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Originally published In Press as doi:10.1074/jbc.M307898200 on September 4, 2003

J. Biol. Chem., Vol. 278, Issue 48, 47491-47497, November 28, 2003
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Ca2+-dependent Modulation of Intracellular Mg2+ Concentration with Amiloride and KB-R7943 in Pig Carotid Artery*

Tadayuki Uetani{ddagger}, Tatsuaki Matsubara{ddagger}, Hideki Nomura§, Toyoaki Murohara{ddagger}, and Shinsuke Nakayama¶||

From the Departments of {ddagger}Cardiology, §Geriatrics, and Cell Physiology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan

Received for publication, July 21, 2003 , and in revised form, September 3, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It has long been recognized that magnesium is associated with several important diseases, including diabetes, hypertension, cardiovascular, and cerebrovascular diseases. In the present study, we measured the intracellular free Mg2+ concentration ([Mg2+]i) using 31P nuclear magnetic resonance (NMR) in pig carotid artery smooth muscle. In normal solution, application of amiloride (1 mM) decreased [Mg2+]i by ~12% after 100 min. Subsequent washout tended to further decrease [Mg2+]i. In contrast, application of amiloride significantly increased [Mg2+]i (by ~13% after 100 min) under Ca2+-free conditions, where passive Mg2+ influx is facilitated. The treatments had little effect on intracellular ATP and pH (pHi). Essentially the same Ca2+-dependent changes in [Mg2+]i were produced with KB-R7943, a selective blocker of reverse mode Na+-Ca2+ exchange. Application of dimethyl amiloride (0.1 mM) in the presence of Ca2+ did not significantly change [Mg2+]i, although it inhibited Na+-H+ exchange at the same concentration. Removal of extracellular Na+ caused a marginal increase in [Mg2+]i after 100–200 min, as seen in intestinal smooth muscle in which Na+-Mg2+ exchange is known to be the primary mechanism of maintaining a low [Mg2+]i against electrochemical equilibrium. In Na+-free solution (containing Ca2+), neither amiloride nor KB-R7943 decreased [Mg2+]i, but they rather increased it. The results suggest that these inhibitory drugs for Na+-Ca2+ exchange directly modulate Na+-Mg2+ exchange in a Ca2+-dependent manner, and consequently produce the paradoxical decrease in [Mg2+]i in the presence of Ca2+.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
From epidemiological statistics and clinical experience, it has long been recognized that magnesium is associated with several important diseases, for example, diabetes mellitus, hypertension, cardiovascular, and cerebrovascular diseases (1–5). Recent advances in techniques for magnesium measurements have made it possible to provide supporting evidence that the intracellular free Mg2+ concentration ([Mg2+]i) is responsive to relevant key hormones and neurotransmitters (6–10).

Amiloride,1 3,5-diamino-6-chloro-N-(diaminomethylene) pyrazinecarboxamide, is a prototype of a large group of compounds with a broad range of unique biological activities. Due to K+-sparing and natriuretic effects, this compound is clinically used as an antihypertensive drug. Furthermore, it inhibits many Na+-dependent transporters, for example Na+-H+, Na+-Ca2+ exchanges etc. (11, 12), this being expected to protect the heart from ischemic changes (13, 14). A more selective Na+-Ca2+ exchange inhibitor, KB-R7943, has recently been synthesized, and evidence for its cardioprotective effect is accumulating (15–17).

It has been pointed out that Na+-Ca2+ exchange shares many characteristic features with Na+-Mg2+ exchange (18). Accordingly, it is interesting to examine possible effects of amiloride and KB-R7943 on [Mg2+]i regulation, and in the present study, we measured [Mg2+]i in pig carotid artery smooth muscle using 31P NMR. Despite low time resolution, this technique enables stable measurements over a long period, and this is advantageous because Mg2+ is considered to be a chronic regulator, such long time measurements are usually required. Further, fluorescent Mg2+ indicators, another option for measurement of [Mg2+]i, were not suitable for this purpose, since amiloride emits light (19).

