REVIEW ARTICLE
Magnesium Research 2006; 19 (4): 225-36
Concentration, compartmentation
and metabolic function of intracellular
free Mg2+
Copyright © 2017 John Libbey Eurotext. Téléchargé par un robot venant de 88.99.165.207 le 16/06/2017.
T. Günther
Charité–Universitätsmedizin Berlin, Campus Benjamin Franklin, Institut für
Molekularbiologie und Biochemie, Arnimallee 22, 14195 Berlin, Germany
Correspondence: T. Günther, Waldhüterpfad 63, D14169 Berlin, Germany. Tel.: (+00 49) 30 813 60 41
Abstract. Intracellular total Mg2+ and free Mg2+ are compartmentalized between
cell organelles and within the cytosol. Different values of [Mg2+]i in the cytosol of the
same cell type were measured by various investigators. A main reason for the
differences is the uncertainty of the dissociation constants used for the Mg furaptra
complex in the fluorescence method and for MgATP when 31P NMR was employed.
The more realistic KD values of Mg furaptra and MgATP measured under in situ
conditions are higher than the KDs used by most investigators. The [Mg2+]is obtained
and the KDs used by various authors were presented. The role of intracellular Mg2+
in metabolic functions and the action of various effectors on [Mg2+]i and [Ca2+]i was
reviewed. Intracellular Mg2+ may have a permissive role supporting the effectorinduced mechanisms that are mediated by Ca2+ as a second messenger.
Key words: magnesium, calcium, compartmentation, furaptra, 31P NMR, Mg2+
electrode, second messenger
doi: 10.1684/mrh.2006.0067
Compartmentation of intracellular Mg2+
Only 1% of total body Mg2+ (about 1 mole) is localized in the extracellular fluid. Of the remainder,
50-60% are adsorbed to hydroxyapatite crystals of
bone and 40-50% are localized intracellularly. Hence,
Mg2+ is an intracellular cation. Total intracellular
Mg2+ in various cell types and tissues amounts to 3-9
mmol/kg wet weight [1]. Due to Mg2+ binding to
DNA, nucleus-containing cells have a higher Mg2+
content than unnucleated cells. Rapidly growing
cells have a higher Mg2+ content than slowly growing
cells due to their higher content of ribosomes and
thus Mg2+ bound to rRNA.
In context with these facts, intracellular Mg2+ is
compartmentalized. The percentage of Mg2+ localized in nuclei, mitochondria, microsomes (ribosomes) and cytosol is shown in table 1. The differences found by various investigators are probably
caused by a different intensity in tissue homogenisation. For a detailed discussion, see [1, 2]. These values are based on the total Mg2+ contents and include
bound Mg2+ and free Mg2+. [Mg2+]i in a cellular compartment depends on total Mg2+, on the concentra-
tion of Mg2+-binding substances, on their Mg2+ affinity, and on the Mg2+ transport activity of the
membranes.
Determination of [Mg2+]i
Methods to measure [Mg2+]i are based on Mg2+binding fluorescent indicators, e.g. furaptra, also
called mag-fura-2, 31P NMR and Mg2+-sensitive
microelectrodes. In some cases null-point and enzymatic methods were used. For a review see [3-5].
Furaptra
In this method, [Mg2+]i is determined according to:
[Mg2+]i = KDMgfuraptra × (R − Rmin) ⁄ (Rmax − R) × Sf⁄Sb
Rmin and Rmax are the excitation wave length ratios
at 335/370 nm for uncomplexed (excess EDTA) and
Mg2+-saturated furaptra respectively. Sf and Sb are
225
T. GÜNTHER
Table 1. Subcellular distribution of Mg2+ in rat liver. Values provided by 3
different investigators in % of total Mg2+ were taken from [1, 2].
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Nuclei
Mitochondria
Microsomes
Cytosol
1
2
3
13.4
21.8
48.0
12.8
16.3
23.2
45.2
14.1
47.8
17.4
13.7
19.2
the fluorescence intensities measured at the 370 nm
excitation wave length for furaptra with excess
EDTA and excess Mg2+ respectively. R is the excitation ratio at 335/370 nm of the sample to be measured. For KD of the Mg furaptra complex, usually a
value of 1.5 mM was used. For uncertainty of KD see
below.
31
P NMR
Many investigators have determined [Mg2+]i according to:
[Mg2+]i = KDMgATP × (ø−1 − 1)
where ø is usually calculated from the chemical shift
differences of the a and b phosphorus peaks of ATP
due to binding of Mg2+. Some investigators also used
other formulae to determine [Mg2+]i from 31P NMR
measurements. For details see [6].
