

Solutions and chemicals. Ca2+-free Tyrode solution contained 0.1 mM EGTA replacing 2.4 mM CaCl2 of normal Tyrode solution. High-Mg2+ Tyrode solution contained 10 mM MgCl2, 0 mM CaCl2, and 0.1 mM EGTA, with NaCl concentration reduced to 128 mM. In one experiment, Mg2+ concentration was raised to 30 mM by simple addition of 20 mM MgCl2 to the high-Mg2+ Tyrode solution without any osmotic compensation. High-K+ (55.9 mM) solutions with various Na+ concentrations ([Na+]; 0-90 mM) were made by equimolar substitution of NaCl with potassium methanesulfonate and sodium methanesulfonate to keep the [K+]×[Cl-] product constant. For Na+-free conditions, Na+ was substituted by equimolar NMDG+. Furaptra (tetrapotassium salt of mag-fura 2) and furaptra-AM (mag-fura 2-AM) were purchased from Molecular Probes (Eugene, OR). EGTA was obtained from Sigma Chemical. Ionomycin (Sigma Chemical) was dissolved from a 10 mM stock solution in DMSO (DOTITE Spectrosol; Dojindo, Kumamoto, Japan). Imipramine · HCl (Nacalai Tesque, Kyoto, Japan) was directly dissolved in the perfusates. All other chemicals were reagent grade.

Curve fitting and statistical analysis. Nonlinear least-squares fitting was carried out with the program Origin (version 5.0J; Microcal Software, Northampton, MA) that uses the Levenberg-Marquardt algorithm. Statistical values were given as means ± SE. The two-tailed Student's t-test was used for statistical comparison with the significance level set at P < 0.05.


    RESULTS.
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS.
DISCUSSION
REFERENCES

All [Mg2+]i measurements were carried out in Ca2+-free conditions (0.1 mM EGTA) to minimize any complications caused by changes in cytoplasmic [Ca2+] ([Ca2+]i; see DISCUSSION) and also to avoid Ca2+ overloading of the cells. Values of basal [Mg2+]i thus measured in the Ca2+-free Tyrode solution showed a Gaussian distribution (not shown) with a mean value of 0.71 mM (±0.01 mM, n = 128).

Effect of extracellular Na+ on [Mg2+]i in intact myocytes. Figure 2 shows the results of our initial experiments, in which [Mg2+]i was continuously measured from the same myocytes. Removal of extracellular Na+, with extracellular [Mg2+] ([Mg2+]o) increased to 10 mM, did not cause any significant change in [Mg2+]i during a period of 30 min (Fig. 2A). Following Na+-free perfusion, cytoplasmic [Na+] ([Na+]i) is expected to fall rapidly to a low level (3), dissipating the Na+ gradient across the cell membrane. High [Mg2+]o was employed so that the inward driving force of Mg2+ and consequently Mg2+ influx should be enhanced. One myocyte treated with even higher [Mg2+]o (30 mM) also showed no clear increase in [Mg2+]i (Fig. 2B), indicating no supporting evidence for the Na+ gradient-dependent Mg2+ transport. However, absence of any clear change in [Mg2+]i in these experiments can also be explained if Mg2+ permeability of the cell membrane is very low and therefore passive Mg2+ influx is so slow (even with the increased driving force) that inhibition of Mg2+ extrusion fails to cause detectable changes in [Mg2+]i in the time scale of these measurements. We therefore carried out experiments in which passive Mg2+ influx was greatly facilitated by the aid of ionomycin, an ionophore for divalent cations.


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Fig. 2.   Cytoplasmic concentration of Mg2+ ([Mg2+]i) measurement with extracellular perfusion of the Na+-free solution containing 10 mM Mg2+ (A) or 30 mM Mg2+ (B). Cells were initially perfused with Ca2+-free Tyrode solution, and extracellular Na+ and Mg2+ concentrations were changed for the periods indicated by the horizontal bars. Values of means ± SE from 3 cells are plotted in A, whereas values from single myocyte are plotted in B.

