Radioautographic study of binding and internalization of corticotropin-releasing factor by rat anterior pituitary corticotrophs

In order to identify the anterior pituitary cell type(s) containing corticotropin-releasing factor (CRF) receptors and to study the internalization processes of this peptide by the target cells, radioautography was performed on rat anterior pituitaries removed at specific intervals (2-60 min) after intracarotid injection of [125I]iodo-CRF into intact and adrenalectomized female rats. In intact animals, all corticotrophs were labeled, whereas in the adrenalectomized animals about 80% of the hypertrophied corticotrophs (adrenalectomy cells) were. In control animals injected with both iodinated CRF and an excess of unlabeled peptide, no specific reaction could be detected. The time-course study in intact animals showed that 2 min after injection most silver grains were found over or within 160 nm of the plasma membrane. At the 5-min time intervals, grains were observed both over the plasma membrane and within the cytoplasm, associated with lysosomes, and the Golgi apparatus. Fifteen minutes after injection, grains were mostly found over lysosomes and the Golgi apparatus, whereas at the longest time intervals (30 and 60 min) almost no labeling could be detected. The results obtained in this study indicate that in the anterior pituitary CRF receptors are restricted to corticotrophs (as identified by electron microscopy) and that, after binding to the plasma membrane, CRF is rapidly internalized to Golgi elements and lysosomes.     

Early glucocorticoid feedback in anterior pituitary corticotrophs: differential inhibition of hormone release induced by vasopressin and corticotrophin-releasing factor in vitro.

Vasopressin and 41-residue corticotrophin-releasing factor (CRF-41) are physiological mediators of the hypothalamic control of pituitary ACTH secretion, whilst adrenocortical glucocorticoids are the major inhibitory factors regulating ACTH output. In the present study it was investigated in vitro whether the characteristics of early glucocorticoid inhibition of stimulated ACTH secretion would differ depending on the nature of the stimulus and the temporal relationship between secretagogue and steroid. The experiments were carried out using perifused segments of rat adenohypophysis obtained from randomly cycling female rats. Repeated pulses (5 min) of CRF-41 or vasopressin were given at 1-h intervals for up to 7 h. The net release of ACTH became stable after the second secretagogue pulse. Administration of 0.1 mumol corticosterone/l 30 min before and during a 5-min pulse of 10 nmol CRF-41/l inhibited CRF-41-stimulated ACTH release to 60% of control. Stimulated hormone release remained suppressed at 90 min after the start of the corticosterone infusion and returned to control levels by 150 min. If corticosterone treatment (35 min total exposure) was started simultaneously with the CRF-41 pulse, no inhibitory effect of the steroid was observed at any subsequent time-point examined (60, 90, 120 and 150 min). In contrast, vasopressin-stimulated ACTH release was inhibited by approximately 50% when corticosterone was applied before, or simultaneously with, a 5-min pulse of 10 nmol vasopressin/l.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hypercorticosteronuria and diminished pituitary responsiveness to corticotropin-releasing factor in obese Zucker rats

Metabolic defects in obese (fa/fa) Zucker rats have previously been shown to be reversed by adrenalectomy; however, hypercorticosteronemia has not been demonstrated. We now report that the total daily excretion of corticosterone and urea nitrogen are significantly greater (P less than 0.01) in obese Zucker rats than in age-matched lean Zucker rats. This excessive excretion of corticosterone is not of autonomous adrenal origin, since dexamethasone treatment (20 micrograms/kg X day) for 2 days induced a proportionate reduction in corticosterone excretion (approximately 50%) in both obese and lean Zucker rats. Corticosterone excretion was further suppressed to levels not different from those in lean rats after 2 days of dexamethasone (40 micrograms/kg X day). Both the peak and total pituitary beta-endorphin secretion in response to an iv bolus of corticotropin-releasing factor (CRF) were diminished in obese Zuckers. The response to CRF in obese Zucker rats was dampened and superimposable on that of dexamethasone-treated lean Zucker rats, suggesting the existence of chronic hypercorticosteronemia as a component of this genetic obesity. These observations provide evidence for a compensatory alteration of the pituitary-adrenal axis. We suggest that corticosterone turnover may be increased in obese Zucker rats.     