KDMgATP represents the dissociation constant of
the MgATP complex. KDMgATP values have been measured for more than 50 years. A selection of these
values is listed in table 2A. More values for KDMgATP
are shown in table 3. Also, when measured by vari-
Abbreviations:
[Mg2+]i
concentration of intracellular free Mg2+,
[Ca2+]i
free Ca2+ and Na+
[Na+]
i
[Mg2+]o
KD
KA
KM
31
P NMR
pMg
TEABr
VSMC
MDCK
CTAL
ANP
PTH
PHA-L
ConA
EGF
AVP
226
concentration of extracellular Mg2+
dissociation constant
association constant
Michaelis constant
31-phosphorus nuclear magnetic resonance
-log [Mg2+]
tetraethylammonium bromide
vascular smooth muscle cells
Madin-Darby canine kidney
cortical thick ascending limb
atrial natriuretic peptide
parathyroid hormone
phytohaemagglutinin-L
concanavalin A
epidermal growth factor
arginine vasopressin
ous investigators under identical conditions of temperature, pH and ionic strength, the values measured
for KD of MgATP show great variability.
Mg2+-sensitive microelectrodes
Mg2+-sensitive microelectrodes are based on the
neutral ionophores ETH 1117 or on the more specific
derivative ETH 5214 with less K+ interference. Values
for [Mg2+]i are obtained by comparing the electrode
potential measured in the cytosol minus membrane
potential with the electrode potential of a calibration
solution which, because of ion interferences, should
be identical to the ionic composition of the cytosol.
[Mg2+]i in cell organelles and cytosol
[Mg2+]i in sarcoplasmic (endoplasmic) reticulum as
measured by furaptra amounted to 1.0 mM [7].
[Mg2+]i in mitochondria is about the same as
[Mg2+]i in the cytosol [8]. Values of 0.67 mM [9], 0.5
mM and 0.8 - 1.5 mM and 0.82 mM, determined by
furaptra in isolated beef heart mitochondria, have
been reported [10]. Since there is a membrane potential of 200 mV inside negative across the mitochondrial membrane, there should be an outside directed
Mg2+ transport mechanism. Mg2+ influx into mitochondria may be mediated by a protein (Mrs2p) that is
inserted into the inner mitochondrial membrane [11].
Tables 3 and 4 present a selection of the values of
[Mg2+]i in the cytosol taken from the enormous number of values reported by various investigators for
different cell types and tissues. More values for
[Mg2+]i measured by various methods are listed in
[14, 68]. As shown in table 3, very different KDs for
Mg furaptra and MgATP were used. The KDs varied
by a factor of up to 4. However, the variation of
[Mg2+]i within the same cell type is less expressed
than was to be expected with respect to the different
KDs e.g. in A7r5 cells, a rat embryonal aortic muscle
derived cell line, or in brain.
When [Mg2+]i was measured in the same cell type
(lymphocytes) by different methods (furaptra, nullpoint, Mg2+ electrode), different values (0.15 mM
(table 4), 0.24 mM, 0.9 mM and 1.35 mM (table 3))
INTRACELLULAR FREE MG2+ CONCENTRATION
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Table 2. A) Various values of the dissociation constant (KD) of the MgATP complex as published by various
authors. Values for log KA (association constant) from [12] were calculated as KD for comparison with the
values provided by other authors.
B) Dissociation constant of MgATP (KD) as a function of pH and temperature according to [14, 20].
C) Dissociation constant of MgATP (KD) as a function of ionic strength at 25 °C and pH 7.2 according to [14].
A)
T (°C)
20
25
25
25
30
25
25
25
37
37
37
37
pH
7.0
7.2
7.2
7.2
7.2
7.2
7.2
Ionic strength (M)
0.1 KCl
0.1 KCl
0.1 KCl
0.1 TEABr
0.1 TEABr
0.1 TEABr
0.14 KCl, 0.014 NaCl
0.14 KCl, 0.01 NaCl
0.13 KCl, 0.02 NaCl
0.14 KCl, 0.014 NaCl
0.15 KCl
0.15 KCl
KD (lM)
144
91.2
56.2
42.6
9.6
37.4
127.5
45
83.3
87.4
38
46
Ref.
[12]
[12]
[12]
[12]
[12]
[13]
[14]
[15]
[16]
[14]
[17]
[18]
B)
KD (lM)
pH
6.7
7.2
7.7
25° C
157.0
127.5
101.0
C)
Ionic strength (M)
0.087
0.156
0.300
were obtained. In erythrocytes, the null- point
method yielded higher values than 31P NMR (0.4 mM
versus 0.2 mM). See also [8]. Also, the same method
used by various investigators yielded very different
values of [Mg2+]i in the same cell type: in resting
platelets 0.266 mM to 0.644 mM, in VSMC 0.31 mM
and 0.62 mM (table 4).
