Chang Ling-Chu, Chen Chi-Ming, Wang Jih-Pyang
Department of Education and Research, Taichung Veterans General Hospital, 160, Chung Kang Road, Sec. 3, Taiwan, ROC.
Biochim Biophys Acta. 2003 Mar 17;1620(1-3):191-8. doi: 10.1016/s0304-4165(02)00532-9.
The expression of phospholipase D (PLD) isoenzymes in neutrophils was investigated using reverse transcription-polymerase chain reaction analysis. Amplification products of predicted size were obtained from rat neutrophils with nucleotide sequences corresponding to PLD1a and PLD2. 1-(3',4'-Dimethoxybenzyl)-6,7-dichloroisoquinoline (DMDI) inhibited the formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated PLD activation in rat neutrophils. The underlying cellular signaling mechanism of DMDI inhibition was investigated. The fMLP-induced protein tyrosine phosphorylation and the membrane translocation of ADP-ribosylation factor (ARF) and Rho A in neutrophils was attenuated by DMDI in a concentration-dependent manner. However, neither the membrane association of protein kinase C-alpha and -beta isoenzymes in fMLP-stimulated cells nor the GTPgammaS- and phorbol 12-myristate 13-acetate-stimulated membrane translocation of ARF and Rho A in a cell-free system was affected significantly by DMDI. These results indicate that the expression of PLD1a and PLD2 mRNA in neutrophils. Attenuation of protein tyrosine phosphorylation and the membrane association of ARF and Rho A probably play a concerted role in the inhibition of PLD by DMDI in rat neutrophils in response to fMLP.
利用逆转录-聚合酶链反应分析研究了中性粒细胞中磷脂酶D（PLD）同工酶的表达。从大鼠中性粒细胞中获得了预测大小的扩增产物，其核苷酸序列与PLD1a和PLD2相对应。1-（3'，4'-二甲氧基苄基）-6,7-二氯异喹啉（DMDI）抑制了大鼠中性粒细胞中N-甲酰甲硫氨酰-亮氨酰-苯丙氨酸（fMLP）刺激的PLD激活。研究了DMDI抑制的潜在细胞信号传导机制。DMDI以浓度依赖性方式减弱了fMLP诱导的中性粒细胞中蛋白酪氨酸磷酸化以及ADP-核糖基化因子（ARF）和Rho A的膜转位。然而，DMDI对fMLP刺激细胞中蛋白激酶C-α和-β同工酶的膜结合以及无细胞体系中GTPγS和佛波醇12-肉豆蔻酸酯13-乙酸酯刺激的ARF和Rho A的膜转位均无显著影响。这些结果表明中性粒细胞中PLD1a和PLD2 mRNA的表达。蛋白酪氨酸磷酸化的减弱以及ARF和Rho A的膜结合可能在DMDI抑制大鼠中性粒细胞中fMLP诱导的PLD过程中起协同作用。
