Hence, we examined the changes in inducible nitric oxide synthase⁄ indu-cible mitochondrial nitric oxide synthase expression and activity, bioener-getics and oxidative stress in heart mi
Trang 1by melatonin in septic mice
Germaine Escames1, Luis C Lo´pez1, Francisco Ortiz1, Ana Lo´pez1, Jose´ A Garcı´a1, Eduardo Ros2 and Darı´o Acun˜a-Castroviejo1,3
1 Instituto de Biotecnologı´a, Departamento de Fisiologı´a, Universidad de Granada, Spain
2 Servicio de Angiologı´a y Cirugı´a Vascular, Hospital Universitario San Cecilio, Granada, Spain
3 Servicio de Ana´lisis Clı´nicos, Hospital Universitario San Cecilio, Granada, Spain
Sepsis-induced multiple organ failure is the major
cause of mortality in critically ill patients, and its
inci-dence is rising [1] The heart and cardiovascular
sys-tems are seriously affected during sepsis [2] Although myocardial impairment in sepsis has been extensively studied, its etiology remains unclear [3] Some reports
Keywords
ATP production; mitochondrial failure;
mitochondrial nitric oxide synthase;
oxidative stress; therapy
Correspondence
D Acun˜a-Castroviejo, Departamento de
Fisiologı´a, Facultad de Medicina, Avenida de
Madrid 11, E-18012, Spain
Fax: +34 958246295
Tel: +34 958246631
E-mail: dacuna@ugr.es
(Received 4 December 2006, revised 9
February 2007, accepted 23 February 2007)
doi:10.1111/j.1742-4658.2007.05755.x
The existence of an inducible mitochondrial nitric oxide synthase has been recently related to the nitrosative⁄ oxidative damage and mitochondrial dys-function that occurs during endotoxemia Melatonin inhibits both inducible nitric oxide synthase and inducible mitochondrial nitric oxide synthase activities, a finding related to the antiseptic properties of the indoleamine Hence, we examined the changes in inducible nitric oxide synthase⁄ indu-cible mitochondrial nitric oxide synthase expression and activity, bioener-getics and oxidative stress in heart mitochondria following cecal ligation and puncture-induced sepsis in wild-type (iNOS+⁄ +) and inducible nitric oxide synthase-deficient (iNOS–⁄ –) mice We also evaluated whether melato-nin reduces the expression of inducible nitric oxide synthase⁄ inducible mitochondrial nitric oxide synthase, and whether this inhibition improves mitochondrial function in this experimental paradigm The results show that cecal ligation and puncture induced an increase of inducible mito-chondrial nitric oxide synthase in iNOS+⁄ +mice that was accompanied by oxidative stress, respiratory chain impairment, and reduced ATP produc-tion, although the ATPase activity remained unchanged Real-time PCR analysis showed that induction of inducible nitric oxide synthase during sepsis was related to the increase of inducible mitochondrial nitric oxide syn-thase activity, as both inducible nitric oxide synsyn-thase and inducible mito-chondrial nitric oxide synthase were absent in iNOS–⁄ –mice The induction
of inducible mitochondrial nitric oxide synthase was associated with mito-chondrial dysfunction, because heart mitochondria from iNOS–⁄ – mice were unaffected during sepsis Melatonin treatment blunted sepsis-induced inducible nitric oxide synthase⁄ inducible mitochondrial nitric oxide syn-thase isoforms, prevented the impairment of mitochondrial homeostasis under sepsis, and restored ATP production These properties of melatonin should be considered in clinical sepsis
Abbreviations
CLP, cecal ligation and puncture; ETC, electron transport chain; GPx, glutathione peroxidase; GRd, glutathione reductase; GSH, glutathione; GSSG, oxidized glutathione; iNOS, inducible nitric oxide synthase; i-mtNOS, inducible mitochondrial nitric oxide synthase; LPO, lipid peroxidation; mtNOS, constitutive mitochondrial nitric oxide synthase; nNOS, neuronal nitric oxide synthase.
