ACTH-treated Rats Research Articles

Adrenocorticotrophic hormone-induced hypertension in the rat: effects of the endothelin antagonist bosentan.

1. The effects of the endothelin antagonist bosentan on adrenocorticotrophic hormone (ACTH)-induced hypertension were examined in the conscious male Sprague-Dawley rat. 2. In order to confirm endothelin antagonism, 18 rats were randomly divided into two groups: receiving either (i) endothelin-1 (0.125, 0.25, 0.5 and 1 nmol/kg, i.v.); or (ii) endothelin-1 at these doses following bosentan (100 mg/kg gavage) and mean arterial pressure recorded (study A). Subsequently, 40 male rats (320 +/- 5 g) were randomly divided into four groups (n = 10): (i) Sham (0.9% saline, s.c.) + 5% acacia gum gavage; (ii) ACTH (500 micrograms/kg per day, s.c.) + 5% acacia gum gavage; (iii) Sham injection + bosentan (100 mg/kg per day) gavage; or (iv) ACTH + bosentan. Six control days (C1-C6) were followed by 11 treatment days (T0-T10). Systolic blood pressure, water intake, urine volume, food intake and bodyweight were measured every second day (study B). 3. Bosentan significantly attenuated the endothelin-1-induced blood pressure rise at 0.125 nmol/kg (P < 0.05), but not at higher doses. 4. Bosentan at a dose which attenuated endothelin-1-induced blood pressure increase had no effect on either blood pressure or metabolic parameters in ACTH-treated rats. 5. These results suggest that endothelin does not play a major role in ACTH-induced hypertension.

Induction of Adrenal Scavenger Receptor BI and Increased High Density Lipoprotein-Cholesteryl Ether Uptake by in Vivo Inhibition of Hepatic Lipase

Hepatic lipase (HL) and scavenger receptor type B class I (SR-BI) have both been implicated in high density lipoprotein (HDL)-cholesteryl ester uptake in cholesterol-utilizing tissues. Inactivation of HL by gene-directed targeting in mice results in up-regulation of SR-BI expression in adrenal gland (Wang, N., Weng, W., Breslow, J. L., and Tall, A. R. (1996) J. Biol. Chem. 271, 21001-21004). The net effect on HDL-cholesteryl ester uptake is not known. We determined the impact of acute in vivo inhibition of rat adrenal HL activity by antibodies on SR-BI expression and on human and rat HDL-[3H]cholesteryl ether (CEth) uptake in the adrenal gland. Rat HDL was isolated from rats in which HL activity had been inhibited for 1 h. The rats were studied under basal conditions (not ACTH-treated) and after previous treatment with ACTH for 6 days (ACTH-treated). Intravenous injection of anti-HL resulted in 70% lowering of adrenal HL activity in both conditions which were maintained for at least 8 h. In not ACTH-treated rats, inhibition of adrenal HL increased adrenal SR-BI mRNA (5.2-fold) and mass (1. 6-fold) within 4 h. HL inhibition resulted in 41% and 14% more adrenal accumulation of human HDL-[3H]CEth during 4 and 24 h, respectively. The adrenal uptake of rat HDL-[3H]CEth increased by 68%, 4 h after the antibody injection. ACTH treatment increased total adrenal HL activity from 3.7 +/- 0.5 milliunits to 34.0 +/- 17. 2 milliunits, as well as adrenal SR-BI mRNA from 2.9 +/- 0.7 arbitrary units (A.U.) to 86.8 +/- 41.1 A.U. and SR-BI mass from 7.7 +/- 1.8 A.U. to 63.16 +/- 46.7 A.U. The human HDL-[3H]CEth uptake by adrenals was also significantly increased from 0.58 +/- 0.11% of injected dose to 7.24 +/- 1.58% of injected dose. Inhibition of adrenal HL activity did not result in further induction of SR-BI expression and did not affect human HDL-[3H]CEth uptake. These findings indicate that SR-BI expression may be influenced by changes in HL activity. HL activity is not needed for the SR-BI-mediated HDL-cholesteryl ester uptake by rat adrenal glands.

Plasma marinobufagenin-like and ouabain-like immunoreactivity in adrenocorticotropin-treated rats.

