When Does Low Oxygen Become Hypoxia? Implications for Nitrite Reduction
Authors: Andrew G. Pinder, Philip E. James
Journal: Circulation Research (2009)

HomeCirculation ResearchVol. 104, No. 3When Does Low Oxygen Become Hypoxia? Implications for Nitrite Reduction Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessLetterPDF/EPUBWhen Does Low Oxygen Become Hypoxia? Implications for Nitrite Reduction Andrew G. Pinder and Philip E. James Andrew G. PinderAndrew G. Pinder Wales Heart Research Institute, Cardiff University School of Medicine, Heath Park, Cardiff, United Kingdom, E-mail and Philip E. JamesPhilip E. James Wales Heart Research Institute, Cardiff University School of Medicine, Heath Park, Cardiff, United Kingdom, E-mail Originally published13 Feb 2009https://doi.org/10.1161/CIRCRESAHA.108.191569Circulation Research. 2009;104:e25–e26To the Editor:Webb and Milsom et al1 present the intriguing finding that endothelial nitric oxide synthase (eNOS) in human umbilical vein endothelial cells (HUVECs) and potentially RBCs is responsible under hypoxic conditions for nitrite (NO2•) reduction to nitric oxide (NO). Based on several practical and theoretical points of view, the conclusions that the authors draw regarding their data are flawed. It is important that these are considered by the wider Circulation Research readership.The authors conclude that lack of oxygen (O2) availability to eNOS promotes NO2• reduction to NO. Whereas the RBC experiments involve measurement of NO produced by RBC under nitrogen and acidic conditions, the HUVEC experiments were carried out at 5% O2 and 5% CO2 (refer to online data supplement that accompanies the article for details). Although there is no question 5% O2 is lower than 95% or 21% O2 (typical in vitro experimental/growth conditions), it cannot be accepted that 5% O2 (equating to ≈50 μmol/L O2) is "hypoxic" to cells in culture over a 30-minute incubation period. Mitochondrial cytochrome c will function optimally until extremely low O2 conditions (Km≈0.1 μmol/L),2 and perhaps more importantly eNOS functions optimally with a Km≈6 to 9 μmol/L.3–4 Limited O2 availability to eNOS is unlikely the cause of the change that the authors have observed under these experimental conditions. The thrust of the article (ie, the reduction of NO2• to NO during hypoxia by eNOS) is not valid for HUVECs.A second issue relates to the fact that NO was not measured in the HUVEC studies but, rather, NO function inferred from cGMP assay. The authors do not show the relevant normoxic cGMP responses to NO2• as a true control. In addition, these authors have previously published data confirming that vascular relaxation responses (soluble guanylate cyclase [sGC]-dependent) are inversely related to O2.5 In broad terms, blood vessels subjected to the same endothelial dependent stimulus or exogenous NO respond with greater relaxation under low O2 conditions.6 It follows, therefore, that HUVEC cells could produce the same amount of NO from eNOS (whether from arginine or reduction of NO2•) under both normoxic and "hypoxic" conditions but the sGC response (and subsequent cGMP detected) would be enhanced in hypoxia only. Moreover, the use of l-NG-monomethyl arginine (LNMMA) would equally inhibit eNOS in both cases (as shown by the authors), thus rendering their interpretation incorrect.Finally, it was recognized some time ago in HUVECs and other primary cultured endothelial cells that within a range of low to moderate O2 conditions (5% O2 as used here), a condition arises in which NADPH oxidase(s) production of superoxide is limited (Km≈30 to 40 μmol/L),7 whereas eNOS production of NO is normal.3–4 Under these conditions, and within a relatively narrow range of O2, there is effectively more NO available without a change in NO production.8 Therefore, in the present studies carried out at 5% O2, it follows that the