Vol.
167,
March
No.
30,
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
1990
COMMUNICATIONS
1406-1412
Pages
CYTOSOLIC FREE MAGNESIUM IN CARDIAC MYOCYTES: IDENTIFICATION OF A Mg2+ INFLUX PATHWAY Gary A. Quamme”
and Simon W. Rabkin
Department of Medicine University of British Columbia University Hospital Vancouver, B.C., Canada Received
February
13,
1990
Regulation of intracellular Mg2+ activity in the heart is not well characterized. Cardiac myocytes were prepared as primary cultures from 7 day old chick embryo hearts and intracellular Mg*+ concentration ([Mg*+],) was determined in single ventricular cells with mag-fura-2. Basal [Mg*+], was 0.48?0.03 mM in normal culture medium. There was no correlation of basal [Mg*+], with cellular contraction or intracellular [Ca’+], (determined with fura-2). Cardiocytes cultured (16 hr) in low Mg (0.16 mM) media contained 0.21?0.05 mM Mg*+ which returned to normal levels when placed in Mg media with a refill time of 20 min. Basal [Ca’+], (121211 nM) and stimulated [Ca”+], (231241 nM) was similar to control cells. Verapamil, 25 uM, reversibly blocked Mg2+ refill. In conclusion, the basal [Mg*+], of isolated cardiomyocytes is considerably below the Mg” electrochemical equilibrium allowing passive Mg*+ influx. The influx pathway for Mg*+ is inhibited 01990 Academic by verapamil and appears to be independent of Ca2+ as assessed by fura-2. FTe**, Inc.
The role of intracellular the introduction concentration [Mg”]
of fura-2,
Ca*+ in cardiac cell function a fluorescent
dye sensitive
and its role in cardiac contraction
has been extensively studied following to Ca*+ changes.
is poorly understood.
in various tissues have ranged from 0.5 to 10.0 mM (1-8).
mag-furapresent
has been developed findings
influences
which
report;
which should enable investigators
1) the basal concentrations
alter intracellular
Mg”;
Intracellular
Estimation
Recently,
of intracellular
a fluorescent
to study Mg2+ regulation
of intracellular
and 3) the identification
Mg*+
probe, (9). The
Mg*+; 2) some regulatory of an apparent
Mg2+ influx
pathway present in cardiac myocytes. MATERIALS
AND METHODS
Materials - fura-2/AM, mag-fura-2, mag-fura-2/AM and Pluronic acid were obtained from Molecular Probes Inc. (Eugene, OR). Trypsin was from Gibco Labs., DNAase and dornase from Worthington, and albumin (fraction V) was from United States Biochemical Corp. All other chemicals were purchased from Sigma. *To whom correspondence should be addressed at Department of Medicine, Hospital-UBC Site, 2211 We&rook Mall, Vancouver, B.C. Canada V6T lW5. 0006-291X/90 Copyright All rights
$1.50
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
1406
University
Vol.
167,
No.
