Reduction of intracellular Mg2+ caused by reactive oxygen species in rat ventricular myocytes.

The concentration of intracellular free Mg2+ ([Mg2+]i) should be maintained strictly for the regulation of cellular functions. Since reactive oxygen species (ROS) are liable to increase in various pathological conditions and induce cellular damage, we investigated whether ROS affect intracellular Mg2+ homeostasis. We measured [Mg2+]i in ventricular myocytes from Wistar rats using the fluorescent indicator, mag-fura-2. The administration of hydrogen peroxide (H2O2) decreased [Mg2+]i in Ca2+-free Tyrode's solution. Intracellular free Mg2+ was also reduced by endogenous ROS as generated by pyocyanin, which was inhibited by pretreatment with n-acetyl cysteine (NAC). The rate of change in [Mg2+]i by 500 µM H2O2 in 5 min (on average, -0.61 µM/s) was independent of extracellular Na+, and intra- and extracellular Mg2+ concentrations. When extracellular Ca2+ was present, the rate of Mg2+ decrease was significantly reduced, on average, by ∼60%. The half-maximal effective concentration of H2O2 on the Mg2+ decrease was estimated to be between 400 and 425 µM. The Mg2+ decrease by H2O2 in the absence of Na+ was inhibited by 200 µM imipramine, a known inhibitor of Na+/Mg2+ exchange. We perfused rat hearts with the Ca2+-free Tyrode's solution containing H2O2 (500 µM, 5 min) on the Langendorff apparatus,. H2O2 stimulation increased Mg2+ concentration in the perfusate, suggesting the H2O2-induced decrease in [Mg2+]i was caused by Mg2+ extrusion. Collectively, these results suggest the existence of a Na+-independent Mg2+ efflux system activated by ROS in cardiomyocytes. The lower [Mg2+]i may in part be attributed to ROS-mediated cardiac dysfunction.

TRPM7 silencing attenuates Mg2+ influx in cardiac myoblasts, H9c2 cells.

TRPM7, a member of the melastatin subfamily of transient receptor potential channels, is suggested to be a potential candidate for a physiological Mg2+ channel. However, there is no direct evidence of Mg2+ permeation through endogenous TRPM7. To determine the physiological roles of TRPM7 in intracellular Mg2+ homeostasis, we measured the cytoplasmic free Mg2+ concentration ([Mg2+]i) in TRPM7-silenced H9c2 cells. [Mg2+]i was measured in a cluster of 8-10 cells using the fluorescent indicator, furaptra. TRPM7 silencing did not change [Mg2+]i in Ca2+-free Tyrode's solution containing 1 mM Mg2+. Increasing the extracellular Mg2+ to 92.5 mM raised [Mg2+]i in control cells (1.56 ± 0.19 mM) at 30 min, while this effect was significantly attenuated in TRPM7-silenced cells (1.12 ± 0.07 mM). The Mg2+ efflux driven by Na+ gradient was unaffected by TRPM7 silencing. These results suggest that TRPM7 regulates the rate of Mg2+ influx in H9c2 cells, although cytoplasmic Mg2+ homeostasis at basal conditions is unaffected by TRPM7 silencing.

Modulation of Mg2+ influx and cytoplasmic free Mg2+ concentration in rat ventricular myocytes.

