Effects of bilateral adrenalectomy on immunoreactive corticotropin-releasing factor in the rat median eminence and intermediate-posterior pituitary

Immunoreactive ACTH (I-ACTH) concentrations in the anterior pituitary (AP), intermediate-posterior pituitary (IP) and plasma, and immunoreactive corticotropin-releasing factor (I-CRF) concentrations in the median eminence (ME) and IP, were determined in adrenalectomized rats from 3 h till 14 days after surgery. Plasma I-ACTH concentrations showed the typical triphasic response over time. AP I-ACTH concentrations decreased immediately after surgery, then increased to high concentrations 3 days after surgery. I-ACTH concentrations in IP did not change through these periods. I-CRF concentrations in ME and IP decreased immediately after surgery, then gradually increased to high concentrations (ME) or to control levels (IP) 14 days after surgery. These results raise the possibility that the I-CRF in IP is of hypothalamic origin.     

Effect of dexamethasone on immunoreactive corticotropin-releasing factor in the rat median eminence and intermediate-posterior pituitary

Immunoreactive ACTH (I-ACTH) concentrations in the anterior pituitary, intermediate-posterior pituitary (IP), and plasma and immunoreactive corticotropin-releasing factor (I-CRF) concentrations in the median eminence and IP were determined in rats receiving dexamethasone for various periods from 16 h to 10 days. Plasma I-ACTH concentrations were decreased 16 h after a single injection of dexamethasone. Anterior pituitary I-ACTH concentrations did not decrease until 4 days after the start of dexamethasone medication. IP I-ACTH concentrations did not change throughout these periods. I-CRF concentrations in median eminence and IP rapidly decreased after dexamethasone administration. These results raise the possibility that the source of I-CRF in the IP is hypothalamic.     

Insulin-induced hypoglycemia increases corticotropin-releasing factor messenger ribonucleic acid levels in rat hypothalamus

To study the effect of acute stress on CRF release and synthesis in rat hypothalamus, ACTH levels in plasma, CRF contents in the median eminence (ME), and CRF mRNA levels in the hypothalamus without ME and cerebral cortex were determined after insulin-induced hypoglycemia. Plasma ACTH levels increased at 30 and 60 min, while ME CRF content decreased at 30 and 60 min, then returned to the control level at 90 min. Hybridization with a cRNA probe revealed a single size class of CRF mRNA in the hypothalamus and cerebral cortex (approximately 1300 nucleotides), and the size of CRF mRNA in these tissues did not change during the experimental period. CRF mRNA levels in the hypothalamus increased to 130% of the control value at 30 min and reached a peak (186% of the control value) at 120 min, but these levels in the cerebral cortex did not change. These results suggest that insulin-induced hypoglycemia stimulates CRF synthesis by increasing CRF mRNA levels in the hypothalamus as well as CRF release, and that release and synthesis of CRF in the cerebral cortex are independent of those in the hypothalamus.     

Insulin-induced hypoglycemia increases proopiomelanocortin messenger ribonucleic acid levels in rat anterior pituitary gland

To study the effect of acute stress on ACTH secretion and synthesis in rat pituitary and hypothalamus, ACTH content and POMC mRNA levels (measured by use of Northern blot analysis) in these tissues as well as the levels of ACTH in plasma and those of CRF in the hypothalamus were determined after insulin-induced hypoglycemia. Plasma ACTH levels increased at 30 and 60 min. ACTH levels in the anterior pituitary lobe (AP) decreased at 30 min, and then returned to control levels at 60 min. No change was seen in the intermediate-posterior pituitary (IP) or the hypothalamus after insulin injection. CRF levels decreased at 30 and 60 min, then returned to control levels at 90 min in the medial basal hypothalamus, including the median eminence. Hybridization with a cDNA probe revealed a single size class of POMC mRNA in AP, IP, and hypothalamus, and the size of POMC mRNA in these tissues did not change during the experimental period. POMC mRNA levels in AP increased at 60 min and reached a peak at 120 min, but those in IP and hypothalamus did not change. These results suggest that 1) insulin-induced hypoglycemia stimulates both secretion and synthesis of ACTH (at least by increasing POMC mRNA levels) in the AP, and 2) the levels of ACTH and POMC mRNA in the IP and hypothalamus are not affected by insulin-induced hypoglycemia.     

