tacrolimus and 2-aminoethoxydiphenyl-borate

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

Transient receptor potential vanilloid 2 (TRPV2) acts as a Ca(2+)-permeable non-selective cation channel that has been reported to be sensitive to temperature, mechanical force, and some chemicals. We recently showed that TRPV2 is critical for maintenance of the thermogenic function of brown adipose tissue in mice. However, the involvement of TRPV2 in the differentiation of brown adipocytes remains unexplored. We found that the expression of TRPV2 was dramatically increased during the differentiation of brown adipocytes. Non-selective TRPV2 agonists (2-aminoethoxydiphenyl borate and lysophosphatidylcholine) inhibited the differentiation of brown adipocytes in a dose-dependent manner during the early stage of differentiation of brown adipocytes. The inhibition was rescued by a TRPV2-selective antagonist, SKF96365 (SKF). Mechanical force, which activates TRPV2, also inhibited the differentiation of brown adipocytes in a strength-dependent manner, and the effect was reversed by SKF. In addition, the inhibition of adipocyte differentiation by either TRPV2 ligand or mechanical stimulation was significantly smaller in the cells from TRPV2KO mice. Moreover, calcineurin inhibitors, cyclosporine A and FK506, partially reversed TRPV2 activation-induced inhibition of brown adipocyte differentiation. Thus, we conclude that TRPV2 might be involved in the modulation of brown adipocyte differentiation partially via a calcineurin pathway.

Topics: Adipocytes, Brown; Animals; Boron Compounds; Calcineurin Inhibitors; Calcium Channels; Cell Differentiation; Cells, Cultured; Cyclosporine; HEK293 Cells; Humans; Imidazoles; Lysophosphatidylcholines; Male; Mice; Mice, Inbred C57BL; Tacrolimus; TRPV Cation Channels

Diabetes mellitus causes multiple cardiovascular complications. Previous studies have shown that prolonged exposure (96 h) of human umbilical vein endothelial cells (HUVECs) to hyperglycemia causes a significant increase in apoptosis. We report here that this increase in apoptosis is associated with an increase in Ca(2+) current (whole cell patch-clamp recorded) resulting from Ca(2+) entry mediated by store-operated channels (SOCs). The number of apoptotic cells after prolonged high glucose (HG, 30 mmol/L) exposure was significantly reduced in the presence of the SOC inhibitor 2-APB or of La(3+). A marked increase (approximately 80%) in Ca(2+)-dependent calcineurin (CN-A) phosphatase activity also occurred after prolonged HG exposure. Prolonged HG exposure-induced increase in CN-A activity was prevented by 2-APB, and selective CN-A phosphatase inhibition by FK506 or calmodulin inhibition by calmidazolium decreased HG-induced apoptosis. Blocking hydrogen peroxide production using catalase or inhibiting the tyrosine kinase pp60(src) during prolonged exposure to HG, resulted in a marked decrease in apoptosis and was further associated with a significant reduction in CN-A phosphatase activity. The results demonstrate a significant role for Ca(2+) entry in HG-induced apoptosis in HUVECs, and suggest that this role is mediated via H(2)O(2) generation and the action of the Ca(2+)-activated protein phosphatase calcineurin.

Topics: Apoptosis; Boron Compounds; Calcineurin; Calcium; Calcium Channels; Calmodulin; Catalase; Endothelium, Vascular; Glucose; Humans; Hydrogen Peroxide; Hyperglycemia; Imidazoles; Patch-Clamp Techniques; Phosphoric Monoester Hydrolases; Proto-Oncogene Proteins pp60(c-src); Tacrolimus; Umbilical Veins

Intracellular Ca(2+) ([Ca(2+)](i)) oscillations seen in interstitial cells of Cajal (ICCs) are considered to be the primary pacemaker activity in the gut. Here, we show evidence that periodic Ca(2+) release from intracellular Ca(2+) stores produces [Ca(2+)](i) oscillations in ICCs, using cell cluster preparations isolated from mouse ileum. The pacemaker [Ca(2+)](i) oscillations in ICCs are preserved in the presence of dihydropyridine Ca(2+) antagonists, which suppress Ca(2+) activity in smooth muscle cells. However, applications of drugs affecting either ryanodine receptors or inositol 1,4,5-trisphosphate receptors terminated [Ca(2+)](i) oscillations at relatively low concentrations. RT-PCR analyses revealed a predominant expression of type 3 RyR (RyR3) in isolated c-Kit-immunopositive cells (ICCs). Furthermore, we demonstrate that pacemaker-like global [Ca(2+)](i) oscillation activity is endowed by introducing RyR3 into HEK293 cells, which originally express only IP(3)Rs. The reconstituted [Ca(2+)](i) oscillations in HEK293 cells possess essentially the same pharmacological characteristics as seen in ICCs. The results support the functional role of RyR3 in ICCs.

Topics: Anesthetics, Local; Animals; Biological Clocks; Boron Compounds; Caffeine; Calcium; Calcium Channel Blockers; Cell Line; Enzyme Inhibitors; Fluorescent Antibody Technique, Indirect; Fluorescent Dyes; Fura-2; Humans; Ileum; Immunohistochemistry; Kinetics; Macrocyclic Compounds; Mice; Microscopy, Fluorescence; Muscle, Smooth; Nifedipine; Oxazoles; Proto-Oncogene Proteins c-kit; RNA, Messenger; Ryanodine; Ryanodine Receptor Calcium Release Channel; Tacrolimus; Tetracaine

Beta-amyloid (Abeta) peptide has been suggested to play important roles in the pathogenesis of Alzheimer's disease (AD). Abeta peptide neurotoxicity was shown to induce disturbance of cellular calcium homeostasis. However, whether modulation of calcium release from the endoplasmic reticulum (ER) can protect neurons from Abeta toxicity is not clearly defined. In the present study, Abeta peptide-triggered ER calcium release in primary cortical neurons in culture is modulated by Xestospongin C, 2-aminoethoxydiphenyl borate or FK506. Our results showed that reduction of ER calcium release can partially attenuate Abeta peptide neurotoxicity evaluated by LDH release, caspase-3 activity and quantification of apoptotic cells. While stress signals associated with perturbations of ER functions such as up-regulation of GRP78 was significantly attenuated, other signaling machinery such as activation of caspase-7 transmitting death signals from ER to other organelles could not be altered. We further provide evidence that molecular signaling in mitochondria play also a significant role in determining neuronal apoptosis because Abeta peptide-triggered activation of caspase-9 was not significantly reduced by attenuating ER calcium release. Our results suggest that neuroprotective strategies aiming at reducing Abeta toxicity should include molecular targets linked to ER perturbations associated with ER calcium release as well as mitochondrial stress.

Topics: Amyloid beta-Peptides; Analysis of Variance; Animals; Blotting, Western; Boron Compounds; Calcium; Carrier Proteins; Caspase 3; Caspases; Cell Death; Cells, Cultured; Cerebral Cortex; Dose-Response Relationship, Drug; Drug Interactions; Embryo, Mammalian; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Enzyme Inhibitors; Heat-Shock Proteins; Immunosuppressive Agents; Indoles; Intracellular Space; L-Lactate Dehydrogenase; Macrocyclic Compounds; Molecular Chaperones; Neurons; Oxazoles; Peptide Fragments; Rats; Rats, Sprague-Dawley; Tacrolimus; Time Factors