Dennis Schulster

The role of protein synthesis in adrenocorticotrophin action: Effects of cycloheximide and puromycin on the steroidogenic response of isolated adrenocortical cells

Abstract The kinetics of the corticosteroidogenic response to adrenocorticotrophin (ACTH) have been investigated using collagenase dispersed adrenocortical cells. Following the addition of ACTH at a concentration that was maximal for steroidogenesis, there was a time-lag of about 3 min before increased steroidogenesis became apparent. This lag was extended (about two-fold) in the presence of a half-maximal concentration of ACTH. Preincubation of cells with submaximal concentrations of both cycloheximide and puromycin extended the time-lag observed following ACTH addition. Increasing doses of cycloheximide or puromycin concomitantly inhibited protein synthesis and steroidogenesis. Moreover cycloheximide, at a dose that halved protein synthesis, also inhibited steroidogenesis by 54–61% for a range of ACTH concentrations (1 × 10 −4 to 1 × 10 −2 I.U./ml). It is concluded that the delay before ACTH-stimulated steroidogenesis is not attributable solely to the time taken for ribosomes to read of from the mRNA strand, the code for protein regulator(s). The results are discussed in terms of steroidogenic mechanisms whereby ACTH either induces de novo protein synthesis or activates a pre-existing, but labile, protein. In this latter scheme the steroidogenic rate observed under various conditions would be directly dependent upon the intracellular level of such an activated protein regulator. The half-life of labile protein, implicated as regulating steroidogenesis, was estimated at 2–4 min in this adrenal cell suspension system.

Corticosteroid and ribonucleic acid synthesis in isolated adrenal cells: Inhibition by actinomycin D

Abstract Both adrenocorticotrophic hormone (ACTH) and cyclic AMP rapidly stimulate corticosterone synthesis in isolated adrenal cells prepared by collagenase disaggregation of decapsulated rat glands. This steroidogenic response is not accompanied by any acute increase in the incorporation of [3H]uridine into acid insoluble RNA; indeed a slight decrease is observed during the incubations. A wide variety of different effects of actinomycin D on adrenal steroidogenesis have previously been reported. The effects of a range of actinomycin D concentrations (1–50 μmol/l) on the steroidogenesis brought about by different concentrations of ACTH (0.1–100. mi.u./ml) and cyclic AMP (1–5 mmol/l) were therefore examined. Actinomycin D (1 μmol/l) inhibits overall RNA synthesis by over 91% but has little or no effect on the cellular response to low concentrations of ACTH, although both basal (non-stimulated) corticosterone output and cyclic AMP stimulated steroidogenesis are appreciably inhibited (by 29–54%). Even at very high doses of actinomycin D (50 μmol/l), which inhibit RNA synthesis by 96% a substantial steroidogenic stimulation is obvious at all concentrations of ACTH and cyclic AMP studied. There is a greater inhibition of stimulated steroidogenesis not only with increasing actinomycin D concentrations, but also with increasing time of cellular exposure to actinomycin D. It is concluded that the acute steroidogenic ACTH mechanism does not require newly synthesized RNA and that if the inhibition by actinomycin D is simply due to an effect on synthesis of various RNA species, then the shortest estimate of the half-life of any RNA involved is 70 min.

On the mechanism of action of cholera toxin on isolated rat adrenocortical cells. Comparison with the effects of adrenocorticotropin on steroidogenesis and cyclic AMP output.

The effects of cholera toxin on isolated rat adrenocortical cells have been investigated. Both steroid and cyclic AMP output from adrenal cells were increased by the toxin in a dose dependent fashion. The concentration of toxin for half maximal stimulation for both of these responses was about 40 ng/ml. Maximal steroidogenesis and cyclic AMP output was obtained with similar concentrations of the toxin. A correlation was observed between the low amounts of cyclic AMP produced in response to all doses of cholera toxin and to physiologically significant concentrations of adrenocorticotropin (ACTH) (less than 0.1 munit/ml; i.e. submaximal for steroidogenesis in this system). This was in direct contrast to the much higher levels of cyclic AMP generated by concentrations of ACTH greater than 1 munits/ml. Time course studies demonstrated a time-lag between toxin addition and steroid response of at least 40 min. Binding of cholera toxin to adrenal cells was rapid and was 90% complete within 15 min at both 37 and 0 degrees C. These data indicate that most of the delay in response to cholera toxin is due to processes subsequent to the initial binding interaction. Following the initial delay the subsequent maximal rate of steroidogenesis brought about by cholera toxin was very similar to that obtained with a concentration of ACTH that was maximal for steroidogenesis. Significant increases in cyclic AMP levels were detected about 20 min before increased steroidogenesis was apparent. Possible explanations for this result are considered. The results presented indicate great potential use for cholera toxin in the study of adrenal steroidogenic control mechanisms, particularly at the level of receptor mechanisms and the role of cyclic AMP.

Phosphodiesterase activity and the potentiation by theophylline of adrenocorticotrophin stimulated steroidogenesis and adenosine 3′,5′-monophosphate levels in isolated rat adrenal cells

Abstract Corticosterone production and adenosine 3′,5′-monophosphate levels in collagenase prepared isolated rat adrenal cells have been measured in response to adrenocorticotrophin in the presence and absence of theophylline. Theophylline (1mM) was found to potentiate the steroidogenic effect of submaximal concentrations of adrenocorticotrophin. This concentration of theophylline was without effect on protein synthesis in this system. Potentiation of adrenocorticotrophin stimulated adenosine 3′,5′-monophosphate levels was also observed in the presence of theophylline (0.5 and 1.0mM). Phosphodiesterase activity in collagenase prepared adrenal cells was 67% of that in intact glands, while the activity in trypsin prepared cells was 37% of that in intact glands.

Activation of adenylate cyclase in bovine adrenal cortex membranes by magnesium ions, guanine nucleotides and corticotropin

1. (1) Adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) activity in plasma membranes from bovine adrenal cortex was labile, but was stabilised in the presence of Mg2+ or 5′-guanylylimidodiphosphate (Gpp(NH)p). The activation of the enzyme by the nucleotide analogue could not be reversed by washing Gpp(NH)p-pretreated membranes. Similarly, Gpp(NH)p-activation was blocked, but not reversed, by EDTA. 2. (2) Maximal activation of adenylate cyclase by saturating concentrations of Gpp(NH)p required 20–30 min at 30°C. Once the activation was complete the enzyme was only minimally stimulated by corticotropin. Conversely, in the presence of both hormone and Gpp(NH)p, at 30°C, a maximum activity was attained in under 5 min. 3. (3) At 20°C, incubation of adrenal membranes for 40–60 min with Mg2+ alone, enhanced adenylate cyclase activity 2.4-fold. Gpp(NH)p-activation was not apparent until after 20 min and required about 2 h for completion. Corticotropin increased the initial rate of Gpp(NH)p-activation by at least 5 times and in the presence of both effectors, activity was maximal with 1 h. 4. (4) The maximum activity elicited by a combination of corticotropin and Gpp(NH)p was 55% greater than that observed in the presence of Gpp(NH)p alone, at both 20 and 30°C. 5. (5) It is concluded that guanine nucleotides, corticotropin and Mg2+ engender new conformation states of adrenal adenylate cyclase which have increased catalytic activity and increased stability to inactivation. An early stage of the guanine nucleotide activation process, possibly the binding, appears to require a minimum level of divalent cation. Whilst corticotropin increased the extent of guanine nucleotide activation the major action of the hormone is to enhance the rate of the activation.

Apparent positive cooperativity of ACTH action on adrenocortical cells: The effect of hormone degradation

The ability of adrenocortical cells to degrade ACTH1--39 and [125I]ACTH has been assessed under various conditions. Under conditions leading to increased hormone degradation there was an elevation of both the ED50 and the value of the Hill coefficient derived from concentration-effect curves for ACTH-stimulated steroidogenesis. Such degradative mechanisms offer a simple explanation for tha apparent positive cooperativity proposed by others for ACTH-receptor-adenylate cyclase interactions.

The effect of cholera toxin on the adrenal weight in hypophysectomized rats

The effects of cholera on adrenal weight in hypophysectomized rats were investigated, in an attempt to demonstrate an ACTH-like, adrenal trophic effect of the toxin. The results suggested that the toxin probably exerts is ACTH-like action on the adrenal via adenylate cyclase. Cholera toxin was also shown to have a thermolytic action, similar to that of ACTH, probably due to stimulation of adrenal glucocorticoid secretion.

Preparation and assessment of antisera to ACTH

Antisera to ACTH were produced in rabbits injected repeatedly at multiple intradermal sites with synthetic [Asp25, Ala26, Gly27]alphah-corticotropin-(1-28)-octacosapeptide-bovine gamma globulin conjugate (octacosapeptide is a sequence analogue of alphah1-28-ACTH). Antibodies to extracted human or porcine ACTH were detected in all of the sera 1 month after immunization. A considerable proportion of the antisera obtained from a single final bleeding 5 months after the primary immunization were suitable for sensitive radioimmunoassay. The antisera were shown to neutralize the steroidogenic activity of ACTH in an isolated rat adrenal cell bioassay system. Titres estimated from antiserum dilution curves and relative avidities from the standard curves were compared. It was possible to detect picogram amounts of ACTH in plasma-free medium with the best antisera. The method described is an effective means of producing anti-sera to the weakly immunogenic N-terminal fragment of the ACTH molecule.