definitive amounts of NO produced may have been exactly the same, whereas the levels of bioavailable NO were effectively increased. Again, eNOS blockade with LNMMA is an inappropriate tool to investigate this phenomenon.The points raised above do not detract from the interesting suggestion by the authors that free NO can be produced by RBCs in normoxia and hypoxia. However, care must be taken when interpreting data from experimental conditions that do not specifically address the question, as in the case of hypoxia affecting eNOS reduction of NO2•. What is more, the points above were not discussed or referenced by the authors. Confirming that eNOS in vascular endothelium is unlikely to reduce NO2• to NO in hypoxia, the authors show tissue experiments demonstrating that LNMMA had no effect on NO generation. In complete agreement with this, in our hands (and others9), eNOS blockade has no effect on nitrite-induced vascular relaxation in the presence or absence of endothelium under true hypoxic conditions (<1% O2).Sources of FundingSupported by British Heart Foundation Programme Grant RG/04/005/14168, Project Grant PG/03/120/16052, and PhD Studentship FS/05/110.DisclosuresNone.1 Webb AJ, Milsom AB, Rathod KS, Chu WL, Qureshi S, Lovell MJ, Lecomte FM, Perrett D, Raimondo C, Khoshbin E, Ahmed Z, Uppal R, Benjamin N, Hobbs AJ, Ahluwalia A. Mechanisms underlying erythrocyte and endothelial nitrite reduction to nitric oxide in hypoxia: role for xanthine oxidoreductase and endothelial nitric oxide synthase. Circ Res. 2008; 103: 957–964.LinkGoogle Scholar2 Wilson DF, Rumsey WL, Green TJ, Vanderkooi JM. The oxygen dependence of mitochondrial oxidative phosphorylation measured by a new optical method for measuring oxygen concentration. J Biol Chem. 1988; 263: 2712–2718.CrossrefMedlineGoogle Scholar3 Whorton AR, Simonds DB, Piantadosi CA. Regulation of nitric oxide synthesis by oxygen in vascular endothelial cells. Am J Physiol. 1997; 272 (6 pt 1): L1161–L1166.MedlineGoogle Scholar4 Rengasamy A, Johns RA. Determination of Km for oxygen of nitric oxide synthase isoforms. J Pharmacol Exp Ther. 1996; 276: 30–33.MedlineGoogle Scholar5 Maher AR, Milsom AB, Gunaruwan P, Abozguia K, Ahmed I, Weaver R, Ashrafian H, Born G, James PE, Frenneaux MP. The role of hypoxia in modifying the vascular response to exogenous nitrite in humans. Circulation. 2008; 117: 670–677.LinkGoogle Scholar6 James PE, Lang D, Tufnell-Barret T, Milsom AB, Frenneaux MP. Vasorelaxation by red blood cells and impairment in diabetes: reduced nitric oxide and oxygen delivery by glycated hemoglobin. Circ Res. 2004; 94: 976–983.LinkGoogle Scholar7 Rossi F. The O2• -forming NADPH oxidase of the phagocytes: nature, mechanisms of activation and function. Biochim Biophys Acta. 1986; 853: 65–89.CrossrefMedlineGoogle Scholar8 James PE, Parton J, Jackson SK, Frenneaux MP. Oxygen indirectly regulates nitric oxide availability. Adv Exp Med Biol. 2003; 510: 139–143.CrossrefMedlineGoogle Scholar9 Dalsgaard T, Simonsen U, Fago A. Nitrite-dependent vasodilation is facilitated by hypoxia and is independent of known NO-generating nitrite reductase activities. Am J Physiol Heart Circ Physiol. 2007; 292: H3072–H3078.CrossrefMedlineGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetailsCited By Keeley T and Mann G (2019) Defining Physiological Normoxia for Improved Translation of Cell Physiology to Animal Models and Humans, Physiological Reviews, 10.1152/physrev.00041.2017, 99:1, (161-234), Online publication date: 1-Jan-2019. Dada J, Pinder A, Lang D, James P and Catapano A (2013) Oxygen Mediates Vascular Smooth Muscle Relaxation in Hypoxia, PLoS ONE, 10.1371/journal.pone.0057162, 8:2, (e57162) February 13, 2009Vol 104, Issue 3 Advertisement Article InformationMetrics https://doi.org/10.1161/CIRCRESAHA.108.191569PMID: 19213959 Originally publishedFebruary 13, 2009 PDF download Advertisement