Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.27.5 (RNase)
17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Activation of adenylyl cyclase by corticotropin-releasing hormone (CRH) stimulates secretion of adrenocorticotropin (ACTH) in rat anterior pituitary corticotropes and in the murine AtT-20 cell line. The stimulation of secretion is mediated by cAMP and is largely dependent on Ca(2+) influx through voltage-gated L-type Ca(2+) channels. To investigate whether CRH and cAMP also increase expression of the L-type Ca(2+) channel in AtT-20 cells, an RNase protection assay was used to measure the alpha(1C) mRNA that encodes the pore-forming subunit of the L-type Ca(2+) channel. The alpha(1C) mRNA level was measured by autoradiographic densitometry and normalized to the beta-actin mRNA level in the same sample. The alpha(1C) mRNA was not changed by 24-hour treatment with CRH (10-500 nM). A 24-hour treatment with 1 mM 8Br-cAMP significantly increased the alpha(1C) mRNA by 40% over its control. The stimulatory effect was blocked by 2 microM actinomycin D and was, therefore, dependent on gene transcription. The measured half-life of the alpha(1C) mRNA, after inhibition of transcription, was 4.7 +/- 0.3 h in control and 5.2 +/- 0.6 h in the presence of 8Br-cAMP. Thus the 8Br-cAMP- induced increase in alpha(1C) mRNA could be due to an increase in alpha(1C) gene transcription or to a transcriptionally regulated increase in a protein that helps to stabilize alpha(1C) mRNA. Finally, to determine if the increased mRNA was followed by an increase in production of L-type Ca(2+) channels, the binding of [(3)H]PN200-110 to Ca(2+) channel proteins was assayed in AtT-20 membrane fragments. 8Br-cAMP increased [(3)H]PN200-110 binding sites by 32% (B(max) 36.0 +/- 1.2 fmol/mg protein in control vs. 47.4 +/- 3.2 fmol/mg protein in 8Br-cAMP-treated cells) but did not change the K(d). These studies show that both alpha(1C) mRNA and L-type Ca(2+) channel protein are increased in AtT-20 cells by cAMP.
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PMID:Expression of the L-type Ca(2+) channel in AtT-20 cells is regulated by cyclic AMP. 1042 88

In response to stress, adrenocorticotropin (ACTH) is secreted from anterior pituitary corticotropes. Corticotropin-releasing hormone (CRH) is a potent stimulator of ACTH secretion. The CRH stimulation of secretion is mediated by cAMP and is largely dependent on Ca(2+) influx through voltage-gated L-type Ca(2+) channels. This study was designed to investigate whether the expression of L-type Ca(2+) channels in the rat anterior pituitary and in corticotropes is regulated by acute stress and CRH. RNase protection assays were used to quantify alpha(1C) mRNA of the L-type Ca(2+) channel. The alpha(1C) mRNA levels from stressed rats increased by 31% in anterior pituitaries of rats after 30 min of exposure to cold stress. Neither 60 min cold stress nor 30 min restraint stress had an effect on alpha(1C) mRNA levels. When alpha(1C) mRNA was detected by in situ hybridization in a population of corticotropes enriched to 90%, 0.5 nM CRH (3 h) stimulated a 36% increase in the average area of label/cell and a 10% increase in the average density of label. Our results suggest that (1) the expression of alpha(1C) subunit mRNA of L-type Ca(2+) channels is increased in the rat anterior pituitary with a stress-specific response that might reflect an increase both in thyrotropes and corticotropes (both are known to be stimulated by cold stress), and (2) the CRH-mediated increase in alpha(1C) mRNA expression in individual rat corticotropes, in vitro, supports the hypothesis that some of the increase in vivo is due to changes in corticotropes.
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PMID:Cold stress and corticotropin-releasing hormone induced changes in messenger ribonucleic acid for the alpha(1)-subunit of the L-type Ca(2+) channel in the rat anterior pituitary and enriched populations of corticotropes. 1042 89

