ARTICLE
Auteur(s) : Atsushi Miyamoto, Hiroshi Moriki, Shigeru
Ishiguro, Akira
Nishio
Department of Veterinary Pharmacology, Faculty of Agriculture,
Kagoshima University, Kagoshima, Japan
It is well known that endotoxin induces vascular hyporeactivity to
some vasoactive agents, such as phenylephrine, in vivo [1-3] and in
vitro [4, 5]. It has been suggested that endotoxin-induced vascular
hyporeactivity to phenylephrine might be dependent on
endotoxin-induced nitric oxide (NO) production, which stimulates
NO-cyclic GMP-potassium channel pathways [5-8]. Prevention of
inducible nitric oxide synthase (iNOS) expression improves survival
in rodent models of endotoxic shock [9, 10].It has been reported
that magnesium ions play an important role in many cell functions
[11], and their deficiency or deficit induce multiple pathological
consequences [12]. Recent reports show that, in Mg-deficient rats,
endotoxin lethality is enhanced [13, 14]. Previous studies by our
group and others showed that endotoxin-induced vascular
hyporeactivity to phenylephrine is also enhanced in Mg-deficient
rat thoracic aortas ex vivo [3] and in vitro [5], and that during
Mg-deficiency in rats, cytokines [14, 15] and NO [16] increase in
plasma.The purpose of the present study was therefore to assess
whether in vitro application of endotoxin directly enhances NO
production in thoracic aortas isolated from Mg-deficient rats, and
also to assess the effects of a specific iNOS inhibitor and
antibodies to endotoxin-receptors on this enhancement.
Materials and methods
Animals
Twenty adult male Wistar rats (8-10-weeks-old) were fed a
Mg-deficient diet (Mg: 0.001%) for 20 days. A control group (15
rats) received a normal diet (Mg: 0.07%). The composition of the
purified diet has been described in detail in a previous report
[17]. The rats were pair-fed and allowed free access to deionized
water. Each rat was housed individually in a stainless steel cage
at an ambient temperature of 22-25 °C under a 12 h
light-dark cycle.
All experiments were performed according to the Guidelines for
Animal Experiments of Kagoshima University.
Reagents
The reagents used in this study were as follows: penicillin G,
streptomycin, Dulbecco’s Modified Eagle Medium (DMEM) (Invitrogen
Co., USA), endotoxin (Escherichia coli 011: B4 lipopolysaccharides
(LPS)) (Sigma Chemical Co., USA), 1400W and
NO2- assay kit F (Wako Pure Chemical
Industries, Ltd., Japan), acetylcholine (Daiichi Pharmaceutical
Co., Japan), mouse anti-human Toll-like receptor 4 (TLR4)
monoclonal antibody, mouse anti-human CD14 monoclonal antibody,
mouse IgG2a isotype control antibody (eBioscience, USA).
Tissue preparation
After feeding with the semi-synthetic diets for 20 days, the rats
were anesthetized with pentobarbital solution (30 mg/kg body
weight, i.p.), and the thoracic aortas were isolated from the rats
under sterile conditions. The aortas were cleaned of all fat and
connective tissue, and cut longitudinally into four segments. To
remove the endothelium, the intimal surface was rubbed gently with
a swab wetted with DMEM. The absence of endothelium was verified by
a lack of relaxation when acetylcholine (1.0 μM) was added at the
end of the experiment. Each segment was preincubated for 1 h
with DMEM supplemented with penicillin (100 U/mL) and streptomycin
(100 μg/mL), and then cultured for 6 h with fresh DMEM in the
presence or absence of LPS.
To examine the effect of a selective iNOS inhibitor, 1400W,
after 1 h preincubation of each vascular segment, 1400W was
added to DMEM 30 min prior to LPS application. To examine the
effects of three kinds of antisera, IgG2a, TLR4 and
CD14, after 1 h preincubation, each antiserum was added to
fresh DMEM 30 min prior to LPS application. After a 6-h
incubation, the culture medium was collected, immediately plunged
into liquid nitrogen and stored at -80 °C until use. All
cultures were performed in a chamber with a 5% (v/v) CO2
atmosphere and 100% humidity at 37 °C.
