Biomedical subjects

G Aguilera

Publications and source records attributed to G Aguilera.

At least 19 recordsLinked to original sources

PubMed2006

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.

Analysis of Variance↗

PubMed2004

Regulation of pituitary corticotropin releasing factor type-1 receptor mRNA binding proteins by modulation of the hypothalamic-pituitary-adrenal axis.

Long-term adrenalectomy (ADX) is associated with marked down-regulation of pituitary corticotropin releasing factor type-1 receptors (CRF-R1) but normal CRF-R1 mRNA levels, suggesting that regulation of receptor levels occurs at post-transcriptional sites. We have reported that adrenal cytosolic proteins, which bind to cis elements in the 5' leader sequence (5'LS) of the rat angiotensin II type 1a receptor (AT(1a)R) mRNA, participate in the regulation of AT(1a)R density by inhibiting AT(1a)R mRNA translation. In this study, we examined anterior pituitary cytosolic proteins that form RNA protein complexes (RPC) with the 5'LS of the CRF-R1 and the AT(1a)R. Competition studies and ultraviolet-crosslinking analysis suggest that formation of CRF-R1 and AT(1a)R 5'LS RPC require at least some proteins that are common to both receptor mRNAs. Pituitaries isolated from male Sprague-Dawley rats six days after ADX showed significant (P < 0.05) increases of 2.9-fold in CRF-R1 5'LS RNA binding protein (BP) activity compared to pituitaries from sham-operated rats; this effect of ADX was prevented by glucocorticoid replacement. By contrast, no differences in the number of pituitary AT(1a)R binding sites or pituitary AT(1a)R 5'LS BP activity were observed between sham and ADX rats, indicating that the effect of ADX on RPC formation was specific for CRF-R1 mRNA. Addition of pituitary cytosolic extracts inhibited in vitro translation of CRF-R1 mRNA by 79% under conditions that had no significant effects on the translation of a control mRNA. The data suggest that CRF-R1 translation is regulated by modulation of the HPA axis through pituitary cytosolic proteins binding to the CRF-R1 5'LS.

5' Untranslated Regions↗

PubMed2003

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.

Amygdala↗

PubMed2003

Single administration of interleukin-1 increased corticotropin releasing hormone and corticotropin releasing hormone-receptor mRNA in the hypothalamic paraventricular nucleus which paralleled long-lasting (weeks) sensitization to emotional stressors.

Single exposure to the proinflammatory cytokine interleukin-1 induces sensitization of the adrenocorticotropin hormone and corticosterone responses to stressors weeks later (hypothalamus-pituitary-adrenal sensitization). Hypothalamus-pituitary-adrenal responses are controlled by corticotropin-releasing hormone and arginine-vasopressin secreted from parvocellular corticotropin-releasing hormone neurons of the hypothalamic paraventricular nucleus and may involve autoexcitatory feedback mechanisms. Therefore, we studied the temporal relationship between resting levels of corticotropin-releasing hormone, corticotropin-releasing hormone-R1 and arginine-vasopressin receptor (V1a, V1b) mRNAs in the paraventricular nucleus and the development of hypothalamus-pituitary-adrenal sensitization to an emotional stressor (novelty). The adrenocorticotropin hormone precursor molecule proopiomelanocortin hnRNA in the pituitary gland served as an index for acute activation. Single administration of interleukin-1 induced sensitization of the hypothalamus-pituitary-adrenal to novelty from 3 to 22 days later, but not after 42 days. Single administration of interleukin-1 induced biphasic increases in corticotropin-releasing hormone and corticotropin-releasing hormone-R1 mRNAs in the paraventricular nucleus: an early peak within 24 h, followed by a delayed (>7 days) increase that peaked after 22 days. Hypothalamic V1a and V1b mRNA levels were unaffected. In contrast, in the pituitary gland, there was an early decrease in corticotropin-releasing hormone-R1 mRNA (from 10.5 to 3 h after interleukin-1) and V1b receptor mRNA (3 to 6 h), which returned to control levels from 24 h onwards. Thus, interleukin-1-induced long-lasting hypothalamus-pituitary-adrenal sensitizations associated with prolonged activation of corticotropin-releasing hormone and corticotropin-releasing hormone-R1 mRNA expression in the paraventricular nucleus, but not with changes in the expression of proopiomelanocortin hnRNA or V1b receptor or corticotropin-releasing hormone R1 mRNAs in the pituitary gland. We propose that transient exposure to immune events can induce long-lasting hypothalamus-pituitary-adrenal sensitization, which at least in part involves long-term hypothalamic adaptations that enhance central corticotropin-releasing hormone signaling.

