蛋白激酶C刺激的中性粒细胞样HL-60细胞中超氧化物生成信号通路的药理学靶向作用：佛波酯、花生四烯酸及激酶、磷酸酶和磷脂酶A2抑制剂的作用

Pharmacological targeting of signaling pathways in protein kinase C-stimulated superoxide generation in neutrophil-like HL-60 cells: effect of phorbol ester, arachidonic acid and inhibitors of kinase(s), phosphatase(s) and phospholipase A2.

作者信息

Mayer A M, Brenic S, Glaser K B

机构信息

Department of Pharmacology, Chicago College of Osteopathic Medicine, Downers Grove, Illinois, USA.

出版信息

J Pharmacol Exp Ther. 1996 Nov;279(2):633-44.

PMID:8930166

Abstract

The purpose of this investigation was to pharmacologically probe the signaling pathways thought to be involved in protein kinase C (PKC)-stimulated superoxide anion (O2-) generation in all-trans retinoic acid-treated human promyelocytic HL-60 cell line (HL-60), targeting PKC, mitogen-activated protein kinase (MAPK), MAPK kinase (MEK), protein serine-threonine phosphatase(s) (PSP), protein tyrosine kinase(s) (PTK) and phosphatase(s) (PTP), secretory phospholipase A2, cyclooxygenase (CO) and 5-lipoxygenase with selected inhibitors. The following agents inhibited phorbol 12-myristate 13-acetate-stimulated O2- generation significantly in the all-trans retinoic acid-treated HL-60 cells (expressed as percentage of control, P < .05): 1) PKC inhibitors: staurosporine (100 nM, 3 +/- 1%); Ro 31-8220 (1 microM, 3 +/- 2%); sphingosine (100 microM, 15 +/- 7%); 2) PSP 1 and 2a inhibitors, okadaic acid (10 microM, 35 +/- 1%); calyculin A (10 microM, 73 +/- 1%); 3) MAPK inhibitor: SB-203580 (100 microM, 62 +/- 1%); 4) PTP inhibitors: phenylarsine oxide (1 microM, 12 +/- 9%); diamide (1 mM, 21 +/- 11%); and 5) secretory phospholipase A2 inhibitors: manoalide (1 microM, 24 +/- 10%); scalaradial (1 microM, 11 +/- 4%). Exogenously added arachidonic acid-stimulated O2- generation in a time- and dose-dependent manner. The following inhibitors enhanced or did not significantly affect phorbol 12-myristate 13-acetate-stimulated O2- generation (expressed as percentage of control): 1) PTK inhibitors: genistein (100 microM, 69 +/- 12%); CGP 53716 (100 microM, 67 +/- 10%); herbimycin A (10 microM, 67.4 +/- 1%); 2) PSP 2b inhibitors: cyclosporin A (30 microM, 71 +/- 5%); FK506 (30 microM, 88 +/- 7%); 3) CO inhibitor: indomethacin (100 microM, 111 +/- 12%); 4) 5-lipoxygenase inhibitor: WY 50,295 (100 microM, 140 +/- 23%); 5) MEK inhibitor: PD98059 (100 microM, 94 +/- 6.7%); and 6) the PTP inhibitor: orthovanadate (100 microM, 131 +/- 25%). Our pharmacological study suggests that, in neutrophil-like HL-60 cells, the signaling pathways leading to PMA-stimulated O2- generation appear to involve PKC, MAPK, phospholipase A2, arachidonic acid, PSP 1 and 2a and PTP. Furthermore, PTK, MEK, CO, 5-lipoxygenase and PSP 2b do not appear to participate in the modulation of PKC-stimulated O2- generation.

摘要

本研究的目的是通过药理学方法探究在全反式维甲酸处理的人早幼粒细胞HL-60细胞系（HL-60）中，被认为参与蛋白激酶C（PKC）刺激的超氧阴离子（O₂⁻）生成的信号通路，使用特定抑制剂靶向PKC、丝裂原活化蛋白激酶（MAPK）、MAPK激酶（MEK）、蛋白丝氨酸 - 苏氨酸磷酸酶（PSP）、蛋白酪氨酸激酶（PTK）和磷酸酶（PTP）、分泌型磷脂酶A₂、环氧化酶（CO）和5 - 脂氧合酶。以下试剂在全反式维甲酸处理的HL-60细胞中显著抑制了佛波酯12 - 肉豆蔻酸酯13 - 乙酸酯刺激的O₂⁻生成（以对照百分比表示，P <.05）：1）PKC抑制剂：星形孢菌素（100 nM，3±1%）；Ro 31 - 8220（1 μM，3±2%）；鞘氨醇（100 μM，15±7%）；2）PSP 1和2a抑制剂，冈田酸（10 μM，35±1%）；花萼海绵诱癌素A（10 μM，73±1%）；3）MAPK抑制剂：SB - 203580（100 μM，62±1%）；4）PTP抑制剂：苯砷酸氧化物（1 μM，12±9%）；二酰胺（1 mM，21±11%）；5）分泌型磷脂酶A₂抑制剂： manoalide（1 μM，24±10%）；scalaradial（1 μM，11±4%）。外源添加的花生四烯酸以时间和剂量依赖的方式刺激O₂⁻生成。以下抑制剂增强或未显著影响佛波酯12 - 肉豆蔻酸酯13 - 乙酸酯刺激的O₂⁻生成（以对照百分比表示）：1）PTK抑制剂：染料木黄酮（100 μM，69±12%）；CGP 53716（100 μM，67±10%）；除莠霉素A（10 μM，67.4±1%）；2）PSP 2b抑制剂：环孢素A（30 μM，71±5%）；FK506（30 μM，88±7%）；3）CO抑制剂：吲哚美辛（100 μM，111±12%）；4）5 - 脂氧合酶抑制剂：WY 50,295（100 μM，140±23%）；5）MEK抑制剂：PD98059（100 μM，94±6.7%）；6）PTP抑制剂：原钒酸盐（100 μM，131±25%）。我们的药理学研究表明，在嗜中性粒细胞样HL-60细胞中，导致PMA刺激的O₂⁻生成的信号通路似乎涉及PKC、MAPK、磷脂酶A₂、花生四烯酸、PSP 1和2a以及PTP。此外，PTK、MEK、CO、5 - 脂氧合酶和PSP 2b似乎不参与PKC刺激的O₂⁻生成的调节。