Loading rat heart myocytes with Mg2+ using low-[Na+] solutions

Abstract

The objective of our study was to investigate how Mg2+ enters mammalian cardiac cells. During this work, we found evidence for a previously undescribed route for Mg2+ entry, and now provide a preliminary account of its properties. Changes in Mg2+ influx into rat ventricular myocytes were deduced from changes in intracellular ionized Mg2+ concentration ([fMg2+]i) measured from the fluorescence of mag-fura-2 loaded into isolated cells. Superfusion of myocytes at 37 degrees C with Ca2+-free solutions with both reduced [Na+] and raised [Mg2+] caused myocytes to load with Mg2+. Uptake was seen with solutions containing 5 mm Mg2+ and 95 mm Na+, and increased linearly with increasing extracellular [Mg2+] or decreasing extracellular [Na+]. It was very sensitive to temperature (Q(10) > 9, 25--37 degrees C), was observed even in myocytes with very low Na+ contents, and stopped abruptly when external [Na+] was returned to normal. Uptake was greatly reduced by imipramine or KB-R7943 if these were added when [fMg2+]i was close to the physiological level, but was unaffected if they were applied when [fMg2+]i was above 2 mm. Uptake was also reduced by depolarizing the membrane potential by increasing extracellular [K+] or voltage clamp to 0 mV. We suggest that initial Mg2+ uptake may involve several transporters, including reversed Na+-Mg2+ antiport and, depending on the exact conditions, reversed Na+-Ca2+ antiport. The ensuing rise of [fMg2+]i, in conjunction with reduced [Na+], may then activate a new Mg2+ transporter that is highly sensitive to temperature, is insensitive to imipramine or KB-R7943, but is inactivated by depolarization.

Figure 1. Consecutive-loading protocol to assess the effects of external [Na⁺] on the rate of [fMg²⁺]_i increase

A myocyte containing mag-fura-2 was superfused at 37°C. The concentration of cytoplasmic ionized Mg²⁺ was calculated from the ratio of fluorescence intensities measured at 510 nm following excitation with light at wavelengths of 340 and 380 nm. Bars at the top of the figure represent solution changes and numbers indicate concentrations (mm). Dotted bars below the trace indicate periods when the superfusate was Ca²⁺ free. Where not specified, solution composition was as in normal Tyrode solution. Changes in [Na⁺] and [Mg²⁺] were balanced with NMDG to keep the osmotic pressure constant. The myocyte was first equilibrated in normal Tyrode solution and then Mg²⁺ loaded twice in succession by superfusion with Ca²⁺-free solutions containing 30 mm Mg²⁺. In the first instance, the solution contained 95 mm Na⁺ and in the second, it was Na⁺ free. Following Mg²⁺ loading, superfusion with normal Tyrode solution caused [fMg²⁺]_i to return to the baseline.

Figure 2. Rate of [fMg²⁺]_i rise depends on external [Na⁺]

Rates of [fMg²⁺]_i rise were measured in experiments utilizing the consecutive-loading protocol (e.g. Fig. 1). Each myocyte was loaded once using a solution containing 30 mm Mg²⁺ and 95 mm Na⁺ (control), and once with 30 mm Mg²⁺ and 0, 10 or 45 mm Na⁺ (test), the order of exposure being randomized. Normalized rates of [fMg²⁺]_i rise (test/control) were calculated for each experiment. Points represent the means ±s.e.m. of n experiments, where n is indicated on the figure. The line drawn by linear regression analysis of all normalized values (r²= 0.63, n = 28) predicts that there should be no change in [fMg²⁺]_i when [Na⁺] is 153 mm (extrapolated dotted line). Dashed lines indicate 95% confidence limits of regression. Mean rate of [fMg²⁺]_i rise with 95 mm Na⁺ and 30 mm Mg²⁺ was 0.25 ± 0.03 mm min⁻¹ (n = 14).

Figure 3. Consecutive-loading protocol to assess the effects of external [Mg²⁺] on the rate of [fMg²⁺]_i increase

A myocyte was twice loaded by removing Ca²⁺ from the superfusate and reducing [Na⁺] to 95 mm. During the first load, superfusate [Mg²⁺] was increased to 30 mm and during the second, to 5 mm. Explanation of bars and composition of solutions as in legend to Fig. 1.

