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

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

Down-regulation of hypothalamic corticotropin-releasing hormone messenger ribonucleic acid (mRNA) precedes early-life experience-induced changes in hippocampal glucocorticoid receptor mRNA

Early-life experiences, including maternal interaction, profoundly influence hormonal stress responses during adulthood. In rats, daily handling during a critical neonatal period leads to a significant and permanent modulation of key molecules that govern hormonal secretion in response to stress. Thus, hippocampal glucocorticoid receptor (GR) expression is increased, whereas hypothalamic CRH-messenger RNA (mRNA) levels and stress-induced glucocorticoid release are reduced in adult rats handled early in life. Recent studies have highlighted the role of augmented maternal sensory input to handled rats as a key determinant of these changes. However, the molecular mechanisms, and particularly the critical, early events leading from enhanced sensory experience to long-lasting modulation of GR and CRH gene expression, remain largely unresolved. To elucidate the critical primary genes governing this molecular cascade, we determined the sequence of changes in GR-mRNA levels and in hypothalamic and amygdala CRH-mRNA expression at three developmental ages, and the temporal relationship between each of these changes and the emergence of reduced hormonal stress-responses. Down-regulation of hypothalamic CRH-mRNA levels in daily-handled rats was evident already by postnatal day 9, and was sustained through postnatal days 23 and 45, i.e. beyond puberty. In contrast, handling-related up-regulation of hippocampal GR-mRNA expression emerged subsequent to the 23rd postnatal day, i.e. much later than changes in hypothalamic CRH expression. The hormonal stress response of handled rats was reduced starting before postnatal day 23. These findings indicate that early, rapid, and persistent changes of hypothalamic CRH gene expression may play a critical role in the mechanism(s) by which early-life experience influences the hormonal stress-response long-term.     

Human decidua and in vitro decidualized endometrial stromal cells at term contain immunoreactive corticotropin-releasing factor (CRF) and CRF messenger ribonucleic acid.

CRF, a hypothalamic neurohormone, has been shown to be present in several tissues outside the brain. During pregnancy, both fetal (placental trophoblast, chorion, and amnion) and maternal (decidua) intrauterine tissues contain immunoreactive CRF. A paracrine/autocrine role of CRF as a regulator of hormonogenesis in human placenta and decidua has been suggested. The expression of CRF mRNA in human decidua was demonstrated in the present study by Northern blot analysis and was found to be higher in specimens collected at term than in those collected during the first and second trimesters of gestation. Furthermore, the presence of CRF was detected immunocytochemically in cultured decidual cells isolated from term decidua as well as in endometrial stromal cells decidualized in vitro by treatment with a mixture of medroxyprogesterone acetate, estradiol, and relaxin. These results indicate that human decidua is an intrauterine extrahypothalamic source of CRF in the maternal compartment and offer new tools to explore the in vitro decidualization processes and the regulation of CRF release from decidual cells.     

Aging impairs estrogenic suppression of hypothalamic proopiomelanocortin messenger ribonucleic acid in the mouse.

Altered neuroendocrine sensitivity to estrogen is a characteristic of reproductive aging in female rodents, but its molecular basis is not well understood. The objective of this study was to determine whether altered modulation of hypothalamic proopiomelanocortin (POMC) mRNA by estradiol (E2) is a component of reduced neuroendocrine sensitivity to estrogen in the aging mouse. Young (4 month-old), middle-aged (13 month-old), and old (25 month-old) C57BL/6J mice were ovariectomized, implanted 2 weeks later with Silastic capsules containing E2 or cholesterol (CHOL), and sacrificed 3 days later. Hypothalamic POMC mRNA was measured by solution hybridization/RNase protection, using a RNA probe complementary to a fragment of mouse POMC mRNA. In the group with CHOL implants, POMC mRNA was 36% lower in middle-aged and old mice compared to young mice. E2 treatment reduced POMC mRNA levels by 44% in young mice but failed to lower POMC mRNA in middle-aged and old animals. These results confirm earlier evidence of reduced levels of POMC mRNA in hypothalami of aging rodents and indicate that the ability of E2 to reduce hypothalamic POMC mRNA is lost by middle age.     

Hypotensive hemorrhage elevates corticotropin-releasing hormone messenger ribonucleic acid (mRNA) but not vasopressin mRNA in the rat hypothalamus.