Among the five members of the melanocortin receptor (MC-R) family, MC2 and MC5 are expressed in peripheral tissues. The receptor MC2 (ACTH receptor) almost exclusively expressed in the adrenal cortex whereas MC5-R is expressed in several organs including the adrenal cortex. Both receptors bind ACTH and activate adenylate cyclase. The aim of this work was to study the spatial distribution of MC5-R among the different zones of the bovine adrenal cortex and to analyze the regulation of its expression by its own ligands, ACTH and alpha-MSH and by angiotensin II (AII). Using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and RNase protection assay, MC5-R was detected only in the glomerulosa zone whereas MC2-R was present in both glomerulosa and fasciculata zones of adult adrenal cortex. Treatments by ACTH, alpha-MSH, or AII increased the MC5-R mRNA level in glomerulosa cells by factors 7, 5, and 4.5, respectively. However, although potentially regulated by hormones, MC5-R is expressed at a level at least 100 times less than MC2-R, suggesting that MC5-R expression might only be at trace levels in grown adults, but could be much higher during embryogenesis.
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PMID:Expression and regulation of melanocortin receptor-5 (MC5-R) in the bovine adrenal cortex. 1068 56

In sheep, the ACTH secretory response to CRH in vivo or in vitro changes as a function of development, with peak responses occurring several weeks before term (145 days of gestation). CRH-stimulated ACTH secretion is mediated via the G protein-coupled CRH type I (CRH R1) receptor. We used a quantitative ribonuclease protection assay and Western immunoblotting to determine messenger RNA (mRNA) and protein levels of the CRH R1 receptor in immature and mature fetuses and adults. In addition, we precociously elevated fetal plasma cortisol levels to determine whether the fetal CRH R1 receptor is sensitive to increases in plasma cortisol. CRH R1 receptor mRNA levels decreased markedly throughout gestation and into the transition to adult life (immature fetus, 1.24+/-0.17; mature fetus, 0.75+/-0.13; adult, 0.18+/-0.093 pg/microg total anterior pituitary RNA). Also, continuous cortisol infusion in immature fetuses significantly decreased CRH R1 mRNA levels by 41%. Similar decreases were noted in protein levels. Thus, the decreased ACTH response to CRH stimulation during late gestation may be related to decreased CRH R1 receptor expression. In addition, plasma cortisol levels may influence corticotroph responsiveness to CRH by decreasing CRH R1 receptor expression.
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PMID:Corticotropin-releasing hormone type I receptor messenger ribonucleic acid and protein levels in the ovine fetal pituitary: ontogeny and effect of chronic cortisol administration. 1091 74

The introduction of a targeted insertion mutation into exon 2 of the gene coding for the glucocorticoid receptor (GR) enabled production of glucocorticoid receptor knock-out (GRKO) mice. GRKO mice on a C57BL/6/129sv mixed genetic background show a variable phenotype, with 90% of -/- mice dying at birth with respiratory insufficiency but 10% of mutant mice surviving to maturity. To investigate the possibility of residual GR expression in surviving GRKO mice we have measured binding of the synthetic glucocorticoid dexamethasone in tissue extracts from adrenalectomized mice. High affinity binding of dexamethasone in protein extracts of liver, kidney, lung and brain from adult GRKO mice is found at levels 30-60% those in wild-type mice, with heterozygotes (+/-) having intermediate levels. PCR and ribonuclease protection analysis showed comparable levels of GR mRNA on the 3' side of the gene-targeted insertional mutation in exon 2 of the GR gene, with almost no GR mRNA detected from exons 1 and 2 on the 5' side of the gene-targeted insertional mutation. Western blot analysis using a C-terminal specific GR antibody detects a 39 kDa GR fragment in extracts from adult GRKO mice. Despite the evidence for expression of a ligand-binding domain fragment of the glucocorticoid receptor these mice are profoundly glucocorticoid resistant, with elevated levels of plasma ACTH and corticosterone. Thymocytes from adult and fetal GRKO mice are resistant to dexamethasone-induced apoptosis and cultured fetal hepatocytes from GRKO mice are completely refractory to glucocorticoid induction of the gluconeogenic enzyme glucose-6-phosphatase. Thus although the surviving adult homozygous GRKO mice express a dexamethasone-binding GR fragment, their classic target tissues remain profoundly glucocorticoid insensitive.
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PMID:GRKO mice express an aberrant dexamethasone-binding glucocorticoid receptor, but are profoundly glucocorticoid resistant. 1122 90