Na+-Mg2+ exchange is thought to be the major mechanism to pump out intracellular Mg2+. Thus, possible inhibitors of Na+-Mg2+ exchange are expected to increase [Mg2+]i. The 31P NMR measurements in the present study, however, revealed that amiloride and KB-R7943 paradoxically decreased [Mg2+]i in a Ca2+-dependent manner. In addition, this effect persisted after washout for several minutes. A selective Na+-H+ exchange inhibitor, 5-(N,N-dimethyl)-amiloride (DMA), did not cause the same changes in [Mg2+]i. The underlying mechanisms are discussed.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation—Pig carotid arteries were collected at an abattoir (Nagoya Meat Hygiene Laboratory, Nagoya, Japan) in physiological saline solution, used as the normal solution for 31P NMR experiments. The arteries were stripped of fat and connective tissue, and cut into segments of ~3 cm in length. The lumen was exposed by cutting the artery segments into two strips along the longitudinal direction. The endothelium was removed by scratching with a cotton-tipped stick. The resultant pig carotid artery strips (~2 g wet weight) were (isometrically) mounted in a sample tube of 10-mm diameter.

31P NMR—The methods employed for the 31P NMR measurements were essentially the same as those used previously (20, 21). An NMR spectrometer (GSX270W, JEOL, Tokyo, Japan) was operated at 109.4 MHz for measurement of phosphorus compounds. The temperature of the sample tube was kept at 32 °C. Radiofrequency pulses corresponding to a flip angle of 30° were applied every 0.5 s, and 31P NMR spectra were normally obtained by accumulating 2500 signals (free induction decays: FIDs) over 25 min. Before Fourier transformation, a broadening factor of 20 Hz was applied to enhance the signal-to-noise ratio. Spectral peak resonances (frequencies) were measured relative to that of phosphocreatine (PCr) in ppm.

Experiments were started after equilibrating preparations under superfusion with normal solution for at least 100 min. In normal solution, six major peaks were observed: phosphomonoesters (PME), inorganic phosphate (Pi), PCr, and the {gamma}-, {alpha}-, and {beta}-peaks of ATP (Fig.1A). Concentrations of phosphorus compounds were estimated by integrating the spectral peaks (Scion Image, Frederick, MD) and by correcting with their saturation factors (Pi, 1.6; PCr, 1.36; {beta}-ATP, 1.7)



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  		FIG. 1.
Effects of amiloride on 31P NMR spectra. Each spectrum was obtained with 2,500 FIDs (free induction decays) accumulated over 25 min (A). After acquiring a control spectrum in normal solution (a), 1 mM amiloride was applied for 100 min, and spectrum b was obtained in the 75–100 min period. Subsequently, amiloride was washed out for 100 min. c, 75–100 min washout. The {beta}-ATP peaks were shown expanded on the right of each spectrum (B). The vertical line indicates the initial chemical shift of the {beta}-ATP peak. See text for assignment of the peaks.

 
Estimation of [Mg2+]i and pHi—Changes in intracellular pH (pHi) were monitored using the chemical shift of the Pi peak (pKa 6.70; chemical shifts of and and 5.72 ppm, respectively; Ref. 20), and employed to correct the [Mg2+]i estimation.

[Mg2+]i was estimated from the observed chemical shift of the {beta}-ATP peak ({delta}o{beta}) (22, 23) in Equation 1,

(Eq. 1)
where {delta}f{beta} and {delta}b{beta} are the chemical shifts of metal-free and Mg2+-bound forms of {beta}-ATP, respectively. We have previously shown {delta}f{beta}(pHi) and {delta}b{beta}(pHi) to be described as sigmoid functions (24) as in Equations 2 and 3.

(Eq. 2)

(Eq. 3)
Recently, McGuigan's group have shown that KD"MgATP" (in µM) at 25 and 37 °C are described as quadratic functions of pH (25) in Equations 4 and 5.

(Eq. 4)

(Eq. 5)
The pH function of KD"MgATP" (pHi) at 32 °C was derived from those at 25 and 37 °C (25) using the van't Hoff isochore in Equations 6 and 7.

(Eq. 6)

(Eq. 7)
TA, TB, and TC are absolute temperatures of 25, 37, and 32 °C, respectively. In the pHi range between 6.5 and 8, this pH function of KD"MgATP" (pH)i at 32 °C provides about 2-fold larger KD"MgATP" values than those obtained using the previously described methods for correcting KD"MgATP" with pHi (21).

