Magnetic resonance study of the transmembrane nitrite diffusion

Nitrite (NO2-), being a product of metabolism of both nitric oxide (NO) and nitrate (NO3-), can accumulate in tissues and regenerate NO by several mechanisms. The effect of NO2- on ischemia/reperfusion injury was also reported. Nevertheless, the mechanisms of intracellular NO2- accumulation are poorly understood. We suggested significant role of nitrite penetration through biological membranes in the form of undissociated nitrous acid (HNO2). This hypothesis has been tested using large unilamellar phosphatidylcholine liposomes and several spectroscopic techniques. HNO2 transport across the phospholipid bilayer of liposomes facilitates proton transfer resulting in intraliposomal acidification, which was measured using pH-sensitive probes. NO2--mediated intraliposomal acidification was confirmed by EPR spectroscopy using membrane-impermeable pH-sensitive nitroxide, AMC (2,2,5,5-tetramethyl-1-yloxy-2,5-dihydro-1H-imidazol-3-ium-4-yl)-aminomethanesulfonic acid (pK 5.25), and by 31P 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 NO2- was observed by direct measurement using chemiluminescence technique. Perfusion of isolated rat hearts with buffer containing 4 μM NO2- was performed. The nitrite concentrations in the effluent and in the tissue, measured after 1 min perfusion, were close, supporting fast penetration of the nitrite through the tissue. Measurements of the nitrite/nitrate showed that total concentration of NOx in myocardium increased from initial 7.8 to 24.7 μM after nitrite perfusion. Physiological significance of passive transmembrane transport of NO2- and its coupling with intraliposomal acidification are discussed.