Magnetic resonance study of the transmembrane nitrite diffusion

Nitrite (NO₂ ^-), being a product of metabolism of both nitric oxide (NO^{{radical dot}}) and nitrate (NO₃ ^-), can accumulate in tissues and regenerate NO^{{radical dot}} 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)-aminome thanesulfonic 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 NO_x 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.