tacrolimus and decamethrin

Calcineurin inhibitors are used in immunosuppressive therapy applied after transplantation, but they are associated with major metabolic side effects including the development of new onset diabetes. Previously, we have shown that the calcineurin inhibiting drugs tacrolimus and cyclosporin A reduce adipocyte and myocyte glucose uptakes by reducing the amount of glucose transporter type 4 (GLUT4) at the cell surface, due to an increased internalization rate. However, this happens without alteration in total protein and phosphorylation levels of key proteins involved in insulin signalling or in the total amount of GLUT4. The present study evaluates possible pathways involved in the altered internalization of GLUT4 and consequent reduction of glucose uptake provoked by calcineurin inhibitors in human subcutaneous adipose tissue. Short- and long-term treatments with tacrolimus, cyclosporin A or another CNI deltamethrin (herbicide) decreased basal and insulin-dependent glucose uptake in adipocytes, without any additive effects observed when added together. However, no tacrolimus effects were observed on glucose uptake when gene transcription and protein translation were inhibited. Investigation of genes potentially involved in GLUT4 trafficking showed only a small effect on ARHGEF11 gene expression (p < 0.05). In conlusion, the specific inhibition of calcineurin, but not that of protein phosphatases, decreases glucose uptake in human subcutaneous adipocytes, suggesting that calcineurin is an important regulator of glucose transport. This inhibitory effect is mediated via gene transcription or protein translation; however, expression of genes potentially involved in GLUT4 trafficking and endocytosis appears not to be involved in these effects.

Topics: Adipocytes; Adult; Aged; Calcineurin; Calcineurin Inhibitors; Cell Membrane; Cyclosporine; Endocytosis; Female; Gene Expression Profiling; Glucose; Glucose Transporter Type 4; Humans; Insulin; Male; Middle Aged; Nitriles; Phosphoprotein Phosphatases; Phosphorylation; Protein Biosynthesis; Pyrethrins; Signal Transduction; Subcutaneous Fat; Tacrolimus; Transcription, Genetic

Glucocorticoid excess induces hyperglycemia, which may result in diabetes. The present experiments explored whether glucocorticoids trigger apoptosis in insulin-secreting cells. Treatment of mouse beta-cells or INS-1 cells with the glucocorticoid dexamethasone (0.1 micromol/l) over 4 days in cell culture increased the number of fractionated nuclei from 2 to 7 and 14%, respectively, an effect that was reversed by the glucocorticoid receptor antagonist RU486 (1 micromol/l). In INS-1 cells, dexamethasone increased the number of transferase-mediated dUTP nick-end labeling-staining positive cells, caspase-3 activity, and poly-(ADP-) ribose polymerase protein cleavage; decreased Bcl-2 transcript and protein abundance; dephosphorylated the proapoptotic protein of the Bcl-2 family (BAD) at serine155; and depolarized mitochondria. Dexamethasone increased PP-2B (calcineurin) activity, an effect abrogated by FK506. FK506 (0.1 micromol/l) and another calcineurin inhibitor, deltamethrin (1 micromol/l), attenuated dexamethasone-induced cell death. The stable glucagon-like peptide 1 analog, exendin-4 (10 nmol/l), inhibited dexamethasone-induced apoptosis in mouse beta-cells and INS-1 cells. The protective effect of exendin-4 was mimicked by forskolin (10 micromol/l) but not mimicked by guanine nucleotide exchange factor with the specific agonist 8CPT-Me-cAMP (50 micromol/l). Exendin-4 did not protect against cell death in the presence of cAMP-dependent protein kinase (PKA) inhibition by H89 (10 micromol/l) or KT5720 (5 micromol/l). In conclusion, glucocorticoid-induced apoptosis in insulin-secreting cells is accompanied by a downregulation of Bcl-2, activation of calcineurin with subsequent dephosphorylation of BAD, and mitochondrial depolarization. Exendin-4 protects against glucocorticoid-induced apoptosis, an effect mimicked by forskolin and reversed by PKA inhibitors.

