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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

Heterotrimeric G-proteins function as signal transducers for a variety of hormone-coupled enzyme systems in eukaryotic cells. In LLC-PK1 renal cells, vasopressin-stimulated adenylylcyclase activity is regulated in part, by the counterbalancing activity of stimulatory G-proteins (Gs) and inhibitory pertussis toxin-sensitive G-proteins (Gi). Two Gi genes encoding the Gi isoforms G alpha i-2 and G alpha i-3 are expressed in LLC-PK1 cells. In polarized cells, these isoforms are topographically segregated to different membranes, which allows for the selective inhibition of adenylylcyclase by G alpha i-2. The genes encoding these isoforms are similarly regulated in these cells during growth and differentiation but differ in response to steroid hormone signals (Holtzman, E.J., Kinane, T.B., West, K., Soper, B.W., Karga, H., Ausiello, D.A., and Ercolani, L. (1993) J. Biol. Chem. 268, 3964-3975). We now demonstrate after stimulating polarized LLC-PK1 cells with forskolin, which raises intracellular cAMP levels 50-fold, G alpha i-2 but not G alpha i-3 protein is increased 3-fold at 12 h and remains elevated above control values by 24 h. In cells stably transfected with G alpha i-2 or G alpha i-3 gene 5'-flanking sequences fused to firefly luciferase cDNA reporter gene, forskolin treatment increased G alpha i-2 transcription 3-fold but inhibited G alpha i-3 transcription by 50% at 12 h. In vivo footprinting of forskolin-treated cells was performed to examine the molecular basis for activation of the G alpha i-2 gene. Protected guanosines were identified in a 135-base pair (bp) area previously associated with enhancer activity of this gene in non-polarized cells. This DNA segment did not contain the classical cAMP response element 5'-TGACGTCA-3'. Utilizing the 135-bp DNA segment as a probe in mobility shift assays, which compared nuclear extracts from cells before and after forskolin treatment, an induced nuclear protein complex was identified. Following systematic reduction and mutation of this DNA segment, a "CCAAT" box motif was identified that bound the induced nuclear protein complex during forskolin-induced G alpha i-2 gene transcriptional activation. Induction of this nuclear protein complex was prevented in forskolin-treated cells by cycloheximide. To demonstrate functional activity of the CCAAT box motif, cells were transiently transfected with plasmids encoding either the minimal 135-bp segment or a multimerized CCAAT box segment fused to a Rous sarcoma minimal promoter/firefly luciferase reporter gene.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:cAMP regulates G-protein alpha i-2 subunit gene transcription in polarized LLC-PK1 cells by induction of a CCAAT box nuclear binding factor. 822 26

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

Studies of bioluminescence in living animals, such as cell-based biosensor applications, require measurement of light at different wavelengths, but accurate light measurement is impeded by absorption by tissues at wavelengths<600 nm. We present a novel approach to this problem--the use of a plastic window in the skin/body wall of mice--that permits measurements of light produced by bioluminescent cells transplanted into the kidney. The cells coexpressed firefly luciferase (FLuc), a vasopressin receptor--Renilla luciferase (RLuc) fusion protein, and a GFP2-beta-arrestin2 fusion protein. Following coadministration of two luciferase substrates, native coelenterazine and luciferin, bioluminescence is measured via the window using fiber optics and a photon counter. Light emission from the two different luciferases, FLuc and RLuc, is readily distinguishable using appropriate optical filters. When coelenterazine 400a is administered, bioluminescence resonance energy transfer (BRET) occurs between the RLuc and GFP2 fusion proteins and is detected by the use of suitable filters. Following intraperitoneal injection of vasopressin, there is a marked increase in BRET. When rapid and accurate measurement of light from internal organs is required, rather than spatial imaging of bioluminescence, the combination of skin/body wall window and fiber optic light measurement will be advantageous.
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PMID:Bioluminescence measurements in mice using a skin window. 1799

Abstract Oxytocin (OT) plays a role in reproduction at the level of the pituitary and mammary glands and uterus. This OT is synthesized in the hypothalamo-neurohypophyseal system (HNS). A number of observations have suggested that estrogens regulate the production of OT in the HNS. In this study the effect of 17beta-estradiol on the activity of the OT gene promoter was examined as well as the effect of 17beta-estradiol in vivo on OT messenger ribonucleic acid (mRNA) and peptide revels in the rat HNS. Vasopressin (VP) and its mRNA were also determined in the in vivo studies. The direct transcriptional stimulation of OT gene expression by 17beta-estradiol was studied in two different heterologous expression systems. When a plasmid having nucleotides -363 to +16 of the rat OT gene fused to the firefly luciferase reporter gene was co-transfected with an estrogen receptor expression vector in P19 embryonal carcinoma cells, luciferase activity was stimulated 80-fold by 17beta-estradiol. In estrogen receptor containing MCF-7 cells transfected with a plasmid having nucleotides -188 to +16 of the rat OT gene fused to the chloramphenicol acetyl transferase gene, 17beta-estradiol induced the expression of the chloramphenicol acetyl transferase gene through the cloned promoter element. After in vivo treatment of ovariectomized rats with 17beta-estradiol, levels of OT mRNA and VP mRNA were measured in microdissected supraoptic and paraventricular nuclei as well as VP and OT levels in these nuclei and the pituitary gland. As compared to non-treated ovariectomized rats there was no difference in contents of OT mRNA and VP mRNA in these hypothalamic nuclei and in levels of the peptides in paraventricular nuclei and the pituitary gland. A 30% reduction of the OT content of the supraoptic nuclei only was found, while the VP content did not change. To explain the results immunocytochemical analyses of the hypothalamus were performed, showing that the estrogen receptor was absent in the magnocellular neurons of the supraoptic and paraventricular nuclei. The results demonstrate that the 5'flanking region of the OT gene confers estrogen-sensitivity to transcription of the OT gene. This potential to respond to estrogens is not used in the OT-producing neurons of supraoptic and paraventricular nuclei probably due to the absence of the estrogen receptor.
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PMID:Regulation of the rat oxytocin gene by estradiol. 1921 99