Adrenocorticotropic Hormone↗

PubMed2003

Lipopolysaccharide endotoxin potentiates the effect of osmotic stimulation on vasopressin synthesis and secretion in the rat hypothalamus.

Vasopressin secreted by magnocellular neurones of the hypothalamic supraoptic and paraventricular nuclei is essential for water balance. In this study, we examined magnocellular neurone responses to osmotic stimulation in vehicle-injected controls or rats receiving an intraperitoneal (i.p.) injection of 250 microg/100 g of lipopolysaccharide (LPS), 3 h or 6 h earlier. LPS injection had no effect on plasma vasopressin concentrations in control rats but it caused marked and transient potentiation of the responses to a single i.p. injection of hypertonic saline (five- and two-fold, 3 and 6 h after LPS, respectively). The enhancement of plasma vasopressin responses was independent of plasma sodium concentrations or changes in blood pressure. Basal vasopressin mRNA expression in the paraventricular and supraoptic nuclei decreased slightly 6 h after LPS injection, without changes in vasopressin transcription as indicated by vasopressin heteronuclear (hn) RNA levels. Parvocellular neurones showed expected increases in vasopressin hnRNA expression following LPS injection and a further increase after i.p. hypertonic saline injection (due to the painful component). In contrast to magnocellular vasopressin mRNA expression, the effects of LPS and hypertonic saline injections in parvocellular neurones were additive and not synergistic. Light microscopic immunohistochemical examination revealed an increase in size of vasopressin but not oxytocin axonal terminals in the neural lobe 3 h after LPS injection. Osmotic stimulation caused marked depletion of vasopressin immunoreactivity in axonal terminals of the neural lobe in both control and LPS-pretreated rats. The changes in vasopressin axon terminals were accompanied by induction of interleukin (IL)-1 beta and IL-6 in the posterior pituitary. The data show that endotoxemia causes morphological and functional alterations of the hypothalamic neurohypophyseal system, resulting in facilitation rather than inhibition of vasopressin synthesis, and secretion in response to osmotic stimulation.

Animals↗

PubMed2003

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.

Animals↗

PubMed2003

Transcriptional and post-transcriptional mechanisms regulating the rat pituitary vasopressin V1b receptor gene.

The number of V1b vasopressin receptors (V1bR) in the anterior pituitary plays an important role during adaptation of the hypothalamic-pituitary-adrenal axis to stress in rats. Regulation of V1bR expression involves transcriptional and translational mechanisms. One of the elements mediating transcriptional activation of the rat V1bR gene is a long stretch of GAGA repeats (GAGA box) in the promoter located near the transcription start point capable of binding a protein complex of 127 kDa present in pituitary nuclear extracts. There is a lack of correlation between changes in V1bR mRNA and the number of VP binding sites, suggesting that V1bR expression depends on the efficiency of V1b R mRNA translation into protein. Two mechanisms by which the 5' untranslated region (5'-UTR) of the rat V1bR mRNA can mediate either inhibition or activation of V1bR mRNA translation have been identified. First, upstream open reading frames (ORF) present in the 5'-UTR repress translation of the major ORF encoding the V1b receptor, and secondly, an internal ribosome entry site (IRES) activates V1bR translation. Stimulation of IRES activity through protein kinase C-mediated pathways results in V1bR mRNA translation increasing V1bR protein levels. The existence of multiple loci of regulation for the V1bR at transcriptional and translational levels provides a mechanism to facilitate plasticity of regulation of the number of pituitary vasopressin receptors according to physiological demand.

