POMC mRNA Expression Research Articles

Interaction between glucocorticoids and corticotropin releasing hormone (CRH) in the regulation of the pituitary CRH receptor in vivo in the rat.

Acute stress causes biphasic changes in corticotropin releasing hormone (CRH) receptor mRNA expression with an early decrease followed by an increase. However, in the absence of glucocorticoids in adrenalectomized rats, stress results in prolonged CRH receptor (CRH-R) mRNA loss, suggesting that interactions between glucocorticoids and hypothalamic factors are critical for regulation of CRH receptor mRNA. To address this question, CRH binding, type-1 CRH-R mRNA, POMC mRNA and POMC hnRNA expression were measured by binding autoradiography and in situ hybridization in pituitaries from intact and adrenalectomized rats. CRH-R mRNA decreased by 59% 5 h after injection of corticosterone (10 mg s.c.) and returned to basal levels by 18 h, a time when plasma corticosterone concentrations were still elevated, and CRH binding and POMC hnRNA were significantly reduced. Elevations in plasma corticosterone in the range of acute stress by injection of 2 mg s.c. caused CRH-R mRNA expression to return to near basal values by 6 h, after a 52% and 39% decrease at 2 h and 4 h. More transient changes were seen after a single injection of CRH (1 microg), with a 44% decrease in CRH-R mRNA and a 175% increase in POMC hnRNA by 2 h, returning to basal values by 4 h. The transient effect of CRH was not due to clearance of CRH from the circulation or receptor desensitization since CRH receptor mRNA expression also recovered after injection of a higher dose (10 microg) or repeated injections of CRH which caused sustained increases in plasma CRH and pituitary POMC hnRNA levels. CRH injection in adrenalectomized rats decreased CRH-R mRNA for up to 6 h, suggesting that glucocorticoids are permissive for the recovery of CRH-R mRNA. Supporting this hypothesis, simultaneous injection of corticosterone and CRH restored CRH-R mRNA expression by 4 h, and increased CRH binding 4 h and 6 h after injection. The data show that interaction between CRH and glucocorticoids counteracts individual inhibitory effects of these regulators alone, and that such effects are likely to contribute to the regulatory pattern of pituitary CRH receptors during acute stress.

Inhibition of fetal adrenal adrenocorticotropin receptor messenger ribonucleic acid expression by betamethasone administration to the baboon fetus in late gestation.

Throughout the majority of intrauterine development, the primate fetal adrenal gland is comprised primarily of fetal zone cells and only late in gestation do definitive zone cells, which express the enzyme delta5-3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) emerge to produce cortisol. The present study was designed to determine whether the induction of definitive zone ACTH receptor messenger RNA (mRNA) levels and components of the steroidogenic pathway known to be expressed specifically in the definitive zone, e.g. the 3beta-HSD enzyme, are dependent upon fetal pituitary ACTH. Fetal pituitaries and adrenal glands were obtained on day 165 (term = day 184) from untreated controls (n = 7) and from baboons in which betamethasone was administered im to the fetus (0.6 mg/100 microl; n = 4) or to the fetus (0.6 mg) and mother (6 mg/ml; n = 4) every other day between days 150 and 164 of gestation. Although fetal pituitary weight was not altered by betamethasone, POMC mRNA levels determined by in situ hybridization were lower (P < 0.05) in betamethasone-treated (0.34 +/- 0.07 arbitrary densitometric units) than in untreated controls (0.63 +/- 0.04). Associated with this decline in pituitary POMC, levels of the major 3.4-kb mRNA transcript for the ACTH receptor expressed as a ratio of beta-actin were approximately 80% lower (P < 0.05) in fetal adrenals of betamethasone-treated baboons (0.12 +/- 0.02) than in untreated controls (0.84 +/- 0.05). In situ hybridization indicated that ACTH receptor mRNA expression in the definitive zone exceeded that in the fetal zone and was reduced by betamethasone. Associated with the decrease in ACTH receptor expression, fetal adrenal weight was suppressed (P < 0.05) by 50% and reflected a marked reduction (P < 0.05) in the size of the cells of the definitive and fetal zones. Betamethasone treatment also induced a decrease (P < 0.05) in the width (microm) of the definitive zone (183 +/- 14 vs. 128 +/- 7; determined by immunohistochemical expression of 3beta-HSD), as well as the levels of the mRNA and protein for 3beta-HSD. Levels of the mRNA for the LDL-receptor and the enzymes 17alpha-hydroxylase-C(17,20) lyase and P450 cholesterol side chain cleavage were also suppressed in adrenals of betamethasone-treated baboons. These findings indicate that treatment of the baboon fetus with betamethasone in late gestation suppressed fetal pituitary POMC mRNA expression and ACTH receptor mRNA levels in the fetal adrenal gland, as well as the hypertrophy and ACTH receptor mRNA and 3beta-HSD mRNA/protein levels in the cells comprising the newly emerging definitive zone. We conclude that ACTH is necessary for the up-regulation of the mRNAs for the ACTH receptor and steroidogenic enzymes in the definitive zone of the primate fetal adrenal gland in late gestation.

