Human red blood cells at work: identification and visualization of erythrocytic eNOS activity in health and disease

Miriam M Cortese-Krott, Ana Rodriguez-Mateos, Roberto Sansone, Gunter Kuhnle, Sivatharsini Thasian-Sivarajah, Thomas Krenz, Patrick Horn, Christoph Krisp, Dirk Wolters, Christian Heiß, Klaus-Dietrich Kröncke, Neil Hogg, Martin Feelisch, Malte Kelm

Research output: Contribution to journalArticlepeer-review

143 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)4229-37
Number of pages9
JournalBlood
Volume120
Issue number20
DOIs
Publication statusPublished - 15 Nov 2012

Keywords

  • Adult
  • Amino Acid Sequence
  • Arginine
  • Chromatography, High Pressure Liquid
  • Citrulline
  • Coronary Artery Disease
  • Endothelium, Vascular
  • Erythrocytes
  • Flow Cytometry
  • Fluoresceins
  • Fluorescent Dyes
  • Humans
  • Immunoprecipitation
  • Mass Spectrometry
  • Microscopy, Confocal
  • Molecular Sequence Data
  • NG-Nitroarginine Methyl Ester
  • Nitric Oxide
  • Nitric Oxide Synthase Type III
  • Oxyhemoglobins
  • Sequence Alignment
  • Sequence Analysis, Protein
  • Sequence Homology, Amino Acid

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