We examined the effect of acute hypotensive hemorrhage on corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) messenger RNAs (mRNAs) in neurons of the rat hypothalamus. Sprague-Dawley male rats were cannulated (femoral artery and vein) and received a 15 ml/kg.3 min hemorrhage on the morning of the fourth day. Time controls received no hemorrhage. After light halothane anesthesia, the rats were decapitated at 1 or 4 h (six to nine rats per group). The hypothalami were removed, frozen, and sectioned at 12 microns. In situ hybridization was performed using two 48-base oligodeoxynucleotide probes for CRH and AVP message, respectively. Hemorrhage led to a fall in arterial blood pressure and heart rate that recovered by 1 h. Plasma ACTH, corticosterone, and AVP were elevated 20, 60, and 90 min after hemorrhage, but returned to near control levels by 4 h. CRH mRNA was significantly elevated 1 and 4 h after hemorrhage, as compared to time controls, in parvocellular neurons of the paraventricular nuclei. However, AVP mRNA was not different from controls at 1 or 4 h after hemorrhage in the magnocellular or parvocellular paraventricular nuclei, or in the supraoptic or accessory nuclei of the hypothalamus. AVP mRNA was also found in neurons of the suprachiasmatic nuclei, but there was no difference in the amount of mRNA between the 1-h hemorrhage and control groups. These data suggest that neural signals, originating for cardiovascular receptors activated by hemorrhage, up-regulate message for CRH but not for AVP in the paraventricular nuclei of the rat hypothalamus.     

Rat hypothalamic corticotropin-releasing factor (CRF) content remains constant despite marked acute or chronic changes in ACTH secretion.

Hypothalamic corticotropin-releasing factor (CRF) activity was only slightly increased by 1 min ether stress and was unaltered by 2.5-10 min ether stress, 15-day adrenalectomy, 11-day hypophysectomy or 3-day dexamethasone treatment. There was a slight tendency for the hypothalamic CRF activity to be higher in the p.m. than in the a.m. Basal hypothalamic deafferentation did not significantly affect hypothalamic CRF content in the a.m. or p.m. compared to intact controls. We conclude that hypothalamic CRF content maintains relative constancy under conditions of marked acute or chronic stimulation or suppression of ACTH secretion.     

Comparative immunoelectron microscopic localization of corticotropin-releasing factor (CRF-41) and oxytocin in the rat median eminence

Comparative ultrastructural localization of corticotropin-releasing factor (CRF) and oxytocin was performed in the rat median eminence of Long Evans and Brattleboro rats. The peroxidase-antiperoxidase technique used on serial ultrathin sections revealed CRF and oxytocin neurosecretory granule colocalization in the same fibers of the internal layer running towards the posterior pituitary. It is probable that both these peptides coexist in the same granules. In the Brattleboro rats, while genetically lacking vasopressin, CRF was nevertheless shown to be present. In these rats, as was demonstrated in the Long Evans rats, CRF distribution paralleled that of oxytocin only in the internal zone of the median eminence.     

Early induction of c-fos precedes increased expression of corticotropin-releasing factor messenger ribonucleic acid in the paraventricular nucleus after immobilization stress.

CRF plays a role in coordinating endocrine, physiological, and behavioral responses to stressful stimuli. Several kinds of stressors have been reported to induce an increase in CRF mRNA expression in the paraventricular nucleus of the hypothalamus (PVN). Recently, the expression of c-fos mRNA has shown promise as a useful tool for metabolic mapping at the cellular level, because various types of stimulation induce c-fos mRNA expression in specific neuron populations in various brain regions. The aim of the present study is to clarify a possible anatomical-temporal correlation between the early induction of c-fos and the enhanced expression of CRF mRNA after stress. Wistar male rats were exposed to immobilization stress for 60 min and killed before and 15, 30, 60, 90, 120, and 180 min after the beginning of immobilization. In situ hybridization was performed by hybridizing sections with 35S-labeled prepro-CRF and c-fos cRNA probes. Relative levels of CRF and c-fos mRNA were compared by estimating the number of grains over the PVN in emulsion-dipped autoradiograms. Rapid induction (within 15 min) of c-fos mRNA was noted in the parvocellular division of the PVN after immobilization stress. The level of c-fos mRNA peaked at 30 min, then gradually declined to the control level within 90 min after the beginning of stress [the number of grains over the PVN: control, 326 +/- 180; 15 min, 2091 +/- 680 (P less than 0.05 vs. control); 30 min, 3385 +/- 239 (P less than 0.05 vs. control)]. The distribution of c-fos mRNA was almost identical to that of CRF mRNA in the PVN. On the other hand, the time course of CRF mRNA induction was delayed to the c-fos mRNA expression. A significant increase in CRF mRNA levels was noted only 120 and 180 min after stress [the number of grains over PVN: control, 3868 +/- 221; 120 min, 5957 +/- 677 (P less than 0.05 vs. control); 180 min, 6600 +/- 450 (P less than 0.05 vs. control)]. The results demonstrate that increased expression of CRF mRNA is preceded by c-fos mRNA induction in the PVN after stress suggesting a role of c-fos in the activation of CRF gene expression.     

