Wang J P
Department of Medical Research, Taichung Veterans General Hospital, Taiwan, Republic of China.
Naunyn Schmiedebergs Arch Pharmacol. 1996 May;353(6):599-605. doi: 10.1007/BF00167177.
1-[6-[[17 beta-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1 H-pyrrole-2,5-dione (U-73122) has been proven to be a useful tool in investigation of phospholipase C (PLC)-coupled signal transduction during cell activation. In the present studies, the inhibition by U-73122 of cytosolic free Ca2+ concentration ([Ca2+]i) of neutrophils was investigated. U-73122 suppressed the [Ca2+]i elevation of neutrophils suspended in Ca(2+)-containing medium challenged by N-formyl-Met-Leu-Phe (fMLP), cyclopiazonic acid (CPA) and ionomycin. The concentrations of U-73122 required for inhibition of CPA- and ionomycin-induced changes with IC50 values 4.06 +/- 0.27 microM and 4.04 +/- 0.44 microM, respectively, is almost 10-times that required for inhibition of the fMLP-induced response (IC50 value 0.62 +/- 0.04 microM). U-73122 also reduced the intracellular Ca2+ mobilization of neutrophils suspended in Ca(2+)-free medium stimulated by fMLP and CPA, but not by ionomycin, with IC50 values 0.52 +/- 0.02 microM and 6.82 +/- 0.74 microM, respectively. 1-[6-[[17 beta-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrr olidinedione (U-73343), a close analog of U-73122 that does not inhibit PLC activity, suppressed the [Ca2+]i elevation of neutrophils challenged by fMLP in Ca(2+)-containing medium, but not in Ca(2+)-free medium, with IC50 value 22.30 +/- 1.61 microM. In Mn(2+)-quench studies, U-73122 suppressed the Mn2+ influx in CPA-activated neutrophils (IC50 value was 7.16 +/- 0.28 microM) as well as in resting neutrophils (IC50 value was 6.72 +/- 0.30 microM). U-73343 also suppressed the Mn2+ influx in resting neutrophils in a concentration-dependent manner. These results suggest that the inhibitory effect of U-73122 on [Ca2+]i of activated neutrophils is attributed partly to the suppression of Ca2+ release from the intracellular Ca2+ stores through PLC inhibition, and partly to the blockade, especially at higher concentrations, of Ca2+ entry from the extracellular space through PLC-independent processes.
1-[6-[[17β-3-甲氧基雌甾-1,3,5(10)-三烯-17-基]氨基]己基]-1H-吡咯-2,5-二酮（U-73122）已被证明是研究细胞激活过程中磷脂酶C（PLC）偶联信号转导的有用工具。在本研究中，研究了U-73122对中性粒细胞胞浆游离钙离子浓度（[Ca2+]i）的抑制作用。U-73122抑制了悬浮于含Ca(2+)培养基中的中性粒细胞在受到N-甲酰甲硫氨酰-亮氨酰-苯丙氨酸（fMLP）、环匹阿尼酸（CPA）和离子霉素刺激时[Ca2+]i的升高。抑制CPA和离子霉素诱导变化所需的U-73122浓度，IC50值分别为4.06±0.27μM和4.04±0.44μM，几乎是抑制fMLP诱导反应所需浓度（IC50值0.62±0.04μM）的10倍。U-73122还降低了悬浮于无Ca(2+)培养基中的中性粒细胞在受到fMLP和CPA刺激时的细胞内Ca2+动员，但对离子霉素刺激无此作用，IC50值分别为0.52±0.02μM和6.82±0.74μM。1-[6-[[17β-3-甲氧基雌甾-1,3,5(10)-三烯-17-基]氨基]己基]-2,5-吡咯烷二酮（U-73343）是U-73122的一种紧密类似物，不抑制PLC活性，它抑制了悬浮于含Ca(2+)培养基中的中性粒细胞在受到fMLP刺激时[Ca2+]i的升高，但在无Ca(2+)培养基中无此作用，IC50值为22.30±1.61μM。在锰淬灭研究中，U-73122抑制了CPA激活的中性粒细胞中的Mn2+内流（IC50值为7.16±0.28μM）以及静息中性粒细胞中的Mn2+内流（IC50值为6.72±0.30μM）。U-73343也以浓度依赖的方式抑制静息中性粒细胞中的Mn2+内流。这些结果表明，U-73122对激活的中性粒细胞[Ca2+]i的抑制作用部分归因于通过抑制PLC来抑制细胞内Ca2+储存释放Ca2+，部分归因于在较高浓度时通过不依赖PLC的过程阻断细胞外空间的Ca2+内流。