Recently, an endogenous digitalis-like factor (EDLF) was shown to be stimulated in corticotropin (ACTH) hypertension in the rat. We have shown that mammalian plasma contains a vasoconstrictor Na,K-ATPase inhibitor, which cross-reacts with an antibody to amphibian EDLF, marinobufagenin. In the present experiment, the effect of 8 days of intramuscular ACTH treatment (0.5 mg/kg/day) of male Fisher 344 x NB rats on blood pressure, plasma ouabain-like and marinobufagenin-like immunoreactivity, and on the activity of Na,K-ATPase in aortic sarcolemma were studied. The ACTH treatment for 8 days resulted in increased systolic blood pressure (151 +/- 12.4 v 121 +/- 4.0 mm Hg, P < .01), inhibition of Na,K-ATPase in aortic sarcolemma (2.99 +/- 0.35 v 5.43 +/- 0.17 micromol ADP/mg(prot)/h), and increases in plasma concentration of marinobufagenin-like (0.44 +/- 0.06 v 0.21 +/- 0.05 nmol/L), but not ouabain-like (0.09 +/- 0.01 v 0.10 +/- 0.04 nmol/L) immunoreactivity. In dissociation enhanced lanthanide fluoroimmunoassay (DELFIA), serial dilutions of plasma from ACTH-treated rats extracted with 25% and 80% acetonitrile, respectively, demonstrated parallelism to the calibration curves of ouabain and marinobufagenin. These findings suggest that an endogenous bufodienolide Na,K-ATPase inhibitor, rather than an endogenous ouabain-like compound, is increased after 8 days of treatment of rats with ACTH.

Neuromedin U enhances proliferation of ACTH-stimulated adrenocortical cells in the rat

Neuromedin U (NMU) is a brain-gut peptide involved in the regulation of the hypothalamic-pituitary-adrenal axis and adrenocortical cell proliferation. In this study, we investigated the effects of NMU8 (three subcutaneous injections of 6.0 nmol/100 g, 24, 16 and 8 h before autopsy) on the adrenal glands of rats treated for 2 or 4 days with a low (2 microg/100 g body weight/24 h) or a high (8 microg) dose of adrenocorticotropic hormone (ACTH). As revealed by RT-PCR, ACTH treatment did not prevent expression of NMUR1 in rat adrenal cortex. At day 4 of ACTH administration, the weight of adrenals was lower than at day 2. NMU8 administration prevented ACTH-induced increases of adrenal weight at day 2 of the experiment. ACTH plasma concentrations were increased in all ACTH-administered rats. NMU8 administration increased ACTH plasma concentration at day 2 of the lower ACTH dose-treated group while it reduced the ACTH plasma level at day 4 in the higher ACTH dose-administered rats. In all groups of ACTH-treated rats, NMU8 changed neither aldosterone nor corticosterone plasma concentrations. In the zona glomerulosa (ZG), NMU8 increased metaphase index at days 2 and 4 in the lower ACTH dose-treated group and had no statistically significant effect in rats treated with the higher ACTH dose. In the zona fasciculata (ZF), NMU8 administration increased metaphase index at day 2 in the lower ACTH dose-treated group but reduced metaphase index at day 4 in the higher dose ACTH-administered rats. NMU8 reduced the number of cells per unit area both in ZG and ZF at day 2 in the higher ACTH dose-treated rats. In the remaining groups NMU8 did not produce statistically significant changes in the number of cells per unit area. Thus, our findings demonstrate that exogenous NMU may stimulate proliferation primarily of the cortical ZG cells in rats administered with ACTH, although at high doses of exogenous corticotropin an opposite effect occurred.

Differential gene expression of cytochrome P450 11beta-hydroxylase in rat adrenal cortex after in vivo activation.

