| In Vitro | In vitro activity: Cyclosporin A binds to the cyclophilin (immunophilin) in T cells, forms a Cyclophilin-Cyclosporin A complex which the binds to and inhibits calcineurin. Cyclosporin A inhibits calcineurin with IC50 of 7 nM, then blocks the nuclear translocation of NF-AT. Cyclosporin A also affects mitochondria by preventing the MTP (mitochondrial permeability transition pore) from opening with an IC50 of 39 nM.
Kinase Assay: Purified bovine brain calcineurin and calmodulin are purchased. Reaction mixtures with purified enzyme contains 100 nM calcineurin, 100 nM calmodulin, and 5 μM 32P-labeled phosphopeptide, in 60 μl (total volume) of assay buffer containing 20 mM Tris (pH 8), 100 mM NaCI, 6 mM MgCl2, 0.5 mM dithiothreitol, 0.1 mg of bovine serum albumin per ml, and either 0.1 mM CaCl2 or 5 mM EGTA. Reaction mixtures with cell lysates contains 20 μl of undiluted lysate, 5 μM 32P-labeled phosphopeptide, and 40 μl of assay buffer. Where indicated, reaction mixtures contains 50 μM peptide 412 or 413 and/or 500 nM okadaic acid, a specific inhibitor of phosphatases 1 and 2A; 500 nM okadaic acid is sufficient for inhibition of Ca2+-independent phosphatases, whereas higher concentrations partially inhibit Ca2+-dependent activity as well. After 15 min at 30°C, reactions are terminated by the addition of 0.5 ml of 100 mM potassium phosphate buffer (pH 7.0) containing 5% trichloroacetic acid. Free inorganic phosphate is isolated by Dowex cation-exchange chromatography and quantitated by scintillation counting as described.
Cell Assay: Immunosuppressive agents are dissolved in ethanol at concentrations 1000-fold more than the concentration desired for cell treatments. Cells (106) are suspended in 1 ml of complete medium in microcentrifuge tubes; 1 μl of ethanol or of the ethanolic solution of Cyclosporin A is added, and the cells are incubated at 37°C for 1 hr. Cells are washed twice with 1 ml of PBS on ice and lysed in 50μl of hypotonic buffer containing 50 mM Tris (pH 7.5); 0.1 mM EGTA; 1 mM EDTA; 0.5 mM dithiothreitol; and 50 μg of phenylmethylsulfonyl fluoride, 50 μg of soybean trypsin inhibitor, 5 μg of leupeptin, and 5 μg of aprotinin per ml. Lysates are subjected to three cycles of freezing in liquid nitrogen followed by thawing at 30°C and then are centrifuged at 4°C for 10 min at 12,000×g. |
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