Inhibition of calcineurin phosphatase activity by the immunosuppressants CyA and FK506 causes both the therapeutic and toxic effects of these drugs. The primary objective of this proposal is to determine how parameters which vary from cell to cell affect CyA and FK506 potency as calcineurin inhibitors. A second objective is to determine if differential abilities to compensate for calcineurin inhibition contribute to the cell type-specific sensitivity to toxic effects of CyA and FK506. In the long term, this information will help reduce the number and severity of CyA/FK506 toxic side effects and expand the usefulness of immunosuppressive therapy with calcineurin inhibitors. Cyclosporin A (CyA) and FK506 have dramatically increased the success rate for graft survival after transplantation. These drugs are used to support virtually every transport performed today, despite severe toxicity of the drugs which can result in withdrawal of treatment and place the graft and patients at risk. Both the therapeutic and toxic effects of these drugs are cell type and tissue selective, with cell type specific differences in potency of CyA/FK506 as calcineurin inhibitors predicted to contribute to the selectivity. Calcineurin inhibition by CyA/FK506 is mechanistically complex, being dependent on the concentrations of both the drug and its protein cofactor (referred to as an immunophilin) in the inhibition. The active species for calcineurin inhibition is a drug-immunophilin complex which binds to calcineurin in a Ca2+-dependent manner. The hypothesis for this study is that the cellular content of immunophilin, and the duration and amplitude of Ca2+ transients, determine the potency of CyA/FK506 as calcineurin inhibitors. It is further hypothesized that CyA toxicity is dependent both on the degree of calcineurin inhibition and the ability of the cell to compensate for loss of calcineurin activity.
Specific aims of this project are: I. To determine if calcineurin binding to CyA-cyclophilin and FK506- FKPB complexes is supported by the Ca2+ concentrations present in non- active cells. II. To quantitate the cyclophilin concentration dependence of calcineurin inhibition by CyA and determine if the cell content of cyclophilin or the type of cyclophilin in the cell affect CyA potency. III. To measure the capacity of tissues and cells to compensate for calcineurin inhibition and the identify unique calcineurin substrates in cells which are sensitive to CyA. It is anticipated that an understanding of the mechanisms underlying calcineurin inhibition will clarify why certain organs are targets for the toxic effects of immunosuppressants, will reveal more and better sites for therapeutic intervention and will indicate which adjuvant therapies might be most successful in ameliorating the CyA/FK506 side effects.
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