tacrolimus and Pituitary-Neoplasms

The selective immunosuppressants cyclosporin A (CsA) and tacrolimus (FK506) are used in the prevention of allogenic transplant rejection and in the therapy of chronic autoimmune inflammatory pathologies. Chronic treatment with CsA leads to secondary functional and trophic alterations of multiple organs and cell systems among which endocrine ones, through insofar uncharacterized mechanisms. With the recent use of FK506 there have been reports of an improved therapeutic efficacy and a reduction of side-effects, as compared to CsA. An intriguing hypothesis is that toxic damage could be due to a systemic CsA activation of arachidonic acid (AA) metabolism, through pathways as yet only partially characterized. The side-effects of both drugs have been poorly studied on cells from tissues other than blood or kidney. We have thus proceeded to study their action on AA release in corticotropic AtT-20/D16-16 cells. The results obtained are as follows: 1) during incubation times > or =12 h, basal AA release is increased by CsA, but not FK506; the acute effect (10 min) of melittin, a PLA2 activator, is significantly potentiated starting from a 30 min pretreatment with CsA but not FK506; manoalide, a PLA2 inhibitor, antagonizes the melittin potentiation of AA release by CsA whereas the inhibition of the melittin stimulus by glucocorticoids is antagonized both by CsA and FK506. 2) during longer (>2 d) incubation times, cell growth is inhibited by CsA but not FK506. These results indicate a role for CsA, not apparent for FK506, in the activation of PLA2 and in the inhibition of cell growth. They also suggest that CsA does not have a direct (i.e. not mediated by the immune system) therapeutic effect in inflammatory processes.

Topics: Animals; Arachidonic Acid; Cell Division; Cell Size; Cell Survival; Cyclosporine; Dexamethasone; Digitonin; Dose-Response Relationship, Drug; Enzyme Activation; Immunosuppressive Agents; Interleukin-1; Melitten; Mice; Phospholipases A; Phospholipases A2; Pituitary Neoplasms; Tacrolimus; Terpenes; Time Factors; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Vitamin E

The effects of immunosuppressant blockers of calcineurin (protein phosphatase 2B) on cAMP formation and hormone release were investigated in mouse pituitary tumor (AtT20) cells. Immunosuppressants enhanced corticotropin-releasing factor- and isoproterenol-evoked cAMP production in proportion with their potency to block calcineurin. Further analysis of cAMP production revealed that intracellular Ca2+ derived through voltage-regulated calcium channels reduces cAMP formation induced by corticotropin releasing-factor or beta 2-adrenergic stimulation and that this effect of Ca2+ is inhibited by blockers of calcineurin. AtT20 cells were found to express at least three species of adenylyl cyclase mRNA-encoding types 1 and 6 as well as a novel isotype, which appeared to be the predominant species. In two cell lines expressing very low or undetectable levels of the novel cyclase mRNA (NCB20 and HEK293 cells respectively), corticotropin-releasing factor-induced cAMP formation was not altered upon blockage of calcineurin activity. These data identify calcineurin as a Ca2+ sensor that mediates the negative feedback effect of intracellular Ca2+ on receptor-stimulated cAMP production. Furthermore, the effect of calcineurin on cAMP synthesis appears to be associated with the expression of a novel adenylyl cyclase isotype, which is highly abundant in AtT20 cells.

Topics: 1-Methyl-3-isobutylxanthine; Adenylyl Cyclases; Adrenergic beta-Agonists; Amino Acid Sequence; Animals; Base Sequence; Calcineurin; Calcium; Calcium Channels; Calmodulin-Binding Proteins; Cell Line; Conserved Sequence; Corticotropin-Releasing Hormone; Cyclic AMP; Cyclosporins; DNA Primers; DNA, Complementary; Feedback; Humans; Immunosuppressive Agents; Isoenzymes; Isoproterenol; Kinetics; Mice; Molecular Sequence Data; Phosphoprotein Phosphatases; Pituitary Gland, Anterior; Pituitary Neoplasms; Polymerase Chain Reaction; RNA, Messenger; Sequence Homology, Amino Acid; Tacrolimus; Tumor Cells, Cultured

Topics: Adenylyl Cyclase Inhibitors; Adrenocorticotropic Hormone; Animals; Calcimycin; Calcineurin; Calcium; Calmodulin-Binding Proteins; Cell Line; Corticotropin-Releasing Hormone; Cyclic AMP; Cyclosporine; Dexamethasone; Dose-Response Relationship, Drug; Egtazic Acid; Feedback; Mice; Nimodipine; Phosphoprotein Phosphatases; Pituitary Gland; Pituitary Neoplasms; Tacrolimus; Tumor Cells, Cultured

The properties of the calcium/calmodulin-dependent protein phosphatase calcineurin and its potential role in stimulus-secretion coupling were examined in AtT20 mouse pituitary corticotrope tumor cells. Protein phosphatase activity was assayed by measuring the liberation of 32P from 32P-casein, adrenocorticotropin secretion was measured by radioimmunoassay. About 60% of the total phosphatase activity was inhibited by 500 nM okadaic acid, suggesting the presence of protein phosphatases 1 and/or 2A. A further 25-30% reduction of phosphatase activity was achieved by chelating free calcium. Addition of the EF-hand protein blocker trifluoperazine or a calcineurin autoinhibitory peptide fragment markedly reduced okadaic acid resistant and calcium-dependent protein phosphatase activity indicating that calcium-dependent 32P release is largely due to calcineurin (protein phosphatase 2B). The remaining 10-15% of total activity was Mg2+ dependent and blocked by NaF, hence possibly due to protein phosphatase 2C. Calcineurin activity was inhibited by the immunosuppressants FK506 and cyclosporin A, either when added to the cell lysates or after preincubation of intact cells with the drugs for 30 min at 37 degrees C. When added to lysates, cyclosporin A inhibited calcium/calmodulin-dependent phosphatase more effectively than FK506. However, when tested on intact cells, FK506 proved 10-fold more potent than cyclosporin A. Both immunosuppressive agents enhanced the calcium-dependent release of adrenocorticotropic hormone into the medium, once more, FK506 was 10-fold more potent than cyclosporin A. Taken together, these data suggest that calcineurin is an inhibitory element in the signal transduction pathway controlling exocytotic secretion in pituitary cells that express voltage-operated calcium channels. This is in direct contrast with leukocytes where voltage-operated calcium channels are not found, and calcineurin is an important element for agonist-induced activation.

Topics: Adrenocorticotropic Hormone; Animals; Calcineurin; Calcium; Calmodulin-Binding Proteins; Cyclosporine; Ethers, Cyclic; Kinetics; Magnesium; Mice; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorus Radioisotopes; Pituitary Neoplasms; Tacrolimus; Trifluoperazine; Tumor Cells, Cultured