In some experiments we also applied simultaneous estimation of [Mg2+]i and pHi from the chemical shifts of {beta}- and {gamma}-ATP (24), in order to reinforce the Ca2+-dependent modulation of [Mg2+]i regulation. For the chemical shift of {gamma}-ATP, an equation analogous to Equation 1 can be written as Equation 8.

(Eq. 8)

(Eq. 9)

(Eq. 10)
[Mg2+]i and pHi were estimated by solving Equations 1 and 8 simultaneously.

Solutions and Chemicals—The normal solution had the following composition (mM): NaCl,137.9; KHCO3 5.9; CaCl2 2.4; MgCl2 1.2; glucose 11.8; HEPES 5 (pH adjusted to 7.4–7.5 at 32 °C). The ionic composition was modified iso-osmotically. For example, in a Na+-free solution, Na+ was replaced with N-methyl-D-glucamine (NMDG) on an equimolar basis. The solutions used for 31P NMR measurements were normally aerated with 95% O2/5% CO2. When bicarbonate was replaced with Cl–, the solution was aerated with 100% O2. Amiloride was purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). 5-(N,N-Dimethyl)-amiloride (DMA) was from Sigma. KB-R7943 ((2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate) was kindly provided by Kanebo Ltd. (Osaka, Japan).

The effect of 1 mM amiloride on [Mg2+]i was examined in the present study (in pig carotid artery). Our previous experiments (21) revealed that this concentration of amiloride sufficiently block Na+-Mg2+ exchange. Preliminarily we examined the effect of KB-R7943 at 1–100 µM on [Mg2+]i. KB-R7943 up to 5 µM caused only a marginal decrease (by ~0.04 mM after 100 min in the presence of extracellular Ca2+). We thus used 50 µM KB-R7943 in the present study.

Statistics—Numerical data are expressed as mean ± S.E. Differences between groups with different experimental protocols were evaluated by use of ANOVA for repeated measures. When a significant difference was indicated between the groups, individual comparisons at the same time point were performed using a Student's unpaired-t test. For all evaluations and comparisons, a p value less than 0.05 was taken as indicating statistical significance.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In the smooth muscle of pig carotid artery 31P NMR was applied to measure intracellular phosphorus compounds. As shown in Fig. 1A, six major peaks were observed in 31P NMR spectra obtained in normal solution. The concentrations of PCr ([PCr]) and Pi ([Pi]) were 35.9 ± 4.0 and 27.2 ± 3.7%, respectively, relative to that of ATP ([ATP]) (n = 21). The [PCr] to [ATP] ratio is comparable to that reported for vascular smooth muscles (22, 26), and agrees well with the notion that this ratio in smooth muscle depends on the physiological function (27), i.e. [PCr]/[ATP] is smaller in tonic than phasic smooth muscles.

The chemical shifts of the Pi and {beta}-ATP peaks were 4.86 ± 0.01 and –16.21 ± 0.01 ppm, respectively (n = 21). From these chemical shifts, pHi and [Mg2+]i were estimated to be 7.01 ± 0.02 pH units and 0.76 ± 0.01 mM, respectively. The chemical shifts of {beta}- and {gamma}-ATP peaks were –2.490 ± 0.003 and –7.556 ± 0.003 ppm, respectively (n = 21). The [Mg2+]i value in pig carotid artery smooth muscle was smaller than those estimated using the same methods in intestinal smooth muscle (taenia caeci: 0.84 mM). This [Mg2+]i value is significantly lower than in phasic smooth muscles (28), suggesting that [Mg2+]i might play a role in characterizing smooth muscle function.

In the PME region, the PME-1 peak was always predominant (29), but a second peak was occasionally distinguishable: PME-1, 6.79 ± 0.01 ppm (n = 21); PME-2, 6.27 ± 0.01 ppm (n = 15). Taking the pHi (~7.0) into consideration (30), the chemical shifts of PME-1 and 2 indicate that they arise from phosphorylethanolamine and phosphorylcholine, respectively.

