TY - JOUR
T1 - Human red blood cells at work
T2 - Identification and visualization of erythrocytic eNOS activity in health and disease
AU - Cortese-Krott, Miriam M.
AU - Rodriguez-Mateos, Ana
AU - Sansone, Roberto
AU - Kuhnle, Gunter G C
AU - Thasian-Sivarajah, Sivatharsini
AU - Krenz, Thomas
AU - Horn, Patrick
AU - Krisp, Christoph
AU - Wolters, Dirk
AU - Heiß, Christian
AU - Kröncke, Klaus Dietrich
AU - Hogg, Neil
AU - Feelisch, Martin
AU - Kelm, Malte
PY - 2012/11/15
Y1 - 2012/11/15
N2 - 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- 3H-arginine to L-3H-citrulline in a Ca2+/calmodul- independent 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.
AB - 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- 3H-arginine to L-3H-citrulline in a Ca2+/calmodul- independent 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.
UR - http://www.scopus.com/inward/record.url?scp=84869786258&partnerID=8YFLogxK
U2 - 10.1182/blood-2012-07-442277
DO - 10.1182/blood-2012-07-442277
M3 - Article
C2 - 23007404
AN - SCOPUS:84869786258
SN - 0006-4971
VL - 120
SP - 4229
EP - 4237
JO - Blood
JF - Blood
IS - 20
ER -