Feedback inhibition of adrenocorticotropic hormone by physiological increases in plasma corticosteroids in conscious dogs.

We have tested the effect of physiological increases in plasma corticosteroids in conscious dogs on the levels of basal and hypoglycemia-stimulated adrenocorticotropic hormone (ACTH) 2 h later. Increases in plasma corticosteroids, produced by infusion of alpha-1-24 ACTH or corticosteroids for 40 min, suppressed basal and stimulated ACTH levels. The magnitude of inhibition produced by an increase in plasma corticosteroids induced by the infusion of ACTH was equivalent to the inhibition produced by the same increase in plasma corticosteroids induced by corticosteroid infusion. The infusions did not affect basal plasma glucose concentrations or the decrease in plasma glucose concentrations after administration of 0.1 U insulin/kg. Basal ACTH concentration was less sensitive than hypoglycemia-stimulated ACTH concentration to corticosteroid-induced suppression. Basal and stimulated secretion were significantly inhibited in all dogs after approximately half-maximal increases in plasma corticosteroids; maximum inhibition occurred after maximal increases in plasma corticosteroids. Therefore, physiological increments in plasma corticosteroids, similar to those produced by acute stress, are effective suppressors of subsequent stress-induced ACTH secretion.

Titration by estrogen receptor activation function-2 of targets that are downstream from coactivators.

Cross-interference (squelching) among nuclear receptors has been proposed to reflect the titration of coactivators that bind the receptors in a hormone-dependent manner. We have tested whether the coactivators are the only target titrated during squelching of one receptor by another, or whether proteins needed for coactivator function are titrated as well. That the coactivators are indeed one target of squelching is apparent. The isolated ligand-binding domain of the estrogen receptor (ER-LBD) squelches transcriptional activation by the thyroid hormone receptor (TR) only when the LBD is bound to ligands that promote coactivator interactions and only when regions of the LBD that promote coactivator interactions are undisturbed. Furthermore, the ER-LBD and the TR compete in vitro for the related p160 coactivators, SRC1a and GRIP1 (glucocorticoid receptor interacting protein 1), or the putative corepressor, RIP140. Finally TR action becomes more potent when coactivator levels are raised. Nonetheless, supplying excess SRC1a or GRIP1 does not abolish squelching by the ER. In fact, squelching becomes even more severe when coactivators are abundant. Supplying combinations of coactivators from the p160 class and the CREB-binding protein (CBP)/p300 class makes squelching most severe. Elevated RIP140 inhibits TR action, but also protects the residual TR action from squelching by the ER-LBD. We conclude that ER-LBD squelches TR both by titrating p160-CBP coactivators and additionally by cooperating with the coactivators to titrate a second factor. The second factor would be needed by the TR for coactivator-mediated transcriptional stimulation.

Estrogen receptor activation function 1 works by binding p160 coactivator proteins.

Estrogen receptor-alpha contains two transactivation functions, a weak constitutive activation function (AF-1) and a hormone-dependent activation function (AF-2). AF-2 works by recruiting a large coactivator complex, composed of one or more p160s, CREB-binding protein (CBP)/p300, and P/CAF (p300 and CBP-associated factor), via direct contacts with the p160s. We report here that independent AF-1 activity also requires p160 contacts. Unlike AF-2, which binds signature NR boxes in the center of the p160 molecule, AF-1 binds to sequences near the p160 C terminus. We propose that the ability of AF-1 and AF-2 to interact with separate surfaces of the same coactivator is important for the ability of these transactivation functions to synergize.

Corticosterone acts on the brain to inhibit adrenalectomy-induced adrenocorticotropin secretion.