Effect of extracellular Na+ on ionomycin-induced rise of [Mg2+]i. Under Ca2+-free conditions, ionomycin is expected to facilitate Mg2+ influx (37), because Mg2+ is the only divalent cation present in the extracellular space. Perfusion of the myocytes with 10 µM ionomycin plus high [Mg2+]o (10 mM) caused a gradual and nearly linear increase in [Mg2+]i, probably due to the increased influx of Mg2+ (Fig. 3). Changes in [Mg2+]i (Delta [Mg2+]i) by 30 min treatment of ionomycin were clearly smaller in the presence of 130 mM extracellular Na+ than in its absence (Fig. 3). The rate of Na+-dependent Delta [Mg2+]i calculated as the difference between the values with or without extracellular Na+ at 30 min (Fig. 3) was, on average, 0.76 mM/30 min or 0.42 µM/s. The ionomycin-induced Delta [Mg2+]i was significantly reduced by lowering [Mg2+]o, as expected from the extracellular origin of the increased [Mg2+]i (Fig. 4). The rise of [Mg2+]i was substantial only in the absence of extracellular Na+ at normal [Mg2+]o (1.2 mM) and was virtually absent at 0.4 mM [Mg2+]o independent of extracellular Na+ (Fig. 4). These observations are qualitatively similar to those in smooth muscle cells of guinea pig tenia (see Fig. 3 of Ref. 35) and are consistent with the hypothesis that Mg2+ is extruded through a Na+ gradient-dependent pathway to cause smaller net influx of Mg2+. This hypothesis was further tested by observation of the Na+ dependence of net Mg2+ efflux in the following series of experiments.


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Fig. 3.   Effects of extracellular Na+ on rise of [Mg2+]i induced by ionomycin plus high extracellular [Mg2+]. Changes in [Mg2+]i are expressed as difference from basal level (Delta [Mg2+]i) and plotted as a function of time. Cells were initially perfused with Ca2+-free Tyrode solution, and 10 µM ionomycin was introduced with raised [Mg2+] (10 mM) either in the presence of 130 mM Na+ (+Na, down-triangle) or in the absence of extracellular Na+ (-Na, open circle ) for the period indicated by horizontal bar. All cells treated with ionomycin in the absence of Na+ and a part of the cells (8/42) treated with ionomycin in the presence of 130 mM Na+ (black-down-triangle ) were then reperfused with Ca2+-free Tyrode solution. Other cells were perfused with solutions of different compositions for the experiments shown in Figs. 5-7. Each symbol represents mean ± SE (n = 42 for down-triangle, n = 8 for black-down-triangle , n = 12 for open circle ).



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Fig. 4.   Extracellular Mg2+ concentration ([Mg2+]o) dependence of ionomycin-induced Delta [Mg2+]i. With [Mg2+]o set at 0.4, 1.2, and 10 mM either in the absence of Na+ (open bars) or in the presence of 130-140 mM Na+ (solid bars), Delta [Mg2+]i values at 30 min after application of 10 µM ionomycin were obtained from the experiments of the type shown in Fig. 3. Each column shows mean ± SE; from left to right, n = 6, 6, 5, 6, 42, and 12.

Net Mg2+ efflux from loaded myocytes. After the [Mg2+]i was raised by treatment with ionomycin and high [Mg2+]o (see above), washout of ionomycin by the Ca2+-free Tyrode solution containing 1.2 mM Mg2+ caused a rapid decrease in [Mg2+]i toward the basal level, indicating net Mg2+ efflux (Fig. 3). Figure 5 shows the results of experiments designed to specifically study the rate of decline of [Mg2+]i from the Mg2+-loaded cells. Myocytes were initially loaded with Mg2+ by treatment with ionomycin in the high-Mg2+ solution containing 10 mM Mg2+ and 130 mM Na+, as described above. Because the effects of ionomycin on cellular Mg2+ loading is quite variable from cell to cell, we appropriately adjusted the concentration (9-12 µM) and treatment time (25-60 min) of ionomycin to achieve similar Mg2+ loading: 0.4-0.7 mM (0.57±0.01 mM, n = 93) above the basal level. Washout of ionomycin and reduction of extracellular Mg2+ back to the normal level quickly decreased [Mg2+]i in the presence of 140 mM extracellular Na+ (open inverted triangles in Fig. 5A). On the other hand, [Mg2+]i did not decrease, or even slightly increase, in the absence of extracellular Na+ (open circles in Fig. 5A); this small increase in [Mg2+]i is probably due to the continued Mg2+ influx driven by membrane potential. [Mg2+]i started to decrease after reintroduction of 140 mM Na+ (open circles in Fig. 5A). The decay of [Mg2+]i in the presence of extracellular Na+ was approximately linear during the first 10 min but slowed thereafter as [Mg2+]i approached the basal level. We therefore analyzed the Delta [Mg2+]i over the initial 10 min in the following analysis.


