Sodium Selenite → 2-(4-(Bis(carboxymethyl)amino)-3-(2-(2-(bis(carboxymethyl)amino)-5-methylphenoxy)ethoxy)phenyl)-1H-indole-6-carboxylic acid

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Reaction Steps

Culture conditions were as described (D. G. Yoder, T. W. Moody, Peptides, 1987, 8:103, except that the medium was modified to RPMI 1640 with 2.5% fetal bovine serum (heat inactivated), 5 mg/l sodium selenite, 5 mg/l human transferrin, 5 mg/l insulin, penicillin (100 U/l) and streptomycin (100 mg/l). To measure intracellular Ca++, cells were incubated with 1 μM indo-1/AM for 60 minutes at 37° C. The cells were then centrifuged at 750×g for 5 minutes and resuspended in 40 mL Buffer F (Dulbecco's phosphate buffered saline containing 0.1% glucose, pH 7.4). The cells were counted then twice washed by centrifugation. The final pellet was resuspended (approximately 1×106 cells/mL) in Buffer F. The cells were thermally equilibrated in a thermostated cuvette (37° C.) for 2 minutes prior to base-line fluorescence measurement. Fluorescence measurements were made with an SLM 8000C spectrofluorimeter equipped with magnetic microstirrer and polarizing filters. Measurements with indo-1-loaded cells were obtained at excitation wavelength of 350 nM and emission wavelength of 480 and 405 nM and the time interval for the measurements was 0.5 seconds. The test compounds were assayed at a single concentration (10-5M) and compared to the response elicited by CCK-8 at 10-6M to determine the % maximal response. Calibrations of [Ca++ ]i were done as described (G. Grynkiewicz, M. Poenie, R. Y. Tsien, J. Biol. Chem., 1985, 260: 3440: [Ca++ ]i =Kd[(R-Rmin)/(Rmax -R)](F480min /F480max) where Rmin and Rmax were the ratios (480/405) obtained in the presence of excess EGTA (10 mM) and digitonin (50 mM), respectively. F480min was the fluorescence value of the calcium-free dye at 480 nm, and F480max was the fluorescence value of the calcium-bound dye at 480 nm. Kd is assumed to be 240 nM, and R is the ratio in the presence and the absence of CCK. Basal calcium levels were 147±3 nM (N=3) and the maximal levels of calcium stimulated with 100 nM CCK-8 were 357±30 nM (N=3). The results of these assays are shown in Table 2.

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