Amiloride Modulates [Mg2+]i in a Ca2+-dependent Manner—Amiloride is known to inhibit numerous Na+ transporters (11, 12), including Na+-Mg2+ exchange (31–33). We thus examined the effects of amiloride on [Mg2+]i regulation in pig carotid artery smooth muscle. As shown in Fig. 1B, the {beta}-ATP peak was significantly (p < 0.05) shifted toward lower frequency (from –16.21 ± 0.01 to –16.25 ± 0.02 ppm, n = 6) 100 min after application of 1 mM amiloride. (Shifts of the {beta}-ATP peak toward lower frequency correspond to decreases in [Mg2+]i and vice versa.) During the subsequent washout of amiloride for 100 min the {beta}-ATP peak did not return to the initial resonance frequency, but rather further shifted toward lower frequency in some of the carotid artery preparations.

Fig. 2 shows changes in [Mg2+]i (filled squares in A) and pHi (filled circles in B) induced by 1 mM amiloride application (n = 6) along with control experiments. Application of 1 mM amiloride decreased [Mg2+]i from 0.75 ± 0.01 mM to 0.66 ± 0.03 mM after 100 min (n = 6), corresponding to the lower frequency shift of the {beta}-ATP peak. The subsequent washout did not restore [Mg2+]i, but rather decreased it for 100 min (0.61 ± 0.03 mM 75–100 min during washout: statistically different from the [Mg2+]i value 75–100 min during application of amiloride, p < 0.05). In two of the six carotid artery preparations, the effect of prolonged washout was examined. [Mg2+]i was restored after 200 min (0.72 mM before application of amiloride; 0.62 mM after 75 min, and 0.68 mM after 200 min of washout). With respect to pHi regulation, pHi was not significantly changed during application and washout of amiloride (filled circles in Fig. 2B). On the other hand, the control experiments show that [Mg2+]i (open squares in Fig. 2A) and pHi (open circles in Fig. 2B) were stable during continuous superfusion with normal solution for 250 min (n = 5).



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  		FIG. 2.
Time course of changes in [Mg2+]i (filled squares in A) and pHi (filled circles in B) during application and washout of amiloride. The carotid artery preparations were superfused with normal solution containing Ca2+. Amiloride (1 mM) was added to the extracellular medium for 100 min (n = 6). In order to show the stability of [Mg2+]i (open squares in A) and pHi (open circles in B), five preparations were used for control experiments (superfused with normal solution throughout 250 min, n = 5). Each point was obtained from the accumulation of 2,500 FIDs over 25 min. Vertical bars represent S.E. values. The dotted line indicates [Mg2+]i value before application of amiloride. Asterisks indicate statistically significant differences (p < 0.05) compared with this [Mg2+]i value (before application of the drug), while crosses indicate statistically significant differences (p < 0.05) compared with the [Mg2+]i before washout of the drug: Only the four data points obtained during washout were used in the latter.

 
ATP is an important Mg2+ buffer in the intracellular environment. Decrease in [ATP] would release Mg2+ and vice versa. However, no significant change was observed during the application and washout of amiloride (Table I, A).


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  		TABLE I
Changes in the concentration of high energy phosphates

Values are expressed relative to [ATP] in control (before application of drugs).

 
The effect of amiloride on [Mg2+]i regulation was also examined in the absence of Ca2+. The time course of change is shown in Fig. 3. Removal of extracellular Ca2+ for 100 min had little effect on [Mg2+]i (0.76 ± 0.01 mM). When 1 mM amiloride was applied to the Ca2+-free solution, the maximal [Mg2+]i increase (0.81 ± 0.03 mM, n = 7) was observed 50–75 min. The data points with statistical significance (p < 0.05) compared with the [Mg2+]i value before amiloride application, are indicated by asterisks. During the subsequent washout of amiloride [Mg2+]i was quickly restored (0.74 ± 0.02 mM during 0–25 min; 0.72 ± 0.02 mM during 75–100 min). Neither pHi nor [ATP] were significantly changed throughout the experiment.