Changes in the hypothalamo-corticotrope axis after bilateral adrenalectomy: evidence for a median eminence site of glucocorticoid action.

Bilateral adrenalectomy (ADX) leads to increased ACTH synthesis and secretion. It is thought that endogenous glucocorticoids exert a feedback mechanism at both pituitary and brain levels. The present study has been performed in order to determine the effect of ADX on the release of hypothalamic neuropeptides with corticotropin-releasing activity (CRA) and if there exists a median eminence site of glucocorticoid action to regulate hypothalamic-pituitary-adrenal (HPA) function. Adrenalectomized and sham-operated male rats were killed at different periods after surgery (2, 5, 7 and 14 days) and trunk blood was collected for ACTH and corticosterone (B) concentrations measurement. Brain (median eminence, ME; and medial basal hypothalamus, MBH) and pituitary (anterior lobe, AP; and neurointermediate lobe, NIL) tissues were dissected in order to evaluate either peptide content or in vitro hormone release. The results indicate that ADX blunted plasma B levels and increased AP ACTH content and secretion in a time-related fashion up to the 14th day. ADX significantly decreased both CRF and CRA contents in the ME at all periods studied; ME arginine-vasopressin (AVP) increased 7 and 14 days after ADX. MBH CRF decreased after ADX, but returned to sham value 2 weeks later; similarly, MBH AVP decreased at all periods after ADX. Removal of endogenous glucocorticoids did not vary neither oxytocin (OXY) content in the ME and MBH nor AVP and OXY contents in the NIL. In our superfusion experiments, we found that ADX increased basal AVP release and did not change spontaneous CRF secretion from ME terminals. Dexamethasone (Dxm, 10 nM) diminished AVP but not CRF output by ME tissues from adrenalectomized rats. A direct relationship was found between ME CRF and 28 mM KCl (hK+)-induced CRF release by MEs from adrenalectomized rats. ME fragments from adrenalectomized rats were hyperresponsive to kH+ stimulation of AVP release. Dxm (10 nM) decreased the hK(+)-evoked CRF and AVP release by MEs from adrenalectomized rats. ADX and dexamethasone treatment did not influence basal and hK(+)-elicited ME OXY release. Additionally, a rapid glucocorticoid inhibitory effect on ACTH secretion by isolated AP cells from both sham and adrenalectomized rats was found, and an in vitro corticotrope hyporesponse to 0.63 nM CRF and 9.25 nM AVP stimulation during several days after ADX.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunoreactive corticotropin and corticotropin-releasing factor in human hypothalamus, adrenal, lung cancer, and pheochromocytoma

Immunoreactive corticotropin-releasing factor (I-CRF) and ACTH (I-ACTH) were examined using RIA, immunoaffinity chromatography, and gel filtration chromatography in human hypothalamus, adrenal (cortex and medulla), lung cancer, and pheochromocytoma. I-CRF and I-ACTH were present in these tissues. Gel filtration of I-ACTH in the adrenal, pheochromocytoma, and lung cancer showed the presence of larger amounts of I-ACTH with large molecular weight forms in contrast to the hypothalamus. Gel filtration of I-CRF in these tissues showed the main peak eluted at the position of synthetic rat CRF. High performance liquid chromatography of this main peak showed two components which eluted in the positions of synthetic rat CRF and oxidized CRF. These elution positions were the same in all tissues and identical with those in the hypothalamus. These results suggest the presence of I-ACTH and I-CRF in these tissues and that CRF outside the brain is identical to hypothalamic CRF.     