With Mg2+ microelectrodes, the first measurements of [Mg2+]i yielded rather high values [49, 50].
Later measurements yielded values for [Mg2+]i in the
range as measured by other methods.
Uncertainty of [Mg2+]i measurements
Because of limited specificity in the measurement
with Mg2+-sensitive electrodes, the calibration solu-
37° C
106.6
87.4
78.1
KD (lM)
61.9
243.0
127.5
tion must be identical with the ionic composition of
the cytosol. This can be done with respect to the
cations. It is not possible for the cytosolic anions.
Therefore, the calibration must be done on the basis
of the activity of Mg2+ and not on the basis of the
concentration of Mg2+. However, there is uncertainty
about the activity coefficient of Mg2+. In an isotonic
solution the activity coefficient may amount to 0.3
[69] or 0.55 [70]. The higher value is the more correct
one. Besides an estimate of the activity coefficient
according to Debye-Hückel for pure salt solutions,
cytoplasma contains proteins that bind water (10%)
without salt (non-solvent water). This effect and the
interaction of Mg2+ with charges of macromolecules
and membranes may lead to an additional reduction
in the activity coefficient [71].
227
T. GÜNTHER
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Table 3. Concentration of intracellular free Mg2+ ([Mg2+]i) in various cell types and tissues as measured with
various methods. The used dissociation constants of Mgfuraptra and MgATP as well as the used Mg2+ sensor
in the Mg2+ electrodes were listed.
Cells / Tissue
Frog muscle fibers
Chicken cardiomyocytes
Rat hepatocytes
VSMC
Guinea pig tenia cecum
A7r5 cells
A7r5 cells
Rat cardiomyocytes
Rat cardiomyocytes
BC3H-1 cells (fibroblasts)
Pancreatic acinar cells
Human lymphocytes
Mouse skeletal muscle
Rat erythrocytes
Human erythrocytes
Human erythrocytes
Human erythrocytes
Rat erythrocytes
Rat brain
Rat brain
Guinea pig brain
Human brain
Human brain
Rat liver
Rat liver
Rabbit uterus
Human skeletal muscle
Human skeletal muscle
Guinea pig heart
Frog skeletal muscle
Guinea pig tenia cecum
Ascites tumor cells
Human platelets
Human platelets
Mouse lymphocytes
Mouse lymphocytes
Frog skeletal muscle
Frog skeletal muscle
Frog skeletal muscle
Rat skeletal muscle
Guinea pig heart
Ferret myocard
Ferret myocard
Pancreatic acinar cells
Snail neurones
228
Method
Furaptra
Furaptra
Furaptra
Furaptra
Furaptra
Furaptra
Furaptra
Furaptra
Furaptra
Furaptra
Furaptra
Furaptra
Mag-indo-1
31
P NMR
31
P NMR
31
P NMR
Null-point
Null-point
31
P NMR
31
P NMR
31
P NMR
31
P NMR
31
P NMR
31
P NMR
31
P NMR
31
P NMR
31
P NMR
31
P NMR
31
P NMR
31
P NMR
31
P NMR
31
P NMR
31
P NMR
Null-point
Null-point
Mg2+ electrode ETH1117
Mg2+ microelectr. ETH1117
Mg2+ microelectr. ETH1117
Mg2+ microelectr. ETH1117
Mg2+ microelectr. ETH1117
Mg2+ microelectr. ETH5214
Mg2+ microelectr. ETH5214
Mg2+ microelectr. ETH1117
Mg2+ microelectr. ETH5214
Mg2+ microelectr. ETH1117
KD
4.6 mM
1.5 mM
1.5 mM
1.5 mM
5.4 mM
5.4 mM
1.5 mM
5.4 mM
5.3 mM
1.58 mM
1.5 mM
2.1 mM
5.1 mM
44.3 lM
38 lM
50 lM
44.3 lM
90 lM
86 lM
20.6 lM
44.3 lM
86 lM
50 lM
29 lM
50 lM
44.3 lM
28.7 lM
45 lM
41 lM
60 lM
38 lM
[Mg2+]i (mM)
1.7
0.48
0.59
0.48
0.98
0.74
0.31
1.13
0.91
0.54
1.39
0.24
1.53
0.193
0.223
0.225
0.4
0.38
0.47
0.56
0.33
0.32
0.35
0.7
0.8
0.4
0.557
0.47
2.5
0.6
0.33
0.44
0.23
0.3
0.9
1.35
3.8
3.3
1.3
0.47
0.72
0.85
3.0
0.58
0.66
Ref.