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Fig. 5.   Effect of extracellular Na+ and imipramine on [Mg2+]i. A: after cells were loaded with Mg2+ by 9-12 µM ionomycin + 10 mM Mg2+, the perfusate was changed, at zero time, to one of the test solutions containing 1.2 mM Mg2+ with various Na+ concentrations for the period indicated by a horizontal bar. Cells were then washed by Ca2+-free Tyrode solution containing 1.2 mM Mg2+ and 140 mM Na+. Changes in [Mg2+]i (ordinate) are expressed as the difference from the level achieved at the end of loading: generally 1.2-1.3 mM [Mg2+]i (for details, see text). The test solution contained 0 mM Na+ (open circle , n = 8), 140 mM Na+ (down-triangle, n = 8), 0 mM Na+ + 200 µM imipramine (, n = 3), or 140 mM Na+ + 200 µM imipramine (black-down-triangle , n = 6). B: imipramine concentration dependence of Delta [Mg2+]i measured at 10 min after application of the test solutions with the protocol shown in A. Solid line indicates least-squares fit of Delta [Mg2+]i data () as a function of imipramine concentration ([X]) by the Hill-type curve with parameters shown in the panel: Delta [Mg2+]i = min + (max - min) {[X]N/(K1/2N + [X]N)}, where max and min denote, respectively, Delta [Mg2+]i in the presence of a saturating concentration of imipramine and in its absence, N is the Hill coefficient, and K1/2 is imipramine concentration that gives a midpoint value of Delta [Mg2+]i between min and max. In the range of independent variable values ([X]), the program iterated the fitting procedure until a set of 4 adjustable variables (max, min, K1/2, and N) that gave the least-squares sum of the error of Delta [Mg2+]i values was found. The 2 dotted lines show the levels of average Delta [Mg2+]i in the absence of imipramine at 0 mM [Na+]o (top) and 140 mM [Na+]o (bottom). In A and B, each symbol represents mean±SE of 3-8 cells.

We examined the effect of imipramine, a known inhibitor of Na+/Mg2+ exchange in erythrocytes (11, 13), on the decay rate of [Mg2+]i (closed symbols in Fig. 5A). Imipramine (200 µM) did not significantly influence Delta [Mg2+]i in the Na+-free solution (closed circles in Fig. 5A), but markedly slowed the decay of [Mg2+]i in the presence of 140 mM Na+ (closed inverted triangles in Fig. 5A). The effect of imipramine was quickly reversed after washout. The relation between imipramine concentration and Delta [Mg2+]i showed that > 90% of the Na+ gradient-dependent net Mg2+ efflux was inhibited by imipramine with a half-inhibitory concentration of ~80 µM (Fig. 5B).

In pilot experiments, we tested other ways that possibly influence the net Mg2+ efflux. Amiloride, a poorly selective blocker of Na+-related transporters, has been reported to inhibit Na+/Mg2+ exchange at millimolar concentrations in erythrocytes (13, 16). We found, however, that fluorescence of amiloride at such high concentrations significantly interfered with the optical measurements. Na+ substitution by Li+, instead of NMDG+, also had serious difficulties because of the direct interaction of Li+ on furaptra; substitution of 30 mM Na+ by 30 mM Li+ caused ~10% decrease in the furaptra R in the solution containing 0-1 mM [Mg2+] (not shown).

