Stress induces parallel changes in corticotrophin-releasing hormone (CRH) Transcription and nuclear translocation of transducer of regulated cAMP response element-binding activity 2 in hypothalamic CRH neurones.

Recent studies in vitro have shown that the cAMP response element-binding (CREB) co-activator, transducer of regulated CREB activity (TORC), is required for transcriptional activation of the corticotrophin-releasing hormone (CRH) gene. To determine the physiological importance of TORC2 regulating CRH transcription during stress, we examined the localisation of TORC2 in CRH neurones, as well as the relationship between changes in CRH heterogeneous nuclear (hn)RNA, nuclear translocation of TORC2 and binding of TORC2 to the CRH promoter. Immunohistochemistry revealed TORC2 immunoreactivity (irTORC2) in the dorsolateral (magnocellular) and dorsomedial (parvocellular) regions of the hypothalamic paraventricular nucleus (PVN). Although staining was mostly cytosolic under basal conditions, there was a marked increase in nuclear irTORC2 in the dorsomedial region after 30 min of restraint, concomitant with increases in CRH hnRNA levels. Levels of nuclear irTORC2 and CRH hnRNA had returned to basal 4 h after stress. Double-staining immunohistochemistry showed TORC2 co-staining in 100% of detected CRH neurones, and nuclear translocation after 30 min of restraint in 61%. Cellular distribution of TORC2 in the dorsolateral PVN was unaffected by restraint. Chromatin immunoprecipitation experiments showed recruitment of TORC2 and phosphorylated CREB (pCREB) by the CRH promoter after 30 min of restraint, but 4 h after stress only pCREB was associated with the CRH promoter. The demonstration that TORC2 translocates to the nucleus of hypothalamic CRH neurones and interacts with the CRH promoter in conjunction with the activation of CRH transcription during restraint stress, provides strong evidence for the involvement of TORC2 in the physiological regulation of CRH transcription.

Vasopressin protects hippocampal neurones in culture against nutrient deprivation or glutamate-induced apoptosis.

Vasopressin (VP) secreted within the brain modulates neuronal function by acting as a neurotransmitter. Recent studies show that VP prevents serum deprivation-induced apoptosis in the neuronal cell line, H32. To determine whether VP is anti-apoptotic in hippocampal neurones, primary cultures of these neurones were used to examine the effect of VP on neuronal culture supplement (B27) deprivation-, or glutamate-induced apoptosis, and the signalling pathways mediating the effects. Removal of B27 supplement from the culture medium for 24 h or the addition of glutamate (3-10µm) decreased neuronal viability (P<0.05) and increased Tdt-mediated dUTP nick-end labelling (TUNEL) staining and caspase-3 activity (P<0.05), which is consistent with apoptotic cell death. VP (10 nm) reduced B27 deprivation- or glutamate-induced cell death (P<0.05). These anti-apoptotic effects of VP were completely blocked by a V1 but not a V2 receptor antagonist, indicating that they are mediated via V1 VP receptors. The anti-apoptotic effect of VP in neurones involves activation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) and inositol trisphosphate/protein kinase B (Akt) signalling pathways. This was shown by the transient increases in phospho-ERK and phospho-Akt after incubation with VP revealed by western blot analyses, and the ability of specific inhibitors to reduce the inhibitory effect of VP on caspase-3 activity and TUNEL staining by 70% and 35%, respectively (P<0.05). These studies demonstrate that VP has anti-apoptotic actions in hippocampal neurones, an effect that is mediated by the MAPK/ERK and phosphatidylinositol-3 kinase/Akt signalling pathways. The ability of VP to reduce nutrient deprivation or glutamate overstimulation-induced neuronal death suggests that VP acts as a neuroprotective agent within the brain.

Inhibition of corticotrophin-releasing hormone transcription by inducible cAMP-early repressor in the hypothalamic cell line, 4B.

