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Blood, 1 April 2006, Vol. 107, No. 7, pp. 2595-2596.
Does eNOS stand for erythrocytic NO synthase?NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
Kleinbongard and colleagues provide provocative and compelling evidence that erythrocytes possess a functional endothelial NO synthase isoform that regulates intravascular NO bioavailability and nitrite levels.
Kleinbongard and colleagues have now expanded this field of research with studies suggesting that red blood cells possess an intrinsic NO synthase: (1) Human red blood cells demonstrate eNOS isoform immunofluorescence surrounding the cytoplasm that colocalizes with glycophorin A. This staining is present in eNOS wild-type mice and absent in eNOS knock outs. (2) Immunogold labeling of eNOS with electron microscopic imaging of ultrathin cryosections of the red blood cell reveals labeled eNOS in the cytoplasmic face of the red cell membrane. (c) eNOS was also detected by Western blotting and eNOS message was measured using reverse transcriptase–polymerase chain reaction (RT-PCR). Additional studies suggest that this eNOS is functional: (1) Enzymatic activity was observed using the arginine-to-citrulline conversion assay. (2) Nitric oxide formation was detected by dialysis of red cells with arginine against an oxyhemoglobin solution and measurement of methemoglobin formation. Nitric oxide was also measured by purging red cells with helium and detecting NO gas by chemiluminescence after arginine addition. (3) Nitric oxide metabolites formed (nitrate, nitrite, and RxNO) following incubation of red cells with arginine. Nitrite formation following arginine exposure did not occur with red cells from eNOS knock-out mice. (4) Activation of red blood cell eNOS modulated red cell filterability and whole blood platelet activation.
A central challenge for all of us working in this controversial field is to understand how NO formed by a red blood cell can survive the rapid and irreversible dioxygenation reaction with oxyhemoglobin that should convert all of the NO to nitrate and inactivate it. Indeed, Kleinbongard and colleagues show that the addition of oxyhemoglobin in small quantities outside of the red blood cells inhibits the effects of arginine on erythrocyte deformability. This is a strange result considering the much higher concentration of oxyhemoglobin within the red cell cytoplasm, where the eNOS enzyme is found. There are a number of possible solutions to this paradox. First, the red cell lipid raft may be analogous to the endothelial caveolae and may tether together complexes of anion or gas transport proteins and eNOS. We have proposed that such a metabolon complex of deoxyhemoglobin, AE1/band 3, carbonic anhydrase, aquaporin, and Rh protein channels may facilitate nitrite protonation, reduction, and export of NO or a NO intermediate.7 A second possibility is that the eNOS is coupled to a "NO oxidase." A metal-based NO oxidase enzyme would effectively compete with vicinal oxyhemoglobin and convert the NO into nitrite or an S-nitrosothiol, which are both bioactive NO signaling molecules able to escape heme inactivation. It is clear that the present finding that erythrocytes possess a functional erythrocytic NO synthase presents challenges and opportunities for the expanding field of NO–red blood cell biology. Footnotes
Comment on Kleinbongard et al, page 2943
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| Copyright © 2006 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
-chain cysteine 93 to form S-nitrosated hemoglobin, which could allosterically deliver an S-nitrosothiol during hemoglobin deoxygenation.