Calcium-dependent biosynthesis of platelet-activating factor by submandibular gland cells

Abstract

Isolated dog submandibular gland cells synthesize platelet-activating factor (PAF) when stimulated with acetylcholine (ACh). This production of PAF was concentration- and time-dependent, and was inhibited by pretreatment with anticholinergic agents. PAF that had accumulated in cells through prior stimulation with ACh vanished rapidly on addition of atropine. Phenylmethanesulphonyl fluoride produced an accumulation of PAF in non-stimulated cells and greatly potentiated further ACh-induced accumulation. PAF production and [14C] arachidonic acid (AA) liberation induced by ACh were increased by higher concentrations of extracellular Ca2+, and ACh failed to stimulate PAF formation in the absence of Ca2+, although ACh still stimulated the liberation of [14C]AA without Ca2+. Both the Ca2+ ionophore ionomycin in intact cells and Ca2+ (at concentrations greater than or equal to 300 nM) in digitonin-permeabilized cells facilitated PAF formation. 1-O-Alkyl-2-lyso-sn-glycero-3-phosphocholine (lyso-PAF):acetyl-CoA acetyltransferase activity rapidly increased in cells incubated with ACh or ionomycin. These results suggest, at least, that the stimulation of a remodelling pathway is involved in the increased PAF synthesis induced by ACh. Dithiothreitol-insensitive cholinephosphotransferase activity was also activated by ACh. However, the activation of both enzymes by ACh was transient, in spite of the fact that ACh-stimulated PAF formation was continuous. This may suggest that additional mechanism(s) other than the activation of these enzymes play an important role in controlling PAF synthesis. The present study provides further evidence that the exocrine submandibular gland cells of dogs have the capacity to increase PAF turnover upon stimulation in a Ca(2+)-dependent manner and retain PAF within the cells partly associated with the membrane and partly released into the cytosol.