We have shown recently that the rapid decline in corticotrophin-releasing hormone (CRH) transcription following activation by stress is associated with induction and binding to the CRH promoter of the repressor isoforms of cAMP responsive element modulator (CREM), inducible cAMP early repressor (ICER). The ability of ICER to inhibit CRH transcription was examined in the hypothalamic cell line, 4B, which expresses CRH. Co-transfection of the inhibitory isoforms of CREM, ICER I and II and CREMbeta, and CRH promoter-luciferase constructs in 4B cells blunted basal and forskolin-stimulated CRH promoter activity, an effect which was abolished by mutation of the CRE of the CRH promoter. Western blot analyses and electromobility gel-shift and super-shift showed increases in endogenous ICER after 3 h of incubation with forskolin. Consistent with an inhibitory effect of CREM on CRH transcription, chromatin immunoprecipitation assays in cells transfected with ICER I revealed recruitment of CREM by the CRH promoter in conjunction with decreases in Pol II association. The study shows that generation of ICER following prolonged stimulation with forskolin, or transfection of an ICER expression vector in hypothalamic cell lines expressing CRH, is associated with CREM binding to the CRH promoter and transcriptional repression. The data support the hypothesis that induction of repressor isoforms of CREM is part of an intracellular feedback mechanism contributing to the termination of CRH transcription during stimulation.

In vitro regulation of corticotropin-releasing hormone.

Studies involving regulation of corticotropin-releasing hormone (CRH) in vitro have been used to validate findings obtained in vivo and more importantly have been used as model systems to better understand signalling mechanisms responsible for the expression of the CRH gene and peptide. Many in vitro studies examining CRH have utilized hypothalamic tissue while a few have focused on the amygdala. Clonal cell lines have also been utilized as models of central nervous system CRH neurons. Stimuli that have been implicated in regulating hypothalamic CRH regulation in vitro include protein kinase A (PKA) and protein kinase C (PKC) activators, glucocorticoids, biogenic amines, cytokines and the gaseous neurotransmitters. Amygdalar CRH levels in vitro are affected by some of the same stimuli that regulate hypothalamic CRH; however there is evidence supporting differential regulation of CRH in these two brain regions by some of the same stimuli. Only a few studies in aggregate have investigated signal transduction mechanisms and these studies have focused on PKA- and glucocorticoid-mediated changes in CRH expression. Thus, much more investigative work in better understanding CRH regulation in vitro is needed.

Corticotropin-releasing hormone (CRH) expression and protein kinase A mediated CRH receptor signalling in an immortalized hypothalamic cell line.

Corticotropin-releasing hormone (CRH) is a 41 amino acid neuropeptide which plays an important role in the stress response in the hypothalamus. We describe the development of an immortalized hypothalamic cell line which expresses CRH. We hypothesized that this cell line would possess the relevant characteristics of parvocellular CRH-expressing neurones such as glucocorticoid receptor (GR) expression and vasopressin (VP) coexpression. For production of hypothalamic cells, embryonic day 19 rat pup hypothalami were dissected and dissociated into tissue culture dishes. They were immortalized by retrovirus-mediated transfer of the SV40 large T antigen gene at 3 days of culture and then screened for expression of CRH following dilution cloning. One cell line was chosen (IVB) which exhibited CRH-like immunoreactivity (CRH-LI) and expressed CRH, VP and CRH1 receptor RNA via the reverse transcriptase-polymerase chain reaction. In addition, the cell line expressed the neuronal marker, microtubule-associated protein-2. We verified that the CRH-LI from IVB cell lysates coeluted with CRH standard via reversed-phase high-performance liquid chromatography (HPLC). Furthermore, oxidation of the lysate converted its HPLC profile to that identical with oxidized CRH standard. In addition, IVB cells exhibited high affinity binding to CRH. Incubation of IVB cells with CRH lead to increases in cAMP levels and protein kinase A activity in a concentration-dependent manner. Incubation of IVB cells with CRH also resulted in increases in phospho-cyclic-AMP response element binding protein (CREB) immunostaining as detected by immunocytochemical analysis. Finally, CRH treatment of IVB cell lines has been linked to CREB-mediated gene expression as determined via the PathDetect CREB trans-reporting system. The characteristics of IVB cells, such as CRH and VP coexpression, GR expression and a biologically active CRH-R1-mediated signalling pathway, suggest that this neuronal cell line may serve as model of parvocellular CRH neurones.

Phlebotomine sandflies and leishmaniasis risks in Colombian coffee plantations under two systems of cultivation.

