A membrane fraction containing H,K-ATPase (EC 3.6.1.36) was prepared from pig gastric mucosa and found to contain phospholipase A2 (EC 3.1.1.4) and lysophospholipase (EC 3.1.1.5) activities. Washing the membranes decreased their protein content by 25%. Recovery profiles of H,K-ATPase, phospholipase A2 and lysophospholipase were similar for membranes washed either with water or with 0.15 or 1.5 M KCl. Nearly identical distribution profiles were obtained for the three enzyme activities after centrifugation of washed vesicle membranes on a linear sucrose gradient. The phospholipase A2 activity was stimulated by calcium and increased further in the presence of calmodulin. The amount of cellular radioactively labelled lysophosphatidylcholine was doubled upon cholinergic stimulation of isolated parietal cells prelabelled with [3H]glycerol or 32Pi. The liberated lyso[32P]phosphatidylcholine had its acyl chain in the sn-1 position, which implies an activation of a phospholipase A2. These findings indicate that secretagogues which increase the cytosolic Ca2+ concentration, i.e. acetylcholine, histamine and gastrin, may activate a phospholipase A2 in the parietal cell.
Fusion of pig gastric H,K-ATPase- and phospholipase A2-containing vesicles in vitro was studied by electron microscopy and by monitoring the change in fluorescence of octadecyl rhodamine B-labelled vesicles. Ca2+ stimulated fusion of the vesicles, and the fusion rate showed a positive correlation with the activity of the phospholipase A2. Both the Ca2(+)-stimulated fusion rate and the Ca2(+)-dependent phospholipase A2 activity were further enhanced by the presence of calmodulin. The present results supported our previous findings (Olaisson et al. 1990) and further indicate that the phospholipase A2 associated with the H,K-ATPase-containing membranes might play a central role in membrane fusion processes in the stimulated parietal cell.
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