Effects of prolonged sodium restriction on the morphology and function of rat adrenocortical autotransplants

Summary Regenerated adrenocortical nodules were obtained by implanting fragments of the capsular tissue of excised adrenal glands into the musculus gracilis of rats (Belloni et al. 1990). Five months after the operation, operated rats showed a normal basal blood level of corticosterone, but a very low concentration of circulating aldosterone associated with a slightly increased plasma renin activity (PRA). Regenerated nodules were well encapsulated and some septa extended into the parenchyma from the connective-tissue capsule. The majority of parenchymal cells were similar to those of the zonae fasciculata and reticularis of the normal adrenal gland, while zona glomerulosa-like cells were exclusively located around septa (juxta-septal zone; JZ). In vitro studies demonstrated that nodules were functioning as far as glucocorticoid production was concerned, while mineralocorticoid yield was very low. Prolonged sodium restriction significantly increased PRA and plasma aldosterone concentration, and provoked a marked hypertrophy of JZ, which was due to increases in both the number and average volume of JZ cells. Accordingly, the in vitro basal production of aldosterone and other 18-hydroxylated steroids was notably enhanced. The plasma level of corticosterone, as well as zona fasciculata/reticularis-like cells and in vitro production of glucocorticoids by regenerated nodules were not affected. These findings, indicating that autotransplanted adrenocortical nodules respond to a prolonged sodium restriction similar to the normal adrenal glands, suggest that the relative deficit in mineralocorticoid production is not due to an intrinsic defect of the zona glomerulosa-like JZ, but is probably caused by the impairment of its adequate stimulation under basal conditions. The hypothesis is advanced that the lack of splanchnic nerve supply and chromaffin medullary tissue in regenerated nodules may be the cause of such an impairment.     

The effects of ageing on the morphology and function of the zonae fasciculata and reticularis of the rat adrenal cortex

Summary The morphological counterpart of the well-known age-dependent marked impairment of glucocorticoid secretion of rat adrenals was investigated by use of morphometric techniques. For this purpose 4-, 8-, 16- and 24-month-old rats were studied. Despite the notable lowering of both basal and ACTH-stimulated production of corticosterone by collagenase-dispersed inner adrenocortical cells, ACTH and corticosterone plasma concentrations displayed significant increases with ageing. Zona fasciculata (ZF) and zona reticularis (ZR) showed a notable hypertrophy in aged rats, which was due to rises in both the average volume and number of their parenchymal cells. The hypertrophy of ZF and ZR cells was in turn associated with increase in the volume of the mitochondrial compartment and proliferation of smooth endoplasmic reticulum, i.e., the two organelles involved in steroid-hormone synthesis. All these morphologic changes, conceivably due to the chronic exposure to high levels of circulating ACTH, are interpreted as a response enabling ZF and ZR to compensate for their age-dependent lowering in glucocorticoid secretion. Stereology also demonstrated that ZF and ZR cells underwent a striking age-related lipid-droplet repletion. Lipid droplets are the intracellular stores of cholesterol esters, the obligate precursors of steroid hormones in rats. This finding is in keeping with the contention that the mechanism underlying the age-dependent decline in rat-adrenal glucocorticoid secretion mainly involves impairments of the utilization of intracellular cholesterol previous to its intramitochondrial transformation to pregnenolone.     

Effects of prolonged cysteamine administration on the rat adrenal cortex: evidence that endogenous somatostatin is involved in the control of the growth and steroidogenic capacity of zona glomerulosa.

A week daily administration of cysteamine (CYS, 300 mg kg-1) lowered plasma aldosterone concentration in rats, without affecting PRA, kalaemia and the plasma levels of ACTH and corticosterone. Prolonged CYS treatment caused a notable hypertrophy of adrenal zona glomerulosa (ZG) and its parenchymal cells, without inducing any apparent change in zona fasciculata morphology. Isolated ZG cells from CYS-treated rats evidenced a notable enhancement in their basal and maximally-stimulated productions of aldosterone and corticosterone. All these effects of chronic CYS administration were completely reversed by the simultaneous infusion of rats with somatostatin (SRIF, 12 micrograms kg-1 h-1). CYS exposure was not found to directly affect the secretory activity of isolated ZG cells from normal rats. Since CYS is known to be a specific depletor of SRIF in different organs of rats, these findings suggest that endogenous SRIF may be involved in the modulation of ZG function.     

