Tesis doctoral de Felix Rodriguez Juarez
Most of the physiological actions of nitric oxide (no) are mediated by the activation of soluble guanylate cyclase (sgc) and the subsequent increase in cgmp levels. No also binds to the binuclear centre of cytochrome c oxidase (cco) and inhibits mitochondrial respiration in competition with oxygen and in a reversible manner. Using hek 293 cells that produce no endogenously from the inducible isoform of the no synthase (inos) in a controlled manner, we determined the relative sensitivity of sgc and mitochondrial respiration at physiological oxygen concentration (30 ¿m). Cgmp production was determined by immunoassay and mitochondrial oxygen consumption by high-resolution respirometry. Our results show that the concentration of no that causes half-activation (ec50) of sgc was approximately 2.9 nm, whereas that required to achieve half-inhibition (ic50) of respiration was 141 nm. In agreement with these data, the no-cgmp signalling transduction pathway was activated at lower no concentrations than the amp-activated protein kinase (ampk) pathway. We conclude that sgc is approximately 50-fold more sensitive than cellular respiration to endogenous no under our experimental conditions. in the second section of this thesis, we performed a detailed study of the oxygen kinetics of cellular respiration in intact cells producing controlled amounts of no. We determined no and oxygen concentrations, and mitochondrial oxygen consumption by high-resolution respirometry over a range of oxygen concentrations, down to nanomolar. Cco maintained residual activity up to 2 ¿m no even at low oxygen concentrations. The concentration of no required to inhibit cco by 50% (ic50) had an unusual parabolic dependence on oxygen concentration. A kinetic model of cco inhibition by no was developed that provided an excellent fit to experimentally determined respiration under hypoxia, and accurately predicted the respiratory responses under hyperoxia. The model takes into account competitive and uncompetitive inhibition by binding of no to the reduced and oxidized forms of cco, respectively, and suggests that dissociation of no from reduced cco may involve its o2-dependent oxidation. in the last group of experiments, we investigated the effects of physiological no concentrations (up to 600 nm) on mitochondrial inner membrane proton conductance at an oxygen concentration of 30 ¿m. Oxygen consumption and mitochondrial inner membrane potential (¿¿m) were simultaneously determined by high-resolution respirometry and the lipophilic cation [3h]tpmp+, respectively, in the presence of oligomycin to inhibit atp synthesis. Membrane potential was progressively decreased by increasing concentrations of rotenone (complex i inhibitor), and its values were determined from the ratio [3h]tpmp+ matrix/[3h]tmpm+ cytosol using the nernst equation at 37 ºc. We conclude that physiological concentrations of endogenous no inhibit mitochondrial oxygen consumption but do not increase proton conductance.
Datos académicos de la tesis doctoral «Efectos del oxido nitrico endogeno sobre la guanilato ciclasa soluble, la respiracion y la conductancia de la membrana mitocondrial interna a los protones en celulas intactas«
- Título de la tesis: Efectos del oxido nitrico endogeno sobre la guanilato ciclasa soluble, la respiracion y la conductancia de la membrana mitocondrial interna a los protones en celulas intactas
- Autor: Felix Rodriguez Juarez
- Universidad: Autónoma de Madrid
- Fecha de lectura de la tesis: 04/12/2009
Dirección y tribunal
- Director de la tesis
- Susana Cadenas álvarez
- Tribunal
- Presidente del tribunal: jorgina Satrústegui gil-delgado
- jordi Muntané relat (vocal)
- Antonio Martinez ruiz (vocal)
- Juan Manuel Serrador peiró (vocal)