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(-5) M) 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-gamma-S, 10(-5) M) 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-gamma-S (10-5 M).5. GTP-gamma-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-gamma-S investigated. Chelerythrine (10-s M) blocked the PMA (10-7 M)-evoked enhancement of GTP-gamma-S (10-4 M)-stimulated ACTH secretion but did not significantly alter GTP-gamma-S(10-4 M)-evoked secretion itself. This suggests that PKC modulates the secretory response to GTP-gamma-S but does not mediate the effects of GTP-gamma-S.6. GTP-gamma-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 cells, PMA is acting at some site distal to calcium entry which modulates the ability of an increase in cytosolic calcium concentration to stimulate ACTH secretion. This site of action is either at the level of or at some stage distal to a GTP-binding protein which mediates the effects of calcium upon secretion.8. PMA, unlike adenosine 3':5'-cyclic monophosphate (cyclic AMP) (Guild, 1991), can stimulate ACTH secretion from permeabilized cells in the absence of added calcium and guanine nucleotides which suggests that PMA and cyclic AMP are acting through distinct mechanisms at this post calcium site of action.