[21]
[22]
[23]
[24]
[25]
[25]
[26]
[25]
[27]
[28]
[29]
[30]
[31]
[11]
[32]
[33]
[34]
[35]
[11]
[36]
[37]
[38]
[11]
[39]
[40]
[41]
[42]
[11]
[43]
[44]
[45]
[46]
[47]
[47]
[48]
[48]
[49]
[50]
[51]
[52]
[53]
[54]
[50]
[55]
[56]
INTRACELLULAR FREE MG2+ CONCENTRATION
Table 4. Alteration of [Mg2+]i by various effectors measured by means of furaptra.
Copyright © 2017 John Libbey Eurotext. Téléchargé par un robot venant de 88.99.165.207 le 16/06/2017.
Cells
MDCK cells
MDCK cells
CTAL cells
CTAL cells
CTAL cells
CTAL cells
Human platelets
Human platelets
Human platelets
Human platelets
Human platelets
Human lymphocytes
Fibroblasts
Fibroblasts
Fibroblasts
Fibroblasts
Fibroblasts
Sub.muc.acini
VSMC
VSMC
VSMC
VSMC
VSMC
VSMC
VSMC
Panc.acinar cells
Panc.acinar cells
Panc.acinar cells
Panc.acinar cells
Panc.acinar cells
[Mg2+]i (mM)
Effector
Resting
Stimulated
0.552
0.538
0.525
0.538
0.540
0.542
0.266
0.614
0.614
0.58
0.58
0.15
0.208
0.208
0.208
0.328
0.328
0.35
0.31
0.31
0.30
0.30
0.62
0.62
0.62
0.82
0.82
0.82
0.82
0.59
0.682
0.362
0.592
0.609
0.497
0.462
0.355
1.270
0.405
0.59
0.40
0.25
0.241
0.304
0.244
0.538
0.538
0.5
2.44 (peak)
0.42
2.36 (peak)
0.43
0.52
0.8 (peak)
0.46
1.06 (peak)
0.61
1.01 (peak)
0.69
0.37
8-Br-cGMP (0.1 mM)
8-Br-cAMP (0.1 mM)
ANP (1 lM)
8-Br-cGMP (0.1 mM)
PTH (1 lM)
Calcitonin (1 lM)
Insulin (200 lU/ml)
Insulin (100 lU/ml)
Thrombin (0.1 U/ml)
Endothelin-1 (1nM)
Angiotensin-II (1 nM)
PHA-L (5 mg/ml)
Insulin (100 ng/ml)
Bombesin (3 nM)
EGF (10 ng/ml)
Bombesin (3 nM)
Endothelin-1 (0.5 lM)
Carbachol (10 lM)
AVP (1 lM)
AVP (1 lM)
Endothelin-1 (1lM)
Endothelin-1 (1 lM)
AVP (10 nM)
Angiotensin-II (10nM)
Angiotensin-II (10 nM)
Acetylcholine (10 lM)
Acetylcholine (10 lM)
Cholecystok. (10 nM)
Cholecystok. (10 nM)
Cholecystok. (01 nM)
There is another common uncertainty. Also KDMg
MgATP
furaptra and KD
must be determined in solutions
identical to the ionic composition of the cytosol.
However the solutions used are composed on the
basis of chloride salts, and are thus different from the
cytosolic anion composition. Due to its content of
inorganic phosphate and phosphate esters, the Mg2+
activity coefficient in the cytosol is lower than in the
chloride-containing solutions. Besides this uncertainty, the values for KDMg furaptra used by different
investigators varied from 1.0-6.8 mM [5, 7, 10]. KD
values estimated in situ yielded higher values
(2.1 mM [9], 5.3 mM [27], 5.4 mM [27] and 6.8 mM [7])
than KDs determined under in vitro conditions (usually 1.5 mM).
Ref.
[57]
[57]
[57]
[57]
[57]
[57]
[58]
[59]
[59]
[60]
[60]
[61]
[62]
[62]
[62]
[63]
[63]
[64]
[65]
[65]
[65]
[65]
[66]
[66]
[66]
[67]
[67]
[67]
[67]
[55]
For KDMgATP the situation is still more complex.
P NMR measures the binding of Mg2+ to phosphoryl groups of ATP. Mg2+ binding is dependent
on pH because the phosphate group dissociates at
a pK value of about 6.8 [12], and is thus dependent
on the intracellular pH. The degree by which the KD
of MgATP is dependent on pH is shown in table 2B.
Besides H+, other cations compete with Mg2+ for
binding to ATP. This encludes a weak binding of K+
and Na+ to ATP (log KA = 0.99 and 0.98 M-1 [12]).
