tacrolimus and calmidazolium

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

A protein phosphatase exclusively dependent upon micromolar amounts of Ca2+ and calmodulin has been identified and partially purified from Leishmania spp. Complete obliteration of its activity is observed in the presence of calmodulin antagonists such as trifluoperazine, fluphenazine and calmidazolium. Relative insensitivity to okadaic acid and lack of activation in the absence of Ca2+ and calmodulin distinguishes this enzyme from PP1, PP2A and PP2C-type protein phosphatases. Cross-reactivity of the enzyme was observed with antibodies that recognize both the A and B chains of calcineurin, a PP2B type Ca2+ and calmodulin-dependent phosphatase from brain. FK506, an immunosuppresive drug that inhibits the enzyme from other sources inhibited the enzyme only in the presence of exogenous FK binding protein, whereas Cyclosporin A inhibited the enzyme in crude preparations. Taken together these results reveal the presence of a Ca2+ and calmodulin-dependent phosphatase from Leishmania. This is the first report of the presence of a PP2B-type protein phosphatase from a pathogenic protozoa.

Topics: Animals; Blotting, Western; Calcineurin; Calcium-Calmodulin-Dependent Protein Kinases; Cyclosporine; Fluphenazine; Humans; Imidazoles; Immunophilins; Leishmania donovani; Protozoan Proteins; Tacrolimus; Tacrolimus Binding Proteins; Trifluoperazine

The nontransformed steroid receptors contain several non-steroid binding proteins, such as hsp90, hsp70, and p59. Recently, we and others have shown that p59 (FKBP59) is an immunophilin which binds two potent immunosuppressants, FK506 and rapamycin. This raises the possibility that FK506 or rapamycin may modify the function of steroid receptors. To develop this line of inquiry, we chose a yeast model system in which the human progesterone receptor form B (hPR-B) was cotransformed with a reporter gene. The reporter contains two copies of a progesterone response element/glucocorticoid response element (PRE/GRE) upstream of the CYC1 promoter which are linked to the lacZ gene of Escherichia coli. We found that FK506 potentiated the ability of progesterone in activating transcription. To gain insight into the mechanism of FK506's regulation of PR action, we questioned whether calcineurin is involved, because it has been shown that FK506 is a specific inhibitor of calcineurin, a Ca(2+)- and calmodulin-regulated phosphatase, through the formation of an FKBP12-FK506-calcineurin-calmodulin complex. We found that 15-O-desmethyl-FK520, an FK506 analogue which is an excellent ligand of FKBP12, but a poor inhibitor of calcineurin, failed to induce the same effect as FK506. We also found that calmidazolium, a calmodulin antagonist, mimicked FK506's action. Furthermore, immunoblot analysis showed that both FK506 and calmidazolium potentiated the effect of progesterone in decreasing the mobility of hPR-B upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This suggests that FK506 and calmidazolium may cooperate with progesterone in increasing the level of hPR-B phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Calcineurin; Calmodulin-Binding Proteins; Carrier Proteins; Gene Expression Regulation; Heat-Shock Proteins; Imidazoles; Phosphoprotein Phosphatases; Polyenes; Receptors, Progesterone; Saccharomyces cerevisiae; Signal Transduction; Sirolimus; Tacrolimus; Tacrolimus Binding Proteins; Transcription, Genetic; Transcriptional Activation

To assess the role of calmodulin in human basophil histamine release, we triggered leukocytes with different secretagogues in the presence of putative inhibitors of calmodulin. Calcium ionophore-induced histamine release was reduced or blocked by calmidazolium, CGS 9343B, felodipine, metofenazate, and Ro 22-4839. H 186/86, a felodipine-related dihydropyridine derivative, blocked A23187-but not ionomycin-triggered histamine release, suggesting a difference in the mode of action of these ionophores. In contrast, leukocyte histamine release triggered by the purported protein kinase C (PKC) activator, 1,2-isopropylidene-3-decanoyl-sn-glycerol (IpOCOC9), was enhanced by calmidazolium, CGS 9349B and metofenazate but not affected by felodipine or Ro 22-4839, whereas the response triggered by 4 beta-phorbol 12-myristate 13-acetate (PMA) was reduced by metofenazate and Ro 22-4839 but not consistently affected by calmidazolium, CGS 9343B or felodipine. The PMA-induced histamine release was enhanced by H 186/86. Anti-IgE- and FMLP-induced responses were either unaffected or slightly enhanced by the examined calmodulin antagonists. In comparison with the calmodulin antagonists, R 59022, an inhibitor of diacylglycerol kinase, failed to reduce calcium ionophore-triggered histamine release, whereas FK506, an inhibitor of peptidyl prolyl cis-trans isomerase (PPI), reduced both anti-IgE- and ionophore-triggered responses. These results indicate that calmodulin constitutes an obligate link in signal transduction pathways leading to human leukocyte histamine release if the trigger is a calcium ionophore but not when responses are induced by anti-IgE, FMLP or PMA; a calmodulin-dependent component may rather balance responses induced by IpOCOC9.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amino Acid Isomerases; Basophils; Benzimidazoles; Calmodulin; Carrier Proteins; Diacylglycerol Kinase; Felodipine; Histamine Release; Humans; Imidazoles; Isoquinolines; Peptidylprolyl Isomerase; Phenothiazines; Phosphotransferases; Pyrimidinones; Tacrolimus; Thiazoles