Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P01185 (vasopressin)
 23,126 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The possible role of cyclic AMP (cAMP) in the regulation of the vasopressin (VP) gene was tested in two cellular expression systems: one cell line with endogenous VP expression and the other which was transiently with a VP promoter-luciferase fusion gene. 8,Bromo-cAMP stimulated the VP mRNA content about 4-fold in the human VP-expressing small cell lung carcinoma cell line GLC-8. The luciferase activity in P19 embryonal carcinoma cells which were transiently transfected with -174 to +44 of the 5'-flanking region of the human VP gene linked to the firefly luciferase gene, was stimulated about 2-fold by the cAMP analogue. The results indicate that cAMP plays a role in the upregulation of the VP gene and hence point to several putative nucleotide motives in the promoter functionally conferring this response.
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PMID:Vasopressin gene expression is stimulated by cyclic AMP in homologous and heterologous expression systems. 217 21

Addition of two commercial luciferin-luciferase reagents caused marked inhibition of the aggregatory response of washed human platelets to thrombin, ADP, vasopressin and platelet-activating factor (PAF). Analysis of the effects of the individual components of one of these reagents revealed that Mg2+, and to a lesser extent bovine serum albumin, was responsible for the observed inhibition. A modified luciferin/luciferase reagent has been designed on the basis of these data for use in washed platelet suspensions which causes minimal inhibition of the aggregatory and secretory responses to thrombin but which gives a near maximal luminescence yield.
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PMID:Inhibition by luciferin-luciferase reagents of aggregatory responses to excitatory agonists in washed platelet suspensions. 400

Genomic clones including the 5' flanking regions of the AQP2 (aquaporin 2) gene were isolated, and the promoter region was examined by transiently transfecting a promoter-luciferase reporter fusion gene into renal cultured epithelial cells. An orientation specific promoter for the AQP2 gene was found within the proximal 3 kb of 5'-flanking region. Minimal basal promoter activity of the AQP2 gene was found within 198 bp upstream from the transcription start site by deletion analysis. Sequencing the transcriptionally active region revealed a typical TATA box, adenosine 3',5'-cyclic monophosphate (cAMP) responsive element (CRE) and three putative CCAAT boxes in the proximal 1.2-kb region. Significantly, a GATA motif, AP1, AP2, and SP1 transcriptional factor consensus sites were also found in this region. Exposure to cAMP-enhancing agents (1 nM vasopressin or 20 mM forskolin and 250 mM 3-isobutyl-1-methylxanthine) showed that these agents increased luciferase activity in a parallel fashion, suggesting that vasopressin-induced AQP2 gene transcription is mediated through increases in intracellular cAMP in at least one renal cell type, the LLC-PK1 cells. The mechanism of cAMP responsiveness of AQP2 gene transcription was further studied using a series of deletion mutants in renal epithelial cells and other cell types. The cAMP regulatory motifs were shown to exist in a 50-bp sequence between -340 and -290 (containing CRE) and a 65-bp sequence (containing an AP2 site) between -150 and the ATG start site in LLC-PK1 cells. In rat inner medullary collecting duct (IMCD) cells, the cAMP regulatory motifs also exist in a 50-bp sequence between -340 and -290 (containing CRE) and in a 10-bp sequence between -160 and -150 (containing an SP1 site). These separate regions may cooperate to confer full cAMP inducibility to the AQP2 gene in a cell-specific manner.
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PMID:cAMP motifs regulating transcription in the aquaporin 2 gene. 876 52

