Effects of scalaradial, a novel inhibitor of 14 kDa phospholipase A2, 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 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 hypothesis that a 14 kDa PLA2 may be important in the regulation of neutrophil degranulation.
Barnette
M S
MS
Department of Inflammation and Respiratory Pharmacology, SmithKline Beecham Laboratories, King of Prussia, PA 19406.
Rush
J
J
Marshall
L A
LA
Foley
J J
JJ
Schmidt
D B
DB
Sarau
H M
HM
D000893:Anti-Inflammatory Agents / Q000494:pharmacology*; D001667:Binding, Competitive; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D015550:Cell Degranulation / Q000187:drug effects*; D006718:Homosteroids / Q000494:pharmacology*; D006801:Humans; D007295:Inositol Phosphates / Q000032:analysis; D007975:Leukotriene B4 / Q000037:antagonists & inhibitors; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000037:antagonists & inhibitors; D009504:Neutrophils / Q000187:drug effects*; D009195:Peroxidase / Q000378:metabolism; D010741:Phospholipases A / Q000037:antagonists & inhibitors*; D054467:Phospholipases A2; D054830:Sesterterpenes; D013729:Terpenes / Q000494:pharmacology*; D019284:Thapsigargin
D000893:Anti-Inflammatory Agents; D006718:Homosteroids; D007295:Inositol Phosphates; D054830:Sesterterpenes; D013729:Terpenes; D007975:Leukotriene B4; D000001:Calcimycin; C071197:scalaradial; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019284:Thapsigargin; D009195:Peroxidase; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium