Steroidogenesis in isolated adrenocortical cells. Correlation with receptor-bound adenosine 3′:5′-cyclic monophosphate

Abstract

Because several groups have recently questioned a mediating role for cyclic AMP in adrenocortical steroidogenesis, we analysed the problem in more detail by measuring three different cyclic AMP pools in cells isolated from decapsulated rat adrenals. Extra-cellular, total intracellular and bound intracellular cyclic AMP were determined by radioimmunoassay in comparison with corticosterone production induced by low corticotropin concentrations. The increase in extracellular and total intracellular cyclic AMP with low corticotropin concentrations was dependent on the presence of a phosphodiesterase inhibitor and short incubation times. Bound intracellular cyclic AMP was less dependent on these two parameters. In unstimulated cells cyclic AMP bound to its receptor represents only a small fraction of the total intracellular cyclic AMP. After stimulation by a concentration of corticotropin around the threshold for corticosterone production, an increase in bound cyclic AMP was observed which correlated very well with steroidogenesis both temporally and with respect to corticotropin concentration. This finding was complemented by measuring a concomitant decrease in free receptor sites. Full occupancy of the receptors was not necessary for maximal steroidogenesis. Binding kinetics of cyclic [(3)H]AMP in concentrations equivalent to the intracellular cyclic AMP concentration suggest the presence of at least three different intracellular cyclic AMP pools. These observations are in agreement with a possible role for cyclic AMP as a mediator of acute steroidogenesis induced by low corticotropin concentrations.

An adrenocorticotropin‐regulated phosphoprotein intermediary in steroid synthesis is similar to an acyl‐CoA thioesterase enzyme

“…mRNA levels were quantified by densitometric scanning of the autoradiograms and related to those of 28 S ribosomal RNA. Corticosterone was measured by radioimmunoassay [7]. Values are expressed as mean ϮSD, n ϭ 3, except for RNA in which three separated RNA samples were pooled before analysis.…”

LH/chorionic gonadotropin signaling pathway involves protein tyrosine phosphatase activity downstream of protein kinase A activation: evidence of an obligatory step in steroid production by Leydig cells

“…In order to test this hypothesis, studies of PTP activity were extended to another steroidogenic system, as Leydig cells, using fresh interstitial testicular and MA-10 cells, an established and well-characterized tumor Leydig cell line (Ascoli 1981, Stocco & Chaudhary 1990, Stocco & Ascoli 1993, Weiss-Messer et al 1998. Testosterone production by Leydig cells in the testis is triggered by luteinizing hormone (LH) through cell surface receptors coupled to a signaling transduction mechanism based on PKAmediated phosphorylation (Dufau et al 1977, Clark et al 1994, a similar pathway to ACTH-activated corticosteroid production in adrenal zona fasciculata (ZF) (Podestá et al 1979, Sala et al 1979.…”

Protein tyrosine phosphatases are involved in LH/chorionic gonadotropin and 8Br-cAMP regulation of steroidogenesis and StAR protein levels in MA-10 Leydig cells

“…While LH is the major physiological agonist in most animals, its action can also be mimicked by chorionic gonadotropin (CG). The signal transduction cascade involved in LH/CG action includes increases in intracellular cAMP levels and activation of protein kinase A (PKA), events that also occur in adrenocorticotropin (ACTH)-stimulated steroid production in adrenal cortical cells (Cooke et al 1976, Dufau et al 1977, Podestá et al 1979, Rae et al 1979, Sala et al 1979, Wang & Ascoli 1990, Swinnen et al 1991. It is well recognized that one of the requirements for full steroidogenic activity in response to LH/CG and ACTH is the PKA-mediated phosphorylation of regulatory proteins (Pon et al 1986, Chaudary & Stocco 1991, Stocco & Clark 1993.…”