Evidence that long-term methionine-enkephalin administration stimulates rat adrenal zona fasciculata

Short-term methionine-enkephalin (DALA) treatment did not induce evident changes in the adrenal zona fasciculata and in the basal corticosterone output of dexamethasone-treated rats administered with maintenance doses of ACTH. Conversely, prolonged (5 days) DALA treatment caused a notable hypertrophy of zona fasciculata cells, along with a significant increase in the plasma concentration of corticosterone. It is suggested that enkephalins exert a trophic action on the rat zona fasciculata.     

Orexins stimulate corticosterone secretion of rat adrenocortical cells, through the activation of the adenylate cyclase-dependent signaling cascade

Orexins-A and B are two novel hypothalamic peptides, which, like leptin and neuropeptide-Y (NPY), are involved in the central regulation of feeding. Since leptin and NPY were found to modulate adrenal function, we have examined whether orexins are able to directly affect rat adrenal steroid secretion. Both orexin-A and orexin-B raised basal corticosterone secretion of dispersed rat zona fasciculata–reticularis (ZF/R) cells, their maximal effective concentration being 10⁻⁸ M. In contrast, orexins did not affect either maximally ACTH (10⁻⁹ M)-stimulated corticosterone production by ZF/R cells or the basal and agonist-stimulated aldosterone secretion of dispersed zona glomerulosa cells. The ACTH-receptor antagonist corticotropin-inhibiting peptide (10⁻⁶ M) annulled corticosterone response of ZF/R cells to ACTH (10⁻⁹ M), but not to orexins (10⁻⁸ M). Orexins (10⁻⁸ M) enhanced cyclic-AMP release by ZF/R cells, and the selective inhibitor of protein-kinase A (PKA) H-89 (10⁻⁵ M) abolished corticosterone responses to both ACTH (10⁻⁹ M) and orexins (10⁻⁸ M). A subcutaneous injection of both orexins (5 or 10 nmol/kg) evoked a clear-cut increase in the plasma concentration of corticosterone (but not aldosterone), the effect of orexin-A being significantly more intense than that of orexin-B. Collectively, these findings suggest that orexins exert a selective and direct glucocorticoid secretagogue action on the rat adrenals, acting through a receptor-mediated activation of the adenylate cyclase/PKA-dependent signaling pathway.     

Effects of streptozotocin-induced experimental diabetes on the morphology and function of the zona fasciculata of rat adrenal cortex

The effects of a severe streptozotocin (STZ)-induced diabetes on the morphology and function of the adrenal zona fasciculata were examined in rats with intact or pharmacologically interrupted hypothalamic-hypophyseal-adrenal axis. In animals with an intact hypothalamic-hypophyseal axis, STZ-diabetes induced hypertrophy of the cells of the zona fasciculata and a rise in the plasma corticosterone concentration. Conversely, in rats in which the hypothalamic-hypophyseal axis had been interrupted, experimental diabetes provoked atrophy of the zona fasciculata cells, and a lowering in the plasma corticosterone level. The effects of STZ-diabetes were completely reversed by insulin infusion in both groups of rats. The hypothesis is discussed that the chronic lack of insulin may directly inhibit the growth and steroidogenic capacity of the rat zona fasciculata and that this effect of experimental diabetes may be masked in rats with an intact hypothalamic-hypophyseal axis by the concurrent enhancement of ACTH release due to chronic stress resulting from the metabolic consequences of prolonged diabetes.     

Effects of prolonged infusion with endothelin-1 on the function and morphology of rat adrenal cortex

A week of SC infusion with endothelin-1 (ET-1) (0.2 microgram.kg-1.hr-1) lowered PRA and raised plasma aldosterone (A) concentration in rats. Kalaemia and the plasma levels of ACTH and corticosterone (B) were not affected. Prolonged ET-1 administration caused a notable hypertrophy of zona glomerulosa (ZG) and its parenchymal cells, without inducing any apparent change in zona fasciculata. Stereology showed that ZG cell hypertrophy was mainly due to the increase in the volume of the mitochondrial compartment and to the proliferation of smooth endoplasmic reticulum (i.e., the two organelles in which the enzymes of steroid synthesis are contained). Isolated ZG cells from ET-1-infused animals evidenced a notable enhancement in their basal production of A and B. The secretory responses of ZG cells to the maximal effective concentrations of their three main stimulators (ACTH, angiotensin-II and K+) displayed comparable increases. These findings indicate that ET-1, when chronically administered, is able to specifically enhance the growth and steroidogenic capacity of rat ZG, and suggest that the mechanism underlying this ET-1 effect involves stimulation of the de novo synthesis of both the steroidogenic enzymes and the membrane framework in which they are located.     