BIOCHEMICAL
3, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Isolation, incubation, and mag-furaloading of cardiac myocytes - Isolated cardiomyocytes were prepared according to previously described methods (10,ll) by trypsin digestion from ventricles of chick embryos (7 days) in Krebs-Ringer buffer with 20 mM MOPS’ supplemented with 0.1% BSA and 1.0 mM glucose, pH 7.4. The isolated cells were seeded on glass coverslips and cultured in medium 818A (20% M199, 73% DBSK buffer, 6% fetal bovine serum, GIBCO) for 24 days. [Ca’+], was determined using fura- as a fluorescent Ca*+ indicator in which cardiomyocytes, (2x10s cells/slide) attached to glass coverslips, were loaded with fura- by incubating them with 10 PM fura-2/AM for 30 min at 21°C in the above culture media. Fura-2/AM was added to the media from a stock solution so that the final concentrations were 10 PM fura-2/AM, 0.125% Pluronic acid, 0.25% BSA in buffer. Loaded cells were washed 2X with a balanced salt solution (NaCl 145 mM, KC1 2.25 mM, CaCl, 2 mM, MgCl, 1 mM, phosphate 1 mM, glucose 18 mM glucose, and Hepes-Tris 20 mM, pH 7.4), incubated 30 min to ensure complete de-esterification and finally washed 1X with fresh solution. The fluorescence signal was monitored at 505 nm with excitation wavelengths alternating between 335 and 385 nm using a Deltascan, Photon Technologies Inc. spectrofluorometer with a Nikkon inverted microscope. The [Ca”], was calculated as described by Grynkiewicz et al. with a Ka of 224 nM for the fura-2Ca2’ complex (12) after correction for fluorescence from extracellular furaand autofluorescence according to previously described methods (13). For the calculation of [Ca”],, we defined the maximum (R,,,) and the minimum (R,;,) fluorescence ratios as the ratios of the fluorescence at 33.5 and 385 nm measured in cells incubated in the above solution containing 2 mM CaCl, and 10 PM digitonin and those in cells incubated in solution containing no Ca2’, 10 PM digitonin, and 20 mM EGTA (pH S), respectively. For measurement of [Mg”], the procedure was similar to Ca2’ with the following exceptions. Cardiomyocytes on a glass coverslips were loaded with mag-fura-2/AM (5 PM) in the above media for 30 min at 21°C. After loading, the cells were washed 2X as above, and kept at room temperature until cytosolic Mg2+ was measured. Free Mg” values were monitored through the fluorescent signals of mag-furaat excitation wavelengths of 340 nm and 385 nm. Cells were permeabilized with 10 ,uM digitonin in the presence of 50 mM Mg to obtain maximal fluorescence (R,,,) of the mag-furaMg*+ complex. This was washed 1X, followed by the addition of 50 mM EDTA and 20 mM Tris buffer at pH 8.5 to determine R,,,. Free Mg’+ was determined as described previously (12) using a Kd of 1.4 mM for the mag-fura-2Mg2+ complex. In all experiments involving Mg” analyzes single traces are shown, but similar results were obtained in at least three separate experiments from independent cell preparations.
RESULTS
AND DISCUSSION
Calibration 340/385
of the mag-fin-a-2
fluorescence
ratio as a function
spectra was similar for de-esterified is in the concentration 2 determined calculations
these conditions
of [Mg2+li.
fluorescence
3401385 ratio at 5 PM.
Mg’+
obtained
was 1.4 mM.
Also shown is the response
or Ca*+.
Figure
1 demonstrates
or Ca2+ concentration.
the
This emission
from lysed cells (12). The useful response constant for Mg*+ to mag-fura-
This constant of mag-fura-
was used in all subsequent to Ca2+ (Fig. 1). The binding
of cation which elicits a half maximal (50% of maximum-
response, of mag-fura-
for Ca*+ is 60 PM with significant
increases in the
Accordingly,
is useful in Mg2+ analyses provided
the concurrent
[Ca’+] is below about 5 PM.
misinterpretation
Mg”’
range of O-10 mM free Mg2+. The binding
under
Ca2+ concentrations
with
of either
mag-fura-
constant, as defined by the concentration minimum)
signals
mag-fura-
Note, the fluorescence
than for maximal
Mg”
response is significantly
concentrations.
of the 340/385 ratio change. 1407
greater for maximal
Thus, care must be taken to avoid
Vol.
167,
No.
BIOCHEMICAL
3, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
0.2
0
0.0001
0.001
0.01
1.0
0.1
1u
1ou
[ 1 mM Figure 1 Selectivity of mag-fura- for Ca”’ and Mg*+. 1p.M mag-fura- was suspended in buffer; KCI, 100 mM; NaCI, 25 mM; Hepes-Tris, 10mM; K2H2 EDTA, 1 mM; pH 7.1 in a chamber mounted on a glass cover slip. The emission was collected at 505 nm. MgSO, or buffered CaCl, was added in appropriate amounts to obtain the given M$+ or Ca*+ concentrations. Sf/b is fluorescence intensity at 0 Mg*+ or 0 Ca*+ relative to that with maximal Mg*+ or Ca*+, respectively, adjusted for background (12). The 340/385 excitation ratio is compared in the presence of either Mg*+ or Ca*+.
