Cellular Mechanisms Regulating Adrenocorticotropin Release

Abstract

Using a tumor cell line of the mouse anterior pituitary (AtT-20/D16-16), the various factors regulating ACTH release and the intracellular mechanisms mediating this response were investigated. CRF, catechola-mines and VIP stimulate ACTH release whereas glucocorticoids and SRIF block secretion. Glucocorticoids block both ACTH synthesis and release. SRIF acts through multiple mechanisms to prevent stimulated ACTH release. Cyclic AMP and Ca⁺⁺ are important second messengers in the receptor mediated release of ACTH but other mediators may also be involved. The interaction of these various CRF-like substances and inhibitors of ACTH release may result in a fine-tune regulation of corticotroph activity Such regulation may be important in the organism response to stress.     

β-Endorphin and ACTH related peptides in primary cultures of rat anterior pituitary cells: evidence for different intracellular pools

Acid extracts of rat anterior pituitary cells and cell-derived culture media were shown to contain three forms of β-endorphin immunoreactive peptides, corresponding in molecular size to the prohormone pro-opiomelanocortin (POMC), β-lipotropin and 3.5 kDa β-endorphin, and essentially two forms of adrenocorticotropin (ACTH) immunoreactivity, representing a 20 kDa intermediate fragment and 4.5 kDa ACTH. Under basal conditions the intracellular peptides contained a high proportion of the bioactive forms of β-endorphin and ACTH whereas the extracellular peptides contained a higher proportion of the inactive precursors. When the cells were incubated for 3 h in the presence of 10⁻⁸ M CRF, the levels of intracellular β-endorphin and ACTH immunoreactivity were reduced by 15–30% and there was a 4–5-fold increase in the level of the secreted peptides; furthermore, unlike the peptides released under basal conditions, the peptides secreted under the influence of CRF contained much higher proportions of 4.5 kDa ACTH and 3.5 kDa β-endorphin, reflecting the intracellular patterns of these peptides. Similar results were obtained when secretion was stimulated by 10⁻⁷ M epinephrine, which produced a 2-fold increase in peptide release. In the presence of 10⁻⁶ M dexamethasone the basal secretion of ACTH and β-endorphin related peptides, and the intracellular levels of these peptides, remained unaltered. The results point to the existence of different intracellular compartments from which peptides at different states of maturation can be released selectively.β-EndorphinACTHPituitary cell cultureProcessingCRFEpinephrine     

Interleukin-1 potentiates basal and AVP-stimulated ACTH secretion in vitro--the role of CRH pre-incubation.

The acute-phase cytokine interleukin-1 (IL-1) is known to activate the hypothalamic pituitary adrenal axis, primarily via corticotropin releasing hormone (CRH). The aim of this study was to determine whether IL-1beta could directly stimulate ACTH secretion from perifused equine anterior pituitary cells, and whether CRH pre-incubation affected corticotroph responsiveness. Isolated equine anterior pituitary cells were pre-incubated with media containing 10 nM CRH or vehicle for 20 hours before being loaded onto columns and perifused with 0.02 nM CRH and 100 nM cortisol. Columns were given a 5-minute pulse of arginine vasopressin (AVP, 10 nM), perifused for 4 hours with 0 (control) or 1 nM IL-1beta, then given a further 5-minute pulse of AVP (10nM). ACTH was measured in 5 minute fractions. In the setting of CRH pre-incubation, cells perifused with IL-1beta for 4 hours showed increased basal ACTH secretion compared to control (114 +/- 6 pM vs. 86 +/- 4 pM [means +/- S.E.M.], p < 0.001) and a significantly greater ACTH response to the final AVP pulse (240 +/- 32% vs. 96 +/- 30%, p = 0.009, expressed as % of ACTH response to the initial AVP pulse). The potentiation of AVP-stimulated ACTH release by IL-1 was not observed in cells pre-incubated with vehicle alone. In conclusion, IL-1 increases ACTH release in equine corticotroph cells pre-incubated with CRH and potentiates responsivity to AVP.     

