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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 28, 2003 as doi:10.1096/fj.02-0692fje. |
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Departamento de Fisiología, Instituto de Biotecnología, Universidad de Granada, Granada, Spain
2Correspondence: Departamento de Fisiología, Facultad de Medicina, Avda. de Madrid 11, E-18012 Granada, Spain. E-mail: dacuna{at}ugr.es
SPECIFIC AIM
Mitochondrial isoform of nitric oxide synthase (mtNOS) is constitutively expressed but whether it may be induced is unclear. Melatonin (aMT) decreases with age, prevents mitochondrial oxidative damage, and increases the activity of the mitochondrial electron transport chain. aMT also inhibits iNOS expression induced by bacterial lipopolysaccharide (LPS). Thus, we examined the age-dependent effects of LPS on the activity and expression of mtNOS and mitochondrial NO production and their relation to the activity of the electron transport chain (ETC). We studied the protective role of melatonin against the age-dependent effects of LPS on mtNOS, NO, and ETC.
PRINCIPAL FINDINGS
1. Effect of aMT on the LPS-induced nitrite production and mtNOS activity
Aging itself increases mitochondrial nitrite levels, and LPS administration further augmented them in lung mitochondria from young and old animals (P<0.001). Treatment with aMT counteracted the effects of endotoxemia, restoring nitrite levels to control levels in all experimental groups. The increase in mitochondrial nitrite was due to mtNOS activation since L-NMMA administration blocked nitrite production due to LPS. Similar changes were found in liver mitochondria from the same animals.
Basal activity of mtNOS in lung mitochondria was almost undetectable, but increased significantly after LPS treatment (Fig. 1
). The effect of LPS on mtNOS activity was significantly higher in aged rats than young animals (P<0.001). Melatonin treatment significantly counteracted LPS-dependent mtNOS activity in young and aged animals (P<0.001), although a residual activity of mtNOS that was significantly higher than basal (P<0.05) remained.
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After LPS, most of the mtNOS was calcium independent (66%), and a 33% of the enzyme activity was blocked after removing calcium (with 10 mM EGTA) or blocking calmodulin (with 100 nM calmidazolium) in the incubation medium. The activity of mtNOS detected in vehicle and LPS+aMT groups was essentially independent of calcium.
2. Effect of aMT on the LPS-induced expression of mtNOS protein
The presence of mtNOS protein was confirmed by Western blot studies in lung mitochondria from young and old rats. A single component corresponding to 130 kDa monomer of mtNOS was detected by immunoblot using an anti-iNOS antibody that was previously demonstrated to react with mtNOS. Mitochondrial NOS also reacts against anti-nNOS, but not against anti-eNOS antibodies. No age differences were found in the amount of mtNOS protein in control mitochondria. However, after LPS administration mtNOS protein content was significantly higher in mitochondria from old animals. Thus, the increase in mtNOS activity was accompanied by a parallel increase in mtNOS protein expression after LPS treatment. A marked reduction in mtNOS protein expression occurred after aMT administration.
3. Effect of aMT on the LPS-induced inhibition of complex I and IV
Aging caused a significant decrease (P<0.001) in the activity of complexes I and IV of the mitochondrial electron transport chain. A significant (P<0.001) reduction in these activities was found after LPS administration. Treatment with aMT significantly (P<0.001) increased the activity of both complexes in control mitochondria. Besides, aMT counteracted (P<0.001) LPS-induced inhibition of complexes I-IV. Results were similar in lung (Fig. 2
) and liver mitochondria.
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CONCLUSIONS AND SIGNIFICANCE
Our results demonstrate, for the first time, that endotoxemia induces the expression and activity of mtNOS, leading to a mitochondrial impairment. The most significant finding of this study is the functional up-regulation of mtNOS after sepsis, with 66% of its activity being calcium independent. The production of NO by mitochondria was inhibitable by the NOS antagonist L-NMMA, demonstrating that NO was produced by mtNOS and not by the other described pathway. Melatonin administration efficiently counteracted LPS-induced mtNOS expression and activity, neutralizing mitochondrial damage. aMT administration also reduced significantly the effects of aging itself on mtNOS/NO system and ETC activity.
