Clinical Science (1995) 89, 293-298 (Printed in Great Britain)
293
Effect of the transmembrane gradient of magnesium and
sodium on the regulation of cytosolic free magnesium
concentration in human platelets
Mitsuisa YOSHIMURA, Tetsuya OSHIMA*, Hideo MATSUURA, Mitsuaki WATANABE,
Yukihito HIGASHI, Norihisa ONO, Hiroyuki HIRAGA, Masayuki KAMBE* and Goro KAjlYAMA
First Department of Internal Medicine and *Department of Clinical laboratory Medicine,
Hiroshima University School of Medicine, Hiroshima. japan
(Received 18 )anuary/l4 March 1995; accepted 24 April 1995)
1. To clarify the mechanism by which cytosolic free
Mg2 concentrations ([Mg2 'Ii) are regulated in
+
human platelets, we investigated platelet membrane
permeability to Mg2 by altering extracellular Mg2+
concentrations, and tested the existence of a
Na+-Mg2 + exchanger by manipulating the transmembrane Na+ gradient.
2. Platelet [Mg2 +Ii was 421 f 52 pmolll in healthy
men. [Mg2+Ii remained constant during a 120min
exposure to nominally zero (low) or 5 mmol/l (high)
external Mg2+ concentrations.
3. Preincubation of platelets with
mol/l ouabain
effectively decreased the transmembrane Na + gradient. The ouabain-induced increase in [Mg2+li was
statistically significant after 30 min of exposure
(14.6 f 2.0%) and reached 24.6 f 4.5% after 60 min.
Similarly, the replacement of extracellular Na+ with
N-methyl-D-glucamine
significantly
increased
and 78.8 f 12.5%,
[Mg2+li by 48.5 f 39%
respectively.
4. These results suggest that [Mg2+li is well
controlled in the presence of large transmembrane
Mg2 concentration gradients, and a Na+-Mg2+
exchanger may be involved in the regulation of
[Mg2 'Ii in human platelets.
+
+
for cellular functions [3]. However, little is known
about the regulation of cytosolic free Mg2+ concentrations ([Mg2 +Ii).
The recent development of the fluorescent Mg2+
indicator mag-fura-2 has facilitated the study of
[Mg2+Ii [4]. With this fluorescent indicator, the
resting values for [Mg2+Ii in human platelets have
been shown to be approximately 250-600 pmol/l [S71, well below the expected values for a thermodynamic equilibrium [S]. It is likely that Mg2+ is
extruded from the cytoplasm against its electrochemical gradient. However, the mechanisms that
maintain this low [Mg2+li are not fully established.
A particularly low permeability of the membrane to
Mg2+ and a Na+-Mg2+ exchange process may be
important in regulating [Mg2+Ii in some kinds of
cell [3, 8-11], but it is possible that these mechanisms may vary with different cell types. In human
platelets, the details of Mg2+ homoeostasis remain
to be elucidated.
We assessed the participation of low Mg2+ permeability in the regulation of [Mg2+li by measuring
the effect of changing extracellular Mg2 concentration on [Mg2+Ii.To examine the existence of the
Na+-Mg2 + exchange process in human platelets,
we studied the effect of manipulating the Na+
gradient across the cell membrane on [Mg2+li.
+
INTRODUCTION
METHODS
Mg2+, the most abundant intracellular divalent
cation, is essential to many cellular functions,
including Ca2+ metabolism, second messenger
systems and all enzymic reactions involving ATP [1,
21. This divalent cation exists in three different
states, being bound to protein, complexed to anions
and ionized. A large portion is involved with ATP,
ADP and AMP. Thus, only 10% of intracellular
magnesium is in the free ionized form, which regulates transport systems and is the most important
Preparation of platelets
Human platelets were obtained from six healthy
Japanese male volunteers who gave their informed
consent. The study was approved by the local
Ethics Committee. None of the subjects had
received any medication for at least 4 weeks before
the study. After an overnight fast, blood was drawn
from the antecubital vein into a syringe containing
3.8% trisodium citrate anticoagulant with a 19-
~
Key words: cytorolic magnesium. mag-fur*2, ouabain, sodium.
