Measurement of phospholipase A 2 and 1-alkylglycerophosphocholine acetyltransferase activities in stimulated alveolar macrophages by HPLC analysis of NBD-labeled ether lipids

Abstract

The importance of phospholipases in cellular signaling and 1-alkylglycerophosphocholine acetyltransferase in the formation of platelet-activating factor (PAF) has stimulated demand for methods to measure these enzyme activities in inflammatory cells. Most of the assays currently used rely on radiolabeled substrates. We have synthesized NBD-labeled ether lipids as substrates for measuring enzyme activities of the PAF cycle and of lysosomal phospholipase A 2 (PLA 2). The fluorescent lipids were incubated with homogenates of stimulated bovine alveolar macrophages. The generated products were separated from the substrates by HPLC on a normal phase and monitored with a fluorescence detector. NBD-lyso-PAF was well accepted by acetyl- and acyltransferases of the cell-free preparations, which metabolized the substrate into NBD-PAF and NBD-alkyl-acylglycerophosphocholines. Homogenates of stimulated cells showed an enhanced production of NBD-PAF. The increased formation of the biological mediator was dependent on the nature of the stimuli and the time of stimulation. Lysosomal PLA 2 was measured with 1-0-(12-NBD-aminododecyl)-2-acyl- sn-glycero-3-phosphocholine as substrate. By varying the pH and the calcium concentration, it was possible to distinguish between the cytosolic PLA 2 and the lysosomal PLA 2 activity. Optimal conditions for the determination of the lysosomal PLA 2 were obtained at pH 4.5 and in the presence of EDTA. Stimulation with particulate agonists induced an enhancement of the lysosomal PLA 2 activity in macrophages.