Technical Reference #422

422.

Effect of FK506 on ATP-induced intracellular calcium oscillations in cow tracheal epithelium Soichiro Kanoh; Mitsuko Kondo; Jun Tamaoki; Hiseki Shirakwa; Kazutetsu Aoshiba; Shunichi Miyazaki; Hideo Kobayashi; Naojazu Nagata; Atsushi Nagai, National Defense Medical College, American Journal of Physiology: Lung Cellular and Molecular Physiology, 276(422), (1999)
Link To Paper

Abstract:
To elucidate the effect of FK506 on Ca21 oscillations inairway epithelium we investigated cultured cow trachealepithelial cells with a Ca21 image-analysis system. ATP (1μM) induced long-lasting Ca21 oscillations having nearlyconstant peak values

Keywords:
airway epithelial; adenosine 5' triphosphate; FK506-binding protein; rapamycin; cyclosporin

Materials & Methods:
Cell culture. Cow tracheae were obtained from a slaughterhouse and tracheal epithelial cells were isolated by protease as previously described (15). Briefly strips of epithelium were pulled off the submucosa washed four times with phosphatebuffered saline (PBS) containing 5 mM dithiothreitol and rinsed two times with PBS. Epithelial tissues were digested with PBS containing 0.05% protease at 4°C overnight. After neutralization of the protease with 5% fetal calf serum (FCS) the cells were pelleted (200 g for 10 min) and suspended in 50% Dulbecco’s modified Eagle’s medium (DMEM) and 50% Ham’s F-12 nutrient mixture that contained 5% FCS 1% nonessential amino acids 100 U/ml of penicillin 100 μg/ml of streptomycin and 50 μg/ml of gentamicin. The isolated cells were plated at a density of 1.0 3 105 cells/cm2 on a glassbottomed petri dish (MatTek Ashland MA) coated with human placental collagen. The medium was changed every 2 days. The cells were cultured for 3–5 or 7–8 days to a subconfluent or confluent stage respectively to observe the difference in ATP-induced Ca21 responses between these different culture conditions. Measurement of [Ca21]i in single cells. The dish on which the cells were grown was washed with Hanks’ balanced salt solution (HBSS) that contained 10 mM HEPES pH 7.4 and was loaded with 10 μM fura 2-AM for 1 h at 37°C. The dish was then washed several times with HEPES-buffered HBSS and mounted on the stage of an inverted microscope (Diaphot 300 Nikon Tokyo Japan). The temperature was kept at 37°C by a ring heater surrounding the dish. For excitation of fura 2 fluorescence ultraviolet (UV) light of 340- or 380-nm wavelength was produced by a xenon lamp and narrow band-pass filters and applied to the cells through a 340 objective lens (Fluor 40 Nikon). Emission fluorescence (F) was led to a silicon-intensifier target camera through a 510 6 10-nm band-pass filter. Ca21 images of the cells were obtained at 3- or 4-s intervals unless otherwise indicated by alternately applying 340- and 380-nm UV light for 0.125 s (four video frames) for each. Data sets were stored on the hard disk of the computer as eight-bit digital images (256 3 256 pixels) and processed to calculate the ratio of 340- to 380-nm fluorescence later. The averaged values of the ratios in individual cells were obtained in an optical field in which 40 cells were sampled simultaneously. A calibration curve between the ratio and [Ca21]i was obtained by measuring the ratios of Ca21-N-(2-hydroxyethyl)EDTA buffer solutions. All these procedures were performed with an image processor (Argus-50/CA system Hamamatsu Photonics Hamamatsu Japan) (10).

Microscopic Technique
Electron Microscopy, Light Microscopy

Cell Type(s)
tracheal epithelial