F 2000

We measured the intracellular calcium levels using fura-2, a fluorescent probe, as described previously^{44 (link)}. Changes in fluorescence were recorded using a fluorescence spectrofluorometer (F-2000; Hitachi, Tokyo, Japan). The [Ca²⁺]i values were estimated according to our previous report^{44 (link)}. Background autofluorescence obtained from the unloaded cells was subtracted from all measurements. Additionally, the inhibitor effectiveness was compared after a 30-min pretreatment.

Measurement of intestinal transit was performed as a terminal experiment. Mice were fasted for 16 h with water available ad libitum. At 30 minutes prior to euthanasia, 0.1 ml of 0.5 mmol/L 70 kDa fluorescein isothiocyanate (FITC)–dextran (Sigma-Aldrich, St. Louis, MO) was administered by oral gavage [26] (link), [27] (link). After 30 minutes, mice were euthanized and the entire gastrointestinal tract, from stomach to anus, was removed. The small intestines were cut into 8 segments of equal length from the stomach to the cecum. The stomach and each segment were flushed with 3 ml of 50 mmol/l Tris buffered saline, the effluent was collected and centrifuged at 1200 rpm for 5 minutes to pellet any suspended debris. Fluorescent intensity in each sample was measured using a fluorescence spectrophotometer (emission wavelength  = 518 nm; excitation wavelength  = 492 nm; F-2000, Hitachi, Inc., Japan). Intestinal geometric center (IGC) was calculated as: (fraction of amount of FITC in each segment) × (segment number). The dextran concentration in each segment was expressed as a fraction of total dextran recovery.

The changes in the intracellular calcium concentration were detected using the fluorescent probe fura-2 [14 (link)]. NISCH-CHO-K1 cells were placed in a buffered physiological saline solution (PSS) as described previously [13 (link)]. Fura 2 (5 mM) was added to 1 mL of the cell suspension (1 × 10⁶ cells) and incubated for 30 min at 37 °C in the dark. The fluorescence was continuously recorded using a fluorescence spectrofluorometer (Hitachi F-2000, Tokyo, Japan). Values of [Ca²⁺]i were then determined, and the background measured in unloaded cells was subtracted from all measurements according to our previous report [13 (link)].

Based on the protocol of Uchiyama and Mihara [45 (link)], the samples were homogenized in 1.15% KCl solution, then mixed with 2-thiobarbitutric acid, followed by incubation in a boiling water bath for 45 min, and then cooled. After centrifugation at 1600× g, 4 ℃ for 10 min, the samples were kept at room temperature for 30 min, and then the supernatants were collected and measured using a Luminescence Spectrometer (Hitachi, F2000, Tokyo, Japan) with excitation at 520 nm and emission at 535 nm.

The fluorescence emission spectra of aromatic amino acids (AAA) in spinach chloroplasts were recorded on fluorescence spectrophotometer F-2000 (Hitachi, Tokyo, Japan) using excitation wavelength λ_ex = 275 nm, excitation slit 20 nm, and emission slit 10 nm. The phosphate buffer used for dilution of the chloroplast suspension was the same as described above. Due to low aqueous solubility the compounds were added to the chloroplast suspension in DMSO solution. The DMSO concentration in all samples was the same as in the control (10%). The chlorophyll concentration in the chloroplast suspension was 10 mg/L.