Adrenal function of rats in hypokinesia

Histological and biochemical examinations of the adrenals and plasma of rats for 3 months exposed to hypokinesia have shown that low motor activity led to a decrease in blood corticosterone level in spite of adrenal cortex hypertrophy. The decreased corticosterone blood level was not indicative of adrenal exhaustion, as the adrenals produced a greater amount of corticosterone in response to additional stress stimulus (5-hour immobilisation of animals in an extended state), as compared to the control. The increased production of corticosterone in response to stress stimulus caused no structural transformations or delipoidization of the cortical substance. This indicated that the reserve potentials of the adrenals increased with the animal adaptation to hypokinesia. The major morphological indication of higher adrenal functional activity in hypokinetic animals was an enhanced destruction of lymphocytes in the thymus cortex, the target organ for corticosteroids produced by the adrenals in response to an additional stress stimulus.     

Evidence that an extrahypothalamic pituitary corticotropin-releasing hormone (CRH)/adrenocorticotropin (ACTH) system controls adrenal growth and secretion in rats

Within two weeks, hypophysectomy induced in rats a striking decrease in the level of circulating ACTH (the concentration of which was at the limit of sensitivity of our assay system), coupled with a net reduction in the plasma corticosterone concentration and an evident adrenal atrophy. Zona fasciculata, the main producer of glucocorticoids, was decreased in volume, due to a lowering in both the number and average volume of its parenchymal cells. Subcutaneous ACTH infusion (0.1 pmol·min^-1), administered during the last week following hypophysectomy, restored the normal blood level of ACTH and completely reversed all effects of hypophysectomy on the adrenals. Subcutaneous infusion for one week with -helical-CRH or corticotropin-inhibiting peptide (1 nmol·min^-1), which are competitive inhibitors of CRH and ACTH, evoked a further significant lowering of plasma corticosterone concentration and markedly enhanced adrenal atrophy in hypophysectomized rats. These findings strongly suggest that an extrahypothalamic pituitary CRH/ACTH system may be involved in the maintenance of the growth and steroidogenic secretory activity of the rat adrenal cortex.     

Adrenocortical responses to adrenocorticotrophin in the rat

The time course of plasma adrenocorticotrophin (ACTH), adrenal cyclic AMP, adrenal corticosterone, and plasma corticosterone was measured in male Sprague-Dawley rats whose endogenous release of ACTH had been blocked (1) following rapid injections of 100 and 300 ng ACTH/100 g body weight, i.v., (2) during prolonged infusions at rates of 1, 2, and 4 ng ACTH/min per 100 g body weight, and (3) after termination of 30-min infusions at rates extending from 0.06 to 8 ng ACTH/min per 100 g body weight. Following injections, the time course of the variables is similar to the one simulated from our models of adrenal cortical secretion, including the simulation of an intermediate variable of our models of the adrenal cortex cell which was presumed to correspond to cyclic AMP. However, during prolonged infusions there is an unexpected overshoot of adrenal cyclic AMP content whereas adrenal and plasma corticosterone concentrations rise to a steady-state value without overshoot. The total amount of cyclic AMP gradually increases following the three increasing infusion rates of ACTH whereas similar levels of plasma corticosterone concentrations are reached at steady state; therefore the saturation of the adrenal cortical secretion is due to a step ulterior to cyclic AMP formation in the steroidogenesis. After 30-min infusions, plasma corticosterone concentration reaches its maximal value following a rate of ACTH input which evokes only a 4-fold increase in adrenal cyclic AMP content; however, there is a 250-fold increase of adrenal cyclic AMP with respect to control value following the higher rates of infusion of ACTH.     

Influence of morphine treatment in pregnant rats on the mineralocorticoid activity of the adrenals in their neonates