To determine the site (brain and/or pituitary) at which corticosterone (Cort) acts to inhibit adrenalectomy-induced ACTH secretion, the following experiments were performed in male rats. Rats in which brain and pituitary feedback sites were to be left intact were subjected to sham hypothalamic lesions. Rats in which only pituitary sites were to be left were subjected to either medial basal hypothalamic (MBH) or para-ventricular nuclei (PVN) lesions. Two days later all rats were adrenalectomized and replaced with varying amounts of Cort (by sc pellet). Lesioned rats also received sc pumps that delivered 0-5 micrograms/day rat CRF. All rats were killed 5 days after adrenalectomy. Sham-lesioned groups exhibited the expected dose-related inhibition of plasma and pituitary ACTH concentrations by Cort, with normal values obtained at plasma Cort levels between 4.4 and 7.7 micrograms/dl. By contrast, rats with MBH and PVN lesions exhibited no ACTH responses to adrenalectomy when CRF infusions were between 0 and 1 micrograms/day. In rats with MBH and PVN lesions receiving 5 micrograms/day CRF, plasma ACTH concentrations were elevated and were not inhibited by plasma Cort values up to 6 micrograms/dl. Plasma ACTH was inhibited in rats with MBH lesions infused with 5 micrograms/day CRF when plasma Cort levels were 30.6 micrograms/dl. These rats also exhibited marked thymic atrophy. We conclude from these results that Cort normally acts only on a brain site to inhibit adrenalectomy-induced increases in ACTH secretion. Only when plasma Cort concentrations are markedly elevated can evidence for pituitary feedback be demonstrated in vivo.

Return of pituitary-adrenal function after adrenal enucleation or transplantation: diurnal rhythms and responses to ether.

Adrenocortical diurnal rhythms and responses to ether vapor were studied in rats 1, 3, and 5 weeks after bilateral adrenal enucleation, autotransplantation, or sham transplantation in order to 1) determine whether diurnal rhythms in the plasma corticosterone concentration and adrenal responsiveness to ACTH are dependent on innervation of the adrenals, 2) compare regeneration of function of transplanted and enucleated adrenals, and 3) investigate adrenal mass-related nonsteroidal inhibition of ether-stimulated ACTH secretion. Rats in both enucleate and transplant groups exhibited significant morning-evening differences in adrenal and plasma corticosterone concentrations and significant adrenocortical responses to ether 3 and 5 weeks, but not 1 week, after surgery. The morning-evening differences in corticosterone concentration occurred in the absence of significant morning-evening variation in the plasma ACTH concentration, supporting our previous finding of a diurnal rhythm in adrenal responsiveness to ACTH. The responsiveness rhythm cannot be dependent on adrenal nerves unless transplanted adrenals receive functionally specific reinnervation within 3 weeks. The processes of regeneration of function after enucleation and after transplantation are similar; there were no differences in plasma or adrenal corticosterone values between rats in enucleate and transplant groups at any time or under any condition tested. As regeneration progressed, plasma ACTH responses to ether declined in both enucleate and transplant groups in the absence of changes in plasma corticosterone feedback. These results support our previous finding of adrenal mass-related nonsteroidal suppression of ACTH responses to ether.

Daily rhythms in adrenal responsiveness to adrenocorticotropin are determined primarily by the time of feeding in the rat.

These experiments were done to determine: 1) whether feeding-related shifts in daily corticosterone rhythms are dependent upon changes in ACTH rhythms, 2) whether restricted feeding of rats results in abnormally high ACTH and corticosterone levels (i.e. stress), and 3) whether changes in either insulin or glucose levels might be the concomitants of feeding that change adrenal responsiveness to ACTH. Young male rats (80--90 g) on a 12-h light, 12-h dark cycle were allowed access to one of three diets for 2 h/day beginning either at lights off or lights on. The diets contained 3%, 4.5%, or 11% fat. A group of rats had ad libitum access to the food containing 4.5% fat. On day 20 of this regimen, rats were killed at 2- to 4-h intervals during the next 24 h, and plasma ACTH, corticosterone, insulin, and glucose were measured. Adrenal weight and corticosterone content were also determined. In none of these experiments was an ACTH rhythm demonstrable by analysis of variance. Neither ACTH levels nor adrenal and plasma corticosterone levels were higher in animals fed 2 h/day than in rats eating ad libitum. Peak corticosterone levels occurred just before feeding, and the restricted feeding paradigm appeard to sharpen the daily rhythms. However, there was also an effect of the light-dark cycle on corticosterone measures. Dietary fat content was directly related to increases in body weight and mean insulin levels and infersely related to adrenal responsiveness to ACTH. The data show that: 1) the time of feeding determines the timing of the corticosterone rhythm in the absence of a rhythm in ACTH, 2) restricted feeding is not a stress, and 3) neither insulin nor glucose has a substantial influence on adrenal responsiveness to ACTH.

Relationships among adrenal weight, corticosterone, and stimulated adrenocorticotropin levels in rats.