Basal intracellular furs-2/AM [Mg”‘],
Mg”
concentrations.
for 20 min at 21°C contained
level of cardiomyocytes
Figure 2 illustrates test whether
a representative
basal Mg”
tracing.
mM, n=27,
ratio change is detected
mag-fura-2.
i.e. a concentration
with 5 JLM magThe mean basal
a range of 0.15 to 0.725 mM. manoeuvres
These included:
of ligands such as angiotensin
all of which altered intracellular
dye to Mg”,
incubated
A variety of physiological
levels was observed with these manoeuvres
of the fluorescent
with
in the basal Mg’+ levels.
(Fig.2), and application
vasopressin (185 nM, n=2),
20-50 PM de-esterified
was 0.47kO.03
there were alterations
cell contraction
Chick cardiomyocytes
were used to
spontaneous
II (231541
[Ca”+],. No alteration
nM, n=3),
and
in the measured
which may reflect the relative
change of 100 pM is required
cardiac
insensitivity
before a 340/355
(Fig. 1).
As free cytosolic Mg ** is about 0.5 mM, it is in the order of 1% of the total intracellular Mg content. and Triton nmol/mg the
Earlier X-100
Accordingly, (present
(mitochondrial
dry weight,
mitochondrial
studies using differential
the fraction
data).
pool plus bound to myofibrils)
there is 21-25 nmol/mg compartment
cell permeablization
and the
(60%)
with digitonin indicated
of total cytosolic Mg which
5-6%
bound
to the
is in the free Mg”
lo-15 (35%) myofibrils
in
(4,15).
form is only about 3%
Thus, relatively small changes in the bound or compartmentalized 1408
pool)
that of the total Mg, 35
in the cytosolic compartment,
remainder
(cytosolic
Mg may lead to
Vol.
167,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
1.0
a 0.8 -
NO ,:
0.4
-
2
“11
‘7.
,5.OmMMy
,
),
0
500
1000
1500
TIME
2000
2500
I
3000
seconds
400
b
300 b z E : z
200
2 z NORMAL 100 ‘“7 ‘1’ LOW Mg
5.0 0
I
0
500
Mq .---___t
I
I
1000
1500
TIME
Figure
mM
tI
2
1
2000
I
2500
I
3000
seconds
Intracellular Mg*+ concentration of single chick cardiomyocytes. [Mg”], was determined with mag-furaand excitation 340/385 nm ratio calibrated. [Ca’+], was determined with furaat 345/390. Cells were cultured in normal media ([Mg], 0.95 mM and [Cal, 2.0 mM) or low Mg media (0.16 and 2.0 mM, respectively) The basal [Mg*+], or [Ca”], was determined and subsequently placed in buffer solution containing high Mg, 5.0 mM. Fluorescence was measured at 1 data points /s with 25 signal averaging and smoothed according to Savitzky and Golay (14).
1409
Vol.
167,
No.
BIOCHEMICAL
3, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
1.0
a 0.6 b 0E
0.6
-
0.4
-
0.2
-
.= I=
E t ST
0
I t
5.0 25
I
I
0
500
uM
mM
Mg
VERAPAMIL I
I
1000
1500
TIME
I
I
I
2000
2500
3000
seconds
400
b
300 b z
E E E
200
.: :: LOW
100
Mg
NORMAL WMg 0 0
~ I 500
25
uM
VERAPAMIL I 1000
TIME
Figure
3
I 1500
I
I
2000
2500
I 3000
seconds
Inhibition oE Mg” influx by verapamil. [Mg”], was determined as given in normal and Mg-depleted cardiomyocytes. Verapamil, 25~M and MgCl,, 5 mM, were added where indicated, verapamil was subsequently removed and [Mg”], allowed to return to normal concentrations in the depleted cells. [CaZ+], was not altered with the above manipulations.
