calcimycin and scalaradial

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

Scalaradial, a marine natural product with anti-inflammatory activity, has been shown to be a selective inhibitor of 14 kDa type II phospholipase A2(PLA2). We have examined the inhibition by scalaradial (0.1 nM to 10 microM) of neutrophil function (degranulation) in response to receptor-mediated activation [N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), 30 nM; leuokotriene B4 (LTB4), 100 nM; platelet-activating factor (PAF), 100 nM] and non-receptor-mediated stimuli [A23187 (1 microM) and thapsigargin (100 nM)]. Furthermore, we evaluated the ability of scalaradial to inhibit the increase in intracellular Ca2+ in response to fMLP, LTB4, A23187, and thapsigargin as well as its ability to prevent either fMLP- or LTB4-mediated elevation in inositol phosphate production (InsP). Scalaradial was a potent inhibitor of both receptor- (IC50 = 50-200 nM) and non-receptor- (IC50 = 40-900 nM) mediated degranulation. Although scalaradial inhibited the mobilization of Ca2+ induced by fMLP, LTB4, and PAF, it did not affect the maximal Ca2+ levels attained with A23187 or thapsigargin. Neutrophil-binding studies with [3H]fMLP and [3H]LTB4 would suggest that the effect of scalaradial on agonist-induced degranulation and increase in intracellular Ca2+ was not at the receptor level because 50-fold higher concentrations were required to have a significant effect on the binding of these agonists. To determine if scalaradial affected phosphatidylinositol selective phospholipase C (PI-PLC) activity, assays were conducted to monitor fMLP- and LTB4-induced formation of InsPs using myo-[3H]inositol-labeled U-937 cells. In these cells, 2.5 to 9-fold higher concentrations of scalaradial were required to inhibit PI-PLC activity than to inhibit agonist-induced degranulation of neutrophils, suggesting that the effects of scalaradial on Ca2+ and degranulation are not the sole result of blocking receptor activation of PI-PLC. Results obtained with receptor-mediated stimuli suggest that scalaradial may have direct effects on Ca2+ channels and InsP turnover, but inhibition of intracellular Ca2+ levels was not required for scalaradial to block degranulation since scalaradial was capable of inhibiting degranulation produced by either A23187 or thapsigargin, without changing the maximal Ca2+ levels obtained with these two stimuli. These results demonstrate that scalaradial can inhibit degranulation in the presence of micromolar intracellular Ca2+ concentration, thus supporting the hypoth

Topics: Anti-Inflammatory Agents; Binding, Competitive; Calcimycin; Calcium; Cell Degranulation; Homosteroids; Humans; Inositol Phosphates; Leukotriene B4; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Peroxidase; Phospholipases A; Phospholipases A2; Sesterterpenes; Terpenes; Thapsigargin