Trang 2have shown that mitochondria are the primary targets
injured in both vital and nonvital organs during
inflammation [4–6] Besides other factors,
mitochond-rial dysfunction in sepsis is directly associated with an
increase in reactive oxygen species and reactive
nitro-gen species [6–10]
It has been shown that mitochondria from several
organs, such as lungs, liver, diaphragm, and hind leg
skeletal muscle, contain an inducible mitochondrial
nitric oxide synthase (i-mtNOS) [9,11–13], which is
encoded by the same gene as cytosolic inducible nitric
oxide synthase (iNOS) [9,11,12,14,15] Moreover,
mito-chondria contain a constitutive NOS (mtNOS) derived
from neuronal NOS (nNOS) [16,17] The expression
and activity of i-mtNOS, but not those of mtNOS,
increase during sepsis [9,11,12] Other studies have
shown induction of mitochondrial NOS in the
dia-phragm and heart of septic rats, although these reports
did not distinguish between constitutive and inducible
forms [8,18]
Increasing evidence suggests that the nitric oxide
(NO) produced by i-mtNOS plays a role in
mitochond-rial dysfunction during sepsis [9,11,12] Because iNOS–⁄ –
mice do not express i-mtNOS, and the mitochondria
of these mice were unaffected by sepsis, it was
sugges-ted that the overproduction of NO by i-mtNOS is the
main factor responsible for mitochondrial nitrosative⁄
oxidative stress and impairment during endotoxemia
[9,12] The induction of i-mtNOS after
lipopoly-saccharide administration leads to an increase in NO
and other reactive species, such as superoxide anion
(O2), hydrogen peroxide (H2O2) and peroxynitrite
(ONOO–), in heart and diaphragm mitochondria
[8,18] Cecal ligation and puncture (CLP) also induces
i-mtNOS in these tissues, increasing mitochondrial lipid
peroxidation (LPO) and the oxidized glutathione⁄
glutathione (GSSG⁄ GSH) ratio, and reducing the
activity of the electron transport chain (ETC)
com-plexes [9,11,12] Although NO is a physiologic
modula-tor of mitochondrial respiration [19,20], high levels of
NO may inhibit the ETC, increasing the formation of
O2 and H2O2 [21] NO reacts with O2 to yield
ONOO–, which in turn impairs the ETC and ATP
syn-thase [19] The parallel failure of the respiratory chain
and oxidative phosphorylation leads to mitochondrial
dysfunction, energy depletion, and cell death A
reduc-tion in the capacity of the mitochondria to produce
ATP may be related to the organ failure in sepsis
[4,5,22]
There is evidence that antioxidants may be useful in
protecting against mitochondrial damage induced by
oxidative and⁄ or nitrosative stress [23] Several reports
have shown that melatonin (aMT) protects against
mitochondrial oxidative stress, due to its antioxidant properties and its ability to enter mitochondria [24– 28] In muscular tissues such as skeletal muscle and diaphragm of septic mice, aMT administration inhi-bited the activity of i-mtNOS, restoring the mito-chondrial GSH pool and the ETC activity in these animals [9,12,29]
Mitochondrial dysfunction is an important patho-physiologic event related to heart failure during sepsis, and i-mtNOS may be directly related to it To address this question, we induced sepsis by CLP in iNOS+⁄ + and iNOS–⁄ – mice, and explored in heart mitochon-dria: (a) the presence and source of i-mtNOS; (b) the relationship between i-mtNOS induction, ETC dys-function, and oxidative phosphorylation activity; (c) the steady-state energy and ATP production; and (d) the protective effect of aMT against mitochondrial damage produced during sepsis
Results
Mitochondrial NOS activities Figure 1 shows that heart mitochondria from iNOS+⁄ +mice contain two mitochondrial NOS iso-forms: a constitutive, Ca2+-dependent form (mtNOS), and an inducible, Ca2+-independent form (i-mtNOS)
In iNOS+⁄ +mice, sepsis induced a significant increase
in i-mtNOS activity, whereas mtNOS activity remained unchanged (Fig 1A) Control iNOS–⁄ – mice exhibited only the constitutive component of mitochondrial NOS that was partially inhibited during sepsis (Fig 1B) aMT administration counteracted sepsis-induced i-mtNOS activity in iNOS+⁄ + mice, without affecting mtNOS activity (Fig 1A) aMT also restored mtNOS activity that had been depressed by sepsis in mutant mice (Fig 1B)
Some considerations should be borne in mind regarding the purity of the mitochondrial preparation used here Heart mitochondria were isolated by differ-ential centrifugation, and purified by Percoll centrifu-gation [9,11,43] To remove contaminants, purified mitochondria were washed with high ionic strength solution (150 mm KCl) This protocol yields a very pure mitochondrial fraction without contaminating organelles and broken mitochondria, as reported else-where [11,12] The lack of mitochondrial contamina-tion with cytosolic NOS was assessed by the absence
of any detectable NOS activity and nitrite levels in the supernatant of the final centrifugation step (data not shown) These data confirm the purity of the mito-chondria used in our experiments, and guarantee the mitochondrial origin of the NOS activity reported
Trang 3here Moreover, the method used for NOS
measure-ment specifically detects mtNOS activity, and the
addi-tion of NG-monomethyl-l-arginine (l-NMMA) (300 lm)
to the reaction mixture of mitochondrial samples from
septic mice blocked the transformation of l-arginine to
l-citrulline, due to mtNOS inhibition (14.67 ± 3.09
versus 1.09 ± 0.87 pmol citrullineÆmin)1Æmg)1protein,
CLP and CLP + l-NMMA, respectively) [9,11,12]
iNOS+⁄ + mice exhibited a slight increase in nitrite
level after sepsis, which was counteracted by aMT
treatment (Table 1) Interestingly, iNOS–⁄ –mice showed
a significant decrease in nitrite level during sepsis,
coin-ciding with the mtNOS activity inhibition, that was
partially counteracted by aMT
iNOS mRNA expression
Figure 2 shows the results obtained in quantitative
RT-PCR experiments Because this is a
semiquantita-tive technique, the data are expressed as the relasemiquantita-tive quantity of mRNA in experimental versus control samples, giving to the control samples a value¼ 1 after deducting basal and background values Sepsis resulted in a significant increase in the transcription of the mRNA encoding iNOS in heart of iNOS+⁄ +mice The expression of iNOS mRNA was not detected in iNOS–⁄ – mice Treatment with aMT absolutely coun-teracted the transcription of iNOS mRNA induced by sepsis
Mitochondrial oxidative stress Sepsis significantly increased LPO levels in heart mito-chondria from iNOS+⁄ + mice, whereas aMT admin-istration reduced LPO below the control values (Table 1) Sepsis, however, did not modify the levels of LPO in iNOS–⁄ – mice, although aMT also reduced them below control values
Fig 1 Total heart mitochondrial NOS
activ-ity comprises constitutive, Ca 2+ -dependent,
and inducible, Ca2+-independent,
compo-nents in iNOS + ⁄ + mice (A) Deficient iNOS
mice, however, show only the constitutive,
Ca2+-dependent component (B) In both
cases, mice were subjected to CLP to
induce sepsis, and killed 24 h later Pure
mitochondrial preparations were used to
determine NOS activity with L -[ 3 H]arginine
as substrate Data represent the means
± SE of six experiments per group C,
con-trol; S, sepsis; S + aMT, sepsis + aMT.