5' Untranslated Regions↗

PubMed2002

A novel approach using transcomplementing adenoviral vectors for gene therapy of adrenocortical cancer.

Current therapies for adrenocortical carcinomas do not improve the life expectancy of patients. In this study, we tested whether a gene-transfer therapy based upon a suicide gene/prodrug system would be effective in an animal model of the disease. We employed E4- and E1A/B-depleted, herpes simplex virus-thymidine kinase-expressing adenoviral mutants that transcomplement each other within tumor cells, hereby improving transgene delivery and efficacy by viral replication in situ. Transcomplementation of vectors increased the fraction of transduced of tumor cells. This increase was accompanied by greater tumor volume reduction compared to non-transcomplementing approaches. Survival time improved with non-replicating vectors plus GCV compared to controls. However, transcomplementation/replication of vectors led to a further significant increment in anti-tumor activity and survival time (p < 0.02). In treated animals, we observed a high number of apoptotic nuclei both adjacent to and distant from injection sites and sites of viral oncolysis. Ultrastructural analyses exhibited nuclear inclusion bodies characteristic of virus production in situ, and provided further evidence that this therapy induced apoptotic cell death within tumor cells. We conclude that the efficacy of suicide gene therapy is significantly amplified by viral replication and, in combination with GCV, significantly reduces tumor burden and increases survival time.

Adenoviridae↗

PubMed2002

Multiple sites of control of type-1 corticotropin releasing hormone receptor levels in the pituitary.

Hypothalamic corticotropin releasing hormone (CRH) stimulates pituitary ACTH secretion through interaction with type 1 CRH receptors (CRH-R1), the number of which varies during alterations of the hypothalamic-pituitary-adrenal (HPA) axis. CRH-R1 are essential for ACTH responses to stress but CRH receptor content in the pituitary does not correlate with corticotroph responsiveness. This indicates that a small number of receptors is sufficient for full ACTH responses probably through post-receptor interaction with vasopressin (VP) signaling. CRH binding and hybridization studies in adrenalectomized, glucocorticoid-treated or stressed rats revealed divergent levels of CRH receptors and CRH-R1 mRNA in the pituitary, with binding reductions but normal or elevated CRH-R1 mRNA levels during alterations of the HPA axis. Western blot analysis of CRH-R1 protein in pituitary membranes from adrenalectomized rats show unchanged CRH-R1 mRNA levels, but reduced CRH binding associated with significant increases in CRH-R1 protein, suggesting that the decrease in binding is due to homologous desensitization and not to reduced receptor synthesis. In contrast, decreased CRH binding following glucocorticoid administration is associated with reduction in CRH-R1 protein suggesting inhibition of CRH-R1 mRNA translation. Regulation of CRH-R1 translation may involve binding of cytosolic proteins, and a minicistron in the 5'UTR of the CRH-R1 mRNA. Post-transcriptional regulatory mechanisms allowing rapid changes in CRH receptor activity are important for adaptation of corticotroph responsiveness to continuous change in physiological demand.

Adrenocorticotropic Hormone↗

PubMed2001

Hypothalamic and amygdaloid corticotropin-releasing hormone (CRH) and CRH receptor-1 mRNA expression in the stress-hyporesponsive late pregnant and early lactating rat.