Na+ dependence of the net Mg2+ efflux was further studied by monitoring the decay of [Mg2+]i at various extracellular [Na+] ([Na+]o) levels between 0 and 140 mM (Fig. 6A). To obtain additional information on the influence of membrane potential, the experiments were repeated at high extracellular [K+] ([K+]o = 55.9 mM, Fig. 6B), in which the cell membrane was expected to depolarize. At both normal [K+]o and high [K+]o, lowering [Na+]o dose dependently reduced the Mg2+ efflux from the loaded cells, as indicated by the slower decline of [Mg2+]i (Fig. 6). Figure 7A shows a more complete analysis of the relation between [Na+]o and the rate of net Mg2+ efflux. At [Na+]o of 0 and 50 mM, the values of Delta [Mg2+]i were significantly more negative at high [K+]o (closed squares) than at normal [K+]o (open squares), suggesting that net Mg2+ efflux was facilitated by high [K+]o or cell membrane depolarization. (Statistical significance was also found between the Delta [Mg2+]i values at 90 mM [Na+]o + 55.9 mM [K+]o and those at 100 mM [Na+]o + 5.9 mM [K+]o.) Figure 7B displays the Na+-dependent Delta [Mg2+]i calculated, at each [K+]o level, by subtraction of Delta [Mg2+]i values in the absence of extracellular Na+ from those in its presence. At normal [K+]o, half activation of the Na+ gradient-dependent Mg2+ efflux occurred at ~80 mM [Na+]o. High [K+]o appeared to cause the shift of the curve toward lower [Na+]o, with no obvious change in the slope (n approx  2).


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Fig. 6.   Effect of extracellular Na+ and K+ on [Mg2+]i. Changes in [Mg2+]i from the loaded level (ordinate) were measured at 5.9 mM [K+]o (A) or 55.9 mM [K+]o (B). After cells were loaded with Mg2+, one of the test solutions containing 1.2 mM Mg2+ with various Na+ concentrations (marked in mM near the symbols) was introduced at zero time for 20 min. Each symbol represents mean±SE of 4-9 cells.



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Fig. 7.   Analysis of Delta [Mg2+]i from the experiments of the type shown in Fig. 6. A: extracellular Na+ dependence of Delta [Mg2+]i measured at 10 min after application of the test solutions containing either 5.9 mM K+ () or 55.9 mM K+ (). Solid line indicates the least-squares fit of the data at 5.9 mM [K+]o by the Hill-type curve (see below). B: Na+-dependent Delta [Mg2+]i calculated by subtraction of the values at zero [Na+]o for each data set and plotted as a function of [Na+]o. Solid line indicates the least-squares fit of the data at 5.9 mM [K+]o () by the Hill-type curve with parameters shown in the panel: Delta [Mg2+]i = max + (min - max) · {[Na]oN/(K1/2N + [Na]oN)}, where min and max denote, respectively, minimum and maximum values of Na+-dependent Delta [Mg2+]i, N is the Hill coefficient, and K1/2 is [Na+]o that gives a midpoint value of the Na+-dependent Delta [Mg2+]i between min and max.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS.
DISCUSSION
REFERENCES

The present experiments used fluorescence signals of a Mg2+ indicator furaptra to study Mg2+ transport across the cell membrane. This method allows noninvasive measurements of [Mg2+]i from single myocytes over a period of hours. A difficulty of [Mg2+]i measurement by furaptra lies in the calibration of fluorescence signals in terms of [Mg2+]i, because properties of furaptra are likely altered in cytoplasmic environments (21). The present calibration method of furaptra fluorescence signals relied on parameter values previously estimated in smooth muscle cells (34) and gave a mean value of the basal [Mg2+]i of 0.71 mM. Although obtained in Ca2+-free Tyrode solution, this value is probably also applicable to the [Mg2+]i in normal Tyrode solution containing 2.4 mM Ca2+, because our previous study showed that removal of extracellular Ca2+ had little influence on [Mg2+]i (18). The value of 0.71 mM is consistent with estimates of a 0.5-1.0 mM range with various methods (see introduction), but somewhat lower than our previous estimate, on average 1.13 mM calibrated under identical conditions in rat cardiac myocytes (34). The difference may be due to cell-to-cell variation in the small number of myocytes (n = 9) in the previous study and was not further considered here.

