tacrolimus and cypermethrin

The immunosuppressive agents cyclosporin A (CsA) and FK-506 have previously been shown to exhibit neurotrophic and neuroprotective properties in vivo. Given that significant clinical expertise exists for both drugs, they represent an attractive starting point for treatment of acute neural injuries. One putative mechanism for neuroprotection by these drugs relates to inhibition of calcineurin activity. However each drug-immunophilin complex can potentially influence additional signal transduction pathways. Furthermore, several non-immunosuppressive immunophilin ligands have been described as possessing neuroprotective properties, suggesting that neuroprotection may be separable from calcineurin inhibition. In the present study, we examined the mechanism of this neuroprotection in facial motor neurons following axotomy-induced injury. Similar to previous studies in rats, CsA and FK-506 enhanced motor neuron survival in mice following acute injury. To examine the mechanism responsible for neuroprotection by these agents, pharmacologic inhibitors of several potential alternate signalling pathways (17-(allylamino)-17-demethoxygeldanamycin, rapamycin, cypermethrin) were evaluated with respect to neuroprotection. Of these, only cypermethrin, a direct calcineurin inhibitor not previously associated with neuronal survival properties, was observed to significantly enhance motor neuron survival following injury. The results demonstrate for the first time that direct inhibition of calcineurin is neuroprotective in vivo. These data support a model in which calcineurin inhibition promotes neuronal survival, distinct from effects upon neurite outgrowth.

Topics: Animals; Animals, Newborn; Axotomy; Calcineurin; Calcineurin Inhibitors; Caspase 3; Cell Survival; Cyclosporine; Enzyme Activation; Immunohistochemistry; Immunosuppressive Agents; Insecticides; Mice; Mice, Inbred ICR; Mice, Inbred Strains; Mice, Knockout; Microscopy, Fluorescence; Motor Neurons; Neuroprotective Agents; Pyrethrins; Signal Transduction; Tacrolimus

The interaction of FK-506 with K(V)1.3, stably expressed in Chinese hamster ovary cells, was investigated with the whole cell patch-clamp technique. FK-506 inhibited K(V)1.3 in a reversible, concentration-dependent manner with an IC(50) of 5.6 microM. Rapamycin, another immunosuppressant, produced effects that were similar to those of FK-506 (IC(50) = 6.7 microM). Other calcineurin inhibitors (cypermethrin or calcineurin autoinhibitory peptide) alone had no effect on the amplitude or kinetics of K(V)1.3. In addition, the inhibitory action of FK-506 continued, even after the inhibition of calcineurin activity. The inhibition produced by FK-506 was voltage dependent, increasing in the voltage range for channel activation. At potentials positive to 0 mV (where maximal conductance is reached), however, no voltage-dependent inhibition was found. FK-506 exhibited a strong use-dependent inhibition of K(V)1.3. FK-506 shifted the steady-state inactivation curves of K(V)1.3 in the hyperpolarizing direction in a concentration-dependent manner. The apparent dissociation constant for FK-506 to inhibit K(V)1.3 in the inactivated state was estimated from the concentration-dependent shift in the steady-state inactivation curve and was calculated to be 0.37 microM. Moreover, the rate of recovery from inactivation of K(V)1.3 was decreased. In inside-out patches, FK-506 not only reduced the current amplitude but also accelerated the rate of inactivation during depolarization. FK-506 also inhibited K(V)1.5 and K(V)4.3 in a concentration-dependent manner with IC(50) of 4.6 and 53.9 microM, respectively. The present results indicate that FK-506 inhibits K(V)1.3 directly and that this effect is not mediated via the inhibition of the phosphatase activity of calcineurin.

Topics: Animals; Calcineurin; Calcineurin Inhibitors; CHO Cells; Cricetinae; Cricetulus; Dose-Response Relationship, Drug; Immunosuppressive Agents; Kinetics; Kv1.3 Potassium Channel; Kv1.5 Potassium Channel; Membrane Potentials; Patch-Clamp Techniques; Potassium Channel Blockers; Pyrethrins; Shal Potassium Channels; Sirolimus; Tacrolimus; Transfection

The endoplasmic reticulum (ER) serves as a critical site of protein synthesis and processing. The temperature-sensitive hamster fibroblast cell line (tsBN7) displays deficient N-linked glycosylation activity at the restrictive temperature and activates cellular apoptosis. Temperature-shifted tsBN7 cells display induction of Grp78 and Gadd153, genes known to be induced by ER stress, and activate apoptosis via the release of cytochrome c from the mitochondria. Cyclosporin A (CsA), a potent blocker of the mitochondrial permeability transition pore (PTP), was sufficient to block cytochrome c release and to rescue tsBN7 cells from apoptosis. CsA-treated cells displayed Grp78 induction at the restrictive temperature, consistent with an ER stress signal being carried to the nucleus, while the apoptosis-associated transcription factor, Gadd153, displayed only a mild induction. Cypermethrin, a type II pyrethroid known to perturb Ca(2+) signaling in neuronal cells, was sufficient to arrest apoptosis under these conditions. This work identifies type II pyrethroids as a valuable new tool in the characterization of cellular stress signaling pathways.