Abbreviations: [Ca1+Ii. cytosolic free Cal+ concentration; [Mg'+]i, cytosolic free Mg'+ concentration; [Na+]i, cytosolic free Na+ concentration; PRP, platelet-rich plasma;
SBFI, scdium-binding benzofuranirophthalate.
Correspondence: Dr Mitsuisa Yoshimura, First Department of Internal Medicine, Hiroshima University School of Medicine, 1-2-3 Kasumi. Minami-ku, Hiroshima 734, Japan.
M. Yoshimura et al
194
gauge needle and the two-syringe technique [12].
Platelet-rich plasma (PRP) was prepared by centrifugation at 1200g for 5min at room temperature.
PRP was further centrifuged at 800g for 1Omin at
room temperature in the presence of 2 pmol/l prostaglandin E,(Sigma Chemical Co., St. Louis, MO,
U.S.A.), to remove plasma. The preparation of cells
was carried out in a HEPES buffer medium containing 145 mmol/l NaCI, 5 mmol/l KCI, ( r 5 mmol/l
MgSO,, 10mmol/l HEPES and 5mmol/l glucose at
pH 7.4. CaCl, was not added to the standard
HEPES solution because it would prevent platelet
activation. The concentration of C a 2 + in the
Ca2+-free medium of platelets was about 1 pmol/l.
CaCI, ( 1 mmol/l) was added to the medium after
loading the cells with fluorescent indicators.
Effects of external Mg2+ and the transmembrane Na'
gradient on [Mg"],
Protocol 1: effects of extracellular Mg2+ concentrations on [Mg2+li. To assess whether changes in
external Mg2 affect [Mg2+Iji, platelet pellets were
resuspended
in
various
concentrations
of
Mg2 +-HEPES buffer solutions containing MgSO,
concentrations
which were nominally
zero
(low-Mg2+), 1 mmol/l (standard), or 5mmol/l
(high-Mg2+), respectively. The cells were then preincubated in different Mg2+ solutions for 60 to
120min at room temperature, and the [ M g 2 + l iwas
measured.
Protocol 2: effects of ouabain and external Na'
concentration (transmembrane Na
gradient) on
cytosolic free Na+ concentration ( [Na+li), cytosolic
free Ca2' concentration ( [CaZ+'Ji),and [Mg2+li.
Platelet pellets were resuspended in standard
HEPES buffer and divided into two batches.
Ouabain ( 10 mol/l) (Sigma Chemical Co., St.
Louis, MO, U.S.A.) was added to one batch to
reduce the transmembrane N a + gradient, while the
control batch received the vehicle only. These suspensions were incubated for 30 to 60min at room
temperature before the cation measurements.
In the other procedure to reduce the transmembrane N a + gradient, platelets were resuspended in
low-Na+-HEPES buffer. We reduced the extracellular N a + concentration to 29 mmol/l by adding four
volumes of Na +-free-HEPES buffer (in which NaCl
was iso-osmotically replaced with N-methyl-Dglucamine) into one volume of standard HEPES
buffer. We selected 29 mmol/l because, below this
concentration, [ N a + l i was reduced [13]. The platelet suspension was maintained at room temperature
for 30 to 60min.