Effect of implantation of dexamethasone adjacent to the paraventricular nucleus on messenger ribonucleic acid for corticotropin-releasing hormone and proopiomelanocortin during late gestation in fetal sheep.

Glucocorticoids act upon the hypothalamic paraventricular nucleus (PVN) and anterior pituitary in a classic negative feedback loop to regulate ACTH biosynthesis and secretion. Evidence exists to indicate that glucocorticoid feedback may be attenuated during late gestation in the sheep fetus to allow the preterm rise in fetal plasma cortisol necessary for parturition in this species. The present studies were undertaken to determine the effect of glucocorticoids placed adjacent to the fetal PVN on messenger RNA (mRNA) for CRH in the PVN and mRNA for POMC in the anterior pituitary during late gestation. We performed our studies at two critical stages during late gestation to determine if gestational age related changes occur in the efficacy of negative feedback regulation of expression of CRH and subsequently POMC. Dexamethasone (DEX) implants were placed bilaterally 2 mm lateral to the fetal PVN at 105 to 107 days gestational age (dGA; group I, n = 4) and 121-123 dGA (group II; n = 4). Gestational-age matched, sham implanted fetuses were used as controls (CONT) for both groups (n = 4 per group). Fetuses were recovered at 126-128 (group I) and 136 dGA (group II). Fetal PVN were isolated by micropunching, and the anterior pituitary was separated from neurointermediate and posterior lobes after necropsy. Total RNA was subjected to Northern analysis using specific complementary DNA probes to CRH and POMC, and specific message was normalized to actin mRNA content in each individual sample. Anterior pituitary POMC mRNA was not different in DEX fetuses compared to CONT for either group I (78 +/- 26% of CONT; mean +/- SEM) or group II (84 +/- 17% of CONT). PVN CRH mRNA content was lower in DEX fetuses in group I (28 +/- 14% of CONT; P less than or equal to 0.01) and group II (65 +/- 12% of CONT; P less than or equal to 0.01). The degree to which DEX suppressed mRNA for CRH was greater in group I compared to group II (P less than or equal to 0.05). We conclude that 1) CRH expression in the PVN of fetal sheep is suppressible by glucocorticoids; 2) suppression can occur directly at the level of the PVN and 3) that the efficacy of negative feedback decreases with increasing gestational age. Furthermore, the lack of effect of hypothalamic administration of DEX on anterior pituitary POMC mRNA indicates that basal expression of POMC in fetal sheep may be independent from support from the PVN at this stage of gestation.     

Lactation inhibits stress-mediated secretion of corticosterone and oxytocin and hypothalamic accumulation of corticotropin-releasing factor and enkephalin messenger ribonucleic acids

The effect of lactation on stress-induced hormone responses and changes in hypothalamic mRNA was assessed in female rats. In control animals the stimulus of ip hypertonic saline resulted in increased plasma levels of corticosterone, oxytocin, and vasopressin and hypothalamic content of CRF and enkephalin mRNA. In lactating females, however, the corticosterone response to this stress failed to reach significance, the plasma oxytocin response was markedly reduced, and the vasopressin response was unaffected. Lactation also resulted in an abolition of the CRF and enkephalin mRNA responses to stress. In contrast, the hypothalamic CRF response to adrenalectomy was unaffected by lactation status. Removal of the pups from their mothers resulted in a return of CRF and enkephalin mRNA responses to stress within 2 days. Lactation is associated with a selective inhibition of normal hypothalamic stress responses.     