Based on results of previous studies, we tested the hypothesis that increased adrenal weight in the absence of increased corticosterone secretion would inhibit the magnitude of the ACTH response to ether vapor. Adrenal hypertrophy was induced by treatment of rats for 3 days with aminoglutethimide, cyanoketone, or metyrapone, three agents that act to inhibit enzymes required for corticosterone synthesis. On the fourth day, resting plasma ACTH and corticosterone levels were normal; however, the logarithm of the ACTH response to ether was directly related to total adrenal weight. This result did not support the hypothesis. Unilateral adrenalectomy and treatment with cyanoketone resulted in total adrenal weight equivalent to that of control rats bearing two adrenals. Resting ACTH levels were normal, but stimulated ACTH was significantly greater in these rats than in controls or in rats with two adrenals, suggesting that there is an interaction between the effects of unilateral adrenalectomy and adrenal enzyme inhibition. As anticipated, adrenal hypertrophy with increased corticosterone production caused by ACTH infusion resulted in a significant negative relationship between resting and stimulated ACTH levels and adrenal weight. We conclude that when adrenals are enlarged by means that prevent excessive corticosterone secretion, there is a mechanism associated with the increase in adrenal weight that correlates directly with the magnitude of stimulated ACTH secretion. We have reexamined the results of our previous results in the light of these experiments.

Drug-induced adrenal hypertrophy provides evidence for reset in the adrenocortical system.

These studies were performed to determine how bilateral adrenal hypertrophy persists despite normal resting ACTH levels in male rats for weeks after a 3-day course of treatment with cyanoketone (CK). This drug blocks corticosterone synthesis by binding to the enzyme 3 beta-ol-dehydrogenase-delta 5, delta 4-isomerase (EC 1.1.1.51). Although plasma ACTH levels were significantly elevated at all times during the course of treatment with CK, ACTH and corticosterone levels of CK-treated rats were normal thereafter compared to those in control rats killed in the morning. Despite these normal ACTH levels, adrenal weight remained elevated (by 100%) for at least 14 days. We determined that increased ACTH secretion was required to initiate and maintain the adrenal growth response to CK. Rats pretreated with hypophysectomy or dexamethasone (1 mg/kg) did not respond with increases in adrenal weight after CK treatment. Sustained elevations in ACTH during CK treatment were required for increased adrenal weight, because rats treated with dexamethasone 3 h after CK injection did not have enlarged adrenals at 24 h. Increased adrenal weight was not sustained after removal of supranormal levels of ACTH achieved by infusion, suggesting that after adrenal hypertrophy is achieved by ACTH, elevated ACTH levels are also required to maintain adrenal enlargement. Finally, ACTH and corticosterone levels were measured in the evening (at the peak of the diurnal rhythm in the adrenocortical system of rats) in CK-treated rats. Evening ACTH levels were significantly elevated in CK-treated rats compared to those in controls 7, 10, and 14 days after the onset of 3 days of treatment with CK; however, corticosterone levels were normal. The effects of CK on the adrenocortical system were exerted via the adrenal, since adrenalectomy normalized the amplitude of the diurnal rhythm of ACTH in CK-treated rats compared to that in adrenalectomized controls. We conclude 1) that adrenal hypertrophy after CK is maintained by increased ACTH secretion which occurs daily in the evening; and 2) that the results provide evidence for a daily reset of the corticosteroid feedback sensor in the adrenocortical system. This conclusion arises from the findings that there is a normal rhythm in corticosterone levels in CK-treated rats and that morning ACTH levels are normal although evening ACTH levels are significantly elevated.

Rapid decreases in adrenal and plasma corticosterone concentrations after drinking are not mediated by changes in plasma adrenocorticotropin concentration.

Rats given water to drink only during a brief daily period respond to water presentation with a rapid decline in plasma corticosterone concentration. To determine whether this response is consequent to a decrease in plasma ACTH concentration or whether it reflects a sudden reduction in adrenal responsiveness to ACTH, we allowed rats access to water for 2 h/day at lights on and measured plasma ACTH and adrenal and plasma corticosterone concentrations at 3- or 5-min intervals after the onset of drinking. Adrenal and plasma corticosterone concentrations decreased significantly within 2-3 min after water presentation in the absence of concomitant changes in plasma ACTH concentration. The effect was apparent by 5 days after initiating the restricted drinking schedule and became stronger with time up to 21 days. Further characterization of the response showed that the in vitro corticosterone secretion of adrenals removed at intervals after water presentation followed the same pattern as the in vivo concentration. When empty water bottles were presented, plasma ACTH and corticosterone concentrations increased. Neither adrenal medullary function nor plasma renin concentration was found to be associated with the decline in adrenal responsiveness to ACTH after drinking. Hemisection of the spinal cord of unilaterally adrenalectomized rats attenuated the corticosterone response regardless of whether the hemisection was contralateral or ipsilateral to the remaining adrenal. These data suggest that the decreases in plasma and adrenal corticosterone concentration occurring after drinking in water-restricted rats are not dependent on changes in plasma ACTH concentrations, but may be related to changes in adrenal blood flow, steroid metabolism and distribution, or neural input to the adrenal cortex.

