Comparative analysis of nitrite uptake and hemoglobin-nitrite reactions in erythrocytes: sorting out uptake mechanisms and oxygenation dependencies

Abstract

Nitrite uptake into red blood cells (RBCs) precedes its intracellular reactions with hemoglobin (Hb) that forms nitric oxide (NO) during hypoxia. We investigated the uptake of nitrite and its reactions with Hb at different oxygen saturations (So(2)), using RBCs with (carp and rabbit) and without (hagfish and lamprey) anion exchanger-1 (AE1) in the membrane, with the aim to unravel the mechanisms and oxygenation dependencies of nitrite transport. Added nitrite rapidly diffused into the RBCs until equilibrium. The distribution ratio of nitrite across the membrane agreed with that expected from HNO(2) diffusion and AE1-mediated facilitated NO(2)(-) diffusion. Participation of HNO(2) diffusion was emphasized by rapid transmembrane nitrite equilibration also in the natural AE1 knockouts. Following the equilibration, nitrite was consumed by reacting with Hb, which created a continued inward diffusion controlled by intracellular reaction rates. Changes in nitrite uptake with So(2), pH, or species were accordingly explained by corresponding changes in reaction rates. In carp, nitrite uptake rates increased linearly with decreasing So(2) over the entire So(2) range. In rabbit, nitrite uptake rates were highest at intermediate So(2), producing a bell-shaped relationship with So(2). Nitrite consumption increased approximately 10-fold with a 1 unit decrease in pH, as expected from the involvement of protons in the reactions with Hb. The reaction of nitrite with deoxyhemoglobin was favored over that with oxyhemoglobin at intermediate So(2). We propose a model for RBC nitrite uptake that involves both HNO(2) diffusion and AE1-mediated transport and that explains both the present and previous (sometimes puzzling) results.

Nitric oxide metabolites in goldfish under normoxic and hypoxic conditions

“…(pHRBC-pHex) (Jensen and Rohde, 2010). The lower pH inside RBCs than outside therefore predicts a ratio lower than 1, implying [ the level of SNO compounds measured with reductive chemiluminescense is ~0.05moll -1 MacAthur et al, 2007), which corresponds with the SNO concentrations in normoxic goldfish RBCs (Fig.6B).…”

“…However, the literature on the ratio between nitrite concentrations in RBCs and plasma is conflicting. Some investigations report higher RBC nitrite levels than plasma nitrite levels (Bryan et al, 2004;Dejam et al, 2005) (Bryan et al, 2005;Feelisch et al, 2008;Jensen and Rohde, 2010) as observed in normoxic goldfish. Nitrite appears to be rapidly transported across the RBC membrane via both nitrous acid (HNO 2 ) diffusion and anion exchanger 1-mediated facilitated NO 2 -diffusion, which predicts that the equilibrium distribution ratio between intracellular (RBC) and extracellular (ex) nitrite is related to the proton distribution according to:…”

Circulating nitric oxide metabolites and cardiovascular changes in the turtleTrachemys scriptaduring normoxia, anoxia and reoxygenation

“…In the turtle, it is possible that nitrite for erythrocyte NO generation might have originated from some of the tissues that were perfused by blood during anoxia. It is known that nitrite permeates cellular membranes and is transported into red cells and tissues upon an elevation of extracellular levels (Bryan et al, 2005;Feelisch et al, 2008;Jensen and Rohde, 2010) or exposure to reduced O 2 levels (Hansen and Jensen, 2010;Sandvik et al, 2012). Measuring nitrite in tissues other than blood in future studies will help to establish the role of nitrite as a source of NO during anoxia in turtles.…”

Features of nitrogen metabolism in fishes

“…Possibly present in the fish-specific heme-containing proteins that perform the function of maintaining the balance of the activity of argininedependent nitrite (nitrate)-reductase contours nitrogen oxide cycle, determine the level of resistance of fish species to oxygen deficiency and the effectiveness of organ protection mechanisms against reperfusion injury (Cossins et al 2009). In turn, the species-specific resistance of fish to high concentrations of exogenous nitrite (Jensen 2007;Jensen and Rohde 2010) may depend on the intensity of the transport of mineral substances in the cells of the gill apparatus, the rate of accumulation of nitrite in the intracellular sector and the efficiency of hemoglobindependent resynthesis NO. In our opinion, should also be noted that the sharp surge in activity intraorganic arginine-dependent NO synthesis can cause growth intraorganic and systemic concentration of endogenous nitrite in fish.…”

“…Experimentally confirmed the possibility of using fish of exogenous nitrite as substrates in reactions resynthesis NO. However, attention is drawn to the following pattern-the authors of most-cited publications recognize that normally the content in the extracellular fluid of fish physiologically active metabolites of NO-endogenous nitrite is constant (Jensen 2007;Tripathi and Krishna 2008;Cossins et al 2009;Jensen and Rohde 2010). Taking note of the reasoned reviewed publications in the importance of the physiological role of nitrite to fish, the existence of specific mechanisms that determine their kinetics in the body, and narrow ranges of concentration in the extracellular fluid, it can be assumed that normally the level of nitrite is a physiological constant.…”