title: Generation of valency hybrids and nitrosylated species of hemoglobin in mice by nitric oxide vasodilators.
abstract: The hemoglobin fraction of blood samples from C57BL/6 male mice was purified by column chromatography and subjected to isolectric focusing (IEF) across a pH gradient. Densitometric scanning of the IEF gel showed the presence of a single peak corresponding to the fully reduced tetramer, H, or (alpha 2+ beta 2+)2. Twenty minutes after an ip injection of 1.1 mmol/kg NaNO2 blood samples treated the same way showed four peaks corresponding to the species: H = 43%, X or (alpha 2+ beta 3+)2 = 10%, Y or (alpha 3+ beta 2+)2 = 33%, and M or (alpha 3+ beta 3+)2 = 14%. In contrast blood samples from control CD-1 male mice showed the presence of three IEF distinct peaks which were all believed to be H valency forms, and six distinct peaks were seen after treatment in vivo with NaNO2. Thus, the C57BL/6 mice yield patterns similar to those observed after in vitro treatment of human red cells with NaNO2 (H. Kruszyna, R. Kruszyna, R. P. Smith, and D. E. Wilcox, 1987b, Toxicol. Appl. Pharmacol. 91, 429-438), and the CD-1 mice are a much less satisfactory model. The appearance and disappearance of the species X, Y, and M over time after ip injection of 1.1 mmol/kg NaNO2 or hydroxylamine HCl were followed in C57BL/6 mice by the technique of IEF. In each case the patterns were consistent with previously established patterns for the respective methemoglobinemias as determined by absorption spectrophotometry, and they were consistent with the suggestion that two pathways exist for the oxidation of H and for the reduction of M which proceed through X and Y, respectively. By using electron paramagnetic resonance (EPR) spectroscopy, we were also able to follow with time the concentration of nitrosylated heme (NO-heme) on reduced subunits in both mouse strains. The peak for the NO-heme coincided in time with the peak methemoglobinemia as determined by either IEF or absorption spectrophotometry. EPR was also used to determine NO-heme in CD-1 mice after injection of a series of NO-vasodilators with and without methylene blue (MB). Low, but clearly detectable amounts of NO-heme were found in the blood of animals given all xenobiotics tested including NaNO2, hydroxylamine HCl, glyceryl trinitrate, hydralazine, sodium nitroprusside, and sodium azide. MB has little effect on the response, and no NO-heme could be detected in control mice.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03756.
authors.forename: R
authors.initials: R
authors.lastname: Kruszyna
authors.forename: H
authors.initials: H
authors.lastname: Kruszyna
authors.forename: R P
authors.initials: RP
authors.lastname: Smith
authors.forename: D E
authors.initials: DE
authors.lastname: Wilcox
mesh_terms: D000818:Animals; D004578:Electron Spin Resonance Spectroscopy; D006454:Hemoglobins / Q000378:metabolism*; D006801:Humans; D008297:Male; D008706:Methemoglobin / Q000378:metabolism; D008751:Methylene Blue / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D009569:Nitric Oxide / Q000378:metabolism*; D012977:Sodium Nitrite / Q000378:metabolism; D014665:Vasodilator Agents / Q000378:metabolism*
chemical_list: D006454:Hemoglobins; D014665:Vasodilator Agents; D009569:Nitric Oxide; D008706:Methemoglobin; D012977:Sodium Nitrite; D008751:Methylene Blue