OR, U.S.A.) together with 0.02% Pluronic F-127
(Molecular Probes, Eugene, OR, U.S.A.) for 30min
at 37°C. Extracellular mag-fura-2/acetoxymethyl
ester was removed by centrifugation at 8001: for
10min at room temperature with 1 pmol/l prostaglandin E,. Platelets were then resuspended at
approximately 1 x 108cells/ml in the same buffer
used for the cell preparation. CaCI, was added to
the cell suspensions at a final concentration of
1 mmol/l, and the platelets were reincubated for
7min at 37°C before recording the fluorescence, in
order to ensure complete de-esterification of magfura-2/acetoxymethyl ester. For fluorescent measurements, 2.5-ml aliquots of the cell suspension were
transferred to a quartz cuvette maintained at 37°C
in a spectrofluorometer (SPEX Fluorolog; SPEX
Industries, Edison, NJ, U.S.A.). The fluorescence
signals were monitored at 510 nm with alternate
excitation with UV light at 340 nm and 380 nm. The
[Mg2+li was calculated from the ratio of the fluorescence at the two excitation wavelengths according
to the formula [4, 14):
+
+
~
Measurement of [Mg"],
Platelet suspensions (approximately 5 x 10' cells/
ml) were loaded with 2 pmol/l mag-fura-2/
acetoxymethyl ester (Molecular Probes, Eugene,
where the K , (dissociation constant) is 1.5 mmol/l,
R is the fluorescence ratio at the excitation wavelengths (340/380nm) of the sample, R,,,, and Rmin,
are the 340/380 nm fluorescence ratios obtained at
saturating and zero Mg2 and C a 2 + , respectively,
and Sf2 and Sb2 are the fluorescence intensities at
380nm for mag-fura-2 with zero and excess Mg2+
and C a 2 + , respectively. R,,,, was determined by
lysing the cells with 50 pmol/l digitonin (Sigma
Chemical Co., St. Louis, MO, U.S.A.) in the
presence of 1 mmol/l Mg2+ and 2mmol/l C a 2 + ,and
Rmin, was then obtained by chelating Mg2+ and
C a 2 + with 6mmol/l EDTA followed by adjustment
of the pH to 8.3 with 7mmol/l Tris. Autofluorescence from unloaded platelets, test agents and
medium was subtracted from measured values. A
correction for extracellular mag-fura-2 leaked from
platelets was made with 3mmol/l EDTA [12, 151 in
the presence of Mg2+ at concentrations of nominally zero and 1 mmol/l, with a slight modification
for mag-fura-2. Elevating external Mg2+ to 5 mmol/
1 required 7mmol/l EDTA to correct for extracellular mag-fura-2 leakage from platelets. These corrections were necessary, because the value for [Mg2+li
would otherwise be overestimated in Ca2+- and
Mg2 +-containing buffer. The ouabain, extracellular
Mg2+ and Na' concentrations did not affect the
intracellular mag-fura-2 concentrations, the extent of
ester hydrolysis of the dye or dye leakage (data not
shown).
All measurements were performed in at least two
replicates. The intra-assay coefficient of variation for
[Mg2+Tji was 2.274, and the day-to-day variation in
one subject was 3.6% (n=5).
In the preliminary study, in order to evaluate
+
Magnesium homoeostasis in human platelets
interference with measurement of [Mg2+li by
changes in [Ca’+li in human platelets, we investigated the fluorescence ratio emitted at 340nm and
380 nm excitation in mag-fura-2-loaded platelets
after the addition of 5 pmol/l arginine vasopressin
(Sigma Chemical Co., St. Louis, MO, U.S.A.)
which led to a significant increase in [Ca’+li
(21.8k 1.2nmol/l to 113.8+ 10.9nmol/l; n = 5 ,
P <0.001). Because arginine vasopressin did not
induce the changes in mag-fura-2 fluorescence in
platelets, we considered that when the change in
[Ca2+Ii was less than lOOnmol/l, the fluorescence
change could be explained by the changes in
CMgZ+Ii.
3:
295
5
10
I5
20
Na+ concn. (mmol/l)
30
25
Fig. I. In situ calibration of fluorescent ratio at 340/385nm of
SBFI-loaded platelets with various Na+ concentrations ranging from 0
to 30mmol/l. Values are means of six experiments.
Measurement of [Ca”],
To measure [Ca”],
platelets incubated with
2 pmol/l fura-2/acetoxymethyl ester (Molecular
Probes, Eugene, OR, U.S.A.) were treated as in the
[MgZ‘Ii determinations. Fluorescence was recorded
at an emission wavelength of 510 nm and excitation
wavelengths of 340 nm and 380 nm. Corrections
were applied for extracellular fura-2 leakage with
10mmol/l EGTA as described [l2], and for autofluorescence by subtracting the fluorescence of the
unloaded platelets. R,,,, was obtained by lysing the
cells with 50pmol/l digitonin in the presence of
1 mmol/l Ca2+, and 10mmol/l EGTA was then
added at pH 8.3 to obtain the Rmin.. [Ca2+li was
calculated using a general formula [14].