Galanin in pituitary adenomas

Tumor galanin content was measured in extracts from human pituitary adenomas using a specific RIA method for monitoring human galanin. Twenty-two out of twenty-four tumors contained galanin with notably high levels in corticotroph adenomas, varying levels in clinically inactive tumors, and low levels in GH secreting adenomas. Tumor galanin and ACTH contents were closely correlated in all tumors. In four young patients with microadenomas and highly active Mb Cushing tumor galanin was inversely related to tumor volume. The molecular form of tumor galanin, studied with reverse-phase HPLC, was homogeneous with the majority of tumor galanin coeluting with standard human galanin. In the tumors analysed with in situ hybridization there was a good correlation between galanin peptide levels and galanin mRNA expression. In some tumors galanin mRNA and POMC levels coexisted, in others they were essentially in different cell populations. Levels of plasma galanin-LI were not related to tumor galanin concentration, and galanin levels were in the same range in sinus petrosus close to the pituitary venous drainage as in peripheral blood. Corticotrophin releasing hormone injections in two patients caused ACTH, but no detectable galanin release into sinus petrosus. Our results demonstrate that corticotroph, but not GH adenomas, express high levels of galanin, in addition to ACTH, and that in some tumors both polypeptides are synthesised in the same cell population. However, galanin levels in plasma were not influenced by the tumor galanin content.     

Modulation of in vitro release of beta-endorphin from the separate lobes of the rat pituitary.

The rate of in vitro release of β-endorphin immunoreactivity from the anterior lobe of rat pituitary increased in response to hypothalamic extract and lys-vasopressin. Lys-vasopressin, at a low concentration, initiated a pronounced (5–6 fold) dose-dependent, parallel increase in the release of β-endorphin and ACTH from the anterior lobe. Corticosterone (5·10^?7 M) did not influence basal but could suppress such stimulated release. These stimulants did not, however, change the rate of release from the intermediate/posterior lobe.Chromatography of incubation media showed that β-endorphin and β-lipotropin were released in parallel from the anterior lobe but only β-endorphin from intermediate/posterior lobe tissue.These findings suggest that the β-endorphin pools in anterior and intermediate lobes differ both in their mechanism of release and in the regulation of this process.     

Regulation of galanin secretion from pituitary cells in vitro by estradiol and GHRH

The effects of estradiol and growth hormone-releasing hormone (GHRH) on galanin release from anterior pituitary cells were examined in vitro. 17-β-Estradiol (0.001–10 nM) increased galanin secretion from anterior pituitary cells in a concentration-dependent manner. Estradiol (10 nM) increased galanin release 300 and 600% from pituitary cells of ovariectomized and male rats, respectively. Immunocytochemical studies demonstrated that estradiol (10 nM) increased the number of galanin-containing cells twofold after 4 days in culture. Growth hormone-releasing hormone (1 and 10 nM) increased and SRIF (1 and 10 nM) decreased galanin release from pituitary cells of ovariectomized and male rats. We conclude that estradiol increases galanin release by a direct effect on pituitary cells, in part by increasing the number of pituitary cells synthesizing galanin. In addition, GHRH stimulates galanin release when estradiol levels are low.     

Perifusion model system to culture bovine hypothalamic slices in series with dispersed anterior pituitary cells