The phlebotomine sandfly fauna of traditional (shaded) and intensified (unshaded) coffee plantations in Colombia was sampled by a variety of methods and the species composition and density under the two systems compared. Twenty species of Lutzomyia sandflies (Diptera: Psychodidae: Phlebotominae) were collected, of which eight were found only in the 'Coffee Axis' ('Eje Cafetero') of the departments of Caldas, Risaralda and Quindio, six were exclusive to the department of Norte de Santander and six occurred in both regions. Four species were collected only in traditional plantations and two exclusively in intensified ones. At least 13 species occurred in both plantation types. Fifteen species are opportunistic man-biters and eight are suspected vectors of leishmaniasis caused by Le. braziliensis, Le. panamensis or Le. mexicana. Seven species were collected inside houses and may be involved in intradomiciliary transmission of Leishmania. The dominant species in Norte de Santander was Lu. spinicrassa, which made up 93.8% of all the sandflies collected in this department. This species was absent from the Eje Cafetero and a number of others among the 15 recorded there might be responsible for Leishmania transmission in this region, including Lu. trapidoi, Lu. yuilli, Lu. gomezi, L. hartmanni and Lu. ovallesi. Sandfly population densities were significantly higher in traditional plantations than in intensified ones. Residents of traditional plantations were able to describe sandflies in significantly more detail than those of intensified plantations, based on seven basic characteristics related to the appearance and biting behaviour of the insects.

Vasopressin and oxytocin neurones of hypothalamic supraoptic and paraventricular nuclei co-express mRNA for Type-1 and Type-2 corticotropin-releasing hormone receptors.

The presence of corticotropin-releasing hormone (CRH) receptors type-1 (CRHR-1) and type-2 (CRHR-2alpha) in the hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei, and the effects of i.c.v. injection of CRH and urocortin on arginine vasopressin (AVP) and oxytocin release, have suggested that CRH ligands have a role in osmoregulation. In this study, double labelling in situ hybridization using 35S-labelled CRHR-1 or CRHR-2alpha and digoxigenin-labelled AVP, oxytocin or CRH riboprobes was employed to examine the localization of CRHR-1 or CRHR-2alpha mRNA in the SON and PVN of control and osmotically stimulated rats. Rats received an i.p. hypertonic saline (1.5 M) injection or isotonic saline injection (controls), or 2% NaCl intake (salt loading) or tap water (controls) for 12 days. While CRHR-1 mRNA was undetectable in the SON and PVN in control rats, its expression was increased markedly at 4 h after i.p. hypertonic saline injection or after 12 days salt loading. Of the cells labelled with digoxigenin-AVP, 53% in the SON and 90% in the PVN coexpressed CRHR-1 mRNA after i.p. hypertonic saline injection. In oxytocinergic neurones, 73% in the SON and 91% in the PVN showed CRHR-1 autoradiographic grains higher than background levels after i.p. hypertonic saline injection. In addition, i.p. hypertonic saline induced CRHR-1 mRNA expression in digoxigenin-CRH stained cells in the parvocellular PVN. CRHR-2alpha transcripts were present in both the SON and PVN under basal conditions, and salt loading, but not acute i.p. hypertonic saline injection, further stimulated this expression. Double labelling in situ hybridization showed colocalization of CRHR-2alpha mRNA with AVP and oxytocin mRNA in the SON. These studies support a role for CRH and urocortin regulating the hypothalamo-neurohypophyseal system, and suggest a direct action of the peptides in the magnocellular neurones.

Prenatal dexamethasone treatment does not prevent alterations of the hypothalamic pituitary adrenal axis in steroid 21-hydroxylase deficient mice.

