The University of Southampton
University of Southampton Institutional Repository

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

Human red blood cells at work: identification and visualization of erythrocytic eNOS activity in health and disease
Human red blood cells at work: identification and visualization of erythrocytic eNOS activity in health and disease
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 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. Employing 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+/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.
0006-4971
4229-4237
Cortese-Krott, Miriam M.
7dc9b44c-847c-4196-8866-a3cc0c1dc357
Rodriguez-Mateos, Ana
874f6e8a-7b38-44af-b32b-4fdbb3019208
Sansone, Roberto
eda52dc1-401d-4ba3-81a8-a3dca81199ea
Kuhnle, Gunter G.C.
ad46444c-97d2-40b3-b192-c37a106f9e9d
Thasian-Sivarajah, Sivatharsini
3dc41453-ee0e-4a98-b84f-ef800828a738
Krenz, Thomas
bdab993e-c70e-4bbb-99c9-4161f3b61d5b
Horn, Patrick
89c09c5f-bfd4-40ab-8f05-3ae25ec717d3
Krisp, Christoph
a7c58c56-3ea3-41e4-b9e7-7dd7e8369f42
Wolters, Dirk
868d710e-c036-4214-bd6d-e6576683bae2
Heiß, Christian
5cd4df8d-fb5d-4d68-831c-6a186b2a320e
Kroncke, Klaus-Dietrich
6dca65ce-bf41-4d07-be29-a86f21ee545a
Hogg, Neil
4f258561-8120-4eb0-a2da-439e83cba361
Feelisch, Martin
8c1b9965-8614-4e85-b2c6-458a2e17eafd
Kelm, Malte
db2bb062-32d7-4b50-9f65-8ba89ffa5f42
Cortese-Krott, Miriam M.
7dc9b44c-847c-4196-8866-a3cc0c1dc357
Rodriguez-Mateos, Ana
874f6e8a-7b38-44af-b32b-4fdbb3019208
Sansone, Roberto
eda52dc1-401d-4ba3-81a8-a3dca81199ea
Kuhnle, Gunter G.C.
ad46444c-97d2-40b3-b192-c37a106f9e9d
Thasian-Sivarajah, Sivatharsini
3dc41453-ee0e-4a98-b84f-ef800828a738
Krenz, Thomas
bdab993e-c70e-4bbb-99c9-4161f3b61d5b
Horn, Patrick
89c09c5f-bfd4-40ab-8f05-3ae25ec717d3
Krisp, Christoph
a7c58c56-3ea3-41e4-b9e7-7dd7e8369f42
Wolters, Dirk
868d710e-c036-4214-bd6d-e6576683bae2
Heiß, Christian
5cd4df8d-fb5d-4d68-831c-6a186b2a320e
Kroncke, Klaus-Dietrich
6dca65ce-bf41-4d07-be29-a86f21ee545a
Hogg, Neil
4f258561-8120-4eb0-a2da-439e83cba361
Feelisch, Martin
8c1b9965-8614-4e85-b2c6-458a2e17eafd
Kelm, Malte
db2bb062-32d7-4b50-9f65-8ba89ffa5f42

Cortese-Krott, Miriam M., Rodriguez-Mateos, Ana, Sansone, Roberto, Kuhnle, Gunter G.C., Thasian-Sivarajah, Sivatharsini, Krenz, Thomas, Horn, Patrick, Krisp, Christoph, Wolters, Dirk, Heiß, Christian, Kroncke, Klaus-Dietrich, Hogg, Neil, Feelisch, Martin and Kelm, Malte (2012) Human red blood cells at work: identification and visualization of erythrocytic eNOS activity in health and disease. Blood, 120, 4229-4237. (doi:10.1182/blood-2012-07-442277). (PMID:23007404)

Record type: Article

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 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. Employing 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+/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.

Text
2012-Cortese-Krott Blood-prepub.pdf - Other
Download (888kB)
Text
__userfiles.soton.ac.uk_Users_nsc_mydesktop_343579feelisch.pdf - Version of Record
Restricted to Repository staff only
Request a copy

More information

e-pub ahead of print date: 24 September 2012
Organisations: Clinical & Experimental Sciences

Identifiers

Local EPrints ID: 343579
URI: http://eprints.soton.ac.uk/id/eprint/343579
ISSN: 0006-4971
PURE UUID: 06fee4b7-1ac3-44ff-9529-4aa1273cfb61
ORCID for Martin Feelisch: ORCID iD orcid.org/0000-0003-2320-1158

Catalogue record

Date deposited: 04 Oct 2012 16:05
Last modified: 15 Mar 2024 03:42

Export record

Altmetrics

Contributors

Author: Miriam M. Cortese-Krott
Author: Ana Rodriguez-Mateos
Author: Roberto Sansone
Author: Gunter G.C. Kuhnle
Author: Sivatharsini Thasian-Sivarajah
Author: Thomas Krenz
Author: Patrick Horn
Author: Christoph Krisp
Author: Dirk Wolters
Author: Christian Heiß
Author: Klaus-Dietrich Kroncke
Author: Neil Hogg
Author: Martin Feelisch ORCID iD
Author: Malte Kelm

Download statistics

Downloads from ePrints over the past year. Other digital versions may also be available to download e.g. from the publisher's website.

View more statistics

Atom RSS 1.0 RSS 2.0

Contact ePrints Soton: eprints@soton.ac.uk

ePrints Soton supports OAI 2.0 with a base URL of http://eprints.soton.ac.uk/cgi/oai2

This repository has been built using EPrints software, developed at the University of Southampton, but available to everyone to use.

We use cookies to ensure that we give you the best experience on our website. If you continue without changing your settings, we will assume that you are happy to receive cookies on the University of Southampton website.

×