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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 5, 2001 as doi:10.1096/fj.00-0509fje. |
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2
* Centro de Investigación Básica de España (CIBE), Merck Sharp & Dohme, Madrid;
Instituto de Bioquímica, Centro Mixto CSIC-UCM, Facultad de Farmacia, Universidad Complutense, 28040 Madrid;
Departamento de Bioquímica, Facultad de Medicina, Campus Universitario, 02071 Albacete; and
Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universitat de Valencia, 46010 Valencia, Spain
2Correspondence: Instituto de Bioquímica, Facultad de Farmacia, 28040 Madrid. Spain. E-mail: boscal{at}eucmax.sim.ucm.es
SPECIFIC AIMS
Duringsepsis, various organs contribute to the release of inflammatory mediators that promote the synthesis of reactive molecules involved in host defense, among them nitric oxide (NO). In vitro experiments suggested that priming with exogenous NO protected hepatocytes against inflammatory injury. To evaluate this response in vivo, we engineered mice carrying a nitric oxide synthase-2 (NOS-2) transgene under the control of the phospho(enol)pyruvate carboxykinase (PEPCK) promoter. These animals allowed us to express NOS-2 specifically in liver under fasting conditions, and the effect of the local synthesis of NO on several models of liver injury was investigated.
PRINCIPAL FINDINGS
1. The NOS-2 transgene was expressed in liver under fasting
conditions
The expression of NOS-2 in transgenic (Tg) animals was assessed
after starvation for 24 and 48 h, and analysis of NOS-2 in liver
samples by Western blot. The protein was undetectable in fed animals,
and a band of the expected size of NOS-2 (130 kDa) was observed at
24 h of starvation. Prolonged starvation up to 48 h did not
result in higher levels of NOS-2. This enzyme was active in
vitro and in vivo because the serum levels of nitrite
plus nitrate increased after starvation of Tg animals with respect to
the wild-type littermates (Wt) and were inhibited after administration
of the NOS-2 inhibitors 1400W and
L-N(6)-(1-iminoethyl)lysine
(L-NIL).
2. Endogenous NO protects against lipopolysaccharide (LPS) toxicity
in D-galactosamine (D-GalN) conditioned mice
To investigate the role of fasting-dependent NO synthesis in liver
function in Tg animals, we analyzed two models of lethal endotoxic
injury: i.p. injection of a high dose of LPS (40 mg/kg body weight) and
i.p. administration of a low dose of LPS (10 µg/kg) or tumor necrosis
factor 
(TNF-) (20 µg/kg) in combination with
D-GalN (800 mg/kg). The survival rates to high doses of LPS
did not show significant differences between Wt and Tg animals,
regardless of their nutritional state. However, as the schematic
diagram shows, a protective effect (70%) was observed in Tg animals
expressing NOS-2, after challenge with a low dose of LPS and
D-GalN. When animals were treated with TNF- and
D-GalN, a significant protection was still observed in the
fasted Tg group (60%). These results suggest that impairment of
proinflammatory cytokine synthesis by NO in liver is a likely mechanism
mediating animal survival, as observed in other models of acute
endotoxic liver injury. To analyze the protection exerted by NO
synthesis on LPS/D-GalN-dependent toxicity, the levels of
the proinflammatory cytokines TNF- and IL-ß were determined at
4 h after i.p. challenge. As Figure 1
shows, a 3.6- and 6.5-fold reduction of TNF- and IL-1ß levels,
respectively, was observed in starved Tg animals. This attenuated
inflammatory response was absent in fed animals or when NOS-2 activity
was inhibited. In parallel to the determination of the serum levels of
proinflammatory cytokines, the activity of hepatic transaminases as
lesion markers was determined. A remarkable reduction in the serum
levels of alanine and aspartarte transaminases (76% and 65%,
respectively) was observed in starved Tg animals, in agreement with the
decreased levels of proinflammatory cytokines.
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3. NF-
B activity is impaired in Tg animals expressing NOS-2
One of the potential mechanisms of protection against LPS/
D-GalN injury is the inhibition of NF-B activity caused
by the endogenous synthesis of NO. To investigate this possibility,
liver extracts were prepared and NF-B activity was evaluated by
electrophoretic mobility shift assays of the nuclear protein fraction.
A significant decrease of NF-B activity was observed in starved Tg
animals treated with LPS/ D-GalN. This impaired activity
was accompanied by the presence in the nucleus of lower amounts of the
Rel proteins p50 and p65, as well as higher levels of cytosolic
IB, an inhibitory protein of NF-B that retains the complex in
the cytoplasm. In agreement with these data, the levels of IB
that parallel the activation of NF-B were significantly lower in
fasted Tg animals after LPS/D-GalN challenge. This decreased NF-B
activity was specific of the fasted Tg animals and was lost in the fed
counterparts (Fig. 2
).
