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:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have developed a multiplex, competitive, reverse-transcriptase polymerase chain reaction (RT-PCR) method which measures absolute levels of renin, angiotensinogen, and the housekeeping transcript elongation factor-1 alpha (EF-1 alpha) mRNA. Sample RNA was simultaneously titrated with serial dilutions of renin, angiotensinogen, and EF-1 alpha competitor RNAs which flanked the endogenous concentrations of target transcripts. The samples were coreverse transcribed in the presence of random primers and resulting first-strand cDNA was coamplified for 10-15 cycles with [32P]-dCTP and primers for renin angiotensinogen, after which EF-1 alpha primers were added. Amplified DNA was separated by electrophoresis on polyacrylamide gel and radioactivity in the bands was quantified by direct radioanalytical scanning. Three conditions were necessary to obtain absolute quantification of renin and angiotensinogen mRNA levels: (a) exogenous competitor RNA was used to control for tube-to-tube variability in the efficiencies of reverse transcription and amplification; (b) Sample RNA was titrated with flanking concentrations of competitor RNA to correct for intraassay differences in the efficiency of amplification due to concentration differences between competitor and target templates; and (c) a housekeeping transcript EF-1 alpha was used to control for tube-to-tube differences in RNA loading and/or degradation. We show that the multiplex RT-PCR method is precise and accurate over approximately three logs of transcript concentration and sensitive to less than 5 and 0.5 fg for renin and angiotensinogen mRNA, respectively. This method will be useful for absolute quantification of target mRNAs, especially when the amount of sample RNA is limited or unknown and/or the gene expression is low.
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PMID:An improved method for absolute quantification of mRNA using multiplex polymerase chain reaction: determination of renin and angiotensinogen mRNA levels in various tissues. 788 70

This study was designed to quantitate cardiac mRNA levels encoding components of the local renin-angiotensin system during the development of volume overload-induced cardiac hypertrophy. Changes in cardiac renin mRNA levels were measured in relation to renin activity in the left ventricle (LV) and in plasma after acute passive stretch of the heart caused by an aortovenocaval shunt in the rat. A quantitative reverse-transcriptase polymerase chain reaction method with competitive internal standards was used to measure mRNA levels in total RNA derived from cardiac tissues after shunt. Seven days after shunt surgery, LV weight was increased by 23%. Renin activities were elevated four- and twofold in plasma and LV, respectively. LV angiotensinogen mRNAs were not significantly increased by shunt surgery; they were twofold higher than phosphoglycerate kinase mRNA from the housekeeping gene PGK-1. By day 7, LV levels for renin mRNA were significantly increased from well below 0.25% to approximately 1% of PGK-1 mRNA. Identity between renin polymerase chain reaction products from kidney and heart cDNAs and absence of "reninlike" amplification products were supported by Southern blotting. Volume overload caused increased expression of the renin gene in the stretched myocardium. This finding is consistent with the concept of a myocardial renin-angiotensin system that can be activated by locally produced renin and contributes to the hypertrophy of cardiac muscle.
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PMID:Stretch-mediated activation of cardiac renin gene. 794 10

At least two distinct genes (AT1A and AT1B) encode type 1 angiotensin II (AT1) receptors in rodents. Receptor binding and Northern blot analysis have clearly demonstrated the presence of AT1 receptors and AT1-receptor mRNA in many tissues but fail to differentiate which type 1 receptor subtype is expressed. A reverse-transcriptase polymerase chain reaction restriction fragment length polymorphism (RT-PCR-RFLP) assay was developed to differentiate the expressed mRNA by subtype. Expression of AT1A was clearly evident in kidney, liver, adrenal gland, ovary, brain, testes, adipose tissue, lung, and heart of adult mice. AT1B was absent from most of these tissues but was detectable in brain, testes, and adrenal gland. No significant differences in expression were evident in kidney, liver, brain, lung, or heart from 16.5- or 18.5-gestation-day fetuses, and only AT1A was evident in placenta. Expression of AT1B was confirmed in adrenal gland, brain, and testes, using a primer set that specifically amplifies only AT1B mRNA. Expression of AT1A and AT1B was also examined in As4.1 cells, a renin-expressing mouse kidney tumoral cell line. Receptor binding and competition assays using AT1- and AT2-receptor antagonists revealed that only AT1 receptors are present on the cell surface. Extremely low levels of AT1-receptor mRNA was detected by Northern blot, and RT-PCR-RFLP analysis revealed that only the AT1A subtype is expressed in this cell line. Despite the high homology between the coding sequence of the AT1A and AT1B genes, they exhibit disparate tissue-specific expression profiles.
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PMID:Differential expression of angiotensin receptor 1A and 1B in mouse. 807 5

