Measurement of the dynamics of stimulation and inhibition of steroidogenesis in isolated rat adrenal cells by using column perfusion

Abstract

Isolated adrenal cells were perfused in a small column by using Bio-Gel polyacrylamide beads as an inert supporting matrix, and the time-course of the response to various stimuli was observed by measuring fluorogenic 11-hydroxycorticosteroids in the effluent. A small but significant response was observed 1 min after stimulation with physiological concentrations of ACTH (adrenocorticotrophin), but the response did not start to build up rapidly for 3-4min and eventually reached a plateau after 9-10min. A similar pattern of events was observed for the decay of the steroid output on removal of ACTH. ACTH analogues, including one with a long duration of action in vivo, were found to produce responses with similar kinetics. However, cyclic AMP caused a more rapid increase in steroidogenesis and its effects were more short-lived after withdrawal. If, as present evidence suggests, cyclic AMP is produced rapidly after ACTH stimulation the delayed build-up of the steroidogenic response to ACTH would indicate that cyclic AMP may not be the intracellular mediator. When inhibitors were applied during ACTH stimulation, aminoglutethimide, which blocks mitochondrial conversion of cholesterol into pregnenolone (3beta-hydroxypregn-5-en-20-one), caused a rapid fall in steroid output (1 min), whereas cycloheximide took longer to achieve its full effect. Nevertheless, the response had fallen by 50% in 2 min, indicating a much shorter half-life than that previously reported for the labile protein implicated in steroidogenesis. In addition the rapid response to cyclic AMP makes it unlikely that steroid production is induced as a result of initiation of protein synthesis. This suggests that the labile protein plays an obligatory but permissive role in the development of the response. Column perfusion has proved to be a simple technique which can readily yield accurate data on responses of cells to stimulants and inhibitors.

Dual effect of antimitotic drugs on steroid secretion in mouse adrenocortical Y-1 tumor cells.

“…Our results clearly show that the inhibition of steroid secretion by anti-mitotic drugs can be observed in Y-1 cells as well as in primary culture cells (11) and that microtubules are involved in the process of steroid secretion in Y-1 cells. We have already characterized biochemical features of adrenocortical microtubules (see Introduction).…”

“…Our results suggest that the enhancement of the ACTH-independent secretion obscurred the inhibition of the ACTHdependnent secretion in these studies. The enhancement of the ACTH independent secretion may be an artefactual feature of Y-1 cells acquired during their adaptation to a continuous culture since this feature is not evident in the experiments with primary culture cells (11). Our results show that this kind of enhancement is more prominent during the lethal condition and that the secretion level is unexpectedly high (Figs.…”

“…4, compare the value of the open circle at time 0 with that of the closed circle at 3-to 4-day) and the ACTH dependence was no longer observed. Although antimitotic drugs are reported to inhibit steroid secretion in the primary culture of adrenocortical cells (11), studies on Y-1 cells have failed to show a clear inhibitory effect of the drugs (15,22). Our results suggest that the enhancement of the ACTH-independent secretion obscurred the inhibition of the ACTHdependnent secretion in these studies.…”

“…Recently much attention has been paid to the cytoskeletons as possible candidates of this transportation system. Both anatomical and physiological studies have shown that microtubules and actin filaments are involved in adrenocortical steroid secretion (4,11,13,14,19,21).…”

The effects of short-term culture and perifusion on LH-dependent steroidogenesis in isolated rat Leydig cells

“…2c). (Purvis & Menard, 1975 (Lowry & McMartin, 1974) or retained in suspension by balancing a flow of medium against an opposing centrifugal force (Schulster & Jenner, 1975 (Segaloff, Puett & Ascoli, 1981) and normal adult Leydig cells (Davies & Platzer, 1981) and suggestions have been made that the decline in the testosterone response is induced by the gonadotrophins. However, it could also be related to the specific perifusion conditions, because our results show that immature Leydig cells attached to plastic and incubated statically with 100 ng LH remain active in steroid production for more than 24 h.…”