Corticotropin-releasing factor (CRF) induces desensitization of the rat pituitary CRF receptor-adenylate cyclase complex

The rise in circulating ACTH levels after adrenalectomy in the rat is associated with a decrease in CRF receptor-binding capacity in the anterior pituitary. To investigate the role of increased hypothalamic CRF release on pituitary CRF receptor regulation after withdrawal of glucocorticoid feedback by adrenalectomy, the effects of chronic CRF infusion and lesions in the medial basal hypothalamus were studied in the rat. Subcutaneous infusion of CRF at 10, 25, 50, and 100 ng/min for 48 h in intact rats caused dose-dependent increases in plasma ACTH levels from the control value of 32.1 +/- 4.3 to 58.0 +/- 4.9, 82.0 +/- 7.1, 135.5 +/- 11.6, and 149.2 +/- 13.2 pg/ml, respectively. In contrast, the pituitary CRF receptor concentration was reduced by 25.3 +/- 4.5%, 38.3 +/- 2.5%, 43.8 +/- 0.9%, and 45.8 +/- 2.0%, respectively. Intravenous infusion of increasing doses of CRF caused a similar increase in plasma ACTH levels, which became maximum at the lowest infusion dose (32.4 +/- 5.4, 138.5 +/- 12.3, 162.0 +/- 18.3, and 167 +/- 19.1 pg/ml for control and 10, 50, and 100 ng/min CRF, respectively). Pituitary CRF receptor concentration was again decreased after iv CRF infusion [by 42 +/- 6.2% with the lowest dose (10 ng/min)], with no further reduction after infusion of 50 and 100 ng/min (49.0 +/- 6.8% and 26.0 +/- 6.2%, respectively)]. The decrease in pituitary CRF receptors after CRF infusion was accompanied by a decrease in CRF-stimulated adenylate cyclase activity, with a 10- to 100-fold increase in the concentration of CRF required for threshold stimulation. In cultured pituitary cells prepared from animals infused with 50 ng/min CRF for 48 h, maximum CRF-stimulated ACTH release was reduced by 29 +/- 3.2% (P less than 0.01; n = 3), with no significant change in sensitivity to CRF (ED50, 0.6 +/- 0.5 and 1.0 +/- 0.5 nM CRF for control and CRF infusion, respectively). The role of endogenous CRF in adrenalectomy-induced pituitary CRF receptor down-regulation was also studied in rats with medial basal hypothalamic deafferentation. The marked loss of pituitary CRF receptors after adrenalectomy was completely prevented by such hypothalamic lesioning, indicating that receptor down-regulation was dependent on the release of CRF or/and other hypothalamic factors. The data demonstrate that while increased CRF levels result in down-regulation and desensitization of pituitary CRF receptors, the differences between adrenalectomy and CRF infusion indicate that additional regulatory factors are involved in the modulation of CRF receptor content and activity after adrenalectomy.     

Characterization of immunoreactive corticotropin and corticotropin-releasing factor in human adrenal and ovarian tumours

The distribution of immunoreactive ACTH (I-ACTH) and corticotropin-releasing factor (I-CRF) in the human adrenal was determined and these peptides were indentified in adrenocortical adenomas from patients with primary aldosteronism. Cushing's syndrome, and ovarian tumours and compared with the results in phaeochromocytomas. I-ACTH and I-CRF, mainly localized in the adrenal medulla from patients without endocrine disorders, showed a good correlation with the epinephrine concentrations. I-ACTH and I-CRF were present in all the above-mentioned tumours. Gel filtration of I-ACTH and I-CRF from these tumours showed the presence of large molecular weight forms of these peptides as well as authentic peptides. The trypsinization study of I-CRF from phaeochromocytoma suggested that such large molecular weight forms were the precursors of authentic CRF. High performance liquid chromatography showed I-CRF in these tumours to be similar to hypothalamic CRF. Phaeochromocytoma tissue slices incorporated [3H]leucine into ACTH and CRF. These findings raised the possibility that I-ACTH and I-CRF are synthesized and processed in phaeochromocytoma.     