1410
J
Vol.
167,
rather
No.
3, 1990
BIOCHEMICAL
large changes in free [Mg”],.
metabolism
AND
of a Mg”
low Mg, 0.16 mM, to deplete lower [Mg”],, of external
0.21+0.05
RESEARCH
This may be highly relevant
because of its role as the natural
Identification
BIOPHYSICAL
influx
cationic
pathway.
mM, n=29,
ligand for ATP
(15). the cells in media containing
[Ca”], was normal, 121+11
these cardiocytes
to replenish
The increase in [Mg”],
gradient
is the prime driving force for Mg*+ influx and abruptly levelled mM,
throughout
suggesting
this procedure
Accordingly,
was linear with time, indicating
a highly
and vasopressin
Ca*+ regulation
appeared
Finally, we used a common influx pathway. inhibited
Figure 3 illustrates
increase in [Mg”],
Mg*+ concentrations, changes occurred
process.
elicited
normal
cardiac myocytes.
to normal values.
Cat+ release
blocker,
to the notion
not shown).
verapamil,
in [Ca”],
electrical
may enter the cell passively down an electrical
is blocked
by verapamil,
pathway
by an
to normal
is regulated.
As the extracellular potential
gradient
These
concentrations
these putative
fluid contains
is in the order of -60 mV,
(16). This Mg*+ influx pathway
occurs in the absence of changes in [Ca’+], [Mg”],
was followed
(fig. 3).
free Mg*+.
magnesium
cell model may prove useful in characterizing
to block the Mg’+
that the cell returned
that the influx
about 0.50 mM free Mg*+, and the transmembrane
closes when normal
spontaneously
(data
Removal of verapamil
The observation
in the absence of alterations
as it abruptly
at normal concentrations,
cells contracted
a normal
electrical
tracing, one of three, in which 25pM verapamil
In summary, a typical cell contains 0.47mM
regulated
that the transmembrane
The
Ca *+-channel
a representative
lends credence
Replacement
in these cells.
inorganic
Mg ‘+ influx in Mg-depleted
immediate
regulated
nM, n=3.
the Mg’+ levels, over about
20 min.
0.55-cO.06
of cellular
Fig. 2 indicates that these cells had a significantly
although
Mg2+ to the media allowed
from the standpoint
Next, we cultured
the cardiocytes.
COMMUNICATIONS
and appears
are reached.
regulatory
to be highly
The Mg-depleted
processes.
ACKNOWLEDGMENTS We acknowledge This was supported
the superb secretarial
assistance of Maureen
by grants from the Medical
Research
McGowan.
Council
of Canada and the B.C.
Heart Foundation. REFERENCES Fry, C.H. (1986) Magnesium
5, 306-312.
Blatter,
LA.,
J.kS.
Brinley,
F.J. Jr., Scarpa, A, and Teiffert,
and McGuigan,
(1986) Q.J. Exp. Physiol. 71, 467-470. T. (1977) J. Physiol 266, 545-565.
Geisbuhler, T., Altschuld, R.A., Trewyn, R.W., Ansei, AZ., (1984) Circ. Res. 54, 536-540. 5.
Murphy, E., Steenberger, 264, 5622-5627.
Lamka, KG., and Brierley, G.P.
C., Levy, LA., Raju, B., and London,
1411
R.E. (1989)
J. Biol. Chem.
Vol.
167,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Cohen, SM., and Burt, CT. (1977) Proc. Natl. Acad. Sci. USA 74, 4271-4274. Gupta, R.K., and Moore, Hess, P., Metzger, Raju, B., Murphy, c540-C548.
R.D. (1980) J. Biol. Chem. 255, 3987-3991.
and P. Weingart,
R. (1982) J. Physiol. 333, 173-188.
E., Levy, L.A., Hall, R.D., and London,
R.E. (19S9)
Am. J. Physiol. 256,
10.