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  		FIG. 3.
Time course of changes in [Mg2+]i (filled squares) and pHi (open circles) upon application of amiloride in Ca2+-free solution (n = 7). The preparations were initially superfused with normal solution for more than 100 min, and then superfused with Ca2+-free solution for 50 min. The dotted line indicates the [Mg2+]i level before application of amiloride. Asterisks indicate statistically significant differences (p < 0.05) compared with this [Mg2+]i value.

 
Previously, we reported simultaneous estimation of [Mg2+]i and pHi from the chemical shifts of {beta}- and {gamma}-ATP. In order to reinforce the results obtained with amiloride, i.e. Ca2+-dependent modulation of [Mg2+]i, we also employed this estimation (see "Materials and Methods"). In Table II, [Mg2+]i and pHi values estimated using this method are compared with those estimated from the chemical shifts of {beta}-ATP and Pi (the estimation mainly used in the present study). Both estimations showed essentially the same changes in [Mg2+]i and pHi, irrespective of Ca2+.


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  		TABLE II
Estimation of [Mg2+]i and pHi

[Mg2+]i and pHi values estimated using two methods: 1) from the chemical shifts of {beta}-and {gamma}-ATP, or 2) from the chemical shifts of {beta}-ATP and Pi (see "Materials and Methods" for details). [Mg2+]i and pHi values for applications of amiloride in the presence of Ca2+ were compared in A. In B, [Mg2+]i and pHi values were for the effects of amiloride in the absence of Ca2+.

 
A Na+-H+ Exchange Inhibitor DMA Had Little Effect on [Mg2+]i —To determine which Na+-transporter is responsible for the decrease in [Mg2+]i, we examined the effect of an amiloride derivative, DMA which has high inhibitory activity toward Na+-H+ exchange (11, 12). In normal solution (containing 5.9 mM ), application of 0.1 mM DMA did not significantly change [Mg2+]i for 100 min (filled squares in Fig. 4). Also, this treatment did not significantly altered either pHi (open circles in Fig. 4) or [ATP] during application and subsequent washout (Table 1, B). When bicarbonate was removed from the extracellular solution (aerated with 100% O2), application of 0.1 mM DMA significantly lowered pHi (from 7.00 ± 0.01 to 6.80 ± 0.01 after 100 min, n = 3), with little influence on [Mg2+]i (0.74 ± 0.05 mM before application; 0.76 ± 0.01 mM 75–100 min during application). These results suggested that pHi was maintained by symporter (34, 35) under superfusion with the normal solution, when Na+-H+ exchange was inhibited by DMA.



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  		FIG. 4.
Values for [Mg2+]i (filled squares) and pHi (open circles) during application and washout of DMA in normal solution (n = 4). The dotted line indicates the [Mg2+]i level before application of DMA. There were no statistical significant difference in [Mg2+]i and pHi values compared with those before application of this drug (p > 0.05).

 
KB-R7943 Produced Essentially the Same Paradoxical Decrease in [Mg2+]i—KB-R7943 is a newly synthesized inhibitor of Na+-Ca2+ exchange (36, 37), and Na+-Mg2+ is known to share many characteristic features with Na+-Ca2+ exchange (18). In normal solution containing 2.4 mM Ca2+, application of 50 µM KB-R7943 decreased [Mg2+]i from 0.76 ± 0.02 to 0.70 ± 0.01 mM (statistically significantly: p < 0.05, n = 5) after 100 min (filled squares in Fig. 5A). The subsequent washout further decreased [Mg2+]i (to 0.65 ± 0.02 mM 25–50 min during washout, statistically different from the [Mg2+]i value 75–100 min during application of KB-R7943, p < 0.05). In the absence of extracellular Ca2+, application of KB-R7943 (50 µM) caused a marginal increase in [Mg2+]i (n = 4, Fig. 5B). (The three [Mg2+]i values 0–75 min during application of KB-R7943, statistically differ from that before application of the drug in the absence of Ca2+. Furthermore, each [Mg2+]i value during application of KB-R7943 is statistically significantly greater (p < 0.05) in the absence than the presence of Ca2+.) Whether the extracellular solution contained Ca2+ or not, no significant change was observed in pHi (open circles in Fig. 5) and [ATP] (Table 1, C) ([ATP] data in the absence of Ca2+ not shown). In the presence or absence of Ca2+, KB-R7943 and amiloride evoked essentially the same responses on the parameters measurable with 31P NMR.