To examine whether TRPM7, a member of the melastatin family of transient receptor potential channels, is a physiological pathway for Mg2+ entry in mammalian cells, we studied the effect of TRPM7 regulators on cytoplasmic free Mg2+ concentration ([Mg2+]i) of rat ventricular myocytes. Acutely isolated single cells were AM-loaded with the fluorescent indicator furaptra, and [Mg2+]i was estimated at 25 °C. After [Mg2+]i was lowered by soaking the cells with a high-K+ and Mg2+-Ca2+-free solution, [Mg2+]i was recovered by extracellular perfusion of Ca2+-free Tyrode's solution that contained 1 mM Mg2+. The initial rate of increase in [Mg2+]i was analyzed as the Mg2+ influx rate. The Mg2+ influx rate was increased by the TRPM7 activator, naltriben (2-50 µM), in a concentration-dependent manner with a half maximal effective concentration (EC50) of 24 µM. This EC50 value is similar to that reported for the activation of recombinant TRPM7 overexpressed in HEK293 cells. Naltriben (50 µM) caused little change in basal [Mg2+]i (~ 0.9 mM) in Ca2+-free Tyrode's solution, but significantly raised [Mg2+]i to 1.31 ± 0.03 mM in 94 min after the removal of extracellular Na+. Re-introduction of extracellular Na+ lowered [Mg2+]i back to the basal level even in the presence of naltriben. Application of 10 µM NS8593, an inhibitor of TRPM7, significantly lowered [Mg2+]i to 0.72 ± 0.03 mM in 50-60 min independent of extracellular Na+. The results suggest that Mg2+ entry through TRPM7 significantly contributes to physiological Mg2+ homeostasis in mammalian heart cells.

Physiological pathway of magnesium influx in rat ventricular myocytes.

Cytoplasmic free Mg(2+) concentration ([Mg(2+)]i) was measured in rat ventricular myocytes with a fluorescent indicator furaptra (mag-fura-2) introduced by AM-loading. By incubation of the cells in a high-K(+) (Ca(2+)- and Mg(2+)-free) solution, [Mg(2+)]i decreased from ? 0.9 mM to 0.2 to 0.5 mM. The lowered [Mg(2+)]i was recovered by perfusion with Ca(2+)-free Tyrode's solution containing 1 mM Mg(2+). The time course of the [Mg(2+)]i recovery was fitted by a single exponential function, and the first derivative at time 0 was analyzed as being proportional to the initial Mg(2+) influx rate. The Mg(2+) influx rate was inversely related to [Mg(2+)]i, being higher at low [Mg(2+)]i. The Mg(2+) influx rate was augmented by the high extracellular Mg(2+) concentration (5 mM), whereas it was greatly reduced by cell membrane depolarization caused by high K(+). Known inhibitors of TRPM7 channels, 2-aminoethoxydiphenyl borate (2-APB), NS8593, and spermine reduced the Mg(2+) influx rate with half inhibitory concentrations (IC50) of, respectively, 17 ?M, 2.0 ?M, and 22 ?M. We also studied Ni(2+) influx by fluorescence quenching of intracellular furaptra by Ni(2+). The Ni(2+) influx was activated by lowering intra- and extracellular Mg(2+) concentrations, and it was inhibited by 2-APB and NS8593 with IC50 values comparable with those for the Mg(2+) influx. Intracellular alkalization (caused by pulse application of NH4Cl) enhanced, whereas intracellular acidification (induced after the removal of NH4Cl) slowed the Mg(2+) influx. Under the whole-cell patch-clamp configuration, the removal of intracellular and extracellular divalent cations induced large inward and outward currents, MIC (Mg-inhibited cation) currents or IMIC, carried by monovalent cations likely via TRPM7 channels. IMIC measured at -120 mV was diminished to ? 50% by 100 ?M 2-APB or 10 ?M NS8593. These results suggest that TRPM7/MIC channels serve as a major physiological pathway of Mg(2+) influx in rat ventricular myocytes.

Mg(2+)- and ATP-dependent inhibition of transient receptor potential melastatin 7 by oxidative stress.

