12301 A ROLE FOR NITRIC OXIDE IN THE NITROSYLATION OF SICKLE CELL HEMOGLOBIN BY HYDROXYUREA DanielB Km Shapiro, Howard Shields,ieremyRupon, L le tt555EXivrrilty
The objective of this work was to understand the mechanism of nitric oxide (NO) transfer from hydroxyurea to sickle cell hemoglobin. Hydroxyurea represents a new treatment for sickle cell anemia. The beneficial effects of hydroxyurea treatment appear to result from an increase in the production of fetal hemoglobin, which reduces the tendency of sickle cell hemoglobin to polymerize. However, some patients benefit from hydroxyurea treatment before their levels of fetal hemoglobin increase, suggesting other mechanisms of action for hydroxyurea. Recent reports also indicate that hydroxyurea may act as a source of nitric oxide and suggest that some of the biological effects attributed to hydroxyurea may be mediated by NO. Using electron paramagnetic resonance (EPR) and absorption spectroscopy, the formation of nitrosyl hemoglobin from the reaction of sickle cell oxy, deoxy, or methemoglobin and hydroxyurea has been demonstrated in vitro. Experiments using synthetic 15N-hydroxyurea indicate the specific transfer of NO from hydroxyurea to the iron atom of these proteins. Addition of cyanide to these reactions produces cyano-methemoglobin and blocks the formation of nitrosyl hemoglobin suggesting that this species arises from a reaction between hydroxyurea and methemoglobin. Results from nitrite/nitrate and gas chromatographic headspace analysis provide further insight into a proposed reaction mechanism, which includes NO or nitroxyl, the one-electron reduced form of NO. These results indicate that the oxidation of hydroxyurea by various heme proteins should be considered as a pathway for in vivo NO or nitroxyl production and that NO or nitroxyl donors may be considered as potential treatments for sickle cell disease.
Recently, Xia and Zweier (PNAS 1997, 94: 12705) using electron pammagnetic resonance spectroscopy (EPR) have questioned the possibility that NO’ or a related molecule is the principal reaction product of NOS. Their conclusions are based on the ability of iron N-methyl-D-glucamine dithiocarbamate (Fe-MGD) to distinguish between NG and NO (while NOS and NO donor have produced an EPR signal, No’ donor Angeli’s salt (AS) has not). However, in our experiments characteristic EPR signal of MGDs-Fe-NO complex has been detected after incubation of Fe-MGD with AS in aerobic conditions (the yield 99 f 3 % at lh). Conversion of AS to NO is not necessary for the generation of the MGDs-Fe-NO complex, based on experiment with superoxide dismutase. Furthermore, Fe-MGD forms NO complex upon reaction with NO donor compound S-nitroso-N-acetyl-penicillamine (SNAP). Fe-MGD does not react with nitrite (thus excluding AS-derived nitrite as the source of NO complex). We conclude that in aerobic conditions dithiocarbamate-iron traps react nonselectively with NO and NO’. Consequently, they are not suited to distinguish between these candidate products of NOS.
IS HOONO’ INVOLVED IN THE REACTION PEROXYNITRITE WITH METHIONINE? sand
NO Or Not NO 9 Andrei M. Komarov’, David A. Winks, Martin Fe&cd, Harald H.H.W. Schmidt4 The George Washington Univ. Med. Ctr., Washington, DC, USA’; Natl. Cancer Inst., Bethesda, MD, USA’; Univ. College London, London, UK3; JuliusMaximilians-Univ., Wtirzburg, Germany4.
PBN INHIBITS BRAIN NITRIC OXIDE FORMATION IN A RAT BACTERIAL MENINGITIS MODEL
Institute of Inorganic Chemistry, ETH, Switzerland 8092 Zurich Based on the nonlinearity of the dependence of k& on [Met] [l] it has been proposed that peroxynitrite forms a reactive intermediate HGGNO’. Kinetic studies were carried out by stopped-flow spectroscopy. We found the reaction to be first-order in peroxynitrite and first-order in methionine (Met) and N-acetylmethionine (N-acMet), at different pH values with Met and N-acMet in at least lo-fold excess. The linear correlation obtained between k& and [Met] and [N-acMet] implies that the activated intermediate HOONO’ is not required to explain our kinetic data. Both peroynitrous acid and peroxynitrite anion react with Met and N-acMet to form sulfoxides with rate constants of: Met:.,,= 1.7M.1x103M~‘s~’and k,,,, = 8.6fl.2 M-Y N-acMet ;k.,,, = [email protected]
M%” and k.,, = 1O.oM.l Me’s” Neither the pH nor the concentration of Met affected the distribution of the nitrite and nitrate or the methionine sulfoxide yields. Approximately two nitrite, two methionine sulfoxides and one nitrate are formed per three peroxynitrite consumed We found that peroxynitrite oxidises Met and N-a&let in a two-electron pathway to the sulfoxide. Any other products reported in [l] are from reactions of excess Met with nitrite after the reaction with peroxynitrite was finished.[l] Pryor et al., Proc. Natl. Acad. Sci USA 1994, 91, 11173-11177.
Y. Kotake,’ H. Endoh, S. Fujii, y. Suzuki, S. Sato, T. Kayama, T. Yoshimura, ‘Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73164, USA. Neurosurgery Department, Yamagata University Medical School, Yamagata, Japan and Bioradical Institute, Yamagata Technopolis Foundation, Yamagata, Japan Phenyl Kter&butylnitrone (PBN) has shown potent protective effect in infant rat bacterial meningitis model (J. Clin. Invest. 98, 2632, 1996). In a similar meningitis model, a high level of nitric oxide (NO) was detected in the brain tissue several hrs after a high ip dose of lipopolysaccharide (LPS). In the present study, using a rat bacterial meningitis model, we show that systemic preadministration of PBN inhibits brain NO formation. Rats were administered ip with PBN (125,250, and 400 mglkg), and 30 min later LPS (SOOugin 21.11) was intra-cysternally injected into an anaesthetized rat placed in a stereotaxicframe. Later, NO in brain was quantified using iron--dithiocarbamate EPR trapping method with Fe-DETC complex as a trapping agent. The results indicate that: 1) the brain EPR signal from NO-complex peaked approximately 8 hours after LPS administration; 2) PBN preadministration (250 mg/kg) inhibited brain NO formation by 85% (6-8 hrs after LPS administration); 3) Lower dose of PBN (125 mglkg) was as effective as higher doses; 4) PBN treatment (250 mg/kg) 3 hrs after LPS administration did not decrease NO formation (supported by NIH GM56878).