Exposure of pregnant rats to morphine, from day 11 to day 18 of gestation, was previously reported to induce both an adrenal atrophy and hypoactivity of the glucocorticoid function in newborns at term, but did not affect, in vitro, the responsiveness of those glands to adrenocorticotrophin hormone (ACTH) concerning corticosterone release. Moreover, these effects were mediated by maternal hormones from the adrenal glands. In the present work, we investigated the effects of a prenatal morphine exposure on the mineralocorticoid activity of the adrenals in neonates. The first aim of the present study was to determine in these newborns 1) the adrenal and plasma aldosterone concentrations at birth time and during the early postnatal period 2) the plasma levels of Na+ and K+ at birth time, 3) the in vitro responsiveness of the newborn adrenals to angiotensin II (A(II)) and ACTH. The second aim of our study was to investigate the mineralocorticoid activity of the adrenals in newborns from adrenalectomized mothers treated with morphine during gestation. According to present data morphine given to intact mothers induced in newborns a severe adrenal atrophy but increased adrenal aldosterone content and plasma aldosterone level. However, prenatal morphine was unable to affect significantly Na+/K+ ratio in both mothers and newborns. In vitro, the adrenals of neonates from morphine-treated mothers were unresponsive to An and ACTH for promoting aldosterone release; in contrast, aldosterone secretion was significantly stimulated by high potassium levels (55 mEq). Maternal adrenalectomy performed one day before the beginning of morphine treatment prevented morphine-induced adrenal atrophy but was unable to affect significantly the adrenal mineralocorticoid function of the offspring. Such data suggest that a prenatal morphine exposure stimulated both aldosterone synthesis and release in neonates. However, this basal hyperfunction did not appear to be coupled with an enhanced adrenal responsivity to AII or ACTH. Prenatal morphine-induced hyperactivity of the mineralocorticoid function of the newborn adrenals, which drastically contrast with hypoactivity of the glucocorticoid one, was independent of adrenal factors from maternal origin.     

Sex differences in adrenocortical structure and function. XXVIII. ACTH and corticosterone in intact, gonadectomised and gonadal hormone replaced rats

Plasma ACTH and corticosterone (B) concentration, ACTH content in the anterior pituitary gland and B content in the adrenals were measured in intact, gonadectomised and testosterone or estradiol replaced rats. Plasma ACTH and B levels and adrenal B content were higher in female than male rats. Neither orchiectomy nor testosterone replacement had an effect on plasma ACTH and B concentration. Orchiectomy did not affect adrenal B content and decreased pituitary ACTH while testosterone significantly lowered ACTH and B content in studied glands. On the other hand ovariectomy did not change pituitary ACTH and adrenal B content and notably lowered concentrations of these hormones in the blood. Estradiol replacement resulted in an increase in plasma ACTH and B concentrations, an effect accompanied by a marked drop in pituitary ACTH and an increase in adrenal B. These findings indicate the distinct sex differences in basal plasma ACTH and B concentrations with higher values in female rats, an effect dependent on the stimulatory action of estradiol on pituitary-adrenocortical axis.     

Effect of melatonin on steroid production by rat adrenals under in vitro superfusion conditions

In vitro superfusion of adrenals from male and female Wistar rats resulted in a gradual decline of the corticosterone content in the samples collected for a period of 80 minutes. ACTH administration either in the beginning or in the end of the perfusion period led to a marked increase in the level of this steroid. Contrary to this, progesterone content in the collected perfusates was constant throughout the experiments and was not influenced by ACTH. The quantities of testosterone secreted under these conditions were below the sensitivity of the assay. Perfusion with melatonin (200 pg/ml) did not affect corticosterone secretion, but resulted in a significant increase of the progesterone content in the recovered fractions. This effect was immediate and was followed by a decline in the progesterone concentration, observed during melatonin perfusion.     

Rat adrenal corticosteroidogenesis; effect of ACTH, cycloheximide and sterol carrier protein.

Corticosterone formation was determined in the reconstructed rat adrenal system which consisted of the mitochondria and post-mitochondrial supernatant fraction (PM-fraction) supported by l-malate, and effect of ACTH and cycloheximide in vivo and cycloheximide, Ca++ and sterol carrier protein (SCP) in vitro were examined. Mitochondria isolated from adrenals of rats which received ACTH 15 min before sacrifice showed an elevated corticosterone formation. Cycloheximide administration 15 min prior to ACTH injection completely blocked the effect of ACTH but in vitro addition of this drug to the incubation mixture did not modify the rate of corticosterone production even at higher concentrations. Since the PM-fraction isolated from adrenals of rats received ACTH or cycloheximide or both did not change the mitochondrial capacity for corticosterone formation, factor(s) which influenced by ACTH administration seemed to be localized in mitochondria. The SCP-bound cholesterol was utilized for corticosterone formation more efficiently than the free cholesterol when added to the incubation mixture, and this might be due to, at least in part, higher rate of binding to the mitochondrial inner membrane of the SCP-bound cholesterol.     

