Platelet activating factor biosynthesis and degradation in rat glomeruli

Abstract

The biosynthesis of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, also known as platelet activating factor (PAF), and its degradation to inactive metabolites were studied in glomerular homogenates from male Sprague-Dawley rats. Homogenates were incubated with various substrates, namely, tritiated acetate (5.3 mumol/L) or tritiated acetyl-coenzyme A (2 mumol/L) or tritiated lyso-PAF (30 nmol/L) at 37 degrees C for 30 minutes in RPMI medium 1640 and were subsequently stimulated with the phospholipase agonist A23187 (9.5 mumol/L) for various time periods. Alkyl-ether lipids were analyzed by thin-layer and high-pressure liquid chromatography and identified by enzymatic and chemical treatment. To assess PAF degradation, glomerular homogenates were incubated with 3H-PAF standard (1 nmol/L) for various time periods. In response to prolonged stimulation (120 minutes) with A23187, glomerular tissue synthesized PAF primarily via an acetyl transferase, utilizing either 3H-lyso-PAF or 3H-acetate/3H-acetyl-coenzyme A. 3H-PAF was rapidly degraded mainly to 3H-lyso-PAF, and this step was inhibited by diisopropyl fluorophosphate. The apparent Km and Vmax values of enzymatic degradation were 16.6 mumol and 100 nmol/min/mg protein, respectively. The presence of PAF biosynthetic and degradative pathways may be of importance in regulating the function of the normal glomerulus and in glomerular inflammatory injury.