ARTICLE
Auteur(s) : Claire Michelet-Habchia, Philippe
Barbereta, Raj Kumar Duttaa, Philippe
Morettoa, Andrée Guiet-Barab, Michel
Barab
a Centre d'Études Nucléaires de
Bordeaux-Gradignan, Interface Physique-Biologie, Le Haut-Vigneau,
BP 120, 33175 Gradignan Cedex, France.
b Laboratoire de Physiologie et Physiopathologie, Université
P. et M. Curie, Bât. A, 4 Place Jussieu, 75252 Paris
Cedex 05, France.
Introduction
Alterations in extracellular and intracellular magnesium in
numerous tissues have been shown to alter ion channel currents. The
magnesium regulation of various ionic channels interferes with
cellular functions. Magnesium ions are known to block the current
through K+ channels (inwardly rectifier, delayed
rectifier, Ca-dependent, ATP-dependent), Na+ channels
and Ca2+ voltage-gated channels and to activate the
current through chloride channels [1]. Magnesium ions interfere
with numerous functions, and particularly extracellular magnesium
ions which are implicated in the blood vessel smooth muscle tone
and reactivity, regulated by membrane potential [2]. The membrane
potential of vascular cells is regulated by ionic channels which
induce depolarization or hyperpolarization. Previous studies [3-5]
have demonstrated the influence of magnesium salts on the membrane
potential of vascular smooth muscle cells (VSMCs) and of vascular
endothelial cells (VECs) of human allantochorial placental vessels
which are representative of the small arteries involved in the
control of fetal and maternal utero-placental resistance, in
contrast to the usually studied umbilical arteries [3]. The
membrane potential of these cells has been investigated and is
modulated by several channels: voltage-sensitive potassium channels
(Kdf), Ca-activated (KCa) and ATP-sensitive
(KATP) potassium channels and voltage-sensitive calcium
channels in VSMCs [6]; Kdf, KCa and
voltage-sensitive calcium channels in VECs [7]. Previous studies
[8] have investigated the possibility of applying nuclear
microprobe analysis to human vascular allantochorial placental
cells in order to reveal the structure of arteries and to generate
maps of the different inorganic ions across the walls of
arteries.
The aim of this study was to investigate the influence of two
magnesium salts (physiological magnesium chloride and magnesium
sulfate, frequently used in the case of pre-eclampsia and/or
eclampsia) [9] added to a survival medium (Hanks's solution) on the
elemental ion distribution (Na, K, Cl, P, Ca, Mg) in VSMCs and in
VECs to investigate the possible relationship with the ionic
movement across the membrane channels.
Material and methods
Sample preparation
Short segments of allantochorial arteries (results obtained
with veins are identical) [3] were sampled in human placenta
collected after normal delivery at term. Samples were immediately
incubated in different media: Hanks' solution, Hanks' solution +
2 mM MgCl2, and Hanks' solution + 2 mM
MgSO4 (the concentration of 2 mM of Mg corresponds
to a middle pharmacological magneso-therapy and to the dose
inducing a measurable effect on the ionic channels3).
Then the samples were cryofixed using the following method: the
samples were quench-frozen in isopentane chilled with liquid
nitrogen and stored in liquid nitrogen until sectioning.
The preparation of placental vessels was carried out according
to a classical scheme: arteries were cut using a cryo-microtome
equipped with a tungsten carbide blade and placed in a cryostat at
– 25°C. The sections (20 µm thick) were directly
collected from the blade using fresh FormvarR films
(50 µg/cm2 areal mass) stretched on pure aluminium
sample holders. The section were left overnight in the cryostat
until complete freeze-drying.
Nuclear microprobe analysis
All sections were analyzed using the CENBG nuclear microprobe.
This experimental setup has been previously presented [10]. The
microanalysis was carried out using a 1.5 MeV proton beam,
focused down to a small (2 µm in diameter) spot on the sample.
A beam current of 150 pA was employed. Particle Induced X-ray
Emission (PIXE) and Rutherford Backscattering Spectometry (RBS)
were employed simultaneously in order to determine both the mineral
content and the organic mass of the analyzed tissues. For each
sample, two analyses were performed: a large scan (
~ 1 mm2) in order to identify the
different strata obtained from the elemental maps of minerals, then
a small scan (
~ – 160 × 150 µm2) was made on the lumen
border to locate the monolayer of endothelial cells.
Data reduction
In order to measure the ionic content of smooth muscle cells and
endothelial cells in arteries, the strata were delimited on the
elemental maps (figure
1). In each lamina, a large region was then chosen for its
homogeneity and local spectra were extracted using a specific
computer treatment of list data files. The endothelial cells on the
lumen border maps were also identified, cell by cell, and local
spectra were also extracted from small regions.
