ARTICLE
Auteur(s) : Antoine Grandvuillemin1, Pascal
Laurant1, Andrzej Mazur2, Yves
Rayssiguier2, Alain
Berthelot1
1EA 3921 Optimisation métabolique et cellulaire, UFR
Médecine-Pharmacie, Besançon, France
2Unité de nutrition humaine; UMR 1019, INRA, Theix,
Saint-Genès Champanelle, France
For 40 years, numerous epidemiological, clinical and
experimental studies have shown a relationship between magnesium
(Mg), metabolism disorders and different pathological states. Mg
seems to be particularly linked to cardiovascular disorders [1, 2]
and the role of Mg in the pathogenesis and treatment of
hypertension is receiving increased attention [3, 4]. There is an
inverse correlation between plasma Mg concentration and blood
pressure [5]. In rats, Mg deficiency results in an increase of
blood pressure [6-9]. Hypomagnesaemia and decreased tissue content
of Mg have also been demonstrated in various experimental models of
hypertension, especially in spontaneously hypertensive rats (SHR)
[10]. Moreover, in this model, an oral Mg supplementation
attenuates blood pressure elevation [11, 12], but it seems to
depend on the age of the animals [13].
Recent dietary surveys have shown that the average Mg intake in
western countries is often below the Recommended Dietary allowances
(RDA = 6 mg/kg/day) [14, 15]. In fact, in developed countries, the
consumption of dietary sources rich in Mg, like legumes, leafy
green vegetables or cereals has been declining since the beginning
of the 20th century. Although this “nutritional deficit”
is the principal origin of Mg deficiency in western populations,
there are some inter-individual differences of Mg homeostasis which
have a genetic origin [16-18]. This hypothesis was subsequently
confirmed by study of mono and dizygotic twins [19] and by the
study of different inbred strains in the mouse [16]. The genetic
system involved in this control is certainly polygenic and presents
a large polymorphism. This hypothesis is consistent with the
significant correlation found by Henrotte et al. [16, 17] between
blood Mg level and human (HLA) or mouse (H-2) tissues antigens, two
antigenic systems coded by the major histocompatibility complex
(MHC), one of the most polymorphic genetic systems described to
date. The same association is also found between MHC or non MHC
genes and levels of Mg in different tissues [17].
To further investigate the mechanism and the biological
significance of the variations of Mg metabolism of genetic origin,
Henrotte et al. [20] selected MGL (Mg Low) and MGH (Mg High) mice.
These two strains present some genetic differences in Mg
homeostasis: MGH mice have higher red blood cell and plasma Mg
concentrations than MGL mice [21-25].
The purpose of the present work was to examine some
cardiovascular hemodynamic and mechanic factors and vascular
reactivity to agonists in MGH and MGL mice.
Material and methods
Mice
MGL and MGH male mice aged 8 weeks were used: (INRA,
Clermont-Ferrand Theix, France). These strains were obtained from a
bidirectionnal selective breeding which had been carried out for 18
generations, from a heterogenous outbreed population constituted of
F2 segregant hybrid between 4 inbred strains: C57BL/6, DBA/2,
C3H/eB, AKR [20].
Upon arrival, the mice were housed in stainless steel cages at a
constant temperature of 22 ± 2°C and a daily 12-h light/dark cycle.
Food (regular mouse food A04, UAR, France) and distilled water were
distributed ad libitum. Before starting the experiments, one week
of adaptation was observed. All experiments were performed
according to the Institute Ethics Committee, in accordance with
decree no. 87-848.
Blood pressure and heart rate measurements
Systolic blood pressure and heart rate were non invasively measured
in 8 unanaesthetized and prewarmed mice from each group by the
tail-cuff method, using a piezoelectric transducer (Pulse sensor,
Kent Scientific, Torrington, CT, USA) connected to a Maclab/8s
system and Chart v. 3/4 software (PHYMEP, Campo Formio, France), as
previously described [26].
Animals followed a training to get used to the protocol over 15
days. Then, 10 measures of blood pressure and heart rate were
performed and the mean of each parameter was calculated.