Glucocorticoid modulation of corticotropin-releasing factor desensitization in cultured rat anterior pituitary cells

The ability of ovine corticotropin-releasing factor (CRF) to stimulate both ACTH release and intracellular cAMP accumulation is rapidly and reversibly desensitized when rat anterior pituitary cells are cultured in the absence of added glucocorticoids. Since this desensitization has not been readily apparent in vivo, where initial CRF exposure results in high levels of ambient glucocorticoids, we examined the effect of glucocorticoids on the desensitization process in vitro. Rat anterior pituitary cells were cultured for 3-5 days in the absence of added steroid hormones. Dexamethasone was then added to some culture wells 24 h before the desensitization experiment began. Desensitization of CRF was achieved by preincubating the cells for 4 h with varying concentrations (10(-11)-10(-7) M) of ovine CRF, washing the cells with medium alone, and then reexposing the cells to CRF. In the absence of glucocorticoid, the ED50 for CRF desensitization (the preincubation dose causing 50% desensitization of subsequent ACTH release) was 3 X 10(-10) M, but cells that had been preexposed to dexamethasone desensitized less readily. With concentrations of dexamethasone of 10(-8) M or greater, no desensitization occurred. When cells were incubated in the absence of added glucocorticoids, CRF-stimulated intracellular cAMP accumulation was diminished by prior exposure to CRF. No decrease in intracellular cAMP accumulation was seen in those cells that had been preincubated with dexamethasone, however. Similar changes in CRF desensitization of ACTH release were observed when cells were incubated with corticosterone, but not with 10(-8) M testosterone, progesterone, aldosterone, or estradiol. These data demonstrate that glucocorticoids profoundly alter the development of CRF desensitization in vitro and suggest that high ambient glucocorticoid concentrations prevent the development of substantial CRF desensitization in vivo.     

Stimulation of cyclic AMP accumulation and corticotropin release by synthetic ovine corticotropin-releasing factor in rat anterior pituitary cells: site of glucocorticoid action.

A 2.5-fold stimulation of cyclic AMP cellular content is measured 60 sec after addition of 100 nM synthetic ovine corticotropin-releasing factor (C-RF; corticoliberin) to rat anterior pituitary cells in culture. A maximal response of cyclic AMP content at 400% above control is observed between 2 and 30 min after addition of the peptide, whereas an 8-fold stimulation of cyclic AMP released into the incubation medium is measured between 10 and 180 min. A linear 7-fold increase of corticotropin release is observed for up to 3 hr. Preincubation from 18 hr with the potent glucocorticoid dexamethasone has no effect on C-RF-induced cyclic AMP accumulation. The same treatment with dexamethasone causes an 80% inhibition of corticotropin release induced by both C-RF and the cyclic AMP derivative 8-bromoadenosine 3',5'-cyclic monophosphate. The present data show that ovine C-RF is a potent stimulator of cyclic AMP accumulation in rat anterior pituitary cells and that the process is insensitive to the action of dexamethasone. The marked inhibition by dexamethasone of corticotropin secretion induced by a cyclic AMP derivative indicates that glucocorticoids exert their potent inhibitory effects on corticotropin secretion at a step distant to cyclic AMP formation.     

Inactivation of early glucocorticoid feedback by corticotropin-releasing factor in vitro.

We have investigated the interaction between hypothalamic ACTH secretagogues and adrenocortical glucocorticoids in rat anterior pituitary tissue using an in vitro perifusion system. Repeated 5 min pulses of 41-residue CRF (CRF-41) or arginine vasopressin (AVP) were applied at 1 h intervals for up to 7 h. Administration of 0.1 microM corticosterone 30 min before and during the 5 min 0.1 nM CRF-41 stimulus at 5 h resulted in a significant inhibition of CRF-41 stimulated ACTH release within 30 min. Inhibition of ACTH release also developed if no CRF-41 stimulus was applied in conjunction with steroid at 5 h. In contrast, if the exposure to corticosterone (0.1 microM, 35 min total duration) was started simultaneously with the application of CRF-41 at 5 h, no inhibition of ACTH release ensued. Similarly, no inhibition of CRF-41-stimulated ACTH release was observed when corticosterone was started simultaneously with a 5 min pulse of cyclic 8-(4-Chlorophenylthio) AMP (8-CPT-cAMP), a cell membrane permeant analog of cAMP. In contrast to CRF-41 and 8-CPT-cAMP, AVP failed to modify glucocorticoid-induced inhibition of AVP- or CRF-41-stimulated ACTH release. Moreover, CRF-41 did not prevent the glucocorticoid-induced inhibition of AVP-stimulated ACTH release. In summary: 1) CRF-41 inactivates early glucocorticoid inhibition of CRF-41-stimulated ACTH secretion, and this is mimicked by a cell membrane permeant analog of cAMP; 2) AVP does not inactivate glucocorticoid-induced inhibition of stimulated ACTH release; 3) the data point to an acute interaction between the cAMP/protein kinase A and glucocorticoid-responsive intracellular pathways. Such differential modulation of feedback inhibition by CRFs may be of functional importance in vivo.     

