tacrolimus and chelerythrine

We investigated the role of inositol 1,4,5-trisphosphate receptors (IP3Rs) activated by preconditioning low-frequency afferent stimulation (LFS) in the subsequent induction of long-term potentiation (LTP) in CA1 neurons in hippocampal slices from mature guinea pigs. Induction of LTP in the field excitatory postsynaptic potential or the population spike by the delivery of high-frequency stimulation (HFS, a tetanus of 100 pulses at 100 Hz) to the Schaffer collateral-commissural pathway to CA1 neuron synapses was suppressed when group I metabotropic glutamate receptors (mGluRs) were activated prior to the delivery of HFS. LTP induction was also suppressed when CA1 synapses were preconditioned 60 min before HFS by LFS of 1000 pulses at 1 Hz and this effect was inhibited when the test stimulation delivered at 0.05 Hz was either halted or applied in the presence of an antagonist ofN-methyl-d-aspartate receptors, group I mGluRs, or IP3Rs during a 20-min period from 20 to 40 min after the end of LFS. Furthermore, blockade of group I mGluRs or IP3Rs immediately before the delivery of HFS overcame the effects of the preconditioning LFS on LTP induction. These results suggest that, in CA1 neurons, after a preconditioning LFS, activation of group I mGluRs caused by the test stimulation results in IP3Rs activation that leads to a failure of LTP induction.

Topics: Animals; Benzoates; Benzophenanthridines; Biophysics; Boron Compounds; CA1 Region, Hippocampal; Dose-Response Relationship, Drug; Electric Stimulation; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Glycine; Guinea Pigs; Immunosuppressive Agents; In Vitro Techniques; Inositol 1,4,5-Trisphosphate Receptors; Long-Term Potentiation; Male; Methoxyhydroxyphenylglycol; Neurons; Tacrolimus

Altered gap junction coupling of cardiac myocytes during ischemia may contribute to development of lethal arrhythmias. The phosphoprotein connexin 43 (Cx43) is the major constituent of gap junctions. Dephosphorylation of Cx43 and uncoupling of gap junctions occur during ischemia, but the significance of Cx43 phosphorylation in this setting is unknown. Here we show that Cx43 dephosphorylation in synchronously contracting myocytes during ischemia is reversible, independent of hypoxia, and closely associated with cellular ATP levels. Cx43 became profoundly dephosphorylated during hypoxia only when glucose supplies were limited and was completely rephosphorylated within 30 minutes of reoxygenation. Similarly, direct reduction of ATP by various combinations of metabolic inhibitors and by ouabain was closely paralleled by loss of phosphoCx43 and recovery of phosphoCx43 accompanied restoration of ATP. Dephosphorylation of Cx43 could not be attributed to hypoxia, acid pH or secreted metabolites, or to AMP-activated protein kinase; moreover, the process was selective for Cx43 because levels of phospho-extracellular signal regulated kinase (ERK)1/2 were increased throughout. Rephosphorylation of Cx43 was not dependent on new protein synthesis, or on activation of protein kinases A or G, ERK1/2, p38 mitogen-activated protein kinase, or Jun kinase; however, broad-spectrum protein kinase C inhibitors prevented Cx43 rephosphorylation while also sensitizing myocytes to reoxygenation-mediated cell death. We conclude that Cx43 is reversibly dephosphorylated and rephosphorylated during hypoxia and reoxygenation by a novel mechanism that is sensitive to nonlethal fluctuations in cellular ATP. The role of this regulated phosphorylation in the adaptation to ischemia remains to be determined.

Topics: Adenosine Triphosphate; Alkaloids; Aminoimidazole Carboxamide; Animals; Antimycin A; Benzophenanthridines; Brefeldin A; Carbazoles; Cell Hypoxia; Cells, Cultured; Connexin 43; Cycloheximide; Deoxyglucose; Flavonoids; Imidazoles; Indoles; JNK Mitogen-Activated Protein Kinases; Maleimides; Myocardial Contraction; Myocytes, Cardiac; Okadaic Acid; Ouabain; Phenanthridines; Phosphorylation; Potassium Cyanide; Protein Processing, Post-Translational; Pyridines; Pyrroles; Rats; Recombinant Fusion Proteins; Ribonucleotides; Staurosporine; Tacrolimus; Tetradecanoylphorbol Acetate

We have recently demonstrated that in quiescent fibroblasts protein kinase C (PKC) epsilon(95) is phosphorylated at Ser(729), Ser(703), and Thr(566) and that upon passage of quiescent cells phosphorylation at Ser(729) is lost, giving rise to PKCepsilon(87). Ser(729) may be rephosphorylated later, suggesting cycling between PKCepsilon(87) and PKCepsilon(95). Here we show that the dephosphorylation at Ser(729) is insensitive to okadaic acid, calyculin, ascomycin C, and cyclosporin A, suggesting that dephosphorylation at this site is not mediated through protein phosphatases 1, 2A or 2B. We demonstrate that this dephosphorylation at Ser(729) requires serum and cell readhesion and is sensitive to rapamycin, PD98059, chelerythrine, and Ro-31-8220. These results suggest that the phosphorylation status of Ser(729) in the hydrophobic domain at Ser(729) is regulated independently of the phosphorylation status of other sites in PKCepsilon, by a mTOR-sensitive phosphatase. The mitogen-activated protein kinase pathway and PKC are also implicated in regulating the dephosphorylation at Ser(729).

Topics: 3T3 Cells; Alkaloids; Animals; Anti-Bacterial Agents; Benzophenanthridines; Blotting, Western; Calcineurin; Cell Adhesion; Cell Line; Culture Media; Cyclosporine; Down-Regulation; Enzyme Inhibitors; Flavonoids; Indoles; Isoenzymes; MAP Kinase Signaling System; Marine Toxins; Methionine; Mice; Models, Biological; Okadaic Acid; Oxazoles; Phenanthridines; Phosphoprotein Phosphatases; Phosphorylation; Plasmids; Precipitin Tests; Protein Binding; Protein Kinase C; Protein Kinase C-epsilon; Protein Structure, Tertiary; Serine; Signal Transduction; Sirolimus; Tacrolimus; Transfection