Involvement of vasopressin in the down-regulation of pituitary corticotropin-releasing factor receptors after adrenalectomy

The role of vasopressin (VP) in the regulation of pituitary corticotropin-releasing factor (CRF) receptors was studied by examining the effects of adrenalectomy and VP infusion on pituitary CRF receptors in genetically VP-deficient rats (di/di) and Long-Evans control rats. Binding studies with [125I]Tyr-ovine CRF in 30,000 X g anterior pituitary membrane-rich fractions revealed similar characteristics for the CRF receptors in Long-Evans and di/di rats, with Kd values of 2.4 +/- 0.6 and 1.9 +/- 0.2 nM, respectively, and receptor concentrations of 278 +/- 31 and 286 +/- 43 fmol/mg, respectively. Two days after adrenalectomy, the pituitary CRF receptor concentration decreased by 72 +/- 4.2% in Long-Evans rats, but by only 20.3 +/- 5.6% in di/di rats. CRF receptor affinity was unchanged after adrenalectomy (Kd = 1.7 +/- 0.5 nM; n = 8). To determine whether VP deficiency is responsible for the smaller decrease in CRF receptor in di/di rats, the effect of exogenous VP infusion (100 ng/min) by sc osmotic minipumps was studied in adrenalectomized di/di rats. Two days after adrenalectomy, pituitary CRF receptors were reduced by 21 +/- 8% in control di/di rats, whereas a 77.7 +/- 1.8% decrease was observed in VP-infused di/di rats, comparable to the effect of adrenalectomy in Long-Evans rats. VP infusion also caused a significant 35 +/- 2% decrease in CRF receptors in the pituitaries of sham-operated di/di rats, with no change in CRF receptor affinity. In Sprague-Dawley rats, VP or CRF infusion (100 ng/min) decreased pituitary CRF receptors by 14 +/- 1.9% and 46 +/- 3%, respectively. However, the combined infusion of both peptides caused a 65% +/- 4.2 decrease, similar to that observed after adrenalectomy. In vitro incubation of quartered pituitaries with VP or CRF for 4 h reduced CRF receptors by 23.1 +/- 8.2% and 38.2 +/- 3.8%, respectively, while simultaneous preincubation with both peptides was followed by a decrease of 55.3 +/- 5.3%. These findings indicate that increased hypothalamic release of VP contributes to the down-regulation of pituitary CRF receptors after adrenalectomy.     

Stimulation of adrenocorticotropin secretion by insulin-induced hypoglycemia in the developing rat involves arginine vasopressin but not corticotropin-releasing factor.

In the neonatal rat, the response of the hypothalamo-pituitary-adrenal axis to stressful stimuli is markedly decreased during the first 2 weeks of life. This peculiar period was named "stress hyporesponsive period." In this report, we studied the effect of insulin-induced hypoglycemia, known as a strong stimulator of the corticotroph function in the adult rat. Rats (8- or 20-day-old) were injected ip with 3 IU/kg synthetic insulin and were killed at various times. In 20-day-old rats, hypoglycemia induced a rapid drop in blood glucose concentrations accompanied by a stimulation of ACTH and corticosterone secretion which reached maximal values within 30 min. On the opposite, in 8-day-old rats, despite a rapid decrease in blood glucose levels, insulin injection induced a gradual rise of plasma ACTH and corticosterone concentrations which peaked at 90 min. This delayed response of the hypothalamo-pituitary-adrenal axis to hypoglycemia in the youngest rats does not seem to be due to a difference of sensitivity to insulin-induced hypoglycemia since injection of increasing doses of insulin (0.3, 0.75, or 3 IU/kg body wt) induced a dose-related decrease of blood glucose concentrations and a rise in plasma ACTH and corticosterone levels, comparable in the two age group studied. Basal or hypoglycemia-stimulated absolute corticosterone values were much lower in 8-day-old rats than in 20-day-old animals, suggesting an immaturity of the adrenal glands in the youngest animals. Daily ACTH injection, starting 3 days before the experiment, had a trophic effect on the adrenal glands leading to a more important increase of corticosterone levels after hypoglycemia in 8-day-old rats. Our results confirm that there is an immaturity of the adrenal glands in young rats, probably due to the low plasma ACTH levels during the neonatal period. To determine the respective role of the two major hypothalamic ACTH secretagogues, we studied the effect of passive immunization against CRF or arginine vasopressin (AVP) on plasma ACTH response after hypoglycemia. Passive immunization against AVP decreased significantly hypoglycemia-stimulated ACTH secretion in both 8- and 20-day-old rats, while no change of plasma ACTH response to insulin injection was observed after passive immunization against CRF. This results suggest that CRF does not seem to be involved in the regulation of ACTH secretion after hypoglycemia in the young rat while AVP seems to be the main hypothalamic stimulatory factor for anterior pituitary corticotrophs response to hypoglycemia during the postnatal period.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of adrenalectomy and dexamethasone administration on the level of prepro-corticotropin-releasing factor messenger ribonucleic acid (mRNA) in the hypothalamus and adrenocorticotropin/beta-lipotropin precursor mRNA in the pituitary in rats