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4. Apoptosis was impaired in fasted Tg animals after LPS/
D-GalN challenge
Because
partof the cytotoxic effects due to LPS/D-GalN administration
has been attributed to induction of liver apoptosis, the levels of
procaspase-3, the activity of caspase-3, and the oligo-nucleosomal
fragmentation of DNA were measured in liver extracts, and the
nucleotidyltransferase terminal labeling (TUNEL) was assayed in liver
sections. Our data show that the levels of procaspase-3 were maintained
in fasted Tg animals (up to 7 h after LPS/D-GalN
challenge), whereas a significant decrease (70%) was observed in the
corresponding Wt animals. This decrease in procaspase-3 was paralleled
by the appearance of caspase-3 activity, and by the occurrence of
internucleosomal degradation of DNA when examined in agarose gels.
Inhibition of NOS-2 activity with L-NIL suppressed the
protective effects of NO synthesis in Tg animals. Moreover, TUNEL
assays of liver sections obtained 7 h after LPS/D-GalN
challenge showed an intense nuclear staining in starved Wt animals,
together with a marked alteration in the tissue structure. However,
this condition was absent in Tg animals that exhibited a normal tissue
morphology. It is interesting that treatment of animals with
L-NIL suppressed the protective effect exerted by the
endogenous synthesis of NO.
CONCLUSIONS
The expression of NOS-2 after fasting the Tg animals promoted
moderate changes in parameters related to oxidative stress (a decrease
of glutathione levels and a moderate increase of
8-hydroxydeoxyguanosine). However, this NO synthesis did not affect the
life span or involve other systemic alterations when compared with the
corresponding Wt animals. Administration of a high dose of LPS was
lethal for both Wt and Tg animals, independent of the nutritional
status. This lethality might be due to the insult exerted by LPS over
other organs, likely the renal and vascular systems. However, in
D-GalN-conditioned mice that exhibit an enhanced liver
response to inflammatory factors, intrahepatic synthesis of NO
protected against the lethal injury elicited by moderate doses of LPS
and TNF- and this effect was lost when the activity of NOS-2 was
pharmacologically inhibited. The LPS/D-GalN induced
lethality appears to be caused by TNF--dependent hepatocyte
apoptosis because nonspecific caspase inhibitors protected against
lethal injury. Indeed, NO inhibits liver caspase-3 and -8 activities
through a S-nitrosylation mechanism, and these caspases appear to be
the most relevant executioners in this organ. The efficiency of this
protective mechanism was evident when using a model of
adenovirus-mediated gene transfer of NOS-2, which abrogated the
apoptosis dependent on TNF- and actinomycin D. Moreover, in a model
of concanavalin A-induced hepatitis, administration of chemical donors
of NO improved survival through a mechanism that involved the
inhibition of caspase-dependent processing of the Th1 response, which
caused Fas-dependent hepatocyte death.
Analysis of the mechanisms mediating the protection exerted by hepatic
NO synthesis included the inhibition of NF-B activation, evidenced
at short times after LPS challenge, and therefore an impairment of the
release of TNF-, IL-ß, and presumably other inflammatory factors
dependent on NF-B activity. The contribution of proinflammatory
cytokines to lethality in the LPS/D-GalN model, in
particular TNF-, has been well-established since survival to LPS
administration was observed in mice lacking TNF- or the p55 receptor
of TNF-. Also, pretreatment of animals with a moderate nonlethal
dose of LPS, which induces a moderate expression of NOS-2, reduced the
lethality of a further administration of LPS/D-GalN,
including a reduction in the synthesis of TNF-. Opposite to the
protective effect by preexistent NO, the NO produced as result of the
inflammatory expression of NOS-2 plays an important role in the
pathogenesis of septic shock.
The impairment of NF-B activity by intrahepatic NO was caused by
different convergent mechanisms: an up-regulation of IB protein
levels, a fact observed systematically in fasted Tg animals; an
inhibition of IB phosphorylation caused by the inhibitory effect of
NO on IB kinase-2 activity; and an inappropriate targeting of IB
caused by the inhibition of the proteasome activity by NO. Consistent
with these suggestions are the data obtained by using a "tet-off"
transgenic system to express a degradation-resistant mutant of
IB. These animals allow a regulated disruption of NF-B
activity in postnatal liver that has no effect on the normal physiology
of the organ but severely impairs the ability to clear infection from
the liver, which indicates that NF-B acts as an integrator of innate
immunity in this organ. In addition to these effects of NO on IB
targeting, it has been described that pretreatment of cultured
hepatocytes with NO-donors protects against TNF--dependent
hepatotoxicity by up-regulating heat shock protein 70 (hsp70)
expression. Moreover, this increase of hsp70 appears to depend on an
enhancement of GSSG concentration, a situation observed in fasted Tg
animals.
Taken together, these data suggest that the availability of a molecule delivering NO in the hepatocyte, as result of hepatic metabolism, might constitute a useful drug for the therapeutic management of various liver diseases.
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FOOTNOTES
1 To read the full text of this article, go to
http://www.fasebj.org/cgi/doi/10.1096/fj.00-0509fje ; to cite this
article, use FASEB J. (January 5, 2001)
10.1096/fj.00-0509fje 
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