Although Delta [Mg2+]i measured in the present study was thought to reflect, in the most part, Mg2+ flux across the cell membrane (Fig. 4), alteration of intracellular Mg2+ binding or Mg2+ sequestration by organelles could also contribute to Delta [Mg2+]i. We therefore minimized the changes in [Ca2+]i and cytoplasmic pH that should affect Mg2+ buffering (23); changes in [Ca2+]i are expected to be negligible and changes in pH should also be minimal in Ca2+-free conditions (9). The previous study from this laboratory (18) has shown that, after ~10 min perfusion with a 0.1 mM EGTA-containing solution, neither Ca2+ release from the sarcoplasmic reticulum (by 25 mM caffeine) nor intracellular acidosis of ~0.4 pH unit (by 5% CO2) has little influence on [Mg2+]i measured with furaptra. Ca2+-free conditions should also minimize Mg2+ uptake by mitochondria, as shown by electron probe microanalysis (4), and Mg2+ uptake by the sarcoplasmic reticulum as a counter ion of Ca2+ release (33). We also avoided experimental conditions that induce Mg2+ release from intracellular organelles by stimulation of cAMP production (29, 30) or muscarinic stimulation (38). It is possible, however, that quantification of transmembrane Mg2+ flux based on the measured Delta [Mg2+]i suffers, because of uncertainties related to intracellular binding and sequestration of Mg2+.

Absence of Na+-dependent changes in [Mg2+]i in intact myocytes. While removal of extracellular Na+ (which should inhibit Na+ gradient-dependent Mg2+ extrusion) in the presence of high [Mg2+]o (which should increase leak influx of Mg2+) failed to show any significant changes in [Mg2+]i (Fig. 2), a large increase in cell membrane permeability to Mg2+ unmasks the Na+ gradient-dependent Mg2+ transport (Fig. 3). It is thus conceivable that very controversial findings reported concerning cardiac myocytes (see introduction) may result from variable Mg2+ permeability of the cell membrane in different experimental conditions (or cell conditions). Handy et al. (17) reported that Na+ withdrawal in the presence of 5 mM Mg2+ caused a small but clear increase in [Mg2+]i (~28 µM/min) in rat ventricular myocytes at 37°C. The difference in the results between the present study and Handy et al. (17) could be due simply to the difference in experimental temperature (37°C vs. 25°C), but other experimental conditions that somehow alter the cell membrane permeability to Mg2+ could also be involved.

Removal of extracellular Na+ should reverse the transmembrane electrochemical gradient of Na+ and could reverse the direction of Na+/Mg2+ exchange to raise [Mg2+]i, i.e., Mg2+ influx associated with Na+ efflux (15). The present results, although providing no evidence for the reversal, do not necessarily exclude the possible reversal of Na+ gradient-dependent Mg2+ transport. After removal of extracellular Na+, [Na+]i probably falls to a low level within several minutes (3), and transport may not be driven in the reversed direction by this low [Na+]i. Therefore, it is likely that extracellular Na+-dependent Delta [Mg2+]i shown in the present study is also influenced by changes in [Na+]i, as noted in our previous study (35).

Evidence for Na+/Mg2+ exchange. Extracellular Na+ suppressed net Mg2+ influx into ionomycin-treated cells and facilitated net Mg2+ efflux from Mg2+-loaded cells. Imipramine markedly inhibited net Mg2+ efflux only in the presence of extracellular Na+. These results are most likely explained by Na+ gradient-dependent Mg2+ efflux (or Na+/Mg2+ exchange). The putative Na+-Mg2+ exchange may play a role in long-term regulation of [Mg2+]i to prevent Mg2+ overloading of cardiac myocytes.

