| Summary: | Thesis (Ph.D.)--Boston University === This study concerns two aspects of adrenal cortical hormone production; both are intended to contribute to an ultimate understanding of the mechanism of adrenocorticotrophic hormone (ACTH) action. Since the corticosteroidogenic process is stimulated by ACTH, the chemical transformations involved in hormone synthesis must be understood. The first phase of this study utilized radiotracer techniques to resolve uncertainties which existed concerning the roles of 11β-hydroxyprogesterone and 11-deoxycorticosterone (DOC) as intermediaries in the formation of cortisol and corticosterone from progesterone. For this purpose, trace amounts of 11β-hydroxyprogesterone-4-c^14 and DOC-21-C^14 were added to incubations of cell-free bovine adrenocortical tissue prepared in a variety of media. The cortisol and corticosterone, which are produced from both endogenous precursors and suitable exogenous precursors by these homogenates, were isolated from lipid extracts of the incubation mixtures by column and paper chromatography. Measurement of the amount and radioactive content of the corticoids formed permitted evaluation of the degree of conversion of DOC-21-C^14 and 11β-hydroxyprogesterone-4-C^14 to cortisol and corticosterone, relative to that of a known intermediary, progesterone, incubated under identical conditions. It was found that DOC is converted in good yield to corticosterone, but not in detectable amounts to cortisol. The results support the postulation that cortisol and corticosterone are formed from progesterone via separate pathways. With regard to 11β-hydroxyprogesterone, a limited conversion of this steroid to both cortisol and corticosterone was observed; however, the degree of this conversion relative to that of progesterone indicated that 11β-hydroxyprogesterone is not a major intermediary in the formation of these corticoids from progesterone.
The second aspect of this study was stimulated by observations of previous investigators that the responsivity of adrenocortical tissue to ACTH in vitro is related to the structural integrity of the tissue preparation. To investigate this possible relationship, a preparation of bovine adrenal cortical tissue was devised which is intermediate in structural organization between homogenized and sliced tissue. This preparation, which consisted of small clumps of cells, was incubated under a variety of conditions and compared to similar studies carried out on adrenocortical slices. The corticosteroid production of these preparations was determined utilizing paper chromatographic techniques.
Evidence was obtained which indicated that certain cofactors and substrates which are necessary for corticoid formation were removed or destroyed in the preparation of these clumps of adrenocortical cells. Apparently as a consequence of this loss, the tissue no longer produced corticoids in significant amounts. However, corticosteroidogenic capabilities were restored in the presence of glucose by the addition of adenosine triphosphate (ATP), diphosphopyridine nucleotide (DPN) and fumarate to the incubation medium. Each of these cofactors contributed to the maximal corticosteroidogenic response, and ATP and DPN could be replaced by triphosphopyridine nucleotide (TPN). This cell-clump preparation did not respond to the ACTH either in the presence or absence of cofactor-fumarate supplementation. In contrast, adrenal cortical slices were shown to respond both to cofactor-fumarate supplementation and to ACTH; however, these effects were not additive. These results indicate that cofactor availability is rate-limiting in the process of corticosteroidogenesis, and are consistent with the concept that the mechanism by which ACTH stimulates corticosteroidogenesis may in some manner be concerned with an increased supply of cofactors to specific localized sites of enzyme activity within adrenocortical cells.
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