Inhibition of Fetal Adrenal Adrenocorticotropin Receptor Messenger Ribonucleic Acid Expression by Betamethasone Administration to the Baboon Fetus in Late Gestation

Throughout the majority of intrauterine development, the primate fetal adrenal gland is comprised primarily of fetal zone cells and only late in gestation do definitive zone cells, which express the enzymeΔ 5-3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD) emerge to produce cortisol. The present study was designed to determine whether the induction of definitive zone ACTH receptor messenger RNA (mRNA) levels and components of the steroidogenic pathway known to be expressed specifically in the definitive zone, e.g. the 3β-HSD enzyme, are dependent upon fetal pituitary ACTH. Fetal pituitaries and adrenal glands were obtained on day 165 (term = day 184) from untreated controls (n = 7) and from baboons in which betamethasone was administered im to the fetus (0.6 mg/100 μl; n= 4) or to the fetus (0.6 mg) and mother (6 mg/ml; n = 4) every other day between days 150 and 164 of gestation. Although fetal pituitary weight was not altered by betamethasone, POMC mRNA levels determined by in situ hybridization were lower (P < 0.05) in betamethasone-treated (0.34 ± 0.07 arbitrary densitometric units) than in untreated controls (0.63 ± 0.04). Associated with this decline in pituitary POMC, levels of the major 3.4-kb mRNA transcript for the ACTH receptor expressed as a ratio of β-actin were approximately 80% lower (P < 0.05) in fetal adrenals of betamethasone-treated baboons (0.12 ± 0.02) than in untreated controls (0.84 ± 0.05). In situ hybridization indicated that ACTH receptor mRNA expression in the definitive zone exceeded that in the fetal zone and was reduced by betamethasone. Associated with the decrease in ACTH receptor expression, fetal adrenal weight was suppressed (P < 0.05) by 50% and reflected a marked reduction (P < 0.05) in the size of the cells of the definitive and fetal zones. Betamethasone treatment also induced a decrease (P < 0.05) in the width (μm) of the definitive zone (183 ± 14 vs. 128 ± 7; determined by immunohistochemical expression of 3β-HSD), as well as the levels of the mRNA and protein for 3β-HSD. Levels of the mRNA for the LDL-receptor and the enzymes 17α-hydroxylase-C17,20 lyase and P450 cholesterol side chain cleavage were also suppressed in adrenals of betamethasone-treated baboons. These findings indicate that treatment of the baboon fetus with betamethasone in late gestation suppressed fetal pituitary POMC mRNA expression and ACTH receptor mRNA levels in the fetal adrenal gland, as well as the hypertrophy and ACTH receptor mRNA and 3β-HSD mRNA/protein levels in the cells comprising the newly emerging definitive zone. We conclude that ACTH is necessary for the up-regulation of the mRNAs for the ACTH receptor and steroidogenic enzymes in the definitive zone of the primate fetal adrenal gland in late gestation.

