Effects of protein kinase C activators upon the late stages of the ACTH secretory pathway of 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 phorbol 12‐myristate 13‐acetate (PMA)‐mediated enhancement of calcium‐evoked adrenocorticotrophin (ACTH) secretion. 2 PMA stimulated ACTH secretion from intact cells in a concentration‐dependent manner. Other phorbol esters; phorbol 12, 13‐dibutyrate (PDBu) and phorbol 12, 13‐didecanoate (PDD) and diacylglycerol analogues; 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG) and 1, 2‐dioctanoyl‐sn‐glycerol (DOG) also stimulated ACTH release from intact AtT‐20 cells. This would suggest that activation of protein kinase C (PKC) stimulates ACTH secretion from AtT‐20 cells. 3 Calcium stimulated ACTH secretion from electrically‐permeabilized cells over the concentration‐range of 10−7 m to 10−5 m. PMA (10−7 m) enhanced the amount of ACTH secreted at every concentration of calcium investigated. The PKC inhibitor, chelerythrine (10−5m) blocked the PMA (10−7 m)‐evoked enhancement of calcium (10−5 m)‐stimulated ACTH secretion but did not alter significantly the calcium (10−5 m)‐evoked secretion itself. This suggests that PKC modulates the secretory response to increases in intracellular calcium but does not mediate the effects of calcium. 4 Guanosine 5′‐O‐(3‐thiotriphosphate) (GTP‐γ‐S, 10−5m) stimulated ACTH secretion from permeabilized cells in the absence of calcium and was additive with calcium‐evoked ACTH secretion up to a maximum value which could be achieved by calcium acting alone. This suggests that a GTP‐binding protein mediates the secretory response to increases in the intracellular calcium. PMA (10−7 m) enhanced ACTH secretion stimulated by the combination of calcium and GTP‐γ‐S (10−5 m). 5 GTP‐γ‐S stimulated ACTH secretion from permeabilized cells in a concentration‐dependent manner with a threshold of 10−6 m. PMA (10−7 m) increased the amount of ACTH secretion evoked by every concentration of GTP‐γ‐S investigated. Chelerythrine (10−5m) blocked the PMA (10−7 m)‐evoked enhancement of GTP‐γ‐S (10−4 m)‐stimulated ACTH secretion but did not significantly alter GTP‐γ‐S (10−4 m)‐evoked secretion itself. This suggests that PKC modulates the secretory response to GTP‐γ‐S but does not mediate the effects of GTP‐γ‐S. 6 GTP‐γ‐S (10−8‐10−4 m) stimulated ACTH secretion from permeabilized cells either in the presence or absence of ATP (5 mM) indicating that its effects on secretion are ATP‐independent. 7 The results of the present study support the hypothesis that, in AtT‐20 cell