A major difficulty in the clinical management of congenital adrenal hyperplasia (CAH) is adjustment of glucocorticoid doses to suppress ACTH and androgens without causing iatrogenic hypercortisolism. The possibility that structural alterations of the adrenal or a dysfunction of the hypothalamic pituitary adrenal (HPA) axis caused by glucocorticoid deficiency during fetal life contribute to this problem was studied in 21-hydroxylase deficient mice caused by deletion of the cytochrome P-450 21-hydroxylase gene. Homozygotes showed about 200-fold elevations in plasma progesterone, hyperplastic adrenal cortices lacking zonation, and structural alterations of adrenocortical mitochondria. Histochemical studies showed increases in hypothalamic CRH messenger RNA (mRNA) and immunoreactive (ir) CRH, and pituitary POMC mRNA in homozygous mice. VP mRNA levels in PVN perikarya were normal, but irVP in parvicellular terminals of the median eminence was increased in homozygotes. Prenatal dexamethasone treatment (0.5 to 2 microg/day) prevented the increases in CRH mRNA, whereas dexamethasone only partially decreased POMC mRNA levels, and had no effect on serum progesterone levels. The data suggest that intrauterine glucocorticoid deficiency in CAH causes hyperactivity of the hypothalamic-pituitary-corticotroph axis and insensitivity to glucocorticoid feedback. These studies in 21-hydroxylase deficient mice may provide new insights on the mechanism, clinical manifestations and management of some types of human CAH.

Evidence for arginine vasopressin as the primary activator of the HPA axis during adjuvant-induced arthritis.

1. Adjuvant-induced arthritis (AA) is an experimental inflammation of the joints that results in chronic activation of the hypothalamo-pituitary-adrenal (HPA) axis. 2. In this study the role of hypothalamic corticotrophin-releasing factor (CRF) and arginine vasopressin (AVP) in the regulation of the HPA axis in this condition both in Sprague-Dawley (SD), and Piebald-Viral-Glaxo (PVG) rats has been further characterized. 3. The increase in AVP peptide content of portal blood (as early as day 11), just prior to the onset of arthritis is confirmed and further increases, peaking at day 16 are shown, coincident with the progression of inflammation in the PVG rats. 4. The increase in AVP is associated with a significant increase in the expression of AVP but not CRF mRNAs in the medial parvocellular division of the hypothalamic paraventricular nucleus (PVN) of arthritic SD rats. 5. In the presence of maximal inflammation of SD rats there was a significant decrease in the maximum binding of [125I]-Tyr-oCRF to anterior pituitary membranes, whereas AVP receptor concentration in anterior pituitary membranes from both PVG and SD rats showed a significant increase with respect to controls. 6. The basal adrenocorticotrophin (ACTH) secretion in vitro was similar in both control and arthritic SD rats but that from arthritic PVG rat pituitaries was significantly greater than the respective controls (436 +/- 91 v 167 +/- 23 pg/tube). The ACTH response of pituitaries of arthritic PVG rats to CRF or the combination of CRF and AVP was significantly higher compared with the controls, although the ACTH response of arthritic SD rat pituitaries was unchanged. 7. The results are consistent with the view that activation of the parvocellular vasopressin system has an important role in the adaptation of the HPA axis to experimentally-induced chronic stress of arthritis.

Regulation of hypothalamic and pituitary corticotropin-releasing hormone receptor messenger ribonucleic acid by adrenalectomy and glucocorticoids.

The effects of adrenalectomy and glucocorticoids on the regulation of corticotropin-releasing hormone (CRH) receptor expression in the hypothalamic paraventricular nucleus (PVN) and pituitary were studied by in situ hybridization in the rat using a complementary RNA probe directed toward the coding region of the type 1 CRH receptor. Eighteen hours after adrenalectomy, CRH receptor messenger RNA (mRNA) expression in the PVN was significantly increased, whereas longer term adrenalectomy (4 and 6 days) had no effect. This transient effect of adrenalectomy was prevented by glucocorticoid replacement. In intact rats, 4 h after immobilization for 1 h or a single ip hypertonic saline injection, CRH receptor mRNA in the PVN markedly increased (P < 0.01), an effect that was unchanged by adrenalectomy (4 or 6 days) or dexamethasone injection (100 micrograms at -14 and 50 micrograms at -1 h) before stress. In the pituitary, CRH receptor mRNA levels decreased transiently after adrenalectomy (-62% after 18 h), returning to basal levels 4 or 6 days after adrenalectomy. The early decrease was prevented by glucocorticoid replacement. In intact rats, dexamethasone (100 micrograms, sc) caused a significant decrease in pituitary CRH receptor mRNA levels 2-10 h after injection, returning to basal levels after 15 h. On the other hand, dexamethasone (5-300 micrograms, sc) had no effect on pituitary CRH receptor mRNA levels 18 h after injection. The data show that although stress stimulation of CRH mRNA in the PVN is glucocorticoid independent, basal levels are likely to be under dual, transcriptional and posttranscriptional, control by glucocorticoids. In the pituitary, changes in hypothalamic CRFs probably play a major role in the control of CRH receptor mRNA levels during manipulations of circulating glucocorticoids levels. In addition, the inability of long term adrenalectomy and glucocorticoid administration to modify pituitary CRH receptor mRNA levels suggests that CRH receptor down-regulation observed under these experimental conditions depends mainly on translational and post-translational events rather than receptor mRNA levels.

