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Updated: Aug 21, 2026
Preparation of Rat Skeletal Muscle Homogenates for Nitrate and Nitrite Measurements
Published on: July 29, 2021
Mitochondrial nitric oxide metabolism in rat muscle during endotoxemia
Silvia Alvarez1, Alberto Boveris
1Laboratory of Free Radical Biology, School of Pharmacy and Biochemistry, University of Buenos Aires, C1113AAD Buenos Aires, Argentina. salvarez@ffyb.uba.ar
Abstract:
In this study, heart and diaphragm mitochondria produced 0.69 and 0.77 nmol nitric oxide (NO)/min mg protein, rates that account for 67 and 24% of maximal cellular NO production, respectively. Endotoxemia and septic shock occur with an exacerbated inflammatory response that damages tissue mitochondria. Skeletal muscle seems to be one of the main target organs in septic shock, showing an increased NO production and early oxidative stress. The kinetic properties of mitochondrial nitric oxide synthase (mtNOS) of heart and diaphragm were determined. For diaphragm, the KM values for O2 and L-Arg were 4.6 and 37 microM and for heart were 3.3 and 36 microM. The optimal pH for mtNOS activity was 6.5 for diaphragm and 7.0 for heart. A marked increase in mtNOS activity was observed in endotoxemic rats, 90% in diaphragm and 30% in heart. Diaphragm and heart mitochondrial O2*- and H2O2 production were 2- to 3-fold increased during endotoxemia and Mn-SOD activity showed a 2-fold increase in treated animals, whereas catalase activity was unchanged. One of the current hypotheses for the molecular mechanisms underlying the complex condition of septic shock is that the enhanced NO production by mtNOS leads to excessive peroxynitrite production and protein nitration in the mitochondrial matrix, causing mitochondrial dysfunction and contractile failure.
Insights
Mitochondrial nitric oxide synthase (mtNOS) activity significantly increases in heart and diaphragm during endotoxemia, contributing to septic shock. This enhanced nitric oxide (NO) production by mitochondria may cause dysfunction.
Area of Science:
- Biochemistry
- Physiology
- Pathology
Background:
- Sepsis involves an inflammatory response damaging mitochondria, particularly in skeletal muscle.
- Increased mitochondrial nitric oxide (NO) production and oxidative stress are observed in septic shock.
- Mitochondrial nitric oxide synthase (mtNOS) plays a role in cellular NO homeostasis.
Purpose of the Study:
- To investigate the kinetic properties of mtNOS in heart and diaphragm.
- To determine the impact of endotoxemia on mtNOS activity and mitochondrial function.
- To explore the role of mtNOS in the pathophysiology of septic shock.
Main Methods:
- Measurement of mitochondrial nitric oxide (NO) production rates.
- Determination of kinetic parameters (KM values for O2 and L-Arg) and optimal pH for mtNOS.
- Assessment of mitochondrial superoxide (O2*-) and hydrogen peroxide (H2O2) production.
- Enzymatic activity assays for Mn-superoxide dismutase (Mn-SOD) and catalase.
Main Results:
- Heart and diaphragm mitochondria produce significant amounts of NO, contributing to cellular NO levels.
- Endotoxemia markedly increased mtNOS activity in diaphragm (90%) and heart (30%).
- Mitochondrial O2*- and H2O2 production increased 2- to 3-fold, with a 2-fold rise in Mn-SOD activity during endotoxemia.
Conclusions:
- Mitochondrial nitric oxide synthase (mtNOS) activity is upregulated during endotoxemia.
- Enhanced mitochondrial NO production and oxidative stress may contribute to mitochondrial dysfunction in septic shock.
- mtNOS-derived peroxynitrite formation is a potential mechanism for mitochondrial damage in sepsis.