The nature of mtNOS is unclear. Although a constitutive, calcium-dependent isoform of mtNOS was identified using an antibody against eNOS, other authors identified mtNOS as an eNOS- or iNOS-like isoenzyme. Our data suggest that mtNOS is an iNOS-like isoenzyme constitutively expressed.
Recently, mtNOS isoform was identified as nNOS with post-translational modifications including acylation with myristic acid, which may serve for subcellular targeting. The authors concluded that mitochondria from rat liver contain a constitutive mtNOS derived from nNOS
. However, the authors show that mtNOS cross-reacted with the antibodies against iNOS. Our results support the presence of an iNOS-like mtNOS isoenzyme constitutively expressed in rat lung mitochondria that shows immunoreactivity against anti-nNOS antibody. Since constitutive isoforms of NOS such as nNOS can be induced by endotoxin administration, the presence of an induction of mtNOS after LPS is not surprising. Nevertheless, 66% of the mtNOS activity induced by LPS is calcium-calmodulin independent. Thus, the possibility that under sepsis an inducible isoform of mtNOS different from constitutive mtNOS may be synthesized and directed toward mitochondria cannot be discarded.
Intramitochondrial NO levels reversibly inhibit complex IV activity competing with O2, signifying that under normal physiological conditions NO serves as a brake or tonic inhibitor of mitochondrial respiration. But high levels of NO and ONOO- (formed by the reaction between NO and O2•-) irreversibly inhibit ETC complexes decreasing ATP production. During endotoxemia, high ONOO- production produces GSH depletion leading to mitochondrial dysfunction. It has been reported that aMT restored the mitochondrial GSH pool, increasing GSH/GSSG redox cycling through the activation of glutathione peroxidase and reductase. These effects were accompanied by a dose-dependent increase in the activity of complexes I and IV of the ETC, resulting in increased ATP production Moreover, aMT is a scavenger of NO and ONOO-, reducing these nitrogen reactive species. Inhibition of mtNOS plus the scavenging ability of NO and ONOO- may account for the protective effects of the indoleamine against endotoxemia, including the recovery of ETC activity and ATP production (Fig. 3
).
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Our findings suggest that aging is associated with a NO-dependent oxidative damage that was enlarged further after LPS administration. Increased mitochondrial oxidative damage and a decreased expression of mitochondrial mRNA coexist in aging. Our data show that aging itself is associated with increased levels of NO without significant changes in mtNOS expression and/or activity. However, after LPS administration, changes in mtNOS expression and activity were significantly higher in older animals. These changes were followed by an increase mitochondrial NO induced by LPS. Increased NO levels with age were accompanied by a reduction in the activity of the mitochondrial complexes I and IV. Administration of aMT counteracted the age-dependent reduction in complexes I and IV, and neutralized LPS-induced ETC inhibition.
This study clearly demonstrates that mtNOS may be induced in an age-dependent manner under pathophysiological conditions such as sepsis, leading to mitochondrial failure. The data support a role for aMT to prevent mitochondrial damage due to endotoxemia (Fig. 3)
. The ability of NO production by single-cell organisms together with the presence of aMT at a unicell level suggests that both molecules constitute an evolutionarily preserved mitochondrial regulatory mechanism. Mortality associated with septic shock is the major cause of death among patients in intensive care units. Melatonin prevents multiple organ dysfunction syndrome and circulatory failure during endotoxemia, protects mitochondria against damage in experimental sepsis, and reduces mortality in septic newborn. Considering these effects of aMT and its virtual absence of toxicity, the use of aMT to preserve mitochondrial energetic should be borne in mind for septic patients.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0692fje; to cite this article, use FASEB J. (March 28, 2003) 10.1096/fj.02-0692fje 
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  S. C. Land and C. Rae iNOS initiates and sustains metabolic arrest in hypoxic lung adenocarcinoma cells: mechanism of cell survival in solid tumor core Am J Physiol Cell Physiol, October 1, 2005; 289(4): C918 - C933. [Abstract] [Full Text] [PDF]
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