Regulation of pituitary ACTH secretion during chronic stress.

Maintenance of adequate levels of response of the hypothalamic-pituitary-adrenal axis during chronic stress is important for survival. Three basic patterns of response can be identified depending on the type of stress: (a) desensitization of ACTH responses to the sustained stimulus, but hyperresponsiveness to a novel stress despite elevated plasma glucocorticoid levels, as occurs in physical-psychological paradigms; (b) no desensitization of ACTH response to the repeated stimulus and hyperresponsiveness to a novel stress, as occurs during repeated painful stress and insulin hypoglycemia; and (c) small and transient increases in ACTH, but sustained elevations of plasma corticosterone and diminished ACTH responses. The level of response of the pituitary corticotroph is determined by differential regulation of the hypothalamic regulators, corticotropin-releasing hormone (CRH) and vasopressin (VP), and the sensitivity of the negative glucocorticoid feedback. While osmotic stimulation increases VP expression in magnocellular neurons of the paraventricular (PVN) and supraoptic nuclei of the hypothalamus, chronic stress paradigms with high pituitary responsiveness are associated with activation of CRH and CRH/VP parvicellular neurons of the PVN, predominantly of the VP-containing population. While moderate increase of CRH output is important for stimulation of POMC transcription, the increase of the VP:CRH secretion ratio appears to be important in maintaining the secretory capacity of the pituitary corticotroph during chronic stimulation. Decreased sensitivity of the glucocorticoid feedback, probably due to interaction of glucocorticoid receptors with transcription factors induced by CRH and VP, is critical for the maintenance of ACTH responses in the presence of elevated plasma glucocorticoid levels during chronic stress. Although both CRH and VP receptors are activated and undergo regulatory variations during chronic stress, only the changes in VP receptor levels are parallel to the changes in pituitary ACTH responsiveness. The inhibitory effect of chronic osmotic stimulation on ACTH secretion in spite of high circulating levels of VP is probably the result of diminished activity of parvicellular PVN neurons and downregulation of pituitary VP receptors. Although the exact interaction between regulatory factors and the molecular mechanisms controlling the sensitivity of the corticotroph during adaptation to chronic stress remain to be determined, it is clear that regulation of the proportional secretion of CRH and VP in the PVN, modulation of pituitary VP receptors, and the sensitivity to feedback inhibition play a critical role.

Activity of the hypothalamic pituitary adrenal axis and sympathoadrenal system during food and water deprivation in the rat.

It has been previously shown that chronic water deprivation or hypertonic saline intake, osmotic stress models with concomitant decrease in food intake, decrease hypothalamic CRH mRNA levels and ACTH responses to acute stimulation. To determine the contribution of food restriction to the effects of osmotic stimulation, the function of the hypothalamic pituitary adrenal axis was analyzed in rats subjected to food deprivation, water deprivation or their combination for 60 h. In all three groups, basal levels of plasma corticosterone were increased, while ACTH and catecholamines were unchanged. Basal plasma vasopressin levels were normal in food deprived rats, but significantly increased in water deprived and simultaneously food and water deprived rats. In contrast to the 25% reduction of plasma ACTH responses to 30 min immobilization by water deprivation, food deprivation had no inhibitory effect and prevented the decreased ACTH responsiveness caused by water deprivation. In control rats, plasma corticosterone levels increased 22.5-fold 30 min after immobilization, and this response was significantly potentiated in the water deprived, food deprived and combined food and water deprived groups. The elevation in plasma catecholamines in response to acute immobilization was also enhanced in both water deprived and food deprived rats. In situ hybridization studies showed a 35% increase in VP mRNA levels in the PVN after water deprivation, whereas food deprivation caused a slight decrease and prevented the stimulatory effect of water deprivation. CRH mRNA in the PVN was reduced by 27% after food deprivation and by 67% after water deprivation, but simultaneous food and water deprivation caused a significantly smaller reduction similar to that in food deprivation alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of the hypothalamic pituitary adrenal axis during chronic stress: responses to repeated intraperitoneal hypertonic saline injection.