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  		FIG. 5.
Changes in [Mg2+]i (filled squares) and pHi (open circles) upon application of KB-R7943 in normal (A, n = 5) and Ca2+-free solution (B, n = 4). The preparations used in B were initially superfused with normal solution for more than 100 min, and then superfused with Ca2+-free solution for 50 min. Asterisks indicate statistically significant difference (p < 0.05, versus the [Mg2+]i levels before application of the drug, indicated by dotted lines). Each dotted line indicates [Mg2+]i value before application of KB-R7943. Asterisks indicate statistically significant differences (p < 0.05) compared with the [Mg2+]i value before application of the drug, while crosses indicate statistically significant differences (p < 0.05) compared with the [Mg2+]i value before washout: Only the four data points obtained during washout were used in the latter.

 
Effect of Na+ Removal on [Mg2+]i Regulation—Fig. 6A shows effects of blocking Na+-dependent transporters by removal of extracellular Na+. Unlike the guinea pig taenia caeci case (24), the Pi peak was resolvable in pig carotid artery throughout the experiment. pHi was thus estimated from the chemical shift of Pi. In the presence of Ca2+, total substitution of extracellular Na+ with NMDG caused a small increase in [Mg2+]i (from 0.76 ± 0.02 to 0.86 ± 0.06 mM, n = 4) after 200 min, while pHi gradually decreased to 6.76 ± 0.04 (from 7.01 ± 0.01). The subsequent readmission of Na+ fully restored both [Mg2+]i and pHi within 50 min. These changes in [Mg2+]i and pHi agree well with the previous results observed in the guinea pig taenia caeci, in which Na+-Mg2+ exchange in the plasma membrane is the primary mechanism to maintain low [Mg2+]i (20, 26).



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  		FIG. 6.
Changes in [Mg2+]i (filled squares) and pHi (open circles) during removal and readmission of Na+ (in the presence of 2.4 mM Ca2+). Extracellular Na+ was substituted with equimolar NMDG (A, n = 4). In B, amiloride (1 mM) was added to the Na+-free solution of the same composition (n = 4). Each dotted line indicate [Mg2+]i value before removal of extracellular Na+. Asterisks indicate statistically significant differences (p < 0.05) compared with the [Mg2+]i value before Na+ removal.

 
In order to confirm that amiloride decreases [Mg2+]i through modulation of Na+-dependent [Mg2+]i regulatory mechanism(s), the drug (1 mM) was applied simultaneously with removal of extracellular Na+ (in the presence of 2.4 mM Ca2+). Fig. 6B shows changes in [Mg2+]i and pHi during application and subsequent washout. During exposure to the Na+-free solution containing 1 mM amiloride for 100 min, [Mg2+]i did not decrease, but rather increased (from 0.76 ± 0.02 to 0.81 ± 0.02 mM, n = 4, p < 0.05). In the same time pHi gradually decreased, but the fall of pHi during Na+ removal was significantly less in the presence of amiloride (p < 0.05, unpaired Student's t test). The subsequent readmission of extracellular Na+ along with washing out amiloride, restored pHi consistently, while this treatment transiently decreased [Mg2+]i to below the initial concentration (to 0.72 ± 0.02 mM during 50–75 min). In one carotid artery preparation, essentially the same results for [Mg2+]i and pHi were obtained with KB-R7943. These results agree well with the notion that Na+-coupled transporters are involved in the amiloride- and KB-R7943-induced paradoxical decreases in [Mg2+]i.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Two pathways have been proposed for Mg2+ transport across the plasma membrane in smooth muscle: 1) Na+ (gradient)-dependent Mg2+ extrusion (Na+-Mg2+ exchange); 2) Na+-independent passive Mg2+ flux, depending on the Mg2+ concentration gradient, and blocked by extracellular Ca2+ (20, 38). The latter pathway may correspond to the Mg2+- and Ca2+-permeable channel recently identified (39) or ion channels of similar ionic permeability. In the presence of extracellular Ca2+, i.e. the normal solution in the present study, the former, Na+-Mg2+ exchange is considered to be the major Mg2+ pathway.