Brain substrates activated by electroacupuncture of different frequencies (I): comparative study on the expression of oncogene c- fos and genes coding for three opioid peptides

Low and high frequency electroacupuncture (EA)-produced analgesia have been shown to be mediated by different brain substrates and different opioid peptides. In this study, Fos-like immunoreactivity (FLI) and in situ hybridization of the three opioid mRNAs were used to examine the effect of low (2 Hz) and high (100 Hz) frequency EA on neuronal activities and the expression of opioid genes. 2 Hz and 100 Hz EA induced a markedly different spatial patterns of Fos expression in the rat brain, suggesting there are distinct neuronal pathways underlying EA of different frequencies. Likewise, 2 Hz and 100 Hz EA exert differential effects on opioid gene expression: while 2 Hz EA induced a more extensive and intensive preproenkephalin (PPE) mRNA expression than 100 Hz EA, it had no effect on preprodynorphin (PPD) mRNA expression which was significantly increased by 100 Hz EA stimulation. In contrast, EA of both frequencies did not affect POMC mRNA expression.

Glutamate enhances the adenylyl cyclase-cAMP system-induced beta-endorphin secretion and POMC mRNA expression in rat hypothalamic neurons in culture: NMDA receptor-mediated modulation

l-Glutamate, a major excitatory amino acid of the central nervous system, plays important roles as neurotransmitter and neuromodulator in the brain. Increasing evidence suggests that glutamate may also involve in the regulation of the neuroendocrine system at the hypothalamus. Employing long term monolayer hypothalamic cell cultures prepared from neonatal rats, we reported here that whereas glutamate significantly enhanced forskolin-, or N 6,2′-O-dibutyrrladenosine-3′5′-cyclic monophosphate [(Bu) 2cAMP]-stimulated immunoreactive (ir)-β EP release form cultures treated daily for 4 consecutive days, the excitatory amino acid alone produced little effect. This potentiation of glutamate was time-related and dose-dependent with an E max value of the amino acid being approximately 50 μM; at this concentration glutamate augmented ir-β EP secretion about 1.8 times ( P<0.05) that induced by 2 μM forskolin alone. Similar effects were also observed for POMC and mRNA levels in cultures subjected to 6 h of the above treatment regime. This potentiating effect of glutamate appears to be mediated specifically through NMDA receptor as it can be mimicked by NMDA but not by kainic acid or quisqualic acid, and blocked by the NMDA receptor antagonist 2-amino-5-phosphonovalerate (APV), but not by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA glutamate receptor antagonist. Interestingly, glutamate was found not to enhance high doses of forskolin (10 μM) or (Bu) 2 cAMP (100 μM) stimulated β EP release and POMC mRNA levels in hypothalamic cell cultures. These novel findings suggests that (i) hypothalamic β EP neurons are subjected to direct glutamatergic modulation, and (ii) the modulating effect of glutamate is mediated, at least in part, through the adenylyl cyclases-cAMP system. Our novel findings have provided a direct evidence supporting the assertion that cortical glutamatergical system may directly involve in the functional modulation of hypothalamic β EP producing neurons.

Progesterone, but not estrogen, modulates the cAMP system mediated ir-β-endorphin secretion and POMC mRNA expression from rat hypothalamic cells in culture

It is now evident that hypothalamic β-endorphin (βEP) modulates reproductive physiology at the central level by inhibiting the function of neurons producing gonadotropin-releasing hormone (GnRH). Increasing evidence suggests that gonadal steroids, which play an important role in the long-loop negative feedback on the hypothalamus-pituitary-gonadal axis, may exert its indirect inhibitory action through modulating the production and release of hypothalamic βEP. However, it remains unclear whether progesterone or estrogen alone or their combination is important to exert this effect. Employing long-term monolayer neonatal hypothalamic cell cultures, we reported here that whereas progesterone significantly enhanced forskolin-, N 6,2′- O-dibutyryladenosine-3′5′-cyclic monophosphate [(Bu) 2cAMP]-, 3-isobutyl-1-methylxanthine (IBMX)- or cholera toxin-stimulated immunoreactive (ir)-βEP release from cultures treated daily for 4 consecutive days, the steroid alone produced little effect. This potentiation of progesterone was time-related and dose-dependent with an EC 50 value of the steroid being approximately 25 nM; at this concentration the steroid increased ir-βEP secretion about 1.6 times ( P < 0.05) that induced by 5 μM forskolin alone. Similar effects were also observed for POMC mRNA levels in cultures subjected to 6 h of the above treatment regime. This potentiating effect appears specific as it can be mimicked by progestin, a progesterone receptor agonist and blocked by the progesterone receptor antagonist RU38486, but not RU28318, a mineralocorticoid receptor antagonist. Furthermore, β-estradiol alone failed to exert a significant effect on basal, forskolin-induced or on forskolin and progesterone co-stimulated βEP release or POMC mRNA levels in hypothalamic cell cultures. Hence, our present results substantiate findings from in vivo studies suggesting that progesterone may play an important role in augmenting hypothalamic βEP production and release to inhibit GnRH secretion. Our present findings are thus consistent with the notion that in the central nervous system, progesterone, but not estrogen, appears to be the major ovarian steroid that exerts an indirect long-loop negative feedback on the hypothalamus-pituitary-gonadal axis of the female rat.