Chronic osmotic stress inhibits, while repeated physical stress can increase pituitary ACTH responsiveness to a novel stress. The interaction between these effects was studied in rats subjected to repeated i.p. injection of hypertonic saline, a strong aversive stimulus with osmotic and painful and psychological stress components, for 14 days. Hypertonic saline injection caused marked drinking responses, transient increases in plasma vasopressin (VP), and marked increases in VP mRNA and irVP in magnocellular cell bodies in the hypothalamus. Parvicellular activity was also enhanced as indicated by increases in VP immunostaining in the external zone of the median eminence and CRH mRNA and irCRH in the PVN. Plasma ACTH levels increased 10-fold after 30 min hypertonic saline injection, returning to basal levels in 4 h, and there was no desensitization of the ACTH responses after repeated injections (from basal values of 76 +/- 10 to 782 +/- 57, 788 +/- 83 and 779 +/- 31 pg/ml 30 min after the first, 4th and 14th injection, respectively). Basal ACTH levels were normal 24 h after the last injection, but pituitary POMC mRNA levels were increased by 95%, and ACTH responses to a novel stress (15 min immobilization) were significantly larger than in controls (P < 0.01) despite increases in morning plasma corticosterone levels (1.5 +/- 0.4 and 9.2 +/- 3.1 micrograms/dl in controls and stressed rats, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of the hypothalamic-pituitary-adrenal axis during water deprivation.

The contribution of the magnocellular vasopressinergic system to the regulation of ACTH secretion was studied by analysis of hypothalamic-adrenal axis function in rats subjected to water deprivation for 48 h. Water deprivation resulted in marked increases in plasma osmolarity and vasopressin (VP) levels and hypothalamic VP mRNA and immunoreactive (ir) VP in magnocellular neurons. While CRH mRNA levels in the paraventricular nucleus were decreased, irCRH accumulation in paraventricular nucleus neurons after colchicine treatment was normal or increased. Similarly, the irCRH content in the median eminence and its release under stress were similar in control and water-deprived rats. While basal plasma ACTH levels were similar in both groups (34.5 +/- 3.8 and 39.8 +/- 3.3 pg/ml), levels stimulated by CRH injection (10 micrograms, i.v.) or 15-min immobilization stress were reduced by 47% (P < 0.01) and 43% (P < 0.05), respectively, in water-restricted rats. The decreased ACTH responses were not prevented by injection of CRH (7.5 micrograms/day, sc) during the period of water deprivation. In contrast to the ACTH responses, basal and CRH-stimulated plasma corticosterone levels were significantly elevated (P < 0.001), and the responses to acute stress were normal. The inhibition of ACTH secretion was not due to increased glucocorticoid feedback, since similar blunted ACTH responses to acute immobilization stress were observed in adrenalectomized rats receiving corticosterone replacement. Despite similar levels of pituitary POMC mRNA, pituitary ACTH content was reduced in water-deprived rats, suggesting a posttranscriptional inhibition of POMC synthesis or processing. The data demonstrate that osmotic activation of the magnocellular VP system is accompanied by reduced responsiveness of the corticotrophs, an effect that is not due to increased glucocorticoid feedback or hypothalamic CRH deficiency. These findings suggest that the magnocellular vasopressinergic system does not play an important role in the regulation of ACTH secretion during chronic osmotic stimulation.

Desensitization of the hypothalamic-pituitary-adrenal axis following prolonged administration of corticotropin-releasing hormone or vasopressin.