RNA blot hybridization analysis with cloned rat CRF precursor (prepro-CRF) cDNA as a probe showed that prepro-CRF mRNA existed in rat hypothalamic and extrahypothalamic brain tissue, whereas it was undetectable in the pituitary and adrenal. To study the effect of glucocorticoid on the level of prepro-CRF mRNA in the hypothalmus and that of ACTH/beta-lipotropin (beta LPH) precursor mRNA in the pituitary, effects of adrenalectomy and dexamethasone administration were studied in rats. Adrenalectomy markedly raised mRNA coding for ACTH/beta LPH precursor in the anterior pituitary, but not in the neurointermediate pituitary lobe. Hypothalamic pre-pro-CRF mRNA increased only to 152% of the control value, 7 days after adrenalectomy. The administration of dexamethasone (200 micrograms/day for 7 days) started immediately after adrenalectomy lowered the ACTH/beta LPH precursor mRNA level in the anterior pituitary to 19% of the intact control value, whereas the level of prepro-CRF mRNA in the hypothalamus decreased only to 102%. These results suggest that glucocorticoids exert their feedback effect at the level of gene expression on both hypothalamic CRF neurons and pituitary corticotropes. Although the possibility that CRF neurons insensitive to glucocorticoid in the hypothalamus might blunt the change in the prepro-CRF mRNA could not be ruled out, it is also possible that the effect of glucocorticoids on the pituitary is dominant.     

Effect of passive immunization against corticotropin-releasing factor (CRF) on the postadrenalectomy changes of CRF binding sites in the rat anterior pituitary gland

The concentration of corticotropin-releasing factor (CRF)-binding sites decreases in the rat anterior pituitary after adrenalectomy; this change may be related either to a direct effect of the circulating glucocorticoids at the pituitary level or to a desensitization of CRF receptors through an increased CRG release in hypophysial portal blood. In order to examine the latter possibility we have measured plasma adrenocorticotropin hormone (ACTH) levels and the number of anterior pituitary CRF binding sites in sham-operated and 24-hour adrenalectomized rats after blockade of endogenous CRF by passive immunization with an antiserum anti-rat CRF (CRF-AS), or after injection of normal rabbit serum (NRS). In NRS-injected rats, after sham operation, plasma ACTH concentration increased (227 +/- 34 vs. 118 +/- 19 pg/ml in controls) without change in CRF-binding sites capacity (20.7 +/- 2.6 vs. 24.6 +/- 3.5 fmol/mg protein in controls). Adrenalectomy induced a large rise in plasma ACTH (785 +/- 89 pg/ml) and a decrease in the number of CRF-binding sites (12.2 +/- 1.7 fmol/mg protein). After CRF-AS injection, plasma ACTH was normalized in sham-operated animals (149 +/- 24 pg/ml) and significantly reduced in adrenalectomized rats (472 +/- 76 pg/ml); the adrenalectomy-induced decrease in the number of CRF-binding sites was unaffected by the CRF-AS administration (12.2 +/- 1.7 fmol/mg protein). The administration of dexamethasone to adrenalectomized rats significantly reduced plasma ACTH concentrations (23.3 +/- 10.6 pg/ml) and prevented the loss in CRF-binding sites capacity (20.7 +/- 1.3 fmol/mg protein).(ABSTRACT TRUNCATED AT 250 WORDS)     

THE BIOACTIVITY OF IMMUNOREACTIVE ADRENOCORTICOTROPHIN IN HUMAN BLOOD IS DEPENDENT ON THE SECRETORY STATE OF THE PITUITARY GLAND