Mg determination
Nine mice from each group were anaesthetized by an intraperitoneal
injection of sodium pentobarbital (60 mg/kg). A maximum amount
of blood was collected at the abdominal aorta level and immediately
centrifuged at 4 000 g for 10 minutes at 4°C.
Plasma was then drawn and frozen at - 80°C until analysis.
Moreover, the kidneys and heart were dissected, weighed and frozen
at - 80°C.
Samples of organs were defrosted, weighed then dried at 105°C
during 28 hours. They were weighed again and dissolved in nitric
acid (65%) at 80-120°C until total dissolution. Solutions were
preserved at 4°C until analysis. Mg determinations were carried out
by atomic absorption spectrometry (Perkin Elmer 3300, Norwalk,
USA), with a range prepared from a standard solution (Merck
standard, Darmstadt, Germany) and with an appropriate dilution for
each matrix in a solution of lanthanum oxide (0.1%).
Study of mesenteric arteries
Dissection and artery settings
Five mice from each group were killed by neck elongation and the
first mesenteric handle was removed and placed in cold
physiological salt solutions (PSS) of the following compositions
(mmol/L): NaCl, 120; NaHCO3, 25; KCl, 4.7;
KH2PO4, 1.2; MgSO4, 1.2;
CaCl2, 1.6; glucose, 11; ethylenediaminetetraacetic acid
(EDTA), 0.026. One segment of the third-order branch of a
mesenteric artery was used per mouse and was carefully dissected
and cleaned of all adherent connective tissues under a dissecting
microscope.
An arterial segment (2-3 mm in length) was slipped onto two
glass microcannulae in a servo-controlled pressurized flow chamber
[27] which contained PSS. PSS was bubbled with 95% O2–5%
CO2 and maintained at 37°C as previously described [28].
One cannula was fixed, whereas the other could be positioned as
appropriate. Both ends of the arterial segment were secured to the
microcannula with nylon ties. The axial length of the arterial
segment was adjusted by carefully moving the cannula until the
vascular walls were parallel without any stretch. Intraluminal flow
was initiated using a peristaltic pump (Living system
instrumentation, Burlington, USA). Preheated and pregassed PSS was
perfused to maintain an intraluminal pressure equal to 40 mmHg.
This pressure was chosen in preliminary studies since arterial
segments elicited a maximal decrease in lumen diameter with 10
μmol/L norepinephrine when exposed to this intravascular pressure
(data not shown). After intravascular pressure and flow had been
established, the arterial segments were checked for leaks, which
were identified by a reduction in the preset intraluminal pressure.
The arterial segments were then equilibrated for 1 h.
Experimental protocol
Morphological measurements (external diameter, lumen diameter,
media thickness) in vessels were made from the transillumination
image with a microcomputer-based video imaging system at
4 points along a portion of each vessel, and the mean value
was calculated. The media cross-sectional area (CSA) was calculated
by subtraction of the luminal cross-sectional area from total
cross-sectional area: CSA =
π(De2-Dl2)/4, where
De was the external diameter and Dl was the
lumen diameter of blood vessel. Then, the viability of the segment
was evaluated. An arterial segment was considered viable if it
constricted and developed tone in response to an extraluminal
application of 10 μmol/L norepinephrine in PSS and then to an
extraluminal of high potassium PSS (NaCl, 1.8; NaHCO3,
25; KCl, 100; KH2PO4, 1.2; MgSO4,
1.2; CaCl2, 1.6; glucose, 11; EDTA, 0.026). The
integrity of the vascular endothelium was verified by dilation in
response to an extraluminal application of acetylcholine (1 μmol/L)
in PSS containing 10 μmol/L norepinephrine. After each activation,
the arterial segment was perfused with PSS and allowed to regain
its resting diameter. Finally, cumulative concentration-response
curves to norepinephrine (3.10-8 – 3.10-5
mol/L) and vasopressin (10-10 – 3.10-7mol/L)
were performed by extraluminal application of the drugs. The
mesenteric artery was stimulated at each concentration until the
maximal decrease in lumen diameter was obtained. The maximal
contraction (Emax) induced by the two vasoactive agents
was expressed as the greatest percentage decrease in lumen
diameter: Emax = 100
(Di-Dmax)/Di, where Di
was the resting lumen diameter and Dmax was the smallest
lumen diameter recorded in response to norepinephrine and
vasopressin. The concentration of norepinephrine and vasopressin
causing half-maximal constriction (EC50) was calculated
and expressed as pD2 (negative log molar
EC50). The pD2 and Emax values were
determined by fitting the original concentration response curve
using the Graph Pad Inplot program (GraphPad Software Inc, version
4.03, San Diego, CA, USA).
