Magnetic resonance study of the transmembrane nitrite diffusion

Abstract

Nitrite (NO 2 -), being a product of metabolism of both nitric oxide (NO • ) and nitrate (NO 3 -), can accumulate in tissues and regenerate NO • by several mechanisms. The effect of NO 2 -on ischemia/ reperfusion injury was also reported. Nevertheless, the mechanisms of intracellular NO 2 -accumulation are poorly understood. We suggested significant role of nitrite penetration through biological membranes in the form of undissociated nitrous acid (HNO 2 ). This hypothesis has been tested using large unilamellar phosphatidylcholine liposomes and several spectroscopic techniques. HNO 2 transport across the phospholipid bilayer of liposomes facilitates proton transfer resulting in intraliposomal acidification, which was measured using pH-sensitive probes. NO 2 --mediated intraliposomal acidification was confirmed by EPR spectroscopy using membrane-impermeable pHsensitive nitroxide, 2,2,5,5-tetramethyl-1-oxyl-2,5-dihydro-1H-imidazol-3-ium-4-yl)-aminomethanesulfonic acid (pK 5.25), and by 31 P-NMR spectroscopy using inorganic phosphate (pK 6.9). Nitrite accumulates inside liposomes in concentration exceeding its concentration in the bulk solution, when initial transmembrane pH gradient (alkaline inside) is applied. Intraliposomal accumulation of NO 2 -was observed by direct measurement using chemiluminescence technique. Perfusion of isolated rat hearts with buffer containing 4 μM NO 2 -was performed. The nitrite concentrations in the effluent and in the tissue, measured after 1 minute perfusion were close, supporting fast penetration of the nitrite through the tissue. Measurements of the nitrate/nitrate showed that total concentration of NO x in myocardium increased from initial 7.8 μM to 24.7 μM after nitrite perfusion. Physiological significance of passive transmembrane transport of NO 2 -and its coupling with intraliposomal acidification are discussed.

Nitric oxide metabolites in goldfish under normoxic and hypoxic conditions

“…We find higher nitrite concentrations in brain, heart and gill (Fig.8) than in liver, kidney and white skeletal muscle (Fig.7), and both the absolute tissue nitrite concentrations and the tissue differences compare well with findings in rats (Bryan et al, 2005). If HNO 2 diffusion is a main mechanism for nitrite transport across tissue cell membranes, then pH will influence cellular nitrite concentrations (Samouilov et al, 2007;Jensen and Rohde, 2010). A higher intracellular pH in goldfish brain and heart tissues than in muscle, as reported in channel catfish (Cameron and Kormanik, 1982), would consequently contribute to a higher nitrite concentration in the former tissues.…”

Regulation of nitrite transport in red blood cells by hemoglobin oxygen fractional saturation

“…Data were analyzed by a two-tailed Student's t-test. *P Ͻ 0.05. suggest that despite being predominantly (Ͼ99.9%) in the anionic form at pH 7.4, the addition of sodium nitrite to the extracellular compartment results in the acidification of the liposomal interior, consistent with the generation of nitrous acid (HNO 2 ) that can freely diffuse across the bilayer (54).…”

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

“…Nitrous acid has a pK a ϳ 3.3 and will only be present at very low concentrations at physiological pH (mass law considerations reveal a HNO 2 concentration of 95 nM at pH 7.8 and 239 nM at pH 7.4 if NO 2 Ϫ is 3 mM). This does, however, not preclude its relative rapid diffusion across the membrane, as documented with phospholipid bilayer of liposomes (40). HNO 2 diffusing into the RBCs will dissociate to H ϩ and NO 2 Ϫ inside the RBCs, and the diffusion will continue until [HNO 2 ] is the same inside and outside the cells (40 Ϫ ] RBC refers to packed cell volume, and values become higher if correctly referred to cell water.…”