In situ hybridization histochemistry was used to monitor the expression of 3beta-hydroxysteroid dehydrogenase, delta4-isomerase (3betaHSD) and cytochrome P450 11beta-hydroxylase (P45011beta) messenger RNA (mRNA) in adult rat adrenals after stimulation in vivo. In Exp 1, adrenals were collected from rats injected with saline or ACTH for 1, 2, 3, or 4 days. Adrenal sections from saline-treated rats showed uniform expression of 3betaHSD mRNA that extended from the adrenal capsule to the medullary border. In contrast, P45011beta mRNA showed high levels in the outer fasciculata and low levels in the inner fasciculata/reticularis. In response to ACTH, the integrated density of 3betaHSD hybridization did not increase until 4 days. The integrated density of P45011beta hybridization increased in ACTH-treated rats between 1-4 days due to increased hybridization in the inner fasciculata/reticularis. In Exp 2, rats were treated with ACTH or saline, and adrenals were harvested at 4, 8, or 24 h. The hybridization density of 3betaHSD did not change after ACTH or saline injection. Increased expression of P45011beta mRNA was observed at 4 and 8 h, but not 24 h post-ACTH. In Exp 3, to determine the response to acute stress, adrenals were collected from rats 24 h after surgical laparotomy. The integrated density of 3betaHSD labeling did not change, whereas both hybridization area and mean density of P45011beta increased. Increased expression of P45011beta mRNA was observed in the inner fasciculata similar to that observed after ACTH injection. In addition, adrenal cells were more responsive to ACTH in vitro after surgical stress. These results suggest that the rat adrenal cortex can respond to acute stress by up-regulation of the expression of steroidogenic enzyme genes and that this occurs in part by increasing the number of cells actively expressing P45011beta mRNA. The adrenal response after stress most likely results at least in part from stimulation by ACTH. These findings suggest that changes in adrenal steroidogenesis in response to ACTH may result from recruitment of steroidogenic cells to synthesize and secrete corticosteroids.

Adrenocorticotropin-induced hypertension in rats: Role of ouabain-like compound

We examined the role of ouabain-like compound (OLC) in hypertension associated with corticotropin (ACTH) excess in rats. Physiological saline solution (1 mL/kg) or synthetic ACTH-Z (0.5 mg/kg) was injected intramuscularly for 15 days to 14 control and 13 male Wistar rats. Significant increases in blood pressure and plasma sodium/potassium ratio, and decreases in plasma potassium concentration and urinary sodium/potassium ratio were observed in ACTH-treated rats. The plasma OLC level was higher in ACTH-treated group (control; 76 ± 13, ACTH; 202 ± 48 pmol/L, P < .05). Plasma OLC level correlated with systolic blood pressure (SPB; r = 0.53 P < .01). Urinary OLC excretion was also higher in ACTH-treated group (control; 0.95 ± 0.01, ACTH; 3.32 ± 0.67 pmol/day, P < .01). A significant relation was also found between urinary OLC excretion and SBP (r = 0.66, P < .01). Plasma potassium concentration negatively correlated with SBP (r = −0.48, P < .01) and urinary sodium/potassium ratio also correlated inversely with urinary OLC excretion (r = −0.55, P < .01). Measurement of OLC levels after the fractionation of urine by reverse-phase high performance liquid chromatography showed that the major OLC peak in urine from both groups coincided with that of authentic ouabain. These results suggest the contribution of OLC to ACTH-induced hypertension in rats.

Adrenocorticotropin-Induced Hypertension in Rats Role of Ouabain-like Compound

We examined the role of ouabain-like compound (OLC) in hypertension associated with corticotropin (ACTH) excess in rats. Physiological saline solution (1 mL/kg) or synthetic ACTH-Z (0.5 mg/kg) was injected intramuscularly for 15 days to 14 control and 13 male Wistar rats. Significant increases in blood pressure and plasma sodium/potassium ratio, and decreases in plasma potassium concentration and urinary sodium/potassium ratio were observed in ACTH-treated rats. The plasma OLC level was higher in ACTH-treated group (control; 76 +/- 13, ACTH; 202 +/- 48 pmol/L, P < .05). Plasma OLC level correlated with systolic blood pressure (SPB; r = 0.53, P < .01). Urinary OLC excretion was also higher in ACTH-treated group (control; 0.95 +/- 0.01, ACTH; 3.32 +/- 0.67 pmol/day, P < .01). A significant relation was also found between urinary OLC excretion and SBP (r = 0.66, P < .01). Plasma potassium concentration negatively correlated with SBP (r = -0.48, P < .01) and urinary sodium/potassium ratio also correlated inversely with urinary OLC excretion (r = -0.55, P < .01). Measurement of OLC levels after the fractionation of urine by reverse-phase high performance liquid chromatography showed that the major OLC peak in urine from both groups coincided with that of authentic ouabain. These results suggest the contribution of OLC to ACTH-induced hypertension in rats.

