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:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Conditions for the highly specific selection of a cell type by the use of lectin-coated magnetic beads are reported for the isolation of inner medullary collecting duct (IMCD) cells from a heterogeneous inner medullary cell suspension, containing both single cells and tubular fragments of variable size. The lectin Dolichos Biflorus Agglutinin (DBA), which binds in rat inner medulla exclusively to IMCD cells, was coupled via the avidin-biotin system to beads. By isolating DBA-bead-IMCD cells in a magnetic field (positive selection) from a suspension containing about 50% IMCD, a fraction of 98 +/- 1% purity was obtained; recovery of cells was up to 90%. Suspensions negative on reverse-transcriptase polymerase chain reaction for vimentin as a marker of contaminating interstitial and vascular cells could be received by repeating this procedure and additional trypsinization. On the other hand, it was possible to reduce the portion of IMCD cells in the suspension by one isolation step to 1.5 +/- 0.9% (negative selection). Performing this step twice resulted in virtually pure suspensions. No significant effects of this isolation technique on cell viability, growth characteristics, and biochemical parameters were observed. Therefore, this method appears to be a powerful tool for the highly specific separation of heterogeneous cell populations.
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PMID:Highly specific separation of heterogeneous cell populations by lectin-coated beads: application for the isolation of inner medullary collecting duct cells. 980 26

The NaCl-reabsorbing collecting duct epithelium develops by budding and branching of the embryonic ureter. The expression of Na+ channels during this branching morphogenesis was studied in the outermost branches of rat ureteric buds (UB; embryonic day E15 to postnatal day P6) and in cortical collecting ducts (CCD; days P7-P28) in primary monolayer culture. Expression of both Na+ channel mRNA and of Na+-selective membrane conductance were estimated by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and by patch-clamp recording, respectively. UB and CCD uniformly represented a principal-like cell type in culture. Messenger RNA encoding the alpha-ENaC subunit was detected in oligo-dT primed cDNA (5 ng) of embryonic UB cells (E15-17) after 30 PCR cycles. The abundance of alpha-ENaC mRNA, when normalized by reference to beta-actin, was higher by a factor of 2 in postnatal (P1-6) UB and by a factor of 5 in CCD cells (P7-14) compared with the embryonic stage. Highly Na+-selective, low-conductance channels were identified in apical patches from both UB and CCD monolayers, but only CCD cells exhibited macroscopic, amiloride-sensitive Na+ currents in whole-cell patch-clamp recordings. We conclude that alpha-ENaC mRNA and functional Na+ channel protein are expressed already before morphogenesis of the CCD is completed and prior to the onset of epithelial NaCl reabsorption.
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PMID:Expression of the epithelial sodium channel (ENaC) during ontogenic differentiation of the renal cortical collecting duct epithelium. 991 8

Previous in vivo studies in cardiomyopathic hamsters suggested that the expression of vasopressin (AVP) V2 mRNA is up- regulated by angiotensin II. The present study was performed to determine whether angiotensin II plays a role in regulating the expression of AVP V2 mRNA and aquaporin-2 (AQP2) mRNA in the inner medullary collecting duct (IMCD) of the male Wistar rat. The expression of AVP V2 mRNA and AQP2 mRNA in the IMCD was measured by competitive reverse-transcriptase polymerase chain reaction (RT-PCR). Six groups of experiments were performed. In the first group, we incubated IMCD with 3 different doses of angiotensin II (10(-11), 10(-9) and 10(-7) mol/L). Angiotensin II caused a significant increase in the AVP V2 mRNA in a dose-dependent manner but its effect on AQP2 mRNA was modest. This effect of angiotensin II was inhibited by angiotensin II receptor antagonist, [Sar1,Ile8]-angiotensin II. To examine the role of PKA in mediating an increase in AVP V2 mRNA expression, we incubated IMCD with 10(-7) and 10(-11) M of angiotensin II in the presence of a specific protein kinase A (PKA) inhibitor, Rp diasteroisomer of adenosine 3'-5'-cylic monophosphothionate (Rp-cAMPS). The angiotensin II-induced upregulation of V2 mRNA was abolished. In the fourth group, we examined the effect of protein kinase C (PKC) inhibition on V2 mRNA expression. The upregulation of V2 mRNA induced by angiotensin II was greatly exaggerated when IMCD was incubated with angiotensin II and RO-31-8220 (PKC inhibitor). In the fifth and sixth groups of studies, we determined the direct effect of PKA and PKC on regulating the expression of V2 mRNA and AQP2 mRNA in the IMCD, respectively. Dibutryl cAMP stimulated an upregulation in the expression of V2 mRNA and AQP2 mRNA, whereas phorbol esters suppressed the expression of V2 mRNA. These results suggested that PKA stimulates and PKC suppresses the expression of V2 mRNA in the IMCD of the kidney.
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PMID:Angiotensin II upregulates the expression of vasopressin V2 mRNA in the inner medullary collecting duct of the rat. 1264 65