Organisms respond to infection in a complex manner involving bidirectional interactions between the neuroendocrine and immune systems. Many of the bioactive endocrine/immune factors are synthesized in a precursor form and are expected to be activated by prohormone convertases (PCs). Since patients with both type 1 and type 2 diabetes have an increased incidence and severity of infections, we hypothesized that in a condition of hyperglycemia, these processing enzymes would be activated in an immune tissue, the spleen. To test this hypothesis, we treated rats with intraperitoneal streptozotocin (STZ) (50 mg/kg/day) daily for 5 days and measured splenic PC1 and PC2 mRNA by ribonuclease protection assay. We found that PC1 mRNA was increased 6.0+/-0.02-fold (P<0.05) and PC2 mRNA was increased 1.80+/-0.01-fold (P<0.005) in the spleen of rats that received STZ compared to rats that received vehicle. Western blot indicated that the 75-kDa form of PC1 was the only form of PC1 present in the spleen and that this form increased with STZ treatment. Immunohistochemistry revealed that PC1 was found in both the white pulp (T-lymphocytes) and red pulp (monocytes and macrophages) and that its increase in immunoreactivity occurred primarily in the white pulp. PC2 and pro-opiomelanocortin (POMC, a possible splenic substrate for PC1/PC2) immunoreactivity was found predominantly in the red pulp. STZ induced an increase in splenic PC1 and POMC, but not PC2 protein levels. We conclude that in the STZ model of diabetes, splenic PCs are induced, which could lead to an increased activation of many immune-derived hormones. We speculate that this up-regulation of prohormone converting enzymes may be related to the increased infections seen in patients with both type 1 and type 2 diabetes.
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PMID:Up-regulation of splenic prohormone convertases PC1 and PC2 in diabetic rats. 1173 Sep 86

Responsiveness of the hypothalamo-pituitary-adrenal axis is decreased during pregnancy. Therefore, the objective of the present study was to determine if responsiveness at the level of individual corticotrophs to corticotropin-releasing hormone (CRH) or arginine vasopressin (AVP) is decreased during pregnancy in sheep. Anterior pituitaries (APs) were collected from pregnant and nonpregnant ewes. Half of the APs were dispersed, and cells were placed on immobilon and treated with vehicle, CRH (10 nM), or AVP (100 nM) for 2 h. Cells were then fixed and incubated with ACTH or pro-opiomelanocortin (POMC) antibodies. The percentage of cells staining positive for immunoreactive (ir) ACTH or POMC, the percentage of cells secreting irACTH or POMC, and the area of irACTH or POMC secretion were measured. RNA was extracted from the other half of the APs to quantify CRH type 1 (CRH-R1) and vasopressin type 1b (V1b) receptor mRNA by ribonuclease protection assay. CRH treatment increased the percentage of corticotrophs with relatively large areas of irACTH and POMC secretion in nonpregnant, but not in pregnant, ewes. AVP treatment significantly increased the percentage of irACTH- and POMC-secreting cells in nonpregnant, but not in pregnant, ewes. V1b receptor mRNA, but not CRH-R1 receptor mRNA, was significantly decreased during pregnancy. These results suggest that corticotroph responsiveness to CRH and AVP is decreased during pregnancy in sheep. Therefore, reduced corticotroph responsiveness may contribute to stress hyporesponsivity during pregnancy.
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PMID:Attenuation of corticotropin-releasing hormone and arginine vasopressin responsiveness during late-gestation pregnancy in sheep. 1202 Oct 66

In the present study, it was hypothesized that the adrenocorticotrophin hormone receptor (ACTH-R) would be up-regulated in the adrenal gland of the sheep fetus following infusion of physiological amounts of ACTH, as shown for adrenal cortical cells in culture. In chronically catheterized sheep, an intravenous infusion of ACTH(1-24) was given to 6 fetuses for 24 h at a rate of 0.5 microg h(-1), starting on Day 126 or 127 of gestation (term approximately 147 days). Four control fetuses received an infusion of vehicle (saline). Total RNA was extracted from the fetal adrenal glands by the guanidinium thiocyanate method. Expression of specific mRNAs was determined by ribonuclease protection assay using cRNA probes directed against: ACTH-R; the steroid enzymes side-chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), 17apha-hydroxylase (P450c17) and 21beta-hydroxylase (P450c21); and beta-actin. Ratios of mRNA expression to beta-actin mRNA expression (arbitrary units) were calculated to correct for differences in RNA quality between samples. The concentration (mean +/- SEM) of immunoreactive cortisol in fetal plasma was greater after ACTH infusion than after vehicle infusion (47 +/- 3 v. 13 +/- 2 ng mL(-1) respectively; P<0.001). Adrenal expression of P450scc and P450c21 mRNA increased after ACTH infusion (P<0.05), whereas expression of P450c17 and 3beta-HSD mRNA was unchanged. There was no difference in ACTH-R mRNA expression between ACTH- and vehicle-infused fetuses (254 +/- 48 v. 305 +/- 76 arbitrary units respectively). It was concluded that ACTH is able to increase plasma cortisol concentrations in the sheep fetus by up-regulating cortisol synthesis in the adrenal gland, but that in vivo this does not require up-regulation of ACTH-R mRNA.
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PMID:Adrenocorticotrophic hormone (ACTH) stimulation of sheep fetal adrenal cortex can occur without increased expression of ACTH receptor (ACTH-R) mRNA. 1205 14