Increase in free cholesterol content of the adrenal cortex after stress: radioautographic and biochemical study

The purpose of this investigation was to quantify free cholesterol biochemically and in radioautographs of 3H-digitonin cholesterol complex in fasciculata cells of control and stressed rat adrenal cortex. Stress was induced by ether, laparotomy, and adrenal and intestinal handling. Control rats were anesthetized with nembutal. All animals were killed ten minutes from the beginning of anesthesia. The adrenals were excised and either fixed in glutaraldehyde containing 3H-digitonin or homogenized for biochemical determination of free cholesterol. The plasma corticosterone level of each animal was measured. The fixed adrenals were processed, using different methods of dehydration and embedment, for light and electron microscopic radioautography. The mean number of silver grains (mean) per unit area of zona fasciculata was counted from light microscopic radioautographs. Crystals of cholesterol-digitonide complex were more numerous in stressed fasciculata cells, particularly over SER. Silver grains were localized over or close to the crystals. The mean for stressed rats was significantly higher than control values, indicating more free cholesterol in fasiculata cells of stressed rats. The results were not affected by either the method of dehydration or the type of embedding medium used. The morphologic results were substantiated by biochemical findings of increase in free cholesterol in adrenals of stressed rats. Plasma corticosterone was significantly high in stressed rats. The increase in free cholesterol in stimulated fasciculata cells is consistent with a previously reported increase in cholesterol esterase activity after ACTH stimulation.     

Effect of jet aircraft noise upon in vitro adrenocortical response to ACTH in feral Mus musculus.

Paired adrenals from feral male $\text{Mus musculus}$ trapped from fields adjacent to the Memphis (TN) International Airport and from suitable control areas were incubated in the presence or absence of ACTH. Incubation media were assayed fluorometrically for corticosterone. Control animals exhibited lower basal secretion rates of corticosterone when compared to their noise exposed counterparts. However, ACTH elicited a significantly greater increase in corticosterone secretion in controls as compared to the noise exposed group. The data suggest a noise related decline in adrenal cortical responsiveness to ACTH.     

Involvement of the suprachiasmatic nucleus in diurnal ACTH and corticosterone responsiveness to stress

We explored the contribution of the suprachiasmatic nucleus (SCN) in ACTH and corticosterone (CORT) diurnal responsiveness of the rat to restraint stress applied either in the morning (AM) or in the evening (PM). Ablation of the SCN caused the diurnal rhythmicity of the CORT response to disappear but had no effects on AM vs. PM differences in the ACTH response. Stress-response curves in SCN-lesioned rats that had prestress levels of CORT either in the AM range or in the PM range, when compared with those obtained for AM and PM controls, showed that the SCN differentially regulates the stress response depending on the underlying secretory activity of the adrenal cortex. When basal CORT secretion is at its lowest, the SCN inhibits CORT responsiveness to stress by controlling pituitary corticotrophs; but when it is at its highest, it has a permissive action that will bypass the hypophysis and reach the adrenals to adjust the response of the gland to ACTH.     

Inhibitory effect of ouabain on epinephrine-induced stimulation of adrenal corticosterone secretion in rats.

Chronic administration of ouabain (3 mg/Kg body weight, subcutaneously, once daily for consecutive 15 days) definitely inhibited epinephrine-induced increase of adrenal corticosterone secretion. The inhibition rate increased along with frequency of ouabain administration. Increase in adrenal corticosterone synthesis and secretion by ACTH (20-50 mU/rat) administration was partially suppressed by pretreatment with chronic ouabain administration. A slight but significant increase of adrenal corticosterone secretion caused by epinephrine administration in hypophysectomized rats was also inhibited by pretreatment with ouabain administration. Chronic administration of neither phentolamine (1 mg/rat, intraperitoneally, once daily for consecutive 15 days) nor propranolol (3 mg/Kg body weight, subcutaneously, once daily for consecutive 15 days) caused significant changes in adrenal corticosterone secretion in response to ACTH as well as to epinephrine. Chronic administration of ouabain in rats causes not only elevated secretion of ACTH from anterior pituitary but also functional change in adrenals leading to suppression of corticosterone secretion in response to ACTH or epinephrine administration.     

