EFFECTS OF MASTOPARAN 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 the effects of mastoparan upon the late stages of the adrenocorticotrophin (ACTH) secretory pathway. 2 M astoparan (10(-8)-10(-5) M), an activator of heterotrimeric guanosine 5'-triphosphate binding proteins (G-proteins), stimulated ACTH secreti on from electrically-permeabilized AtT-20 cells in a concentration-dep endent manner in the effective absence of calcium ions with a threshol d of 10(-6) M. Guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) (10(- 8)-10(-4) M) also stimulated ACTH secretion from electrically-permeabi lized AtT-20 cells in a concentration-dependent manner in the effectiv e absence of calcium ions with a threshold of 10(-6) M. This GTP-gamma -S-evoked secretion is consistent with previous studies which demonstr ated that a G-protein, termed G(E), mediates calcium evoked ACTH secre tion from AtT-20 cells. GTP-gamma-S-evoked secretion however was not a s great as that obtained in response to mastoparan. 3 Both mastoparan (10(-5) M) and GTP-gamma-S (10(-4) M) stimulated ACTH secretion from e lectrically-permeabilized AtT20 cells in a time-dependent manner. A ti me of 30 min was adopted as the standard incubation period for the stu dy of both mastoparan and GTP-gamma-S-stimulated ACTH secretion from p ermeabilized AtT-20 cells. 4 Mastoparan (10(-8)-10(-5) M) stimulated A CTH secretion from permeabilized AtT-20 cells to the same extent in th e presence and absence of the protein kinase C (PKC) inhibitor, cheler ythrine chloride (10(-5) M). 5 Mastoparan (10(-8)-10(-5) M)-stimulated ACTH secretion from permeabilized AtT-20 cells was significantly redu ced in the presence of guanosine 5'-O-(2-thiodiphosphate) (GDP-beta-S, 10(-4) M). 6 The mastoparan analogue, Mas-7 (10(-8)-10(-5) M) stimula ted ACTH secretion from permeabilized AtT-20 cells to a greater extent than mastoparan (10(-8)-10(-5) M) however, the mastoparan analogue Ma s-17 (10(-8)-10(-5) M) had no effect upon ACTH secretion from permeabi lized AtT-20 cells. 7 Mastoparan (10(-8)-10(-5) M) stimulated ACTH sec retion from permeabilized AtT-20 cells in the presence and absence of ATP, normally present in the standard permeabilization medium at a con centration of 5 mM. Mastoparan (10(-8)-10(-5) M)-stimulated ACTH secre tion as well as control secretion was reduced when ATP was omitted. 8 The results of the present study demonstrate that mastoparan stimulate d ACTH secretion from permeabilized AtT-20 cells and displayed charact eristics consistent with calcium ion- and GTP-gamma-S-stimulated ACTH secretion from permeabilized AtT-20 cells. This suggests that in perme abilized AtT-20 cells, mastoparan directly activates G(E) and that thi s G-protein may be a heterotrimeric G-protein. This study also suggest s mastoparan may be a useful alternative to GTP-gamma-S as a means of directly activating G(E).