scholarly journals Overexpression of the Epithelial Na+Channel γ Subunit in Collecting Duct Cells

2005 ◽  
Vol 280 (18) ◽  
pp. 18348-18354 ◽  
Author(s):  
Kenneth A. Volk ◽  
Russell F. Husted ◽  
Rita D. Sigmund ◽  
John B. Stokes
Keyword(s):  
Collecting Duct ◽  
Na Channel ◽  
Γ Subunit ◽  
Duct Cells ◽  
Collecting Duct Cells ◽  
Epithelial Na Channel

scholarly journals Novel Regulation of the Epithelial Na + Channel by Soluble Adenylyl Cyclase in Kidney Collecting Duct Cells

2008 ◽  
Vol 22 (S1) ◽  
Author(s):  
Kenneth R. Hallows ◽  
Nuria M. Pastor‐Soler ◽  
Robert S. Edinger ◽  
Nicholas M. Oyster ◽  
Huamin Wang ◽  
...  
Keyword(s):  
Adenylyl Cyclase ◽  
Collecting Duct ◽  
Na Channel ◽  
Soluble Adenylyl Cyclase ◽  
Kidney Collecting Duct ◽  
Duct Cells ◽  
Collecting Duct Cells ◽  
Epithelial Na Channel

Whole cell and unitary amiloride-sensitive sodium currents in M-1 mouse cortical collecting duct cells

1996 ◽  
Vol 270 (4) ◽  
pp. C998-C1010 ◽  
Author(s):  
M. L. Chalfant ◽  
T. G. O'Brien ◽  
M. M. Civan
Keyword(s):  
Collecting Duct ◽  
Cell Permeability ◽  
Na Channel ◽  
Na Channels ◽  
Cortical Collecting Duct ◽  
Ionic Selectivity ◽  
Whole Cell ◽  
Duct Cells ◽  
Excised Patches ◽  
Collecting Duct Cells

Amiloride-sensitive whole cell currents have been reported in M-1 mouse cortical collecting duct cells (Korbmacher et al., J. Gen. Physiol. 102: 761-793, 1993). We have confirmed that amiloride inhibits the whole cell currents but not necessarily the measured whole cell currents. Anomalous responses were eliminated by removing external Na+ and/or introducing paraepithelial shunts. The amiloride-sensitive whole cell currents displayed Goldman rectification. The ionic selectivity sequence of the amiloride-sensitive conductance was Li+ > Na+ >> K+. Growth of M-1 cells on permeable supports increased the amiloride-sensitive whole cell permeability, compared with cells grown on plastic. Single amiloride-sensitive channels were observed, which conformed to the highly selective low-conductance amiloride-sensitive class [Na(5)] of epithelial Na+ channels. Hypotonic pretreatment markedly slowed run-down of channel activity. The gating of the M-1 Na+ channel in excised patches was complex. Open- and closed-state dwell-time distributions from patches that display one operative channel were best described with two or more exponential terms each. We conclude that 1) study of M-1 whole cell Na+ currents is facilitated by reducing the transepithelial potential to zero, 2) these M-1 currents reflect the operation of Na(5) channels, and 3) the Na+ channels display complex kinetics, involving > or = 2 open and > or = 2 closed states.

scholarly journals Physical and functional interaction of Rnf2 with Af9 regulates basal and aldosterone-stimulated transcription of the α-ENaC gene in a renal collecting duct cell line

2013 ◽  
Vol 33 (5) ◽  
Author(s):  
Zhi-Yuan Yu ◽  
Qun Kong ◽  
Bruce C. Kone
Keyword(s):  
Glutathione Transferase ◽  
Collecting Duct ◽  
Chromosome 9 ◽  
Na Channel ◽  
Central Component ◽  
Mrna Levels ◽  
Functional Interaction ◽  
Α Subunit ◽  
Duct Cells ◽  
Collecting Duct Cells