Circulating endothelin (ET) levels are elevated in heart failure and positively correlated with severity of heart failure. Recent studies demonstrated arginine vasopressin (AVP) V2 mRNA expression was upregulated in the inner medullary collecting duct (IMCD) of cardiomyopathic hamsters (CM). The goal of the present studies was to determine if ET-1 is involved in upregulating the expression of AVP V2 mRNA in the IMCD of CM by using a mixed ETA/ETB receptor antagonist bosentan. Our results showed plasma ET-1 levels increased in CM hamsters and related with the severity of heart failure. The competitive reverse-transcriptase polymerase chain reaction (RT-PCR) method was used to quantify the expression of AVP V2 and aquaporin 2 (AQP2) mRNA in the IMCD. AVP V2 mRNA expression was elevated in placebo-treated CM hamsters and decreased significantly with 14 days of bosentan treatment. Similar results were seen with AQP2 mRNA. The effect of bosentan in normalizing the expression of AVP V2 and AQP2 mRNA in the IMCD of CM was confirmed by in situ hybridization studies. Bosentan treatments reduced the intensitites of the signals in the IMCD of CM hamsters to that seen in normal hamsters. This study demonstrated that AVP V2 and AQP2 mRNA are upregulated in CM hamsters and these upregulations are attenuated by bosentan treatment, suggesting that ET-1 plays a role in upregulating the expression of AVP V2 mRNA in CM hamsters.
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PMID:Attenuation of renal vasopressin V2 receptor upregulation by bosentan, an ETA/ETB receptor antagonist. 1450 20

We examined the direct epithelial effects of the major product of arachidonic acid metabolism in the kidney, prostaglandin E(2) (PGE(2)), on ion transport and signal transduction in the hormone-sensitive Madin-Darby canine kidney (MDCK) C7 subclone as a model of renal collecting duct principal cells. MDCK C7 cells were grown on microporous permeable filter supports and mounted in Ussing-type chambers. Reverse transcriptase (RT)-PCR and sequencing were used to determine E-prostanoid (EP) receptor expression. Basolateral and, about 14-fold less potent, apical addition of PGE(2) increased short-circuit current (I(sc)) in a concentration-dependent manner. This ion transport was biphasic with a rapid peak not detectable under chloride-free conditions. The remaining, stably elevated current was unaffected by furosemide, hydrochlorothiazide, ethylisopropanol amiloride, and 5-nitro-2-(3-phenyl-propyl-amino)benzoic acid (NPPB). In contrast, apical amiloride (10 microM) significantly decreased I(sc), indicating sodium reabsorption. The effect of PGE(2) was attenuated in the presence of vasopressin. Agonists acting by cAMP elevation like dibutyryl-cAMP and theophylline also induced an amiloride-sensitive ion transport with similar kinetics as PGE(2). Moreover, PGE(2) rapidly increased intracellular cAMP levels. RT-PCR demonstrated mRNA expression of the epithelial sodium channel (ENaC), and of the EP2 receptor in MDCK C7 cells. Accordingly, EP2 receptor agonist butaprost mimicked PGE(2) epithelial action. In conclusion, PGE(2) induces amiloride-sensitive sodium reabsorption in MDCK C7 monolayers. This ion transport is most likely mediated by EP2 receptor activation leading to increased intracellular cAMP levels. Therefore, PGE(2) might also contribute to Na(+) reabsorption in the mammalian collecting duct.
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PMID:Prostaglandin E2 stimulates sodium reabsorption in MDCK C7 cells, a renal collecting duct principal cell model. 1458 Mar 65