Immunoreactive proopiomelanocortin (POMC) peptides and POMC-like messenger ribonucleic acid are present in many rat nonpituitary tissues

Immunoreactive (IR) POMC peptides have been found in several rat nonpituitary tissues. We found IR-ACTH, IR-beta-endorphin (beta END), and IR-gamma MSH in extracts from the following eight rat nonpituitary tissues, listed in order of decreasing POMC peptide concentrations: testis, duodenum, kidney, colon, liver, lung, stomach, and spleen, but not in adrenal or muscle extracts. Concentrations were very low and ranged from less than 0.00003% to 0.0005% of pituitary levels. In testis, duodenum, and colon, IR-gamma MSH and IR-beta END concentrations were only 5-37% of IR-ACTH levels. Gel filtration chromatography showed that IR-ACTH and IR-beta END coeluted in a major peak of 15,000 daltons, which is slightly larger than expected for a C-terminal peptide containing rat ACTH and beta-lipotropin. There were also a minor higher mol wt peak of IR-ACTH and IR-beta END and a minor IR-beta END peak that eluted in the position of mature beta END. There was no peak of IR-ACTH that corresponded to the size of mature ACTH. To determine whether these nonpituitary tissues also contained a POMC-like mRNA, which would confirm that the peptides were synthesized locally within the tissues, we examined poly(A) RNA prepared from 10 nonpituitary tissues and total RNA from pituitary by Northern blot hybridization for the presence of a POMC-like mRNA with an exon 3 riboprobe. Pituitary contained a single POMC mRNA species of about 1000 nucleotides. A short POMC-like mRNA of about 800 bases was found in all nonpituitary tissues, except spleen and muscle. Compared to POMC mRNA levels in pituitary, the concentration of POMC-like mRNA was 0.5% in testis and 0.03-0.07% in the other tissues. The ratio of POMC-like mRNA to IR-POMC peptide concentrations in nonpituitary tissues was at least 1000 times greater than that in the pituitary. We conclude that the POMC gene is expressed in many nonpituitary tissues and that either the short POMC-like mRNA is translated much less efficiently or POMC peptides are released or degraded much more rapidly in nonpituitary tissues than in the pituitary.     

Stress elevates corticotropin-releasing factor (CRF) and CRF-binding protein mRNA levels in rainbow trout (Oncorhynchus mykiss)

The objectives of this study were to characterize rainbow trout (Oncorhynchus mykiss) corticotropin-releasing factor (CRF)-binding protein (CRF-BP) cDNA and to examine the variations in CRF-BP and CRF mRNA levels in response to different intensities of stress. Trout were physically disturbed by a single or three consecutive periods of chasing until exhaustion followed by 2 h of recovery. The pituitary CRF-BP and preoptic area CRF1 mRNA contents were significantly increased only after repeated chasing events. Physical disturbance increased plasma cortisol levels with the largest change occurring in the group of trout that were exposed to repeated chasing events. Trout were also individually isolated in 120 l tanks or confined to 1.5 l boxes for 4, 24 or 72 h. CRF-BP mRNA levels in confined fish were greater than those of isolated fish at 72 h although there were no differences compared with the control group. CRF1 mRNA levels in the preoptic area were greater and remained elevated for a longer period in confined compared with isolated trout. Isolation led to a transient increase in plasma cortisol levels, but the higher cortisol values developed in the confined fish suggest that this treatment was more stressful than isolation. These results demonstrate that the intensity and duration of stress are important factors regulating CRF and CRF-BP mRNA levels in rainbow trout. We hypothesize that pituitary CRF-BP is involved in regulating the activity of the stress axis, possibly by reducing access to CRF1 receptors in the corticotropes.     

Forskolin-induced immunoreactive atrial natriuretic peptide (ANP) secretion and pro-ANP messenger ribonucleic acid expression of hypothalamic neurons in culture: modulation by glucocorticoids.

