Hedley L, Phagoo S B, James I F
Sandoz Institute for Medical Research, London, England.
Anal Biochem. 1996 May 1;236(2):270-4. doi: 10.1006/abio.1996.0166.
Changes in intracellular calcium concentration are important in mediating a wide variety of physiological responses. Recently there has been renewed interest in the use of aequorin, a protein from jellyfish that emits light when calcium is bound, to measure calcium levels in cells. We have loaded populations of cells from the human glioma line, U373MG, with aequorin. Lysis of aequorin-loaded but not control cells with detergent resulted in a luminescence signal that was dependent on extracellular calcium. Aequorin-loaded cells responded to substance P, histamine, or the calcium ionophore, ionomycin, with an increase in luminescence. Signals in response to detergent, ionomycin, or substance P could be detected up to 48 h after cells were loaded with aequorin. Other neurokinin-1 agonists but not agonists at neurokinin-2 or neurokinin-3 receptors produced luminescence signals. Neurokinin-1 antagonists inhibited the substance P-induced signal. The aequorin-loading procedure worked well with U373MG cells but not with AR42J, CHO, IMR-90, or WI-38 cells.
细胞内钙浓度的变化在介导多种生理反应中起着重要作用。最近，人们对使用水母蛋白水母发光蛋白重新产生了兴趣，这种蛋白在与钙结合时会发光，用于测量细胞中的钙水平。我们已将人胶质瘤细胞系U373MG的细胞群体加载了水母发光蛋白。用去污剂裂解加载了水母发光蛋白的细胞而非对照细胞会产生依赖于细胞外钙的发光信号。加载了水母发光蛋白的细胞对P物质、组胺或钙离子载体离子霉素有反应，发光增强。在细胞加载水母发光蛋白后长达48小时都能检测到对去污剂、离子霉素或P物质的反应信号。其他神经激肽-1激动剂而非神经激肽-2或神经激肽-3受体的激动剂产生发光信号。神经激肽-1拮抗剂抑制P物质诱导的信号。水母发光蛋白加载程序对U373MG细胞效果良好，但对AR42J、CHO、IMR-90或WI-38细胞则不行。
