title: 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 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 (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 microM NO(2)- 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 microM after nitrite perfusion. Physiological significance of passive transmembrane transport of NO(2)- and its coupling with intraliposomal acidification are discussed.
authors.affiliation: Center for Biomedical EPR Spectroscopy and Imaging, The Davis Heart and Lung Research Institute, The Department of Internal Medicine, The Ohio State University College of Medicine, Columbus, OH 43210, USA. Alex.Samouilov@osumc.edu
authors.forename: A
authors.initials: A
authors.lastname: Samouilov
authors.forename: Ya Yu
authors.initials: YY
authors.lastname: Woldman
authors.forename: J L
authors.initials: JL
authors.lastname: Zweier
authors.forename: V V
authors.initials: VV
authors.lastname: Khramtsov
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D004058:Diffusion; D004578:Electron Spin Resonance Spectroscopy; D005260:Female; D006863:Hydrogen-Ion Concentration; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D015394:Molecular Structure; D009206:Myocardium / Q000378:metabolism; D009573:Nitrites / Q000737:chemistry / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D008081:Liposomes; D009573:Nitrites; D010743:Phospholipids