Data and statistical analysis
All values indicated in the tables, figures and texts are presented
as means ± SEM. Statistical evaluation of the data was performed by
unpaired Student t-test (GraphPad Instat, GraphPad Software Inc).
Results
Mg concentrations, blood pressure and heart rate
In plasma, the Mg concentration was significantly higher in MGH
than in MGL mice (table 1). The Mg level
in kidney was also higher in the MGH group than in the MGL group;
however, it was not significantly different between these two
groups for the heart (table 1). Blood
pressure and heart rates were similar between the two groups (table 2).
Table 1 Magnesium concentration in plasma and dried
organs of MGH and MGL mice.
|
Strain
|
MGH
|
MGL
|
|
Plasma (mmol/L)
|
0.79 ± 0.02
|
0.68 ± 0.03**
|
|
Heart (μmol/g)
|
45.67 ± 1.35
|
45.78 ± 1.07
|
|
Kidney (μmol/g)
|
24.34 ± 0.36
|
22.91 ± 0.40*
|
Table 2 Arterial pressure and heart rate of MGH and MGL
mice.
|
MGH
|
MGL
|
|
Arterial pressure (mmHg)
|
127 ± 7
|
115 ± 10
|
|
Heart rate (bpm)
|
590 ± 14
|
627 ± 24
|
Mesenteric arteries study
Mesenteric artery morphologies
MGH mice presented some significant differences as compared with
the MGL group, i.e. smaller media thickness, smaller lumen diameter
and external diameter and smaller media cross-sectional area (CSA).
The media/lumen ratio was no different between the two groups (table 3).
Table 3 Morphologic parameters of pressurized
mesenteric resistances arteries of MGH and MGL mice.
|
Weight (g)
|
Media thickness (μm)
|
Lumen diameter (μm)
|
Vessel diameter (μm)
|
Media/lumen ratio (%)
|
CSA (μm2)
|
|
MGH
|
32 ± 1
|
7.4 ± 0.4
|
151.0 ± 5.9
|
165.9 ± 5.8
|
9.95 ± 0.80
|
3693 ± 239
|
|
MGL
|
30 ± 1
|
9.4 ± 0.5*
|
200.9 ± 11.8**
|
219.6 ± 12.7**
|
9.35 ± 0.27
|
6283 ± 720**
|
Norepinephrine-induced constriction
Extra-luminally applied norepinephrine induced a
concentration-dependent constriction of the isolated mesenteric
arteries (figure
1A). The constriction (higher maximal response
Emax: 54.40 ± 4.30% for MGL and 54.23 ± 2.97% for MGH)
and the sensitivity (pD2) (5.59 ± 0.09 for MGL and 5.67
± 0.05 for MGH) were not significantly different between the two
studied strains.
Vasopressin-induced constriction
Extra-luminally applied vasopressin induced a
concentration-dependent constriction of the isolated mesenteric
arteries (figure
1B). The constriction was significantly greater in MGH than
in MGL mice. The Emax to vasopressin was significantly
higher in MGH than in MGL mice (39.33 ± 4.80% for MGL vs. 55.56 ±
3.65% for MGH, p < 0.05) whereas the pD2 value tended
to be higher in MGH than in MGL mice (pD2: 8.73 ± 0.04
for MGH and 8.50 ± 0.10 for MGL; p < 0.06).
Discussion
The metabolic variation between MGH and MGL mice was accompanied by
modifications of the vascular morphology of mesenteric resistance
arteries and higher vasopressin-induced vasoconstriction. Despite
these differences, the arterial blood pressure was similar between
the two groups. The MGL mice had a lower Mg plasma concentration
than MGH mice. Mg status cannot be only determined with magnesemia.
