The effects of calyculin A upon calcium-, guanine nucleotides- and phorbol 12-myristate 13-acetate-stimulated ACTH secretion from AtT-20 cells.

1. The mouse AtT-20/D16-16 anterior pituitary tumour cell line was used as a model system for the study of protein phosphatase involvement in the late stages of the secretory pathway for adrenocorticotrophin (ACTH) secretion. The effects of the type 1 and 2 phosphatase inhibitor calyculin A upon calcium-, guanine nucleotide- and phorbol 12-myristate 13-acetate (PMA)-stimulated ACTH secretion from electrically-permeabilized AtT-20 cells were studied. 2. Calyculin A (1 nM-1 microM) inhibited both calcium (10 microM)- and guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) (100 microM)-evoked ACTH secretion from permeabilized cells in a concentration-dependent manner. These effects were maximal with 100 nM calyculin A. 3. ACTH secretion was stimulated from electrically-permeabilized cells when the cytosolic free calcium ion concentration, controlled by calcium-EGTA buffers, was raised over the concentration range of 100 nM to 10 microM. This calcium-stimulated ACTH secretion was inhibited by co-incubation with calyculin A (100 nM). 4. GTP-gamma-S (10 nM-100 microM) stimulated ACTH secretion from permeabilized cells at concentrations greater than 1 microM GTP-gamma-S. Co-incubation with calyculin A (100 nM) inhibited this stimulation of ACTH secretion observed at these concentrations of GTP-gamma-S. 5. PMA (100 nM) significantly stimulated ACTH secretion from permeabilized cells in the absence of either calcium and guanine nucleotides and this action was enhanced by calyculin A (100 nM). Furthermore, an inhibition of GTP-gamma-S (100 microM)-stimulated ACTH secretion observed in the presence of calyculin A (100 nM) was not observed in the presence of PMA (100 nM). 6. The results of the present study indicate that dephosphorylation by phosphatases plays an important role in stimulus-secretion coupling in AtT-20 cells and is involved in mediating the effects of GE upon the secretory apparatus in these cells. Furthermore, the point of regulation of the secretory response by PKC which underlies the ability of PKC to amplify the calcium/GE system may lie distal to both GE and these phosphatases.