Simultaneous measurement of intracellular pH and Ca2+ using the fluorescence of SNARF-1 and fura-2

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Abstract
Upon cell stimulation with hormones and other mitogens, a variety of biochemical and physiological responses occur within the first few minutes. Changes in both intracellular pH (pHin) and intracellular Ca2+ concentration ([Ca2+]in) are prominent and play a major role in the signal transduction mechanism leading to the physiological response, i.e., secretion, neurotransmission, proliferation, or differentiation. However, it is not clear whether these ions work independently in the activation pathway leading to a particular physiological response. The fluorescence characteristics of most Ca2+ indicators are pH sensitive, and quantitative estimates of [Ca2+]in cannot be made without knowledge of pHin. Thus it is desirable to have a technique to simultaneously monitor these two ions with relatively high time resolution. Here we have developed experimental conditions that allow us to use optimum emission conditions for a pH fluorescent indicator SNARF-1 and optimum excitation conditions for the Ca2+ indicator fura-2. The fluorescence spectra of these compounds are sufficiently different to allow simultaneous measurement of pH and Ca2+ both in vitro and in situ. We have observed simultaneous changes in both pHin and [Ca2+]in in BALB/c 3T3 cells on treatment with the nonfluorescent Ca2+ ionophore 4-bromo-A23187. This temporal relationship between pHin and Ca2+ gives further credence to the interrelationship between these two second messengers in the expression of physiological responses.