Although the effects of the various secretagogues on corticotropin (ACTH) secretion have been well studied, their effects on the POMC gene expression have not been thoroughly characterized. In this study, we established a new model system using the AtT20 mouse corticotroph tumor cell line transfected stably with a plasmid containing 0.7 kb of the rat POMC 5' promoter-luciferase fusion gene. The responsiveness to exogenous CRH improved markedly when the cells were cultured with low serum medium (1% FBS) compared with serum rich medium (10%). Using this culture condition, we examined the effects of not only CRH but also other secretagogues such as catecholamines, vasopressin, and angiotensin II, upon the transcriptional activity of the POMC gene. CRH stimulated POMC promoter activity (3.5-fold increase) as well as cAMP generation and ACTH secretion in a dose- and time-dependent manner, with the maximal effect being observed 3-5 h after the start of incubation. Catecholamines, especially epinephrine (10 nM and above), also stimulated all parameters, although less potently than CRH, and the effect was mimicked by the beta-, but not alpha-adrenergic, agonist, suggesting the involvement of the beta-adrenergic receptor. The combined effects of epinephrine and CRH were greater in all parameters than those of CRH alone, and the effects of both hormones were completely blocked by H89, an inhibitor of protein kinase A. Vasopressin and angiotensin II showed minimal effects on POMC expression. Our results suggest that 1) catecholamines, as well as CRH, positively regulate the POMC gene at physiological concentrations; 2) the cAMP-PKA system is the common intracellular signaling pathway for CRH and catecholamines; and 3) vasopressin and angiotensin II also have weak but significant stimulatory effects on POMC promoter activity.
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PMID:Regulation of the rat proopiomelanocortin gene expression in AtT-20 cells. I: Effects of the common secretagogues. 911 88

To study the transcriptional regulation of the vasopressin gene in vitro, 3 kb of the 5' regulatory region of the rat vasopressin gene was isolated and subcloned, along with a series of various deletion mutants, into vectors containing the luciferase reporter gene. After transfecting these genes transiently into the human choriocarcinoma cell line JEG-3 along with a glucocorticoid receptor (GR) expression vector, transcriptional activity was quantitated using the luciferase assay. Forskolin, 8-bromo-cAMP, and protein kinase A catalytic subunit expression all markedly increased transcription from the 3-kb promoter. Analyses with deletion mutants of the promoter showed that two cAMP-responsive element (CRE)-like sequences (-227 to -220 bp and -123 to -116 bp) contribute to this positive regulation. Expression of KCREB, a dominant negative mutant of the cAMP-responsive element binding protein (CREB), suggested the involvement of CREB. Transfection of the activator protein 2 (AP2) DNA consensus sequence partially blocked transcription. Dexamethasone suppressed forskolin-stimulated expression. The negative effect of glucocorticoid was GR dependent and may be mediated by a mechanism not involving GR binding to DNA because it was independent of the putative glucocorticoid-responsive element previously reported in the vasopressin promoter (-622 to -608 bp) and was preserved in the shorter promoter constructs in which no glucocorticoid-responsive element-like sequence was found. Our data suggest that several trans-acting factors including CREB, AP2, and GR are likely to be involved in vasopressin gene transcription and that the positive and negative regulation of vasopressin gene transcription is complex.
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PMID:Positive and negative regulation of the rat vasopressin gene promoter. 938 10

Prolonged stimulation of rat A7r5 aortic smooth muscle cells with 3 microM vasopressin, or of hamster DDT1 MF-2 smooth muscle cells with 10 microM bradykinin or 100 microM histamine led within 4 h to a 40-50% down-regulation of the type 1 InsP3 receptor (InsP3R-1) and of the type 3 InsP3 receptor (InsP3R-3). InsP3R down-regulation was a cell- and agonist-specific process, since several other agonists acting on PLC-coupled receptors did not change the expression level of the InsP3R isoforms in these cell types and since no agonist-induced down-regulation of InsP3Rs was observed in HeLa cells. Down-regulation of InsP3Rs was prevented by an inhibitor of proteasomal protease activity, N-acetyl-Leu-Leu-norleucinal (ALLN). The Ca2+ channel blocker verapamil (2 microM) also induced InsP3R-1 down-regulation (43%) in A7r5 cells, which was inhibited by ALLN. In A7r5 cells transiently transfected with a cDNA construct, bearing a luciferase coding sequence under control of the rat InsP3R-1 promoter, reduced luciferase activity could be demonstrated upon stimulation of cells with vasopressin or verapamil. Thus, besides enhanced protein degradation, a reduction of InsP3R promoter activity might contribute to the down-regulation of InsP3Rs in A7r5 cells. We next investigated the effect of InsP3R down-regulation on Ca2+ responses in A7r5 cells. A rightward shift in the dose-response curve for InsP3-induced Ca2+ release was observed in permeabilized monolayers of vasopressin-pretreated A7r5 cells (EC50 630 nM and 400 nM for pretreated and non-pretreated cells, respectively). The Ca2+ responses to threshold doses of vasopressin were markedly reduced in intact vasopressin-pretreated cells. We conclude that prolonged agonist-exposure leads to down-regulation of InsP3Rs in A7r5 and DDT, MF-2 smooth muscle cells. The mechanism of down-regulation likely involves proteasomal degradation and reduction of InsP3R promoter activity. Moreover, down-regulation of InsP3Rs resulted in desensitization of Ca2+ release from InsP3 sensitive stores.
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PMID:Agonist-induced down-regulation of type 1 and type 3 inositol 1,4,5-trisphosphate receptors in A7r5 and DDT1 MF-2 smooth muscle cells. 957 6