Figure 4. Rate of [Mg²⁺]_i rise depends on external [Mg²⁺]

Rates of [fMg²⁺]_i rise were measured in consecutive-loading experiments (e.g. Fig. 3), each myocyte being loaded using solutions containing 95 mm Na⁺ and: control, 30 mm Mg²⁺; test, 5 or 15 mm Mg²⁺. The order of exposure was randomized. Normalized rates of [fMg²⁺]_i rise (test/control) were calculated for each experiment. Points represent the means ±s.e.m. of n experiments, where n is indicated on the figure. The line drawn by linear regression analysis of all normalized values (r²= 0.93, n = 18) predicts that there should be no change in [fMg²⁺]_i when external [Mg²⁺] is −0.1 mm (extrapolated dotted line). Dashed lines indicate 95% confidence limits of regression.

Figure 5. [fMg²⁺]_i can rise dramatically in Na⁺-depleted myocytes

A myocyte was superfused with a Na⁺-free Tyrode solution (but containing 1 mm Ca²⁺) for 13 min to reduce its internal [Na⁺] to a very low level. Calcium was then removed from the solution for 3 min before [Mg²⁺] was increased to 30 mm. After 4 min in this high-[Mg²⁺] medium, the cell was superfused with normal Tyrode solution. Explanation of bars and composition of solutions as in legend to Fig. 1.

Figure 6. Imipramine fails to prevent [fMg²⁺]_i rise in a Mg²⁺-loaded myocyte

A myocyte was Mg²⁺ loaded by superfusing it with Ca²⁺- and Na⁺-free Tyrode solution containing 30 mm Mg²⁺. When [fMg²⁺]_i approached 2 mm, the superfusate was switched to a ‘Mg²⁺-clamp’ solution that contained 1 mm Mg²⁺ (Ca²⁺ and Na⁺ free) to prevented further rise in [fMg²⁺]_i. After 5 min, superfusate [Mg²⁺] was increased to 30 mm for 5 min and then the myocyte was superfused with normal Tyrode solution. Imipramine (0.2 mm) was added to superfusates from the start of the Mg²⁺-clamp period until the end of the second loading phase. Rates of [fMg²⁺]_i rise during the two load periods were measured and compared. Explanation of bars and composition of solutions as in legend to Fig. 1.

Figure 7. Effects of KB-R7943 on [fMg²⁺]_i rise

Myocytes were superfused with Ca²⁺- and Na⁺-free loading solution containing 30 mm Mg²⁺. With some cells (A), 20 μm KB-R7943 (KBR) was added to the superfusate when [fMg²⁺]_i was well above 2 mm. With others (B), 20 μm KB-R7943 was added to the superfusate once it was clear that [fMg²⁺]_i was increasing, but well before it reached 2 mm. After sufficient time to establish the rate of [fMg²⁺]_i change, the cells were superfused with normal Tyrode solution. In both cases, [fMg²⁺]_i returned to the initial preload level. Explanation of bars and composition of solutions as in legend to Fig. 1.

Figure 8. High external [K⁺] slows the rate of [fMg²⁺]_i rise

After equilibration in normal Tyrode solution and superfusion with Ca²⁺-free Tyrode solution for 3 min, a myocyte was superfused with a high-[K⁺] loading solution containing (mm): Mg²⁺, 30; Na⁺, 70; and K⁺, 70 for 10 min. Superfusate [K⁺] was then reduced to 6 mm (replaced by NMDG) for 5 min, increased to 70 mm for 4 min, and then reduced to 6 mm for the rest of the experiment. Superfusate [Mg²⁺] was reduced to 1 mm at 27 min to stabilize [fMg²⁺]_i before superfusion with normal Tyrode solution started at 32 min. Rates of [fMg²⁺]_i rise were measured at both values of [K⁺]. Explanation of bars and composition of solutions as in legend to Fig. 1.

Figure 9. Membrane depolarization slows [fMg²⁺]_i rise

A myocyte containing mag-fura-2 was patch clamped in whole-cell configuration. Its membrane potential (V) was maintained at −80 mV while it was superfused with normal Tyrode solution and then with Ca²⁺-free Tyrode solution. On changing to a Na⁺- and Ca²⁺-free Tyrode solution containing 30 mm Mg²⁺, and with the potential still clamped at −80 mV, [fMg²⁺]_i started to rise rapidly. When [fMg²⁺]_i reached about 2 mm, the potential was clamped to 0 mV for 2 min, after which it was returned to −80 mV for the rest of the experiment. Patch pipette resistance was 3 MΩ in normal Tyrode solution when filled with internal solution, and series resistance was < 10 MΩ at the beginning of the experiment. The upper panel shows membrane potential and the middle panel clamp current (I_m). Explanation of bars and composition of solutions as in legend to Fig. 1.