*P < 0.05, **P < 0.01, ***P < 0.001 versus
C; # P < 0.05, ### P < 0.001 versus S.
Table 1 Effects of sepsis and aMT treatment on the mitochondrial nitrite and LPO levels, and on the activity of the mitochondrial ATPase in wild-type and iNOS knockout mice C, control; S, sepsis; S + aMT, sepsis + aMT Sepsis was induced by CLP, and the animals were killed
24 h later Nitrite, nmolÆmg protein)1; LPO, nmolÆmg protein)1; ATPase, nmol PiÆmin)1Æmg protein)1 Data are means ± SE, n ¼ 6.
*P < 0.05 versus C; P < 0.05 versus S.
Trang 4Figure 3A shows that glutathione peroxidase (GPx)
activity increased in heart mitochondria of iNOS+⁄ +
mice after sepsis, and this increase was preserved after
aMT administration In these animals, mitochondrial glutathione reductase (GRd) activity decreased during sepsis, whereas aMT treatment increased it to above control values (Fig 3B) Heart mitochondria from iNOS–⁄ – mice did not show changes in GPx and GRd activities with any treatment (Fig 3) Basal GPx activ-ity was lower in iNOS– ⁄ – than in iNOS+ ⁄ + mice (Fig 3A)
The mitochondrial level of GSH decreased and that
of GSSG increased in hearts from iNOS+⁄ +mice after CLP (Fig 4A,B), raising the GSSG⁄ GSH ratio (Fig 4C) Sepsis also reduced total glutathione levels in iNOS+⁄ + mice (Fig 4D) Treatment with aMT increased GSH levels and reduced GSSG levels in iNOS+⁄ + mice, normalizing the GSSG⁄ GSH ratio (Fig 4A–C) aMT also increased the total glutathione pool in this mouse strain (Fig 4D) No changes in glu-tathione levels were found in heart mitochondria
of iNOS–⁄ – mice under any experimental conditions (Fig 4A–D)
ETC complexes and ATPase activities Figure 5A–D shows that, after CLP, the activity of the four ECT complexes was significantly reduced in iNOS+⁄ + mice aMT administration increased the activity of these complexes above the control values (Fig 3A–D) The activity of the ETC complexes in heart mitochondria from iNOS–⁄ –mice was unaffected
by sepsis The basal activities of complex I and plex II were significantly lower, and those of com-plex III and IV were significantly higher, in iNOS–⁄ –
Fig 2 Effects of aMT treatment on CLP-induced mRNA levels
of iNOS Ten nanograms of RNA extracted from mouse heart
was used, and quantification of iNOS mRNA was performed by
real-time RT-PCR The relative level was calculated as
the ratio of inflammatory mRNA expression to b-actin mRNA
expression ***P < 0.001 versus C;###P < 0.0001 versus S Each
value represents the mean ± SE for three independent
experi-ments.
Fig 3 Changes in heart mitochondrial GPx (A) and GRd (B) activities after sepsis and aMT treatment in iNOS + ⁄ +
and iNOS – ⁄ – mice Data represent the means ± SE of six experiments per group C, control; S, sep-sis; S + aMT, sepsis + aMT *P < 0.05 and
**P < 0.01 versus C;##P < 0.005 versus S; + P < 0.05 versus iNOS +⁄ + mice.
Trang 5than in iNOS+⁄ + mice (Fig 5A–D) The activity of
the ETC complexes was also unchanged by aMT
treat-ment in iNOS–⁄ –mice No changes in ATPase activity
were observed in any mouse strain under sepsis, and aMT treatment only slightly decreased ATPase activity
in iNOS+⁄ +mice (Table 1)
Fig 4 GSH level (A), GSSG level (B),
GSSG ⁄ GSH ratio (C) and GSH + GSSG level
(D) in heart mitochondria of iNOS+⁄ +and
iNOS – ⁄ – mice Data represent the
mean ± SE of six experiments per group C,
control; S, sepsis; S + aMT, sepsis + aMT.
