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Diffusion of peroxynitrite across erythrocyte membranes
A Denicola1, J M Souza, R Radi
1Department of Physical Biochemistry, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay.
Summary
Peroxynitrite anion (ONOO-) crosses red blood cell membranes via anion channels and passive diffusion. This reactive species contributes to oxidative damage, and understanding its transport is key to studying cellular injury.
Area of Science:
- Biochemistry
- Cell Biology
- Physiology
Background:
- Peroxynitrite anion (ONOO-) is a biologically relevant reactive species implicated in oxidative tissue damage.
- Mechanisms of peroxynitrite diffusion across biological membranes are not well understood.
- Erythrocytes are a key model for studying membrane transport due to their well-defined structure.
Purpose of the Study:
- To investigate the mechanisms of peroxynitrite diffusion across erythrocyte membranes.
- To determine the role of anion channels and passive diffusion in peroxynitrite transport.
- To understand the implications of peroxynitrite transport for intracellular oxidative damage.
Main Methods:
- Studied peroxynitrite reaction kinetics with oxyhemoglobin in solution and within erythrocytes.
- Measured oxyhemoglobin oxidation yields in intact erythrocytes, erythrocyte lysates, and DIDS-treated erythrocytes.
- Investigated the effect of pH on peroxynitrite-induced hemoglobin oxidation and nitration.
- Utilized 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) as an inhibitor of anion exchange.
Main Results:
- Peroxynitrite rapidly oxidizes oxyhemoglobin to methemoglobin in solution and within erythrocytes.
- Intracellular oxidation yields were approximately 40% of solution yields due to cytosolic component competition.
- Membrane diffusion was not a significant barrier, with similar oxidation in intact cells and lysates.
- DIDS inhibited oxyhemoglobin oxidation by up to 50%, indicating anion channel involvement, particularly at alkaline pH.
- Peroxynitrite also caused hemoglobin nitration, enhanced in thiol-depleted erythrocytes.
- No DIDS inhibition was observed at pH 5.5, where peroxynitrite exists primarily as the protonated form (ONOOH).
Conclusions:
- Peroxynitrite anion (ONOO-) crosses the erythrocyte membrane via two distinct pathways.
- The anionic form of peroxynitrite utilizes DIDS-inhibitable anion channels for transport.
- The protonated form (ONOOH) diffuses passively across the erythrocyte membrane.
- These findings elucidate peroxynitrite transport mechanisms critical for understanding its role in cellular oxidative stress and damage.