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. Mastoparan (10(-8)-10(-5) M), an activator of heterotrimeric guanosine 5'-triphosphate binding proteins (G-proteins), stimulated ACTH secretion from electrically-permeabilized AtT-20 cells in a concentration-dependent manner in the effective absence of calcium ions with a threshold of 10(-6) M. Guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) (10(-8)-10(-4) M) also stimulated ACTH secretion from electrically-permeabilized AtT-20 cells in a concentration-dependent manner in the effective absence of calcium ions with a threshold of 10(-6) M. This GTP-gamma-S-evoked secretion is consistent with previous studies which demonstrated that a G-protein, termed GE, mediates calcium evoked ACTH secretion from AtT-20 cells. GTP-gamma-S-evoked secretion however was not as 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 electrically-permeabilized AtT20 cells in a time-dependent manner. A time of 30 min was adopted as the standard incubation period for the study of both mastoparan and GTP-gamma-S-stimulated ACTH secretion from permeabilized AtT-20 cells. 4. Mastoparan (10(-8)-10(-5) M) stimulated ACTH secretion from permeabilized AtT-20 cells to the same extent in the presence and absence of the protein kinase C (PKC) inhibitor, chelerythrine chloride (10(-5) M). 5. Mastoparan (10-8 10-5 M)-stimulated ACTH secretion from permeabilized AtT-20 cells was significantly reduced 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) stimulated ACTH secretion from permeabilized AtT-20 cells to a greater extent than mastoparan (10-8 10-5 M) however, the mastoparan analogue Mas-17 (10-8- 10-5 M) had no effect upon ACTH secretion from permeabilized AtT-20 cells.7. Mastoparan (10-8-10-5 M) stimulated ACTH secretion from permeabilized AtT-20 cells in the presence and absence of ATP, normally present in the standard permeabilization medium at a concentration of 5 mM. Mastoparan (10-8- 10-5 M)-stimulated ACTH secretion as well as control secretion was reduced when ATP was omitted.8. The results of the present study demonstrate that mastoparan stimulated ACTH secretion from permeabilized AtT-20 cells and displayed characteristics consistent with calcium ion- and GTP-y-gamma-S-stimulated ACTH secretion from permeabilized AtT-20 cells. This suggests that in permeabilized AtT-20 cells, mastoparan directly activates GE and that this G-protein may be a heterotrimeric G-protein. This study also suggests mastoparan may be a useful alternative to GTP-gamma-S as a means of directly activating GE.
McFerran
B W
BW
Molecular Endocrinology Group, School of Biological and Medical Sciences, University of St. Andrews.
Guild
S B
SB
D000324:Adrenocorticotropic Hormone / Q000378:metabolism*; D000470:Alkaloids; D000818:Animals; D053119:Benzophenanthridines; D015550:Cell Degranulation / Q000187:drug effects; D002463:Cell Membrane Permeability / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004558:Electric Stimulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000187:drug effects / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D036341:Intercellular Signaling Peptides and Proteins; D008407:Mast Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D010455:Peptides; D010617:Phenanthridines / Q000494:pharmacology; D010903:Pituitary Gland, Anterior / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D010911:Pituitary Neoplasms / Q000473:pathology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D014407:Tumor Cells, Cultured; D014862:Wasp Venoms / Q000494:pharmacology*
D000470:Alkaloids; D053119:Benzophenanthridines; D004791:Enzyme Inhibitors; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D010617:Phenanthridines; D014862:Wasp Venoms; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C037500:mastoparan; D000324:Adrenocorticotropic Hormone; C016299:chelerythrine; D011493:Protein Kinase C; D019204:GTP-Binding Proteins