Summary Dispersed bovine anterior pituitary cells were incubated either in static or perifusion cultures to assess basal growth hormone release as well as stimulatory and inhibitory effects of growth hormone-releasing hormone and somatostatin, respectively, on growth hormone release. Total concentrations of growth hormones over a 12-hour incubation period were fivefold greater in perifused than in static cultures (2034 ± 160 vs. 387 ± 33 ng/12 h). A dose-dependent increase in growth hormone secretion in response to challenge with growth hormone-releasing hormone (10⁻¹² to 10⁻⁸ M) for 1 h was observed in both static and perifusion cultures; however, perifused cells were more responsive to the same concentration of neuropeptide than those in static culture. Concentrations of somatostatin (10⁻¹² to 10⁻⁸ M) for 1 h did not inhibit basal growth hormone secretion in either static or perifusion cultures. To establish model, slices of the hypothalamus, immediately adjacent to the sagittal midline, were perifused in series with anterior pituitary cells, and media effluent was assayed for growth hormone concentrations. Release of growth hormone was pulsatile and seemed to mimic the episodic pattern of bovine secretion. Hypothalamic slices were placed in one chamber of the perifusion system, and basal secretion of growth hormone-releasing hormone and somatostatin was pulsatile in media effluent. Tissue viability of hypothalamic slices and anterior pituitary cells was evaluated by KCl depolarization. Tissues were viable for at least 120 h. Thus, this hypothalamo-pituitary dual chamber perifusion system is a valid in vitro model to study regulation of growth hormone secretion.     

Cholecystokinin octapeptide releases growth hormone from the pituitary in vitro

Cholecystokinin-octapeptide (CCK-8)(10^?6 to 10^?8M) produced a marked increase in growth hormone (GH) release from incubated rat anterior pituitary quarters and from cultured GH₃ pituitary tumor cells. Although several CCK-8 analogues also caused GH release, bombesin, secretin and pancreatic polypeptide had no effect on GH secretion $\text{in vitro}$. In the GH₃ cell line, CCK-8 (10^?7M) reversed the inhibitory effect of somatostatin (10^?5M) on GH release. As CCK immunoreactivity has been demonstrated to be present in the hypothalamus, these results suggest that CCK-8 may be a physiologically important growth hormone releasing factor.     

Primary culture of normal pituitary cells of carp (cyprinus carpio) for the study of gonadotropin release

Summary A method for preparing enzymaticlaly dispersed pituitary cell cultures of carp (Cyprinus carpio) is described. The cultures have been used to assay a synthetic analog of gonadotropin releasing hormone (GnRH) and to determine the specificity of steroids able to affect gonadotropin (GtH) release in vitro. Time course secretion studies indicated that by 48 h incubation, in the presence of 500 pM GnRH, cumulative secretion of gonadotropin (719 ng±90/2.5×10⁵ cells) had exceeded that of controls (446 ng±106/2.5×10⁵ cells). Estradiol-17β, progesterone, testosterone, and 11-ketotestosterone showed different inhibitory effects on pituitary basal GtH release. Based on the results, it was concluded that carp pituitary cell cultures can be applied to investigations of several aspects of the hypothalamo-hypophysial-gonadal system. This investigation was supported by the Deutsche Forschungsgemeinschaft, Bonn, FRG.     

Effect of different concentrations of serotonin, histamine and insulin on the hormone (serotonin and ACTH) production of Tetrahymena in nutrient-free physiological milieu

Cell populations of Tetrahymena pyriformisGL were kept in nutrient-free (Losina) milieu and treated with different (10⁻⁶–10⁻²¹ M) concentrations of serotonin, histamine or insulin for 30 min. Following that the hormone (serotonin and adrenocorticotropin (ACTH) content of the cells were measured by immunocytochemical flow cytometric method. Serotonin reduced histamine when applied in 10⁻¹² and 10⁻¹⁵ M concentrations, while elevated ACTH levels when applied in 10⁻⁶, 10⁻⁹ and 10⁻²¹ M concentrations. Histamine reduced serotonin concentration at 10⁻⁹–10⁻²¹ M concentrations and increased ACTH in 10⁻⁶ M. Insulin elevated both hormones’ content in each concentration except at 10⁻¹² M. The results demonstrate that (1) in nutrient-free conditions the hormonal effects differ from that of nutrient-rich (tryptone + yeast) condition; (2) there is an optimal hormone concentration, which causes the strongest effect and this is different for each hormones; (3) the hormone receptors of Tetrahymena are very sensitive; as they react to zeptomolar concentrations. Such small concentration is even more effective than higher ones. Since hormones must become highly diluted in the natural environment of Tetrahymena, it seems that such low concentrations are the actual physiological concentrations.     