For all PIXE analyses, the X-ray spectra were fitted with the Gupix
software [11]. For large scans, the RBS spectra were treated using
the Rumpin software [12]. By using the areal mass of the tissues
given by the RBS data, concentrations of Na, K, Cl, P, S, Ca and Mg
could be calculated. For endothelial cell monolayers however, the
statistics of RBS spectra was not sufficient to use a reliable
Rumpin treatment. Simple elemental ratios were thus calculated,
using sulphur as a reference element, as its content remained
constant in all analysed tissues. This procedure has already been
successfully employed in such analytical conditions.
Statistical comparisons between various specimens were carried
out with Student's test. The values of the significance level (p)
of 0.05 and less were considered as significant.
Results
Vascular smooth muscle cells (VSMC)
The ionic concentrations measured in VSMC are presented in figure 2. The histograms
compare, for each ion, the artery walls incubated in Hanks'
solution + 2 mM MgCl2 and those incubated in Hanks'
solution + 2 mM MgSO4 to those incubated in Hanks'
solution taken as a reference. Whatever the medium, the ionic
concentrations remained constant
(0.06 < p < 0.9) except a significant
increase of Na (p < 0.05) with MgCl2 and of
Mg (p < 0.005) concentrations in Hanks' solution +
MgCl2 and in Hanks' solution + MgSO4.
Vascular Endothelial cells (VEC)
The data obtained in VEC are presented in figure 3. Results are expressed
in terms of concentration ratios against sulphur taken as a
reference. Measurements indicated a significant increase of the Na
(p < 0.01) and Mg (p < 0.01)
concentration in Hanks' solution + MgCl2 and a
significant increase of Mg (p < 0.01) concentration in
Hanks' solution + MgSO4 compared to Hanks' solution
alone. The other elemental ion concentrations, whatever the medium,
remained constant (0.09 < p < 0.9).
Discussion
In human allantochorial placental vessels, the previous
electrophysiological studies [3, 4] have indicated that
MgCl2 and MgSO4 depolarize VSMCs with
different thresholds, indicating that MgCl2 influences
cell membrane potential directly while MgSO4 interferes
first with the endothelial cells, which may act as an intermedary
between Mg2+ ions and VSMCs. This effect has been
confirmed on VECs: MgCl2 and MgSO4 induce
depolarization and the influence of MgCl2 is higher and
more direct than that of MgSO4.
The membrane potential of VSMCs of human allantochorial
placental vessels is the major factor which explains the
excitation-contraction coupling. Indeed, the smooth muscle cells of
these vessels may be considered as tonic because they only respond
to excitatory stimuli with graded depolarization. This
depolarization may be a consequence of various factors,
particularly the secretion of endothelium-derived depolarized
factors (EDDFs).
In 1997 [13], a scheme was proposed to explain the effects of Mg
salts on the membrane potential of human allantochorial placental
vessel cells: 1- a direct action of external Mg2+ ions
on the membrane potential by secretion of EDDFs which depolarize
the membrane; 2- an indirect action on the membrane potential by: *
a regulation of K+ and Ca2+ channels; * an
action on the internal Mg2+ concentration; * an
interaction with internal Na+ and Ca2+
concentrations; and * a regulation of the
Na+/Ca2+ exchanger.
Previous electrophysiological studies [5, 14] have confirmed the
regulation of K+ and Ca2+ channels by
external Mg salts. Indeed, in VSMCs incubated in a physiological
medium+MgCl2, Kdf, KCa and
KATP channels are blocked (Kdf are open at
low MgCl2 concentration), while in VECs, Kdf
and KCa are blocked. In a physiological
medium+MgSO4, the same observations occur, except the
Kdf open at low concentrations. This observation also
occurs to Ca2+ — voltage gated channels which are
blocked [14, 15].
The present microanalysis study indicates that in VSMCs and in
VECs, the two Mg salts have no effect on the concentrations of K,
Cl, P and Ca, but that MgCl2 significantly increases Na
and Mg concentrations while MgSO4 increases only Mg
concentration.
These results confirm the blockage of the potassium and calcium
channels by Mg salts (the influx and the outflux of K and Ca ions
remain constant and their concentrations have not modified).
Moreover, the data indicate an interaction between Mg salts and
internal Na concentration in VSMCs and internal Mg concentration in
VSMCs and in VECs, corroborating the hypothesis scheme [13].
Internal Ca concentration is not modified, therefore it is
difficult to consider a relationship between Mg ions and
Na+/Ca2+ exchanger, but it seems possible to
consider the existence of an Na+/Mg2+
exchanger, demonstrated in numerous cell membranes [16], exchanger
which might be the major element of the regulation of Na and Mg
distribution in VSMCs and in VECs.
Conclusion
The results of the present study indicate the importance of
Mg2+ in the regulation of intracellular ion
concentrations in VSMCs and in VECs of allantochorial placental
vessel cells, indicating the relationship between Mg2+
ions and ionic channels, Na and Mg internal concentrations and with
a possible Na+/Mg2+ exchanger. Further
research should be undertaken to establish whether the effects of
Mg on the intracellular ion concentrations is dependent on the
Mg-salt dose.
References
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