De Haan, J.R. (1967)
Dev. Biol. 16, 216-230.
11.
Rabkin,
12.
Grynkiewicz,
13.
Quamme, 165
14.
Savitsky, A., and Golay, M.J.E. (1964) Analytical
15.
Altschuld, R.A., and Brierley, G.P. (1989) In: Isolated Adult Cardiomyocytes. Eds. Piper, H.M., and Isenberg, G., CRC Press Inc., Boca Raton, Florida, Vol. 1, pp83-95.
16.
Quamme,
S.W., and Sanga, P. (1987) G., Poenie, G., Pfeilschifter,
J. Mol. Cell Cardiol.
M., and Tsien, R.Y. (1985) J., and Murer,
19, 1073-1083.
J. Biol. Chem. 260, 3440-3450.
H. (1989) Biochem.
Chemistry
36, 1627-1639.
G.A., and Dirks, J.H. (1953) Rev. Physiol. Biochem.
1412
Biophys. Acta., 1013, 159-
Pharmacol.
97, 69-110.
167,
March
No.
30,
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
1990
COMMUNICATIONS
1406-1412
Pages
CYTOSOLIC FREE MAGNESIUM IN CARDIAC MYOCYTES: IDENTIFICATION OF A Mg2+ INFLUX PATHWAY Gary A. Quamme”
and Simon W. Rabkin
Department of Medicine University of British Columbia University Hospital Vancouver, B.C., Canada Received
February
13,
1990
Regulation of intracellular Mg2+ activity in the heart is not well characterized. Cardiac myocytes were prepared as primary cultures from 7 day old chick embryo hearts and intracellular Mg*+ concentration ([Mg*+],) was determined in single ventricular cells with mag-fura-2. Basal [Mg*+], was 0.48?0.03 mM in normal culture medium. There was no correlation of basal [Mg*+], with cellular contraction or intracellular [Ca’+], (determined with fura-2). Cardiocytes cultured (16 hr) in low Mg (0.16 mM) media contained 0.21?0.05 mM Mg*+ which returned to normal levels when placed in Mg media with a refill time of 20 min. Basal [Ca’+], (121211 nM) and stimulated [Ca”+], (231241 nM) was similar to control cells. Verapamil, 25 uM, reversibly blocked Mg2+ refill. In conclusion, the basal [Mg*+], of isolated cardiomyocytes is considerably below the Mg” electrochemical equilibrium allowing passive Mg*+ influx. The influx pathway for Mg*+ is inhibited 01990 Academic by verapamil and appears to be independent of Ca2+ as assessed by fura-2. FTe**, Inc.
The role of intracellular the introduction concentration [Mg”]
of fura-2,
Ca*+ in cardiac cell function a fluorescent
dye sensitive
and its role in cardiac contraction
has been extensively studied following to Ca*+ changes.
is poorly understood.
in various tissues have ranged from 0.5 to 10.0 mM (1-8).
mag-furapresent
has been developed findings
influences
which
report;
which should enable investigators
1) the basal concentrations
alter intracellular
Mg”;
Intracellular
Estimation
Recently,
of intracellular
a fluorescent
to study Mg2+ regulation
of intracellular
and 3) the identification
Mg*+
probe, (9). The
Mg*+; 2) some regulatory of an apparent
Mg2+ influx
pathway present in cardiac myocytes. MATERIALS
AND METHODS
Materials - fura-2/AM, mag-fura-2, mag-fura-2/AM and Pluronic acid were obtained from Molecular Probes Inc. (Eugene, OR). Trypsin was from Gibco Labs., DNAase and dornase from Worthington, and albumin (fraction V) was from United States Biochemical Corp. All other chemicals were purchased from Sigma. *To whom correspondence should be addressed at Department of Medicine, Hospital-UBC Site, 2211 We&rook Mall, Vancouver, B.C. Canada V6T lW5. 0006-291X/90 Copyright All rights
$1.50
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
1406
University
Vol.
167,
No.
