Immunophilin regulation of neurotransmitter release

Background: The immunophilins are proteins that mediate actions of immunosuppressant drugs such as FK506 and cyclosporin A by binding to calcineurin, inhibiting its phosphatase activity, and increasing the phosphorylation level of transcription factors required for interleukin 2 formation. Though concentrations in the brain greatly exceed levels in immune tissues, no function has been previously established for nervous system immunophilins. Nitric oxide (NO) has been implicated in neurotransmitter release. FK506 appears to inhibit NO production by maintaining NO synthase in a highly phosphorylated and thereby inactivated state. Accordingly, we examined effects of FK506 and cyclosporin A on neurotransmitter release in PC12 cells treated with nerve growth factor (NGF) and in rat brain stratal synaptosomes. Materials and Methods: We monitored effects of immunophilin ligands on [H-3]-neurotransmitter release from PC12 cells differentiated with NGF. Rat brain striatal synaptosomes were loaded with radiolabeled transmitters and treated with FK506 or cyclosporin A prior to initiating neurotransmitter release with N-methyl-D-aspartate (NMDA) or potassium depolarization. Striatal synaptosomes were also loaded with P-32-orthophosphate and treated with FK506. P-32-labeled synaptic vesicle proteins were isolated from these synaptosomes in an attempt to relate specific FK506-dependent phosphorylation of vesicle proteins with the effects of FK506 on neurotransmitter release. Identification of proteins targetted by FK506 was made by immunoblot analysis and immunoprecipitation. Results: Low nanomolar concentrations of the immunosuppressant drugs FK506 and cyclosporin A (CsA) inhibit transmitter release from PC-12 cells and from NMDA-stimulated brain synaptosomes. By contrast, the immunosuppressants augment depolarization-induced transmitter release from synaptosomes. synapsin I, a synaptic vesicle phosphoprotein, displays enhanced phosphorylation in the presence of FK506. Conclusions: Inhibition of transmitter release in PC-12 cells and NMDA-treated synaptosomes by immunosuppressants may reflect augmented phosphorylation of NO synthase, reducing its catalytic activity. This fits with the requirement of NO for transmitter release in PC12 cells and NMDA-treated synaptosomes. Stimulation by immunosuppressants of transmitter release in potassium depolarized synaptosomes may result from augmented phosphorylation of synapsin I, whose phosphorylation is known to facilitate transmitter release. Thus, immunophilins may modulate release of numerous neurotransmitters both by influencing NO formation and the phosphorylation state of synaptic vesicle-associated proteins.