Simultaneous measurement of Ca2+ and pH by laser cytometry using fluo-3 and SNARF-1.

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.

我们提出一种新的便捷方法，利用激光细胞计数术，同时使用氟-3（用于测量细胞内钙离子浓度[Ca2+]i）和SNARF - 1（用于测量细胞内pH值pHi）的混合物，在等激发（488nm） - 双发射（氟 - 3为530nm，SNARF - 1为>630nm）的条件下，测量细胞内钙离子浓度[Ca2+]i和细胞内pH值pHi。通过使用这种技术，我们测量了ATP刺激的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细胞在[Ca2+]i升高的同时发生碱化；相反，在HSG细胞中，即使观察到[Ca2+]i升高，也完全没有发生碱化。使用BAPTA（一种Ca2+螯合剂）和氨氯吡嗪（一种Na+/H+交换体抑制剂），我们发现pHi的升高需要[Ca2+]i的升高，但[Ca2+]i的升高并不总是需要pHi的升高。从我们的结果我们得出结论，在ATP刺激的信号转导中，[Ca2+]i的升高优先于pHi的升高。