This study investigated the expression of corticotropin releasing hormone (CRH) and its receptor CRHR-1, and arginine vasopressin (AVP) mRNAs during the stress hyporesponsive periods of late pregnancy and lactation (day-3) and in virgin stress-responsive females. In situ hybridization histochemistry showed that basal CRH mRNA in the paraventricular nucleus (PVN) decreased in pregnant and increased in lactating rats (compared with virgin controls), whereas it increased after restraint stress only in virgin rats. Basal PVN CRHR-1 mRNA increased markedly in all groups but reached lower levels in pregnant rats. Basal AVP mRNA in the parvocellular PVN was higher in lactating rats, and in contrast to CRH mRNA, it increased after stress in all groups. In medial preoptic area (MPOA) CRH mRNA levels were higher in lactating females compared with virgin and pregnant rats, and unexpectedly they decreased markedly after stress only in virgin rats. CRH mRNA levels in the central and medial nuclei of the amygdala were higher in lactating rats than in virgin or pregnant ones, and stress had no effect in either group. These data suggest that these stress hyporesponsive periods: (1) do not depend on basal CRH mRNA expression in the PVN; (2) appear to have intact stress-activated afferent pathways to the PVN, as shown by preservation of CRHR-1 and AVP responses to stress, but the information may be differently processed; (3) are associated with an alteration in a CRH mediated pathway from the MPOA.

Amygdala↗

PubMed2001

Hypothalamic pituitary adrenal axis and hypothalamic-neurohypophyseal responsiveness in water-deprived rats.

The differential effects of osmotic stimulation on magnocellular and parvocellular hypothalamic neurons were studied by analysis of corticotropin-releasing hormone (CRH) and vasopressin (VP) expression in controls and 48-h water-deprived rats subjected to either restraint for 1 h or a single lipopolysaccharide injection (250 microg/100 g). Water deprivation reduced basal CRH mRNA levels but the increments following 4 h of restraint or 6 h lipopolysaccharide (LPS) injection were similar to those in controls. In contrast, water deprivation had no effect on basal VP heteronuclear RNA (hnRNA) and mRNA levels in parvocellular neurons, but responses to restraint or LPS injection were reduced. VP expression in magnocellular paraventricular and supraoptic nuclei, and plasma sodium and vasopressin were higher in water-deprived rats, changes which were unaffected by restraint. LPS injection reduced VP mRNA but not hnRNA levels in magnocellular neurons and increased plasma vasopressin levels only in water-deprived rats independently of changes in plasma sodium. This was accompanied by an increase in vasopressin mRNA content in the posterior pituitary. The data show that the blunted ACTH responses to acute stress during chronic osmotic stimulation are correlated with the inability of parvocellular neurons to increase VP rather than CRH expression. In addition, LPS-induced endotoxemia causes disturbances of the magnocellular vasopressinergic system with an unexpected potentiation of osmotic simulated VP secretion. The lack of increase in VP transcription after LPS and changes in VP mRNA distribution suggest that endotoxemia affect the secretory process at the levels of the neurohypophyseal axon terminal.

Analysis of Variance↗

PubMed2001

Regulation of pituitary corticotropin releasing hormone receptors.

Corticotropin releasing hormone (CRH) stimulates pituitary ACTH secretion through type-1 CRH (CRH1) receptors. Stimulation of the hypothalamic pituitary adrenal (HPA) axis as well as increased corticotroph responsiveness during stress and adrenalectomy are associated with marked pituitary CRH binding downregulation. The presence of CRH1 receptors in the pituitary are essential to maintain ACTH secretion. Downregulation of CRH binding is associated with normal or elevated levels of CRH1 receptor mRNA and this may contribute to the maintainence of permissive levels of CRH1 receptors in the pituitary. Injection of either CRH or glucocorticoids in rats in vivo induces CRH binding and CRH1 receptor mRNA downregulation, whereas their simultaneous administration causes only transient CRH1 receptor mRNA loss. Vasopressin increases CRH1 receptor mRNA levels. This suggest that interactions between CRH, vasopressin and glucocorticoids accounts for CRH1 receptor mRNA upregulation during stress. The lack of correlation between CRH binding and CRH1 receptor mRNA indicates that the major sites for pituitary CRH1 receptor regulation are at the post-transcriptional level.