Histaminergic activation of the hypothalamic-pituitary-adrenal axis.

Centrally administered histamine (HA) stimulates the secretion of adenohypophysial POMC-derived peptides, which subsequently cause release of corticosterone. The effect of HA on POMC-derived peptide release is indirect, and it is possible that hypothalamic neurons containing corticotropin-releasing hormone (CRH), arginine vasopressin (AVP), or oxytocin (OT) are involved in the mediation of this response. We studied the effect of HA on: 1) expression of CRH, AVP, and OT messenger RNA (mRNA) at the hypothalamic level; 2) expression of c-fos and POMC mRNA at the pituitary level; and 3) peripheral plasma levels of AVP, OT, ACTH, beta-endorphin (beta-END), and corticosterone. HA (270 nmol) infused intracerebroventricularly increased the expression of CRH, AVP, and OT mRNA in the paraventricular nucleus as well as that of OT mRNA in the supraoptic nucleus of the hypothalamus. At the pituitary level the expression of mRNA for c-fos and POMC increased in the anterior but not in the intermediate lobe in response to HA. Plasma levels of AVP, OT, ACTH, beta-END, and corticosterone all increased in response to central HA administration. Circulating levels of AVP and OT peaked after 5 min, ACTH and beta-END after 15 min, whereas corticosterone levels were highest after 30 min. In concert with our earlier discoveries, the present data support the hypothesis that HA-induced secretion of ACTH and beta-END is mediated via central activation of hypothalamic neuroendocrine neurons containing CRH, AVP, and/or OT.

Histaminergic activation of the hypothalamic-pituitary-adrenal axis

Centrally administered histamine (HA) stimulates the secretion of adenohypophysial POMC-derived peptides, which subsequently cause release of corticosterone. The effect of HA on POMC-derived peptide release is indirect, and it is possible that hypothalamic neurons containing corticotropin-releasing hormone (CRH), arginine vasopressin (AVP), or oxytocin (OT) are involved in the mediation of this response. We studied the effect of HA on: 1) expression of CRH, AVP, and OT messenger RNA (mRNA) at the hypothalamic level; 2) expression of c-fos and POMC mRNA at the pituitary level; and 3) peripheral plasma levels of AVP, OT, ACTH, beta-endorphin (beta-END), and corticosterone. HA (270 nmol) infused intracerebroventricularly increased the expression of CRH, AVP, and OT mRNA in the paraventricular nucleus as well as that of OT mRNA in the supraoptic nucleus of the hypothalamus. At the pituitary level the expression of mRNA for c-fos and POMC increased in the anterior but not in the intermediate lobe in response to HA. Plasma levels of AVP, OT, ACTH, beta-END, and corticosterone all increased in response to central HA administration. Circulating levels of AVP and OT peaked after 5 min, ACTH and beta-END after 15 min, whereas corticosterone levels were highest after 30 min. In concert with our earlier discoveries, the present data support the hypothesis that HA-induced secretion of ACTH and beta-END is mediated via central activation of hypothalamic neuroendocrine neurons containing CRH, AVP, and/or OT.