Corticosterone-induced changes in hypothalamic corticotropin-releasing factor (CRF) content after stress.

Central and peripheral humoral responses of the adrenocortical system were measured for 2 h after the application of several stimuli. Two min after the onset of the stresses of sham-adrenalectomy or laparotomy with intestinal traction there was a 4-6 fold increase in hypothalamic CRF content as compared to control content, This is the usual CRF response to stress. In contrast, after adrenalectomy or manipulation of the pedicles of adrenal glands; CRF content at 2 min was only slightly increased above baseline values. This finding suggests that touching the adrenal vascular and nervous supply results in a direct neural input to the hypothalamus that is qualitatively different from most other stimuli. At times later than 2 min after stress, whem plasma corticosterone levels rise in the intact rat, the patterns of CRF and ACTH responses that were observed after adrenalectomy were determined by whether corticosterone replacement therapy was given. Without corticosterone replacement, the CRF and ACTH responses to adrenalectomy resembled those of laparotomy with intestinal traction. When corticosterone was administered 2 and 40 min after adrenalectomy, the CRF and ACTH responses resembled those of sham-adrenalectomy. At 20 min, CRF content was low after laparotomy with intestinal traction or adrenalectomy and high after shan-adrenalectomy or adrenalectomy with corticosterone replacement. Plasma ACTH peaked by 20 min, and remained high for 2 h after the first 2 stimuli, and was significantly decreased from the 20 min peak by 40 min after application of the latter stimuli. CRF content increased to a second peak 80 min after laparotomy with intestinal traction or adrenalectomy. This rise in CRF must represent increased formation of the releasing factor because ACTH levels were elevated and constant for the preceding 60 min. After sham-adrenalectomy or adrenalectomy with corticosterone replacement, CRF content and ACTH are low at 80 min. Measurement of circulating ACTH levels in conjunction with CRF content after these stimuli have yielded sufficient information to assign mechanisms of altered synthesis and secretion to explain the observed changes in CRF content. Corticosterone damps the adrenocortical system response to the stimuli of sham-adrenalectomy or adrenalectomy with corticosterone replacement by two mechanisms. Firstly, it acts to inhibit CRF secretion probably via rate-sensitive feedback. Secondly, it acts to inhibit the second wave of CRF formation that is observed 80 min after stress is applied, probably via the proportional feedback mechanism.

Adrenal sensitivity to adrenocorticotropin varies diurnally.

To further characterize diurnal changes in the rhythm in adrenal responsiveness to ACTH, we have measured ACTH distribution volume, MCR, and t 1/2. These do not change between morning and evening in groups of untreated, dexamethasone-pretreated, or hypophysectomized female rats. To characterize the nature of the change in adrenal responsiveness to ACTH, dexamethasone-pretreated rats were infused for 2 h with a variety of doses of ACTH in the morning and evening. The adrenal response to an infusion rate of ACTH that maximally stimulated the adrenals (200 pg/100 g BW.min) was the same in the morning and evening, showing that adrenal capacity does not change. However, infusion of ACTH at lower rates (50-100 pg/100 g BW.min) revealed that the slope of the steroid response curve increased between morning and evening, demonstrating a diurnal change in adrenal sensitivity to ACTH. These results together with previous data showing that the magnitude and time course of the adrenal cAMP response to ACTH changes diurnally strongly suggest that ACTH receptor affinity or coupling with adenylate cyclase changes diurnally. In other experiments, plasma ACTH and corticosterone levels were determined in groups of young and adult male and adult female untreated rats killed at 4-h intervals around the clock. Peak sensitivity to ACTH was found at lights-out, and trough sensitivity was found at lights-on, suggesting that the experimentally demonstrated rhythm occurs normally.