In the present studies the chronic effects of glucocorticoids and drugs activating cAMP-dependent pathways on the production and secretion of immunoreactive (ir) ANP from long term monolayer cultures of neonatal rat hypothalamic neurons were examined. Forskolin treatment increased ir-ANP release in a time-dependent and dose-related manner, with an EC50 of approximately 30 microM; at a lower dose of 10 microM, forskolin doubled ir-ANP release (P less than 0.01) compared to that in control cultures (mean +/- SEM, 9.6 +/- 0.3 pg/well; n = 4). While dexamethasone (DM) alone did not affect basal secretion of ir-ANP, 10 nM of the glucocorticoid significantly enhanced the effect of forskolin (10 microM) by raising ir-ANP release approximately 3 times that induced by forskolin alone (P less than 0.001). This potentiation of DM was both time dependent and dose responsive, with an EC50 of 1 nM; this effect was significantly suppressed by 100 nM RU38486, a glucocorticoid or type II receptor antagonist, but not by RU28318, a mineralocorticoid receptor antagonist. In addition, forskolin (10 microM) or DM (10 nM) alone significantly increased ir-ANP production approximately 1.4 times (P less than 0.05) and 1.3 times (P less than 0.05) over that of control cultures, respectively, whereas concurrent treatment with forskolin and DM increased ir-ANP production by approximately 1.8 times (P less than 0.01). These changes were reflected by a corresponding increment in the abundance of pro-ANP mRNA in the cultures, as demonstrated by Northern blot analysis. We conclude from the present findings that glucocorticoid- and cAMP-dependent pathways may modulate the function of ANP neurons in rat hypothalami by regulating the secretion and production of the neuropeptide at the genomic level.     

Colocalization of neurotensin messenger ribonucleic acid (mRNA) and progesterone receptor mRNA in rat arcuate neurons under estrogen-stimulated conditions.

In the female rat, estrogen and progesterone directly or indirectly regulate the activity of neurotensin (NT)-synthesizing neurosecretory cells located in the hypothalamic arcuate nucleus (ARC). To determine whether these NT neurons are subject to direct regulation by ovarian steroids, estrogen-inducible messenger RNA (mRNA) encoding nuclear progesterone receptor (PR) was used as a cellular marker for nuclear estrogen receptor (ER) as well as PR, and double label in situ hybridization was employed to determine the extent to which NT/neuromedin N mRNA and PR mRNA are colocalized in ARC neurons under estrogen-stimulated conditions. In estradiol-treated ovariectomized rats, approximately 80% of NT/neuromedin N mRNA-expressing cells in sections through the dorsomedial division of the ARC and approximately 60% of such cells in sections through the ventrolateral division of the ARC were found to contain PR mRNA. Depending on the ARC division and rostrocaudal level, double labeled cells accounted for approximately 20-50% of PR mRNA-containing cells. These results indicate that under estrogen-stimulated conditions the majority of NT neurons in the ARC express both PR and ER, as previous studies of this region indicate that estrogen-inducible PR occurs only in cells that also express ER. In the rat, NT neurons appear to be a major ARC cell type subject to direct regulation by estrogen and progesterone.     

Tissue distribution and regulation of insulin-like growth factor (IGF)-binding protein-3 messenger ribonucleic acid (mRNA) in the rat: comparison with IGF-I mRNA expression.

Insulin-like growth factor-binding protein-3 (IGFBP-3) is the most abundant IGFBP in rat and human sera. The present study demonstrates the expression of the rat IGFBP-3 gene in a large number of tissues and coexpression, but not necessarily equal expression, with IGF-I mRNA. Tissues with a major abundance of IGFBP-3 were kidney, antrum of stomach, placenta, uterus, and liver. Changes in hepatic and renal levels of IGFBP-3 mRNA were analyzed after hypophysectomy (with and without GH treatment) and in the developing postnatal rat. These results were compared to changes in IGF-I mRNA levels under the same physiological conditions. Using S1 nuclease analysis, IGFBP-3 mRNA was present in the kidney and liver of 1-day-old rats and rose significantly in both organs by week 1. Thereafter, levels remained relatively constant, particularly in the liver. This is in marked contrast to the hepatic IGF-I pattern, which showed a continual rise up to 8 weeks. Hepatic IGFBP-3 gene expression was partially GH dependent, with IGFBP-3 mRNA levels falling (approximately 50%) after hypophysectomy and rising slightly after GH treatment. These changes were much less dramatic than those in IGF-I mRNA. In contrast, the renal levels of IGFBP-3 mRNA increased after hypophysectomy, (approximately 100%), but did not decrease with GH treatment. These data suggest that IGFBP-3 mRNA abundance is regulated differently in different tissues, and in at least some tissues is less sensitive to regulation than is IGF-I mRNA.