Determination of NO production
The stable end product of NO, NO2-, was
estimated in the culture medium using a fluorometric
NO2- assay kit. This assay was performed
according to the manufacturer’s instructions using a Cytofluor
Multi-Well Plate Reader Series 4000 (PE Applied Biosystems, USA),
and the data were extrapolated from a standard curve prepared for
each assay.
Statistical analysis
Results are expressed as the mean ± SEM of n observations,
where n represents the number of animals studied. Statistical
analyses were performed using the Student’s t test or Bonferroni
test after one-way analysis of variance. Statistical significance
was established when the probability level was equal to or less
than 5%.
Results
General
As described in our early studies [18-21], the Mg-deficient rats
used in this study showed a low plasma magnesium level (0.51 ± 0.05
mEq/L) after 20 days, however, plasma levels of calcium (4.95 ±
0.05 mEq/L), potassium (5.51 ± 0.30 mEq/L) and sodium (148.2 ± 0.71
mEq/L) were not significantly different from those of the controls.
They also developed hyperemia of the ears after about 8 days on
Mg-deficient diet.
NO production from thoracic aortas
To test NO production in vitro, thoracic aortas isolated from
control and Mg-deficient rats were cultured for 6 h with or
without LPS.
As shown in ( figure 1 ), LPS
increased NO production in a concentration-dependent manner. The
degree of this increment was significantly higher in Mg-deficient
rat aortas than in control rat aortas in the presence of 0.1 and
1.0 μg/mL LPS. The enhanced NO production was not affected by
endothelium denudation ( (figure 2) ).
1400W attenuates increased NO production by LPS
To confirm the participation of iNOS in LPS-induced NO production,
the effect of a highly selective iNOS inhibitor, 1400W [22], on
LPS-induced NO production was studied.
As shown in ( figure 3 ), treatment
of thoracic aortas with 1400W significantly attenuated LPS-induced
NO production. However, a significantly higher production of NO was
observed in Mg-deficient rat aortas than in control aortas in the
presence of 1400W.
CD14 and TLR4 antibodies attenuate increased NO production by
LPS
It has been suggested that LPS-mediated signaling via CD14 and TLR4
is responsible for iNOS gene induction [23].
To determine the involvement of the above LPS receptors in
LPS-induced NO production, the effects of anti-CD14- and
anti-TLR4-antibodies were examined.
As shown in ( figure 4 ),
LPS-induced NO production was significantly attenuated by
anti-CD14- or anti-TLR4 antibodies. Treatment with a mouse
isotype-matched control antibody (anti-IgG2a) did not affect
LPS-induced NO production.
LPS-induced NO production in the presence of anti-CD14- or
anti-TLR4 antibodies was higher in Mg-deficient rat aortas than in
control aortas.
Discussion
Our previous ex vivo study showed that endotoxin-induced
hyporeactivity to phenylephrine was enhanced in Mg-deficient rats,
and suggested that some cytokines, such as interleukin-1β and tumor
necrosis factor-α, might contribute to this enhancement [3].
The present results show that endotoxin directly stimulates NO
production by the thoracic aorta. These data partly support those
of previous studies, in which endotoxin was shown to stimulate iNOS
expression and NO production in aortas and vascular smooth muscle
cells [24-27].