Transplantation of the pituitary pars distalis induces the corticotrophs to store melanocyte-stimulating hormone, an effect reversed by the administration of corticotropin-releasing factor.

Corticotrophs of the pituitary pars distalis do not contain immunohistochemically detectable alpha-melanocyte-stimulating hormone (MSH). After grafting the glands beneath the renal capsule for 25 days, this hormone could be demonstrated in corticotrophs, coexisting with corticotropin. The administration of corticotropin-releasing factor deprived these cells of the content of MSH but did not apparently affect the presence of adrenocorticotropin (ACTH). It is suggested that the histological appearance of MSH in the corticotrophs may be due to a diminished rate of corticotropin release, which may provide time for splitting the former hormone in amounts larger than the negligible ones normally present in the pars distalis, or to the hypothetical fact that some hypothalamic factor may inhibit the cleavage of ACTH into MSH.     

The neuronal growth-associated protein (GAP)-43 is expressed by corticotrophs in the rat anterior pituitary after adrenalectomy

The neuronal growth-associated protein (GAP)-43 has been localized in both long fibers and punctate clusters by immunocytochemistry within the rat anterior pituitary (AP). After adrenalectomy (ADX), GAP-43 immunoreactivity (GAP-43-ir) is greatly increased and is associated with corticotrophs at the light microscopic level. We have undertaken an electron microscopic study to determine the cellular localization of GAP-43 in the post-ADX AP. Using preembedding immunocytochemistry, we found GAP-43-ir localized exclusively to the cytoplasmic surface of the plasmalemma within a subset of endocrine cells with ultrastructure typical of degranulated corticotrophs at 4 d after ADX. We combined preembedding immunoelectron microscopy for GAP-43 with immunogold labeling for ACTH and found that GAP-43-ir was invariably present only in cells containing ACTH-positive granules. The density of GAP-43-ir was highest within extensive processes emanating from the soma, suggesting that these processes are the basis for the punctate clusters of GAP-43 staining seen surrounding corticotrophs in the light microscope. We also observed rare synaptic-like contacts between GAP-43-ir processes and distant cell bodies. GAP-43 mRNA was detected in extracts of the AP 4 d after ADX using RT-PCR, and quantitative PCR confirmed that GAP-43 mRNA was significantly up-regulated in the AP in response to ADX. We postulate that increased expression of GAP-43 may stimulate process outgrowth and intercellular communication by activated corticotrophs.     

Expression of type 2 iodothyronine deiodinase in corticotropin-secreting mouse pituitary tumor cells is stimulated by glucocorticoid and corticotropin-releasing hormone

We identified the presence of iodothyronine deiodinase in AtT-20 mouse pituitary tumor cells that secrete corticotropin. Iodothyronine deiodinating activity in AtT-20 cells fulfills all the characteristics of type 2 iodothyronine deiodinase (D2), including the inhibition by thyroid hormones, the insensitivity to inhibition by 6-propyl-2-thiouracil, and the low Michaelis-Menten constant value for T4. Northern analysis using mouse D2 cRNA probe demonstrated the hybridization signal of approximately 7.0 kb in size in AtT-20 cells. D2 activity and D2 mRNA were stimulated by glucocorticoid in a dose-dependent manner but were not stimulated by testosterone or beta-estradiol. D2 expression was stimulated by (Bu)2cAMP, and CRH in a dose-dependent manner in the presence of dexamethasone. These results suggest the previously unrecognized role of local thyroid hormone activation by D2 in the regulation of pituitary corticotrophs.     