Feedback inhibition of adrenocorticotropin and vasopressin responses to hypoxia by physiological increases in endogenous plasma corticosteroids in dogs.

We examined the influence of increases in plasma corticosteroids produced by ACTH infusion on subsequent ACTH and vasopressin (AVP) responses to hypoxia in anesthetized dogs. Basal and stimulated ACTH levels were inhibited by increases in corticosteroids. Moderate increases in corticosteroids (5.2 micrograms/dl) caused a 50% reduction in the subsequent integrated ACTH response to hypoxia. Maximal increases in corticosteroids eliminated the integrated ACTH response to hypoxia. In addition, AVP responses to hypoxia were attenuated by prior maximal elevations in corticosteroids. Physiological elevation of corticosteroids inhibits subsequent ACTH and AVP responses to hypoxia.

Pituitary-adrenal function in rats chronically exposed to cold.

Exposure to cold for 2 weeks was used to assess the effects of a sustained stimulus on pituitary-adrenal function in male rats. The diurnal peak in plasma and adrenal corticosterone was advanced by 4 h during the first 24 h of exposure to cold but returned to its usual time (2000 h) by the next day. Plasma ACTH and corticosterone levels were generally greater at all times during the 24-h cycle in animals exposed to cold for up to 2 weeks, with the greatest increase occurring consistently at the time of peak. When rats exposed to cold for 1 week were returned to a normal 24 C environment, plasma corticosterone tended to increase. Plasma ACTH and plasma and adrenal corticosterone responses to a superimposed acute provocative stimulus (ip saline injection) were faster, greater, and more sustained in rats exposed to cold for 3 or 7 days. Similarly, the compensatory adrenal hypertrophy response to unilateral adrenalectomy was greater in cold-exposed rats. Such animals were also more resistant to pituitary-adrenal suppression by prednisolone. In contrast, there was no change in the sensitivity of the adrenal to exogenous ACTH. The results suggest that chronic exposure to cold causes a sustained activation of central mechanisms that regulate pituitary ACTH secretion as well as extra-pituitary mechanisms that regulate adrenal size; it reduces the effectiveness of negative feedback mechanisms, but does not alter those involved in the regulation of adrenal rhythmicity or adrenal sensitivity to ACTH.

Inhibition of the adrenocorticotropin and corticosteroid responses to hypoglycemia after prior stress.

We have tested the efficacy of prior stress-induced increases in corticosteroids on inhibition of the ACTH response to hypoglycemia induced 2 h later. Five dogs were studied in each of five experiments. In each experiment, there were two stimulus periods; in the first, 5% dextrose, 3 or 10 micrograms/kg X min nitroprusside, or 0.05 or 0.10 U/kg insulin was administered, and in the second, 0.10 U/kg insulin was administered. Whereas prior infusion of 5% dextrose did not affect the subsequent ACTH response to 0.10 U/kg insulin, prior stimulation of the pituitary-adrenal axis had a significant effect on the subsequent ACTH response to 0.10 U/kg insulin. The integrated ACTH response to hypoglycemia was significantly reduced by prior stimulation by either 10 micrograms/kg/min nitroprusside or 0.10 U/kg insulin, although the intensity of the hypoglycemia during the second stimulus period was not altered by any of the prior stimuli. Overall, the magnitude of the suppression of the pituitary-adrenal response to hypoglycemia was significantly related to the increase in plasma corticosteroids produced by the first stimulus, and was consistent with the degree of corticosteroid feedback inhibition of ACTH observed in previous studies after ACTH or corticosteroid infusion. Therefore, we conclude that prior stimulation of the hypothalamo-pituitary-adrenal axis of conscious dogs by hypotension or hypoglycemia inhibits subsequent responses in proportion to the corticosteroid feedback signal produced.

Constant corticosterone replacement normalizes basal adrenocorticotropin (ACTH) but permits sustained ACTH hypersecretion after stress in adrenalectomized rats.