Measurement of “a’],
For measurement of [Na+Ii, platelets were loaded
with 2 pmol/l sodium-binding benzofuranisophthalate (SBFI)/acetoxymethyl ester (Molecular Probes,
Eugene, OR, U.S.A.), and a procedure similar to
that for [Mg2+Ii measurements was followed. SBFIloaded cells (n=6) were excited at 340nm and
385nm and emission was measured at 500nm.
Immediately after the fluorescent recording, the suspension was centrifuged at 3000g for 5min at 10°C
and the fluorescence of the supernatant was measured and subtracted from the total fluorescence to
correct for the extracellular dye leakage. Autofluorescence was also subtracted from the total measurement. We then calculated the 340/385 nm excitation
ratio of the resultant fluorescent intensities. Calibration of [Na+li was performed in situ by suspending SBFI-loaded cells in solutions of known N a +
concentration (0-30 mmol/l), which were prepared
by mixing different amounts of Na+-gluconate and
K+-gluconate HEPES (145mmol/l N a + and K + ,
115mmol/l gluconate and 30mmol/l CI-) in the
presence of 2 pmol/l Na’ ionophore gramicidin D
(Sigma Chemical Co., St. Louis, MO, U.S.A.) (Fig.
1) C161.
Table I. Eflects of extracellular Mgl+ on [Mgl+]i in human platelets.
Values are expressed as means +SEM from six experiments. Individual
values are means from two t o four replicate samples.
Extracellular
Mgl+ concn.
Low (nominally zero)
Standard (I mmol/l)
Hinh (5 mmolll)
[Mg’+]i ( ~ m o l l l )
Omin
60min
+
421
52
397 36
420 & 57
427f61
l2Omin
431 +52
396 & 26
429 f49
Statistical analysis
Values are expressed as means+SEM. Analysis of
variance with repeated measures and the Wilcoxon
matched-pairs signed rank test were applied for
group comparisons. In all cases, statistical significance was accepted as P c O . 0 5 .
RESULTS
OProtocol I: effects of extracellular Mg2+ concentrations
on [Mf+Ii
The basal [Mg2+li in platelets was 421 f 5 2
pmolll (Table 1). Nominally MgZ+-freemedium did
not alter [Mg2+Ii within 120min, and an increase
in external Mgz+ to 5mmol/l did not affect
[Mg’+li. It was difficult to detect any changes in
[Mg2+li when the external Mg2+ concentration
varied between nominally zero and 5 mmol/l.
Protocol 2 effects of reduction of transmembrane Na’
gradient on cytosolic cation concentrations
Baseline [Na ‘Ii in platelets was 8.0 f 0.8 mmol/l
(Fig. 2). After treatment with 10-4mol/l ouabain to
inhibit Na+-K+-ATPase, [Na+li was significantly
increased at 30 min and reached 25.8 1.7 mmol/l
after 60min, confirming that the Na’ transmembrane gradient was reduced. Similarly, [Ca’ +Ii,
+
2%
M. Yoshimura et al
30
8001
1
OJ,
0
200
30
60
1,
I
0
30
60
Time (min)
Time (min)
50 1
B
Fig. 3. Time course of effects of ouabain (lO-'mol/l) and low-Na+
solution (29mmol/l) on [Mg2+]i in human platelets. [Mg'+], started
to increase significantly after 30min within each group. 0, Ouabain; 0,
low-Na' buffer; 0,
vehicle. Statistical significance: *P<O.O5. Values are
expressed as means SEM from six experiments.
T
I
30
60
glucamine significantly elevated [Mg2'Ii to 573 k
59 pmol/l after 30 rnin and 732 k 39 pmol/l after
60 min, above timed control values by 48.5 f3.9% at
30 rnin and 78.8 k 12.5% at 60 min, respectively.