Chronic stress induces adrenal hyperplasia and hypertrophy in a subregion-specific manner

The adrenal gland is an essential stress-responsive organ that is part of both the hypothalamic-pituitary-adrenal axis and the sympatho-adrenomedullary system. Chronic stress exposure commonly increases adrenal weight, but it is not known to what extent this growth is due to cellular hyperplasia or hypertrophy and whether it is subregion specific. Moreover, it is not clear whether increased production of adrenal glucocorticoid after chronic stress is due to increased sensitivity to adrenocorticotropic hormone (ACTH) vs. increased maximal output. The present studies use a 14-day chronic variable stress (CVS) paradigm in adult male rats to assess the effects of chronic stress on adrenal growth and corticosterone steroidogenesis. Exogenous ACTH administration (0-895 ng/100 g body wt) to dexamethasone-blocked rats demonstrated that CVS increased maximal plasma and adrenal corticosterone responses to ACTH without affecting sensitivity. This enhanced function was associated with increased adrenal weight, DNA and RNA content, and RNA/DNA ratio after CVS, suggesting that both cellular hyperplasia and hypertrophy occurred. Unbiased stereological counting of cells labeled for Ki67 (cell division marker) or 4,6-diamidino-2-phenylindole (nuclear marker), combined with zone specific markers, showed that CVS induced hyperplasia in the outer zona fasciculata, hypertrophy in the inner zona fasciculata and medulla, and reduced cell size in the zona glomerulosa. Collectively, these results demonstrate that increased adrenal weight after CVS is due to hyperplasia and hypertrophy that occur in specific adrenal subregions and is associated with increased maximal corticosterone responses to ACTH. These chronic stress-induced changes in adrenal growth and function may have implications for patients with stress-related disorders.     

Stereological and functional investigations on isolated adrenocortical cells: Zona fasciculata/reticularis cells of chronically ACTH-treated rats

Summary The morphology and function of isolated inner (zona fasciculata/reticularis) adrenocortical cells of rats pretreated with ACTH for 3, 6, 9 or 12 days were investigated. ACTH treatment induced a notable time-dependent enhancement in the steroidogenic capacity (corticosterone production) and growth of inner cells. The volumes of cells, mitochondrial compartment, membrane space [the cellular space occupied by smooth endoplasmic reticulum (SER) membranes] and lipid-droplet compartment, as well as the surface area of mitochondrial cristae and SER tubules, were increased in relation to the duration of ACTH pretreatment, and showed a highly significant positive linear correlation with both basal and stimulated corticosterone production. The acute exposure of isolated cells to ACTH provoked a striking lipid-droplet depletion, the extent of which was linearly and positively correlated with stimulated corticosterone secretion. The hypertrophy of the mitochondrial compartment and SER are interpreted as the morphological counterpart of the enhanced steroidogenic capacity of inner adrenocortical cells, inasmuch as the enzymes of steroid synthesis are located in these two organelles, and it is well known that chronic ACTH exposure stimulates the de novo synthesis of many of them in vivo. The rise in the number of lipid droplets, in which cholesterol is stored, is interpreted as being due to the fact that, under chronic ACTH treatment, the processes leading to cholesterol accumulation in adrenocortical cells (exogenous uptake and endogenous synthesis) exceed those of its utilization in basal steroid secretion. Cholesterol accumulated in lipid droplets as a reserve material may be rapidly utilized after acute ACTH exposure to meet the needs of the enhanced steroidogenic capacity of adrenocortical cells.     