Differential regulation of brain and pituitary corticotropin-releasing factor receptors by corticosterone

The regulatory actions of CRF during the neuroendocrine response to stress are mediated by specific receptors within the nervous system and the anterior pituitary gland. Glucocorticoids exert negative feedback inhibition on ACTH secretion by interacting at the pituitary corticotrophs and the central nervous system. To determine whether glucocorticoids influence ACTH secretion by regulating the concentration of CRF receptor sites, binding of [125I]Tyr-oCRF to pituitary and brain membrane-rich particles was studied after glucocorticoid treatment. Corticosterone administration (0.5-150 mg/day) for 1-4 days in adult male rats caused a dose-dependent decrease in the number of CRF receptors in the anterior pituitary in parallel with the reduction in ACTH secretion. In the brain, binding studies in membrane-rich fractions or by autoradiography in slide-mounted frozen sections revealed no changes in CRF receptors in the cortex, hippocampus, amygdala, septal area, and olfactory bulb, although circulating corticosterone levels were higher than during stress. The selective down-regulation of anterior pituitary CRF receptors after corticosterone administration, without alterations in brain CRF receptors, is similar to the change in CRF receptors previously reported after adrenalectomy and indicates that receptor regulatory mechanisms in secretory cells differ from those in neural tissue. Furthermore, the decrease in pituitary CRF receptors after physiological increases in circulating glucocorticoids may contribute to the inhibitory effects of adrenal steroids on ACTH secretion.     

Demonstration of desacetyl alpha-melanocyte-stimulating hormone in fetal and adult human anterior pituitary corticotrophs

Immunocytochemistry, radioimmunoassay and high-performance liquid chromatography (HPLC) techniques have been used in combination to investigate the presence of immunoreactive (ir)-alpha-MSH in the normal human pituitary gland, and to investigate the possible origin of these cells from the fetal pars intermedia. Two separate antisera to alpha-MSH were employed in immunocytochemistry to distinguish between authentic alpha-MSH and the desacetyl form. Only desacetyl alpha-MSH was detected in the pituitary gland of fetal and adult man, in both the pars (zona) intermedia and the pars anterior. In the fetus, a large proportion of the ACTH-containing cells of the anterior lobe also contained ir-alpha-MSH, while ir-alpha-MSH containing cells were more sparse in adults. Radioimmunoassay of acid extracts of adult pituitary tissue showed alpha-MSH levels representing less than 0.05% of the ACTH content of the gland. HPLC analysis of these extracts confirmed that only the desacetyl form was present. These results suggest that alpha-MSH peptides are synthesized by anterior lobe cells of the human pituitary gland, which are not derived from the fetal pars intermedia. Possible regulatory mechanisms affecting cells which contain ir-alpha-MSH are discussed, and by comparison with the intermediate lobe of other species it is concluded that there is little evidence for a true intermediate lobe in the human pituitary gland.     

Heterogeneity between brain and pituitary corticotropin-releasing factor receptors is due to differential glycosylation

Chemical affinity cross-linking studies have identified brain and pituitary CRF receptors with similar pharmacological characteristics but different mol wts (anterior pituitary, 75,000; brain, 58,000). In order to determine whether the heterogeneous nature of CRF receptors was inherent in the protein, we examined the glycoprotein nature of both types of CRF receptors using lectin affinity chromatography and treatments with exo- and endoglycosidases. CRF receptors in both the cerebral cortex and anterior pituitary adsorbed to and specifically eluted from Concanavalin-A- and wheat germ agglutinin-immobilized lectin affinity columns, indicating that both forms of the receptor are glycoproteins containing complex and high-mannose carbohydrate moieties. Cerebral cortical CRF receptors were sensitive to both neuraminidase and alpha-mannosidase treatment while pituitary CRF receptors were only affected by neuraminidase treatment, suggesting that CRF receptors in brain and pituitary differed slightly in the nature of their glycosylation units. After treatment of cerebral cortical or anterior pituitary CRF receptors with the endoglycosidase, N-glycanase, the mol wts were markedly decreased; the mol wt of the anterior pituitary CRF receptor was decreased from 75,000 to approximately 40,000-45,000 while in a corresponding manner, the cortical receptor was decreased from 58,000 to approximately 40,000-45,000. Limited proteolysis after deglycosylation with N-glycanase using the proteinases Staphylococcus aureus V8 (S. aureus V8) or papain, generated virtually identical peptide fragments from anterior pituitary- or cerebral cortex- labeled CRF receptor proteins. In summary, these data support the hypothesis that the ligand binding subunit of the CRF receptor in both brain and pituitary resides on a polypeptide of 40,000-45,000 and appears to be identical in both tissues. Differences observed in the mobility of the two proteins were found to be due to differences in the posttranslational modification of the proteins in the two tissues.     