DEHYDROEPIANDROSTERONE DOES NOT PREVENT ADRENOCORTICOTROPHIN‐INDUCED HYPERTENSION IN CONSCIOUS RATS

1. We tested the hypothesis that dehydroepiandrosterone (DHEA), which prevents dexamethasone-induced hypertension in rats, may block adrenocorticotrophin (ACTH) hypertension, which has been presumed due to corticosterone excess. The blood pressure and metabolic effects of DHEA (18 mg/kg per day) were examined in sham and ACTH-treated (0.5 mg/kg per day) conscious Sprague-Dawley rates (n = 20). 2. ACTH but not sham injection increased blood pressure, water intake and urine output and decreased bodyweight. 3. DHEA administration for 10 days did not alter blood pressure or metabolic effects in either sham or ACTH-treated rats. 4. DHEA, which is known to block dexamethasone-induced hypertension, did not modify ACTH-induced hypertension in the rat. This suggests either that ACTH-induced hypertension is not simply a consequence of glucocorticoid activity or that the action of DHEA in dexamethasone hypertension is not through blocking the glucocorticoid receptor.

ACTH does not control neonatal seizures induced by administration of exogenous corticotropin-releasing hormone.

ACTH has been used extensively for treatment of massive infantile spasms (MIS) and other intractable seizures. The mechanisms by which ACTH exerts anticonvulsant effects are unknown. ACTH is a neuropeptide with both endocrine and neuromodulatory properties; its efficacy against MIS could derive from intrinsic anticonvulsant properties or from hormonal effects, either directly or through glucocorticoids. We tested ACTH activity against exogenous corticotropin-releasing hormone (CRH)-induced seizures in the infant rat model. CRH was administered into the cerebral ventricles of 85 infant rats aged 5-13 days. ACTH was used either 20-60 min before CRH administration or "chronically" (pretreatment with four doses of ACTH every 6 h, before CRH administration). In a separate group of rat pups, we measured plasma corticosterone to ascertain ACTH availability. Administration of CRH, an age-specific endogenous convulsant, resulted in a prolonged series of seizures after 2- to 55-min latency. There was no difference in latency between controls (9.5 +/- 1.2 min) and ACTH-treated rats (12.4 +/- 2.8 min for combined acute and chronic groups). CRH-induced seizure duration (88.2 +/- 9 vs. 74.7 +/- 9.4 min) and severity of seizures was also unchanged by ACTH treatment. ACTH reached the circulation and caused significant increase in plasma glucocorticoids. ACTH does not block the convulsant action of exogenous CRH in infant rats. An alternative mechanism for the anticonvulsant effect of ACTH may be suppression of synthesis and secretion of an endogenous convulsant, i.e., CRH.

Rat liver contains a limited number of binding sites for hepatic lipase.

The binding of hepatic lipase to rat liver was studied in an ex vivo perfusion model. The livers were perfused with media containing partially purified rat hepatic lipase or bovine milk lipoprotein lipase. The activity of the enzymes was determined in the perfusion media before and after passage through the liver. During perfusion with a hepatic-lipase-containing medium the lipase activity in the medium did not change, indicating that there was no net binding of lipase by the liver. In contrast, more than 80% of the lipoprotein lipase was removed from the medium. This lipoprotein lipase activity could be recovered into the perfusion medium completely by heparin perfusion of the liver. If livers, first depleted of hepatic lipase by heparin, were subsequent perfused with a hepatic-lipase-containing medium, 90 +/- 24 m-units of the lipase activity was bound per g of liver (up to 1000 m-units/total liver). However, heparin treatment of the liver decreases the ability of the liver to re-bind hepatic lipase by 80%. Perfusion of rat livers with 0.3 M NaCl released 60% of the lipase activity into the medium. Upon subsequent perfusion of these livers with hepatic-lipase-containing media, 541 +/- 164 m-units of hepatic lipase could be bound per g of liver (up to 5000 m-units/total liver). The binding of hepatic lipase was also studied in livers of corticotropin (ACTH)-pre-treated rats. In these rats also, hepatic lipase bound only to livers which had been pre-perfused with heparin or 0.3 M NaCl. After heparin pre-perfusion, 88 +/- 12 m-units of hepatic lipase could be bound per g of liver, similar to that with livers of control rats not treated with ACTH. After prior salt perfusion, however, the capacity of the livers of ACTH-pre-treated rats to bind hepatic lipase was 212 +/- 60 m-units/g of liver. This is less than in livers of control rats (541 +/- 164 m-units/g of liver). These results indicate that in rat liver the binding of hepatic lipase is heterogeneous in character and consists of heparin-resistant and heparin-sensitive components. The hepatic-lipase binding capacity of the liver is saturable and fully utilized under various conditions. The heparin-sensitive binding capacity is lowered in ACTH-treated rats, whereas the heparin-resistant binding is unaffected. We postulate that the functional hepatic lipase activity can be regulated by changes in the binding capacity of the liver.