**P < 0.01 versus C;#P < 0.05 versus S.
Fig 5 Complex I (A), II (B), III (C) and IV (D) activities in heart mitochondria of iNOS + ⁄ + and iNOS – ⁄ – mice Data represent the mean ± SE
of six experiments per group C, control; S, sepsis; S + aMT, sepsis + aMT *P < 0.05 and **P < 0.01 versus C; # P < 0.05, # P < 0.05,
## P < 0.01 and ### P < 0.001 versus S; + P < 0.05, ++ P < 0.01 and +++ P < 0.001 versus iNOS + ⁄ +
mice.
Trang 6Mitochondrial ATP production
To assess whether sepsis modifies the bioenergetic
sta-tus of heart mitochondria, ATP production was
deter-mined ATP production was significantly reduced in
iNOS+⁄ + but not in iNOS–⁄ – mice during sepsis,
whereas aMT administration restored the ability of
mitochondria to produce ATP in the former (Fig 6)
After the ATP production assay, the amount of AMP
in the samples was less that 3% of the total
nucleo-tides, discounting extramitochondrial ATP production
by adenylate kinase in our assays The experimental
procedure used here allowed us to detect ATP inside
(pellet, fraction p2) and outside (supernatant,
frac-tion s1) the mitochondria The results indicated that
92–98% of the ATP produced was detected outside the
mitochondria
Animal survival
To determine the mortality of CLP-induced sepsis in
our experimental paradigm, and to assess whether the
improvement in mitochondrial function after aMT
treatment was followed by a reduction in mortality,
mice survival was analyzed Figure 7 shows the
survi-val curves obtained from untreated and aMT-treated septic mice The half-life of iNOS+⁄ + animals with sepsis was 26.5 h, increasing up to 35 h when they were treated with aMT Moreover, there was 100% mortality at 32.5 h in septic mice, whereas aMT treat-ment increased survival up to 50 h
A significant improvement in survival was observed
in iNOS–⁄ – mice (Fig 7) Untreated animals with sep-sis showed a half-life of 67.5 h, and aMT treatment increased it up to 129.5 h Also, there was 100% mor-tality at 90 h in septic mice, whereas aMT administra-tion increased this time up to 150 h
Discussion
The results of this study demonstrate the presence of two NOS isoforms with constitutive and inducible kin-etic properties in heart mitochondria During inflam-mation, i-mtNOS activity is increased, but not that of mtNOS The induction of i-mtNOS depends on iNOS expression, because mitochondria from iNOS–⁄ – mice lack i-mtNOS These data, and the results obtained from iNOS mRNA expression, suggest that i-mtNOS found in heart mitochondria derives from the cytosolic iNOS and is encoded by the same gene Sepsis was also accompanied by increased oxidative stress and inhibition of the ETC complexes, leading to a reduction
in ATP Because heart mitochondria from iNOS–⁄ –
Fig 6 Changes in mitochondrial ATP production in heart of
iNOS + ⁄ +
and iNOS – ⁄ –
mice, using succinate as substrate Data rep-resent the mean ± SE of six experiments per group C, control;
S, sepsis; S + aMT, sepsis + aMT *P < 0.05 and **P < 0.01
versus C; ## P < 0.01 versus S.
Fig 7 Survival curves obtained from untreated and aMT-treated septic iNOS +⁄ + and iNOS – ⁄ – mice The total number of animals used in this study was 20 in each group.
Trang 7mice were unaffected by endotoxemia and they do not
express i-mtNOS, mitochondrial impairment during
sepsis was probably related to i-mtNOS induction in
iNOS+⁄ + mice aMT treatment counteracted
sepsis-induced iNOS mRNA expression, a finding related to
the reduction of i-mtNOS aMT also prevented
mito-chondrial dysfunction, increasing ATP production, and
the survival of septic mice
Since the discovery of NOS activity in the
mitochon-dria [16,24,30], several reports have shown the presence
of this enzyme in different tissues with properties of
endothelial NOS, nNOS, and⁄ or iNOS [14,31,32] In
different models of sepsis and tissues, the existence of
both mtNOS and i-mtNOS isoforms has been reported
[9,11,12] The constitutive isoform was identified in
liver mitochondria as an nNOS isoform that was
post-translationally modified [11,17,33] Recent reports
also support the existence of i-mtNOS in mitochondria
from different tissues [9,11–13,15] Whereas the
ab-sence of mtNOS in nNOS knockout mice suggested
that mtNOS is derived from cytosolic nNOS [16], the
absence of i-mtNOS in iNOS knockout mice supported
its relationship with cytosolic iNOS [9,12]
NO is particularly important in the regulation of
cardiac function [10] It is involved in vascular and
nonvascular effects, including regulation of
cardiomyo-cyte contractility, in which mitochondrial respiration
and bioenergetics play an important role [15]
Produc-tion of excessive quantities of NO leads to profound
cellular disturbances and myocardial dysfunction
[8,15,18,34] Mitochondrial dysfunction is a
conse-quence of inflammation [6], and the induction of
i-mtNOS in heart mitochondria may be responsible for
mitochondrial failure during sepsis [9,11,12] The
