Proenkephalin derived peptides in canine neutrophils

Abstract

Opioids and neutrophils are proposed to interact in cardiac ischemic preconditioning and in ischemia reperfusion injury. Since neutrophils are potentially both source and target for the opioids, canine neutrophils were isolated and their enkephalin content was characterized by radioimmunoassay (RIA) utilizing antibodies specific for the C-terminus of Methionine-Enkephalin (ME) and ME-Arg-Phe (MEAP). After culture, purified neutrophils and media were separated and protease activity was terminated in a boiling bath. Peptides were extracted and separated by size exclusion chromatography into large (e.g. proenkephalin), intermediate (e.g. peptide B and F) and small (e.g. ME and MEAP) fractions. The fractions were dried under vacuum, reconstituted in buffer and the enkephalins were determined by RIA. ME-assays primarily identified intermediate-sized peptides in cells and media with limited ME in the media and limited large precursors in the cells. The MEAP-assay identified primarily large and intermediate sized peptides in the cells. Very little MEAP was detected. The absence of MEAP and the predominance of precursors suggest that enkephalin processing in neutrophils may differ from that in cardiomyocytes. The data further suggest that neutrophils could represent a substantive source of cardiac enkephalin during ischemia and reperfusion. LG supported by NIH MORE program R25 GM 064365, AHA TX Affiliate