The peripheral injection of alcohol stimulates the activity of the hypothalamic-pituitary-adrenal (HPA) axis, but the ready penetration of this drug in most bodily compartments has made it difficult to identify its specific sites of action. Here we determined whether alcohol can directly influence the corticotropes. We first determined whether alcohol acted within the brain to stimulate neurons in the paraventricular nucleus (PVN) of the hypothalamus, which synthesizes corticotropin-releasing factor (CRF) and vasopressin (VP). To test this hypothesis, we injected alcohol intracerebroventricularly (icv; 5 microl of 200-proof; 86 micromol) and compared these results with those obtained after its ip administration (3.0 g/kg). Although not causing neuronal damage and not leading to detectable levels of the drug in the general circulation, icv alcohol significantly up-regulated PVN CRF heteronuclear RNA levels and increased plasma ACTH levels, a change comparable to the one observed in the ip model. To determine whether alcohol stimulated the corticotropes independently of CRF and/or VP, we injected the drug ip or icv and measured changes in anterior pituitary proopiomelanocortin (POMC) transcripts and ACTH release in the presence or absence of endogenous CRF and/or VP. Intracerebroventricular and ip alcohol significantly increased POMC primary transcript levels, measured by ribonuclease protection assay, over a time-course that corresponded to ACTH release. Both the POMC and the ACTH responses were completely abolished by removal of CRF and VP. Collectively, these results indicate that alcohol-induced activation of the corticotropes does not represent a direct influence of the drug on the pituitary but requires CRF and VP.
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PMID:Site of action of acute alcohol administration in stimulating the rat hypothalamic-pituitary-adrenal axis: comparison between the effect of systemic and intracerebroventricular injection of this drug on pituitary and hypothalamic responses. 1520 75

CRH-binding protein (CRH-BP) binds CRH with high affinity and inhibits CRH-mediated ACTH release from anterior pituitary corticotrope-like cells in vitro. In female mouse pituitary, CRH-BP is localized not only in corticotropes, but is also expressed in gonadotropes and lactotropes. To investigate the functional significance of gonadotrope CRH-BP, we examined the molecular mechanisms underlying GnRH-regulated CRH-BP expression in alphaT3-1 gonadotrope-like cells. CRH-BP is endogenously expressed in alphaT3-1 cells, and quantitative real-time RT-PCR and ribonuclease protection assays demonstrate that GnRH induces a 3.7-fold increase in CRH-BP mRNA levels. GnRH also induces intracellular CRH-BP (2.0-fold) and secreted CRH-BP (5.3-fold) levels, as measured by [125I]CRH:CRH-BP chemical cross-linking. Transient transfection assays using CRH-BP promoter-luciferase constructs indicate that GnRH regulation involves protein kinase C-, ERK- and calcium-dependent signaling pathways and is mediated via a multipartite GnRH response element that includes activator protein 1 and cAMP response element (CRE) sites. The CRE site significantly contributes to GnRH responsiveness, independent of protein kinase A, representing a unique form of multipartite GnRH regulation in alphaT3-1 cells. Furthermore, EMSAs indicate that alphaT3-1 nuclear proteins specifically bind at activator protein 1 and CRE sites. These data demonstrate novel regulation of pituitary CRH-BP, highlighting the importance of the pituitary gonadotrope as a potential interface between the stress and reproductive axes.
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PMID:Gonadotropin-releasing hormone (GnRH) positively regulates corticotropin-releasing hormone-binding protein expression via multiple intracellular signaling pathways and a multipartite GnRH response element in alphaT3-1 cells. 1597 7


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