The activation of phosphatidylinositol-hydrolyzing phospholipase A2 during prostaglandin synthesis in transformed mouse BALB/3T3 cells

• Published in: The Journal of biological chemistry Vol. 256; no. 10; pp. 5215 - 5219
• Main Authors: Hong, S L, Deykin, D
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 25.05.1981
• Subjects: Animals; Bradykinin - pharmacology; Calcimycin - pharmacology; Cell Transformation, Neoplastic - metabolism; Cells, Cultured; Enzyme Activation; Kinetics; Mice; Mice, Inbred BALB C; Phosphatidylinositols - biosynthesis; Phospholipases - metabolism; Phospholipases A - metabolism; Phospholipases A1; Phospholipases A2; Prostaglandins - biosynthesis; Prostaglandins E - biosynthesis; Thrombin - pharmacology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(19)69389-0

To investigate the type of phospholipase activated by agents that stimulate prostaglandin synthesis, we used transformed mouse cells whose phospholipids were doubly labeled with [14C]inositol and [3H]arachidonic acid. [14C]Inositol was incorporated mostly into the phosphatidylinositol and [3H]arachidonic acid was distributed into the various phospholipids. When these cells were incubated with bradykinin, a stimulator of prostaglandin synthesis, the release of 3H radioactivity from cellular phospholipids and the synthesis of prostaglandin were initiated within seconds and reached a maximum in 40 to 70 s. Analysis of the intracellular lipids revealed a concomitant increase of radioactivity associated with lysophosphatidylinositol, which was detectable within 5 s of incubation with bradykinin and reached a maximum between 40 and 70 s. Lysophosphatidylinositol which could be formed either from a phospholipase A1 or phospholipase A2 reaction, was identified by its chromatographic properties and conversion to glycerophosphorylinositol. We found that the 3H/14C ratio of purified lysophosphatidylinositol was 1/11 of that of phosphatidylinositol, which indicated that lysophosphatidylinositol formed in response to bradykinin is 1-acyl-sn-glycero-3-phosphorylinositol and most probably is formed from a phospholipase A2 deacylation of phosphatidylinositol (a phospholipase A1 deacylation would result in the formation of lysophosphatidylinositol of a 3H/14C ratio similar to phosphatidylinositol). Furthermore, we did not detect between control and stimulated cells any significant difference in the level of several phospholipase C metabolites including inositol phosphate, diglyceride, and phosphatidic acid. These results suggest that phospholipase C is probably not activated. The formation of lysophosphatidylinositol was also stimulated by thrombin and ionophore A23187, both activators of prostaglandin synthesis. Dexamethasone, a lipase inhibitor, inhibited the appearance of lysophosphatidylinositol, whereas aspirin and low concentrations of indomethacin, the cyclooxygenase inhibitor, did not inhibit. The results presented in ths paper provide evidence that a phospholipase A2-hydrolyzing phosphatidylinositol is activated when intact cells are stimulated for prostaglandin synthesis.