scalaradial and arachidonyltrifluoromethane

The role of phospholipase A2 (PLA2) and its metabolite arachidonic acid (AA) in the proliferation and differentiation of HL-60 cells was investigated. Addition of either 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) or retinoic acid (RA) to HL-60 cells for 2 h inhibited PMA-stimulated PLA2 activity measured by [3H]AA release. The inhibitor of PLA2 activity, p-bromophenacyl bromide (BPB), significantly inhibited the proliferation of HL-60 cells and of fibroblast L929 and Swiss 3T3 cells in a dose-dependent manner. The effect of BPB on proliferation is probably through its inhibitory effect on PLA2 activity, since the same doses of BPB which inhibited proliferation also inhibited PLA2 activity determined by [3H]AA release. The importance of PLA2 activity for cell growth was further supported by the effect of two other PLA2 inhibitors, AACOCF3 and scalaradial, which inhibited HL-60 proliferation in a dose-dependent manner. BPB, AACOCF3 and scalaradial significantly increased the doubling time to 32.4 h, 34.0 h and 31.8 h, respectively, compared with 24.6 h in the control. The inhibitory effect of BPB on HL-60 proliferation was reversed by addition of exogenous free AA to HL-60 cells, indicating the importance of this metabolite for the proliferation process. This reversible effect is specific for AA since it was not achieved by other fatty acids like linolenic acid (LA) or oleic acid (OA). Addition of free AA to HL-60 cells did not induce differentiation, as expected. Although BPB, AACOCF3, or scalaradial inhibited proliferation, they did not induce differentiation nor affect the differentiation induced by 1,25(OH)2D3 or RA. These results implicate that PLA2 activity has no regulatory role in differentiation of HL-60 cells. The differential effect of PLA2 inhibitors on proliferation and differentiation of HL-60 cells suggests that these two processes function under different regulatory mechanisms.

Topics: 3T3 Cells; Acetophenones; Animals; Anti-Inflammatory Agents; Arachidonic Acid; Arachidonic Acids; Blotting, Northern; Calcitriol; Cell Differentiation; Cell Division; Enzyme Inhibitors; Granulocytes; HL-60 Cells; Homosteroids; Humans; Mice; Phospholipases A; Phospholipases A2; Sesterterpenes; Terpenes; Tetradecanoylphorbol Acetate; Tretinoin

Platelet-activating factor (PAF) production is carefully controlled in inflammatory cells. The specific removal of arachidonate (AA) from 1-O-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine (GPC), thought to be mediated by CoA-independent transacylase (CoA-IT), is required to generate the PAF precursor 1-O-alkyl-2-lyso-GPC in human neutrophils. Exposure of A23187-stimulated human monocytes to the CoA-IT inhibitors SK&F 98625 and SK&F 45905 inhibited PAF formation (IC50s of 10 and 12 microM, respectively), indicating that these cells also need CoA-IT activity for PAF production. Because CoA-IT activity transfers arachidonate to a 2-lyso phospholipid substrate, its activity is obligated to an sn-2 acyl hydrolase to form the 2-lyso phospholipid substrate. SB 203347, an inhibitor of 14 kDa phospholipase A2 (PLA2), and AACOCF3, an inhibitor of 85 kDa PLA2, both inhibited AA release from A23187-stimulated human monocytes. However, AACOCF3 had no effect on A23187-induced PAF formation at concentrations as high as 3 microM. Further, depletion of 85 kDa PLA2 using antisense (SB 7111, 1 microM) had no effect on PAF production, indicating a lack of a role of 85 kDa PLA2 in PAF biosynthesis. Both SB 203347 and the 14 kDa PLA2 inhibitor scalaradial blocked PAF synthesis in monocytes (IC50s of 2 and 0.5 microM, respectively), suggesting a key role of 14 kDa PLA2 in this process. Further, A23187-stimulated monocytes produced two forms of PAF: 80% 1-O-alkyl-2-acetyl-GPC and 20% 1-acyl-2-acetyl-GPC, which were both equally inhibited by SB 203347. In contrast, inhibition of CoA-IT using SK&F 45905 (20 microM) had a greater effect on the production of 1-O-alkyl (-80%) than of 1-acyl (-14%) acetylated material. Finally, treatment of U937 cell membranes with exogenous human recombinant (rh) type II 14 kDa PLA2, but not rh 85 kDa PLA2, induced PAF production. Elimination of membrane CoA-IT activity by heat treatment impaired the ability of 14 kDa PLA2 to induce PAF formation. Taken together, these results suggest that a 14 kDa PLA2-like activity, and not 85 kDa PLA2, is coupled to monocyte CoA-IT-induced PAF production.

Topics: Acyltransferases; Anti-Inflammatory Agents; Arachidonic Acid; Arachidonic Acids; Benzenesulfonates; Calcimycin; Enzyme Inhibitors; Homosteroids; Humans; Monocytes; Neutrophils; Phospholipases A; Phospholipases A2; Platelet Activating Factor; Recombinant Proteins; Sesterterpenes; Sulfonamides; Terpenes; Urea