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1、RED CELLSRedbloodcellsexpressafunctionalendothelialnitricoxidesynthasePetra Kleinbongard, Rainer Schulz, Tienush Rassaf, Thomas Lauer, Andre Dejam, Thomas Jax, Intan Kumara, Putrika Gharini, Svetlana Kabanova, Burcin Ozu yaman, Hans-Georg Schnu rch, Axel Go decke, Artur-A. Weber, Mirko Robenek, Hors
2、t Robenek, Wilhelm Bloch, Peter Ro sen, and Malte KelmThe synthesis of nitric oxide (NO) in the circulation has been attributed exclu- sively to the vascular endothelium. Red blood cells (RBCs) have been demon- strated to carry a nonfunctional NO syn- thase (NOS) and, due to their huge hemo- globin
3、content, have been assumed to metabolize large quantities of NO. More recently, however, RBCs have been iden-tified to reversibly bind, transport, and release NO within the cardiovascular sys- tem. We now provide evidence that RBCsfrom humans express an active and func- tionalendothelial-typeNOS(eNO
4、S),which is localized in the plasma membrane and the cytoplasm of RBCs. This NOS is regu- lated by its substrateL-arginine, by cal- cium, and by phosphorylation via PI3 kinase. RBC-NOS activity regulates de- formability of RBC membrane and inhib- itsactivationofplatelets.TheNOS-depen- dent conversio
5、n ofL-arginine in RBCs is comparable to that of cultured human endothelial cells. RBCs in eNOS?/?micein contrast to wild-type mice lack NOS protein and activity, strengthening the evidence of an eNOS in RBCs. These data show an eNOS-like protein and activity in RBCs serving regulatory functions in R
6、BCs and platelets, which may stimulate new approaches in the treatment of NOdeficiency states inherent to several vas- cular and hematologic diseases. (Blood. 2006;107:2943-2951) 2006 by TheAmerican Society of HematologyIntroductionNitric oxide (NO) is a signaling molecule of major importance presen
7、t in various cell types.1,2It modulates not only the function of the vascular wall but also that of blood cells, such as platelets and leukocytes. NO is synthesized by a family of NO synthases (NOSs) through the conversion ofL-arginine toL-citrulline, using molecu- lar oxygen. Until recently, the ex
8、pression pattern of NOS isoformsappeared to be cell specific. Constitutively expressed neuronal andendothelial NOS (referred as NOS1 and NOS3) were identified inand cloned from neuronal and endothelial cells at first. Inducible NOS (NOS2) was originally isolated from activated macro- phages.3,4In th
9、e vascular system under resting conditions, NO synthesis has been attributed exclusively to the vascular endothe- lium expressing a NOS3 (eNOS) isoform. Initially thought to be a simple calmodulin-regulated enzyme, it is clear that eNOS has evolved to be tightly controlled by cofactors and posttrans
10、lationalmodifications, phosphorylation on multiple residues, and regulated protein-protein interactions.5,6 To date, human blood and, in particular, hemoglobin-carrying red blood cells (RBCs) have been considered as a major sink of NO.7,8Although early reports postulated a NOS resident in RBCs,9subs
11、equent studies were unable to confirm an active NOS within RBCs.10Current information on the NOS isoform, its localization, and functional activity within RBCs is still inconsistent and subject to considerable debate. Most importantly, a NOS-dependent forma-tion and release of NO-related species fro
12、m RBCs has not been shown so far. In fact, the diffusion-limited chemical inactivation of NO by intra-erythrocytic hemoglobin would suggest that even if RBCs contain NOS, NO production from such would represent a futile vestigial function derived from an earlier stem-cell precursor (prior to RBC hem
13、oglobinization). Indeed, the characterization and proof of a functional NOS in RBCs has been hampered by the high content of hemoglobin. First, the complex and oxygen-sensitive biochemistry of NO with intracellular and extracellular proteins8demands a composite analysis of the various constituents o
14、f the circulating NO pool to assess RBC-based NOS activity in blood. Second, the extraordi- nary high protein content of RBCs precludes standard proceduresfor characterization of NOS protein. Therefore RBC-specificapproaches have to be elaborated to allow identification and localization of NOS and i
15、ts regulation.Using immunofluorescence confocal microscopy, standard thin- section and immunogold cryosection, freeze-fracture electron microscopy, Western blotting, and reverse transcriptasepoly- merase chain reaction (RT-PCR) we provide unequivocal evidence that human RBCs express an active NOS. A
16、ctivity of NOS was analyzed by the release of NO and NO-related species and the conversion rate of the NOS substrateL-arginine. In human RBCs, NOS-derived NO regulates deformability of RBC membrane andFrom the Department of Medicine, Medical Clinic I, University Hospital Rheinisch- Westfa lische-Technische-Hochschule-Aachen (RTWH), Aachen; Department of Medicine, Division of Cardiology, Pulmonary Diseases and Angiology, Heinrich-Heine-University,Du sseldorf;DepartmentsofCardiovascular