Effects of streptozotocin-induced diabetes on lymphocyte POMC and growth hormone gene expression in the rat

Diabetes in the rat is associated with a change in the profiles of several neuroendocrine hormones resulting in poor growth and decreased immune function. Since lymphocytes can also serve as a source of neuroendocrine hormones, we have examined whether the change in hormone profiles are accompanied by an impairment of lymphocyte GH and POMC gene expression in the immune system. Diabetes was induced by the administration of streptozotocin (STZ; 10 mg/100 g body weight) and 3 days later GH and ACTH protein and mRNA were determined. The results show a modest diminution of GH RNA in the spleen of diabetic animals whereas the expression of POMC mRNA and ACTH by the thymus was enhanced. The expression of POMC in the spleen appeared unaltered while the increase of POMC RNA in the thymus was evident after the first day of STZ treatment. STZ had no direct effect on GH or POMC expression in the spleen or thymus cells in vitro. Insulin does not appear to be involved in the expression of lymphocyte GH or POMC. The administration of insulin to the diabetic animals had no significant effect on the expression of GH or POMC by the immune cells. In addition, lymphocytes do not appear to serve as a source of insulin or are the expression of genes for lymphocyte GH or ACTH altered by insulin in vitro. Taken together, the findings are the first to report on the expression of neuroendocrine genes in lymphocytes during diabetes. The mechanism for the inhibition of GH and stimulation of POMC expression by lymphocytes in diabetic animals is unknown, but it is tempting to speculate an important role in the development of the autoimmunity that characterizes this complex disease.

Anterior pituitary proopiomelanocortin expression is decreased in hypertensive rat strains.

Studies comparing neuroendocrine differences between the spontaneously hypertensive rat (SHR) and the normotensive Wistar-Kyoto (WKY) strains have suggested altered anterior pituitary corticotrope expression of POMC associated with the development of hypertension in SHR animals. One major difficulty in comparing the SHR and WKY strains is that the two strains exhibit genetic differences unrelated to blood pressure status, because inbred in the SHR genome is a profile of behavioral characteristics different from those in the WKY, including hyperactivity in a novel environment and hyperreactivity in responding to stress. The present studies examine two new inbred rat strains, the WKHT and WKHA, which independently express the hypertension and behavioral traits, respectively. Together with the SHR and WKY, these genetically related, homozygous strains permit a more definitive means of examining the neuroendocrine correlates of either hypertension or behavior. The adult (5-month-old) male anterior pituitary gland content of the POMC peptides beta-endorphin and ACTH was decreased approximately 50% in the SHR and WKHT strains compared to that in the WKY strain, whereas hormone levels in the WKHA strain were not significantly different from those in the WKY strain. Reduced POMC peptide levels were, therefore, specifically associated with the hypertensive trait. Hormone content in prehypertensive weanling (5- to 7-week-old) SHR and WKHT animals was also reduced approximately 35% compared to that in WKY animals. Northern blot analysis identified a 45% decrease in POMC mRNA expression in the hypertensive SHR and WKHT strains, which paralleled the changes in tissue hormone content. Using both immunocytochemistry and in situ hybridization histochemistry, the number of labeled cells per unit area of tissues section was reduced approximately 45% in anterior pituitary tissues from SHR and WKHT rats compared to that in WKY tissues. The levels of POMC mRNA per cell, determined by quantitative densitometry, were not statistically different in the anterior pituitaries of WKHT, SHR, and WKY rats. The decrease in hormone content and POMC mRNA levels may, thus, reflect decreased anterior pituitary gland corticotrope populations. Although POMC peptide levels in the anterior pituitaries of adult WKHA animals were not significantly different from those in WKY animals, the morphological studies demonstrated a 30% increase in the corticotrope population in the WKHA strain. In contrast, POMC mRNA levels in WKHA animals were decreased 30%, and the amount of POMC mRNA per corticotrope was decreased approximately 35% compared to that in WKY, SHR, and WKHT tissues.(ABSTRACT TRUNCATED AT 400 WORDS)

Anterior pituitary proopiomelanocortin expression is decreased in hypertensive rat strains