BIOCHEMICAL
3, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Isolation, incubation, and mag-furaloading of cardiac myocytes - Isolated cardiomyocytes were prepared according to previously described methods (10,ll) by trypsin digestion from ventricles of chick embryos (7 days) in Krebs-Ringer buffer with 20 mM MOPS’ supplemented with 0.1% BSA and 1.0 mM glucose, pH 7.4. The isolated cells were seeded on glass coverslips and cultured in medium 818A (20% M199, 73% DBSK buffer, 6% fetal bovine serum, GIBCO) for 24 days. [Ca’+], was determined using fura- as a fluorescent Ca*+ indicator in which cardiomyocytes, (2x10s cells/slide) attached to glass coverslips, were loaded with fura- by incubating them with 10 PM fura-2/AM for 30 min at 21°C in the above culture media. Fura-2/AM was added to the media from a stock solution so that the final concentrations were 10 PM fura-2/AM, 0.125% Pluronic acid, 0.25% BSA in buffer. Loaded cells were washed 2X with a balanced salt solution (NaCl 145 mM, KC1 2.25 mM, CaCl, 2 mM, MgCl, 1 mM, phosphate 1 mM, glucose 18 mM glucose, and Hepes-Tris 20 mM, pH 7.4), incubated 30 min to ensure complete de-esterification and finally washed 1X with fresh solution. The fluorescence signal was monitored at 505 nm with excitation wavelengths alternating between 335 and 385 nm using a Deltascan, Photon Technologies Inc. spectrofluorometer with a Nikkon inverted microscope. The [Ca”], was calculated as described by Grynkiewicz et al. with a Ka of 224 nM for the fura-2Ca2’ complex (12) after correction for fluorescence from extracellular furaand autofluorescence according to previously described methods (13). For the calculation of [Ca”],, we defined the maximum (R,,,) and the minimum (R,;,) fluorescence ratios as the ratios of the fluorescence at 33.5 and 385 nm measured in cells incubated in the above solution containing 2 mM CaCl, and 10 PM digitonin and those in cells incubated in solution containing no Ca2’, 10 PM digitonin, and 20 mM EGTA (pH S), respectively. For measurement of [Mg”], the procedure was similar to Ca2’ with the following exceptions. Cardiomyocytes on a glass coverslips were loaded with mag-fura-2/AM (5 PM) in the above media for 30 min at 21°C. After loading, the cells were washed 2X as above, and kept at room temperature until cytosolic Mg2+ was measured. Free Mg” values were monitored through the fluorescent signals of mag-furaat excitation wavelengths of 340 nm and 385 nm. Cells were permeabilized with 10 ,uM digitonin in the presence of 50 mM Mg to obtain maximal fluorescence (R,,,) of the mag-furaMg*+ complex. This was washed 1X, followed by the addition of 50 mM EDTA and 20 mM Tris buffer at pH 8.5 to determine R,,,. Free Mg’+ was determined as described previously (12) using a Kd of 1.4 mM for the mag-fura-2Mg2+ complex. In all experiments involving Mg” analyzes single traces are shown, but similar results were obtained in at least three separate experiments from independent cell preparations.
RESULTS
AND DISCUSSION
Calibration 340/385
of the mag-fin-a-2
fluorescence
ratio as a function
spectra was similar for de-esterified is in the concentration 2 determined calculations
these conditions
of [Mg2+li.
fluorescence
3401385 ratio at 5 PM.
Mg’+
obtained
was 1.4 mM.
Also shown is the response
or Ca*+.
Figure
1 demonstrates
or Ca2+ concentration.
the
This emission
from lysed cells (12). The useful response constant for Mg*+ to mag-fura-
This constant of mag-fura-
was used in all subsequent to Ca2+ (Fig. 1). The binding
of cation which elicits a half maximal (50% of maximum-
response, of mag-fura-
for Ca*+ is 60 PM with significant
increases in the
Accordingly,
is useful in Mg2+ analyses provided
the concurrent
[Ca’+] is below about 5 PM.
misinterpretation
Mg”’
range of O-10 mM free Mg2+. The binding
under
Ca2+ concentrations
with
of either
mag-fura-
constant, as defined by the concentration minimum)
signals
mag-fura-
Note, the fluorescence
than for maximal
Mg”
response is significantly
concentrations.
of the 340/385 ratio change. 1407
greater for maximal
Thus, care must be taken to avoid
Vol.
