探討在嗜中性白血球 CYL-26z 抑制 phospholipase D 活化，artocarpol A 促進生成超氧自由基及formyl peptide 活化phosphoinositide 3-kinase的作用機制

Abstract

Neutrophils play a critical role in the first-line defense against invading bacteria via degranulation and respiratory burst. However, the released proteases and the generated reactive oxygen species may also responsible for tissue destruction in inflammatory conditions. The search for potential synthetic compounds and natural products and study of their molecular mechanisms to regulate degranulation and respiratory burst in neutrophils would be useful for anti-inflammatory chemotherapy. In the present study, the inhibition of degranulation by a synthetic compound, 5-[4-acridin-9-ylamino]phenyl]-5-methyl-3-methylenedihydrofuran-2-one (CYL-26z), and the stimulation of respiratory burst by artocarpol A (ART), a natural phenolic compound isolated from Artocarpus rigida, were examined in rat neutrophils. CYL-26Z showed concentration-dependent inhibition of formyl-Met-Leu-Phe (fMLP)-stimulated phospholipase D (PLD) activity in neutrophils but did not affect the phorbol 12-myristate 13-acetate (PMA)-induced response. CYL-26z caused a slight but significant attenuation of the fMLP-induced global protein tyrosine phosphorylation. CYL-26z blocked the membrane recruitment of protein kinase C-a (PKC-a) but failed to affect the membrane association of PKC-bI and -bII. CYL-26z inhibited the translocation of RhoA, but not ADP-ribosylation factor (Arf), to the membrane, the dissociation of RhoA-Rho guanine nucleotide dissociation inhibitor (GDI) complex and the RhoA activation in fMLP-stimulated neutrophils. CYL-26z effectively attenuated the membrane association of RhoA but not Arf in response to GTPgS in a cell-free system. CYL-26z inhibited the membrane expression of CD11b, CD45 and CD63 in response to fMLP stimulation. These results indicate that the CYL-26z inhibition of fMLP-stimulated neutrophil degranulation is, at lest partly, attributable to the attenuation of PLD activity via the blockade of RhoA activation. ART stimulated superoxide anion generation in neutrophils. Pretreatment of cells with 4-(4-fluorophenyl)-2-(4-methyl-sulfinyl-phenyl)-5-(4-pyridyl)-1H-imidazole (SB 203580), a p38 mitogen-activated protein kinase (MAPK) inhibitor, 2-[1- (3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (GF 109203X), a broad PKC inhibitor, 1-[6-((17b-3-Methoxyestra-1,3,5(10)-trien-17-yl)amino)- hexyl]-1H-pyrrole-2,5-dione (U-73122), a phospholipase C (PLC) inhibitor, 2- aminoethyldiphenyl borate (2-APB), an inositol 1,4,5-trisphosphate (IP3) receptor blocker, and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY 294002), a phosphoinositide 3-kinase (PI3K) inhibitor, but not with 2'-amino-3'-methoxyflavone (PD98059) and 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126), both MKK (MAP/ERK kinase) inhibitors, attenuated ART-induced response. ART induced the phosphorylation and activation of p38 MAPK but not the phosphorylation of extracellular signal-regulated kinase (ERK). ART stimulated the [Ca2+]i elevation in the presence or absence of external Ca2+, and increased the cellular IP3 levels. ART increased the recruitment of PKC-a, -bI, and -bII to the plasma membrane of neutrophils, and stimulated Ca2+-dependent PKC activation in the cytosol preparation. ART stimulated the dual phosphorylation (Thr308 and Ser473) of protein kinase B (PKB/Akt) and the formation of phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3), whereas LY 294002 inhibited these effects in neutrophils. Rat neutrophils expressed both class IA PI3K (p85, p110a, p110b, and p110d) and class IB PI3K (p110g) proteins. Detectable membrane-association of class IA PI3Ks, class IB PI3K and Ras was seen as early as 1.5 min, 0.5 min and 1.5 min, respectively, after stimulation with ART. The kinetics of ART-induced Ras activation paralleled the kinetics of class IA PI3Ks recruitment to membrane caused by ART, and the p85 and p110g immunoprecipitates contained Ras. ART stimulated Src family kinase activation. Both Src kinase activity and PI(3,4,5)P3 formation in ART-stimulated neutrophils were inhibited by 4-amino-1-tert-butyl-3-(1'-naphthyl)pyrrazolo [3,4-d]pyrimidine (PP1 analog), a Src family kinase inhibitor. PP1 analog also attenuated the ART-stimulated superoxide anions generation in neutrophils. ART induced the serine phosphorylation and the membrane association of p47phox, whereas GF 109203X inhibited both responses. These results indicate that the ART stimulation of superoxide anion generation involved the activation of p38 MAPK, PLC/Ca2+, PKC and PI3K signaling pathways in neutrophils. In summary, CYL-26z inhibition of PLD activation, through the blockade of RhoA activation, is attributed to the fMLP-induced degranulation. ART stimulation of superoxide anion generation is attributable to the activation of PLC/Ca2+, PKC, p38 MAPK and PI3K signaling pathways in rat neutrophils.