Several reporter gene assays have been described where gene transcription is activated as a consequence of a specific signal transduction event, such as activation of adenylyl cyclase (1.2). Reporter genes typically consist of specific responsive elements placed upstream of a minimal promoter, which together control the expression of a readily detectable reporter protein, such as luciferase. We have developed a dual glow-signal firefly and Renilla luciferase assay, which allows the simultaneous measurement of two reporter genes in the same well of a 96-well plate. In this report we demonstrate the use of this assay for the simultaneous analysis of agonist activity at two G-protein coupled receptors which signal through activation of the G-protein alpha sub-unit, G alpha S. Chinese hamster ovary (CHO) cells stably transfected with a cAMP responsive firefly luciferase reporter were further transfected with the human Vasopressin V2 receptor. Similarly, CHO cells stably transfected with a cAMP responsive Renilla luciferase reporter were further transfected with the human beta 2-adrenoceptor. The two cell lines were mixed in individual wells of a 96-well plate and a number of compounds were screened to determine their activity at both receptors. Stimulation with vasopressin and beta 2-adrenoceptor agonists resulted in the activation of the firefly and Renilla luciferases respectively. Stimulation with forskolin, which directly stimulates adenylyl cyclase, caused the activation of both reporter genes, and stimulation with a range of further compounds with no activity at either receptor did not generate a reporter response. The dual luciferase assay allows the simultaneous screening of two receptors in a 96-well format resulting in significant time and cost savings.
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PMID:Development of a dual glow-signal firefly and Renilla luciferase assay reagent for the analysis of G-protein coupled receptor signalling. 1007 73

It is now widely accepted that ATP functions as a signalling substance in the nervous system. The presence of P2 receptors mediating the action of extracellular ATP in brain regions involved in hormonal regulation raises the possibility that a similar role for ATP might also exist in the neuroendocrine system. In this study, the release from the rat isolated neurohypophysis preparation of endogenous ATP, oxytocin and vasopressin (AVP) were measured simultaneously using luciferin-luciferase and RIA techniques. After 70 min preperfusion, electrical field stimulation caused a rapid increase in the amount of ATP in the effluent and the release of AVP and oxytocin also increased stimulation-dependently. Inhibition of voltage-dependent Na+ channels by tetrodotoxin (1 microM) reduced the stimulation-evoked release of AVP and oxytocin; however, the evoked release of ATP remained unaffected. The effect of endogenous ATP on the hormone secretion was tested by suramin (300 microM), the P2 receptor antagonist. Suramin significantly increased the release of AVP, and the release of oxytocin was also enhanced. ATP, when applied to the superfusing medium, decreased the release of AVP, but not that of oxytocin, and its effect was prevented by suramin. ATP (60 nmol), added to the tissues, was readily decomposed to ADP, AMP and adenosine measured by HPLC combined with ultraviolet light detection, and the kinetic parameters of the enzymes responsible for inactivation of ATP (ectoATPase and ecto5'-nucleotidase) were also determined (Km=264+/-2.7 and 334+/-165 microM and vmax=6.7+/-1.1 and 2.54+/-0.24 nmol/min per preparation (n=3) for ectoATPase and ecto5'-nucleotidase respectively). Taken together, our data demonstrate the stimulation-dependent release, P2 receptor-mediated action and extracellular metabolism of endogenous ATP in the posterior lobe of the hypophysis and indicate its role, as a paracrine regulator, in the local control of hormone secretion.
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PMID:Local regulation of vasopressin and oxytocin secretion by extracellular ATP in the isolated posterior lobe of the rat hypophysis. 1007 81

