Atsumi T, Sugita K, Kohno M, Takahashi T, Ueha T
Department of Oral Physiology, Meikai University School of Dentistry, Saitama, Japan.
Cytometry. 1996 Jun 1;24(2):99-105. doi: 10.1002/(SICI)1097-0320(19960601)24:2<99::AID-CYTO1>3.0.CO;2-C.
We present a new convenient method for simultaneous measurement of intracellular calcium concentration ([Ca2+]i) and intracellular pH (pHi) using laser cytometry with a mixture of fluo-3 (for [Ca2+]i) and SNARF-1 (for pHi), with iso excitation (488 nm)-dual emission (530 nm for fluo-3 and > 630 nm for SNARF-1). By using this technique, we measured the changes in [Ca2+]i and pHi in A-431 human epidermoid carcinoma cells and HSG human salivary gland cells stimulated by ATP. We found that alkalization in A-431 cell occurred with the elevation of [Ca2+]i; in contrast, alkalization in HSG cells did not occur at all, even though the elevation of [Ca2+]i was observed. Using BAPTA (a chelating agent of Ca2+) and amiloride (an inhibitor of the Na+/H+ exchanger), we found that the elevation of pHi requires the elevation of [Ca2+]i but that the elevation of [Ca2+]i does not always require a rise in pHi. From our results we conclude that elevation of [Ca2+]i takes precedence over the elevation of pHi in ATP-stimulated signal transduction.
我们提出了一种新的便捷方法，使用激光细胞术，通过混合使用Fluo-3（用于测量细胞内钙浓度[Ca2+]i）和SNARF-1（用于测量细胞内pH值[pHi]），在等激发（488nm）-双发射（Fluo-3为530nm，SNARF-1大于630nm）条件下同时测量细胞内钙浓度[Ca2+]i和细胞内pH值（pHi）。通过使用该技术，我们测量了ATP刺激下A-431人表皮样癌细胞和HSG人唾液腺细胞中[Ca2+]i和pHi的变化。我们发现，A-431细胞中随着[Ca2+]i升高出现碱化；相反，HSG细胞中尽管观察到[Ca2+]i升高，但根本未出现碱化。使用BAPTA（一种Ca2+螯合剂）和氨氯吡脒（一种Na+/H+交换体抑制剂），我们发现pHi升高需要[Ca2+]i升高，但[Ca2+]i升高并不总是需要pHi升高。根据我们的结果，我们得出结论，在ATP刺激的信号转导中，[Ca2+]i升高优先于pHi升高。
