Sensitive superoxide detection in vascular cells by the new chemiluminescence dye L-012

Sohn, Hae-Young; Gloe, Torsten; Keller, Matthias; Schoenafinger, Karl and Pohl, Ulrich (1999): Sensitive superoxide detection in vascular cells by the new chemiluminescence dye L-012. In: Journal of Vascular Research, No. 6: pp. 456-464

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-l,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 mu M) there was an 2,8-fold increase of L-012 chemiluminescence, whereas incubation with angiotensin II (100 nM) did not result in a measurable increase. Addition of vanadate (100 mu 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 similar to 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. Copyright(C) 1999 S. Karger AG. Basel.

• Item Type: Journal article
• Form of publication: Publisher's Version
• Faculties: Medicine
• Subjects: 600 Technology > 610 Medicine and health
• ISSN: 1018-1172
• Alliance/National Licence: This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively.
• Language: English