The physical and functional interaction of Rnf2 (RING finger protein 2), a central component of the PRC (Polycomb repressive complex) 1 and Af9 (ALL1-fused gene from chromosome 9 protein), an aldosterone-sensitive transcription factor, in regulating basal and aldosterone-stimulated transcription of the α-ENaC (epithelial Na+ channel α-subunit) gene was explored in mIMCD3 CD (collecting duct) cells. Since Rnf2 lacks DNA-specific binding activity, other factors must mediate its site-specific chromatin recruitment. Rnf2 and Af9 co-localized in the nucleus and co-immunoprecipitated. A GST (glutathione transferase)–Af9 carboxy-terminal fusion protein directly interacted with in vitro translated Rnf2 in GST pull-down assays. Rnf2 knock down enhanced basal and aldosterone-stimulated α-ENaC mRNA levels and α-ENaC promoter activity. ChIP/QPCR (chromatin immunoprecipitation/quantitative PCR) assays demonstrated enrichment of Rnf2, H2AK119 (mono-ubiquitinated histone H2A lysine 119), and H3K27me3 (histone H3 lysine 27 trimethylated), a PRC2 chromatin mark, at multiple α-ENaC promoter subregions corresponding to regions of known Af9 enrichment, under basal conditions. Sequential ChIP confirmed Rnf2–Af9 co-occupancy of the α-ENaC promoter. Aldosterone provoked early and sustained depletion of Rnf2, ubiquitinated H2AK119, and trimethylated H3K27 associated with the subregions of the α-ENaC promoter. Thus, Af9 mediates site-selective physical and functional recruitment of Rnf2 to the α-ENaC promoter to constrain basal α-ENaC transcription in collecting duct cells, and aldosterone reverses this process.

Aldosterone regulation of sodium channel gamma-subunit mRNA in cortical collecting duct cells

1996 ◽  
Vol 271 (1) ◽  
pp. C423-C428 ◽  
Author(s):  
D. L. Denault ◽  
G. Fejes-Toth ◽  
A. Naray-Fejes-Toth
Keyword(s):  
Nested Pcr ◽  
Collecting Duct ◽  
Na Channel ◽  
Mrna Levels ◽  
Cortical Collecting Duct ◽  
Gamma Subunit ◽  
Subunit Mrna ◽  
Duct Cells ◽  
Pcr Product ◽  
Collecting Duct Cells

Specific regulatory mechanisms of aldosterone-stimulated Na+ reabsorption through the apical amiloride-sensitive channel are unknown. In this study, we examined the effects of aldosterone on Na+ channel gamma-subunit mRNA levels in cultured rabbit cortical collecting duct cells. With the use of reverse transcriptase-polymerase chain reaction (RT-PCR) with RNA isolated from aldosterone-treated cells and degenerate primers, a 446-base pair (bp) PCR product was amplified and further characterized by nested PCR and sequencing. The nested PCR yielded a predicted 164-bp product. Sequencing of the 446-bp PCR product revealed 83% nucleotide and 91% amino acid identity to the rat colonic Na+ channel gamma-subunit. The relative abundance of Na+ channel mRNA was determined by quantitative PCR after a 24-h aldosterone treatment. The results demonstrate that Na+ channel gamma-subunit mRNA levels were significantly higher (2.6 +/- 0.42) in aldosterone-treated cultures vs. the controls. This increase, however, is less than the aldosterone-induced increase (3.2 +/- 2.0) in the amiloride-sensitive short-circuit current. These results indicate that Na+ channel gamma-subunit mRNA levels are increased by aldosterone and that this increase is likely to be responsible, at least in part, for the aldosterone-induced Na+ current in the kidney.

Norepinephrine stimulates the epithelial Na+ channel in cortical collecting duct cells via α2-adrenoceptors

2015 ◽  
Vol 308 (5) ◽  
pp. F450-F458 ◽  
Author(s):  
Morag K. Mansley ◽  
Winfried Neuhuber ◽  
Christoph Korbmacher ◽  
Marko Bertog
Keyword(s):  
Collecting Duct ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Nerve Fibers ◽  
Good Evidence ◽  
Fine Tuning ◽  
Na Channel ◽  
Cortical Collecting Duct ◽  
Duct Cells ◽  
Collecting Duct Cells

There is good evidence for a causal link between excessive sympathetic drive to the kidney and hypertension. We hypothesized that sympathetic regulation of tubular Na+ absorption may occur in the aldosterone-sensitive distal nephron, where the fine tuning of renal Na+ excretion takes place. Here, the appropriate regulation of transepithelial Na+ transport, mediated by the amiloride-sensitive epithelial Na+ channel (ENaC), is critical for blood pressure control. To explore a possible effect of the sympathetic transmitter norepinephrine on ENaC-mediated Na+ transport, we performed short-circuit current ( Isc) measurements on confluent mCCDcl1 murine cortical collecting duct cells. Norepinephrine caused a complex Isc response with a sustained increase of amiloride-sensitive Isc by ∼44%. This effect was concentration dependent and mediated via basolateral α2-adrenoceptors. In cells pretreated with aldosterone, the stimulatory effect of norepinephrine was reduced. Finally, we demonstrated that noradrenergic nerve fibers are present in close proximity to ENaC-expressing cells in murine kidney slices. We conclude that the sustained stimulatory effect of locally elevated norepinephrine on ENaC-mediated Na + absorption may contribute to the hypertensive effect of increased renal sympathetic activity.