These elevations in [Mg2 'Ii significantly developed
in a time-dependent manner.
Time (min)
Fig. 2. Time course of effects of ouabain on [Na+Ii and [ C a l l i in
human platelets. Incubation with 10-'mol/l ouabain (0)and vehicle (0)
were started at time 0. Ouabain significantly increased [Na+Ii and [Ca'+Ii
at 30 and 60min in a timedependent manner. Statistical significance:
*P<O.O5 compared with timed control. Values are expressed as means
fSEM from six experiments.
DISCUSSlON
The resting platelet [Mg2'li that we measured
with the fluorescent Mg2 indicator mag-fura-2
(421 k 52pmol/l) is consistent with that in previous
reports [5-71. This value is much lower than that
expected if Mg2 was distributed passively across
the plasma membrane [8]. Hence, several mechanisms must maintain [Mg2+Ii at a low concentration against the electrochemical gradient. Furthermore, because Mg2 affects platelet activity [ 17,
181, [Mg2'li may be strictly regulated.
Mg2+ passes through the cell membrane more
slowly than other cations [3, 191. However, these
results remain controversial. It has been suggested
that the low permeability of the membrane to
Mg2+plays some part in the regulation of [Mg"],
in several kinds of cell [3, 81. There is reportedly
little or no change in [Mg2'Ii at extracellular
Mg2+ concentrations below lOmmol/l [8, 20, 211,
and even in the absence of Mg2' in diverse cells
[lo, 201. However, in the taenia of guinea-pig
caecum, an increase in permeability to Mg2+ is
achieved only when both Mg2+ and Ca2+ are
removed from the external solution, because external Ca2' effectively prevents membrane permeability to Mg2+ [S, 223. On the other hand, several
studies have shown that both an elevation and
reduction in extracellular Mg2 concentration can
alter [Mg2+li rapidly in vascular smooth muscle
cells [23] and cardiac cells [24]. In our study,
[Mg"],
remained nearly constant in platelets
during exposure to external Mg2' concentrations of
+
+
which was 27.9 f2.8 nmol/l in control platelets,
significantly increased with incubation time to
37.2 f2.5 nmol/l after 60 min. This change can be
explained by the presence of a Na+-Ca2'
exchanger in human platelets c-131. Moreover,
[Na+li and [Ca2'li were evaluated every 30min
in low-Na' solution. [Na'Ii was 8.1 f I.Ommol/l
at baseline, 8.0 f0.9 mmol/l at 30 min and
8.0 f 1.1 mmol/l at 60 min, respectively (n = 5). Lowering external Na' to 29mmol/l did not produce
any changes in [Na+Ii within 60min. Similarly,
[Ca2+li remained constant during a 60min exposure to Iow-Na' concentrations (26.8 3.2 nmol/l at
baseline,
27.2 k 3.0 nmol/l
at
30 min
and
27.0 k 3.4 nmol/l at 60 min, respectively; n = 5).
Fig. 3 demonstrates the time course of the effects
of ouabain ( 10-4mol/l) and low-Na+ solution
(29 mmol/l) on [Mg2+li. After ouabain administration, [Mg2+li was significantly increased from
389 f59 pmol/l to 462 k 63 pmol/l at 30 min, and to
523 +_ 66 pmol/l at 60min. [Mg2 'Ii of ouabaintreated cells was 14.6 f2.0% and 24.6 f4.5%
greater than the timed controls after 30min and
60 min, respectively. Similarly, the reduction in the
extracellular Na' concentration with N-methyl-D-
*
+
+
297
Magnesium homoeortasis in human platelets
nominally zero or 5mmol/l for 120min. Thus, low
MgZ permeability may be important in regulating
[Mg2+Iiin human platelets.
