Steroidogenic activity of high molecular weight forms of corticotropin.
The high molecular weight forms of adrenocorticotropic hormone (ACTH) produced by mouse pituitary tumor cells (AtT-20/D-16v) were separated from each other by gel filtration; their ability to stimulate steroidogenesis by isolated rat adrenal cortical cells was studied. Pools of pro-ACTH/endorphin. ACTH biosynthetic intermediate, and glycosylated ACTH(1--39) were obtained; on the basis of NaDodSO4-polyacrylamide gel electrophoresis, over 97% of the immunoactive ACTH was found to have the expected molecular weight. Suspension of isolated rat adrenal cortical cells were incubated overnight in tissue culture medium and used in a 2-h steroid production assay. Synthetic human ACTH(1--39) [hACTH(1--39)] was used as a bioassay and immunoassay standard; 60 pM hACTH(1--39) stimulated half-maximal production of fluoregenic steroid. The amount of pro-ACTH/endorphin, ACTH biosynthetic intermediate, or glycosylated (ACTH(1--39) added was estimated with an ACTH(17--24) immunoassay. All three high molecular weight forms of ACTH are capable of stimulating the same maximal level of steroidogenesis as hACTH(1--39). Glycosylated ACTH(1--39) is equipotent with hACTH(1--39); ACTH biosynthetic intermediate and pro-ACTH/endorphin are, respectively, 100- and 300-fold less potent than hACTH(1--39). Steroid production in response to all four forms of ACTH is linear in time. All of the different forms of ACTH stimulate the synthesis of corticosterone and related steroids; no significant production of cortisol or aldosterone was observed. beta-Lipotropin (beta LPH) and 16K fragment, which comprise the non-ACTH regions of pro-ACTH/endorphin and are secreted by the pituitary tumor cells, did not stimulate or interfere with steroidogenesis. Brief incubations of pro-ACTH/endorphin and ACTH biosynthetic intermediate with trypsin generated lower molecular weight forms of ACTH and increased biological activity 50-fold; thus, the decreased steroidogenic potency of these forms of ACTH is thought to be due to structural constraints on the ACTH(1--39)-like sequence in these larger precursor molecules
D000302:Adrenal Cortex / Q000187:drug effects / Q000378:metabolism*; D000305:Adrenal Cortex Hormones / Q000096:biosynthesis*; D000324:Adrenocorticotropic Hormone / Q000494:pharmacology*; D000818:Animals; D001681:Biological Assay; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004723:Endorphins / Q000494:pharmacology; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D008970:Molecular Weight; D010911:Pituitary Neoplasms; D011284:Pregnenolone / Q000378:metabolism; D051381:Rats; D014357:Trypsin
D000305:Adrenal Cortex Hormones; D004723:Endorphins; D011284:Pregnenolone; D000324:Adrenocorticotropic Hormone; D014357:Trypsin