Torfs Herbert, Poels Jeroen, Detheux Michel, Dupriez Vincent, Van Loy Tom, Vercammen Linda, Vassart Gilbert, Parmentier Marc, Vanden Broeck Jozef
Laboratory for Developmental Physiology and Molecular Biology, Zoological Institute K.U. Leuven, Naamsestraat 59, 3000 Leuven, Belgium.
Invert Neurosci. 2002 Apr;4(3):119-24. doi: 10.1007/s10158-001-0013-2. Epub 2001 Nov 1.
The bioluminescent Ca(2+)-sensitive reporter protein, aequorin, was employed to develop an insect cell-based functional assay system for monitoring receptor-mediated changes of intracellular Ca(2)(+)-concentrations. Drosophila Schneider 2 (S2) cells were genetically engineered to stably express both apoaequorin and the insect tachykinin-related peptide receptor, STKR. Lom-TK III, an STKR agonist, was shown to elicit concentration-dependent bioluminescent responses in these S2-STKR-Aeq cells. The EC(50) value for the calcium effect detected by means of aequorin appeared to be nearly identical to the one that was measured by means of Fura-2, a fluorescent Ca(2)(+)-indicator. In addition, this aequorin-based method was also utilised to study receptor antagonists. Experimental analysis of the effects exerted by spantide I, II and III, three potent substance P antagonists, on Lom-TK III-stimulated S2-STKR-Aeq cells showed that these compounds antagonise STKR-mediated responses in a concentration-dependent manner. The rank order of inhibitory potencies was spantide III > spantide II > spantide I.
生物发光的Ca(2+)敏感报告蛋白水母发光蛋白被用于开发一种基于昆虫细胞的功能检测系统，以监测受体介导的细胞内Ca(2+)浓度变化。果蝇Schneider 2（S2）细胞经过基因工程改造，可稳定表达脱辅基水母发光蛋白和昆虫速激肽相关肽受体STKR。STKR激动剂Lom-TK III在这些S2-STKR-Aeq细胞中可引发浓度依赖性生物发光反应。通过水母发光蛋白检测到的钙效应的EC(50)值似乎与通过荧光Ca(2+)指示剂Fura-2测量的值几乎相同。此外，这种基于水母发光蛋白的方法还被用于研究受体拮抗剂。对三种强效P物质拮抗剂spantide I、II和III对Lom-TK III刺激的S2-STKR-Aeq细胞所产生的影响进行的实验分析表明，这些化合物以浓度依赖性方式拮抗STKR介导的反应。抑制效力的顺序为spantide III > spantide II > spantide I。