exist-ence of an iNOS isoform was also recently confirmed
in heart mitochondria from rats [13] Normally, the
induction of i-mtNOS produces a significant increase
in NO and nitrite [9,11,12] The lack of a significant
increase in nitrite in iNOS+⁄ +mice after sepsis
repor-ted here could be explained by two main mechanisms
In mitochondria, the major oxidative decay pathway
of NO is its reaction with O2 to form ONOO–[20] In
turn, ONOO– reacts with a variety of biomolecules
[35] Moreover, ONOO– can react with H4-biopterin
(BH), a cofactor necessary for NO synthesis by NOS,
leading to formation of the BH3radical [36], and
caus-ing NOS inactivation [37,38] An alternative
explan-ation for the lack of changes in nitrite under sepsis is
the presence of an NOS-independent NO source in
mitochondria Alterations in the redox state of the
ETC lead to the formation of reactive nitrogen species,
including NO and ONOO–, and thus to nitrite [39] In
turn, mitochondrial nitrite reductase can recycle NO
from nitrite, masking the nitrite increase during sepsis [40] The existence of elevated nitrite levels in mito-chondria from other tissues under conditions of sepsis [9,11,12] suggests that the presence of a nitrate reduc-tase with higher activity in heart mitochondria than in the other tissues could explain the lack of changes in nitrite reported here However, differences in the relat-ive activities of nitrate reductases in mitochondria from different tissues have not yet been found
The reactive species produced as a consequence of i-mtNOS induction during sepsis are highly toxic, and they can impair the mitochondrial ETC and oxidative phosphorylation [19–21] Our results show a significant inhibition of the four complexes of the respiratory chain
in septic iNOS+⁄ +mice Similar results were reported for other tissues [5,7–9,11,12] However, septic iNOS–⁄ – mice did not show alterations in ECT activity during sepsis, suggesting that the oxidative⁄ nitrosative stress derived from i-mtNOS induction is responsible for ETC dysfunction in inflammation Unlike the situation with ETC complexes, our results do not show changes in ATPase activity in septic mice It was recently shown that the ETC complexes, but not ATPase, are damaged during the early and acute phases of Chagasic cardiomy-opathy [41] In these phases, the innate inflammatory response corresponds with iNOS induction and a subse-quent increase in NO These results suggest that ATPase
is more resistant to oxidative⁄ nitrosative stress than ETC complexes, probably because ETC complexes, unlike ATPase, have redox centers such as Fe–S that are very sensitive to NO [42]
ETC coupled with oxidative phosphorylation is responsible for the production of 90–95% of the total ATP synthesized in the cell [26] Thus, ETC damage may alter the synthesis of ATP without any effect on ATPase Our results show a reduced ability of the mitochondria to produce ATP during sepsis, which may reduce cardiomyocyte contractility [43,44] Be-cause the activity of the respiratory complexes was not affected in septic iNOS–⁄ – mice, ATP production by heart mitochondria was not altered in this mouse strain Thus, the reduction in the production of ATP found in diaphragm and heart after endotoxin admin-istration [5,22,45] probably reflects mitochondrial impairment due to i-mtNOS induction by the toxin Heart mitochondria from iNOS–⁄ – mice show lower complex I and II activities and higher complex III and
IV activities than iNOS+⁄ + mice, a finding also des-cribed in diaphragmatic and skeletal muscle mitochon-dria [9,12] Whereas the latter was attributed to the lack of the inhibitory effect of the NO derived from i-mtNOS, which is absent in iNOS–⁄ –mice, the reasons for the former phenomenon remains unclear [9,12] In
Trang 8any case, the data regarding ATP production suggest
that the low activities of complex I and II in iNOS–⁄ –
mice are compensated by the higher activities of
com-plex III and IV, allowing normal mitochondrial
homeostasis
Mitochondrial ETC impairment leads to electron
leakage and formation of O2 through the partial
reduc-tion of oxygen by one electron Subsequent reducreduc-tion by
one or two electrons can yield H2O2, and HO,
respect-ively [46] However, the main source of mitochondrial
H2O2is superoxide dismutase activity [47], whereas HO
can be derived from H2O2and ONOO–decomposition,
although the latter is a minor process [35] The small
effect on complex I compared with the strong inhibition
of complex III produced during sepsis in iNOS+⁄ +
mice suggests that the latter was the most important
source of free radicals in our experimental model NO
and O2 react to produce ONOO– in mitochondria
[7,19,20], increasing ETC damage [19,20] and LPO
activity [48] Besides causing direct oxidative damage,
ONOO–can produce nitration, and to a lesser extent
ni-trosation, of mitochondrial components indirectly [35]
The free radical pathways of ONOO–are mainly
initi-ated secondary to the reaction of ONOO– with CO2,