From the Na+ dependence of the ionomycin-induced Delta [Mg2+]i (Fig. 3), we could estimate transmembrane Mg2+ flux using values assumed for a cell surface-to-volume ratio of 0.63 µm-1 [surface area 1.23×104 µm2 (24); volume 1.95×104 µm3 (5)], a cytoplasm-to-cell volume ratio of 0.5 (32), and a cytoplasmic Mg2+ buffering capacity of 2.5 (23). With these values taken from the literature, Na+-dependent suppression in Delta [Mg2+]i of 0.42 µM/s (see RESULTS) would correspond to Mg2+ flux (net efflux) of 0.083 pmol · cm-2 · s-1. With the Na+-dependent Delta [Mg2+]i value of 28 µM/min estimated by Handy et al. (17) in rat ventricular myocytes at 5 mM [Mg2+]o and 37°C, a calculation under otherwise identical conditions yields a similar Mg2+ flux value of 0.092 pmol · cm-2 · s-1. A value of Na+ gradient-dependent Mg2+ flux somewhat lower than, but within the same order of magnitude of, the present estimate was reported previously in smooth muscle cells of guinea pig tenia at 10 mM [Mg2+]o and 25°C [0.026 pmol · cm-2 · s-1 (35)]. Thus our present results are in reasonable agreement with earlier measurements. An estimate of the Na+ gradient-dependent Mg2+ efflux could also be obtained from the Na+ dependence of [Mg2+]i decay from the loaded myocytes (Fig. 5). From the Na+-dependent Delta [Mg2+]i of -0.69 mM/10 min (a value at 140 mM [Na+]o in Fig. 7B), a calculated value for the Na+ gradient-dependent Mg2+ efflux would be 0.23 pmol · cm-2 · s-1 at 1.2 mM [Mg2+]o and 25°C. A 2.7 times greater value than that estimated above from the Na+ dependence of the ionomycin-induced Delta [Mg2+]i (0.083 pmol · cm-2 · s-1) may be due to lower [Mg2+]o (1.2 vs. 10 mM) and/or higher [Mg2+]i in the loaded myocytes; note that the rate of [Mg2+]i decay is significantly slowed as [Mg2+]i approaches the basal level (Fig. 5).

Handy et al. (17) reported an almost complete inhibition of Na+-dependent Delta [Mg2+]i by 10 µM imipramine in rat ventricular myocytes. It is not clear if the difference in temperature (37°C vs. 25°C) could entirely explain the much higher concentration of imipramine (a half-inhibitory concentration of ~80 µM) required in the present study (Fig. 5B). Our estimate is, however, roughly comparable to reported IC50 values of the agent for Na+/Mg2+ exchange in human erythrocytes [25 µM (11)] or ferret erythrocytes [<500 µM (13)]. The Hill coefficient of 2 to best explain the relation between imipramine concentration and Delta [Mg2+]i suggests the binding of two (or more) imipramine molecules to a putative transporter molecule.

The Hill coefficient of 2 was also obtained for the relation between [Na+]o and the rate of Mg2+ efflux (reflected in Delta [Mg2+]i), suggesting two (or more) Na+ binding sites on the transporter. If Mg2+ is extruded in exchange for two (or more) Na+, Na+/Mg2+ exchange should carry no net current that may be insensitive to cell membrane potential (or net inward current that may be inhibited by cell membrane depolarization). On the contrary, however, the leftward shift of the relation between [Na+]o and the rate of Mg2+ efflux by high [K+]o (Fig. 7B) suggest facilitation of Na+/Mg2+ exchange by cell membrane depolarization. These conflicting results are puzzling and could be explained if high [K+]o substantially lowers [Na+]i by facilitation of Na+-K+-ATPase, Na+/Mg2+ exchange being driven by the larger driving force for Na+. Alternatively, extracellular K+ may be directly involved in Mg2+ transport (28) in addition to the effect on membrane potential and [Na+]i. Further studies are required to stoichiometrically determine the exchange in cardiac myocytes.

In conclusion, the present results demonstrate the existence of a Na+ gradient-dependent Mg2+ efflux activity in rat cardiac myocytes. Being half-maximally activated by ~80 mM [Na+]o and inhibited by imipramine, this Mg2+ transport is consistent with Na+/Mg2+ exchange. Our results do not provide clear evidence for reversal of Mg2+ transport, i.e., Na+ gradient-dependent Mg2+ influx, in our experimental conditions.


    ACKNOWLEDGEMENTS

We thank Prof. Satoshi Kurihara of the Department of Physiology of the Jikei University School of Medicine for helpful comments and Prof. J. Patrick Barron of the International Medical Communications Center of Tokyo Medical University for reading the manuscript.


    FOOTNOTES

This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture, Japan.

Present address of M. Tashiro: Dept. of Internal Medicine, The Jikei Univ. School of Medicine, 3-25-8 Nishishinbashi, Minato-ku, Tokyo 105-8461, Japan.

Address for reprint requests and other correspondence: M. Konishi, Dept. of Physiology, Tokyo Medical Univ., 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan (E-mail: mkonishi{at}tokyo-med.ac.jp).

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.

Received 14 April 2000; accepted in final form 6 July 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS.
DISCUSSION
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