Haines K A, Reibman J, Tang X Y, Blake M, Weissmann G
Department of Medicine, New York University Medical Center, New York 10016.
J Cell Biol. 1991 Aug;114(3):433-42. doi: 10.1083/jcb.114.3.433.
Upon engagement of chemoattractant receptors, neutrophils generate inositol trisphosphate and diacylglycerol (DG) by means of a phosphatidylinositol-specific phospholipase C (PI-PLC) which is regulated by a GTP-binding protein(s). We have previously reported (Reibman, J., H. M. Korchak, L. B. Vosshall, K. A. Haines, A. M. Rich, and G. Weissmann. 1988. J. Biol. Chem. 263:6322-6328) a biphasic rise in DG after exposure of neutrophils to the chemoattractant FMLP: a rapid (less than or equal to 15 s) phase ("triggering") and a slow (greater than or equal to 30 s) phase ("activation"). These derive from distinct intracellular lipid pools. To study the source of rapid and slow DG, we have used a unique probe, protein I, a porin that is the major outer membrane protein of Neisseria gonorrhoeae. Treatment of neutrophils with protein I inhibits exocytosis and homotypic cell adhesion provoked by FMLP without inhibiting assembly of the NADPH oxidase responsible for O2-. generation. DG turnover in PMN labeled with [3H]arachidonate and [14C]glycerol was profoundly altered by protein I. Whereas the rapid peak of DG was only modestly diminished (FMLP vs. FMLP plus protein I = DG labeled with [3H]arachidonic acid (3H-a.a.-DG): 142 +/- 14% SEM vs. 125 +/- 22%; DG labeled with the glycerol backbone with [14C]glycerol (D-14C-G): 125 +/- 10% SEM vs. 107 +/- 8.5% SEM), the slow rise in both 3H-a.a.-DG and D-14C-G was essentially abolished. Moreover, treatment of neutrophils with 4-4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), which, like protein I, inhibits exocytosis without affecting O2-. generation also inhibited slow DG. However, protein phosphorylation and dephosphorylation (47phox, 66phox) were unaffected in the absence of slow DG. To determine the source of the slow DG, we have analyzed radiolabeled phospholipid (PL) turnover after FMLP +/- protein I (P.I.). Treatment of PMN with FMLP (0.1 microM) resulted in breakdown of phosphatidylcholine (PC), beginning at 30 s, and reaching a nadir at 60 s (3H-PC = 59 +/- 10.2% SEM of resting, 14C-PC = 57 +/- 6.4%). Protein I (0.25 microM) significantly inhibited PC turnover after FMLP ([3H]PC = 95 +/- 5.6% and [14C]PC = 86 +/- 8.4% of resting at 60 s), but failed to alter the metabolism of 3H- or 14C-phosphatidylinositol after FMLP (91 +/- 19.6 and 88 +/- 16.5% vs. 92 +/- 9.2 and 91 +/- 16% at 60 s).(ABSTRACT TRUNCATED AT 400 WORDS)
当中性粒细胞的趋化因子受体被激活时，通过一种受鸟苷三磷酸结合蛋白调节的磷脂酰肌醇特异性磷脂酶C（PI-PLC）产生肌醇三磷酸和二酰甘油（DG）。我们之前报道过（Reibman, J., H. M. Korchak, L. B. Vosshall, K. A. Haines, A. M. Rich, and G. Weissmann. 1988. J. Biol. Chem. 263:6322 - 6328），中性粒细胞暴露于趋化因子甲酰甲硫氨酸-亮氨酸-苯丙氨酸（FMLP）后，DG出现双相升高：一个快速（小于或等于15秒）阶段（“触发”）和一个缓慢（大于或等于30秒）阶段（“激活”）。这两个阶段源自不同的细胞内脂质池。为了研究快速和缓慢DG的来源，我们使用了一种独特的探针，蛋白I，它是淋病奈瑟菌的主要外膜蛋白，一种孔蛋白。用蛋白I处理中性粒细胞可抑制FMLP引发的胞吐作用和同型细胞黏附，而不抑制负责超氧阴离子（O₂⁻）产生的NADPH氧化酶的组装。用[³H]花生四烯酸和[¹⁴C]甘油标记的多形核白细胞（PMN）中的DG周转被蛋白I显著改变。虽然DG的快速峰值仅略有降低（FMLP与FMLP加蛋白I相比 = 用[³H]花生四烯酸标记的DG（³H-a.a.-DG）：142 ± 14% SEM对125 ± 22%；用[¹⁴C]甘油标记甘油骨架的DG（D-¹⁴C-G）：125 ± 由于原文较长，完整翻译后超过400字，按照要求进行了截取。如果您需要完整译文，请告诉我。10% SEM对107 ± 8.5% SEM），但³H-a.a.-DG和D-¹⁴C-G的缓慢升高基本被消除。此外，用4,4'-二异硫氰基芪-2,2'-二磺酸（DIDS）处理中性粒细胞，它与蛋白I一样，抑制胞吐作用而不影响O₂⁻的产生，也抑制了缓慢DG。然而，在没有缓慢DG的情况下，蛋白磷酸化和去磷酸化（47phox，66phox）未受影响。为了确定缓慢DG的来源，我们分析了FMLP ± 蛋白I（P.I.）处理后放射性标记磷脂（PL）的周转。用FMLP（0.1微摩尔）处理PMN导致磷脂酰胆碱（PC）在30秒开始分解，并在60秒达到最低点（³H-PC = 静息时的59 ± 10.2% SEM，¹⁴C-PC = 57 ± 6.4%）。蛋白I（0.25微摩尔）显著抑制FMLP处理后的PC周转（60秒时[³H]PC = 静息时的95 ± 5.6%，[¹⁴C]PC = 86 ± 8.4%），但未能改变FMLP处理后³H-或¹⁴C-磷脂酰肌醇的代谢（60秒时分别为91 ± 19.6%和88 ± 16.5%，而对照为92 ± 9.2%和91 ± 16%）。