167,
No.
BIOCHEMICAL
3, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
0.2
0
0.0001
0.001
0.01
1.0
0.1
1u
1ou
[ 1 mM Figure 1 Selectivity of mag-fura- for Ca”’ and Mg*+. 1p.M mag-fura- was suspended in buffer; KCI, 100 mM; NaCI, 25 mM; Hepes-Tris, 10mM; K2H2 EDTA, 1 mM; pH 7.1 in a chamber mounted on a glass cover slip. The emission was collected at 505 nm. MgSO, or buffered CaCl, was added in appropriate amounts to obtain the given M$+ or Ca*+ concentrations. Sf/b is fluorescence intensity at 0 Mg*+ or 0 Ca*+ relative to that with maximal Mg*+ or Ca*+, respectively, adjusted for background (12). The 340/385 excitation ratio is compared in the presence of either Mg*+ or Ca*+.
Basal intracellular furs-2/AM [Mg”‘],
Mg”
concentrations.
for 20 min at 21°C contained
level of cardiomyocytes
Figure 2 illustrates test whether
a representative
basal Mg”
tracing.
mM, n=27,
ratio change is detected
mag-fura-2.
i.e. a concentration
with 5 JLM magThe mean basal
a range of 0.15 to 0.725 mM. manoeuvres
These included:
of ligands such as angiotensin
all of which altered intracellular
dye to Mg”,
incubated
A variety of physiological
levels was observed with these manoeuvres
of the fluorescent
with
in the basal Mg’+ levels.
(Fig.2), and application
vasopressin (185 nM, n=2),
20-50 PM de-esterified
was 0.47kO.03
there were alterations
cell contraction
Chick cardiomyocytes
were used to
spontaneous
II (231541
[Ca”+],. No alteration
nM, n=3),
and
in the measured
which may reflect the relative
change of 100 pM is required
cardiac
insensitivity
before a 340/355
(Fig. 1).
As free cytosolic Mg ** is about 0.5 mM, it is in the order of 1% of the total intracellular Mg content. and Triton nmol/mg the
Earlier X-100
Accordingly, (present
(mitochondrial
dry weight,
mitochondrial
studies using differential
the fraction
data).
pool plus bound to myofibrils)
there is 21-25 nmol/mg compartment
cell permeablization
and the
(60%)
with digitonin indicated
of total cytosolic Mg which
5-6%
bound
to the
is in the free Mg”
lo-15 (35%) myofibrils
in
(4,15).
form is only about 3%
Thus, relatively small changes in the bound or compartmentalized 1408
pool)
that of the total Mg, 35
in the cytosolic compartment,
remainder
(cytosolic
Mg may lead to
Vol.
167,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
1.0
a 0.8 -
NO ,:
0.4
-
2
“11
‘7.
,5.OmMMy
,
),
0
500
1000
1500
TIME
2000
2500
I
3000
seconds
400
b
300 b z E : z
200
2 z NORMAL 100 ‘“7 ‘1’ LOW Mg
5.0 0
I
0
500
Mq .---___t
I
I
1000
1500
TIME
Figure
mM
tI
2
1
2000
I
2500
I
3000
seconds
Intracellular Mg*+ concentration of single chick cardiomyocytes. [Mg”], was determined with mag-furaand excitation 340/385 nm ratio calibrated. [Ca’+], was determined with furaat 345/390. Cells were cultured in normal media ([Mg], 0.95 mM and [Cal, 2.0 mM) or low Mg media (0.16 and 2.0 mM, respectively) The basal [Mg*+], or [Ca”], was determined and subsequently placed in buffer solution containing high Mg, 5.0 mM. Fluorescence was measured at 1 data points /s with 25 signal averaging and smoothed according to Savitzky and Golay (14).
