A new chemiluminescence paradox: selective inhibition of isoluminol-amplified activity in phagocytes by peptides from annexin AI.

Chemiluminescence systems enhanced by either isoluminol or luminol in combination with a peroxidase are sensitive methods for the detection of reactive oxygen species (ROS) generated by phagocyte NADPH oxidase. The two amplifying substrates are structurally very similar, differing only in the position of the amino group in the aromatic ring of the molecules. This difference renders isoluminol a less lipophilic molecule that is less permeable to biological membranes. The use of isoluminol is consequently restricted to studies dealing with the secretion of oxygen metabolites. In this study we show that synthetic peptides derived from the N-terminal domain of the calcium-regulated protein annexin AI interfere with the detection of radicals in an isoluminol-amplified, but not in a luminol-amplified, system. The annexin AI-derived peptides reduce the light output with isoluminol excited by superoxide and horseradish peroxidase (HRP) in formyl-methionyl-leucyl-phenylalanine- and phorbol myristate acetate-stimulated cells, as well as by hydrogen peroxide and HRP. The precise mechanism for the inhibition is not known. The results presented strongly suggest that a reduced cellular response detected with isoluminol-amplified chemiluminescence should be confirmed with an alternative technique to determine release of superoxide anions and hydrogen peroxide.