leading to the rapid formation of carbonate and
nitro-gen dioxide radicals The ONOO–⁄ CO2 pathway
becomes highly relevant in mitochondria, as these are
the main organelles in which CO2 is produced, due to
the decarboxylation reactions Although ONOO– can
yield HO•, it is a rather minor pathway in mitochondria,
as most ONOO–will react directly with either target or
CO2 In any case, ONOO– promotes, to some degree,
mitochondrial LPO activity; this could be initiated by
nitrogen dioxide and HO•radicals Carbonate radicals,
however, are poor direct initiators of LPO, due to their
negative charge, which limits their diffusion to the
hydrophobic domains of membrane phospholipids [35]
These mechanisms explain the LPO increase found in
heart mitochondria from septic iNOS+⁄ +mice, a
find-ing related to the i-mtNOS induction, because iNOS–⁄ –
mice did not show changes in LPO levels
Mitochondrial GPx activity increased in iNOS+⁄ +
mice, reflecting a compensatory mechanism to reduce
oxidative stress during sepsis However, the
GSSG⁄ GSH ratio remains elevated under these
condi-tions, because the reduced activity of GRd, probably
due to oxidative damage [53], prevents the recovery of
GSH from GSSG Moreover, total glutathione levels
in these mitochondria were also reduced, probably
reflecting inhibition of GSH transport into the
mito-chondria [49] The lack of mitomito-chondrial oxidative
stress and the presence of a normal GSH pool in septic
iNOS–⁄ – mice further support the idea that i-mtNOS
induction is the main event related to oxidative stress during sepsis in heart mitochondria
Different types of antioxidants with beneficial effects against mitochondrial oxidative stress have been pro-posed [23] One of these molecules is aMT, an indoleamine with excellent antioxidant and anti-inflammatory properties in the cell and mitochondria [24,27,28,50,51] First, aMT inhibits the expression and activity of both cytosolic iNOS and mitochondrial i-mtNOS in septic rats and mice [11,29,37], allowing the animals to recover from multiorgan failure Sec-ond, aMT directly scavenges reactive oxygen species and reactive nitrogen species [24,26–28,52], induces the expression of antioxidative enzymes [8], and restores mitochondrial GSH homeostasis [53] Third, aMT increases the activity of the ETC and ATP production
in vitro and in vivo [54,55] Our results demonstrate these protective effects of aMT in heart mitochondria
of septic iNOS+⁄ +mice aMT treatment counteracted iNOS expression, reducing the activity of i-mtNOS, increased the activity of the ETC complexes over the control values, and normalized the levels of LPO Reducing the levels of free radicals with aMT prevents them from causing oxidative damage to GRd [53], nor-malizing the GSSG⁄ GSH ratio Low oxidative status also prevents the mitochondrial transition pore open-ing and uncouplopen-ing, a condition associated with ATP hydrolysis [27,56,57] In fact, aMT restored the ability
of heart mitochondria to produce ATP In these circumstances, cardiomyocytes could have enough energy for muscle contraction, avoiding myocardial dysfunction, and probably heart failure, in sepsis The significant increase in survival of mice treated with aMT further supports this observation It is inte-resting that aMT had minor effects on heart mito-chondria from iNOS–⁄ – mice As reported in other pathophysiologic conditions, it seems that aMT can upregulate mitochondrial function when it is impaired [9,11,12,24,49,53,55], but the indoleamine had minor effects under normal conditions The half-life and maximum survival time of septic iNOS–⁄ – mice were significantly higher than those of wild-type animals Moreover, aMT treatment significantly increased the half-life and maximum survival time of mutant mice Because iNOS–⁄ –mice with sepsis did not show signifi-cant mitochondrial damage, they probably died by a mechanism different from iNOS-dependent dysfunc-tion, such as cyclooxygenase-2 induction Because aMT treatment had only minor effects on mitochon-dria of iNOS–⁄ – mice, the inhibitory effect of aMT on cyclooxygenase-2 expression may explain the signifi-cant improvement in survival of iNOS–⁄ – mice This hypothesis, however, remains to be studied
Trang 9In summary, this study demonstrates that, besides
mtNOS, mouse heart mitochondria contain an
i-mtNOS isoform that is induced during sepsis Among
other consequences, mitochondrial dysfunction,
oxida-tive stress and reduced ability to produce ATP follows
an increase in i-mtNOS These alterations could
con-tribute to the myocardial dysfunction that often occurs
during sepsis The parallel increases in iNOS
expres-sion and i-mtNOS activity, the inhibition of both
iNOS mRNA expression and i-mtNOS activity after
aMT treatment, and the lack of i-mtNOS expression in
mitochondria from iNOS–⁄ – mice, suggest that the
enzyme is encoded by the same gene that encodes
cyto-solic iNOS Moreover, the absence of any sign of
