THE EFFECTS OF CALYCULIN-A UPON CALCIUM-STIMULATED, GUANINE NUCLEOTIDES-STIMULATED 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 (AC TH) secretion. The effects of the type 1 and 2 phosphatase inhibitor c alyculin A upon calcium-, guanine nucleotide- and phorbol 12-myristate 13-acetate (PMA)-stimulated ACTH secretion from electrically-permeabi lized AtT-20 cells were studied. 2 Calyculin A (1 nM - 1 mu M) inhibit ed both calcium (10 mu M)- and guanosine 5'-O-(3-thiotriphosphate) (GT P-gamma-S) (100 mu M)-evoked ACTH secretion from permeabilized cells i n a concentration-dependent manner. These effects were maximal with 10 0 nM calyculin A. 3 ACTH secretion was stimulated from electrically-pe rmeabilized cells when the cytosolic free calcium ion concentration, c ontrolled by calcium-EGTA buffers, was raised over the concentration r ange of 100 nM to 10 mu M. This calcium-stimulated ACTH secretion was inhibited by co-incubation with calyculin A (100 nM). 4 GTP-gamma-S (1 0 nM-100 mu M) stimulated ACTH secretion from permeabilized cells at c oncentrations greater than 1 mu M GTP-gamma-S. Go-incubation with caly culin 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 e ither calcium and guanine nucleotides and this action was enhanced by calyculin A (100 nM). Furthermore, an inhibition of GTP-gamma-S (100 m u M)-stimulated ACTH secretion observed in the presence of calyculin A (100 nM) was not observed in the presence of PMA (100 nM). 6 The resu lts of the present study indicate that dephosphorylation by phosphatas es plays an important role in stimulus-secretion coupling in AtT-20 ce lls and is involved in mediating the effects of G(E) upon the secretor y apparatus in these cells. Furthermore, the point of regulation of th e secretory response by PKC which underlies the ability of PKC to ampl ify the calcium/G(E) system may lie distal to both G(E) and these phos phatases.