Nitric Oxide (NO) is a gas that has multiple signalling and effector functions in mammalian tissues. Hemoglobin scavanges NO and this limits NO's biological activity; conversely, it has recently been proposed that reversible NO binding to the beta- globin cysteine 93 is allosterically linked to oxygenation and this contributes to the control of oxygen delivery in tissues. We have investigated NO transport and metabolism by hemoglobin in human subjects in vitro and in vivo. We have developed new, highly sensitive chemical and spectrophotometric assays for S- nitrosohemoglobin (SNO-Hb) and nitrosyl (heme) hemoglobin (Hb-Fe II-NO). We find that inhalation of NO at 80 ppm by normal subjects leads to formation of significant amounts of nitrosylated hemoglobin with a large arteriovenous gradient, largely due to formation of methemoglobin >> Hb-Fe II-NO SNO- Hb. The primary cause of the A-V difference is the Hb-FE II- NO. In addition, we find an A-V gradient in red cell nitrate and nitrate levels. These results confirm the existence of the SNO-Hb pathway but suggest that reversible binding to ferrous heme is a more important mechanism. Similar results have been obtained with sickle cell patients; in vitro red cell studies with NO show the dominance of methemoglobin formation. We are also using a variety of physiological measures of blood flow- including MRI and reflectance spectroscopy - to examine the effects of NO inhalation on regional blood flow. We have also begun measurements in transgenic/knockout mice manifesting a sickle cell disease-like phenotype. In parallel experiments we have established quantitative mRNA (real-time), protein (Western) and chemiluminescence assays for nitric oxide synthase (NOS) enzymes and their products to identify the tissues in which NO production is occurring in these experimental subjects. We have recently shown that inhaled NO will change blood flow in the arm after NOS synthase inhibition. We expect that these studies will clarify the complex interaction of NO and hemoglobin and establish how hemoglobin participates in the delivery and metabolism of NO. They should also clarify the therapeutic potential of NO in diseases such as sickle cell disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Intramural Research (Z01)
Project #
1Z01DK025093-03
Application #
6532100
Study Section
(LCB)
Project Start
Project End
Budget Start
Budget End
Support Year
3
Fiscal Year
2001
Total Cost
Indirect Cost
Name
U.S. National Inst Diabetes/Digst/Kidney
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Deans, Katherine J; Minneci, Peter C; Suffredini, Anthony F et al. (2007) Randomization in clinical trials of titrated therapies: unintended consequences of using fixed treatment protocols. Crit Care Med 35:1509-16
Power, Gordon G; Bragg, Shannon L; Oshiro, Bryan T et al. (2007) A novel method of measuring reduction of nitrite-induced methemoglobin applied to fetal and adult blood of humans and sheep. J Appl Physiol 103:1359-65
Crawford, Jack H; Isbell, T Scott; Huang, Zhi et al. (2006) Hypoxia, red blood cells, and nitrite regulate NO-dependent hypoxic vasodilation. Blood 107:566-74
Holly, M K; Dear, J W; Hu, X et al. (2006) Biomarker and drug-target discovery using proteomics in a new rat model of sepsis-induced acute renal failure. Kidney Int 70:496-506
Pelletier, Mildred M; Kleinbongard, Petra; Ringwood, Lorna et al. (2006) The measurement of blood and plasma nitrite by chemiluminescence: pitfalls and solutions. Free Radic Biol Med 41:541-8
Kim-Shapiro, Daniel B; Schechter, Alan N; Gladwin, Mark T (2006) Unraveling the reactions of nitric oxide, nitrite, and hemoglobin in physiology and therapeutics. Arterioscler Thromb Vasc Biol 26:697-705
Kleinbongard, Petra; Dejam, Andre; Lauer, Thomas et al. (2006) Plasma nitrite concentrations reflect the degree of endothelial dysfunction in humans. Free Radic Biol Med 40:295-302
Dejam, Andre; Hunter, Christian J; Pelletier, Mildred M et al. (2005) Erythrocytes are the major intravascular storage sites of nitrite in human blood. Blood 106:734-9
Huang, Zhi; Shiva, Sruti; Kim-Shapiro, Daniel B et al. (2005) Enzymatic function of hemoglobin as a nitrite reductase that produces NO under allosteric control. J Clin Invest 115:2099-107
Piknova, Barbora; Gladwin, Mark T; Schechter, Alan N et al. (2005) Electron paramagnetic resonance analysis of nitrosylhemoglobin in humans during NO inhalation. J Biol Chem 280:40583-8

Showing the most recent 10 out of 33 publications