1% of total Mg of the body is located in plasma, while the rest is
found in organs (45%) and bones (54%) [29]. A high proportion of
blood Mg is also located in erythrocytes [30]. We have previously
shown that MGH mice also presented a higher Mg concentration in
erythrocytes and in the kidney. This difference for the kidney was
also observed in the present work. In addition, previous studies
[21, 22, 25] showed less Mg urinary excretion in the MGH strain as
compared to MGL ones.
In the present study, blood pressure was not different between
the MGH and MGL mice. Some authors have already studied the effect
of hypermagnesemia on blood pressure, with some epidemiological
studies showing that a diet rich in Mg is inversely correlated to a
decrease in blood pressure in normotensive subjects [31]. In the
same way, Altura et al. [32] reviewed some epidemiological studies
which showed that a diet rich in Mg decreased the frequency of
hypertensive diseases. In animal models of hypertension, oral
supplementation of Mg results in a decrease in the elevation of
blood pressure in secondary hypertension (DOCA-salt hypertension)
and in SHR [12, 33]. However, in normotensive rats, oral Mg
supplementation has no effect on blood pressure [34]. In the
present study we have shown no relationship between magnesemia
(genetically determined) and blood pressure.
Knowing that catecholamines are important in the regulation of
blood pressure, the absence of altered vascular reactivity to
norepinephrine in the isolated and perfused mesenteric arteries
between MGH and MGL supports the fact that the vascular contractile
function is not related to the genetically determined Mg status in
these mice. In a same way, it has been demonstrated that increasing
blood pressure is associated with an increase of the media/lumen
ratio [35]. In our study, the media/lumen ratio was not different
between these two strains. All these findings seem indicate that
MGH and MGL mice present no alteration of their cardiovascular
functions despite the well-known cardiovascular effects of Mg.
Some differences, however, appear between MGH and MGL mice in
vasopressin-induced vasoconstriction and the morphology of
mesenteric arteries. In fact, the effect of vasopressin was more
important in the mesenteric arteries from MGH mice than in those
from MGL mice. The difference between MGH and MGL mice lies in the
maximum effect (Emax) and not in the sensitivity
(pD2). This may be a consequence in the differences of
Mg metabolism between MGH and MGL mice. However, the variations of
response to vasopressin have no consequence on the arterial blood
pressure. The real impact of this observation on the cardiovascular
system of these mice is still unknown. In vitro, high extracellular
Mg levels decrease vasopressin-induced contraction in rat aorta
[36]. In resistant mesenteric arteries, however, high extracellular
Mg concentration decreases vasopressin-induced vasoconstriction in
hypertensive but not normotensive rats [28].
The mesenteric arteries of MGH mice present morphological
differences as compared to MGL mice, with a smaller lumen and total
diameter of the vessel, a smaller media thickness and
cross-sectional area. Because blood pressure was measured at only
one point and this experiment was performed in young mice, the real
impact of this vascular remodelling on the cardiovascular system
between these mice strains is actually unknown. During the aging
process, blood pressure and cardiovascular function change, with an
increase of the risks of cardiovascular diseases, like
atherosclerosis and hypertension [37]. Mg may protect against these
pathologies by preventing the structural modifications of the
arteries observed during cardiovascular diseases (i.e. increase of
the vessel diameter or media thickness) or by an anticalcic action
[32]. Because MGH mice present less important vascular remodeling,
they might be protected against atherosclerosis and hypertension
during aging. Some other experiments in these strains are expected
to clarify this theoretical hypothesis.
In conclusion, our study focuses on the vascular function of MGH
and MGL mice, models of genetic variations of magnesium status. The
two strains present some variations in the morphology and
reactivity of the mesenteric arteries which are not associated with
changes in blood pressure levels. More studies in MGH and MGL
strains are needed to characterize the cardiovascular functions of
this model, and the effect of the variations of Mg metabolism of a
genetic origin.
