Red Blood Cell Function in Nitric Oxide Biotransport

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DESCRIPTION (provided by applicant): Our long-term goal is to quantify nitric oxide (NO) transport in the microcirculation in physiology and pathophysiology. In vascular wall, endothelium-derived nitric oxide reaches smooth muscle where it can activate soluble guanylate cyclase (sGC) and modulate vascular tone. The level of bioavailable NO are maintained by its production and consumption in the microcirculation. Despite intense research on the transport of NO from the production site (endothelium) to the target site (smooth muscle), question remains on whether and how significant amount of endothelium-derived NO reaches smooth muscle in the presence of efficient NO scavenger red blood cells (RBCs) in vascular lumen. The proposed research will provide functional relationship between hemodynamic and biochemical parameters in NO and RBC interaction. The Specific Aims for the proposed research are 1) to determine the role of oxygenation on NO interaction with RBC and 2) to quantify the differences in NO end-products formation from NO interaction with free Hb and RBC Hb. Based on the preliminary data, the hypotheses we will test are that (a) NO interaction with oxygenated RBCs will result in higher nitrite formation rate as compared to nitrite formation rate of NO interaction with deoxygenated RBCs, (b) oxygenated RBCs will have higher total NO concentration as compared to total NO concentration of deoxygenated RBCs, and (c) hemoglobin bound in the RBC membrane (RBC-hemoglobin) will results in higher total NO concentration and thus will have higher NO bioavailability as compared to that of free hemoglobin solution or RBC solution containing small amount of free Hb. For testing these hypotheses, we will use a novel bioreactor that simulates in vivo interactions of NO and RBCs. We will compare interaction products of NO with oxygenated- and deoxygenated- RBCs, presence of free hemoglobin with RBCs, and free hemoglobin. We will measure the reaction products of NO including intermediate products (s-nitrosohemoglobin & nitrosyl-Hb) and end-products (nitrite & nitrate) using chemiluminescence method. We will measure the gaseous and aqueous NO concentration using an NO electrode. We will additionally use UV/Vis spectrophotometric spectrum to quantify these concentration. By designing a new experimental model for studying biochemical interactions of NO and RBCs, we will advance the knowledge of biomedical researchers on these molecular interactions. This may lead to evaluation of therapeutics in areas as diverse as sickle cell anemia, pulmonary hypertension, septic shock, and blood substitutes. PUBLIC HEALTH RELEVANCE: Nitric oxide (NO) plays important roles in numerous physiological functions including regulation of vascular tone, neurotransmission, immune response and inhibition of platelet aggregation. The proposed research will significantly advance our fundamental understanding of NO metabolism in the microcirculation and provide biomedical researchers a new experimental model for studying biochemical interactions of NO and RBCs. The proposed research will also quantify the effect of free Hb on the NO metabolism. This knowledge is critical to the development of hemoglobin based oxygen carrier and the understanding the effects of chronic hemolysis in sickle cell disease and pulmonary hypertension.