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 (P MA)-mediated enhancement of calcium-evoked adrenocorticotrophin (ACTH) secretion. 2 PMA stimulated ACTH secretion from intact cells in a con centration-dependent manner. Other phorbol esters; phorbol 12,13-dibut yrate (PDBu) and phorbol 12,13-didecanoate (PDD) and diacylglycerol an alogues; 1-oleoyl-2-acetyl-sn-glycerol (OAG) and 1,2-dioctanoyl-sn-gly cerol (DOG) also stimulated ACTH release from intact AtT-20 cells. Thi s would suggest that activation of protein kinase C (PKC) stimulates A CTH secretion from AtT-20 cells. 3 Calcium stimulated ACTH secretion f rom electrically-permeabilized cells over the concentration-range of 1 0(-7) M to 10(-5) M. PMA (10(-7) M) enhanced the amount of ACTH secret ed 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 sig nificantly the calcium (10(-5) M)-evoked secretion itself. This sugges ts that PKC modulates the secretory response to increases in intracell ular calcium but does not mediate the effects of calcium. 4 Guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S, 10(-5) M) stimulated ACTH secr etion from permeabilized cells in the absence of calcium and was addit ive with calcium-evoked ACTH secretion up to a maximum value which cou ld be achieved by calcium acting alone. This suggests that a GTP-bindi ng protein mediates the secretory response to increases in the intrace llular calcium. PMA (10(-7) M) enhanced ACTH secretion stimulated by t he combination of calcium and GTP-gamma-S (10(-5) M). 5 GTP-gamma-S st imulated ACTH secretion from permeabilized cells in a concentration-de pendent manner with a threshold of 10(-6) M. PMA (10(-7) M) increased the amount of ACTH secretion evoked by every concentration of GTP-gamm a-S investigated. Chelerythrine (10(-5) 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 secreti on 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-gam ma-S (10(-8)-10(-4) M) stimulated ACTH secretion from permeabilized ce lls either in the presence or absence of ATP (5 mM) indicating that it s effects on secretion are ATP-independent. 7 The results of the prese nt study support the hypothesis that, in AtT-20 cells, PMA is acting a t some site distal to calcium entry which modulates the ability of an increase in cytosolic calcium concentration to stimulate ACTH secretio n. This site of action is either at the level of or at some stage dist al 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 c ells in the absence of added calcium and guanine nucleotides which sug gests that PMA and cyclic AMP are acting through distinct mechanisms a t this post calcium site action.