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-dpyridazine-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 microM) 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 microM) 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 approximately 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.