Topics: Animals; Cell Line; Dexamethasone; Exenatide; Humans; Insecticides; Insulin; Insulin Secretion; Islets of Langerhans; Kinetics; Lizards; Mice; Microscopy, Fluorescence; Mifepristone; Nitriles; Peptides; Pyrethrins; Tacrolimus; Venoms

Depolarization has been known to play an important role in the neuronal damage that occurs following cerebral ischemia. In the present study, we investigated the roles of calmodulin (CaM) and CaM-dependent enzymes in depolarization-induced neuronal cell death. Treatment of primary cortical neurons with 10 microM veratridine, a voltage sensitive Na(+) channel activator, induced cell death as indicated by lactate dehydrogenase leakage from neurons. CaM antagonists (calmidazolium, trifluoperazine, W-7, and W-5) inhibited cell death induced by veratridine in a concentration-dependent manner. CaM kinase II (CaMKII) inhibitors (KN-62, KN-93, and myristoylated autocamtide-2 related inhibitory peptide), but not inhibitors of nitric oxide synthase or calcineurin, prevented veratridine-induced neuronal cell death. Veratridine rapidly activated CaMKII in neurons, and CaM antagonists and a CaMKII inhibitor suppressed the CaMKII activation. These results suggest that the CaM-CaMKII pathway contributes to depolarization-evoked cell death in neurons.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Benzylamines; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Calmodulin; Cell Death; Cells, Cultured; Cerebral Cortex; Egtazic Acid; Enzyme Inhibitors; Fetus; Kinetics; Membrane Potentials; Neurons; NG-Nitroarginine Methyl Ester; Nifedipine; Nitriles; Pyrethrins; Rats; Sulfonamides; Tacrolimus; Trifluoperazine; Veratridine

The present study was undertaken to examine the physical and physiological interaction of protein phosphatase 2B, calcineurin, with the ryanodine receptor (RyR) in rat cardiac tissue and neonatal cardiomyocytes. The presence of calcineurin, the RyR and FK506-binding protein (FKBP)12.6 in rat cardiac sarcoplasmic reticulum (SR) was identified by Western blot analysis. The possible interactions between calcineurin, the RyR and FKBP12.6 were further studied by co-immunoprecipitation using CHAPS-solubilized cardiac-membrane fractions (CSMFs) or SR preparations. Physical interactions between the RyR and calcineurin were found in the CSMF in the presence of added 100 microM Ca(2+); however, the interactions were interrupted in the presence of 20 mM EGTA, 1 microM rapamycin or 1 microM FK506, suggesting that the interaction is Ca(2+)-dependent, and is mediated by FKBP12.6. The Ca(2+)-dependent interaction between FKBP12.6 and the RyR was also found by co-immunoprecipitation. Effects of calcineurin inhibitors were tested on neonatal-rat-heart cardiomyocytes. Treatment of neonatal cardiomyocytes with 20 microM deltamethrin, 10 microM cyclosporin A (CsA), or 10 mciroM FK506 led to Ca(2+) oscillations in originally quiescent cardiomyocytes. Preincubation of cardiomyocytes with 20 microM rapamycin which dissociates FKBP12.6 from the RyR, evoked Ca(2+) oscillations, probably due to the leakiness of the RyR. However, Ca(2+) oscillations by rapamycin were not further affected by 10 microM CsA or 10 mciroM deltamethrin, suggesting that only RyR-associated calcineurin could regulate the channel activities. In spontaneously Ca(2+)-oscillating cardiomyocytes, CsA or FK506 treatments increased the frequency of oscillations. In 10 microM ryanodine-treated cardiomyocytes, CsA failed to induce Ca(2+) oscillations. These data show evidence that calcineurin associated with the RyR could modulate Ca(2+) release in rat heart.

Topics: Animals; Animals, Newborn; Blotting, Western; Calcineurin; Calcium; Calcium Channels; Cells, Cultured; Chelating Agents; Cholic Acids; Cyclosporine; Detergents; Egtazic Acid; Fluorescent Antibody Technique; Immunosuppressive Agents; Insecticides; Microscopy, Confocal; Myocardium; Nitriles; Phosphorylation; Precipitin Tests; Protein Binding; Pyrethrins; Rats; Rats, Sprague-Dawley; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Sirolimus; Tacrolimus; Tacrolimus Binding Proteins; Time Factors