The Na+-Mg2+ exchange mechanism is considered to maintain low [Mg2+]i against the electrochemical gradient across the plasma membrane (18, 20). We have previously shown that amiloride, an inhibitor for Na+-Ca2+ exchange, also blocks Na+-Mg2+ exchange, and consequently elevate [Mg2+]i under Ca2+-free conditions in which passive Mg2+ flux pathways are accelerated (24). The present 31P NMR measurements in pig carotid artery smooth muscle unexpectedly revealed decrease during applications of Na+-Ca2+ exchange inhibitors, i.e. amiloride or KB-R7943, to the normal extracellular solution (containing 2.4 mM Ca2+). Using 31P NMR, we could also continuously monitor two factors which may affect [Mg2+]i by changing Mg2+ buffering: 1) ATP itself is known as an important intracellular Mg2+ buffer, 2) while pHi has also been proposed to modulate the intracellular Mg2+ buffering capacity (40). However, neither of these significantly changed throughout the experiments with amiloride (Fig. 2B, Fig. 5A, and Table I). The fact of no significant pHi change also rules out possible [Mg2+]i modulation via co-operation of Na+-dependent transporters, like [Ca2+]i regulation in cardiac myocytes, i.e. coupling of Na+-H+ and Na+-Ca2+ exchange via [Na+]i (41). We should therefore consider other mechanisms to explain the paradoxical decrease in [Mg2+]i.

Alteration of Na+-gradient across the plasma membrane would also affect Na+-Mg2+ exchange. In intestinal smooth muscle, using 87Rb NMR we have previously shown amiloride (1 mM) to have little effect on intracellular Rb+ accumulation, an index of Na+-K+ pump activity. Further, intracellular Mg2+ is depleted through a passive Mg2+ flux pathway during exposure to Mg2+-free and Ca2+-free solutions even in the presence of 1 mM amiloride (21). On the other hand, Iwamoto et al. (36) have reported that Na+-K+ pump activity is hardly impaired by KB-R7943 (up to 30 µM). This drug also does not significantly influence Na+-H+ exchange or passive 22Na uptake (36).

It has recently been reported that KB-R7943 suppress the Na+-independent passive Mg2+ pathways at concentrations (~20 µM) similar to those used in the present study (42). This effect may account for the paradoxical decrease in [Mg2+]i upon applications of KB-R7943 in the presence of Ca2+. Amiloride might have a similar effect on the passive Mg2+ pathway. However, both KB-R7943 and amiloride significantly increases [Mg2+]i when extracellular Ca2+ is removed. Furthermore, removal of extracellular Na+ prevented the paradoxical decrease in [Mg2+]i induced by these drugs, even in the presence of Ca2+ (the data for KB-R7943 not shown). Taken together, it is suggested that Na+-dependent Mg2+ pathways, presumably Na+-Mg2+ exchange is involved in the paradoxical decrease in [Mg2+]i.

KB-R7943 is an isothiourea derivative, structurally distinct from amiloride derivatives, but has been shown to block Na+-Ca2+ exchange (36, 37). These drugs showed similar Ca2+-dependent modulation of [Mg2+]i, i.e. paradoxical decrease in [Mg2+]i in the presence of extracellular Ca2+ (Figs. 2A and 5A) and rise in [Mg2+]i in the absence of Ca2+ (Figs. 3 and 5B). DMA, a Na+-H+ exchange inhibitor and a derivative of amiloride, did not produce such Ca2+-dependent modulation of [Mg2+]i regulation, suggesting involvement of some interaction of divalent cations. A non-selective divalent cation-binding site has recently been shown to play a role in intracellular Mg2+-dependent regulation of maxi K+ channels (43, 44). It is considered that a similar divalent cation binding site of Na+-Mg2+ exchanger may account for effects of Na+-Ca2+ exchange inhibitors on Na+-Mg2+ and their Ca2+-dependent modulations.