To prevent the interaction of K+ and Na+ with ATP,
0.1 M tetraethylammonium salt was taken to adjust
ionic strength. The effect of the ionic strength on
the dissociation constant of MgATP is shown in
table 2C.
31
229
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T. GÜNTHER
A more realistic determination of KD of MgATP
was performed using an Mg2+-sensitive electrode
and a background solution containing 140 mM K+
and 14.6 mM Na+. A value of 87.4 lM was obtained at
pH 7.2 and 37° C [14, 20]. The same value (86 lM) was
obtained by means of 31P NMR under approximately
in vivo conditions [39].
When the more realistic KD of MgATP was used to
recalculate the values of [Mg2+]i measured by other
investigators, the values for [Mg2+]i increased to a
factor of 2.8 [14]. Values of 0.7 to 1.7 mM were
obtained for 14 determinations of [Mg2+]i in various
tissues [14].
should be enriched at the inner side of the negatively
charged plasma membrane.
Taken together, besides the uncertainty in the average values of [Mg2+]i in the cytosol, there is an
unequal distribution of free Mg2+ in the cytosol by a
factor ranging between 3 and 10. The high [Mg2+]i
cannot be explained by enrichment of free Mg2+ in
cell organelles (e.g. mitochondria, endoplasmic
reticulum) because [Mg2+]i in these cell organelles
was about the same as the average [Mg2+]i in the
cytosol (see above). The mechanism causing the heterogeneous distribution of intracellular free Mg2+ is
not defined.
Microheterogeneity of [Mg2+]i in the cytosol
Metabolic function of intracellular Mg2+
The above reported values of [Mg2+]i are average
values over total cytosol. Compartmentation of cytosolic Mg2+ was not considered. However, Mg2+ is not
uniformly distributed within the cytosol. Mg2+ is
enriched at negatively charged phospholipids of cellular and intracellular membranes and at the surface
of negatively charged macromolecules. For a
detailed discussion see [2]. The differently enriched
Mg2+ relates to free Mg2+. There is experimental
evidence for a different distribution of free Mg2+
within the cytosol.
Furaptra applied in cultured rat aortic smooth
muscle cells and using a KD of 1.5 mM yielded an
[Mg2+]i of 0.2 mM in the peripheral area, 0.6 mM in
the perinuclear region and about 0.6 mM in the
nuclear region [72]. A heterogeneous distribution of
intracellular free Mg2+ in rat VSMC was also found by
other investigators [73] using furaptra. Using a KD of
1.45 mM, [Mg2+]i in the nuclear region was 0.32 mM.
[Mg2+]i in the perinuclear region, which contains the
highest density of intracellular organelles, amounted
to 0.4-1.13 mM and the non- nuclear cytoplasmic area
had an [Mg2+]i of 0.77 mM. The peripheral region
near the plasma membrane had the lowest [Mg2+]i.
By using mag-indo-1, other authors [74] found a
mean [Mg2+]i of 1.4 mM in human tracheal gland
cells. Within a single cell, [Mg2+]i was uniformly distributed within the nucleoplasm. Cytosolic [Mg2+]i
varied from 0.34 mM to 3 mM in another opposing
region in the same cell.
Since positively charged Mg2+ ions are enriched at
negatively charged membranes and macromolecules, it must be expected that the negatively
charged furaptra is rejected by these structures,
resulting in erroneous values of [Mg2+]i. This may be
a reason why a low [Mg2+]i was measured with furaptra near the plasma membrane, although Mg2+
In view of these facts, it is uncertain to define an
absolute and exact value of [Mg2+]i within a defined
space of the cytosol. Besides compartmentation of
free Mg2+ in the cytosol, cytosolic metabolic pathways such as glycogenolysis and glycolysis are also
compartmentalized [75, 76].
Since Mg2+ is enriched at negatively charged membranes, an exact knowledge of [Mg2+]i at the inner
surface of cell membranes would be essential to
define the role of intracellular Mg2+ in the regulation
of membrane-bound metabolic pathways and in the
modulation of K+ and Ca2+ channels [77] as well as
the role of intracellular Mg2+ in the feedback inhibition of Mg2+ influx via TRPM7 in the regulation of
[Mg2+]i [78, 79].
Generally, metabolic pathways are regulated by
changing the activity of the rate-limiting enzymes.
Usually these are allosteric enzymes with a complex
regulation by activation and feedback inhibition by
various metabolites, end products of biosynthesis
and by effectors. Moreover, these enzymes can be
regulated by phosphorylation-dephosphorylation.
