Activation of Hsp90/NOS and increased NO generation does not impair mitochondrial respiratory chain by competitive binding at cytochrome C Oxidase in low oxygen concentrations

Abstract

Nitric oxide (NO) is known to regulate mitochondrial respiration, especially during metabolic stress and disease, by nitrosation of the mitochondrial electron transport chain (ETC) complexes (irreversible) and by a competitive binding at O 2 binding site of cytochrome c oxidase (CcO) in complex IV (reversible). In this study, by using bovine aortic endothelial cells, we demonstrate that the inhibitory effect of endogenously generated NO by nitric oxide synthase (NOS) activation, by either NOS stimulators or association with heat shock protein 90 (Hsp90), is significant only at high prevailing pO 2 through nitrosation of mitochondrial ETC complexes, but it does not inhibit the respiration by competitive binding at CcO at very low pO 2 . ETC complexes activity measurements confirmed that significant reduction in complex IV activity was noticed at higher pO 2 , but it was unaffected at low pO 2 in these cells. This was further extended to heat-shocked cells, where NOS was activated by the induction/activation of (Hsp90) through heat shock at an elevated temperature of 42°C. From these results, we conclude that the entire attenuation of respiration by endogenous NO is due to irreversible inhibition by nitrosation of ETC complexes but not through reversible inhibition by competing with O 2 binding at CcO at complex IV.

Hyperthermia-induced Hsp90·eNOS Preserves Mitochondrial Respiration in Hyperglycemic Endothelial Cells by Down-regulating Glut-1 and Up-regulating G6PD Activity

“…Such an enhanced NO generation was demonstrated to have serious implications in cellular respiration, and we have elucidated a possible mechanism of this Hsp90⅐eNOS-induced modulation of cellular respiration involving both irreversible/reversible inhibitory roles of NO on the mitochondrial oxidative metabolism (22). In this work we demonstrate that in a hyperglycemic setting the same hyperthermiainduced eNOS activation by Hsp90 also increases NO generation and reduces ROS, but this is accompanied by an increase in mitochondrial respiration.…”

“…Fig. 1 (21)(22)(23) in the presence of 30 mM glucose despite the reduced respiration. The osmotic control (5 mM glucose ϩ 25 mM mannitol) showed OCR and parameters close NG (Fig.…”

“…Mitochondrial ETC Enzymatic Activities-Mitochondrial electron transfer chain complexes (I-IV) activities were measured as described before (22,23). Normoglycemia and hyperglycemia-treated control and heat-shocked BAECs were homogenized in ice-cold 3 mM HEPES buffer (pH 7.2), 0.5 mM EGTA, 0.25 M sucrose, and 2.5% protease inhibitor mixture.…”

“…The activity of succinate-cytochrome c reductase was assayed by assessing ferricytochrome c reduction. The assay mixture contained 50 mM phosphate buffer, pH 7.4, 0.3 mM EDTA, 50 M KCN, 19.8 mM succinate, and 50 M ferricytochrome c (22).…”

Modulating Nitric Oxide: Implications for Cytotoxicity and Cytoprotection

“…Additionally, • NO and its derivatives have the capacity to induce the peroxidation of phospholipids and the oxidation of thiol groups present on mitochondrial membrane proteins. These processes ultimately contribute to the release of apoptogenic factors into the cytosol [183][184][185][186][187].…”