Using reverse-transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry we investigated the ontogeny of renin, angiotensinogen and angiotensin converting enzyme (ACE) in the mesonephros at 27 and 41 days of gestation, and the metanephros at 41 and 64 days of gestation in ovine fetuses (term is 145 to 150 days). The volume and composition of fetal urine, stored as allantoic fluid were measured in 12 fetuses at 27 days, and 13 fetuses at 41 days. Renin, angiotensinogen and ACE were identified in both meso- and metanephroi at 41 days but not in the mesonephros at 27 to 30 days. Allantoic fluid volumes were 21 +/- 3 and 45 +/- 5 ml at 27 to 30 days and 41 days, respectively. This fluid was significantly different in composition to that of amniotic fluid or maternal plasma. The results suggest that the mesonephros can substantially modify its glomerular filtrate by 27 days of gestation, and can produce local angiotensin II by 41 days.
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PMID:Ontogeny of hormonal and excretory function of the meso- and metanephros in the ovine fetus. 891 29

D2-like receptors in the kidney have been suggested to be important in the regulation of renin release but the D2-like subtype(s) expressed in juxtaglomerular (JG) cells is not known. Therefore, we determined which of the D2-like family of dopamine receptors is located in primary cultures of rat juxtaglomerular (JG) cells. Reverse transcriptase-polymerase chain reaction (RT-PCR) identified D3 and D4 but not D2Long mRNA in JG cells (n = 3). D3 receptor function was demonstrated by a concentration-dependent inhibition of forskolin-stimulated cAMP production by LY-171555 (a non-selective D2-like receptor agonist) and PD-128593 (a partially selective D3 agonist) (n = 3-7/group). The stimulatory action of LY-171555 and PD-128593 we blocked by the non-selective D2-like antagonist YM-09151. We conclude that D3 and D4 dopamine receptor subtypes are expressed in JG cells; the receptor subtype linked to the inhibition of cAMP in JG cells remains to be established.
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PMID:Dopamine D3 receptors in rat juxtaglomerular cells. 902 38