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1.0
a 0.6 b 0E
0.6
-
0.4
-
0.2
-
.= I=
E t ST
0
I t
5.0 25
I
I
0
500
uM
mM
Mg
VERAPAMIL I
I
1000
1500
TIME
I
I
I
2000
2500
3000
seconds
400
b
300 b z
E E E
200
.: :: LOW
100
Mg
NORMAL WMg 0 0
~ I 500
25
uM
VERAPAMIL I 1000
TIME
Figure
3
I 1500
I
I
2000
2500
I 3000
seconds
Inhibition oE Mg” influx by verapamil. [Mg”], was determined as given in normal and Mg-depleted cardiomyocytes. Verapamil, 25~M and MgCl,, 5 mM, were added where indicated, verapamil was subsequently removed and [Mg”], allowed to return to normal concentrations in the depleted cells. [CaZ+], was not altered with the above manipulations.
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large changes in free [Mg”],.
metabolism
AND
of a Mg”
low Mg, 0.16 mM, to deplete lower [Mg”],, of external
0.21+0.05
RESEARCH
This may be highly relevant
because of its role as the natural
Identification
BIOPHYSICAL
influx
cationic
pathway.
mM, n=29,
ligand for ATP
(15). the cells in media containing
[Ca”], was normal, 121+11
these cardiocytes
to replenish
The increase in [Mg”],
gradient
is the prime driving force for Mg*+ influx and abruptly levelled mM,
throughout
suggesting
this procedure
Accordingly,
was linear with time, indicating
a highly
and vasopressin
Ca*+ regulation
appeared
Finally, we used a common influx pathway. inhibited
Figure 3 illustrates
increase in [Mg”],
Mg*+ concentrations, changes occurred
process.
elicited
normal
cardiac myocytes.
to normal values.
Cat+ release
blocker,
to the notion
not shown).
verapamil,
in [Ca”],
electrical
may enter the cell passively down an electrical
is blocked
by verapamil,
pathway
by an
to normal
is regulated.
As the extracellular potential
gradient
These
concentrations
these putative
fluid contains
is in the order of -60 mV,
(16). This Mg*+ influx pathway
occurs in the absence of changes in [Ca’+], [Mg”],
was followed
(fig. 3).
free Mg*+.
magnesium
cell model may prove useful in characterizing
to block the Mg’+
that the cell returned
that the influx
about 0.50 mM free Mg*+, and the transmembrane
closes when normal
spontaneously
(data
Removal of verapamil
The observation
in the absence of alterations
as it abruptly
at normal concentrations,
cells contracted
a normal
electrical
tracing, one of three, in which 25pM verapamil
In summary, a typical cell contains 0.47mM
regulated
that the transmembrane
The
Ca *+-channel
a representative
lends credence
Replacement
in these cells.
inorganic
Mg ‘+ influx in Mg-depleted
immediate
regulated
nM, n=3.
the Mg’+ levels, over about
20 min.
0.55-cO.06
of cellular
Fig. 2 indicates that these cells had a significantly
although
Mg2+ to the media allowed
from the standpoint
Next, we cultured
the cardiocytes.
COMMUNICATIONS
and appears
are reached.
regulatory
to be highly
The Mg-depleted
processes.
ACKNOWLEDGMENTS We acknowledge This was supported
the superb secretarial
assistance of Maureen
by grants from the Medical
Research
McGowan.
Council
of Canada and the B.C.
Heart Foundation. REFERENCES Fry, C.H. (1986) Magnesium
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Altschuld, R.A., and Brierley, G.P. (1989) In: Isolated Adult Cardiomyocytes. Eds. Piper, H.M., and Isenberg, G., CRC Press Inc., Boca Raton, Florida, Vol. 1, pp83-95.
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Quamme,
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H. (1989) Biochem.
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