To unravel the molecular regulation of renal transcellular Ca(2+) transport, a murine distal convoluted tubule (mpkDCT) cell line derived from distal convoluted tubules (DCT) microdissected from a SV-PK/Tag transgenic mouse was characterized. This cell line originated from DCT only, as mRNA encoding for the DCT marker thiazide-sensitive Na(+)/Cl(-) cotransporter was expressed, whereas mRNA encoding for the connecting tubule and collecting duct marker aquaporin-2 was not detected, as determined by reverse-transcriptase PCR. mpkDCT cells expressed mRNA encoding the Ca(2+) channels TRPV5 and TRPV6 and other key players necessary for transcellular Ca(2+) transport, i.e., calbindin-D(9k), calbindin-D(28k), plasma membrane Ca(2+)-ATPase isoform 1b, and Na(+)/Ca(2+) exchanger 1. Primary cultures of DCT cells exhibited net transcellular Ca(2+) transport of 0.4 +/- 0.1 nmol.h(-1).cm(-2), whereas net transcellular Ca(2+) transport across mpkDCT cells was significantly higher at 2.4 +/- 0.4 nmol.h(-1).cm(-2). Transcellular Ca(2+) transport across mpkDCT cells was completely inhibited by ruthenium red, an inhibitor of TRPV5 and TRPV6, but not by the voltage-operated Ca(2+) channel inhibitors felodipine and verapamil. With the use of patch-clamp analysis, the IC(50) of ruthenium red on Na(+) currents was between the values measured for TRPV5- and TRPV6-expressing HEK 293 cells, suggesting that TRPV5 and/or TRPV6 is possibly active in mpkDCT cells. Forskolin in combination with IBMX, 1,25-dihydroxyvitamin D(3), and 1-deamino-8-d-arginine vasopressin increased transcellular Ca(2+) transport, whereas PMA and parathyroid hormone had no significant effect. In conclusion, the murine mpkDCT cell line provides a unique cell model in which to study the molecular regulation of transcellular Ca(2+) transport in the kidney in vitro.
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PMID:Characterization of a murine renal distal convoluted tubule cell line for the study of transcellular calcium transport. 1462 1

Recent studies in our laboratory have demonstrated that bosentan, a mixed endothelin ET(A)/ET(B) receptor antagonist, prevented the upregulation of the arginine vasopressin (AVP) V(2) receptor in the inner medullary collecting duct (IMCD) of cardiomyopathic hamsters. These results suggested that endothelin-1 (ET-1) is involved in the upregulation of AVP V(2) receptors. Studies were performed to detect the effect of ET-1 on the expression of AVP V(2) receptors and the ET receptor mediating these effects within the IMCD of the rat. Rat IMCD tissue was isolated and incubated with the following: ET-1, or ET-1 in combination with ET(A) and ET(B) receptor antagonists BQ-123 and BQ-788, respectively, and sarafotoxin c (S6c), an ET(B) receptor-specific agonist. Tissue samples were then analyzed using quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blotting. ET-1 treatment resulted in increased V(2) mRNA from a control level of 186.8 +/- 15.0 amol/microg total RNA to 430.7 +/- 49.0 amol/microg total RNA (P <.003). ET-1/ET(A) treatment resulted in no significant decrease in V(2) mRNA expression 335.0 +/- 38.0 amol/microg total RNA. Whereas ET-1/ET(B), and ET-1/ET(B)/ET(A) treatment resulted in V(2) mRNA approaching control 256.0 +/- 15.0 amol/microg total RNA, and 215.6 +/- 42.3 amol/microg total RNA. However, ET-3 treatment produced no significant changes in V(2) receptor mRNA expression. Sarafotoxin treatment corroborated both the ET-1 and ET receptor antagonist data, demonstrating striking significant increases in V(2) receptor mRNA and protein expression. S6c treatment increased V(2) mRNA expression from a control level of 199 +/- 17.3 amol/microg total RNA to 284.3 +/- 42.1 amol/microg total RNA (P < 05). Western blotting revealed that changes in V(2) mRNA expression in the various treatment conditions were similar to changes in protein expression. Overall, these data indicate that in the IMCD ET-1 increases AVP V(2) receptor expression and these changes are mediated by the ET(B) receptor.
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PMID:Endothelin upregulates the expression of vasopressin V2 mRNA in the inner medullary collecting duct of the rat. 1533 81

