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

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 ce lls (human umbilical vein endothelial cells) and rat aortic segments. Follo wing 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 chemilumines cence, 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 dete ction of an enhanced superoxide production after stimulation with angiotens in 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 super oxide formation which was predominantly produced by an NAD(P)H oxidase. Sho rt-Term vanadate-enhanced L-012 chemiluminescence represents a highly sensi tive assay making it possible to detect small changes of superoxide formati on in intact vascular cells. Copyright(C) 1999 S. Karger AG. Basel.