A highly sensitive bioassay, using preincubated purified isolated rat adrenal cells, has been developed for measuring plasma ACTH. This bioassay enables detection of ACTH in plasma of about 0.9 pmol/l. Bioactive ACTH (B-ACTH) plasma levels were determined during insulin-induced hypoglycaemia in 12 female subjects and the values were compared with immunoreactive ACTH (I-ACTH) levels. The mean (+/- SD) basal B-ACTH level amounted to 1.7 +/- 0.8 pmol/l, the mean I-ACTH to 2.9 +/- 1.4 pmol/l. The highest mean B-ACTH plasma value was found 30 min after insulin injection: 14.7 +/- 15.7 pmol/l (I-ACTH: 12.8 +/- 9.9 pmol/l). By 90 min the B-ACTH level had returned to baseline (1.6 +/- 0.8 pmol/l), whereas the I-ACTH level was still significantly higher (5.4 +/- 2.7 pmol/l) than at time zero. Remarkably, the B-ACTH to I-ACTH ratio (B/I ratio) showed a biphasic profile during the insulin tolerance test, the ratio increasing from 0.60 +/- 0.77 at time zero to 1.08 +/- 0.35 at the ACTH peak, and decreasing after that to a lower value of 0.33 +/- 0.11 at 90 min. From these results it is concluded: (1) in the morning hours a considerable amount of circulating I-ACTH has no steroidogenic activity; (2) the B/I ratio temporarily increases immediately after insulin injection but gradually decreases afterwards to values half the baseline level at 90 min. Whereas this decrease at 90 min can be explained by differences in disappearance rates, the increase of B-ACTH relative to I-ACTH at 30 min indicates that estimations of immunoreactive ACTH reflect the biological activity of newly released ACTH with greater precision than at steady state level. Thus, the B/I-ACTH ratio can be used as a tool for measuring the state of the pituitary releasing activity.     

Onset of glucocorticoid responsiveness of anterior pituitary corticotrophs during development is scheduled by corticotropin-releasing factor

Expression of the CRF gene in the hypothalamus and that of the POMC gene in the anterior pituitary are reduced during the first week of life in the rat. During this so-called stress nonresponsive period (SNRP), stimuli such as ether vapors, electroshocks, and hypoxia do not elicit ACTH secretion from the pituitary, as occurs later in development. The current hypothesis to explain the SNRP is an increased negative glucocorticoid feedback on POMC and CRF synthesis and/or release during this time. To test this hypothesis we studied the effects of adrenalectomy (ADX) on anterior pituitary POMC mRNA expression. In 7-day-old rats POMC mRNA levels were increased only 3-fold 48 h post-ADX, compared to a 7-fold increase in 14-day-old animals. This blunted effect of endogenous glucocorticoid removal on pituitary POMC mRNA could be due to decreased up-regulation of CRF after removal of glucocorticoids or normal up-regulation of CRF but decreased pituitary responsiveness to CRF relative to those in 14-day-old animals. Therefore, we studied in vitro beta-endorphin release from pituitaries obtained from 7- and 14-day-old rats. CRF stimulated basal beta-endorphin release to the same extent in pituitaries from both groups. The inhibition by corticosterone of CRF-stimulated beta-endorphin secretion was also indistinguishable in pituitaries obtained from 7- or 14-day-old rats. Since the responsiveness of the 7-day-old pituitary was normal, the blunted enhancement of POMC biosynthesis after ADX must be mediated at the level of the hypothalamus. Indeed, in situ hybridization showed that while in 14-day-old rats ADX induced a significant increase [190 +/- 10% (+/- SE) of control; n = 5; P less than 0.0005] in hypothalamic mRNA levels, ADX did not change the expression of the CRF gene in the paraventricular nucleus of 7-day-old rats, indicating a lack of glucocorticoid modulation of hypothalamic CRF synthesis. Finally, we studied the effects of 48 h CRF treatment on the post-ADX increase in POMC mRNA levels in 7-day-old rats. Daily injections of 200 ng CRF/rat induced an increase in anterior pituitary POMC mRNA concentrations [669 +/- 139% (+/- SE) of control; n = 6; P less than 0.02 vs. adrenalectomized vehicle-treated rats] comparable to that in adrenalectomized untreated 14-day-old rats. In conclusion, our data indicate that the glucocorticoid regulation of hypothalamic CRF gene expression is not mature during the first week of life, i.e. within the so-called SNRP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of corticotropin-releasing factor (CRF) receptors in the rat pituitary gland: effects of adrenalectomy on CRF receptors and corticotroph responses