Transient receptor potential melastatin 7 (TRPM7) is a Ca(2+)- and Mg(2+)-permeable nonselective cation channel that contains a unique carboxyl-terminal serine/threonine protein kinase domain. It has been reported that reactive oxygen species associated with hypoxia or ischemia activate TRPM7 current and then induce Ca(2+) overload resulting in neuronal cell death in the brain. In this study, we aimed to investigate the molecular mechanisms of TRPM7 regulation by hydrogen peroxide (H2O2) using murine TRPM7 expressed in HEK293 cells. Using the whole-cell patch-clamp technique, it was revealed that the TRPM7 current was inhibited, not activated, by the application of H2O2 to the extracellular solution. This inhibition was not reversed after washout or treatment with dithiothreitol, suggesting irreversible oxidation of TRPM7 or its regulatory factors by H2O2 under whole-cell recording. Application of an electrophile, N-methylmaleimide (NMM), which covalently modifies cysteine residues in proteins, also inhibited TRPM7 current irreversibly. The effects of H2O2 and NMM were dependent on free [Mg(2+)]i; the inhibition was stronger when cells were perfused with higher free [Mg(2+)]i solutions via pipette. In addition, TRPM7 current was not inhibited by H2O2 when millimolar ATP was included in the intracellular solution, even in the presence of substantial free [Mg(2+)]i, which is sufficient for TRPM7 inhibition by H2O2 in the absence of ATP. Moreover, a kinase-deficient mutant of TRPM7 (K1645R) was similarly inhibited by H2O2 just like the wild-type TRPM7 in a [Mg(2+)]i- and [ATP]i-dependent manner, indicating no involvement of the kinase activity of TRPM7. Thus, these data suggest that oxidative stress inhibits TRPM7 current under pathological conditions that accompany intracellular ATP depletion and free [Mg(2+)]i elevation.

Magnesium homeostasis in cardiac myocytes of Mg-deficient rats.

To study possible modulation of Mg(2+) transport in low Mg(2+) conditions, we fed either a Mg-deficient diet or a Mg-containing diet (control) to Wistar rats for 1-6 weeks. Total Mg concentrations in serum and cardiac ventricular tissues were measured by atomic absorption spectroscopy. Intracellular free Mg(2+) concentration ([Mg(2+)]i) of ventricular myocytes was measured with the fluorescent indicator furaptra. Mg(2+) transport rates, rates of Mg(2+) influx and Mg(2+) efflux, were estimated from the rates of change in [Mg(2+)]i during Mg loading/depletion and recovery procedures. In Mg-deficient rats, the serum total Mg concentration (0.29±0.026 mM) was significantly lower than in control rats (0.86±0.072 mM) after 4-6 weeks of Mg deficiency. However, neither total Mg concentration in ventricular tissues nor [Mg(2+)]i of ventricular myocytes was significantly different between Mg-deficient rats and control rats. The rates of Mg(2+) influx and efflux were not significantly different in both groups. In addition, quantitative RT-PCR revealed that Mg deficiency did not substantially change mRNA expression levels of known Mg(2+) channels/transporters (TRPM6, TRPM7, MagT1, SLC41A1 and ACDP2) in heart and kidney tissues. These results suggest that [Mg(2+)]i as well as the total Mg content of cardiac myocytes, was well maintained even under chronic hypomagnesemia without persistent modulation in function and expression of major Mg(2+) channels/transporters in the heart.

[Magnesium and cardiac function].

Free magnesium ion (Mg(2 + )) is involved in numerous processes of cardiac function. However, mechanism of regulation by Mg(2 + ) has not been fully understood. Extracellular Mg(2 + ) can act on the external surface of the cell membrane, whereas intracellular Mg(2 + ) can exert its effects via many different sites : various enzymes, intracellular organella and internal surface of the cell membrane. In this article, we will briefly review the extracellular and intracellular effects of Mg(2 + ) on each step of E-C coupling of cardiac myocytes, in an attempt to integrate them into cardiac function.

KB-R7943 inhibits Na+-dependent Mg2+ efflux in rat ventricular myocytes.

