On the mechanism by which vascular endothelial cells regulate their oxygen consumption

Abstract

Two enzymes, soluble guanylyl cyclase and cytochrome c oxidase, have been shown to be exquisitely sensitive to nitric oxide (NO) at low physiological concentrations. Activation of the soluble guanylyl cyclase by endogenous NO and the consequent increase in the second messenger cyclic GMP are now known to control a variety of biological functions. Cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, is inhibited by NO. However, it is not clear whether NO produced by the constitutive NO synthase interacts with cytochrome c oxidase, nor is it known what the biological consequences of such an interaction might be. We now show that NO generated by vascular endothelial cells under basal and stimulated conditions modulates the respiration of these cells in response to acute changes in oxygen concentration. This action occurs at the cytochrome c oxidase and depends on inf lux of calcium. Thus, NO plays a physiological role in adjusting the capacity of this enzyme to use oxygen, allowing endothelial cells to adapt to acute changes in their environment.Evidence in favor of a role of endogenous nitric oxide (NO) as a modulator of cell respiration has been derived from experiments in cells activated with cytokines and bacterial products in which NO is generated continuously in large quantities by the inducible NO synthase (NOS). In these conditions, NOinduced inhibition of cell respiration is persistent and attributable to nonselective inhibition of various mitochondrial enzymes, including complexes I-IV in the respiratory chain. Such inhibition contributes to the pathological actions of NO (1). On the other hand, experiments in animals have suggested that inhibition of endogenous generation of NO increases whole body oxygen consumption (2, 3), and bradykinin (Bk) and carbachol have been shown to reduce oxygen consumption in skeletal and cardiac muscle in a manner that could be prevented by an inhibitor of NOS (3-5).Exogenous administration of low concentrations of NO inhibits cytochrome c oxidase (complex IV in the mitochondrial respiratory chain) in a variety of cells and isolated mitochondria. Such inhibition is competitive with oxygen and is fully reversible (6-9) even after several hours (10). These findings suggest that endogenous NO may regulate cell respiration. To test this hypothesis, we have analyzed the effect of endogenous NO, generated under basal conditions and after stimulation with Bk and ATP, on respiration in porcine aortic endothelial cells. Our results show that endogenously released NO, by acting on cytochrome c oxidase, is responsible for the physiological regulation of respiration in these cells. MATERIALS AND METHODSMaterials. Culture media and fetal calf serum were from GIBCO. Fura-2 acetoxymethylester was from Calbiochem. Other reagents were from Sigma.Cell Culture and Preparation. Endothelial cells were prepared from fresh porcine thoracic aortae obtained from the abattoir, were cultured overnight in DMEM 20% fetal calf serum, and then were grown to confluence...

Endothelial cell respiration is affected by the oxygen tension during shear exposure: role of mitochondrial peroxynitrite

“…In agreement with a study by Moncada's group (17), who showed that the V O 2 of cultured ECs is dependent on PO 2 only when the cells are activated to synthesize NO, no difference in V O 2 was found between static BAECs at either 5% or 21% O 2 (Figs. 1A and 2 and Table 1).…”

“…At the end of shear exposure, ECs were harvested and centrifuged, and the pellet was resuspended at a density in the order of 10 7 cells/ml in modified Krebs-Henseleit buffer (in mM: 117.3 NaCl, 4.7 KCl, 1.3 MgSO4, 1.2 CaCl2, 1.2 KH2PO4, and 25 NaHCO3, pH 7.4) supplemented with 20 mM glucose, in agreement with earlier respirometry studies (17). EC respiration was measured immediately using EPR oximetry with lithium phthalocyanine (LiPc) as the probe in a Bruker ER-300 X-band EPR spectrometer fitted with a TM 110 microwave cavity, as described previously (34,36,53).…”

“…1A and 2 and Table 1). In their study, EC treatment with the neuropeptide bradykinin, which activates eNOS, induced production of NO that interacted at the level of the ETC complex IV and inhibited respiration; inhibition was greater at lower PO 2 that resembled the physiological range in tissues (17). In the present study, endogenous NO production due to shear exposure inhibited respiration, but the effect was more pronounced at 21% O 2 compared with that at lower PO 2 levels, because of ONOO Ϫ formation in the mitochondria of ECs sheared at 21% O 2 .…”

“…Specifically, when ECs were preincubated and sheared in the presence of either the eNOS inhibitor N G -nitro-L-arginine methyl ester (L-NAME) or the ONOO Ϫ scavenger uric acid (UA), the flow effect on complexes I and II/III was attenuated and the effect on complex IV was abolished. Inhibition of complex IV is expected in NO-producing cells (17). However, shear-induced inhibition affected all complexes (including complex I), was accompanied by nitrotyrosine staining in the mitochondria, and was prevented by UA, indicating that endogenous eNOS-derived NO diffuses to the mitochondria where it reacts with O 2 ⅐ Ϫ generated by the ETC resulting in mitochondrial ONOO Ϫ formation, and the latter is responsible for the inhibition of ETC at multiple sites (30).…”

Superoxide-dependent consumption of nitric oxide in biological media may confound in vitro experiments

“…The present study shows that, over a range of concentrations covering those encountered in itro following constitutive or inducible NO synthase activity [22][23][24], NO can be consumed as a result of the presence of two common constituents of cellculture media : riboflavin and Hepes buffer. The combination of the two ingredients under normal laboratory lighting conditions leads to a greatly amplified quench of the NO signal.…”