Adrenocorticotropic Hormone↗

PubMed2001

Regulation of corticotropin-releasing hormone (CRH) transcription and CRH mRNA stability by glucocorticoids.

1. The increases in corticotropin-releasing hormone (CRH) mRNA following long-term adrenalectomy are associated with low levels of CRH gene transcription, suggesting that glucocorticoids regulate CRH mRNA at the posttranscriptional level. In this study we determined the time course of transcriptional activation after early adrenalectomy by intronic in situ hybridization, and evaluated the effects of glucocorticoids on CRH mRNA stability. 2. Plasma corticosterone was undetectable 3 h after adrenalectomy, but CRH hnRNA increased only by 12 h, and remained elevated for the next 72 h. CRH mRNA increased 18 h after adrenalectomy and reached a plateau lasting from 2 to 6 days, despite very low CRH hnRNA levels. 3. Assessment of CRH mRNA stability, by incubation of slide-mounted hypothalamic sections in an intracellular-like medium at 37 degrees C, prior to measuring CRH mRNA levels by in situ hybridization, revealed a half-life (t1/2) of 11.5 min in sham-operated rats, and a slower decrease adrenalectomized rats (t1/2--26.3 min). Corticosterone administration for 3 days markedly decreased CRH mRNA t1/2 in both sham-operated and adrenalectomized rats (6.5 and 5.0 min, respectively). 4. The data show that adrenalectomy causes transient increases in CRH mRNA transcription, followed by decreases in the rate of CRH mRNA degradation. This suggests that glucocorticoids regulate CRH mRNA at two sites, by inhibiting transcription and by decreasing mRNA stability.

Adrenal Cortex Hormones↗

PubMed2001

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.

Adolescent↗

PubMed2001

Effect of repeated lipopolysaccharide administration on tissue cytokine expression and hypothalamic-pituitary-adrenal axis activity in rats.

The effects of chronic immune challenge on cytokine expression and hypothalamic-pituitary-adrenal axis (HPA) axis responses to stress were studied in Wistar rats after administration of increasing doses of lipopolysaccharide (LPS). Repeated LPS (R-LPS) decreased body weight and increased adrenal weight and pituitary pro-opiomelanocortin mRNA levels. LPS injection increased plasma adrenocorticotropic hormone (ACTH) and corticosterone but the effect was attenuated in R-LPS. Plasma corticosterone but not ACTH responses to restraint were also reduced in R-LPS. Basal and restraint-stimulated corticotropin releasing hormone (CRH) mRNA levels were lower in R-LPS, but responses to a new LPS injection were similar to controls. In contrast, type 1 CRH receptor (CRH-R1) mRNA responses to both LPS and restraint were blunted in R-LPS. Vasopressin mRNA levels in parvocellular neurones were higher in R-LPS, and increased further after restraint but not after a new LPS injection. Glucocorticoid receptor (GR) levels in the paraventricular nucleus (PVN) increased after a single LPS or R-LPS (24 h after the last injection) but declined after a new injection in R-LPS. Interleukin (IL)-1beta and IL-6 mRNAs increased in the pituitary, spleen and circumventricular organs after single or R-LPS, suggesting that cytokines may contribute to the activation of the HPA axis though pathways from the circumventricular organs as well as paracrine effects in the pituitary. The data show that (i) adaptation of the HPA axis during repeated LPS injection involves increases in vasopressin : CRH expression ratios in parvocellular neurones; (ii) that hypothalamic CRH and vasopressin responses to acute stimulation are independent of CRH-R1 expression in the PVN; and (iii) there is a dissociation between pituitary and adrenal responses to acute stress suggesting a decrease of adrenal sensitivity to ACTH.

Adrenocorticotropic Hormone↗

PubMed2001

Inhibition of corticotropin releasing hormone type-1 receptor translation by an upstream AUG triplet in the 5' untranslated region.

