sirolimus and herbimycin

Glial fibrillary acidic protein (GFAP) is expressed upon cAMP-mediated induction of differentiation of glial progenitor cells into type II astrocytes. The protein is regulated by hormones, growth factors and cytokines but the signal transduction pathways involved in the regulation of GFAP expression are largely unknown. Specific protein kinase inhibitors were used to study their effect on the expression of GFAP in rat C6 glioma cells. Herbimycin A, a selective protein tyrosine kinase inhibitor, reduced GFAP mRNA and protein expression upon cAMP analog or beta-adrenergic receptor-mediated induction of differentiation. The latter inhibitor attenuated the elevation of cAMP by adenylate cyclase and abolished the activity of phosphatidylinositol 3-kinase (PI 3-K). These data indicate that GFAP expression is regulated by protein tyrosine phosphorylations, modulating the cAMP concentration and PI 3-K activity in C6 glioma cells.

Topics: Adenylyl Cyclases; Adrenergic beta-Agonists; Androstadienes; Animals; Anti-Bacterial Agents; Benzoquinones; Cell Differentiation; Chromones; Cyclic AMP; Enzyme Inhibitors; Flavonoids; Gene Expression; Glial Fibrillary Acidic Protein; Glioma; Indoles; Isoproterenol; Isoquinolines; Lactams, Macrocyclic; Maleimides; Morpholines; Phosphatidylinositol 3-Kinases; Protein-Tyrosine Kinases; Quinones; Rats; Rifabutin; Sirolimus; Sulfonamides; Tumor Cells, Cultured; Wortmannin

Endothelium-derived hyperpolarizing factor (EDHF) mediates NO/prostacyclin-independent relaxation in the coronary circulation. Because hemodynamic stimuli modulate endothelial gene expression and because coronary arteries are subjected to pronounced variations in vessel distension, we determined the effects of cyclic stretch on the expression and activity of the coronary EDHF synthase/cytochrome P450 (CYP) 2C8/9. In cultured porcine coronary and human umbilical vein endothelial cells, acute application of cyclic stretch (6%, 1 Hz, 10 minutes) elicited the generation of 8,9-epoxyeicosatrienoic acid (EET), 11,12-EET, and 14,15-EET. Prolonged stretch (4 to 36 hours) increased the expression of CYP 2C mRNA and protein 5- to 10-fold and was accompanied by a 4- to 8-fold increase in EET generation. A corresponding increase in CYP 2C mRNA and protein was also observed in pressurized segments of porcine coronary artery perfused under pulsatile conditions (8%, 1 Hz) for 6 hours. Although in cultured endothelial cells, cyclic stretch elicited the rapid activation of tyrosine kinases as well as Akt and the p38 mitogen-activated protein kinase, the mechanism by which cyclic stretch induces the expression of CYP 2C could not be elucidated, because inhibitors of these pathways induced CYP 2C expression in cells maintained under static conditions. These results have identified coronary EDHF synthase/CYP 2C as a novel mechanosensitive gene product in native and cultured endothelial cells. Because this enzyme generates both EETs and superoxide anions, this finding has wide-reaching implications for vascular homeostasis in conditions of manifest endothelial dysfunction.

Topics: 8,11,14-Eicosatrienoic Acid; Androstadienes; Animals; Benzoquinones; Cells, Cultured; Coronary Vessels; Cytochrome P-450 Enzyme System; Cytochrome P450 Family 2; Endothelium, Vascular; Enzyme Inhibitors; Humans; Ionomycin; Lactams, Macrocyclic; Oxygenases; Patch-Clamp Techniques; Periodicity; Pulsatile Flow; Quinones; Rifabutin; Sirolimus; Stress, Mechanical; Swine; Umbilical Veins; Vasodilation; Wortmannin

We have developed an in vitro system to model the interactions of drugs used to treat transplant rejection. This system consists of stimulation of human lymphocytes with a primary mitogen (anti-T-cell receptor complex antibodies (OKT3 or wt31)) and treatment with a primary immunosuppressive drug (ISD) (Cyclosporine A (CsA) or FK-506)). This is later followed by stimulation with a secondary mitogen (Interleukin-2 or anti-CD28), and treatment with a second ISD. This system allows a variety of concentrations and compounds to be rapidly tested. We have used this system to study the effect of various compounds when used as either primary or secondary ISDs. Our results show that when CsA is used as the primary ISD, further proliferation can be inhibited by rapamycin, mycophenolic acid, or suramin. When FK-506 is the primary ISD, inhibition of proliferation by rapamycin is variable depending on the primary and secondary mitogens. If rapamycin is the primary ISD, both CsA and FK-506 show antagonistic interactions. These results suggest that the order in which combinations of ISDs are administered in transplantation may have significant effects on the clinical outcome.

Topics: Antibodies, Monoclonal; Benzoquinones; CD28 Antigens; Cells, Cultured; Cyclosporine; Drug Interactions; Humans; Immunosuppressive Agents; Interleukin-2; Lactams, Macrocyclic; Lymphocytes; Mycophenolic Acid; Polyenes; Quinones; Receptors, Antigen, T-Cell; Rifabutin; Sirolimus; Suramin; Tacrolimus