Relative sensitivity of soluble guanylate cyclase and mitochondrial respiration to endogenous nitric oxide at physiological oxygen concentration.
Nitric oxide (NO) is a widespread biological messenger that has many physiological and pathophysiological roles. Most of the physiological actions of NO are mediated through the activation of sGC (soluble guanylate cyclase) and the subsequent production of cGMP. NO also binds to the binuclear centre of COX (cytochrome c oxidase) and inhibits mitochondrial respiration in competition with oxygen and in a reversible manner. Although sGC is more sensitive to endogenous NO than COX at atmospheric oxygen tension, the more relevant question is which enzyme is more sensitive at physiological oxygen concentration. Using a system in which NO is generated inside the cells in a finely controlled manner, we determined cGMP accumulation by immunoassay and mitochondrial oxygen consumption by high-resolution respirometry at 30 microM oxygen. In the present paper, we report that the NO EC50 of sGC was approx. 2.9 nM, whereas that required to achieve IC50 of respiration was 141 nM (the basal oxygen consumption in the absence of NO was 14+/-0.8 pmol of O2/s per 10(6) cells). In accordance with this, the NO-cGMP signalling transduction pathway was activated at lower NO concentrations than the AMPKs (AMP-activated protein kinase) pathway. We conclude that sGC is approx. 50-fold more sensitive than cellular respiration to endogenous NO under our experimental conditions. The implications of these results for cell physiology are discussed.
Rodríguez-Juárez
Félix
F
CNIC (Centro Nacional de Investigaciones Cardiovasculares), Biology of Nitric Oxide Laboratory, Melchor Fernández Almagro 3, 28029 Madrid, Spain.
Aguirre
Enara
E
Cadenas
Susana
S
D055372:AMP-Activated Protein Kinases; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002478:Cells, Cultured; D003576:Electron Transport Complex IV / Q000502:physiology*; D006162:Guanylate Cyclase / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D006834:Hydrazines / Q000494:pharmacology; D008840:Microfilament Proteins / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology*; D009097:Multienzyme Complexes / Q000378:metabolism; D009569:Nitric Oxide / Q000502:physiology*; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D010100:Oxygen / Q000502:physiology*; D010101:Oxygen Consumption / Q000187:drug effects*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D000098662:Vasodilator-Stimulated Phosphoprotein
D015815:Cell Adhesion Molecules; D006834:Hydrazines; D008840:Microfilament Proteins; D009097:Multienzyme Complexes; D010750:Phosphoproteins; D000098662:Vasodilator-Stimulated Phosphoprotein; D009569:Nitric Oxide; C084012:1,1-diethyl-2-hydroxy-2-nitrosohydrazine; D052247:Nitric Oxide Synthase Type II; D003576:Electron Transport Complex IV; D017346:Protein Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases; D006162:Guanylate Cyclase; D010100:Oxygen