Use of semipermeable polyurethane hollow fibers for pituitary organ culture

Summary A new model for organ culture of endocrine tissue is described. Rat anterior pituitary fragments were cultured for 4 wk within semipermeable polyurethane isocyanate hollow fibers. Growth hormone and prolactin, two of the anterior pituitary hormones, were released into the medium during the entire culture period. Electron microscopy of the pituitary fragments after 2 wk in in culture showed a rim of viable tissue in all specimens examined. Individual cells, from this outer rim, exhibited excellent organelle preservation and numerous secretory granules. Experiments involving potassium depolarization and 10⁻⁶ M dopamine provided evidence for the normal responsiveness of the cultured pituitary tissue to both stimulatory and inhibitory factors. These studies illustrate the potential utility of the described organ culture system for further investigations of endocrine physiology.     

The effect of somatostatin and of prolyl-leucyl-glycinamide (MIF) on ACTH release in dispersed pituitary cells.

The hypothalamic releasing and release-inhibiting peptides have multiple effects on more than one pituitary hormone. In this study the action of the two hypothalamic inhibiting factors, somatostatin (GH-IH) and MSH release-inhibiting factor, prolyl-leucyl-glycinamide (MIF), on ACTH release were studied. Increasing concentrations of GH-IH and MIF were added to 1 ml of a suspension of dispersed anterior pituitary cells from male rats. Both GH-IH and MIF (10^?5 to 10^?11 M) were without effect on basal ACTH secretion of normal and of adrenalectomized rats. However, both peptides, within certain concentration ranges, inhibited the ACTH release stimulated by rat hypothalamic extracts or by arginine vasopressin. The most effective concentrations were 35 nM MIF or 6 nM GH-IH. Beyond these concentrations no further suppression was observed. Our results indicate that somatostatin and MIF can inhibit ACTH release, but only in a state of steroid deprivation and within a limited dose range.     

In vitro stimulation of prolactin release by vasoactive intestinal peptide

The effect of vasoactive intestinal peptide (VIP) on anterior pituitary hormone release was examined in a variety of in vitro preparations. Synthetic VIP was capable of stimulating increased prolactin (PRL) release from male rat hemipituitaries in doses as low as 10⁻⁹ M only when the enzyme inhibitor bacitracin was present in the incubation medium. Natural porcine VIP was similarly capable of stimulating PRL release, but only at higher doses (10⁻⁶ M). Additionally, synthetic VIP was capable of stimulating PRL release from dispersed anterior pituitary cells harvested from adult male and lactating female rats and from an enriched population of lactotrophs obtained by unit gravity sedimentation of similar dispersed cells from infantile female rats. No effect of VIP on luteinizing hormone, growth hormone or thyroid stimulating hormone release was seen. These findings taken in concert with the presence of VIP in the hypothalamus, pituitary and hypophyseal portal plasma of the rat suggest a physiological role for VIP in the control of PRL secretion.     

Adenosine regulates the release of adrenocorticotropic hormone (ACTH) from cultured anterior pituitary cells

We have recently shown the presence of adenosine receptors coupled to adenylate cyclase in anterior pituitary and in the present studies we have investigated the effects of adenosine on ACTH release. The R-site specific analogs of adenosine such as N-Ethylcarboxamide adenosine (NECA), L-N⁶-phenylisopropyl adenosine (PIA), 2-chloro-adenosine (2-Cl-Ado) all stimulated ACTH release in a dose-dependent manner. NECA was the most potent analog and stimulated ACTH release by about 170% with an apparent Ka of 0.1 µM, whereas PIA and 2-Cl-Ado were less potent and stimulated the release by about 110% and 125% with an apparent Ka of 0.2 and 0.4 µ-M respectively. The stimulation of ACTH release by NECA was inhibited by 3-isobutyl-1-methylxanthine (IBMX). On the other hand, adenosine deaminase (ADA) treatment of the cells also stimulated ACTH release as well as adenylate cyclase activity by about 2-fold, suggesting that endogenous adenosine plays an inhibitory role in the release of ACTH. Other agents, such as corticotropin-releasing factor (CRF), vasoactive intestinal peptide (VIP) and forskolin (FSK) also stimulated ACTH release from these cells. In addition, the stimulation by an optimal concentration of NECA was almost additive with maximal stimulation caused by VIP and FSK. These data suggest that adenosine modulates ACTH release from anterior pituitary through its interaction with adenosine receptors coupled to adenylate cyclase.Abbreviations NECA N-Ethylcarboxamideadenosine - PIA L-N⁶-Phenylisopropyladenosine - 2-Cl-Ado 2-chloroadenosine - FSK Forskolin - VIP Vasoactive Intestinal Peptide - CRF Corticotropin Releasing Factor - ADA Adenosine Deaminase - IBMX 3-Isobutyl-1-methylxanthine     