During evolution, the regulation of metabolic pathways was adapted to the requirements of the cell
with respect to energy supply as well as intermediates and end products for biosynthesis.
What is the role of intracellular Mg2+ in these
mechanisms? All reactions of Mg2+ with ligands such
as low molecular substances (ATP, etc.), proteins,
enzymes, and nucleic acids obey the law of mass
action, yielding a sigmoidal curve when plotted as a
function of pMg. With respect to Mg2+-dependent
enzymes at high [Mg2+]i, enzymes are usually inhibited by Mg2+. Activation and inhibition of an enzyme
by Mg2+ as a function of pMg results in a bell-shaped
curve [1]. Activation by Mg2+ occurs at the ascending
part of the bell-shaped curve up to the pMg optimum.
230
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INTRACELLULAR FREE MG2+ CONCENTRATION
Alterations of [Mg2+]i near or within the range of the
pMg optimum of a Mg2+-dependent rate-limiting
enzyme have no significant effect on the rate of a
metabolic pathway.
Since the exact absolute value of [Mg2+]i in the
cytosol is uncertain (see above) and as there are
considerable differences in the values of [Mg2+]i for
the same cell type (tables 3, 4), it is uncertain at
which degree of activation (part of the bell-shaped
pMg dependency) the Mg2+-dependent rate-limiting
enzymes are operating.
For some glycolytic enzymes, the pMg optimum is
at pMg 3 [80, 81]. However, the Mg2+ affinity and the
pMg optimum of enzymes can change. This effect
was shown for isolated glycolytic enzymes, when
their Mg2+ dependency was determined at various
constant Mg2+: ATP or Mg2+ : ADP ratios [81]. This
effect may occur in intact cells. The synthesis of ATP
and the utilization of ATP in the cytosol is compartmentalized [75, 76]. In organisms, there is an additional mechanism that can change the affinity of
Mg2+ to enzymes. For example, the addition of glucagon to liver cells resulted in a dramatic decrease in
the KM of adenylyl cyclase for Mg2+ [82].
In all Mg2+-dependent enzymes, which use MgATP
as a substrate, an alteration of [Mg2+]i has only a
minor effect on the enzyme activity because ATP is
nearly saturated with Mg2+ (to about 90%). Moreover,
an alteration of MgATP affects enzyme activity
according to Michaelis-Menten kinetics and depends
on the KM of MgATP and the MgATP concentration.
Intracellular Mg2+ is buffered at a high level [20, 83].
Thus, intracellular Mg2+ is not suitable for a regulatory function. Its metabolic function can be tested by
artificially changed [Mg2+]i in experiments with
intact cells. In such experiments it was found that
[Mg2+]i did not play a regulatory role in erythrocyte
glycolysis [84].
When cells [85] or isolated mitochondria [86] were
drastically Mg2+-depleted by means of A23187, respiration was reduced. In these experiments, [Mg2+]i in
mitochondria was not measured. Because of the
complex effects under the experimental conditions,
it cannot be decided whether [Mg2+]i in mitochondria has a regulatory or a permissive function. [Ca2+]i
in mitochondria was also changed in these experiments and may have affected respiration. For a discussion of the interaction of H+, Ca2+, inorganic
phosphate, spermine and various cofactors with
Mg2+ in respiration and oxidative phosphorylation,
see [87].
The synthesis of purine and pyrimidine precursors
of nucleic acids depends on Mg2+, and at each step of
DNA replication, RNA transcription and RNA trans-
lation Mg2+ is required for enzyme function. Mg2+ is a
cofactor in all these processes. Regulation of enzyme
activity through changes in [Mg2+]i has not been
observed [88].
Computer models have shown that Mg2+ does not
regulate cardiac metabolism [89].
A drastic reduction in the cellular Mg2+ content by
means of A23187 yielded a reduction in the biosynthesis of proteins, DNA and RNA, respiration and
glycolysis [85]. Again these experiments did not
prove that intracellular Mg2+ has a regulatory function. They may indicate a permissive function of
intracellular Mg2+. Alternatively to conclusions
about a permissive role of intracellular free Mg2+,
other authors have suggested a regulatory function
of intracellular free Mg2+ [89-92].
Mg2+ content in malignant cells
Rapidly dividing normal cells have higher contents of
Mg2+, K+, Na+ and Cl- than slowly growing normal
cells [93]. In rapidly dividing tumor cells, the concentrations of intracellular Na+ and Cl- were elevated,
whereas intracellular total Mg2+ and K+ contents
were significantly lower than in rapidly dividing normal cells. Injection of tumorous mice with amiloride
reduced [Na+]i and cell proliferation without significantly changing total Mg2+ and K+ content. It was
concluded that an early brief surge in [Ca2+]i is essential in mitogenic stimulation followed by an increase
in [Na+]i and pHi [93].