scholarly journals Human amiloride-sensitive epithelial Na+ channel γ subunit promoter: functional analysis and identification of a polypurine-polypyrimidine tract with the potential for triplex DNA formation

2000 ◽  
Vol 347 (1) ◽  
pp. 105-114 ◽  
Author(s):  
Scott D. AUERBACH ◽  
Randy W. LOFTUS ◽  
Omar A. ITANI ◽  
Christie P. THOMAS
Keyword(s):  
Core Promoter ◽  
Collecting Duct ◽  
Regulatory Element ◽  
Na Channel ◽  
Triplex Dna ◽  
Polypyrimidine Tract ◽  
Mobility Shift ◽  
Γ Subunit ◽  
Flanking Region ◽  
Epithelial Na Channel

The mRNA for the epithelial Na+ channel γ subunit (γENaC) is regulated developmentally in the lung, colon and distal nephron and in response to Na+ deprivation and systemic corticosteroids in the distal colon. Because such regulation is likely to be at the level of gene transcription, we examined the function of the promoter and other 5ʹ flanking elements of the human γENaC gene. The proximal 5ʹ flanking region contains two GC boxes but does not contain a TATA box. A 450 bp human γENaC fragment (-459 to +40) directed the expression of luciferase in H441 cells and primer extension analysis in transfected cells confirmed the correct initiation of human γENaC-luciferase chimaeric transcripts. By deletional analysis, GC boxes at -21 and -52 were found to be critical for this promoter activity. To begin to identify transcription factors that bind to the core promoter, a double-stranded oligonucleotide that corresponded to this region was synthesized and tested in a gel mobility-shift assay. Incubation of this radiolabelled oligonucleotide with nuclear extracts from H441 and FRTL5 cells resulted in the formation of four specific and distinct DNA-protein complexes. On the basis of antibody ‘supershift’ assays, one of these factors corresponds to Sp1, whereas the other three correspond to Sp3. Further upstream, an approx. 300 nt (-1143 to -839) polypurine-polypyrimidine tract (PPy tract) containing internal mirror repeats was identified. When contained in a supercoiled plasmid, the approx. 1200 nt 5ʹ flanking region was sensitive to S1 endonuclease, which was consistent with the formation of an intramolecular triplex DNA (‘H-DNA’) structure with an unpaired single strand. High-resolution mapping with S1 endonuclease and sequencing of S1-generated clones confirmed that all S1-sensitive sites were within the PPy tract. Finally, a negative regulatory element was identified between -1525 and -1296 that functioned in lung, colon and collecting duct cell lines.

Synthesis of the Na-K-ATPase γ-subunit is regulated at both the transcriptional and translational levels in IMCD3 cells

2005 ◽  
Vol 288 (1) ◽  
pp. F76-F81 ◽  
Author(s):  
Juan M. Capasso ◽  
Christopher J. Rivard ◽  
Tomas Berl
Keyword(s):  
Collecting Duct ◽  
Pi3 Kinase ◽  
Transcriptional Level ◽  
Cortical Collecting Duct ◽  
Subunit Protein ◽  
Subunit Mrna ◽  
Γ Subunit ◽  
Duct Cells ◽  
Subunit Expression ◽  
Collecting Duct Cells

We previously reported that hypertonicity-mediated upregulation of the γ-subunit of Na-K-ATPase is dependent on both the JNK and the PI3 kinase pathways ( Proc Natl Acad Sci USA 98: 13414, 2001). The present experiments were undertaken to explore the mechanisms whereby these pathways regulate the expression of the γ-subunit in inner medullary collecting duct cells (IMCD3). Inhibition of JNK with SP-600125 (20 μM), a concentration that causes an ∼95% inhibition of hypertonicity-stimulated JNK activation, markedly decreased the amount of the γ-subunit in response to 550 mosmol/kgH2O for 48 h. This was accompanied by a parallel decrease in the γ-subunit mRNA. The rate at which the γ-subunit mRNA decreased was unaffected by actinomycin D. In contrast, inhibition of PI3 kinase with LY-294002 results in a marked decrease in the amount of γ-subunit protein but without alteration in γ-subunit message. The rate at which the γ-subunit protein decreased was unaffected by cyclohexamide. Transfection of IMCD3 cells with a γ-subunit construct results in the expression of both γ-subunit message and protein. However, in cortical collecting duct cells (M1 cells) such transfection resulted in expression of only the message and not the protein. We conclude that JNK regulates the γ-subunit at the transcriptional level while PI3 kinase regulates γ-subunit expression at the translational level. There is also posttranscriptional cell specificity in the expression of the γ -subunit of Na-K-ATPase.

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