The transmembrane N a + gradient functions as a
source of energy for the Na+-Mg2+ exchange process [3]. We modified the N a + gradient by raising
the intracellular Na+ concentrations by inhibition
of the Na+-K+ pump with ouabain, or by reducing
the extracellular N a + concentrations. [MgZ+Iiwas
elevated by about 25% in the presence of the
Na+-K+ pump inhibitor ouabain and by about
80% in a low-Na+ solution after 60min, indicating
the presence of a Na+-dependent Mg2+ transport
system in platelets. However, it has been suggested
[25] that, in cultured chicken heart cells, an increase
in [Mg2+li caused by addition of ouabain or by
Na+ removal is largely due to a secondary effect of
an increase in intracellular C a Z + through
Na+-Caz+ exchange, because the effect of reducing
the N a + gradient on [Mg”],
is inhibited in
Ca2+-free medium. The authors speculated that the
increased intracellular CaZ displaces Mg2 bound
to common intracellular binding sites and leads to
an increase in [Mg”],. However, our data show
that, in human platelets, the rise in [CaZ+Iicaused
by adding ouabain is about 10nmol/l after 60min,
which is too low to affect [MgZ+li,whereas the rise
in [Mg”], is about lOOpmol/l. Moreover, most
cytosolic Mg2+ is bound to ligands such as ATP
[6], but the binding constants for these interactions
are approximately i0-5moi/i, a value too low to
influence [Mg”], [26]. It is therefore likely that
intracellular competition between Mg2 and CaZ
for binding sites is not a major factor in modulating
[MgZ+liin human platelets. The increase in cytosolic Ca2+ levels probably does not affect the magfura-2 fluorescence, because the K , of mag-fura-2
for Ca2+ is about 53pmol/l [4], well below the
changes in Ca2+.Rather, the changes in [Mg2+], in
platelets are probably due to the Na+-MgZ+
exchange process. Because a specific inhibitor of the
Na+-dependent Mg2+ transporter does not exist,
the existence of the Na+-Mg2+ exchanger cannot
be demonstrated directly. Molecular genetics may
prove useful in this regard.
In the measurement of intracellular cation concentrations with fluorescent indicators, errors in the
correction for extracellular dye leakage can lead to
misleading results [13, 151. Failure to correct for
extracellular dye leakage leads to overestimation of
the cation concentrations. In this study, to minimize
the time-dependent leakage of fluorescent agents,
dye-loading was performed after a 30 to 60min
preincubation with ouabain and Iow-Na’ medium,
and the corrections for dye leakage were carried out
with chelating agents or centrifugation. Therefore,
we consider this error in measurement to be relatively small.
In conclusion, the [MgZ+], was about 400pmol/l
in resting human platelets and remained constant
regardless of the extracellular Mg2 concentration.
+
+
+
+
+
+
Reducing the N a + gradient significantly increased
the [MgZ+Ii. [MgZ+Ii is well controlled in the
presence of large transmembrane Mg2 concentration gradients, and a Na+-Mg2+ exchanger is
probably the major regulator of [MgZ+liin human
platelets.
+
ACKNOWLEDGMENTS
This study was supported, in part, by a Clinical
Pathology Research Foundation of Japan, Kurozumi Medical Foundation, and a Grant-in-Aid for
Scientific Research (No. 06304028) from the Ministry of Education, Science and Culture of Japan.
REFERENCES
I. Elin RJ. Assessment of magnesium status. Clin Chem 1987; 3 3 1965-70.
2. White JR, Campbell RK. Magnesium and diabetes: a review. Ann Pharmacother
1993; 27: 775-90.
3. Flatman PW. Mechanisms of magnesium transport. Annu Rev Physiol 1991; 5 3
259-7 I.
4. Raju B, Murphy E, Levy LA. A fluorescent indicator for measuring cytosolic
free magnesium. Am J Physiol 1989 W: C540-8.
5. Hwang DL, Yen CF, Nadler JL. Insulin increased intracellular magnesium
transport in human platelets. J Clin Endocrinol Metab 1993; 76: 549-53.
6. Matsuno K, Koyama M, Takeda H, et al. Cytosolic free magnesium
concentration in human platelets. Thromb Res 1993; 69 131-7.