All the angiotensin peptides originate from angiotensinogen, a glycoprotein synthesized by several tissues, including the brain and the anterior pituitary. In the rat, immunohistochemistry has been used to localize angiotensinogen in gonadotropes and in uncharacterized cells surrounding sinusoids. Both cell types are capable of secreting angiotensinogen in cell culture; only the gonadotropes contain angiotensin II (AngII) and are capable of secreting it in culture. It has been asserted that the perisinusoidal cells are the only source of angiotensinogen for the generation of AngII by gonadotropes. Our current data favor the existence of a complete intracellular renin-angiotensin system (RAS) in gonadotropes and a separate extracellular system which utilizes the high concentration of angiotensinogen from perisinusoidal cells. Furthermore, we postulate that gonadotrope AngII serves mainly reproductive functions, while the proximity of angiotensinogen-secreting cells to folliculostellate cells, and their access to the intercellular sinusoidal and follicular spaces, places the extracellular RAS in a strategic position to affect pituitary growth and the mediation of acute-phase immune responses. In the rat brain, angiotensinogen is expressed by the 16-18th day of fetal life and by areas generally concerned with vasopressor, electrolyte, and fluid homeostasis. Antisense deoxyoligonucleotides to angiotensinogen mRNA lower blood pressure in hypertensive rats and inhibit in vitro growth of neuroblastoma cells, indicating a significant role for angiotensinogen in mitogenic and homeostatic functions. It is commonly agreed that astrocytes express angiotensinogen. Neuronal angiotensinogen has also been demonstrated by immunohistochemistry, as a secretion from neuronal cell cultures, and by reverse-transcriptase polymerase chain reaction. The fate of secreted astrocytic and neuronal angiotensinogen remains obscure. Angiotensinogen is regulated in a tissue-specific manner with smaller or absent responses observed for brain tissue. By using astrocyte and neuronal cultures the actions on angiotensinogen production of growth hormone, IGF-1, inflammatory lipopolysaccharide, and phorbol ester have been examined. Recent observations show that angiotensinogen is regulated positively or negatively by glucocorticoids and that a positive synergism between cAMP and glucocorticoids exists. On the basis of analogous systems for other proteins, a scheme involving glucocorticoid receptors, CREB, and AP-1 transcription factors is formulated to explain glucocorticoid-cAMP interactions. These transcriptional interactions may form a significant functional link between the RAS and adrenergic mechanisms.
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PMID:Novel perspectives on pituitary and brain angiotensinogen. 910 Dec 59

The hypothesis, based on previous in vivo data, that angiotensin AT1 receptors are regulated by GH or insulin-like growth factor I (IGF-I) has been investigated in this study using primary cultures of rat astrocytes as a model of AT1 receptor expression. At a dose of 1 ng/ml GH, there was an increase in AT1 density within 4 h and a maximum increase of 361 +/- 57% of the control value at 12 h. At 24 h, receptor density was still 176 +/- 23% that in the control. Astrocytes incubated with 1 ng/ml rat IGF-I for 24 h showed no change in AT1 receptor density. Reverse transcriptase-PCR was used to show that astrocytes express both the AT1a receptor subtype and, to a much lesser extent, the AT1b subtype. Treatment with 1 ng/ml recombinant bovine GH for 12 h increased the messenger RNA of the AT1a receptor by 170%, without affecting the AT1b receptor. Inhibition of protein synthesis by cycloheximide and of transcription by the adenosine analog dichlororibofuranosylbenzimidazole both prevented the increase in AT1 receptor density following GH treatment, indicating that the action of GH is transcriptional. In summary, we have shown that GH up-regulates, directly and not via IGF-I, angiotensin receptors of the AT1a subtype in astrocytes by a transcriptional mechanism. The long latency of the response and the dependency on transcription relegate the AT1a gene to the class of GH-regulated genes identified as delayed stable genes. This mechanism of AT1 activation may be one way in which GH activates the renin-angiotensin system and initiates consequential cardiovascular and angiogenic effects.
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PMID:Growth hormone regulates AT-1a angiotensin receptors in astrocytes. 932 27