Na(+)-dependent Mg(2+) efflux activity was studied with the fluorescent Mg(2+) indicator furaptra in the presence of various potential antagonists known to inhibit other transporters and channels. Among the compounds tested, KB-R7943, an inhibitor of Na(+)/Ca(2+) exchange, most potently inhibited the Na(+)/Mg(2+) exchange with half inhibitory concentrations (IC(50)) of 21 µM: (25°C) and 16 µM: (35°C). These IC(50) values were a factor of three to four lower than those of imipramine, a widely used inhibitor of Na(+)/Mg(2+) exchange. Apart from the inhibitory effect on Na(+)/Mg(2+) exchange, relatively high concentrations of KB-R7943 (100 µM: at 25°C and ≥20 µM: at 35°C), in combination with prolonged UV-illumination, caused cell shortening, probably because of the phototoxicity of the compound and the formation of rigor crossbridges. We conclude that KB-R7943 may be a useful tool to study cellular Mg(2+) homeostasis if care is taken to minimize its phototoxicity.

Metabolic inhibition strongly inhibits Na+-dependent Mg2+ efflux in rat ventricular myocytes.

We measured intracellular Mg2+ concentration ([Mg2+]i) in rat ventricular myocytes using the fluorescent indicator furaptra (25 degrees C). In normally energized cells loaded with Mg2+, the introduction of extracellular Na+ induced a rapid decrease in [Mg2+]i: the initial rate of decrease in [Mg2+]i (initial Delta[Mg2+]i/Deltat) is thought to represent the rate of Na+-dependent Mg2+ efflux (putative Na+/Mg2+ exchange). To determine whether Mg2+ efflux depends directly on energy derived from cellular metabolism, in addition to the transmembrane Na+ gradient, we estimated the initial Delta[Mg2+]i/Deltat after metabolic inhibition. In the absence of extracellular Na+ and Ca2+, treatment of the cells with 1 microM carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, an uncoupler of mitochondria, caused a large increase in [Mg2+]i from approximately 0.9 mM to approximately 2.5 mM in a period of 5-8 min (probably because of breakdown of MgATP and release of Mg2+) and cell shortening to approximately 50% of the initial length (probably because of formation of rigor cross-bridges). Similar increases in [Mg2+]i and cell shortening were observed after application of 5 mM potassium cyanide (KCN) (an inhibitor of respiration) for > or = 90 min. The initial Delta[Mg2+]i/Deltat was diminished, on average, by 90% in carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone-treated cells and 92% in KCN-treated cells. When the cells were treated with 5 mM KCN for shorter times (59-85 min), a significant decrease in the initial Delta[Mg2+]i/Deltat (on average by 59%) was observed with only a slight shortening of the cell length. Intracellular Na+ concentration ([Na+]i) estimated with a Na+ indicator sodium-binding benzofuran isophthalate was, on average, 5.0-10.5 mM during the time required for the initial Delta[Mg2+]i/Deltat measurements, which is well below the [Na+]i level for half inhibition of the Mg2+ efflux (approximately 40 mM). Normalization of intracellular pH using 10 microM nigericin, a H+ ionophore, did not reverse the inhibition of the Mg2+ efflux. From these results, it seems likely that a decrease in ATP below the threshold of rigor cross-bridge formation (approximately 0.4 mM estimated indirectly in the this study), rather than elevation of [Na+]i or intracellular acidosis, inhibits the Mg2+ efflux, suggesting the absolute necessity of ATP for the Na+/Mg2+ exchange.

Effects of intracellular and extracellular concentrations of Ca2+, K+, and Cl- on the Na+-dependent Mg2+ efflux in rat ventricular myocytes.

