Sensitive Superoxide Detection in Vascular Cells by the New Chemiluminescence Dye L-012

Abstract

The detection superoxide production in vascular cells is usually limited by a low sensitivity of available assays. We tested the applicability of the luminol derivate L-012 [8-amino-5-chloro-7-phenylpyridol[3,4-d]pyridazine-1,4(2H,3H)dione] to measure superoxide production in cultured endothelial cells (human umbilical vein endothelial cells) and rat aortic segments. Following stimulation with the protein kinase stimulator phorbol 12-myristate 13-acetate (PMA, 1 μ M) there was an 2.8-fold increase of L-012 chemiluminescence, whereas incubation with angiotensin II (100 n M) did not result in a measurable increase. Addition of vanadate (100 μ M) considerably increased the chemiluminescence (up to 17-fold) after PMA and made possible the detection of an enhanced superoxide production after stimulation with angiotensin II (by 1.7-fold). This was due to a ∼9-fold increase in signal intensity of L-012 in the presence of vanadate. Prolonged incubation with vanadate also led to a tyrosine phosphorylation-dependent increase in superoxide formation which was predominantly produced by an NAD(P)H oxidase. Short-term vanadate-enhanced L-012 chemiluminescence represents a highly sensitive assay making it possible to detect small changes of superoxide formation in intact vascular cells.

Journal

Journal ID (publisher-id): JVR

Journal ID (nlm-ta): J Vasc Res

Journal ID (doi): 10.1159/issn.1018-1172

Title: Journal of Vascular Research

Publisher: S. Karger AG

ISSN (Print): 1018-1172

ISSN (Electronic): 1423-0135

Publication date Subscription-year: 1999

Publication date (Print): December 1999

Publication date (Electronic): 24 December 1999

Volume: 36

Issue: 6

Pages: 456-464

Affiliations

^aInstitute of Physiology, Ludwig Maximilians University Munich, and ^bHoechst Marion Roussel, Hoechst AG, Frankfurt, Germany

Article

Publisher ID: 25688 Other ID: J Vasc Res 1999;36:456–464

DOI: 10.1159/000025688

PubMed ID: 10629421

Copyright statement: © 1999 S. Karger AG, Basel

License:

Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

Page count

Figures: 7, References: 23, Pages: 9

Product

Self URI (application/pdf): https://www.karger.com/Article/Pdf/25688

Self URI (text/html): https://www.karger.com/Article/FullText/25688

Self URI (journal page): https://www.karger.com/SubjectArea/Cardiovascular-System

Categories

Subject: Research Paper