ACTH and corticosterone response to naloxone and morphine in normal, hypophysectomized and dexamethasone-treated rats

The effect of opiate receptors blocker naloxone on ACTH and corticosterone secretion in normal, dexamethasone-treated and hypophysectomized rats was studied. A dose-related increase in plasma corticosterone level was found at 45 min after s.c. injection of naloxone in a dose range of 0.25-2.0 mg kg-1. The rise in plasma corticosterone was preceded by a slight increase in plasma ACTH. Acute morphine administration in a relatively low dose (6 mg kg-1 s.c.) induced a significant rise in both plasma ACTH and corticosterone levels. Dexamethasone treatment was followed by low basal corticosterone level, by total inhibition of the stress response and response to morphine injection, while the response to ACTH administration was normal. Under these circumstances as well as in rats 6 days after hypophysectomy, naloxone failed to increase plasma corticosterone levels. It is concluded that a direct stimulation of corticosteroid biosynthesis in adrenal cortex is not involved in the mechanism of naloxone-induced activation of pituitary-adrenocortical function.     

Sex-differences in adrenocortical responsiveness during development in rats

It is known that the stress hyporesponsive period (SHRP), which seems to be related to an immature hypothalamo-pituitary-adrenal (HPA) regulatory system, occurs during the first 2 weeks after birth in rats. In the present study, we investigated the effects of sex-steroid hormones on adrenocortical responsiveness to adrenocorticotropic hormone (ACTH) in neonatal rats. The levels of cyclic adenosine 3',5'-monophosphate (cAMP), corticosterone, and adenylate cyclase activity increased with the dose of ACTH in adrenal cells of males and females in vitro. The ACTH responsiveness in adrenal cells increased with age (7-35 days of age), that is, the loss in responsiveness to ACTH just after birth began to recover in 14-35-day-old rats, but the responsiveness in 14-day-old rats was attenuated in males compared with females. Although castration markedly augmented the responsiveness in male rats, testosterone-replacement in the castrated male rats inhibited the enhancement. Furthermore, the responsiveness in 14-day-intact female rats was suppressed by treatment with testosterone. Expression levels of ACTH receptor mRNA in adrenals increased with age in the female rat, but not in the male. Castration enhanced the level of ACTH receptor mRNA to three-fold of that in intact male rats at 14 days of age, but replacement treatment with testosterone in castrated male rats lowered the elevated levels. Testicular androgens are thought to evoke a gender-specific response in neonates, and the temporal decrease of adrenal ACTH-responsiveness might be due to the topically immature adrenal system as well as the central nervous system in mammals.     

Molecular genetic analysis of glucocorticoid signalling in development

A null mutation of the glucocorticoid receptor was generated by homologous recombination. Mutant newborn mice showed impaired lung development, hypertrophy of the adrenal cortex and a strongly reduced size of the adrenal medulla. Phenylethanolamine N-methyltransferase (PNMT) was undetectable in the adrenals of the mutant mice. Serum levels of corticosterone were moderately and ACTH levels were strongly elevated in the mutants. A weaker but significant increase of corticosterone and ACTH was observed already in heterozygous animals. This points to a dysregulation of the HPA axis due to defective feedback regulation via the glucocorticoid receptor. Liver gluconeogenetic enzymes were reduced to a variable degree. Whereas survival of heterozygous mutants was not affected, most of the homozygous mutant mice died during the perinatal period.     

Testosterone receptor blockade after trauma and hemorrhage attenuates depressed adrenal function

Although the testosterone receptor antagonist flutamide restores the depressed immune function in males after trauma and hemorrhage, it remains unknown whether this agent has any salutary effects on adrenal function. To study this, male rats underwent laparotomy and were bled to and maintained at a blood pressure of 40 mmHg until 40% of the shed blood volume was returned in the form of Ringer lactate. Animals were then resuscitated and flutamide (25 mg/kg body wt) was administered subcutaneously. Plasma adrenocorticotropic hormone (ACTH) and corticosterone, as well as adrenal corticosterone and cAMP were measured 20 h after resuscitation. In additional animals, ACTH was administered and ACTH-induced corticosterone release and adrenal cAMP were determined. The results indicate that adrenal contents of corticosterone and cAMP were significantly decreased and morphology was altered after hemorrhage. Administration of flutamide improved corticosterone content, restored cAMP content, and attenuated adrenal morphological alterations. Flutamide also improved the diminished ACTH-induced corticosterone release and adrenal cAMP response at 20 h after hemorrhage and resuscitation. Furthermore, the diminished corticosterone response to ACTH stimulation in the isolated adrenal preparation was improved with flutamide. These results suggest that flutamide is a useful adjunct for improving adrenal function in males following trauma and hemorrhage.     