Intracellular Mg2+ concentration ([Mg2+]i) was measured in rat ventricular myocytes with the fluorescent indicator furaptra (25 degrees C). After the myocytes were loaded with Mg2+, the initial rate of decrease in [Mg2+]i (initial Delta[Mg2+]i/Deltat) was estimated upon introduction of extracellular Na+, as an index of the rate of Na+-dependent Mg2+ efflux. The initial Delta[Mg2+]i/Deltat values with 140 mM [Na+]o were essentially unchanged by the addition of extracellular Ca2+ up to 1 mM (107.3+/-8.7% of the control value measured at 0 mM [Ca2+]o in the presence of 0.1 mM EGTA, n=5). Intracellular loading of a Ca2+ chelator, either BAPTA or dimethyl BAPTA, by incubation with its acetoxymethyl ester form (5 microM for 3.5 h) did not significantly change the initial Delta[Mg2+]i/Deltat: 115.2+/-7.5% (seven BAPTA-loaded cells) and 109.5+/-10.9% (four dimethyl BAPTA loaded cells) of the control values measured in the absence of an intracellular chelator. Extracellular and/or intracellular concentrations of K+ and Cl- were modified under constant [Na+]o (70 mM), [Ca2+]o (0 mM with 0.1 mM EGTA), and membrane potential (-13 mV with the amphotericin-B-perforated patch-clamp technique). None of the following conditions significantly changed the initial Delta[Mg2+]i/Deltat: 1), changes in [K+]o between 0 mM and 75 mM (65.6+/-5.0% (n=11) and 79.0+/-6.0% (n=8), respectively, of the control values measured at 140 mM [Na+]o without any modification of extracellular and intracellular K+ and Cl-); 2), intracellular perfusion with K+-free (Cs+-substituted) solution from the patch pipette in combination with removal of extracellular K+ (77.7+/-8.2%, n=8); and 3), extracellular and intracellular perfusion with K+-free and Cl--free solutions (71.6+/-5.1%, n=5). These results suggest that Mg2+ is transported in exchange with Na+, but not with Ca2+, K+, or Cl-, in cardiac myocytes.

Intracellular and extracellular concentrations of Na+ modulate Mg2+ transport in rat ventricular myocytes.

Apparent free cytoplasmic concentrations of Mg2+ ([Mg2+]i) and Na+ ([Na+]i) were estimated in rat ventricular myocytes using fluorescent indicators, furaptra (mag-fura-2) for Mg2+ and sodium-binding benzofuran isophthalate for Na+, at 25 degrees C in Ca2+-free conditions. Analysis included corrections for the influence of Na+ on furaptra fluorescence found in vitro and in vivo. The myocytes were loaded with Mg2+ in a solution containing 24 mM Mg2+ either in the presence of 106 mM Na+ plus 1 mM ouabain (Na+ loading) or in the presence of only 1.6 mM Na+ to deplete the cells of Na+ (Na+ depletion). The initial rate of decrease in [Mg2+]i from the Mg2+-loaded cells was estimated in the presence of 140 mM Na+ and 1 mM Mg2+ as an index of the rate of extracellular Na+-dependent Mg2+ efflux. Average [Na+]i, when estimated from sodium-binding benzofuran isophthalate fluorescence in separate experiments, increased from 12 to 31 mM and 47 mM after Na+ loading for 1 and 3 h, respectively, and decreased to approximately 0 mM after 3 h of Na+ depletion. The intracellular Na+ loading significantly reduced the initial rate of decrease in [Mg2+]i, on average, by 40% at 1 h and by 64% at 3 h, suggesting that the Mg2+ efflux was inhibited by intracellular Na+ with 50% inhibition at approximately 40 mM. A reduction of the rate of Mg2+ efflux was also observed when Na+ was introduced into the cells through the amphotericin B-perforated cell membrane (perforated patch-clamp technique) via a patch pipette that contained 130 mM Na+. When the cells were heavily loaded with Na+ with ouabain in combination with intracellular perfusion from the patch pipette containing 130 mM Na+, removal of extracellular Na+ caused an increase in [Mg2+]i, albeit at a very limited rate, which could be interpreted as reversal of the Mg2+ transport, i.e., Mg2+ influx driven by reversed Na+ gradient. Extracellular Na+ dependence of the rate of Mg2+ efflux revealed that the Mg2+ efflux was activated by extracellular Na+ with half-maximal activation at 55 mM. These results contribute to a quantitative characterization of the Na+-Mg2+ exchange in cardiac myocytes.