Studies comparing neuroendocrine differences between the spontaneously hypertensive rat (SHR) and the normotensive Wistar-Kyoto (WKY) strains have suggested altered anterior pituitary corticotrope expression of POMC associated with the development of hypertension in SHR animals. One major difficulty in comparing the SHR and WKY strains is that the two strains exhibit genetic differences unrelated to blood pressure status, because inbred in the SHR genome is a profile of behavioral characteristics different from those in the WKY, including hyperactivity in a novel environment and hyperreactivity in responding to stress. The present studies examine two new inbred rat strains, the WKHT and WKHA, which independently express the hypertension and behavioral traits, respectively. Together with the SHR and WKY, these genetically related, homozygous strains permit a more definitive means of examining the neuroendocrine correlates of either hypertension or behavior. The adult (5-month-old) male anterior pituitary gland content of the POMC peptides beta-endorphin and ACTH was decreased approximately 50% in the SHR and WKHT strains compared to that in the WKY strain, whereas hormone levels in the WKHA strain were not significantly different from those in the WKY strain. Reduced POMC peptide levels were, therefore, specifically associated with the hypertensive trait. Hormone content in prehypertensive weanling (5- to 7-week-old) SHR and WKHT animals was also reduced approximately 35% compared to that in WKY animals. Northern blot analysis identified a 45% decrease in POMC mRNA expression in the hypertensive SHR and WKHT strains, which paralleled the changes in tissue hormone content. Using both immunocytochemistry and in situ hybridization histochemistry, the number of labeled cells per unit area of tissues section was reduced approximately 45% in anterior pituitary tissues from SHR and WKHT rats compared to that in WKY tissues. The levels of POMC mRNA per cell, determined by quantitative densitometry, were not statistically different in the anterior pituitaries of WKHT, SHR, and WKY rats. The decrease in hormone content and POMC mRNA levels may, thus, reflect decreased anterior pituitary gland corticotrope populations. Although POMC peptide levels in the anterior pituitaries of adult WKHA animals were not significantly different from those in WKY animals, the morphological studies demonstrated a 30% increase in the corticotrope population in the WKHA strain. In contrast, POMC mRNA levels in WKHA animals were decreased 30%, and the amount of POMC mRNA per corticotrope was decreased approximately 35% compared to that in WKY, SHR, and WKHT tissues.

Pituitary adenylate cyclase-activating polypeptide regulation of AtT-20/D16v corticotrope cell proopiomelanocortin expression and secretion.

The hypothalamic peptides pituitary adenylate cyclase-activating polypeptides (PACAPs) may play central roles in the regulation of anterior pituitary gland function. The two pro-PACAP-derived peptides, PACAP38 and PACAP27, are posttranslationally processed from a common precursor molecule and share amino acid sequence homology with vasoactive intestinal peptide (VIP). To further evaluate and compare the physiological roles of these peptides, we have examined the short and long term effects of PACAP38, PACAP27, and VIP on POMC-related peptide production in the mouse AtT-20/D16v corticotrope cell line. Short term (0.5- to 5-h) treatment of AtT-20/D16v cells with PACAP38, PACAP27, or VIP elicited similar concentration-dependent and biphasic stimulation of ACTH release. Half-maximal stimulation of the higher potency phase was attained with 1-2 nM peptide; maximal secretion was observed at peptide concentrations greater than 100 nM. Similar 2- to 3-fold maximal stimulation of ACTH secretion was elicited by all three bioactive peptides; linear sustained ACTH secretion was observed. Long term (12- to 72-h) treatment of AtT-20/D16v cultures with these peptides resulted in substantially decreased rates of cellular division, with a concomitant increase in cell size and formation of cell processes characteristic of cellular differentiation. These morphological changes coincided with a sustained 2-fold increase in AtT-20 corticotrope intracellular hormone content and secretion. Northern blot analysis demonstrated a parallel induction of POMC mRNA expression by the PACAP38, PACAP27, and VIP peptides. These results suggest that AtT-20/D16v cells possess primarily the type II PACAP receptor subtype, which binds PACAP38, PACAP27, and VIP with apparent equal affinity. Furthermore, the long term effects of these peptides implicate a potentially significant role for PACAP and VIP peptides in the mediation of altered pituitary gland functions. In this vein, the PACAPs may prove to be unique regulators of neuroendocrine function and development.

Regulation of intermediate pituitary corticotropin-releasing hormone receptors by dopamine.