Modulation of aldosterone synthase messenger ribonucleic acid levels by dietary sodium and potassium and by adrenocorticotropin.

Recent evidence suggests that an aldosterone synthase (AS) separate from the 11 beta-hydroxylase (11 beta-OHase) mediates the final step(s) in aldosterone synthesis in the rat. We have compared changes in AS and 11 beta-OHase mRNA levels with experimental maneuvers known to stimulate or suppress aldosterone secretion. In Exp 1, male rats were fed regular rat chow (group 1), a low sodium, high potassium diet (group 2), or a high sodium, low potassium diet (group 3). Northern analysis of adrenal capsular (zona glomerulosa) and decapsulated adrenal core (fasciculata-reticularis) tissues was performed with specific oligonucleotide probes for AS and 11 beta-OHase mRNAs, normalized with a cDNA probe for 18S ribosomal RNA (rRNA). There was a marked increase in capsular AS mRNA in group 2 rats compared to levels in group 1 (P < 0.0001) and group 3 (P < 0.0001) rats. Capsular As mRNA decreased (P < 0.05) in group 3 compared to that in group 1 rats. Adrenal core AS mRNA levels were quite low with all three diets. In contrast, capsular and core 11 beta-OHase mRNA levels did not change significantly with diet. In Exp 2, two groups served as controls (groups 1 and 3), and two groups received sc injections of repository ACTH (groups 2 and 4). Control and ACTH-treated rats were killed 3 h (groups 1 and 2) or 24 h (groups 3 and 4) after initial ACTH administration. Capsular and core AS and 11 beta-OHase mRNA levels were evaluated with and without normalization with 18S rRNA, cyclophilin, and glyceraldehyde phosphate dehydrogenase housekeeping probes. Capsular AS mRNA increased at 3 h (P < 0.05), but decreased at 24 h (P < 0.01). Nonnormalized adrenal capsular and core 11 beta-OHase mRNA levels were unchanged at 3 h, but increased significantly at 24 h (P < 0.05) in capsular tissue of ACTH-treated rats. However, in ACTH-treated rats, 18S rRNA and cyclophilin mRNA levels increased at 24 h in both capsular and core tissue (P < 0.01), and glyceraldehyde phosphate dehydrogenase mRNA levels increased in capsular tissue (P < 0.00001), resulting in a lack of change in normalized 11 beta-OHase mRNA. Changes in cholesterol side-chain cleavage were similar to those in 11 beta-OHase mRNA. These data provide further evidence that a separate AS plays a significant role in modulating aldosterone secretion in rodents.

Effects of chronic corticotrophin treatment on aldosterone metabolism in the rat.

Chronic treatment with high doses of ACTH leads to marked reduction in aldosterone biosynthesis and secretion both in vivo and in vitro. In contrast, it has been reported that peripheral plasma aldosterone levels may be elevated following prolonged ACTH treatment. The present study attempts to determine the reason(s) for this apparently paradoxical finding. ACTH treatment (40 micrograms/100 g body weight) of male Sprague-Dawley rats for 7 days caused a decrease of more than 90% in aldosterone secretion into the adrenal vein in vivo and aldosterone production by intact adrenal capsules incubated in vitro. In contrast, peripheral plasma aldosterone levels appeared to be increased when measured by radioimmunoassay using two different polyclonal antibodies (antibody 1 (AB1) raised against aldosterone-3-carboxymethyloxime-bovine serum albumin (BSA) and antibody 2 (AB2) raised against aldosterone-21-hemisuccinate-BSA). However, when a highly specific monoclonal antibody (raised against aldosterone-3-carboxymethyloxime-BSA and showing low cross-reactivity to aldosterone metabolites) was used, peripheral plasma aldosterone levels appeared to be reduced in ACTH-treated rats. Following chromatographic fractionation of peripheral plasma, significantly more material with aldosterone-like immuno-reactivity, but which was less polar than authentic aldosterone in chromatographic mobility, was detected in the fractions using antibodies AB1 and AB2. The absence of this material from fractions of adrenal vein plasma leads us to infer that this material is generated in the peripheral circulation, probably as a result of hepatic metabolism. In addition, the overall metabolic clearance rate (MCR) of [3H] aldosterone was found to be significantly decreased following prolonged ACTH treatment. We conclude that the seemingly discrepant findings with regard to the effects of chronic ACTH treatment on peripheral plasma aldosterone levels and the secretion of aldosterone in vivo can be reconciled by (1) the changes in the overall MCR of aldosterone and (2) the generation of increased quantities of aldosterone metabolites such as 5 alpha-dihydroaldosterone and 3 alpha, 5 beta-tetrahydroaldosterone which show significant cross-reactivity with some aldosterone antibodies.

