Does nitric oxide allow endothelial cells to sense hypoxia and mediate hypoxic vasodilatation?<i>in vivo</i> and <i>in vitro</i> studies

Edmunds, Nick; Moncada, Salvador; Marshall, Janice M.

doi:10.1113/jphysiol.2002.023663

Cited by 41 publications

(55 citation statements)

References 35 publications

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“…The conserved influences of NO extend from the regulation of basic cellular processes such as intermediary metabolism (2), cellular proliferation (3), and apoptosis (4) to systemic processes such as hypoxic vasoregulation (5). Mammalian NO production has been attributed to the enzymatic activity of NO synthases, nitrate (NO 3 Ϫ )/nitrite (NO 2 Ϫ ) reductases and non-enzymatic NO 2 Ϫ reduction (6).…”

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confidence: 99%

Tissue Processing of Nitrite in Hypoxia

Feelisch

Fernandez

Bryan

et al. 2008

Journal of Biological Chemistry

194

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Although nitrite (NO 2؊ ) and nitrate (NO 3 ؊ ) have been considered traditionally inert byproducts of nitric oxide (NO) metabolism, recent studies indicate that NO 2 ؊ represents an important source of NO for processes ranging from angiogenesis through hypoxic vasodilation to ischemic organ protection. Despite intense investigation, the mechanisms through which NO 2 ؊ exerts its physiological/pharmacological effects remain incompletely understood. We sought to systematically investigate the fate of NO 2 ؊ in hypoxia from cellular uptake in vitro to tissue utilization in vivo using the Wistar rat as a mammalian model. We find that most tissues (except erythrocytes) produce free NO at rates that are maximal under hypoxia and that correlate robustly with each tissue's capacity for mitochondrial oxygen consumption. By comparing the kinetics of NO release before and after ferricyanide addition in tissue homogenates to mathematical models of NO 2 ؊ reduction/NO scavenging, we show that the amount of nitrosylated products formed greatly exceeds what can be accounted for by NO trapping. This difference suggests that such products are formed directly from NO 2 ؊ , without passing through the intermediacy of free NO. Inhibitor and subcellular fractionation studies indicate that NO 2 ؊ reductase activity involves multiple redundant enzymatic systems (i.e. heme, iron-sulfur cluster, and molybdenumbased reductases) distributed throughout different cellular compartments and acting in concert to elicit NO signaling. These observations hint at conserved roles for the NO 2 ؊ -NO pool in cellular processes such as oxygen-sensing and oxygen-dependent modulation of intermediary metabolism. Nitric oxide (NO)3 is the archetypal effector of redox-regulated signal transduction throughout phylogeny, from microorganisms to plants and animals (1). The conserved influences of NO extend from the regulation of basic cellular processes such as intermediary metabolism (2), cellular proliferation (3), and apoptosis (4) to systemic processes such as hypoxic vasoregulation (5). Mammalian NO production has been attributed to the enzymatic activity of NO synthases, nitrate (NO 3 Ϫ )/nitrite (NO 2 Ϫ ) reductases and non-enzymatic NO 2 Ϫ reduction (6). The NO produced is believed to act directly as a signaling molecule by binding to the heme of soluble guanylyl cyclase or nitrosating peptide/protein cysteine residues (7). More recently, it has become apparent that NO 2 Ϫ , previously considered an inert byproduct of NO metabolism present in plasma (50 -500 nM) and tissues (0.5-25 M), is, under some conditions, also a source of NO/nitrosothiol signaling (6,8). Although the importance of NO 2 Ϫ has received increasing appreciation (9) as being central to processes including exercise (10), hypoxic vasodilation (11), myocardial preconditioning (12, 13), and angiogenesis (14), controversy surrounds the chemistry, kinetics, and tissue specificity of NO 2 Ϫ bioactivity (15, 16). Perhaps the greatest uncertainty pertains to the role of heme moieties in NO 2...

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confidence: 99%

Tissue Processing of Nitrite in Hypoxia

Feelisch

Fernandez

Bryan

et al. 2008

Journal of Biological Chemistry

194

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“…1 mM NO-saturated solution was added to the cell suspension immediately before sampling into the EPR microtube, and respiration was measured. Although in cells the NO generation is in the order of nanomolar (maximum about 50 nM), the addition of a higher concentration was necessary because NO reacts rapidly with O 2 (Edmunds et al 2003). Knowing that the half life of NO is very short, we measured the change in NO concentration in the cell suspension with time using the Clark electrode, and the results are summarized in Fig.…”

Section: Resultsmentioning

confidence: 99%

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

Presley

Vedam

et al. 2009

Cell Stress and Chaperones

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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.

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“…The hypoxia-induced, adenosine-mediated, arteriolar dilatation is also NO dependent (Skinner & Marshall, 1996;Edmunds & Marshall, 2001, 2003. Our most recent studies indicate that this NO dependency arises not only because adenosine acts via A 1 receptors on the endothelium to stimulate NO synthesis (Ray et al 2002), but because NO is required to allow the adenosine to be released from the endothelial cells in systemic hypoxia (Edmunds et al 2003).…”

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confidence: 98%

“…Our most recent studies indicate that this NO dependency arises not only because adenosine acts via A 1 receptors on the endothelium to stimulate NO synthesis (Ray et al 2002), but because NO is required to allow the adenosine to be released from the endothelial cells in systemic hypoxia (Edmunds et al 2003). NO competes with O 2 for the same binding site on mitochondrial cytochrome oxidase (complex IV) of the electron transport chain, and raises the apparent K m of complex IV for O 2 , so reducing O 2 consumption (Brown & Cooper, 1994;Clementi et al 1999).…”

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confidence: 99%

The Role of Free Radicals in the Muscle Vasodilatation of Systemic Hypoxia in the Rat

Pyner

Coney²,

Marshall³

2003

Experimental Physiology

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Muscle vasodilatation evoked by systemic hypoxia is adenosine mediated and nitric oxide (NO) dependent: recent evidence suggests the increased binding of NO at complex IV of endothelial mitochondria when O2 level falls leads to adenosine release. In this study on anaesthetised rats, the increase in femoral vascular conductance (FVC) evoked by systemic hypoxia (breathing 8% O2 for 5 min) was reduced by oxypurinol which inhibits xanthine oxidase (XO): XO generates O2− from hypoxanthine, a metabolite of adenosine. By contrast, infusion of superoxide dismutase (SOD), which dismutes O2− to hydrogen peroxide (H2O2), potentiated the hypoxia‐evoked increase in FVC. However, NO synthesis inhibition reduced the hypoxia‐evoked increase in FVC and it was not further altered by SOD. In other studies, the spinotrapezius muscle was pre‐loaded with hydroethidine (HE), or dihydrorhodamine (DHR) which fluoresce in the presence of O2− and H2O2, respectively. In muscle loaded with HE, systemic hypoxia increased fluorescence in endothelial cells of arterioles, whereas in muscle loaded with DHR, fluorescence was diffusely located in and around arteriolar endothelium. We propose that in systemic hypoxia, O2− generated by the XO degradation pathway from adenosine released by endothelial cells, and released by endothelial mitochondria by increased binding of NO to complex IV, is dismuted to H2O2, which facilitates hypoxia‐induced dilatation.

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Does nitric oxide allow endothelial cells to sense hypoxia and mediate hypoxic vasodilatation?in vivo and in vitro studies

Cited by 41 publications

References 35 publications

Tissue Processing of Nitrite in Hypoxia

Tissue Processing of Nitrite in Hypoxia

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

The Role of Free Radicals in the Muscle Vasodilatation of Systemic Hypoxia in the Rat

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