Immunoneutralization of corticotropin-releasing hormone prevents the diurnal surge of ACTH

To determine whether CRH is required for the evening rise in plasma ACTH, rats were injected at 0800 hr with CRH antiserum (anti-CRH) or normal rabbit serum (NRS). Blood samples were taken through venous catheters at 0800 hr before treatment and at 1300, 1700, and 2100 hr. Plasma was assayed for immunoreactive ACTH and corticosterone. There was no significant difference in pretreatment values between the two groups. Immunoneutralization of CRH abolished the rise in plasma ACTH seen at 1700 hr in the NRS group but had little effect on earlier levels. The diurnal elevation in plasma corticosterone continued after anti-CRH treatment, but peak levels occurred earlier. Plasma ACTH and corticosterone were significantly correlated at the time of the diurnal surge, but not at 0800 hr or 1300 hr in the NRS controls or at any time point in the anti-CRH group. These results suggest that CRH is required for the diurnal surge of plasma ACTH. They also confirm previous observations by others that the adrenal cortex does not require active CRH or a diurnal surge of ACTH in order to exhibit a significant diurnal increase in secretion of corticosterone, and that factors other than CRH may be relatively more active than CRH in regulation of ACTH secretion during the time of circadian inactivity.     

Adrenal splanchnic innervation contributes to the diurnal rhythm of plasma corticosterone in rats by modulating adrenal sensitivity to ACTH

Activity of the hypothalamic-pituitary-adrenal axis is characterized by a diurnal rhythm with an AM nadir and PM peak. Splanchnic nerve transection disrupts the diurnal rhythm in plasma corticosterone; however, there is a controversy as to whether the nerve-mediated effect is 1) via inhibition in the AM vs. excitation in the PM, or 2) involves changes in adrenal sensitivity to ACTH. The present studies were designed to address these issues. Adult male rats were anesthetized and underwent bilateral transection of the thoracic splanchnic nerve or sham transection. One week after surgery, rats were killed in the AM or PM with collection of nonstress plasma for measurement of corticosterone and ACTH. Plasma corticosterone was increased in the PM relative to the AM; however, plasma corticosterone in the PM was attenuated by splanchnic nerve transection, without affecting plasma ACTH. This decrease in PM plasma corticosterone after nerve-transection was 1) associated with decreased adrenal responsivity to ACTH, 2) associated with decreased adrenal cAMP content, 3) prevented by adrenal demedullation, and 4) not affected by removal of adrenal capsaicin-sensitive afferent fibers. Repeated serial blood sampling from individual rats confirmed the excitatory effect of splanchnic innervation in the PM. These results support the hypothesis that the adrenal splanchnic innervation modulates the diurnal rhythm in plasma corticosterone by increasing adrenal responsivity to ACTH and augmenting steroidogenesis in the PM and suggest that alterations in adrenal corticosterone secretion obscured by pulsatile secretion are more clearly revealed with repeated serial blood sampling.     

Effect of the growth hormone-secreting tumor StW5 on pituitary and adrenal gland function in rats

A growth hormone-secreting tumor (StW5 was implanted into male rats and resulted in a tripling of adrenal weight concomitant with a 30% decrement in pituitary weight. Plasma concentrations of corticosterone in tumor-bearing (TB) rats were significantly elevated at rest or after ACTH injections or the stress of either anesthesia. The rise in plasma concentrations of corticosterone was due mainly to the large increment in adrenal size although a significant increase in adrenal responsiveness to ACTH was demonstrated in vitro. In addition, plasma corticosterone concentrations were higher in TB rats despite both a doubling of the blood volume and a 50% increase in liver capacity to metabolize corticosterone. Pituitary ACTH content was significantly lower in TB rats, but these pituitary glands could still release near-normal quantities of ACTH as shown both by in vitro incubations and adrenal corticosterone output following ether stress.     

Steroid production by quartered and capsular rat adrenal gland in response to haemorrhage.

60 min after rapid bleeding (1.5--2.0 per cent of b. w.) both aldosterone and corticosterone production rate by quartered rat adrenals were found to be elevated. However, no difference was observed in the rate of aldosterone and corticosterone production by capsular adrenals of sham operated and hypovolaemic rats. Corticosterone production rate by decapsulated adrenals was much more higher after haemorrhage than in the control group. The same alterations could be observed incubating adrenal tissue with ACTH (0.3 mug per ml). Steroid production rate by quartered adrenals of sodium deficient rats was not affected by high in vitro concentration of angiotensin II (2.5 mug per ml). It is concluded that the effect of acute blood loss on corticosteroid biosynthesis of the rat is mediated by ACTH alone.