7. Touyz RM, SchiHrin EL. The effect of angiotensin II on platelet intracellular
free magnesium and calcium ionic concentration in essential hypertension.
J Hypertens 1993; II: 551-8.
8. Nakayama S, Tomita T. Regulation of intracellular free magnesium
concentration in the taenia of guinea-pig caecum. J Physiol (London) 1991; 435:
559-12.
9. Feray JC. Garay R. An Na+-stimulated Mg’’-transport system in human red
blood cells. Biochim Biophyr Acta 1986 8% 76-84.
10. Blatter LA. Estimation of intracellular free magnesium using ion-selective
microelectrodes: evidence for an Na/Mg exchange mechanism in skeletal
muscle. Magnesium Trace Elem 1991-1992 10 67-79.
I I. Gunther T, Vormann J. Activation of Na+/Mg”’ antiport in thymocytes by
CAMP. FEBS Lett 1991; 297: 1324.
12. Oshima T, Young EW, Bukoski RD, McCarron DA. Abnormal calcium handling
by platelets of spontaneously hypertensive rats. Hypertension 1990; I5 6061 I.
13. Oshima T, lshida T, Matsuura H, et al. Lack of effect of ouabain on calcium
homeostasis in rat platelets: comparative study with human platelets. Am J
Physiol 1994; 266: R651-7.
14. Grynkiewicz G, Poenie M, Tsien RY. A new generation of calcium indicators
with greatly improved fluorescence properties. J Biol Chem 1985; 160:
3440-50.
15. Ng LL. Davies JE, Garrido MC. lntracellular free magnesium in human
lymphocytes and the response t o lectins. Clin Sci 1991;
53947.
16. Borin M, Siffert W. Stimulation by thrombin increases the cytosolic free Na+
Concentration in human platelets. J Biol Chem 1990 265 19543-50.
17. Nadler JL, Shaw S. Malayan S, Natarajan RD, Luong H, Rude RK. lntracellular
free magnesium deficiency plays a key role in increased platelet reactivity in
I diabetes mellitus. Diabetes Care 1992; 1 5 83541.
Type 1
18. Kinlough-Rathbone RL, Chahil A, Mustard IF. Effect of external calcium and
magnesium on thrombin-induced changes in calcium and magnesium of pig
platelets. Am J Physiol 1973; 214: 941-5.
19. Flatman PW. Magnesium transport across cell membranes. J Membr Biol 1984;
w): 1-14.
20. DAngelo EKG, Singer HA, Rembold CM. Magnesium relaxes arterial smooth
muscle by decreasing intracellular Cal+ without changing intracellular Mg”.
J Clin Invest 1992; 89: 1988-94.
21. Hall SK, Fry CH. Buri A, McGuigan ]AS. Use of ion-sensitive microelectrodes
to study intracellular free magnesium concentration and its regulation in
mammalian cardiac muscle. Magnesium Trace Elem 1991-1992; 10 80-9.
22. Nakayama S, Tomita T. Depletion of intracellular free Mg” in Mg’+- and
Ca’+-free solution in the taenia isolated from guinea-pig caecum. J Physiol
1990; 421: 363-78.
298
M. Yoshimura et al.
23. Zhang A, Cheng TPO, Altura BT. Altura BM. Extracellular magnesium
regulates intracellular free Mg” in vascular smooth muscle cells. Pfliigers Arch
1992 421: 391-3.
24. Altura BM, Barbour RL, Dowd TL. Wu F. Altura BT, Gupta RK.
Low extracellular magnesium induces intracellular free Mg deficits,
ischemia, depletion of high-energy phosphates and cardiac failure in intact
working rat hearts: A I’P-NMR study. Biochim Biophys Acta 1993; 1182:
329-32.
25. Freudenrich CC,Murphy E. Liu 5, Lieberman M. Magnesium homeostasis in
cardiac cells. Mol Cell Biochem 1992 1 1 4 97-103.
26. Quamme GA, Dai LJ. Rabkin SW. Dynamics of intracellular free Mg’+ changes
in a vascular smooth muscle cell line. A m J Physiol 1993; 265: H281-8.