Effects of corticotrophin-releasing hormone on corticotrophs in anterior pituitary gland allografts in hypophysectomized,orchidectomized hamsters

Summary We investigated the effects of corticotrophin-releasing hormone (CRH) on the percentage of anterior pituitary gland (APG) cells which are corticotrophs as well as the size and shape of corticotrophs. Pituitary glands were removed from 7-week-old male hamsters and placed beneath the renal capsules of hamsters that had been hypophysectomized and orchidectomized 3 weeks previously. Beginning 6 days after each host had received a single allograft, each was injected subcutaneously twice daily with 4 g CRH or vehicle for 16 days. Six hosts in each group were decapitated 16 h after the last injection. Sections of anterior pituitary tissue were stained for ACTH and with hematoxylin. The percentage of corticotrophs among APG cells was greater in allografts exposed to exogenous CRH (20%) than in allografts exposed to vehicle (15%). Exposure to exogenous CRH increased the cross-sectional area of corticotroph cells in allografts to values greater than those measured for corticotrophs in allografts exposed to vehicle, without altering the shape of cells. Results of subsequent studies suggested that hamsters with allografts injected with vehicle do not release ACTH and that exogenous CRH causes an abrupt release of ACTH from allografts. These results indicate that CRH releases ACTH from ectopic corticotrophs and that administration of CRH can increase corticotroph size and the percentage of APG cells that are corticotrophs.     

Effect of sodium valproate on the secretion of proopiomelanocortin derived peptides from cultured rat anterior pituitary cells

We examined effects of sodium valproate, a gamma amino butyric acid (GABA)-transaminase inhibitor, on the secretion of immunoreactive (IR)-ACTH and IR-beta-endorphin/LPH from cultured rat anterior pituitary cells to determine whether sodium valproate has a direct action on the secretion of ACTH and its related peptides from the cultured rat anterior pituitary gland. During the 3 h incubation, the basal secretion of IR-ACTH and IR-beta-endorphin/LPH decreased to 50.8% and 58.3%, respectively, of the control concentration after adding 10(-7) M sodium valproate into the incubation media and to 67.7% and 69.3%, respectively, of the control levels with 10(-8) M sodium valproate. However, sodium valproate at a concentration of 10(-6) M or 10(-9) M did not affect the basal concentration of IR-ACTH and IR-beta-endorphin/LPH. Sodium valproate at a concentration of 10(-7) M significantly attenuated the stimulated release of IR-ACTH and IR-beta-endorphin/LPH by 10(-9) or 10(-10) M of ovine corticotrophin releasing factor. These results indicate that sodium valproate could directly effect rat anterior pituitary cells to suppress both basal and stimulated release of proopiomelanocortin derived peptides and this supports the hypothesis that sodium valproate has a direct effect at the pituitary corticotroph in reducing plasma ACTH.     

