Release of Type II phospholipase A2 immunoreactivity and phospholipase A2 enzymatic activity from human placenta W Farrugia, G E Rice, M H Wong, K F Scott and S P Brennecke 'Perinatal Research Centre, Department of Perinatal Medicine, The Royal Women's Hospital, Carlton, Victoria 3053, Australia, 2Garvan Institute of Medical Research, Department of Medicine, University of New South Wales, St Vincent's Hospital, Darlinghurst, New South Wales 2010, Australia and 'Department of Obstetrics and Gynaecology, University of Melbourne, The Royal Women's Hospital, Carlton, Victoria 3053, Australia (Requests for offprints should be addressed to W Farrugia) Abstract The aim of this study was to determine whether Type II phospholipase A2 (PLA2) is released from late pregnant human placental tissue. Placental explants were incubated in vitro and the release of immunoreactive (ir) Type II PLA2 and PLA2 enzymatic activity into the medium was determined. Both irType II PLA2 and PLA2 enzymatic activity accumulated in the incubation medium in a time-dependent manner (P<0\m=.\0001). This release was not associated with a loss of cell membrane integrity, as indicated by measurement of the intracellular enzyme, lactate dehydrogenase, in the incubation medium. The concentration of irType II PLA2 and PLA2 enzyme activity present in incubation medium were significantly correlated (P<0\m=.\01). Consistent with the hypothesis that Type II PLA2 may be stored in secretory granules within human placental tissue, incubation in the presence of a membrane depolarising concentration of KCl (60 mm) caused the release of irType II PLA2 2\m=.\0-fold (P<0\m=.\001). PLA2 enzyme activity released into the incubation medium displays biochemical characteristics consistent with those previously reported for secretory PLA2 iso- zymes, that is, a requirement for millimolar concentrations of calcium for optimal enzyme activity, inhibited by reducing agents, such as dithiothreitol and insensitive to heat inactivation. The data obtained in this study establish that irType II PLA2 is released from term placenta, when incubated in vitro. The release of this extracellularly-active PLA2 isozyme may contribute to gestational and labour\x=req-\ associated increases in glycerophospholipid metabolism and prostaglandin formation. Journal of Endocrinology (1997) 153, 151\p=n-\157 Introduction Although the mechanisms that initiate human labour remain to be clearly established, biologically active metabo¬ lites of cell membrane phospholipids (e.g., platelet-activating factor (PAF) and eicosanoids, including hydroxyeicosatetrae- noic acids (HETEs) and prostaglandins) have been impli¬ cated in both the onset (Romero et al. 1987) and progression of human labour (Challis et al. 1976, Liggins et al. 1977, Johnston et al. 1993, Olson et al. 1993). One of the princi¬ pal groups of enzymes involved in the formation of phospholipid-derived mediators is the phospholipase A2 (PLA2) family of enzymes. These enzymes hydrolyse the sn-2-acyl-ester bond of 1,2 diacyl-sn-3 phosphoglycerides, liberating equimolar amounts of 1-acyl lysophosphatides and fatty acids (Slotboom et al. 1978). These phospholipid metabolites may be further processed to form potent phospholipid-derived mediators including PAF and eicosa¬ noids. PLA2 enzymatic activity is considered to be a rate- limiting step in the formation of such phospholipid-derived mediators in some tissues (Flower & Blackwell 1976). Two major functional groups of PLA2 have been identified: extracellular PLA2 (14-18 kDa) and intracellu¬ lar PLA2 (31-110 kDa) (Loeb & Gross 1986, Davidson & Dennis 1990). The membrane-associated intracellular or cytoplasmic enzyme is found in low abundance, is depen¬ dent on the presence of increased intracellular calcium for activation and may play a role in signal transduction (Clarke et al. 1990, Kramer et al. 1991). Four types of extracellular PLA, isozymes have been identified in mam¬ malian tissues to date: Type I (pancreatic phospholipase; de Haas et al. 1970, Davidson & Dennis 1990), Type II (pro-inflammatory phospholipase; Seilhamer et al. 1989, Davidson & Dennis 1990), Type IV (Chen et al. 1994) and the recently identified and only partially characterised 42 kDa PLA2 from human placenta (Bulh et al. 1995). At present, the involvement of only one of these PLA2 isozymes (Type II) in human pregnancy and labour- associated events has been studied in any detail. Type II PLA2 messenger RNA, immunoreactive (ir) content and enzymatic activity have been identified and localised in several human gestational tissues including amnion,