EFFECTS OF SCALARADIAL, A NOVEL INHIBITOR OF 14 KDA PHOSPHOLIPASE A(2), ON HUMAN NEUTROPHIL FUNCTION

Scalaradial, a marine natural product with anti-inflammatory activity, has been shown to be a selective inhibitor of 14 kDa type II phospholipase A(2)(PLA(2)). We have examined the inhibition by scalaradial (0.1 nM to 10 mu M) of neutrophil function (degranulation) in response to receptor-mediated activation [N-formyl-L-methionyI-L-leucyl-L-phenylalanine (fMLP), 30 nM; leuokotriene B-4 (LTB(4)), 100 nM; platelet-activating factor (PAF), 100 nM] and non-receptor-mediated stimuli [A23187 (1 mu M) and thapsigargin (100 nM)]. Furthermore, we evaluated the ability of scalaradial to inhibit the increase in intracellular Ca2+ in response to fMLP, LTB(4), A23187, and thapsigargin as well as its ability to prevent either fMLP- or LTB(4)-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, LTB(4), and PAF, it did not affect the maximal Ca2+ levels attained with A23187 or thapsigargin. Neutrophil-binding studies with [H-3]fMLP and [H-3]LTB(4) 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 LTB(4)-induced formation of InsPs using myo-[H-3]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 hypothesis that a 14 kDa PLA(2) may be important in the regulation of neutrophil degranulation.