Corticotropin releasing factor stimulates adrenocorticotropin and beta-endorphin release from AtT-20 mouse pituitary tumor cells

Corticotropin releasing factor (CRF) was tested for its ability to stimulate ACTH and β-endorphin secretion from clonal $\text{AtT-20}\text{D16-16}$ mouse pituitary tumor cells. Release of both hormones was stimulated 4 to 5-fold over the basal release at nanomolar concentrations of synthetic CRF. CRF analogues stimulated $\text{ACTH}\text{β-endorphin}$ release with the same order of potency in the tumor cells as in primary cultures of anterior pituitary cells. A 90-min exposure to CRF elicited a 29–35% increase in total ACTH and β-endorphin immunoreactivity in tumor cell cultures. Dexamethasone markedly inhibited CRF-stimulated and basal ACTH and β-endorphin release. $\text{AtT-20}\text{D16-16}$ cells may serve as a good model system for studying the biochemistry of CRF receptor-mediated events involved in $\text{ACTH}\text{β-endorphin}$ release and synthesis.     

Endogenous ouabain-like factor (OLF) secretion is modulated by nicotinic mechanisms in rat adrenocortical cells

This study tested the hypothesis that rat adrenocortical secretion of endogenous ouabain-like factor (OLF) is regulated by nicotinic mechanisms. OLF secreted by dispersed cell suspensions of zona glomerulosa (ZG) and fasciculata/reticularis (ZFR) cells was found to co-elute with authentic ouabain by reverse phase HPLC; OLF concentrations in cell supernatants were measured by radioimmunoassay. Nicotine (10⁻⁶ − 10⁻³ M) stimulated significant OLF secretion in rat adrenocortical cells. Acetylcholine (10⁻⁷ − 10⁻⁴ M) and eserine (10⁻⁷ − 10⁻³ M) stimulated OLF secretion in ZG cells at lower concentrations and stimulated at higher concentrations. Acetylcholine had no effect on ZFR secretion of OLF, but eserine stimulated OLF secretion. ACTH (10⁻⁸ M) strongly potentiated the OLF stimulatory effect of nicotine in ZG cells; however significant interactions between nicotine and ACTH or angiotensin II on OLF secretion in ZFR cells were not apparent. The ganglionic blockers hexamethonium and mecamylamine further potentiated the effect of nicotine, implicating nicotinic acetylcholine receptors (nAChRs) in regulation of OLF secretion. The α7-receptor antagonist methyllycaconitine (MLA) dose-dependently inhibited the effect of nicotine in the ZG cells, and in ZFR cells MLA potentiated nicotine-induced OLF secretion. These data suggest that nicotinic regulation may underlie OLF secretion by rat adrenocortical cells, and strongly suggest presence of functional nicotinic acetylcholine receptors on these cells.     

Culture requirements for optimal expression of 1α,25-dihydroxyvitamin D3-enhanced thyrotropin secretion

Summary An effect of the hormone, 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] on hormone secretion by normal rat pituitary cells was investigated in vitro. Based on previous findings using GH₄C₁ cells, dispersed anterior pituitary cell cultures were prepared and maintained in serum-free conditions for up to 6 d. Under these circumstances, there was no effect of 1,25(OH)₂D₃ to alter medium or cell-associated levels of thyrotropin (TSH), prolactin (PRL), or growth hormone (GH). Cultures maintained under these conditions had lower medium and cell-associated hormone levels and lesser responses to agonists than cultures maintained in serum-supplemented medium. In the presence of 10% charcoal-treated fetal bovine serum, treatment with 10⁻⁸ M 1,25(OH)₂D₃ for 24 h selectively increased TRH (10⁻¹⁰ to 10⁻⁷ M)-induced TSH secretion (P<0.001), with maximal enhancement observed at 10⁻⁹ M TSH-releasing hormone (TRH). Enhancement of TSH secretion by 1,25(OH)₂D₃ was detected after 15 min exposure to TRH. There was no effect on agonist-induced PRL or GH secretion or on cell-associated hormone levels. The effect was evident after 24 h treatment with 1,25(OH)₂D₃, and decreased thereafter. Several other steroid hormones had no effect on 10⁻⁹ M TRH-induced TSH secretion. These data contrast with the effect of 1,25(OH)₂D₃ in GH cells. They suggest that 1,25(OH)₂D₃ may act selectively in the normal pituitary to modulate TSH secretion.