title: Human red blood cells at work: identification and visualization of erythrocytic eNOS activity in health and disease.
abstract: A nitric oxide synthase (NOS)-like activity has been demonstrated in human red blood cells (RBCs), but doubts about its functional significance, isoform identity and disease relevance remain. Using flow cytometry in combination with the nitric oxide (NO)-imaging probe DAF-FM we find that all blood cells form NO intracellularly, with a rank order of monocytes > neutrophils > lymphocytes > RBCs > platelets. The observation of a NO-related fluorescence within RBCs was unexpected given the abundance of the NO-scavenger oxyhemoglobin. Constitutive normoxic NO formation was abolished by NOS inhibition and intracellular NO scavenging, confirmed by laser-scanning microscopy and unequivocally validated by detection of the DAF-FM reaction product with NO using HPLC and LC-MS/MS. Using immunoprecipitation, ESI-MS/MS-based peptide sequencing and enzymatic assay we further demonstrate that human RBCs contain an endothelial NOS (eNOS) that converts L-(3)H-arginine to L-(3)H-citrulline in a Ca(2+)/calmodulin-dependent fashion. Moreover, in patients with coronary artery disease, red cell eNOS expression and activity are both lower than in age-matched healthy individuals and correlate with the degree of endothelial dysfunction. Thus, human RBCs constitutively produce NO under normoxic conditions via an active eNOS isoform, the activity of which is compromised in patients with coronary artery disease.
authors.affiliation: Cardiovascular Research Laboratory, Department of Cardiology, Pulmonology and Angiology, Heinrich Heine University of Düsseldorf, 40225 Düsseldorf, Germany.
authors.forename: Miriam M
authors.initials: MM
authors.lastname: Cortese-Krott
authors.forename: Ana
authors.initials: A
authors.lastname: Rodriguez-Mateos
authors.forename: Roberto
authors.initials: R
authors.lastname: Sansone
authors.forename: Gunter G C
authors.initials: GG
authors.lastname: Kuhnle
authors.forename: Sivatharsini
authors.initials: S
authors.lastname: Thasian-Sivarajah
authors.forename: Thomas
authors.initials: T
authors.lastname: Krenz
authors.forename: Patrick
authors.initials: P
authors.lastname: Horn
authors.forename: Christoph
authors.initials: C
authors.lastname: Krisp
authors.forename: Dirk
authors.initials: D
authors.lastname: Wolters
authors.forename: Christian
authors.initials: C
authors.lastname: Heiß
authors.forename: Klaus-Dietrich
authors.initials: KD
authors.lastname: Kröncke
authors.forename: Neil
authors.initials: N
authors.lastname: Hogg
authors.forename: Martin
authors.initials: M
authors.lastname: Feelisch
authors.forename: Malte
authors.initials: M
authors.lastname: Kelm
mesh_terms: D000328:Adult; D000595:Amino Acid Sequence; D001120:Arginine / Q000097:blood; D002851:Chromatography, High Pressure Liquid; D002956:Citrulline / Q000097:blood; D003324:Coronary Artery Disease / Q000097:blood / Q000201:enzymology* / Q000473:pathology; D004730:Endothelium, Vascular / Q000473:pathology; D004912:Erythrocytes / Q000201:enzymology*; D005434:Flow Cytometry; D005452:Fluoresceins / Q000032:analysis; D005456:Fluorescent Dyes / Q000032:analysis; D006801:Humans; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000097:blood; D052250:Nitric Oxide Synthase Type III / Q000037:antagonists & inhibitors / Q000097:blood* / Q000502:physiology; D010108:Oxyhemoglobins / Q000378:metabolism; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid
chemical_list: C503301:4-amino-5-methylamino-2',7'-difluorofluorescein diacetate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D010108:Oxyhemoglobins; D002956:Citrulline; D009569:Nitric Oxide; D001120:Arginine; D052250:Nitric Oxide Synthase Type III; D019331:NG-Nitroarginine Methyl Ester