The effects of chronic SRIH-14 and octreotide administration on the pituitary-adrenal axis in adult male rats

The effects of chronic treatments with SRIH-14 and octreotide on pituitary corticotropes (ACTH cells) and on the adrenal cortex of male Wistar rats were examined. Adult males received two daily s.c. injections of 20 microg/100 g of body weight of either SRIH-14 or octreotide for 28 consecutive days. ACTH cells were studied using a peroxidase-antiperoxidase immunocytochemical procedure. Morpho-metry was used to evaluate the changes in cell and nuclear volumes (microm3) and volume densities (%) of ACTH-immunoreactive cells. The adrenal cortex was analyzed by histological and morphometric methods. A significant (p<0.05) decrease in body weight and in the absolute weights of the pituitary and adrenal glands was observed in both treated groups. Morphometric parameters of ACTH cells in both treated groups were not significantly (p>0.05) different than in control rats. The absolute volumes of the adrenal gland and adrenal cortex were significantly (p<0.05) decreased in both treated groups. The absolute and relative volumes of the zona glomerulosa (ZG), as well as the cellular and nuclear volumes of the ZG were significantly (p<0.05) decreased in the both treated groups. In rats treated with SRIH-14 and octreotide, the absolute and relative volumes of the zona fasciculata (ZF) and zona reticularis (ZR), as well as their stereological parameters, did not change significantly (p>0.05). The aldosterone levels in the SRIH-14 and ocreotide-treated groups were significantly (p<0.05) decreased - by 13% and 19%, respectively. The concentration of ACTH and corticosterone did not change significantly. Together, these findings show that SRIH-14 and octreotide administration affected the morphological characteristics of the adrenal ZG in a similar manner, and brought about a decrease in plasma aldosterone concentration. These treatments did not affect pituitary ACTH cells or adrenal ZF and ZR functioning.     

Effect of long-term inhibition of hydroxy-methylglutaryl coenzyme A reductase by mevinolin on the zona fasciculata of rat adrenal cortex. A combined morphometric and biochemical study

The effects of a 7-day infusion with mevinolin, a potent competitive inhibitor of hydroxy-methylglutaryl coenzyme A (HMG-CoA) reductase, on the adrenal zona fasciculata were examined in normal and dexamethasone/ACTH-treated rats. In both groups of animals, the drug caused a lowering in plasma and intra-adrenal cholesterol concentrations, as well as a slight decrease in the blood level of corticosterone. Morphometry of zona fasciculata cells showed that specific mevinolin-induced changes (i.e. those occurring in both groups of rats and therefore not due to enhanced release of ACTH following decrease in circulating corticosterone) are severe lipid-droplet depletion and a conspicuous increase in smooth endoplasmic reticulum (SER) and peroxisomes. The hypothesis is discussed that these morphological changes express a compensatory response of zona fasciculata cells to counteract the mevinolin-induced inhibition of cholesterol synthesis in both liver and adrenal cortex.     

Effect of beta-endorphin on the steroid production of isolated zona glomerulosa and zona fasciculata cells

Small doses of β-endorphin (10^?11?10^?5M) decrease corticosterone production of zona fasciculata cells but fail to influence steroid production of zona glomerulosa cells. 10^?4M β-endorphin increases corticosterone production of both zones. The stimulating effect of ACTH on zona fasciculata corticosterone- and zona glomerulosa aldosterone production was decreased by β-endorphin (10^?9?10^?7M). Conclusion: β-endorphin might modulate both basal and ACTH stimulated corticosterone secretion.     

Proliferation and distribution of adrenocortical cells in the gland of ACTH- or dexamethasone-treated rats

The effects of prolonged (7-day) ACTH and dexamethasone administrations on rat adrenocortical-cell turnover have been investigated by combined stereological and metaphase-arrest techniques. ACTH was found to increase the number of parenchymal cells in each adrenal zone; however, ACTH altered the cell distribution in the cortex, lowering their percentage in the zona glomerulosa (ZG) and zona fasciculata (ZF) and enhancing it in the zona reticularis (ZR). The cell birth-rate was markedly raised by ACTH exclusively in ZG and ZF. Dexamethasone notably decreased the number of ZF and ZR cells, without altering that of ZG cells. Moreover, dexamethasone increased the percentage of parenchymal cells in ZG and ZF, and lowered it in ZR. In the adrenal cortices of dexamethasone-administered animals, metaphases were virtually absent. These data indicate that ACTH increases the cell birthrate in ZG and possibly ZF, and enhances the centripetal migration of newly-formed cells and their accumulation in ZR. Dexamethasone inhibits both proliferation of adrenocortical cells in the outer cortical layers and their centripetal migration into ZR. Moreover, it appears to cause parenchymal-cell loss in the inner adrenocortical layers.     