Regulation of pituitary vasopressin V1b receptors plays a critical role in regulating pituitary adrenocorticotropic hormone (ACTH) secretion during adaptation to stress. The objective of this study was to isolate the promoter regulatory region of the V1b receptor gene to better understand the molecular mechanisms involved in V1b receptor regulation. Screening of a rat genomic library using probes directed to the coding region and to the 5'UTR of the rat V1b receptor resulted in the isolation of several clones containing the 5'upstream regions of the V1b receptor cDNA. Sequencing of an 11.2 Kb fragment revealed 8.2 Kb upsteam of the reported cDNA sequence, which contains a putative promoter regulatory region. The 3' end of the clone contained 1472 base pairs corresponding to the recognized cDNA sequence, followed by 1506 bp of unknown sequence located at the end of the sixth transmembrane domain, probably corresponding to an intron, characteristic of these family of receptors. An additional 161 bp intron was found in the 5'UTR, similar to that described in the rat oxytocin receptor gene. 5'RACE and RNase protection analysis mapped two major putative transcription start points at -830 and -861 bp from the starting methionine. Analysis of the putative promoter region showed no indication of a proximal TATA box, but the presence of a CACA box, a GAGA box, several AP-1 and AP-2 sites and a cluster of Sp1 sites upstream of the AP-2 sites. A luciferase construct containing a 2.1-kb of putative promoter, and part of the 5'UTR including the first intron, showed promoter activity when transfected into COS-7, CHO and PC12 cell lines but not in AtT-20 cells. A similar construct without the intron and distal 5'UTR sequence has no promoter activity in the same cell lines. In summary, the V1b receptor gene contains at least 3 exons and 2 introns. The 5'flanking sequence contains several potential sites for transcriptional regulation, and induced luciferace activity only in constructs containing intron 1, suggesting that the latter is important for receptor gene activation. The data provide bases for future analysis of the regulatory elements controlling V1b receptor transcription.
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PMID:Isolation and characterization of the promoter region of the rat vasopressin V1b receptor gene. 1079 83

In the kidney, water reabsorption is mainly regulated by the binding of arginine vasopressin to vasopressin type 2 (V2) receptors. These receptors are expressed selectively in principal cells of the collecting ducts. To identify molecular mechanisms responsible for the cell-specific expression of the V2 receptor, we have analyzed the proximal promoter of the corresponding gene. We report the identification of a 33-bp enhancer [collecting duct tissue-specific element 1 (CSE1)] that induced high levels of expression of the luciferase reporter gene in three collecting duct cell lines, but not in other renal cell lines. In gel shift assays, CSE1 bound a DNA-binding protein expressed selectively in collecting duct cell lines, and a 7-bp mutation, which abolished the activity of CSE1 in transient transfection experiments, also abolished the binding of this protein. Furthermore, decoy experiments performed using CSE1 showed that this sequence was involved not only in the expression of a construct containing 4.2 kb of the V2 receptor proximal promoter, but also in the expression of the endogenous V2 receptor gene. CSE1 appears to act mostly by counteracting the inhibitory effects of a strong ubiquitous repressor element that we called CIE1. Collectively, these results identify the first functional collecting duct-specific cis-acting element.
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PMID:Identification of a short cis-acting element in the human vasopressin type 2 receptor gene which confers high-level expression of a reporter gene specifically in collecting duct cells. 1104 82


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