Mahlangu Dick A F D, Dix James A
Department of Chemistry, State University of New York, Box 6000, Binghamton, NY 13902-6000, USA.
Anal Biochem. 2004 Feb 1;325(1):28-34. doi: 10.1016/s0003-2697(03)00142-8.
A method is introduced to measure chloride permeability in cultured epithelial cells using 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ) and 6-methoxy-N-ethylquinolinium iodide quinolinium (MEQ) as fluorescent chloride-sensitive probes. The method involves growing cells in multiwell plates, incubating cells with SPQ or MEQ, and then exchanging intracellular or extracellular halide ions with nitrate. The resulting time course of SPQ or MEQ fluorescence is followed by repetitive readings with a multiwell fluorescence plate reader. Exchange times are extracted by fitting the time course with a single exponential function of time. The method was validated by measuring the effect of chloride channel activators and blockers in A6 and MDCK cells. The baseline iodide/nitrate exchange time was 200-300 s. Isoproterenol (a modulator of cAMP-activated chloride channels) increased the exchange rate by a factor of 1.4+/-0.1; A23187 (a modulator of calcium-activated chloride channels) increased the rate by 3.4+/-0.4; bradykinin (also a modulator of calcium-activated chloride channels) increased the rate by 2.0+/-0.4; forskolin (a direct stimulator of adenylate cyclase) increased the rate by 2.7+/-0.3. Diphenylamine-2-carboxylate (a chloride channel blocker) decreased the rate by 0.12+/-0.03. These results indicate that our method is a valid indicator of halide-nitrate exchange in cultured epithelial cells.
介绍了一种利用6-甲氧基-N-(3-磺丙基)喹啉鎓(SPQ)和6-甲氧基-N-乙基喹啉鎓碘化物(MEQ)作为荧光氯敏感探针来测量培养上皮细胞中氯通透性的方法。该方法包括在多孔板中培养细胞，用SPQ或MEQ孵育细胞，然后用硝酸盐交换细胞内或细胞外的卤离子。用多孔荧光板读数器重复读取，记录SPQ或MEQ荧光的时间进程。通过将时间进程与时间的单指数函数拟合来提取交换时间。通过测量氯通道激活剂和阻滞剂对A6和MDCK细胞的作用，验证了该方法的有效性。基线碘化物/硝酸盐交换时间为200-300秒。异丙肾上腺素（一种环磷酸腺苷激活氯通道的调节剂）使交换速率提高了1.4±0.1倍；A23187（一种钙激活氯通道的调节剂）使速率提高了3.4±0.4倍；缓激肽（也是一种钙激活氯通道的调节剂）使速率提高了2.0±0.4倍；福斯可林（一种腺苷酸环化酶的直接刺激剂）使速率提高了2.7±0.3倍。二苯胺-2-羧酸盐（一种氯通道阻滞剂）使速率降低了0.12±0.03。这些结果表明，我们的方法是培养上皮细胞中卤化物-硝酸盐交换的有效指标。