To characterize further the effects of providing a constant corticosterone signal after bilateral adrenalectomy, we have compared the effects of bilateral adrenalectomy with no replacement (ADX) and with replacement with a corticosterone pellet implanted sc at surgery (B-PELLET) to those of sham-adrenalectomy (SHAM) on pituitary and plasma ACTH concentrations during the first 3 postoperative days. In ADX rats, plasma ACTH concentrations were elevated at all times compared to those in the SHAM group; pituitary ACTH content decreased during the first 12 h, then increased and was not different from that in the SHAM group thereafter. Replacement of corticosterone at the time of adrenal surgery in B-PELLET rats resulted in no differences in pituitary and plasma ACTH concentrations from SHAM values, suggesting that immediate steroid replacement prevents the major adrenalectomy-induced changes in central regulatory components governing basal activity of the adrenocortical system. Although B-PELLET rats had normal basal morning ACTH concentrations 5 days after surgery, they exhibited augmented and sustained ACTH responses to five different ACTH-releasing stimuli (injection, restraint, chlorpromazine, and, under pentobarbital anesthesia, morphine or sham adrenalectomy). The circulating corticosterone concentrations were maintained at relatively constant, low levels (3-6 micrograms/dl). Because these concentrations appear to restore basal morning ACTH concentrations to normal, but do not restore the ACTH response to stress to normal, we conclude that a different corticosterone signal is required to normalize stress-induced ACTH responses.

Circadian variations in plasma corticosterone permit normal termination of adrenocorticotropin responses to stress.

We previously reported that adrenalectomized rats given constant corticosterone via a sc pellet (B-PELLET) hypersecrete ACTH in response to stress. Although lacking a feedback signal, B-PELLET rats do not secrete ACTH indefinitely after stress; plasma ACTH levels in these animals returned to those in sham-operated (SHAM) rats within 1-4 h after 2-min restraint. To distinguish between the requirement for circadian or stress-induced increases in corticosterone, we compared changes in ACTH and corticosterone levels after stress in SHAM and B-PELLET rats with those in cyanoketone-treated rats (CK) and adrenalectomized rats given corticosterone in their drinking fluid (B-WATER). B-WATER rats exhibited sustained increases in plasma corticosterone after lights-off, correlating with the nocturnal feeding period. Morning plasma corticosterone levels in B-WATER rats were constant and even lower than those in B-PELLET rats; however, B-WATER rats did not differ from SHAM rats in their ACTH response to ip injection. CK rats, which have an approximately normal circadian corticosterone rhythm but do not have significant corticosterone responses to acute stimuli, also exhibited plasma ACTH levels similar to those of SHAM rats at all times after 5-min restraint. Compared with SHAM and B-WATER rats in the same experiment, B-PELLET rats tended to hypersecrete ACTH 60 min after 5 min of restraint, but only had significantly elevated plasma ACTH relative to both groups 45 min after 10 min of restraint. We conclude that circadian, rather than stress-induced, increases in corticosterone may be sufficient for normal termination of ACTH responses to stress.

Circadian patterns of stress-induced ACTH secretion are modified by corticosterone responses.

To test whether there is a circadian rhythm in the ACTH response to stress, young female rats were exposed to a variety of ACTH-releasing stimuli at 0600 and 1800 h and changes in circulating ACTH and corticosterone were measured. The results of these experiments suggested that after the high intensity stimuli of laparotomy with intestinal traction or 250 mug histamine ip/100 g BW, the morning ACTH response was greater than the evening response. However, the ACTH response to ip saline was greater in the evening in one experiment and greater in the morning in a second experiment. Plasma corticosterone responses were faster and greater in the morning in the first experiment and in the evening in the second experiment. The ACTH response to 125 mug histamine ip/100 g BW was greater in the evening and the change in corticosterone was greater in the morning. Thus, after low intensity stimuli, the ACTH responses appeared to depend on both the lag time prior to the corticosterone response, and its magnitude. To test this possibility, rats were adrenalectomized and the ACTH response was measured 7.5 and 15 min after the start of surgery and after injection with either 2% EtOH-saline, or 50 mug corticosterone at operation, or with 30 mug corticosterone at 5 min. Compared with ACTH levels in rats treated with vehicle, ACTH levels were decreased 7.5 min after 50 mug corticosterone at operation (P less than 0.01), but not after 30 mug corticosterone at 5 min. ACTH levels were slightly reduced 10 min after 30 mug corticosterone at 5 min compared with those of rats injected with vehicle at operation (P less than 0.05). These results are consistent with the interpretation that corticosterone secretion modifies stress-induced ACTH secretion via the fast-feedback effect. Comparison of the ACTH responses to acute adrenalectomy plus injection with EtOH-saline at 0600 and 1800 h demonstrated that, in the absence of a corticosterone response to the stress, the ACTH response is greater in the morning that in the evening (P less than 0.01). Finally, this group of experiments suggests strongly that the responsivenss of rat adrenal glands to ACTH increases markedly between 0600 and 1800 h.