The influence of an upstream open reading frame (ORF) in the 5'-untranslated region (UTR) of the mRNA on corticotropin-releasing hormone receptor type 1 (CRHR1) translation was studied in constructs containing the 5'-UTR of CRHR1, with or without an ATG-to-ATA mutation in the upstream ORF, and the main ORF of luciferase or CRHR1. Upstream mutation in luciferase constructs increased luciferase activity when transfected into COS-7 or AtT20 cells compared with the native 5'-UTR. Transfection of CRHR1 constructs containing the upstream mutation into AtT20 or LVIP2.0zc reporter cells, resulted in higher (125)I-Tyr-oCRH binding and corticotropin-releasing hormone-stimulated cAMP production, without changes in CRHR1 mRNA levels (measured by RNase protection assay). In vitro translation of luciferase or CRHR constructs with or without mutation of the upstream ATG, and Western blot analysis with anti-luciferase and anti-CRHR1 antibodies confirmed that mutation of the upstream ATG increases translation of the main ORF. The mechanism by which the upstream ORF inhibits translation may involve translation of the upstream peptide, because in vitro translation, or transfection into LVIP2.0zc cells of a fusion construct of the upstream ORF and green fluorescent protein (GFP) yielded a band consistent with the molecular size of GFP protein. The study shows that the upstream AUG in 5'-UTR of CRHR1 mRNA inhibits receptor expression by inhibiting mRNA translation and suggests the short open reading frame in the 5'-UTR plays a role in regulating translation of the CRH receptor.

5' Untranslated Regions↗

PubMed2001

Direct stimulation of arginine vasopressin gene transcription by cAMP in parvocellular neurons of the paraventricular nucleus in organotypic cultures.

The regulation of arginine vasopressin (AVP) gene transcription in the paraventricular nucleus (PVN) was studied in rat hypothalamic organotypic cultures using intronic in situ hybridization. While AVP heteronuclear (hn) RNA was not detected in the PVN under basal conditions, a marked induction of AVP hnRNA was observed after 2 and 3 h incubation of slices with forskolin. In contrast to the stimulatory effects of forskolin, phorbol 12-myristate 13-acetate (PMA) was completely ineffective in inducing AVP hnRNA in the PVN at any time examined (1-3 h). Forskolin-induced AVP hnRNA expression was unaffected by blockage of neurotransmission by the sodium channel inhibitor, tetrodotoxin, indicating that forskolin acts directly on AVP cells in the PVN. Dual staining in situ hybridization of forskolin-stimulated hypothalamic sections using both radio labeled AVP hnRNA and digoxigenin-labeled CRH mRNA probes revealed colocalization of both transcripts, indicating AVP hnRNA is expressed in the parvocellular neurons. The data demonstrate that cAMP directly activates AVP gene transcription in parvocellular neurons of the PVN.

Animals↗

PubMed2000

Vasopressinergic regulation of the hypothalamic-pituitary-adrenal axis: implications for stress adaptation.

In addition to its role on water conservation, vasopressin (VP) regulates pituitary ACTH secretion by potentiating the stimulatory effects of corticotropin releasing hormone (CRH). The pituitary actions of VP are mediated by plasma membrane receptors of the V1b subtype, coupled to calcium-phospholipid signaling systems. VP is critical for adaptation of the hypothalamic-pituitary-adrenal (HPA) axis to stress as indicated by preferential expression of VP over CRH in parvocellular neurons of the hypothalamic paraventricular nucleus, and the upregulation of pituitary VP receptors during stress paradigms associated with corticotroph hyperresponsiveness. V1b receptor mRNA levels and coupling of the receptor to phospolipase C are stimulated by glucocorticoids, effects which may contribute to the refractoriness of VP-stimulated ACTH secretion to glucocorticoid feedback. The data suggest that vasopressinergic regulation of the HPA axis is critical for sustaining corticotroph responsiveness in the presence of high circulating glucocorticoid levels during chronic stress.

Adaptation, Physiological↗