The stimulation of ACTH release from anterior pituitary cells by corticotropin-releasing factor (CRF) is mediated by specific, high affinity receptors with a Ka of 10(9) M-1 for ovine CRF. The relationship between ACTH secretion and CRF receptor activation was analyzed in normal and adrenalectomized rats by comparison of ACTH release with changes in CRF receptors and adenylate cyclase activity. The marked increase in plasma ACTH levels that occurred after adrenalectomy (from 71 to 478 pg/ml after 4 days) was accompanied by a progressive decrease in pituitary CRF receptor concentration [by 29 +/- 1%, 75 +/- 2%, 77 +/- 6%, and 80 +/- 4% (+/- SE) after 1, 2, 3, and 4 days, respectively]. Most of this decrease was due to receptor down-regulation rather than occupancy by endogenous CRF, since high dose infusions of CRF (300-500 ng/min) for 30 min before pituitary membrane preparation reduced CRF-binding sites by only 40%. The marked reduction in CRF receptors after adrenalectomy was accompanied by comparable decreases in maximal CRF-stimulated adenylate cyclase activity and sensitivity to CRF (ED50, 3.8 +/- 2.8 vs. 58 +/- 3.7 X 10-9 M CRF in control and 2-day-adrenalectomized rats, respectively). Fluoride-stimulated adenylate cyclase activity was unchanged at 24 h, but was decreased by 28 +/- 7% at later times. Such decreases in CRF receptors and adenylate cyclase activity in adrenalectomized rats were prevented by dexamethasone treatment. In cultured anterior pituitary cells from 4-day-adrenalectomized rats, CRF-stimulated cAMP production was decreased by 40%. However, in contrast to the decreases in CRF receptors and cAMP production, there was a 3-fold increase in CRF-stimulated ACTH release, with no change in sensitivity to CRF. The ability of corticotrophs to maintain increased ACTH release, in conjunction with reduced CRF receptors and CRF-stimulated adenylate cyclase, indicates that elevated ACTH secretion can be maintained by occupancy and activation of only a small number of CRF receptors. This finding also suggests that synergistic interactions between CRF and other regulators of ACTH release may contribute to the sustained increase in ACTH secretion that follows adrenalectomy.     

Ontogeny of Hypothalamic Corticotropin-Releasing Factor and Anterior Pituitary Pro-opiomelanocortin Expression in Male and Female Offspring of Alcohol-Exposed and Adrenalectomized Dams

This study was designed to determine whether the previously described sexually dimorphic changes in rat hypothalamic corticotrop-in-releasing factor (CRF) and anterior pituitary pro-opiomelanocortin (POMC) mRNA expression in response to fetal alcohol exposure (FAE) are present prepubertally and whether they are altered by maternal adrenalectomy. Hypothalamic CRF and anterior pituitary POMC mRNA levels were determined in male and female offspring of adrenalectomized (ADX) and sham-adrenalectomized (Sham) dams exposed to alcohol (FAE) or a pair-fed (PF) control diet during the last 2 weeks of gestation. CRF and POMC mRNA levels were measured by Northern blotting at 1, 7, 14, and 21 days of age. In offspring of control PF dams, CRF mRNA levels increased faster in females, increasing by day 7, followed by a decrease at days 14 and 21, whereas in males there was a gradual increase from days 1 to 21. FAE altered the ontogenic profile of CRF mRNA in female offspring by delaying and exaggerating the rise of CRF expression to day 14, but produced no effect in males. Maternal adrenalectomy, combined with FAE, resulted in an early rise of CRF mRNA on day 14 in male offspring. In females, the combined ADX/FAE treatment resulted in significantly increased CRF mRNA levels, compared with those of ADX/PF offspring, on days 7 and 14. By day 21, these differences in CRF mRNA levels between the ADX/FAE and ADX/PF offspring had disappeared. POMC mRNA levels generally increased by day 7, followed by a dramatic decrease by day 14 and another increase by day 21. FAE male offspring showed decreased levels of POMC mRNA, whereas females were not affected. Maternal adrenalectomy reversed this inhibition in male offspring, resulting in POMC mRNA levels similar to those measured in male offspring of PF control animals. In contrast, POMC mRNA levels of female offspring of ADX dams decreased in response to FAE. These data suggest that the previously observed switch from suppressed to enhanced POMC expression in FAE males is the result of developmental events beyond weaning. Because this sexually dimorphic regulation of CRF and POMC expression by prenatal alcohol exposure and maternal adrenalectomy occurs before the presence of adult levels of sex steroids, this suggests an organizational effect on the developing hypothalamic-pituitary-adrenal function.     

