Mogami H, Lloyd Mills C, Gallacher D V
The Physiological Laboratory, Crown Street, Liverpool University, P. O. Box 147, Liverpool L69 3BX, U.K.
Biochem J. 1997 Jun 1;324 ( Pt 2)(Pt 2):645-51. doi: 10.1042/bj3240645.
It is recognized in many cellular systems that the receptor/G-protein activation of phospholipase C and Ins(1,4,5)P3 production is the transduction pathway regulating the release of Ca2+ from internal stores. Ca2+ signals can now be monitored at the level of single cells but the biochemical detection of Ins(1,4,5)P3 cannot match this resolution. It is often difficult or impossible to directly attribute responses evoked in single cells by putative phospholipase C-coupled agonists to changes in Ins(1,4,5)P3 levels. U73122 is an aminosteroid that is reported to act as a specific inhibitor of phospholipase C and it has become an important tool in establishing the link between phospholipase C activation and cellular Ca2+ signalling. In the present study we use both patch-clamp electrophysiology and the imaging of fluorescent Ca2+ indicators to investigate the effect of U73122 in mouse pancreatic acinar cells. The study reveals that U73122 has effects other than the inhibition of phospholipase C. U73122 can directly activate ion channels. It can itself promote the release of Ca2+ from intracellular stores in permeabilized cells and in intact cells it triggers a release of Ca2+ that is initiated specifically at the secretory pole of these morphologically and functionally polarized cells. We also present evidence that U73122 can potentiate the response to Ins(1,4,5)P3; this is seen both in permeabilized cells and in patch-clamp protocols in which cells are internally dialysed with submaximal concentrations of Ins(1,4,5)P3. The effects of U73122 are therefore multiple and not specific for the inhibition of phospholipase C. Importantly, all the effects described influence Ca2+ signalling yet in many experimental protocols some of these effects can go unnoticed and might in error be attributed simply to the inhibition of Ins(1,4,5)P3 production.
在许多细胞系统中，人们认识到磷脂酶C的受体/G蛋白激活以及肌醇-1,4,5-三磷酸（Ins(1,4,5)P3）的产生是调节细胞内钙库释放Ca2+的转导途径。现在可以在单细胞水平监测Ca2+信号，但Ins(1,4,5)P3的生化检测无法达到这种分辨率。通常很难或不可能将假定的磷脂酶C偶联激动剂在单细胞中引发的反应直接归因于Ins(1,4,5)P3水平的变化。U73122是一种氨基类固醇，据报道它作为磷脂酶C的特异性抑制剂发挥作用，并且它已成为建立磷脂酶C激活与细胞Ca2+信号传导之间联系的重要工具。在本研究中，我们使用膜片钳电生理学和荧光Ca2+指示剂成像来研究U73122对小鼠胰腺腺泡细胞的影响。该研究表明，U73122除了抑制磷脂酶C外还有其他作用。U73122可以直接激活离子通道。它本身可以促进通透细胞内细胞内钙库释放Ca2+，并且在完整细胞中它会引发Ca2+释放，这种释放特别在这些形态和功能极化细胞的分泌极开始。我们还提供证据表明U73122可以增强对Ins(1,4,5)P3的反应；这在通透细胞和膜片钳实验中都可以看到，在膜片钳实验中细胞用亚最大浓度的Ins(1,4,5)P3进行内部透析。因此，U73122的作用是多方面的，并非特异性抑制磷脂酶C。重要的是，所描述的所有作用都影响Ca2+信号传导，但在许多实验方案中，其中一些作用可能未被注意到，并且可能错误地仅仅归因于对Ins(1,4,5)P3产生的抑制。