When rapidly dividing HL60 cells, a promyelocytic
leukemia cell line, were transformed to neutrophiliclike cells by incubation with retinoic acid, the total
Mg2+ content of the cells was reduced by 19%. The
total Mg2+ content in mitochondria and cytoplasm
(cytoplasm was defined as the region excluding
mitochondria and nuclei) were reduced by 18%, and
the Mg2+ content of nuclei was unchanged [94]. The
reduction of total Mg2+ content in cytoplasm may be
caused by a reduction of ATP by 31% and ADP by 40%
[94], and by an increase in Na+/Mg2+ antiport [95]. An
alteration of Mg2+ bound to ribosomes was not investigated. Total cytoplasmic Ca2+ content in the transformed cells was reduced by 40%, and the K+/Na+
ratio in nuclei was reduced by about 28% [94]. The
complex mechanisms by which the contents of the
various ions were changed during the differentiation
process of the cells and their metabolic significance
are not defined.
231
T. GÜNTHER
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Action of effectors on [Mg2+]i and [Ca2+]i
[Mg2+]i in nucleated cells is somewhat constant. It
can be changed significantly by effectors. The development of furaptra and fura-2 offered the possibility
of rapidly and continuously measuring [Mg2+]i and
[Ca2+]i and their alterations by various effectors.
When intracellular Mg2+ has a regulatory function,
[Mg2+]i must be changed by effectors at a reasonable
concentration within a reasonable time period and to
a reasonable extent. The alterations of [Mg2+]i after
addition of various effectors are listed in table 4.
Remarkably, different investigators measured different values of [Mg2+]i in resting platelets and a different increase of [Mg2+]i by insulin as well as controversial effects by thrombin in these cells. Thrombin
decreased [Mg2+]i in human platelets from 0.614 mM
to 0.405 mM [59], whereas other authors found an
increase in [Mg2+]i in platelets from 0.54 mM to 1.33
mM by thrombin [96].
The increases in [Mg2+]i induced by various effectors at the concentrations listed amounted maximally to a factor of 2, corresponding to an alteration
in pMg of 0.3. A small part of the increase in [Mg2+]i
by some effectors may include an increase in [Ca2+]i
because of the unspecificity of furaptra. Due to the
uncertainty of an absolute value of [Mg2+]i, the alterations in [Mg2+]i must be considered as relative.
Of the effectors listed in table 4, insulin had no
effect on [Ca2+]i [58, 59]. Insulin may increase Mg2+
uptake into platelets. In VSMC, vasopressin [65, 66]
and endothelin-1 [65] induced rapid peaks of [Mg2+]i
and [Ca2+]i followed by a decrease in [Mg2+]i and
[Ca2+]i. The peak and sustained values of [Mg2+]i are
listed in table 4. The reduction in [Mg2+]i may be
caused by Mg2+ efflux via Na+/Mg2+ antiport [65].
Mg2+- and Ca2+-free medium decreased the
vasopressin-mobilized [Ca2+]i by 60.8% and prevented the increase in [Mg2+]i [65]. Based on these
results, the authors concluded that vasopressin and
endothelin-1 mobilized Mg2+ in VSMC through the
action of the intracellular second messenger Ca2+.
However, in A7r5 cells, a VSMC line, 100 nM vasopressin had no effect on [Mg2+]i [26].
In fibroblasts, bombesin induced an [Mg2+]i and
[Ca2+]i peak [62, 63], followed by a sustained
decrease in [Mg2+]i and by oscillations of [Ca2+]i.
Endothelin-1 (0.5lM) had a similar effect on [Mg2+]i
as 3 nM bombesin. There was a considerable variation in the [Mg2+]i response to bombesin,
endothelin-1 [63] and EGF [97] among different individual cells of the same cell type.
Some effectors have a cell -specific action, e.g.
endothelin-1 had no effect on [Mg2+]i and [Ca2+]i in
232
platelets [60]. 8-Br-cGMP increased [Mg2+]i in MDCK
cells [57] and had no effect on [Mg2+]i in platelets
[59].
The increase in [Mg2+]i by AVP and endothelin-1
[65] affected [Ca2+]i and [Mg2+]i simultaneously and
in the same direction. In VSMC, AVP and endothelin
[65] and in pancreatic acinar cells, acetylcholine and
cholecystokinin induced short peaks of [Mg2+]i followed by a decrease in [Mg2+]i [67]. In a more
detailed study with pancreatic acinar cells, 100 pM
cholecystokinin and 100 lM acetylcholine decreased
[Mg2+]i from 0.58 mM to 0.47 mM. This may be caused
by Mg2+ uptake into the endoplasmic reticulum [55]
or by Mg2+ efflux via Na+/Mg2+ antiport [65, 67].