Another possibility we should also consider is Mg2+ transport via the Na+-Ca2+ exchanger itself. There is numerous literature for Na+-dependent Ca2+ movements across the plasma membrane in vascular and non-vascular smooth muscles (45, 46). Expression of NCX1 has recently been shown in intestinal smooth muscle (47). Also, Konishi and co-workers (48) have reported that high-(extracellular) Mg2+-induced [Mg2+]i rise in fibroblasts is attenuated by transfection of NCX1 or NCX3 (the degree of attenuation: NCX1 > NCX3), but that this attenuation is not seen in Na+-free solutions. These results indicate that Mg2+ might be extruded via Na+-Ca2+ exchange, although this possibility is less likely in intact smooth muscle for the following reasons. Firstly, the Na+-dependent Mg2+ extrusion rate in fibroblasts seems relatively low compared with the paradoxical decrease in [Mg2+]i with amiloride or KB-R7943, taking the expression level (by ~15-fold (49)) of NCX1 into consideration. Secondly, experiments on Na+-dependent Mg2+ extrusion have been carried out in the absence of extracellular Ca2+ (48). In the presence of Ca2+, NCX exchangers presumably transport Ca2+ participating in their original role. Thirdly, the resting [Mg2+]i (estimated in normal solution) is not altered by transfection of NCX (48). It seems likely that its level is determined by another Na+-dependent transporter protein for Mg2+.

The present results that amiloride and KB-R7943 affected [Mg2+]i regulation but DMA did not, prompt us to speculate that many drugs known for affecting Na+-Ca2+ exchange might have similar effects on Na+-Mg2+ exchange, and might consequently decrease [Mg2+]i. It is well known that Mg2+ deficiency is an important risk factor for ischemic cardiovascular and cerebrovascular diseases (1, 4, 5). Further, it has frequently been pointed out that Mg2+ homeostasis is affected by diabetes mellitus, a critical complication for ischemic diseases (2, 3, 6, 50). Clinically, amiloride at low concentrations (<1 µM) is used as K+-sparing diuretics to treat patients with hypertension and heart failure (51). The therapeutic concentration does not seem high enough to modulate the properties of Na+-Mg2+ exchange, however, chemical modification of amiloride reducing the inhibitory effects on Na+-Ca2+ exchange, like 6-iodoamiloride (52), would produce a more preferable drug in terms of [Mg2+]i homeostasis, thereby broadening the therapeutic concentration range. In addition, it may be noteworthy that due to inhibition of reverse mode Na+-Ca2+ exchange, both KB-R7943 and amiloride can be also used as cardioprotective against Ca2+ overload. It is speculated that the inhibitory effect of KB-R7943 on the reverse mode Na+-Ca2+ exchange might be a key to solve their Ca2+-dependent action on [Mg2+]i regulation.

In conclusion, using 31P NMR we here demonstrate that amiloride and KB-R7943 modulate [Mg2+]i in a Ca2+-dependent manner. [Mg2+]i significantly decreases in the presence of Ca2+ (normal conditions), whereas [Mg2+]i increases only in its absence. These treatments had little effect on ATP and pHi. It is suggested that these drugs directly affect Na+-Mg2+ exchange to produce the Ca2+-dependent modulation of [Mg2+]i. Also, it is likely that many inhibitory agents for Na+-Ca2+ have similar effects on [Mg2+]i regulation.


   FOOTNOTES
 
* This work was supported in part by research grants from Cardiovascular Diseases (11C-1) from the Ministry of Health and Welfare (Japan). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence should be addressed: Dept. of Cell Physiology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan. Tel.: 81-52-744-2045; Fax: 81-52-744-2048; E-mail: h44673a{at}nucc.cc.nagoya-u.ac.jp.

1 The abbreviations used are: amiloride, 3,5-diamino-6-chloro-N-(diaminomethylene)pyrazinecarboxamide; PCr, phosphocreatine; PME, phosphomonoesters. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Peter W. Flatman (Edinburgh University) and Masato Konishi (Tokyo Medical University, Japan) for useful discussions and improving the manuscript, and also to Nagoya Meat Hygiene Laboratory (Nagoya, Japan) for a continuous supply of pig carotid artery. KB-R7943 is a kind gift from Kanebo Ltd. (Osaka, Japan).



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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