mit-ochondrial dysfunction and the lack of i-mtNOS
expression in iNOS–⁄ – mice further support the role of
i-mtNOS in mitochondrial impairment during sepsis
Administration of aMT to septic iNOS+⁄ + mice
nor-malized mitochondrial function, restoring their ability
to produce ATP, and increasing mice survival These
properties, together with the prevention of
endotoxin-induced circulatory failure in rats [58,59], and the
mor-tality reduction in septic newborns after aMT therapy
[60], suggest that the use of the indoleamine in septic
patients should be seriously considered
Experimental procedures
Chemicals
l-[2,3,4,5-3H]arginine monohydrochloride (58 CiÆmmol)1)
was obtained from Amersham Biosciences Europe GmBH
(Barcelona, Spain) Liquid scintillation cocktail (Ecolume)
was purchased from ICN (Madrid, Spain) All other
chemicals, of the purest available grade, were obtained from
Sigma-Aldrich (Madrid, Spain) unless otherwise specified
Experimental animals
All procedures involving animals were carried out under an
approved protocol and in accordance with the Spanish
Government Guide and the European Community Guide
for animal care iNOS knockout B6.129P2-Nos2tm1Lau mice
(iNOS–⁄ –) and their respective wild-type control C57⁄ Bl ⁄ 6
mice (iNOS+⁄ +) were obtained from Jackson’s Laboratory
through Charles River Laboratories (Barcelona, Spain)
The animals were housed in the university’s facility with a
12 h : 12 h light⁄ dark cycle (lights on at 07:00 h) at
22 ± 2C, and given regular chow and tap water Both
iNOS+⁄ + and iNOS–⁄ – mice 12–14 weeks of age were
grouped (n¼ 18 animals ⁄ group) as follows: (a) control
group; (b) sepsis group; and (c) sepsis + aMT group
Sepsis was induced by CLP [61] under intraperitoneal
equithesin anesthesia (1 mLÆkg)1) Four doses of aMT (30 mgÆkg)1) were injected as follows: one dose 30 min before surgery (intraperitoneal); the second dose just after surgery (subcutaneous); and the remaining doses 4 h and
8 h after surgery (subcutaneous) Animals were killed 24 h after CLP, except for the survival studies
Preparation of cardiac mitochondria
Pure mitochondria were isolated from 12–14-week-old wild-type and knockout mouse hearts by differential centrifuga-tion and density gradient centrifugacentrifuga-tion with Percoll as follows [9,11] All procedures were carried out in the cold Briefly, cardiac muscle was excised, washed with saline, treated with proteinase K (1 mgÆmL)1) for 30 s, washed with buffer A (250 mm mannitol, 0.5 mm EGTA, 5 mm He-pes, 0.1% BSA, pH 7.4, 4C), and homogenized (1 : 10,
w⁄ v) in buffer A at 800 r.p.m at 4 C with a Teflon pestle The homogenate was aliquoted, and centrifuged at 600 g for 5 min at 4C (twice) (rotor type F34-6-38 Eppendorf 5810R centrifuge), and the supernatants were centrifuged at
10 300 g for 10 min at 4C (rotor type F1255 Beckman TL-100 centrifuge) Then, the mitochondrial pellets were suspended in 0.5 mL of buffer A, and placed in ultracentri-fuge tubes containing 1.4 mL of buffer B (225 mm manni-tol, 1 mm EGTA, 25 mm Hepes, 0.1% BSA, pH 7.4, 4C) and 0.6 mL of Percoll The mixture was centrifuged at
105 000 g for 30 min at 4C (rotor type F1255 Beckman TL-100 centrifuge) The fraction corresponding to a pure mitochondrial fraction was collected, washed twice with buffer A at 10 300 g for 10 min at 4C (rotor type F1255 Beckman TL-100 centrifuge) to remove the Percoll, and washed again with a high ionic strength solution of KCl (150 mm) to yield a highly pure mitochondrial preparation without contaminating organelles and broken mitochondria [28,50] Aliquots of these pure mitochondrial fractions were frozen to) 80 C The purity of the mitochondrial prepara-tions was assessed as described elsewhere [9,11]
Mitochondrial NOS activity measurement
Measurement of constitutive, Ca2+-dependent, and indu-cible, Ca2+-independent, mitochondrial NOS activities was done as previously described [11,62] Briefly, an aliquot of frozen mitochondria was thawed and homogenized (0.1 gÆmL)1) in 25 mm Tris buffer (pH 7.6) containing 0.5 mm dithiothreitol, 10 lgÆmL)1 pepstatin, 10 lgÆmL)1 leupeptin,
10 lgÆmL)1 aprotinin and 1 mm phenylmethanesulfonyl fluoride at 4C (rotor type F34-6-38 Eppendorf 5810R cen-trifuge) The homogenate was centrifuged at 2500 g for
5 min at 4C, and the supernatant was used immediately for determination of NOS activity; one aliquot was frozen
at) 80 C for protein determination [63] Ten microliters of the supernatant (2 mgÆmL)1 protein) were added to the
Trang 10incubation mixture (100 lL, final volume) prewarmed at
37C, and containing (final concentration) 25 mm Tris,
1 mm dithiothreitol, 30 lm H4-biopterin, 10 lm FAD,
0.5 mm inosine, 0.5 mgÆmL)1 BSA, 0.1 mm CaCl2, 10 lm
l-arginine, and 40 nm l-[3H]arginine (pH 7.6) The reaction
was started by the addition of 10 lL of NADPH (0.75 mm
final concentration), and continued for 30 min at 37C To
determinate the Ca2+-independent activity of NOS