Inhibition of NO synthesis has an additive effect on hypertension induced by ACTH in conscious rats.

1. The haemodynamic and metabolic effects of oral intake of approximately 30 mg/kg per day N-nitro-L-arginine (NOLA) were examined in sham and adrenocorticotrophin (ACTH, 0.5 mg/kg per day) treated conscious Sprague-Dawley rats (n = 33). 2. NOLA administration produced an increase in systolic blood pressure of 24 +/- 6 mmHg (P < 0.001), but did not alter food or water intake, urine volume or electrolyte excretion in rats not treated with ACTH. 3. Compared with sham injection, ACTH-treated rats demonstrated an increase in systolic blood pressure (water + sham, 3 +/- 1 mmHg; water + ACTH, 16 +/- 3 mmHg; P < 0.001), loss of bodyweight, and increases in water intake and urine volume. 4. The magnitude of the blood pressure rise in ACTH-treated rats was greater in those receiving NOLA than in those drinking water only (water + ACTH, 16 +/- 3 mmHg; NOLA + ACTH, 37 +/- 3 mmHg; P < 0.05). Metabolic changes were similar. 5. Inhibition of nitric oxide is unlikely to be a major determinant of ACTH-induced hypertension in the rat, since NOLA increased blood pressure whether or not ACTH was administered, indicating an additive effect of ACTH and NOLA administration.

Arachidonic acid biosynthesis in non-stimulated and adrenocorticotropin-stimulated Sertoli and Leydig cells.

The biosynthesis of arachidonic acid in Sertoli and Leydig cells isolated from the testes of mature rats has been investigated. Both types of cells incorporated [2-14C]eicosatrienoic acid from the incubation medium and transformed it into arachidonic acid. The administration of adrenocorticotropin (ACTH) to the rats decreased the delta 5 desaturating activity in the isolated testicular cells, while ACTH produced no changes in the uptake of the substrate. Similar results were obtained when ACTH was added to the incubation medium of cells isolated from non-hormone treated rats. The total fatty acid composition of the Sertoli cells isolated from ACTH-treated rats showed a significant increase in the relative percentage of 18:2n-6 and a decrease in the C20 and C22 polyenes. This may indicate that ACTH exerts an inhibitory effect on delta 6, delta 5 and delta 4 desaturase activities. Addition of corticosterone to the incubation medium also produced a significant decrease in arachidonic acid biosynthesis. Because ACTH is known to stimulate the release of corticosterone in vivo, both hormones may act cumulatively in the regulation of arachidonic acid metabolism in Sertoli and Leydig cells.

Effect of chronic ACTH treatment on the physical state of lipid droplets in rat adrenocortical cells

A histophysical method has been adapted to determine the thermotropic phase transitions of adrenocortical lipid droplets using a polarizing microscope equipped with a cold/hot stage. Cryosections of freshly-removed, unfixed adrenals, derived from control (untreated), and 14 days ACTH-treated rats were examined. The lipid droplets in the zona fasciculata and zona reticularis of the untreated rats were birefringent at room temperature (22°C). The birefringence of zona glomerulosa lipids selectively increased in the temperature range from −10 to −15°C. In cryosections prepared from ACTH-treated rats, thermotropic phase transitions of the lipid droplets appeared at a temperature range between −30 and −40°C in each cortical zone. The chemical analysis of the isolated lipids revealed that the relative amount of triglycerides in the zona fasciculata lipids increased, while that of free and esterified cholesterol decreased after chronic ACTH treatment. Present data suggest that the increased fluidity of lipid droplets promotes lipid mobilization in response to the enhanced demand of the chronically stimulated adrenocortical cells. Viscosity-dependent mobilization of free cholesterol from lipid droplets is not a rate-limiting process in adrenal steroidogenesis, but rather may represent an important control of the availability of precursor from lipid stores.