References
1 Saris NE, Mervaala E, Karppanen H,
Khawaja JA, Lewenstam A. Magnesium. An update on
physiological, clinical and analytical aspects. Clin Chim Acta
2000; 294: 1-26.
2 Maier JA. Low magnesium and atherosclerosis: an
evidence-based link. Mol Aspects Med 2003; 24: 137-46.
3 Belin RJ, He K. Magnesium physiology and pathogenic
mechanisms that contribute to the development of the metabolic
syndrome. Magnes Res 2007; 20: 107-29.
4 Touyz RM. Transient Receptor Potential Melastatin 6 and 7
Channels, Magnesium Transport and Vascular Biology: Implications in
Hypertension. Am J Physiol Heart Circ Physiol 2008; 294:
H1103-H1118.
5 Laurant P, Hayoz D, Brunner H,
Berthelot A. Dietary magnesium intake can affect mechanical
properties of rat carotid artery. Br J Nutr 2000; 84: 757-64.
6 Rayssiguier Y, Mbega JD, Durlach V,
Gueux E, Durlach J, Giry J, Dalle M,
Mazur A, Laurant P, Berthelot A. Magnesium and blood
pressure. I. Animal studies. Magnes Res 1992; 5: 139-46.
7 Laurant P, Dalle M, Berthelot A,
Rayssiguier Y. Time-course of the change in blood pressure
level in magnesium-deficient Wistar rats. Br J Nutr 1999; 82:
243-51.
8 Laurant P, Touyz RM. Physiological and
pathophysiological role of magnesium in the cardiovascular system:
implications in hypertension. J Hypertens 2000; 18: 1177-1191.
9 Adrian M, Chanut E, Laurant P, Gaume V,
Berthelot A. A long-term moderate magnesium-deficient diet
aggravates cardiovascular risks associated with aging and increases
mortality in rats. J Hypertens 2008; 26: 44-52.
10 Berthelot A, Luthringer C, Exinger A. Trace
elements during the development of hypertension in the
spontaneously hypertensive rat. Clin Sci (Lond) 1987; 72:
515-8.
11 Berthelot A, Esposito J. Effects of dietary
magnesium on the development of hypertension in the spontaneously
hypertensive rat. J Am Coll Nutr 1983; 2: 343-53.
12 Touyz RM, Milne FJ. Magnesium supplementation
attenuates, but does not prevent, development of hypertension in
spontaneously hypertensive rats. Am J Hypertens 1999; 12:
757-65.
13 Sado T, Oi H, Sakata M, Yoshida S,
Kawaguchi R, Kanayama S, Shigetomi H, Haruta S,
Tsuji Y, Ueda S, Kitanaka T, Yamada Y,
Kobayashi H. Aging impairs the protective effect of magnesium
supplementation on hypertension in spontaneously hypertensive rats.
Magnes Res 2007; 20: 196-9.
14 Galan P, Preziosi P, Durlach V, Valeix P,
Ribas L, Bouzid D, Favier A, Hercberg S.
Dietary magnesium intake in a French adult population. Magnes Res
1997; 10: 321-8.
15 King DE, Mainous 3rd AG, Geesey ME,
Woolson RF. Dietary magnesium and C-reactive protein levels. J
Am Coll Nutr 2005; 24: 166-71.
16 Henrotte JG, Colombani J, Pineau M,
Dausset J. Role of H-2 and non-H-2 genes in the control of
blood magnesium levels. Immunogenetics 1984; 19: 435-48.
17 Henrotte JG, Pla M, Dausset J. HLA- and
H-2-associated variations of intra- and extracellular magnesium
content. Proc Natl Acad Sci USA 1990; 87: 1894-8.
18 Schmitz C, Deason F, Perraud AL. Molecular
components of vertebrate Mg2+-homeostasis regulation. Magnes Res
2007; 20: 6-18.
19 Darlu P, Rao DC, Henrotte JG, Lalouel JM.
Genetic regulation of plasma and red blood cell magnesium
concentrations in man. I. Univariate and bivariate path analyses.
Am J Hum Genet 1982; 34: 874-87.
