Thyrotropin-releasing Hormone-induced Changes in Intracellular [ Ca 2 + ] Measured by Microspectrofluorometry on Individual quin 2-10 aded Cells

We have developed an accurate and practical method for measuring intracellular Ca 2+ concentration ([Ca2÷]~) in single cells in monolayer culture using the fluorescent Ca2+-binding dye quin2. Quin2 was loaded into cells as a membrane-permeant ester which is hydrolyzed in the cytoplasm to the impermeant free acid, which is the indicator form (Tsien, R. Y., T. Pozzan, and T. J. Rink, 1982, J. Cell Biol., 94:325-334). The method involves the measurement of fluorescence at 340nm excitation (134o), where dye fluorescence is dependent on Ca 2+, and at 360-nm excitation (136o), where dye fluorescence is independent of Ca 2+. The ratio of these two values (1340/1360) is thus related to the concentration of Ca 2+ but independent of dye concentration and can be used as a measure of [Ca2+]. To test the ratio method in the microscope, we measured [Ca2+]i in GH3 cells in monolayer culture. We found a resting [Ca2+]i of 44 + 28 nM (mean _ SD, n = 34), as compared with a suspension value (Gershengorn, M., and C. Thaw, 1983, Endocrinology, 113:1522-1524) of 118 + 18 nM. We also measured [Ca2÷]~ during stimulation of the cells with thyrotropin-releasing hormone (TRH) and found a 2.4-fold increase above resting levels within 20 s, a trough at 73% of resting at 90-100 s, and a peak slightly above resting at 3 min. Depolarization of the plasma membrane with KCI produced a sustained increase in [Ca2+]~. All of these data are in good agreement with the results of Gershengorn and Thaw on suspension cultures. When measuring both resting [Ca2+]i and the effects of TRH and KCI on small groups of cells, we found some variation among experiments. Using an image intensifier-video camera, we videotaped cells during TRH stimulation. Digital image analysis of these pictures demonstrated that there was a large variation in responsiveness from cell to cell. The microscope ratio method offers the possibility of resolving regions of differing [Ca 2+] within the tLytoplasm. C a 2+ has been implicated as the mediator of numerous important cellular processes, acting as a second messenger for various hormones and growth factors (l, 10), as a modulator of differentiation (3), in stimulus-contraction coupling in muscle (8), and in stimulus-secretion coupling in neurons and secretory cells (4). It is thus important to have accurate and practical methods for measuring intracellular C a 2+ concentration ([Ca2+]i) and its changes in response to a variety of physiologic and pharmacologic stimuli. There are several methods for measuring [Ca 2÷] that are currently in use. These methods have provided valuable information, but each is subject to experimental limitations. For example, Ca2+-sensi tive microelectrodes can only be used in cells large enough to be impaled, and they only monitor [Ca 2÷] near the electrode tip. Also, impalement with a microelectrode may perturb the cell's calcium homeostasis (17). Photoproteins (e.g., aequorin) and bis-azo dyes (e.g., arsenazo III) share two disadvantages: (a) They can only be introduced into cells by disruptive methods (such as microinjection or osmotic lysis [7]). (b) They have limited specificity for Ca 2÷, since they are affected by Mg 2÷, ionic strength, temperature (photoproteins), and pH (bis-azo dyes) (17). One of the best [Ca2+]i measurement methods currently in use employs fluorescent tetracarboxylate dyes that bind Ca 2÷ with consequent changes in fluorescence emission intensity (15). The most widely used is quin2, which can be loaded Abbreviations used in this paper, quin2, methoxyquinoline derivative of bis (o-aminophenoxyl)ethane-N,N,N',N'-tetraacetic acid; quin2/AM, tetraacetoxymethyl ester of quin2; BSS, balanced salt solution; TRH, thyrotropin-releasing hormone; 134o, intensity of fluorescence emission at 492 nm with 340-nm excitation; I36o, intensity of fluorescence emission at 492 nm with 360-nm excitation; R, the ratio 1~o/I36o. THE JOURNAL OF CELL BIOLOGY VOLUME 99 SEPTEMBER 1984 1167 1172 © The Rockefeller University Press 0021-9525/84/09/1167/'06 $1.0