Heterogeneous pools of cholesterol side-chain cleavage activity in adrenal mitochondria from adrenocorticotropic hormone-treated rats: Reconstitution of the isocitrate response with succinate and low concentrations of isocitrate

Cholesterol side-chain cleavage in isolated adrenal mitochondria requires unique energy requirements that may determine not only electron transport to P450 but also cholesterol availability. In mitochondria from ACTH-treated rats, two approximately equal pools of reactive cholesterol are indicated by the partial effectiveness of succinate (SU; Type A), and the metabolism of residual cholesterol by 1 m m isocitrate (IC; Type B). Type A metabolism is associated with relatively few initial cholesterol-P450 scc complexes and is rapidly and selectively lost when mitochondria are preincubated without an energy source. We now show that cholesterol metabolism supported by IC resolves into equal high and low affinity components (EC 50 = 10 and 250 μ m) exhibiting, respectively, Type A and Type B characteristics. SU and 50 μ m IC, in combination, provided nearly the same activity characteristics as 1 m m IC, including resistance to preincubation and increased turnover of cholesterol-P450 scc complexes. Much higher (three to six times) and more sustained pregnenolone formation was seen, with all reductants, following either enhancement of the reactive cholesterol pool or addition of 20-α-hydroxycholesterol, indicating that adrenocorticotropic hormone-mitochondria are limited by substrate availability. ATP generation was most effectively supported by SU, and IC was maximally active at 50 μ m, emphasizing differences between respiratory and steroidogenic energy requirements. ATP production and the maintenance of uniform suppression after in vivo cycloheximide treatment indicate the integrity of the mitochondrial interaction with all reductants. Inhibitors of SU oxidation (KCN, malonate) strongly inhibited SU-supported cholesterol metabolism but had little effect on SU synergism with IC. Fumarate (but not α-ketoglutarate or oxaloacetate) was equally effective as a synergist, but was totally ineffective as a reductant. SU or fumarate, therefore, act by a nonreductive pathway to boost NADPH production from low concentrations of IC. This decrease in apparent K m for IC may be mediated by stimulation of mitochondrial uptake of the reductant through the specific transporters.

Highly selective inhibition of estrogen biosynthesis by CGS 20267, a new non-steroidal aromatase inhibitor

CGS 20267 is a new non-steroidal compound which potently inhibits aromatase in vitro (IC 50 of 11.5 nM) and in vivo (ED 50 of 1–3 μg/kg p.o.). CGS 20267 maximally inhibits estradiol production in vitro in LH-stimulated hamster ovarian tissue at 0.1 μM with an IC 50 of 0.02 μM and does not significantly affect progesterone production up to 350 μM. In ACTH-stimulated rat adrenal tissue in vitro, aldosterone production was inhibited with an IC 50 of 210 μM (10,000 times higher than the IC 50 for estradiol production); no significant effect on corticosterone production was seen at 350 μM. In vivo, in ACTH-treated rats, CGS 20267 does not affect plasma levels of corticosterone or aldosterone at a dose of 4 mg/kg p.o. (1000 times higher than the ED 50 for aromatase inhibition in vivo). In adult female rats, a 14-day treatment with 1 mg/kg p.o. daily, completely interrupts ovarian cyclicity and suppresses uterine weight to that seen 14 days after ovariectomy. In adult female rats bearing estrogen-dependent DMBA-induced mammary tumors, 0.1 mg/kg p.o. given daily for 42 days caused almost complete regression of tumors present at the start of treatment. Thus compared to each other, CGS 16949A and CGS 20267 are both highly potent in inhibiting estrogen biosynthesis in vitro and in vivo. The striking difference between them is that unlike CGS 16949A, CGS 20267 does not affect adrenal steroidogenesis in vitro or in vivo, at concentrations and doses several orders of magnitude higher than those required to inhibit estrogen biosynthesis.

Heterogeneous pools of cholesterol side-chain cleavage activity in adrenal mitochondria from ACTH-treated rats: Differential responses to different reducing precursors