20 Henrotte JG, Motta R, Biozzi G. Selection of
two lines of mice for high and low red blood cell magnesium
concentration, called MgH (high) and MgL (low). Mouse News Letters
1988; 81: 84-5.
21 Henrotte JG, Franck G, Santarromana M,
Clavel M, Mouton D, Motta R. Mice selected for low
(MGL) and high (MGH) red blood cell magnesium levels: recent
result. Magnes Res 1993; 6: 304-5.
22 Feillet-Coudray C, Coudray C, Wolf FI,
Henrotte JG, Rayssiguier Y, Mazur A. Magnesium
metabolism in mice selected for high and low erythrocyte magnesium
levels. Metabolism 2004; 53: 660-5.
23 Feillet-Coudray C, Trzeciakiewicz A,
Coudray C, Rambeau M, Chanson A, Rayssiguier Y,
Opolski A, Wolf FI, Mazur A. Erythrocyte magnesium
fluxes in mice with nutritionally and genetically low magnesium
status. Eur J Nutr 2006; 45: 171-7.
24 Ozgo M, Bayle D, Zimowska W, Mazur A.
Effect of a low magnesium diet on magnesium status and gene
expression in the kidneys of mice selected for high and low
magnesium erythrocyte levels. Magnes Res 2007; 20: 148-53.
25 Rondón LJ, Groenestege WM, Rayssiguier Y,
Mazur A. Relationship between low magnesium status and TRPM6
expression in the kidney and large intestine. Am J Physiol Regul
Integr Comp Physiol 2008; 294: R2001-R2007.
26 Massiera F, Bloch-Faure M, Ceiler D,
Murakami K, Fukamizu A, Gasc JM,
Quignard-Boulange A, Negrel R, Ailhaud G,
Seydoux J, Meneton P, Teboul M. Adipose
angiotensinogen is involved in adipose tissue growth and blood
pressure regulation. FASEB J 2001; 15: 2727-9.
27 Halpern W, Kelley M. In vitro methodology for
resistance arteries. Blood Vessels 1991; 28: 245-51.
28 Laurant P, Touyz RM, Schiffrin EL. Effect of
magnesium on vascular tone and reactivity in pressurized mesenteric
resistance arteries from spontaneously hypertensive rats. Can J
Physiol Pharmacol 1997; 75: 293-300.
29 Rude RK. Magnesium metabolism and deficiency. Endocrinol
Metab Clin North Am 1993; 22: 377-95.
30 Günther T. Total and free Mg2+ contents in erythrocytes:
a simple but still undisclosed cell model. Magnes Res 2007; 20:
161-7.
31 Joffres MR, Reed DM, Yano K. Relationship of
magnesium intake and other dietary factors to blood pressure: the
Honolulu heart study. Am J Clin Nutr 1987; 45: 469-75.
32 Altura BM, Zhang A, Altura BT. Magnesium,
hypertensive vascular diseases, atherogenesis, subcellular
compartmentation of Ca2+ and Mg2+ and vascular contractility. Miner
Electrolyte Metab 1993; 19: 323-36.
33 Berthon N, Laurant P, Hayoz D,
Fellmann D, Brunner HR, Berthelot A. Magnesium
supplementation and deoxycorticosterone acetate-salt hypertension:
effect on arterial mechanical properties and on activity of
endothelin-1. Can J Physiol Pharmacol 2002; 80: 553-61.
34 Laurant P, Kantelip JP, Berthelot A. Dietary
magnesium supplementation modifies blood pressure and
cardiovascular function in mineralocorticoid-salt hypertensive rats
but not in normotensive rats. J Nutr 1995; 125: 830-41.
35 Laurant P, Hayoz D, Brunner HR,
Berthelot A. Effect of magnesium deficiency on blood pressure
and mechanical properties of rat carotid artery. Hypertension 1999;
33: 1105-10.
36 Turlapaty PD, Altura BM. Effects of
neurohypophyseal peptide hormones on isolated coronary arteries:
Role of magnesium ions. Magnesium 1982; 1: 122-8.
37 Ribera-Casado JM. Ageing and the cardiovascular system.
Z Gerontol Geriatr 1999; 32: 412-9.
|
Printable version
Version PDF
