新合成 carbazole 類化合物 LCY50 以濃度依存性的方式抑制 formyl-methionyl-leucyl-phenylalanine (fMLP) 刺激鼠的嗜中性白血球生成超氧自由基，其 IC50 值為 10.7 ± 0.8 μM，但此抑制作用無時間依存性的關係。LCY50 抑制 fMLP 刺激嗜中性白血球產生超氧自由基的作用具有可逆性。此外，LCY50 不會影響 phorbol 12-myristate 13-acetate (PMA) 刺激嗜中性白血球超氧自由基的生成。在反應過程中 LCY50 不會引起 lactate dehydrogenase (LDH) 的釋出，因此對嗜中性白血球不具細胞毒性。LCY50 不會抑制dihydroxyfurmaric acid (DHF)自體氧化產生超氧自由基。在非完整細胞系統的反應中，LCY50 不會抑制 arachidonic acid 活化細胞質與細胞膜分劃混合液產生超氧自由基。這些結果顯示，LCY50 不為超氧自由基之直接清除劑，亦不會直接抑制 NADPH oxidase 之活性。LCY50 不會影響 fMLP 刺激嗜中性白血球所產生的 extracellular signal-regulated kinase (ERK) 、 p38 mitogen-activated protein kinase (MAPK)、AktSer473 及AktThr308 磷酸化作用。在嗜中性白血球中，LCY50 可以增加細胞內 cyclic AMP 的含量。然而 LCY50 抑制 fMLP 刺激嗜中性白血球產生超氧自由基的作用不會被 KT5720 及 SQ22536 所對抗。LCY50 抑制 fMLP 刺激嗜中性白血球所引起的 phospholipase D (PLD) 活性，其 IC50 值為 1.4 ± 0.4 μM。此外 LCY50 也會抑制 fMLP 刺激嗜中性白血球所產生的 PKCα、βI、δ 及 Rho A膜轉位作用，其 IC50 值分別為 5.6 ± 2.4、8.4 ± 1.0、13.9 ± 2.9 及 10.4 ± 2.9 μM。LCY50 只有稍量抑制 fMLP 刺激細胞所引起的 [Ca2+]i 增加及蛋白質酪氨酸磷酸化的現象。綜合上述的結果顯示，LCY50 可能經由干擾 PLD 及 PKC 訊息傳遞路徑而抑制 fMLP 刺激大白鼠的嗜中性白血球生成超氧自由基的作用。 LCY50 inhibited the formyl-Met-Leu-Phe (fMLP)-induced superoxide anion generation in a concentration-, with IC50 value of 10.7 ± 0.8 μM, but not time-dependent manner in rat neutrophils, while had no effect on phorbol 12-myristate 13-acetate (PMA)-induced response. The inhibition by LCY50 was reversed by washing. LCY50 did not release of lactate dehydrogenase during the reaction time. LCY50 had no effect on the superoxide anion generation during dihydroxyfumaric acid autoxidation and in arachidonic acid stimulated cell-free system. LCY50 did not affect the fMLP-stimulated phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), AktSer473 and AktThr308. LCY50 alone increased cellular cyclic AMP level in neutrophils. However, the inhibition of superoxide anion generation by LCY50 in neutrophils was not reversed by KT5720 and SQ22536. LCY50 effectively blocked the phospholipase D (PLD) activation, with IC50 value of 1.4 ± 0.4 μM, and the membrane recruitment of protein kinase C α (PKCα) (IC50 5.6 ± 2.4 μM), PKCβI (IC50 8.4 ± 1.0 μM), PKCδ (IC50 13.9 ± 2.9 μM) and Rho A (IC50 about 10 μM) in neutrophils stimulated with fMLP. LCY50 only slightly decreased the fMLP-induced [Ca2+]i rise and protein tyrosine phosphorylation. These results indicate that the PKC and PLD signaling pathways probably involved in the inhibition of superoxide anion generation by LCY50 in rat neutrophils.
