NDLI: Diffusion of peroxynitrite across erythrocyte membranes

Content Provider	PubMed Central
Author	Denicola, Ana Souza, José M. Radi, Rafael
Copyright Year	1998
Abstract	Peroxynitrite anion (ONOO−) is a reactive species of increasingly recognized biological relevance that contributes to oxidative tissue damage. At present, however, there is limited knowledge about the mechanisms of peroxynitrite diffusion through biological compartments. In this work we have studied the diffusion of peroxynitrite across erythrocyte membranes. In solution, peroxynitrite rapidly reacts with oxyhemoglobin to yield methemoglobin, with k 2 = (10.4 ± 0.3) × 103 M−1⋅s−1 at pH 7.4 and 25°C. Addition of peroxynitrite to intact erythrocytes caused oxidation of intracellular oxyhemoglobin to methemoglobin. Oxidation yields in red blood cells at pH 7.0 were approximately 40% of those obtained in solution, which results mostly from competition of other cytosolic components for peroxynitrite. Indeed, rather small differences were observed between oxidation yields in lysates compared with intact erythrocytes, in particular at acidic and neutral pH values, indicating that membrane was not precluding peroxynitrite diffusion. Incubation of erythrocytes at pH 7.0 with 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), a specific inhibitor of anion exchange, resulted in up to 50% inhibition of oxyhemoglobin oxidation by peroxynitrite. More protection by DIDS was achieved at alkaline pH, while no effect was observed at pH 5.5, where 95% of peroxynitrite is in the acidic form, ONOOH (pKa = 6.8). In addition, peroxynitrite caused nitration of intracellular hemoglobin, in a process that was enhanced in thiol-depleted erythrocytes. Our results indicate that peroxynitrite is able to cross the erythrocyte membrane by two different mechanisms: in the anionic form through the DIDS-inhibitable anion channel, and in the protonated form by passive diffusion.
Starting Page	3566
File Format	PDF
ISSN	10916490
e-ISSN	10916490
Journal	Proceedings of the National Academy of Sciences of the United States of America
Issue Number	7
Volume Number	95
Language	English
Publisher	The National Academy of Sciences
Publisher Date	1998-03-31
Access Restriction	Open
Rights Holder	The National Academy of Sciences
Subject Keyword	Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

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