The aim of the study was to investigate whether the adrenal renin-angiotensin system plays an independent role in the regulation of mineralocorticoid biosynthesis in the adrenal gland and to explore the mechanisms of this action. Twelve-week-old male Sprague-Dawley rats were studied: 22 rats were maintained on a regular diet; 27 and 22 rats received a low salt diet with and without treatment, respectively, with the angiotensin II (Ang II) AT1-subtype receptor antagonist losartan (10 mg/kg per day). A fraction of each group of rats underwent bilateral nephrectomy (n = 12, 15, and 10, respectively) and was killed 48 hours later. In an additional group of 24 (12 intact and 12 nephrectomized) rats, the effects of the Ang II AT2-subtype receptor antagonist PD123319 were investigated. In intact rats, plasma renin activity (PRA) and adrenal renin activity and expression were progressively raised by salt restriction and losartan, whereas aldosterone synthase mRNA and plasma aldosterone (PA) levels were increased by salt restriction and reduced by losartan. Forty-eight hours after nephrectomy, PRA fell to undetectable levels; in contrast, adrenal renin expression, assessed by semiquantitative reverse-transcriptase polymerase chain reaction (using GAPDH as a standard for gene expression), showed an 18-fold increase and was further increased after salt restriction and losartan (all P < .05). Also, adrenal renin activity was raised after nephrectomy and further increased after salt restriction (P < .05) and losartan. Cytochrome P450 aldosterone synthase expression in the adrenal cortex was stimulated by nephrectomy alone and by nephrectomy combined with low salt intake (P < .05), with consequent increases in PA concentrations. In losartan-treated salt-restricted nephrectomized rats, cytochrome P450 aldosterone synthase expression (P < .05 versus nephrectomy alone and nephrectomy plus salt restriction) and PA concentrations were diminished (P < .05) in spite of the observed increases of adrenal renin expression. The AT2-receptor antagonism did not significantly affect PRA, adrenal renin, and aldosterone biosynthesis and production in either intact or nephrectomized salt-restricted rats. These results demonstrate that the adrenal renin-angiotensin system plays an independent role in the regulation of mineralocorticoid biosynthesis in vivo. This action is mediated primarily via the Ang II AT1-subtype receptors.
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PMID:Role of tissue renin in the regulation of aldosterone biosynthesis in the adrenal cortex of nephrectomized rats. 935 60

In this study we showed, for the first time, the existence of a moderate density of specific angiotensin II (Ang II) binding sites (Kd=3.9+/-1.7 nM and Bmax=467.2 130.0 fmol/mg protein) in plasma membrane preparations from rat thyroid gland. Reverse transcriptase/polymerase chain reactions, using primers based on the cloned AT1 and AT2 receptor subtypes, and pharmacological characterization, using the Ang II receptor subtype antagonists Losartan and PD 123319, revealed that these Ang II binding sites match with the AT1 receptor subtypes. To obtain more information on the molecular structure of this Ang II receptor, immunoblotting analyses were carried out using a polyclonal rabbit anti-AT1 antiserum. Western analysis of fresh plasma membrane preparations from thyroid tissue showed three prominent bands of approximately 60, 45 and 40 kDa which appear to be related to different degrees of glycosylation of the receptor molecule. The functional significance of the Ang II receptors in thyroid gland is currently not known. Nevertheless, since Ang II receptors play a pivotal role in the co-ordinated actions of the renin-angiotensin system (RAS), our findings support a reciprocal regulation of thyroid function by the RAS.
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PMID:Characterization of angiotensin II receptors (binding and mRNA) in the rat thyroid gland. 968 52

Previous studies have suggested the presence of an intrinsic renin-angiotensin system (RAS) in the rat epididymis with functions in epididymal activity and sperm maturation. In the present study, the localization and expression of angiotensinogen, the component of the RAS which is indispensable for intracellular angiotensin generation, were investigated by immunochemistry, hybridization histochemistry and by reverse-transcriptase polymerase chain reaction (RT-PCR). Western blot analysis of protein from the epididymis confirmed the presence of angiotensinogen with the expected molecular mass of about 60 kDa, in agreement with results from other tissues. Immunocytochemistry showed the regional localization of immunoreactivity for angiotensinogen in the rat epididymis. In situ hybridization histochemistry further demonstrated the expression of angiotensinogen mRNA by the epididymal epithelium in a region-specific manner along the length of the rat epididymis. RT-PCR confirmed that the rat epididymis expresses angiotensinogen mRNA. On the other hand, mRNA of renin was not detected in the rat epididymis using Northern blot and RT-PCR analyses. The present study strongly supports the existence of an intrinsic, angiotensin-generating system based on locally formed angiotensinogen as a precursor for angiotensin production. This epididymal RAS may have paracrine or autocrine roles in mediating the epididymal and sperm functions.
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PMID:Angiotensinogen expression by rat epididymis: evidence for an intrinsic, angiotensin-generating system. 1058 Aug 44


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