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The effects of calyculin A upon calcium‐, guanine nucleotides‐ and phorbol 12‐myristate 13‐acetate‐stimulated ACTH secretion from AtT‐20 cells
Author(s) -
McFerran B.W.,
Guild S.B.
Publication year - 1995
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/j.1476-5381.1995.tb14941.x
Subject(s) - gtp' , calcium , egta , secretion , phorbol , endocrinology , medicine , biology , g protein , protein kinase c , chemistry , biochemistry , phosphorylation , signal transduction , enzyme
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 μm) inhibited both calcium (10 jam)‐ and guanosine 5′‐0‐(3‐thiotriphosphate) (GTP‐γ‐S) (100 μ m )‐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 μ m . This calcium‐stimulated ACTH secretion was inhibited by co‐incubation with calyculin A (100 nM). 4 GTP‐γ‐S (10nM‐100μ m ) stimulated ACTH secretion from permeabilized cells at concentrations greater than 1 μm GTP‐γ‐S. Co‐incubation with calyculin A (100 nM) inhibited this stimulation of ACTH secretion observed at these concentrations of GTP‐γ‐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‐γ‐S (100 μ 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 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 G E 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/G E system may lie distal to both G E and these phosphatases.