Cholecystokinin (10 pM) induced [Ca2+]i oscillations. Frequency and amplitude of [Ca2+]i oscillations were increased when [Mg2+]i was decreased by
preincubation at low extracellular Mg2+ concentration [55]. Thus, intracellular Mg2+ can modulate Ca2+
signaling.
The reviewed alterations in [Ca2+]i reflect the general function of Ca2+ as a second messenger. Ca2+
signaling can occur as a single transition, as a sustained plateau or as repetitive oscillations. [Ca2+]i
oscillations are known to be involved in the control
of a number of important cell processes, e.g. regulation of gene transcription, where the efficacy of the
control varies with the amplitude and frequency of
the oscillations [98, 99]. The different patterns of
intracellular Ca2+ reveal a mechanism by which a
multifunctional second messenger as Ca2+ can
achieve specificity in signal transduction [98, 99].
Ca2+ versus Mg2+ as a second messenger
From the results it can be concluded that some effectors (vasopressin, endothelin-1, bombesin, cholecystokinin, acetylcholine, carbachol) primarily increase
[Ca2+]i which secondarily increase [Mg2+]i through
liberation of intracellular Mg2+. The mechanism of
Mg2+ liberation is not defined. From other experiments it has been suggested that bound Mg2+ may be
released through acidification and by displacement
of protein-bound Mg2+ by Ca2+ [100, 101]. In experiments with pancreatic acinar cells, an alteration of
pHi had no effect on cholecystokinin-induced
changes of [Mg2+]i. The changes in [Mg2+]i were
related to the release and reuptake of Mg2+ by the
endoplasmatic reticulum [55]. Competition of Ca2+
with protein-bound Mg2+ may be the probable
mechanism. The function of the effector-induced
increase in [Mg2+]i may enhance Mg2+-dependent
reactions to support the effector-induced mechanisms in the target cells.
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INTRACELLULAR FREE MG2+ CONCENTRATION
The effector-induced alterations of [Mg2+]i were
discussed as evidence that intracellular Mg2+ is a
second messenger [94]. It merely indicates that intracellular Mg2+ can affect the action of Ca2+ as a second messenger. Additional evidence that intracellular Ca2+ but not Mg2+ is the second messenger was
obtained with human lymphocytes stimulated by
Con A. Con A increased [Ca 2+]i and [Mg2+]i. However, [Mg2+]i was only elevated in cells with a high
[Ca2+]i [102]. This result indicates that a sufficient
increase in [Ca2+]i is necessary to induce the
increase in [Mg2+]i.
In the complex interactions between intracellular
Mg2+ and Ca2+, [Mg2+]i has a level at which Ca2+
release is almost maximally inhibited, and Ca2+ storage is almost maximally activated by intracellular
Mg2+. Thus [Mg2+]i provides a minimal [Ca2+]i, so
that effectors can induce pronounced changes in
[Ca2+]i [103].
These results are in agreement with the physicochemical properties of Ca2+.
Because of the larger diameter of Ca2+ compared
with Mg2+, in multidentate chelates with proteins
there is less ligand-ligand repulsion by the negatively
charged chelating groups. This yields 1000 times
lower dissociation constants of Ca2+ with Ca2+binding proteins compared with Mg2+ and to 1000
times lower [Ca2+]i compared with [Mg2+]i. Thus
Ca2+ was favored against Mg2+ as a second messenger during evolution. For details see [99, 104].
Conclusion
Intracellular Mg2+ is buffered by reversibly binding
to ligands such as nucleotides, nucleic acids, proteins, phospholipids and negatively charged low
molecular substances. The exact average value of
[Mg2+]i is uncertain due to methodical difficulties
and higher than usually reported. Intracellular free
Mg2+ and metabolic pathways are compartmentalized. The Mg2+ affinity and pMg optimum of enzymes
can be changed by altering pH, [Ca2+]i, substrates,
end products, effectors and hormones. Therefore,
the exact quantitative role of intracellular Mg2+
under the conditions of an intact cell or tissue cannot
be defined. The increase in [Mg2+]i in various cell
types by effectors is small. Various effectors increase
[Ca2+]i and [Mg2+]i. The alterations in [Ca2+]i are
more expressed than the alterations in [Mg2+]i. The
evidences favor intracellular Ca2+ as a second messenger. The simultaneous increase in [Mg2+]i may
activate Mg2+-dependent reactions to support
effector-induced mechanisms in target cells [19].
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