Side-chain cleavage (SCC) of endogenous cholesterol in adrenal mitochondria isolated from ACTH-treated rats indicates that the size of the reactive cholesterol pool depends on the reducing precursor. At optimal concentrations of reductant, this pool was typically at least 2 times greater for isocitrate than for succinate. Succinate-supported reactions were rapidly completed, were highly sensitive to a 2-min preincubation, and failed to deplete spectrally detected P-450 scc-cholesterol complexes. Cholesterol SCC with 1 mM isocitrate exhibited 2–3 times more fast-phase metabolism, a pronounced slow phase, insensitivity to preincubation, and 60% depletion of spectrally detected cholesterol- P-450 scc complexes. Addition of bovine serum albumin (BSA) and EDTA, either during homogenization or directly to the incubation, prevented preincubation losses in response to succinate and removed most of the difference between succinate and isocitrate activities. This effect of BSA/EDTA was reversed within 5 min by octanoate by a mechanism that was enhanced by Ca 2+. These distinct reductant characteristics suggest that only a subpopulation of mitochondria or of pools of activity within individual mitochondria can support cholesterol SCC with succinate while isocitrate is necessary for the remainder. The rapid responses of succinate-supported metabolism to preincubation or to octanoate suggest depletion of a critical factor for cholesterol metabolism. Metabolism of added 20α-hydroxycholesterol or deoxycorticosterone established that NADPH remained fully available after succinate-supported cholesterol metabolism had stopped or after preincubation. Cessation of pregnenolone formation, therefore, results from a failure to supply cholesterol, not inadequate NADPH. The preincubation effect suggests loss of an energy-dependent component that enhances this supply of cholesterol. One possibility tested was that GTP, an activator of intermembrane cholesterol transfer (Xu et al. (1989) J. Biol. Chem. 264, 17674–17680), was being lost. Added GTP slightly activated succinate-supported pregnenolone production but did not prevent preincubation-induced losses. α-Ketoglutarate, which can generate matrix GTP, is an effective reductant that, in combination with succinate, prevents preincubation-induced losses.

Effects of chronic treatment with adrenocorticotropin on adenylate cyclase activity in the adrenal cortex

The effects of prolonged administration of ACTH in vivo on adenylate cyclase activity in the zona glomerulosa (the capsular fraction) and the zona fasciculata-reticularis (the decapsulated fraction) of rat adrenocortical glands were investigated. Hyperresponsiveness of adenylate cyclase activity to ACTH in vitro was observed for both the capsular and the decapsulated fractions from ACTH-treated rats with a similar magnitude of stimulation by NaF, the guanine nucleotide or forskolin. There were no differences in binding sites for ACTH in membranes between the ACTH- and the saline-treated rats. The augmenting effects of the in vivo ACTH treatment on the enzyme stimulated by ACTH were accompanied by an increased sensitivity to the guanine nucleotide, suggesting that ACTH treatment in vivo was followed by an increased efficacy in coupling of the ACTH receptors to the adenylate cyclase complex associated with the guanine nucleotide regulatory components.

Adrenocorticotrophin-induced hypertension in the rat: haemodynamic, metabolic and morphological characteristics

Adrenocorticotrophin (ACTH) administration has been systematically studied in man and sheep. It raises systolic blood pressure (SBP) in the rat, but this has been little studied. ACTH was injected once daily at 0.5 mg/kg for 12 days in male Sprague-Dawley rats (n = 19). Sham-injected animals were studied in parallel (n = 15). ACTH increased SBP from 94 +/- 4 to 121 +/- 4 mmHg (P less than 0.001), significantly greater (P less than 0.02) than sham injection. The SBP of ACTH-treated rats was significantly higher than that of sham-injected rats when the same animals were measured by both the tail-cuff method (ACTH, 126 +/- 3 mmHg; sham, 99 +/- 3 mmHg) and direct arterial cannulation (ACTH, 137 +/- 2 mmHg; sham, 123 +/- 3 mmHg): P less than 0.005 and P less than 0.001, respectively. There was a loss of body weight, and increased water intake and urine output in ACTH-treated animals compared with both control (P less than 0.001) and sham treatments (P less than 0.02). ACTH increased plasma [Na] (sham, 140 +/- 1 mmol/l; ACTH, 145 +/- 1 mmol/l; P less than 0.001) and urinary Na excretion compared with control (P less than 0.01) and sham injection (P less than 0.05), and also decreased plasma [K] (sham, 4.6 +/- 0.2 mmol/l; ACTH, 3.3 +/- 0.8 mmol/l; P less than 0.01) and increased urinary K excretion (P less than 0.01) compared with control. SBP in adrenalectomized animals (n = 10) was unchanged by ACTH. ACTH increased adrenal, renal, cardiac and brain weights compared with sham injection (P less than 0.05). There were no significant changes in vascular morphology, although ACTH treatment increased glomerular epithelial cell droplets and abolished the adrenal zona glomerulosa.