Functional significance of interrenal cell zonation in the adrenal gland of the duck (Anas platyrhynchos)

Slices of whole adrenal gland tissue, incubated in vitro in the presence of ACTH for 1 h and 2 h produced corticosterone and aldosterone in constant ratio (16:1). Tangential slices taken from the region immediately below the connective tissue capsule and slices taken from deeper regions of the gland consisted primarily of cells conforming to the distinct structural characteristics of the subcapsular zone (SCZ) and inner zone (IZ) tissues respectively. When samples were incubated in the presence of ACTH for 1 h and 2 h, the interrenal cells of the SCZ produced relatively more aldosterone than cells taken from the IZ of the gland. The corticosterone: aldosterone ratio for the IZ after 1 h (68:1) and after 2 h (102:1) were ten times greater than the ratios for the SCZ after 1 h (7:1) and after 2 h (10:1). The SCZ slices were not more than 60 cells thick and consisted of cells arranged in cords. These cells contained irregular nuclei, mitochondria with shelf-like cristae and a moderate abundance of smooth endoplasmic reticulum. In contrast, the production of large amounts of corticosterone by the cells of the IZ was associated with tissue containing more vascular space than the SCZ and the cells contained large round nuclei surrounded by an abundance of smooth endoplasmic reticulum and the mitochondria had tubular rather than shelf-like cristae.     

ACTH-induced ultrastructural changes in the zona fasciculata of the hamster adrenal cortex. Are intraadrenal thrombi regulators of corticosteroid secretion?

Correlated stereological and functional studies were performed on the effect of massive ACTH doses on adrenal cortex of the female hamster. ACTH resulted in a marked increase in adrenal gland weight at day 6 of treatment followed by a drop at day 9. Stereology showed significant enlargement of the zona fasciculata (ZF) cells with the highest value at day 6 and subsequent drop at day 9 of treatment. This hypertrophy was due to a notable increase in the volume of mitochondrial, SER, Golgi apparatus and lipid droplet compartments. Cortisol secretion by adrenal slices and homogenates was also highest at day 6 of ACTH administration and notably lower at day 9. At day 6 of ACTH treatment in outer ZF thrombi were seen. In their vicinity the subendothelial space was dilated and endothelial cells dissociated from the basal lamina. Numerous erythrocytes were also visible among dissociated ZF cells. At day 9 of experiment in outer part of ZF numerous spaces devoid of parenchymal cells appeared. The earlier authors considered the "empty spaces" or "holes" in hyperstimulated adrenal cortex as a sign of holocrine secretion of steroid hormones. The present findings enable us to introduce a new hypothesis on the development of these spaces. In our opinion in hyperstimulated adrenal cortex numerous thrombi may be formed leading thus to the degeneration of adrenocortical cells. Thus, the appearance of the "empty spaces" or "holes" in the gland is not connected with the holocrine secretion but with the regulation of the number of secretory cells in adrenal cortex by the thrombi-dependent mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphology and functional responses of isolated inner adrenocortical cells of rats infused with interleukin-beta.

The effects of the prolonged infusion with interleukin-1 beta (IL-1 beta) (20 pM.kg-1.min-1) on the function and morphology of the isolated inner cells of the rat adrenal cortex were investigated. After 3 and 5 days of IL-1 beta infusion, the level of circulating ACTH was below the control level, while the plasma concentration of corticosterone was strikingly elevated. After 5 days of infusion, isolated inner adrenocortical cells showed an enhanced basal and ACTH-stimulated corticosterone secretion, and showed a conspicuous hypertrophy. The acute exposure to IL-1 beta 10(-6) M did not affect the secretory activity of dispersed cell from either control or IL-1 beta-infused rats. These findings indicate that the prolonged exposure to high levels of circulating IL-1 beta, like those occurring during chronic inflammatory diseases, is able to enhance the growth and steroidogenic (glucocorticoid) capacity of the rat inner adrenocortical zones. Moreover, they suggest that the mechanism underlying this adrenocorticotrophic effect of IL-1 beta does not involve either a stimulation of the hypophyseal ACTH release or a direct stimulatory effect of monokine on adrenocortical cells. It is suggested that IL-1 beta may activate an intra-adrenal paracrine regulatory mechanism.     