Modulation of Mg2+ efflux from rat ventricular myocytes studied with the fluorescent indicator furaptra.

The fluorescent Mg(2+) indicator furaptra (mag-fura-2) was introduced into single ventricular myocytes by incubation with its acetoxy-methyl ester form. The ratio of furaptra's fluorescence intensity at 382 and 350 nm was used to estimate the apparent cytoplasmic [Mg(2+)] ([Mg(2+)](i)). In Ca(2+)-free extracellular conditions (0.1 mM EGTA) at 25 degrees C, [Mg(2+)](i) averaged 0.842 +/- 0.019 mM. After the cells were loaded with Mg(2+) by exposure to high extracellular [Mg(2+)] ([Mg(2+)](o)), reduction of [Mg(2+)](o) to 1 mM (in the presence of extracellular Na(+)) induced a decrease in [Mg(2+)](i). The rate of decrease in [Mg(2+)](i) was higher at higher [Mg(2+)](i), whereas raising [Mg(2+)](o) slowed the decrease in [Mg(2+)](i) with 50% reduction of the rate at approximately 10 mM [Mg(2+)](o). Because a part of the furaptra molecules were likely trapped inside intracellular organelles, we assessed possible contribution of the indicator fluorescence emitted from the organelles. When the cell membranes of furaptra-loaded myocytes were permeabilized with saponin (25 microg/ml for 5 min), furaptra fluorescence intensity at 350-nm excitation decreased to 22%; thus approximately 78% of furaptra fluorescence appeared to represent cytoplasmic [Mg(2+)] ([Mg(2+)](c)), whereas the residual 22% likely represented [Mg(2+)] in organelles (primarily mitochondria as revealed by fluorescence imaging). [Mg(2+)] calibrated from the residual furaptra fluorescence ([Mg(2+)](r)) was 0.6-0.7 mM in bathing solution [Mg(2+)] (i.e., [Mg(2+)](c) of the skinned myocytes) of either 0.8 mM or 4.0 mM, suggesting that [Mg(2+)](r) was lower than and virtually insensitive to [Mg(2+)](c). We therefore corrected furaptra fluorescence signals measured in intact myocytes for this insensitive fraction of fluorescence to estimate [Mg(2+)](c). In addition, by utilizing concentration and dissociation constant values of known cytoplasmic Mg(2+) buffers, we calculated changes in total Mg concentration to obtain quantitative information on Mg(2+) flux across the cell membrane. The calculations indicate that, in the presence of extracellular Na(+), Mg(2+) efflux is markedly activated by [Mg(2+)](c) above the normal basal level (approximately 0.9 mM), with a half-maximal activation of approximately 1.9 mM [Mg(2+)](c). We conclude that [Mg(2+)](c) is tightly regulated by an Mg(2+) efflux that is dependent on extracellular [Na(+)].

Regulation of L-type calcium current by intracellular magnesium in rat cardiac myocytes.

