Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
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Cell Culture:

Rat glioma C6 cells were kindly provided by the Japan Cancer Research Bank (Tokyo, Japan) and grown in Ham's F10 medium supplemented with 15% horse serum and 2.5% fetal calf serum as described previously (22).

Measurement of [Ca2+]i:

C6 cells were incubated with 2 microM fura-2/AM in Krebs-Ringer-Hepes medium (KRH) containing 0.2% bovine serum albumin for 45 min at 37��. The composition of KRH was as follows: 120 mM NaCl, 5.4 mM KCl, 1.0 mM CaCl2, 0.8 mM MgCl2, 11.1 mM glucose, 20 mM Hepes, (pH 7.4). After loading with fura-2, C6 cells were detached from dishes with 5 mM EDTA in Puck's solution, washed, and then resuspended with fresh KRH as described previously (23). Measurement of [Ca2+]i was performed as described previously (22). Briefly, fluorescence of the fura-2-loaded cell suspension was monitored with a Jasco CAF-110 (Japan Spectroscopic Co., Tokyo, Japan) in a cuvette at 35��. The excitation wavelengths were 340 and 380 nm and emission was measured at 510 nm. [Ca2+]i was estimated as described by Grynkiewicz et al. (1986).

Measurement of IP3:

Measurement of IP3 was performed as described previously (24). Briefly, C6 cells were passed on to 35 mm plastic tissue culture dishes and grown in a monolayer for 2 days. The dishes were washed twice with KRH and cells were preincubated with KRH containing 10 mM LiCl for 10 min at 37 C. The mixture after addition of drugs was further incubated for 30 s. The reaction was then terminated by addition of an equal volume of 15% trichloroacetic acid. The preparations were kept on ice for 30 min and then centrifuged. The supernatant was washed three times with 10 volumes of water-saturated diethyl ether to remove trichloroacetic acid from the solution. The resultant solutions were neutralized by titration of pH 7.5 with NaHCO3. After centrifugation, 100 l of supernatant was assayed using an IP3 assay kit (Amersham).

Preparation of permeabilized cells:

Permeabilized C6 cells were prepared as described previously (25). Briefly, C6 cells were detached from dishes with 5 mM EDTA in Puck's solution, and were washed and incubated with buffer composed of 110 mM KCl, 10 mM NaCl, 2 mM MgCl2, 5 mM KH2PO4, 2 mM EGTA, 1 mM dithiothreitol and 20 mM Hepes (pH 7.2), in the presence of saponin (30 microg/ml) for 5 min at 37��. After centrifugation, the cell pellet was washed with an intracellular buffer composed of 110 mM KCl, 10 mM NaCl, 2 mM MgCl2, 5 mM KH2PO4 and 20 mM Hepes (pH 7.2). The final pellet was resuspended (about 5�~107 cells/ml) in intracellular buffer containing creatine kinase (20 units/ml), phosphocreatine (20 mM) and oligomycin (10 microg/ml). The fluorescence of fura-2 free acid (1 microM) was monitored as described in Measurement of [Ca2+]i. When fluorescence declined to a stable state after subsequent addition of ATP (1 mM), drugs were added.

Ca2+ entry into cells:

Ca2+ entry into cells was measured as described previously (25). Briefly, fura-2-loaded cells were washed twice with Ca2+-free KRH containing 0.2 mM EGTA and then transferred to the cuvette. When the elevation of [Ca2+]i induced by drugs reverted to the resting level, 3.2 mM Ca2+ was added to the extracellular medium.

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