Insulin-induced hypoglycemia in conscious dogs. I. Dose-related pituitary and adrenal responses.

We have measured changes in plasma glucose, ACTH, corticosteroids, and vasopressin and hematocrit after five doses of insulin in six conscious dogs. We found insulin dose-related changes for each of these responses (P less than 0.01, by two-way analysis of variance). The increases in plasma ACTH and hematocrit correlated to the decrease in plasma glucose; the increase in plasma vasopressin was more strongly correlated with the increases in plasma Na+ than with the decreases in plasma glucose. Each dog appeared to have a characteristic ACTH response curve; therefore, the relationship between plasma glucose and plasma ACTH responses varied among dogs, but was significant in five of six dogs studied. Maximal plasma corticosteroid responses occurred with submaximal plasma ACTH responses (200-600 pg/ml). A single dose of insulin produced reproducible changes in plasma ACTH when given to five dogs in three separate experiments over a 2- to 6-month period. In these experiments, the measurement of ACTH allowed us to distinguish three levels of response to insulin, whereas measurement of the corticosteroid response allowed us to distinguish only two levels of response.

The adrenocortical system responds slowly to removal of corticosterone in the absence of concurrent stress.

After removal of corticosteroid feedback by surgical or pharmacological adrenalectomy, plasma ACTH increases more rapidly than can be explained by changes in receptor-mediated gene expression. In aminoglutethimide-treated rats, plasma ACTH increased only at doses much higher than those inhibiting plasma corticosterone, suggesting that adrenal enzyme blockers may themselves be stressful. To determine the adrenocortical system response to stressless corticosterone removal, adrenalectomized rats maintained for 5 days on corticosterone in the drinking water were switched to steroid-free fluid (-B) or again given steroid (+B); additional rats were adrenalectomized (ADX). Plasma ACTH did not differ between -B and +B rats until 18-24 h after steroid removal, regardless of whether steroid was withdrawn at the circadian maximum or minimum. Plasma ACTH was similar between -B and ADX rats 0.5-14 days after corticosterone removal, although morning plasma ACTH was more stable in -B rats at 4-7 days. Evening plasma ACTH increased significantly after day 3 in ADX and -B rats. Unlike ADX rats, -B rats did not exhibit pituitary ACTH depletion at 12 and 24 h, but both -B and ADX groups had significantly elevated pituitary ACTH by 6.5 days. We conclude that 1) rapid increases in ACTH secretion after surgical or pharmacological adrenalectomy result from interaction between stress and loss of corticosteroid feedback; 2) no immediate interaction occurs between loss of feedback and circadian stimuli; and 3) the effects of steroid withdrawal may require at least 3 days to be stably expressed.

Apparent dissociation of adrenocorticotropin and corticosteroid responses to 15 ml/kg hemorrhage in conscious dogs.

The blood pressure, heart rate, ACTH, corticosteroid, vasopressin, and renin responses to rapid 15 ml/kg hemorrhage were measured in six conscious healthy dogs with chronically maintained femoral arterial catheters. The hemorrhage decreased the mean arterial blood pressure slightly (P less than 0.001), increased the heart rate (P less than 0.001), and increased arterial plasma levels of ACTH (P less than 0.01), corticosteroids (P less than 0.001), vasopressin (P less than 0.001), and renin activity (P less than 0.001). Overall and in the individual experiments, there appeared to be little correspondence between the ACTH and corticosteroid responses. In none of the experiments was there a clear rise in ACTH above control levels before the first rise in corticosteroids. To ascertain that adrenal secretion of corticosteroids was increased during 15 ml/kg hypovolemia, changes in the clearance and distribution volume of cortisol were estimated by counting tritium extracted from plasma of five dogs infused with [1,2-3H] cortisol to steady state levels before and during hypovolemia. The stimulus caused a 30% reduction from steady state levels of dichloromethane-extractable tritium counts (P less than 0.001). Combined with the observed increase in plasma corticosteroid levels, these results show that the increase in adrenal secretion of corticosteroids after hemorrhage was underestimated by measurement of changes in peripheral plasma levels. The hypothesis that hemorrhage results in an increase in adrenal sensitivity to ACTH is tested in the following paper.