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- Author: M. J. O'HARE x
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SUMMARY
The metabolism of [3H]corticosterone by freshly disaggregated suspensions and deliberately damaged monolayer cultures of rat adrenocortical cells has been examined. In both cases, increased yields of 11-dehydrocorticosterone were obtained compared with intact, undamaged monolayer cultures, in which effective 11-dehydrogenase activity was extremely low. It was concluded that the greater part of the effective 11-dehydrogenase activity of the freshly prepared adrenal cell suspension was due to cells damaged by the disaggregation procedure. The maximum rate at which corticosterone was converted to 11-dehydrocorticosterone by the suspensions was equivalent to at least 50 ng/million cells/h. Undamaged monolayer cultures did not metabolize more than 0·2 ng of corticosterone/million cells/h to 11-dehydrocorticosterone.
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SUMMARY
The patterns of exogenous steroid metabolism by rat adrenocortical zona fasciculata and zona reticularis cells cultured together as a monolayer have been examined. In the continued presence of corticotrophin (ACTH), the cultured cells synthesized corticosterone, 18-hydroxydeoxycorticosterone and deoxycorticosterone from [3H]pregnenolone. In the prolonged absence of ACTH, however, [3H]pregnenolone was metabolized mainly to progesterone, 20α-dihydroprogesterone, 20α-hydroxy-5α-pregnan-3-one and 5α-pregnane-3β,20α-diol with small amounts of 5α-pregnane-3,20-dione, 11β-hydroxyprogesterone and 11β-hydroxy-20α-dihydroprogesterone, while virtually no corticosterone, 18-hydroxydeoxycorticosterone or deoxycorticosterone were produced. While 5-ene-3β-hydroxysteroid dehydrogenase-isomerase activity persisted at a substantial level in unstimulated cells and the 11β- and 18-hydroxylases were reduced but still present, 21-hydroxylase activity was almost completely lost when ACTH was omitted from the culture medium. These changes in enzyme activities were completely reversed by the addition of ACTH, cyclic AMP or dibutyryl cyclic AMP to unstimulated cultures for 4–5 days, during which time no proliferation of adrenal cells was observed.
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SUMMARY
Confluent monolayer cultures of cells from the zona fasciculata and zona reticularis of the normal adult rat adrenal cortex were maintained with or without corticotrophin (ACTH) for up to 4 months, without proliferation of adrenal cells. Proliferating fibroblast-like cells, however, eventually overgrew the adrenal monolayer in cultures both with and without ACTH. Adrenocortical cells in culture, maintained without ACTH, spread rapidly to form a confluent monolayer, whereas cell spreading was markedly inhibited in the presence of ACTH. Exposure of previously unstimulated cells to ACTH or cyclic AMP caused the adrenal cells to retract with loss of confluence, the process being reversed when ACTH or cyclic AMP was withdrawn. Ultrastructural features of cells cultured with ACTH were typical of normal adrenocortical cells; in cultures without ACTH they were similar to those of adrenocortical cells found in the hypophysectomized rat.
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SUMMARY
Quantitative aspects of corticosteroidogenesis were examined in monolayer cultures of cells from the zona fasciculata and zona reticularis of the adult rat adrenal cortex, maintained for up to 4 months. Corticosterone secretion continued at a steady rate in cultures maintained with corticotrophin (ACTH), the output of maximally stimulated cultures being approximately 12 μg/106 adrenocortical cells/day. In the absence of ACTH, a small amount of 20α-hydroxypregn-4-en-3-one, but no detectable corticosterone, was secreted, resulting in a fluorogenic steroid output 1/125 th of that of maximally stimulated cultures. Restimulation with ACTH of cultures maintained for up to 2 months in its absence resulted in maximum levels of corticosterone secretion after 4–5 days of continuous ACTH treatment. The levels of corticosterone secretion attained on restimulation were similar to those observed in cultures maintained with ACTH from the out set. Withdrawal of ACTH resulted in a fall in steroid output which took 10 days to reach final unstimulated levels. Trophic stimulation of corticosteroidogenesis with a similar time-course was obtained with both cyclic AMP and dibutyryl cyclic AMP, the latter being the more effective.