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.     

Reduced hypothalamic POMC and anterior pituitary CRF1 receptor mRNA levels after acute, but not chronic, daily "binge" intragastric alcohol administration

BACKGROUND: Endogenous corticotropin-releasing factor (CRF), its pituitary CRF1 receptor, and proopiomelanocortin (POMC) may be involved in the hypothalamic-pituitary-adrenal (HPA) responses to alcohol. METHODS: Alcohol (1.5 g/kg) or water was administered intragastrically to male Fischer rats after the "binge" pattern regimen, that is, three times daily at 1 hr intervals at the beginning of the light cycle. The levels of CRF, CRF1 receptor, and POMC mRNAs in the hypothalamic-pituitary axis were measured after acute (1 day) or chronic (14 days) binge pattern alcohol administration. Plasma levels of ACTH and corticosterone were measured to examine time-dependent alterations of HPA responses. RESULTS: Plasma ACTH and corticosterone levels were elevated dramatically after 1 day of acute binge pattern alcohol administration. After 14 days of chronic alcohol, however, no elevation in plasma ACTH levels and an attenuated elevation in plasma corticosterone levels were found. CRF mRNA levels in the hypothalamus were not altered after either acute or chronic alcohol administration. CRF1 receptor mRNA levels in the anterior pituitary were decreased significantly after acute administration, with no change after chronic alcohol administration. POMC mRNA levels in the anterior pituitary were not altered by either acute or chronic alcohol administration. In the hypothalamus, POMC mRNA levels were decreased significantly after acute but not chronic binge alcohol administration. CONCLUSIONS: These results suggest that (1) rats exposed to chronic binge alcohol develop tolerance in HPA activity, as shown by no elevation of ACTH and an attenuated corticosterone response to chronic alcohol after initial dramatic elevations by acute alcohol administration; (2) a concurrent acute decrease in CRF1 receptor mRNA levels in the anterior pituitary is associated with increased HPA activity, and (3) alterations of POMC gene expression in the hypothalamic region may have implications for a molecular understanding of the neuroendocrine response to alcohol.     

beta-Lipotropin is the major opioid-like peptide of human pituitary and rat pars distalis: lack of significant beta-endorphin.

beta-Lipotropin is the predominant opioid peptide of the human pituitary and rat pars distalis and is present in concentrations essentially equimolar with corticotropin. When freshly, obtained nonfrozen rat anterior pituitaries were homogenized with 0.2 M HCl, approximately 98% of the immunoreactivity detected utilizing an antiserum that crossreacts equally with beta-lipotropin and beta-endorphin coeluted with 125I-labeled human beta-lipotropin upon molecular sieve chromatography. The remainder of the activity eluted with synthetic human beta-endorphin. Similar results were obtained for human pituitary. HCl homogenization of thawed tissue or homogenization of fresh tissue with acetic acid yielded substantially greater concentrations of beta-endorphin and decreased concentrations of beta-lipotropin. In human subjects, acute anterior pituitary stimulation using either insulin-induced hypoglycemia or vasopressin administration was associated with increased plasma beta-lipotropin and corticotropin levels. At the time of peak concentrations, no significant levels of beta-endorphin were detectable. These data indicate the lack of significant amounts of beta-endorphin in human pituitary. Additionally, there appears to be no specific intrapituitary conversion of beta-lipotropin to beta-endorphin.