The responses of the hypothalamic-pituitary-adrenal axis during chronic stress are characterized by normal or slightly elevated plasma ACTH, increased hypothalamic corticotropin-releasing hormone (CRH) and vasopressin secretion, decreased pituitary CRH receptors and hypersensitivity of the ACTH and glucocorticoid responses to a novel stress. To determine the role of CRH and vasopressin in the pituitary hyperresponsiveness to a superimposed stress, pituitary CRH receptors and plasma ACTH responses were measured in rats receiving minipump infusions of CRH or a combination of CRH and vasopressin (VP), 50 ng/min of each for 50 h. Rats were killed by decapitation with or without exposure to ether vapor for 5 min or immobilization for 15 or 30 min, and blood was collected for ACTH and corticosterone determinations. The pituitary CRH receptor concentration measured by binding 125I-Tyr-oCRH, was reduced by 45 and 80% in CRH- and CRH-plus-VP-infused rats, respectively, with no changes in receptor affinity. Acute stress by ether exposure or immobilization had no effect on pituitary CRH receptors. Adrenal weight was significantly increased, and thymus weight decreased in CRH-infused animals, indicating activation of the pituitary adrenal axis. However, in contrast to the responses following chronic stress, the increases in plasma ACTH in response to an injection of 10 micrograms/kg CRH or acute stress were significantly lower in CRH- and CRH-plus-VP-infused rats. Furthermore the content and release of ACTH from quartered pituitaries were also decreased in chronically treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Participation of alpha 1-adrenergic receptors in the secretion of hypothalamic corticotropin-releasing hormone during stress.

The role of alpha 1-adrenergic receptors in the secretion of corticotropin-releasing hormone (CRH) during stress was studied by immunohistochemical analysis of the CRH content of the median eminence (ME) after intracerebroventricular (icv) administration of the alpha 1-adrenergic agonist, methoxamine, or the antagonist, prazosin, in rats pretreated with colchicine. Immunohistochemical staining was performed by the peroxidase technique on 40 microns free-floating sections using a polyclonal antibody specific for CRH. In the first experimental model, rats were implanted with icv cannulae and adapted to the experimental conditions by daily handling and icv injection of artificial CSF. Colchicine (75 micrograms) was administered through the cannulae 6 h before the experiment, conditions in which axonal transport was blocked with little change in basal immunostaining. Two hours after immobilization stress or a single injection of methoxamine (100 micrograms, icv), there was a marked decrease in CRH immunoreactivity throughout the ME, reflecting release of the neuropeptide into the portal circulation. The decrease in CRH immunostaining following immobilization was largely prevented by icv injection of the alpha 1-adrenergic antagonist, prazosin. In the second experimental model, rats were sacrificed 48 h after icv colchicine injection, conditions in which colchicine acts as a stressor and causes marked depletion of irCRH from the ME. This chronic effect of colchicine was also partially prevented by administration of prazosin, 400-ng injection 5 min prior to colchicine, followed by a continuous icv mini-pump infusion of prazosin, indicating that alpha 1-adrenergic stimulation contributes to the action of colchicine.(ABSTRACT TRUNCATED AT 250 WORDS)

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

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

Further studies on the relationship between potassium and sodium levels and adrenocortical activity.

Steroid production in vitro by dog adrenal cortical slices was measured in the presence of varying concentrations of sodium and potassium ions. Increasing concentrations of potassium produced a significant increase in the intracellular potassium content and in the rate of synthesis of aldosterone. However, the potassium effect on aldosterone secretion may also occur without changes in tissue potassium content. Ouabain significantly diminished intracellular potassium content, but inhibited aldosterone production only at the high dose of 2.5 X 10(-3) M in the presence of an elevated external potassium level. Physiological changes in sodium concentration can modify aldosterone production. The effect was observed with changes as small as 10 mEq/liter sodium. Changes in external potassium and sodium levels modulate aldosterone as well as corticosterone, but not cortisol production. No changes in the intracellular content of potassium were detectable in angiotensin-stimulated tissue. Nevertheless, high doses of ouabain blocked angiotensin-stimulated aldosterone production without affecting cortisol production. The dissociation observed between intracellular potassium levels and aldosterone production suggests that there is not a simple relationship between the two parameters.