Topics: Animals; Apoptosis; Blotting, Western; Caspase 12; Caspase Inhibitors; Caspases; CCAAT-Enhancer-Binding Proteins; Cell Line; Cell Survival; Cricetinae; Cyclosporine; Cytochromes c; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Fibroblasts; Glycosylation; Heat-Shock Proteins; In Situ Nick-End Labeling; Insecticides; Ion Channels; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase 4; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Mitogen-Activated Protein Kinase Kinases; Molecular Chaperones; Nitriles; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Pyrethrins; Signal Transduction; Tacrolimus; Temperature; Thapsigargin; Transcription Factor CHOP; Transcription Factors; Tunicamycin; Ultraviolet Rays

1 The effects of the immunosuppressants, tacrolimus (FK506) and cyclosporin A (CsA), on catecholamine (CA) release were examined in cultured bovine adrenal chromaffin cells. 2 In intact cells, FK506 (1-30 microM) inhibited CA release stimulated by acetylcholine (ACh; 100 microM), 1,1-dimethyl-4-phenyl-piperazinium (DMPP, 10 microM) or high K+ (40 mM). CsA (1-30 microM) had a little inhibitory effect on the ACh- or DMPP-stimulated CA release, whereas it enhanced the high K(+)-stimulated CA release. 3 In beta-escin-permeabilized cells, FK506 inhibited CA release stimulated by Ca2+ (1 and 10 microM) in the presence and absence of MgATP (2 mM). CsA induced CA release under Ca(2+)-free condition and enhanced the Ca(2+)-stimulated CA release in the presence and absence of MgATP. 4 It is known that the Ca(2+)-dependent exocytosis involves at least two distinct steps, ATP-requiring priming stage and ATP-independent fusion step in adrenal chromaffin cells. Therefore, it is suggested that FK506 inhibits the Ca(2+)-dependent exocytosis probably at the fusion step whereas CsA induces CA release from bovine adrenal chromaffin cells.

Topics: Acetylcholine; Animals; Catecholamines; Cattle; Chromaffin Cells; Cyclosporine; Dimethylphenylpiperazinium Iodide; Escin; Immunosuppressive Agents; In Vitro Techniques; Nicotinic Agonists; Potassium; Pyrethrins; Tacrolimus

While effector molecules produced by activated macrophages (including nitric oxide, tumor necrosis factor alpha, interleukin 1, etc.) help to eliminate pathogens, high levels of these molecules can be deleterious to the host itself. Despite their importance, the mechanisms modulating macrophage effector functions are poorly understood. This work introduces two key negative regulators that control the levels and duration of macrophage cytokine production. Vacuolar-type H+-ATPase (V-ATPase) and calcineurin (Cn) constitutively act in normal macrophages to suppress expression of inflammatory cytokines in the absence of specific activation and to inhibit macrophage cytokine responses induced by bacterial lipopolysaccharide (V-ATPase), interferon gamma (V-ATPase and Cn), and calcium (Ca2+) flux (Cn). Cn and V-ATPase modulate effector gene expression at the mRNA level by inhibiting transcription factor NF-kappaB. This negative regulation by Cn is opposite to its crucial positive role in T cells, where it activates NFAT transcription factor(s) leading to expression of interleukin 2, tumor necrosis factor alpha, and other cytokine genes. The negative effects of V-ATPase and Cn on NF-kappaB-dependent gene expression are not limited to the macrophage lineage, as similar effects have been seen with a murine fibroblast cell line and with primary astrocytes.

Topics: Animals; Astrocytes; Calcineurin; Cell Line; Cells, Cultured; Cytokines; DNA-Binding Proteins; Gene Expression Regulation; Insecticides; Interferon-gamma; Interleukin-12; L Cells; Lipopolysaccharides; Macrophages, Peritoneal; Mice; Mice, Inbred Strains; NF-kappa B; NFATC Transcription Factors; Nuclear Proteins; Proton-Translocating ATPases; Pyrethrins; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; T-Lymphocytes; Tacrolimus; Thapsigargin; Transcription Factors; Tumor Necrosis Factor-alpha; Vacuolar Proton-Translocating ATPases

S100betabeta is a calcium binding, neurotrophic protein produced by nonneuronal cells in the nervous system. The pathway by which it enhances neuronal survival is unknown. Here we show that S100betabeta enhances survival of embryonic chick forebrain neurons in a dose-dependent manner. In the presence of suboptimal amounts of S100betabeta, neuronal survival is enhanced by the immunosuppressants FK506 and cyclosporin A at concentrations that inhibit calcineurin, which is present in these cells. Rapamycin, an immunosuppressant that does not inhibit calcineurin, did not enhance cell survival. Cypermethrin, a direct and highly specific calcineurin inhibitor, mimicked the immunophilin ligands in its neurotrophic effect. None of the drugs stimulated neuronal survival in the absence of S100betabeta. In the presence of suboptimal amounts of S100betabeta, FK506, cyclosporin A, and cypermethrin (but not rapamycin) also increased NF-kappaB activity, as measured by immunofluorescence of cells stained with antibody to the active subunit (p65) and by immunoblotting of nuclear extracts. Antioxidant and glucocorticoid inhibitors of NF-kappaB decreased both the amount of active NF-kappaB and the survival of neurons caused by S100betabeta alone or in the presence of augmenting drugs. We conclude that S100betabeta enhances the survival of chick embryo forebrain neurons through the activation of NF-kappaB.

Topics: Animals; Antioxidants; Brain Chemistry; Calcineurin Inhibitors; Calcium-Binding Proteins; Cattle; Cell Nucleus; Cell Survival; Cells, Cultured; Chick Embryo; Coumarins; Cyclosporine; Dexamethasone; Dimerization; Immunophilins; Immunosuppressive Agents; Isocoumarins; Macromolecular Substances; Nerve Growth Factors; Neurons; NF-kappa B; Prosencephalon; Pyrethrins; Pyrrolidines; S100 Calcium Binding Protein beta Subunit; S100 Proteins; Serine Proteinase Inhibitors; Sirolimus; Tacrolimus; Thiocarbamates