It has been shown that chronic cold exposure results in selective CRH receptor up-regulation in the intermediate pituitary. Since the intermediate pituitary is under dopaminergic control, the participation of a dopaminergic mechanism in the effect of cold stress was studied in rats treated with dopaminergic agonists and antagonists. CRH receptors were measured by the binding of radioiodinated Tyr-ovine (o) CRH to neurointermediate pituitary membranes of slide-mounted sections. Cold exposure for 60 h caused the expected increase in CRH binding in neurointermediate lobe membranes. Administration of the dopaminergic agonist bromocriptine did not prevent the effect of cold stress, but increased CRH binding in control rats. The dopaminergic antagonist metoclopramide decreased intermediate pituitary CRH binding in control and cold-exposed rats. Bromocriptine administration for 1-8 days caused a progressive increase in the binding of [125I]Tyr-oCRH in neurointermediate pituitary membranes, despite atrophy of the intermediate zone. Scatchard analysis of the binding data indicated that the changes were due to variations in receptor concentration, without changes in affinity. No changes in anterior pituitary CRH receptors were observed with agonist or antagonist treatment. Autoradiographic analysis of CRH binding after 3 days of treatment with bromocriptine or haloperidol confirmed the results observed in membranes and demonstrated that changes in binding were confined to the intermediate lobe. The functional consequences of the changes in CRH binding were studied by analysis of adenylate cyclase activity in cells and homogenates of intermediate pituitaries of rats treated with bromocriptine. In 18-h cultured intermediate pituitary cells from rats treated with bromocriptine for 3 days, CRH-stimulated cAMP production, measured in the presence of phosphodiesterase inhibitors, was increased to levels only slightly higher than those in cells from control rats. Likewise, CRH-stimulated adenylate cyclase, measured by conversion of [32P]ATP to [32P] cAMP, was not significantly different in homogenates from microdissected intermediate lobes from control and bromocriptine-treated rats. The lack of parallel changes in adenylate cyclase responsiveness suggests only partial receptor coupling, probably reflecting an inhibitory effect of dopamine on components of the adenylate cyclase. This study demonstrates that in contrast to the recognized inhibitory effect on cell division and POMC mRNA expression, dopamine causes up-regulation of CRH receptors in the intermediate pituitary. The qualitatively similar and nonadditive effects of cold stress and dopaminergic agonists suggest that a dopaminergic mechanism may be involved in intermediate pituitary CRH receptor regulation during chronic cold stress.

Epidermal growth factor enhances ACTH secretion and expression of POMC mRNA by corticotropes in mixed and enriched cultures

Previous studies have shown that epidermal growth factor (EGF) stimulates the release of adrenocorticotropin (ACTH) in vivo and in vitro by amplifying the effects of corticotropen-releasing hormone (CRH). The aim of the present studies was to compare responses to EGF by corticotropes in an unseparated culture with those in cultures enriched to 90–93% ACTH-β-endorphin cells (by counterflow centrifugation). Since EGF binding sites had been identified on growth hormone (GH) or prolactin (PRL) cells (9), the enriched corticotrope cultures were studied to learn if the abundant GH or PRL cells found in unseparated cultures were required to mediate the actions of EGF. In unseparated cultures, EGF (1 or 10 ng/ml) or CRH (1–5 nM) increased the percentages of ACTH cells from 9.5 to 15% and the percentage of cells labeled for POMC mRNA from 7.5 to 12% of the population. In enriched cultures, CRH and EGF increased the percentages of cells labeled for POMC mRNA from 70% to 90–94% of the population. EGF alone stimulated ACTH secretion in both the unseparated and enriched cultures by 1.2- to 2.2-fold. EGF amplified the effects of CRH by 30–40% in both unseparated and enriched cultures. In unseparated cultures grown in serum-free media, however, 1 ng/ml EGF did not amplify the effects of CRH and 10 ng/ml EGF partly abolished the CRH stimulation. In contrast, enriched cultures grown in serum-free media continued to respond to the growth factor. the secretory responses of corticotropes in enriched cultures were similar to those of their counterparts in the unseparated cultures. This indicates that the reduction in numbers of GH and PRL cells in the enriched cultures does not interfere with EGF actions on the corticotrope population.

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)