An adrenocorticotropin analog [ACTH (6-39) ] which acts as a potent in vitro adrenocorticotropin antagonist at low calcium concentration and as a weak agonist at high calcium concentration

Ways, D.K.; Mahaffee, D.D.; Ontjes, D.A.

Endocrinology 104(4): 1028-1035


ISSN/ISBN: 0013-7227
PMID: 220022
DOI: 10.1210/endo-104-4-1028
Accession: 068524542

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The biological activity of a synthetic human ACTH analog containing residues 6-39 of the native sequence was characterized in 2 different in vitro assay systems. In broken membranes from rat adrenal cortex, the analog did not stimulate adenylate cyclase but blocked the stimulatory effects of ACTH(1-24) at equimolar concentrations. In this system, the Kd (Kact) for ACTH(1-24) was approximately 0.41 .mu.M, while the Ki of ACTH(6-39) was 0.32 .mu.M. The analog showed no agonist effects in the membrane system over a wide range of Ca concentrations but, in the presence of the GTP analog, Gpp(NH)p AMP production in response to ACTH(1-24). In the intact cell system, a 50- to 100-fold molar excess of inhibitor was required to block steroidogenesis. The apparent Kd of ACTH(1-24) for steroidogenesis was approximately 0.1 nM, while the Ki of the antagonist was 7.0 nM. In cells already stimulated by ACTH, steroidogenesis could be terminated within 15 min by addition of an excess of the inhibitor. Addition of an excess of ACTH(1-24) to inhibited cells caused a prompt resumption of steroid production. With increasing concentrations of Ca2+ in the incubation medium, ACTH(6-39) became a complete agonist with respect to steroidogenesis and a partial agonist with respect to cAMP production. Mg2+ could block the ability of Ca2+ to induce hormonal activity in the analog. ACTH(6-39) appears to be a more potent competitive inhibitor of ACTH than other analogs previously described. The discrepancy in the relative potencies for the inhibition of adenylate cyclase in membranes and the inhibition of steroidogenesis in cells is probably due to the fact that very low submaximal levels of receptor occupancy and cAMP formation are sufficient to induce maximal steroidogenesis. Both guanyl nucleotides and Ca play an essential role in the coupling process whereby the occupied receptor promotes the activation of adenylate cyclase. Under appropriate experimental conditions, both of these agents can greatly amplify the effectiveness causing the inhibitor to become a hormonal agonist.