Further investigations on the atrial natriuretic factor (ANF)-induced inhibition of the growth and steroidogenic capacity of rat adrenal zona glomerulosa in vivo

A prolonged infusion with ANF (20 micrograms/kg/h for 7 days) induced atrophy of zona glomerulosa cells and lowering of basal plasma concentration of aldosterone in rats whose hypothalamo-hypophyseal-adrenal axis and renin-angiotensin system had been interrupted by the simultaneous administration of dexamethasone/captopril and maintenance doses of ACTH/angiotensin II. Chronic ANF treatment also caused comparable reductions in the aldosterone response of zona glomerulosa cells to the acute stimulation with angiotensin II, potassium and ACTH. These data are interpreted to indicate that ANF exerts an inhibitory effect on the growth and secretory activity of rat zona glomerulosa, and that the mechanism underlying this action of ANF does not involve blockade of renin release or ACTH secretion.     

Involvement of cAMP but not PKA in the increase of corticosterone secretion in rat zona fasciculata-reticularis cells by aging

The effects and mechanisms of aging on corticosterone secretion in zona fasciculata-reticularis (ZFR) cells of ovariectomized (Ovx) rats were studied. Young (3-month) and old (24-month) female rats were Ovx for 4 days before decapitation. ZFR cells were isolated and incubated with different hormones or reagents at 37 degrees C for 30 min. Aging increased the basal secretion of corticosterone both in vivo and in vitro. The adrenocorticotropin (ACTH)-, forskolin-, 3-isobutyl-l-methylxanthine (IBMX)-, 8-bromo-adenosine 3',5'-cyclic monophosphate (8-Br-cAMP)-, and ovine prolactin (oPRL)-stimulated release of corticosterone by ZFR cells was greater in old than in young Ovx rats. H89, an inhibitor of protein kinase A (PKA), decreased the production of corticosterone in ZFR cells from young but not old Ovx rats. Forskolin-, or IBMX-induced production of cAMP was greater in old than in young Ovx animals, which correlated with the increase of corticosterone production by aging. The activity of 11 beta-hydroxylase that converts deoxycorticosterone (DOC, 10(-9) or 10(-8) M) to corticosterone in rat ZFR cells was decreased by age. However, the corticosterone production in response to high dose of DOC (10(-7) M) was indifferent between young and old groups. These results suggest that aging increases corticosterone production in Ovx rats via a mechanism in part associated with an increase of adenylyl cyclase activity and a decrease of phosphodiesterase activity, and then an increase of the generation of cAMP, but not related to either PKA activity or 11 beta-hydroxylase.     

Investigations on the effects of long-term administration of a methionine-enkephalin analogue on the adrenal zona fasciculata of rats treated with dexamethasone or dexamethasone and ACTH

Prolonged (7 days) methionine-enkephalin (DALA) treatment provoked a dose-dependent increase in the volume of zona fasciculata cells of dexamethasone-administered rats, along with a notable rise in the plasma concentration of corticosterone and the activity of 11 beta-hydroxylase. Comparable dose-dependent effects were observed after chronic administration of ACTH to dexamethasone-suppressed rats. The chronic administration of the maximum dose of DALA (500 micrograms/kg/day) was found to significantly further the trophic action of ACTH on the zona fasciculata of dexamethasone-treated animals. It is suggested that enkephalins act independently of and synergistically with ACTH in stimulating the growth and steroidogenic capacity of the rat adrenal zona fasciculata.     

Feedback inhibition of adrenocorticotropin release by corticosterone infusions in the adrenalectomized rat.

Advantage was taken of a specific and sensitive bioassay for rat plasma adrenocorticotropin (ACTH) based on the dispersion of rat adrenal cells with trysin, to investigate the relationship between plasma corticosterone concentration and inhibition of ACTH release under steady-state conditions achieved by graded rates (0-5.12 mug/min per 100 g body weight) of intravenous infusion of the steroid for 45 min in 28-day adrenalectomized rats. In contrast to prior reports involving suppression of stress-induced ACTH release, the inhibitory effect of corticosterone was shown, under our experimental conditions, to be exerted also on the basal rate of ACTH secretion. Indeed, a slight though not significant decrease of plasma ACTH concentration was observed with the corticosterone infusion rate of 0.64 mg/min per 100 g body weight, and further progressive and highly significant drops in concentration were recorded for infusion rates of 2.56 and 5.12 mg/min per 100 g body weight. An increase of the metabolic clearance rate of corticosterone, observed as a function of the infusion rate, was ascribed to saturation by the steroid of the plasma transcortin binding sites.