DISSOCIATION OF PLATELET-ACTIVATING-FACTOR PRODUCTION AND ARACHIDONATE RELEASE BY THE ENDOMEMBRANE CA2-ATPASE INHIBITOR THAPSIGARGIN - EVIDENCE FOR THE INVOLVEMENT OF A CA2+-DEPENDENT ROUTE OF PRIMING IN THE PRODUCTION OF LIPID MEDIATORS BY HUMAN POLYMORPHONUCLEAR LEUKOCYTES()

The production of platelet-activating factor (PAF) and the release of [H-3]arachidonate were studied in human polymorphonuclear leukocytes ( PMN) stimulated with thapsigargin, an inhibitor of endomembrane Ca2+-A TPase. Concentrations of thapsigargin as low as 10-25 nM primed PMN fo r both PAF production and [H-3]arachidonate release in response to the chemotactic peptide (fMLP), whereas concentrations in the range 25-20 0 nM induced a time- and dose-dependent production of PAF, which occur red in the absence of both [H-3]arachidonate release and [H-3]phosphat idylethanol formation. Studies in fura-2/AM-loaded cells showed that c oncentrations of thapsigargin that elicited PAF production induced a p rotracted and long lasting elevation of cytosolic free calcium concent ration ([Ca2+]i) between 200 and 700 nM. The lower concentrations prim ed the cells for a late [Ca2+]i elevation in response to fMLP similar to that elicited by cytochalasin B or ionomycin. PAF production showed a good correlation with the increase of [Ca2+]i (r = 0.91) irrespecti ve of the procedure used to grade [Ca2+]i. In contrast, phorbol 12,13- dibutyrate failed to induce both PAF production and elevation of [Ca2]i, but it was a very effective stimulator of [H-3]arachidonate releas e and [H-3]phosphatidylethanol production. These data indicate that PA F production and [H-3]arachidonate release in PMN differ in both bioch emical pathway and modulatory mechanisms. Whereas PAF production seems extremely sensitive to changes in [Ca2+]i, which seems to exert its m odulatory effect at the lyso-PAF:acetyl-CoA acetyltransferase step, [H -3] arachidonate release seems tightly modulated by protein kinase C-d ependent mechanisms and is coincidental with activation of phospholipa se D.