Subpopulations of corticotrophs in the sheep pituitary during late gestation: effects of development and placental restriction

The prepartum surge in fetal plasma cortisol is essential for the normal timing of parturition in sheep and may result from an increase in the ratio of ACTH to proopiomelanocortin (POMC) in the fetal circulation. In fetuses subjected to experimental induction of placental restriction, the prepartum surge in fetal cortisol is exaggerated, whereas pituitary POMC mRNA levels are decreased, and in vitro, unstimulated ACTH secretion is elevated in corticotrophs nonresponsive to CRH. We therefore investigated the changes in the relative proportions of cells expressing POMC, ACTH, and the CRH type 1 receptor (CRHR(1)) shortly before birth and during chronic placental insufficiency. Placental restriction (PR) was induced by removal of the majority of placental attachment sites in five ewes before mating. Pituitaries were collected from control and PR fetal sheep at 140 d (control, n = 4; PR, n = 4) and 144 d (control, n = 6; PR, n = 4). Pituitary sections were labeled with specific antisera raised against POMC, ACTH, and CRHR(1). Three major subpopulations of corticotrophs were identified that expressed POMC + ACTH + CRHR(1), ACTH + CRHR(1), or POMC only. The proportion of pituitary corticotrophs expressing POMC + ACTH + CRHR(1) decreased (P < 0.05) between 140 (control, 60 +/- 1%; PR, 66 +/- 4%) and 144 (control, 45 +/- 2%; PR, 56 +/- 6%) d. A significantly higher (P < 0.05) proportion of corticotrophs expressed POMC + ACTH + CRHR(1) in the pituitary of the PR group compared with controls. This study is the first to demonstrate subpopulations of corticotrophs in the fetal sheep pituitary that differentially express POMC, ACTH, and CRHR(1) and the separate effects of gestational age and placental restriction on these subpopulations of corticotrophs.     

Chronic administration of corticotropin-releasing factor increases pituitary corticotroph number

The effect of chronic administration of CRF on rat pituitary morphology was studied. Experimental animals received CRF (10 micrograms/day) over a period of 52 days by means of sc osmotic pumps changed at 10- to 14-day intervals. The average 0800 h plasma corticosterone levels in the treated animals were significantly greater than control values [7.52 +/- 0.99 (+/- SE) vs. 1.14 +/- 0.5 micrograms/dl; P less than 0.001]. The CRF-treated animals also had a significantly greater adrenal weight (16.44 +/- 1.38 vs. 12.24 +/- 0.85 mg; P less than 0.05) and lower thymus weight (164 +/- 12 vs. 248 +/- 27 mg; P less than 0.005). There was a marked increase in the number of ACTH-producing cells in the anterior pituitaries of the rats that received CRF (13.3 +/- 0.8% vs. 4.5 +/- 0.3% ACTH-producing cells; P less than 0.001), as determined by immunocytochemical methods. Corticotrophs of rats treated with CRF manifested a significant increase in nuclear area (24.0 +/- 0.7 vs. 21.4 +/- 0.4 micron 2; P less than 0.001) and an increased diameter of forming and storage granules (191.1 +/- 1.1 vs. 158.6 +/- 3.5 nm and 196.1 +/- 1.2 vs. 170.1 +/- 3.7 nm, respectively; P less than 0.001). There was no demonstrable increase in ACTH cell area. These data indicate that long term administration of CRF is capable of increasing the number of pituitary corticotrophs. It also supports the view that the corticotroph hyperplasia occurring after adrenalectomy, in unusual cases of ectopic CRF production, and in rare instances of Cushing's disease is a proliferative response to CRF.     

Paradoxical responsiveness of growth hormone to corticotropin-releasing factor in acromegaly

Corticotropin-releasing factor elicited an increase in serum GH in two of six patients with acromegaly. Both patients also responded paradoxically to LHRH administration and one of them also to TRH, further illustrating the dedifferentiation of the receptors of the somatotrophs in acromegaly.