The effects of changing cytosolic [Mg(2+)] ([Mg(2+)](i)) on L-type Ca(2+) currents were investigated in rat cardiac ventricular myocytes voltage-clamped with patch pipettes containing salt solutions with defined [Mg(2+)] and [Ca(2+)]. To control [Mg(2+)](i) and cytosolic [Ca(2+)] ([Ca(2+)](i)), the pipette solution included 30 mM citrate and 10 mM ATP along with 5 mM EGTA (slow Ca(2+) buffer) or 15 mM EGTA plus 5 mM BAPTA (fast Ca(2+) buffer). With pipette [Ca(2+)] ([Ca(2+)](p)) set at 100 nM using a slow Ca(2+) buffer and pipette [Mg(2+)] ([Mg(2+)](p)) set at 0.2 mM, peak l-type Ca(2+) current density (I(Ca)) was 17.0 +/- 2.2 pA pF(-1). Under the same conditions, but with [Mg(2+)](p) set to 1.8 mM, I(Ca) was 5.6 +/- 1.0 pA pF(-1), a 64 +/- 2.8% decrease in amplitude. This decrease in I(Ca) was accompanied by an acceleration and a -8 mV shift in the voltage dependence of current inactivation. The [Mg(2+)](p)-dependent decrease in I(Ca) was not significantly different when myocytes were preincubated with 10 microM forskolin and 300 microM 3-isobutyl-L-methylxanthine and voltage-clamped with pipettes containing 50 microM okadaic acid, to maximize Ca(2+) channel phosphorylation. However, when myocytes were voltage-clamped with pipettes containing protein phosphatase 2A, to promote channel dephosphorylation, I(Ca) decreased only 25 +/- 3.4% on changing [Mg(2+)](p) from 0.2 to 1.8 mM. In the presence of 0.2 mM[Mg(2+)](p), changing channel phosphorylation conditions altered I(Ca) over a 4-fold range; however, with 1.8 mM[Mg(2+)](p), these same manoeuvres had a much smaller effect on I(Ca). These data suggest that [Mg(2+)](i) can antagonize the effects of phosphorylation on channel gating kinetics. Setting [Ca(2+)](p) to 1, 100 or 300 nM also showed that the [Mg(2+)](p)-induced reduction of I(Ca) was smaller at the lowest [Ca(2+)](p), irrespective of channel phosphorylation conditions. This interaction between [Ca(2+)](i) and [Mg(2+)](i) to modulate I(Ca) was not significantly affected by ryanodine, fast Ca(2+) buffers or inhibitors of calmodulin, calmodulin-dependent kinase and calcineurin. Thus, physiologically relevant [Mg(2+)](i) modulates I(Ca) by counteracting the effects of Ca(2+) channel phosphorylation and by an unknown [Ca(2+)](i)-dependent mechanism. The magnitude of these effects suggests that changes in [Mg(2+)](i) could be critical in regulating L-type channel gating.

Effects of membrane potential on Na+ -dependent Mg2+ extrusion from rat ventricular myocytes.

To study Mg2+ transport across the cell membrane, the cytoplasmic concentration of Mg2+ ([Mg2+](i)) in rat ventricular myocytes was measured with the fluorescent indicator furaptra (mag-fura-2) under Ca2+ -free conditions (0.1 mM EGTA) at 25 degrees C. The fluorescence ratio signal of furaptra was converted to [Mg2+](i) using calibration parameters previously estimated in myocytes (Watanabe and Konishi, Pflügers Arch 442: 35-40, 2001). After [Mg2+](i) was raised by loading the cells with Mg2+ in a solution containing 93 mM Mg(2+), the cells were voltage-clamped at a holding potential of -80 mV using the perforated patch-clamp technique with amphotericin B. At the holding potential of -80 mV, the reduction of extracellular Mg2+ to 1.0 mM caused a rapid decrease in [Mg2+](i) only in the presence of extracellular Na(+). The rate of the net Mg2+ efflux appeared to be dependent on the initial level of [Mg2+](i); the decrease in [Mg2+](i) was significantly faster in the myocytes markedly loaded with Mg2+. The rate of decrease in [Mg2+](i) was influenced little by membrane depolarization from -80 to -40 mV, but the [Mg2+](i) decrease accelerated significantly at 0 mV by, on average, approximately 40%. Hyperpolarization from -80 to -120 mV slightly but significantly slowed the decrease in [Mg2+](i) by approximately 20%. The results clearly demonstrate an extracellular Na(+)- and intracellular Mg2+ -dependent Mg2+ efflux activity, which is consistent with the Na(+)-Mg2+ exchange, in rat ventricular myocytes. We found that the apparent rate of Mg2+ transport depends slightly on the membrane potential: facilitation by depolarization and inhibition by hyperpolarization with no sign of reversal between -120 and 0 mV.