(i-mtNOS), 10 mm EDTA was added to the buffer before
the reaction was started Control incubations were
per-formed in the absence of NADPH The reaction was
stopped by adding 400 lL of cold 0.1 m Hepes buffer
con-taining 10 mm EGTA and 1 mm l-citrulline (pH 5.5) The
mixture was decanted onto a 2 mL column packed with
Dowex-50W ion exchange resin (Na+ form), and eluted
with 1.2 mL of water l-[3H]Citrulline was quantified
by liquid scintillation spectroscopy The retention
of l-[3H]arginine by the column was greater than 98%
Enzyme activity was determined as pmoL l-[3
H]citrul-lineÆmin)1Æmg protein)1
Real-time quantitative RT-PCR assay of iNOS
mRNA expression
Quantification of the iNOS mRNA levels was done by
SYBR green two-step real-time RT-PCR (Stratagene Mx
3005P; Stratagene, La Jolla, CA, USA) Total cellular
RNA was isolated from the heart using the RNA isolation
kit Real Total RNA Spin Plus (Durviz, S.L., Valencia,
Spain) Ten nanograms of the total RNA extracted was
used Gene-specific primers for iNOS (forward primer,
5¢-AGACGGATAGGCAGAGATTGG-3¢, and reverse
pri-mer, 5¢-ACTGACACTTCGCACAAAGC-3¢) and b-actin
(forward primer, 5¢-GCTGTCCCTGTATGCCTCTG-3¢,
and reverse primer, 5¢-CGCTCGTTGCCAATAGTGA
TG-3¢) were designed using the beacon designer software
(Premier Biosoft Int., Palo Alto, CA, USA) and obtained
from Thermo Electron GmbH (Ulm, Germany) Real-time
PCR reactions were carried out in a final volume of 25 lL
of reaction mixture containing 10 ng of RNA, 12.5 lL of
2X SYBR Green Master Mix (Stratagene), 75 nm each
spe-cific gene primer, and H2O The samples were run in
tripli-cate in the amplification program, and the mean value was
used as the final expression value A negative control
with-out RNA template was run The PCR program was
initi-ated by 10 min at 95C before 40 thermal cycles, each of
30 s at 95C and 1 min at 55 C Data were analyzed
according to the relative standard curve method,
construc-ted with triplicate serial dilutions (50, 5, 0.5 and 0.05 ng),
and were normalized by b-actin expression
Nitrite determination
Mitochondrial fractions were thawed and suspended in
ice-cold distilled water, and immediately sonicated to break the
mitochondrial membranes Aliquots of these samples were used to calculate nitrite levels following the Griess reaction [64], and expressed in nmoL nitriteÆmg protein)1
Determination of mitochondrial function
The activities of the four respiratory complexes were deter-mined as previously described [65,66], with slight modifica-tions [9,12], and expressed as nmoLÆmin)1Æmg protein)1 Complex V (ATP synthase) activity was measured by following the rate of hydrolysis of ATP to ADP + Pi Ferrous sulfate⁄ ammonium molybdate reagent was utilized
to determinate Pi concentration [67] ATPase activity was expressed in nmoL PiÆmin)1Æmg protein)1
Determination of ATP production
For the determination of ATP production, hearts were excised, washed with saline, treated with proteinase K (1 mgÆmL)1) for 30 s, washed with buffer A (220 mm mannitol, 70 mm sucrose, 1 mm EGTA, 20 mm Hepes, 1% BSA, pH 7.2, 4C), and homogenized (1 : 10, w ⁄ v) in buf-fer A at 800 r.p.m at 4C with a Teflon pestle The homo-genates were centrifuged at 1500 g for 5 min at 4C (rotor type F1255 Beckman TL-100 centrifuge), and the superna-tants were centrifuged again at 23 000 g for 5 min at 4C (rotor type F1255 Beckman TL-100 centrifuge) Then, the mitochondrial pellets were suspended in 1 mL of buffer A, and centrifuged at 10 300 g for 3 min at 4C (rotor type F1255 Beckman TL-100 centrifuge) The resultant pellets (p1) were suspended in respiration buffer (225 mm manni-tol, 75 mm sucrose, 10 mm KCl, 10 mm Tris⁄ HCl, 5 mm potassium phosphate, pH 7.2, saturated with O2, plus 5 mm succinate, 30C), and ATP production was induced adding
125 nmol of ADP After 45 s, the sample was centrifuged
at 13 000 g for 3 min at 2C (rotor type F1255 Beckman TL-100 centrifuge) [68,69], and the ATP content in the pel-let (p2) and supernatant (s1) was measured Ice-cold 0.5 m perchloric acid was rapidly added to the p1, p2 and s1 frac-tions, mixed for 2 min in a vortex mixer, and centrifuged at
25 000 g for 15 min at 2C (rotor type F1255 Beckman TL-100 centrifuge) to precipitate proteins The pellets were frozen to ) 80 C for protein determination [63]; the sup-ernatants were mixed with 8 lL of 5 m potassium carbon-ate to neutralize the pH, and centrifuged at 12 000 g for
10 min at 2C (rotor type F1255 Beckman TL-100 centri-fuge) ATP was measured in the resultant supernatants by HPLC with a 4· 250 mm ProPac PA1 column (Dionex, Barcelona, Spain) [70] After stabilization of the column with the mobile phase, samples (20 lL) were injected onto the HPLC system The mobile phase consisted of water (phase A) and 0.3 m ammonium carbonate (pH 8.9)
(pha-se B), and the following time schedule for the binary gradi-ent (flow rate 1 mLÆmin)1) was used: 5 min, 50% A and 50% B; 5 min, 50% to 100% B, and then 100% B for