Tissue Processing of Nitrite in Hypoxia

Abstract

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...

Mechanistic studies of nitrite reactions with metalloproteins and models relevant to mammalian physiology

“…Nitrite has also been shown to induce vasodilation [11] and to reduce damage to organs during ischemia/reperfusion (I/R) injury [12,13]. Although the mechanisms of these biological functions have not been conclusively established, the role of nitrite may be to generate NO under hypoxic conditions [14]. Nitrite is also an intermediate in the denitrification processes by bacteria and fungi, whereby nitrate is converted to N 2 [15].…”

Different Disappearance Rates of Plasma Nitrite (NO2-) Contribute to Apparent Steady-State Arterio-Venous Differences in Anesthetized Animals

“…19) In addition, a great contribution of tissue as a major component of peripheral compartment is suggested in this study (Fig. 6).…”

“…19) The capacity-and time-dependent kinetics of the NO 2 Ϫ pool in RBCs may not be identical in arterial and venous blood.…”

“…A possible difference in the uptake rate of RBCs could not be large enough to explain such a large A-V difference, and contributions of tissue factors would be a great concern. 18,19) The emerging role of tissue as a reservoir of NO bioactivity via NO 2 Ϫ should be taken into consideration; some reports indicate that plasma NO 2 Ϫ levels may not accurately reflect increases in NO or NO 2 Ϫ levels in biological systems. 3,20,21) However, no alternative measures exist for assessing NO bioactivity in easily accessible biomaterials (such as blood).…”

Nitrite Therapy Ameliorates Myocardial Dysfunction via H 2 S and Nuclear Factor‐Erythroid 2‐Related Factor 2 (Nrf2)‐Dependent Signaling in Chronic Heart Failure

“…Among NO donors, sodium nitrite (NaNO 2 ) represents a promising therapeutic agent for the treatment of HF,17 being an attractive candidate for restoring physiological NO signaling in states of chronic NO insufficiency such as myocardial ischemia. NaNO 2 is rapidly absorbed from the circulation by peripheral tissues and stored in cells until conversion to NO is needed 18, 19, 20. Indeed, NaNO 2 administration has been demonstrated to protect against I/R injury21 in multiple tissues and organs, such as skeletal muscle,22 renal,23, 24 and liver 25, 26, 27.…”