As shown in figures 1 and 2, endotoxin-induced
enhancement of NO production was observed in Mg-deficient rat
aortas, and this enhancement was not affected by removing
endothelial cells. These results suggest that the enhancement of
endo-toxin-induced NO production does not depend on endothelial
nitric oxide synthase (eNOS). The following results strongly
support the non-dependency on eNOS in endotoxin-induced NO
production, which show a selective iNOS inhibitor, 1400W,
suppressed endotoxin-induced NO production to basal levels in
control rat thoracic aortas. In Mg-deficient rat thoracic aortas,
however, the suppression with 1400W was significantly weaker than
in control rats, and endotoxin-induced NO production was not
suppressed to basal levels ( (figure 3) ). These
results suggest that endotoxin induces much more iNOS activity in
thoracic aortas from Mg-deficient rats than in those from control
rats. The result was similar to previous data in which NOS activity
in lungs was not significantly different between control and
Mg-deficient rats, but its induction by endotoxin was significantly
higher in Mg-deficient rats than control rats [16]. In the absence
of endotoxin, NO production from Mg-deficient rat thoracic aortas
was not significantly different from control aortas. The data
suggest that the amount of iNOS in thoracic aortas might not be
affected by Mg-deficiency. Our previous data showed that iNOS mRNA
level in thoracic aorta was higher in Mg-deficient rats than
control ones [5]. In this experiment, however, the NO production
from Mg-deficient rat’s aorta without stimulation of endotoxin was
not higher than that from control rats. The precise reason is
unclear, but it might be dependent on the higher sensitivity of the
assay method of mRNA than that of NO. Endotoxin enhanced vascular
hyporeactivity to phenylephrine much more in Mg-deficient rats’
thoracic aortas than control ones, and its effect was observed at
0.01 μg/mL endotoxin [5]. However, in this experiment enhanced
production of NO was not observed at this dose of endotoxin ( (figure 1) ).
This difference might be dependent on the higher sensitivity to NO
in Mg-deficient rat aorta [20, 21].
A recent study using cDNA array technology showed that most
genes (78%) in rat neutrophils were expressed at a level more than
two-fold higher in Mg-deficient rats compared to control rats [28].
These data suggest the possibility that endotoxin receptors on the
thoracic aorta might increase during Mg-deficiency. Some studies
have indicated that TLR4 and CD14 might mediate a response to
endotoxin [29-32]. Recent reports have shown that both CD14 and
TLR4 are required for endotoxin-induced iNOS induction in chicken
macrophages [32].
Therefore, we speculated that during Mg-deficiency the
expression of endotoxin receptors might be upregulated. To confirm
this speculation, the effect of specific antibodies to TLR4 and
CD14 on NO production from the thoracic aorta was examined. As
shown in ( figure 4 ), both
antibodies significantly suppressed endotoxin-induced NO
production. However, the degree of suppression was significantly
weaker in Mg-deficient rat aortas than in control aortas. These
data strongly suggest that a greater number of endotoxin receptors
leads to stronger signal transduction and ultimately higher iNOS
expression and activity in Mg-deficient rat aortas than in control
rats. Data reported recently, which showed the presence of CD14 on
vascular smooth muscle cells [33], support, at least in part, the
above suggestion.
It has been reported that severe Mg deficiency for a short
period induces a decrease in blood pressure [34-36]. In this
experimental condition for 20 days on Mg-deficient diet, blood
pressure in Mg-deficient rats might be decreased by increasing NO
production via macrophages and neutrophiles [16, 37, 38]. On the
other hand, it is well known that endotoxin induces hypotension via
NO production [6-10]. So it is possible to speculate that an
additive effect of NO production by Mg deficiency and endotoxin
administration enhances the lethality by endotoxin during Mg
deficiency.
It has been suggested that Mg deficiency induces
hyperlipidaemia, which plays an important role in the pathogenesis
of vascular injury [39]. Mg deficiency was shown to enhance
vascular endothelial injury, thus promoting the development and
progression of atherosclerosis [40, 41]. As shown in ( figure 2 ), however,
endototoxin-induced NO production of Mg-deficient rat aortas was
not affected by removing endothelial cells. From the present
results, the endothelial cells might be injured and endothelial
dysfunction for spontaneous NO production might be occurring.
However, the relation between NO production and atherosclerosis
during Mg deficiency is unclear.
Further studies should focus on the complex relationship between
NO, vascular hyporeactivity and atherosclerosis for a better
understanding of the pathophysiology of endotoxin shock during Mg
deficiency.
Conclusion
The present data demonstrate that endotoxin directly stimulates NO
production by the thoracic aorta, and that its production is
enhanced in Mg-deficient rats. The data also suggest that endotoxin
receptors might contribute to this enhancement.
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