The activity of the epithelial sodium (Na(+)) channel (ENaC) in the aldosterone-sensitive distal nephron (ASDN) needs to be tightly regulated to match urinary Na(+) excretion with dietary Na(+) intake. The ubiquitin-protein ligase Nedd4-2, which in vitro interacts with ENaC subunits and reduces ENaC cell surface abundance and activity by ubiquitylation of the channel, may participate in the control of ENaC. This study confirms in vivo by reverse-transcriptase-PCR that Nedd4-2 is expressed throughout the nephron and is detectable by immunoblotting in kidney extracts. By immunohistochemistry, Nedd4-2 was found to be strongly expressed in the ASDN, with low staining intensity in the late distal convoluted tubule and early connecting tubule (where apical ENaC is high) and gradually increasing detection levels toward the collecting duct (CD; where apical ENaC is low). Compared with high-Na(+) diet (5% Na(+)), 2 wk of low-Na(+) diet (0.01% Na(+)) drastically reduces Nedd4-2 immunostaining and increases apical ENaC abundance in ASDN. Reduced Nedd4-2 immunostaining is not dependent on increased apical Na(+) entry in the CD, because it is similarly observed in mice with intact and with suppressed apical ENaC activity in the CD. Consistent with a role of mineralocorticoid hormones in the long-term regulation of Nedd4-2, 5-d treatment of cultured CD (mpkCCD(cl4)) cells with 1 microM aldosterone leads to reduction of Nedd4-2 protein expression. It is concluded that Nedd4-2 abundance is regulated by Na(+) diet, by a mechanism that likely involves aldosterone. This regulation may contribute to adaptation of apical ENaC activity to altered Na(+) intake.
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PMID:Dietary sodium intake regulates the ubiquitin-protein ligase nedd4-2 in the renal collecting system. 1657 85

K(+) channels play an important role in renal collecting duct cell function. The current study examined barium (Ba(2+))-sensitive whole-cell K(+) currents (IKBa) in mouse isolated collecting duct principal cells. IKBa demonstrated strong inward rectification and was inhibited by Ba(2+) in a dose- and voltage-dependent fashion, with the K (d) decreasing with hyperpolarization. The electrical distance of block by Ba(2+) was around 8.5%. As expected for voltage-dependent inhibition, the association constant increased with hyperpolarization, suggesting that the rate of Ba(2+) entry was increased at negative potentials. The dissociation constant also increased with hyperpolarization, consistent with the movement of Ba(2+) ions into the intracellular compartment at negative potentials. These properties are not consistent with ROMK but are consistent with the properties of Kir2.3. Kir2.3 is thought to be the dominant basolateral K(+) channel in principal cells. This study provides functional evidence for the expression of Kir2.3 in mouse cortical collecting ducts and confirms the expression of Kir2.3 in this segment of the renal tubule using reverse-transcriptase polymerase chain reaction. The conductance described here is the first report of a macroscopic K(+) conductance in mouse principal cells that shares the biophysical profile of Kir2.3. The properties and dominant nature of the conductance suggest that it plays an important role in K(+) handling in the principal cells of the cortical collecting duct.
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PMID:A Kir2.3-like K+ conductance in mouse cortical collecting duct principal cells. 1709 Dec 15

Aldosterone and endothelin-1 (ET-1) act on collecting duct cells of the kidney and are important regulators of renal sodium transport and cardiovascular physiology. We recently identified the ET-1 gene (edn1) as a novel aldosterone-induced transcript. However, aldosterone action on edn1 has not been characterized at the present time. In this report, we show that aldosterone stimulated edn1 mRNA in acutely isolated rat inner medullary collecting duct cells ex vivo and ET-1 peptide in rat inner medulla in vivo. Aldosterone induction of edn1 mRNA occurred in cortical, outer medullary, and inner medullary collecting duct cells in vitro. Inspection of the edn1 promoter revealed two putative hormone response elements. Levels of heterogeneous nuclear RNA synthesis demonstrated that edn1 mRNA stimulation occurred at the level of transcription. RNA knockdowns corroborated pharmacological studies and demonstrated both mineralocorticoid receptor and glucocorticoid receptor participated in this response. Aldosterone resulted in dose-dependent nuclear translocation and binding of mineralocorticoid receptor and glucocorticoid receptor to the edn1 hormone response elements. Hormone receptors mediated the association of chromatin remodeling complexes, histone modification, and RNA polymerase II at the edn1 promoter. Direct interaction between aldosterone and ET-1 has important implications for renal and cardiovascular function.
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PMID:Aldosterone modulates steroid receptor binding to the endothelin-1 gene (edn1). 1963 49


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