Guanosine nucleotide-dependent activation of the amiloride-blockable Na+ channel

1989 ◽  
Vol 256 (5) ◽  
pp. F965-F969
Author(s):  
H. Garty ◽  
O. Yeger ◽  
A. Yanovsky ◽  
C. Asher
Keyword(s):  
Membrane Vesicles ◽  
Na Channel ◽  
Bladder Epithelium ◽  
Stimulatory Action ◽  
Fourfold Increase ◽  
Cyclic Monophosphate ◽  
Sustained Effect ◽  
22Na Uptake ◽  
Gamma S ◽  
Epithelial Na Channel

Effects of guanosine nucleotides on the epithelial Na+ channel were studied in apical membrane vesicles derived from the toad bladder epithelium. Trapping 10 microM guanosine-5'-O-(thiotriphosphate) (GTP gamma S) in vesicles evoked two- to fourfold increase in the amiloride-sensitive (Na+ channel-mediated) 22Na+ uptake. The nucleotide had no significant effect on the amiloride-insensitive 22Na+ uptake or the valinomycin-mediated 86Rb+ uptake in the same membranes. The stimulatory action of GTP gamma S was mimicked by 5'-guanylylimidiodiphosphate (GppNHp) and could at least partly be reversed by guanosine-5'-O-(thiodiphosphate) (GDP beta S) (10-fold excess). GTP itself and adenosine-5'-O-(thiotriphosphate) (ATP gamma S) had no sustained effect on Na+ transport in vesicles. Thus it appears that the epithelial Na+ channel is directly or indirectly regulated by the occupancy of a guanosine-specific site, probably the alpha subunit of a G protein. The possibility that GTP gamma S acts indirectly by activating a membrane-bound, GTP-dependent enzyme the product of which modulates the channel conductance was assessed by measuring 22Na+ fluxes in membrane vesicles prepared to contain products of such enzymes. None of the reagents tested [adenosine 3',5' cyclic monophosphate (cAMP), guanosine 3',5y cyclic monophosphate (cGMP), inositol 1,4,5-trisphosphate (IP3), and diacylglycerol (DAG)] increased the tracer flux in vesicles or altered its response to GTP gamma S.

Inhibition of amiloride-sensitive Na+ channel by isothiouronium derivatives

1996 ◽  
Vol 271 (5) ◽  
pp. C1457-C1462
Author(s):  
A. Avigdor ◽  
C. Asher ◽  
D. M. Tal ◽  
S. J. Karlish ◽  
H. Garty
Keyword(s):  
Membrane Vesicles ◽  
Distal Colon ◽  
Covalent Modification ◽  
Na Channel ◽  
Plasma Membrane Vesicles ◽  
High Affinity ◽  
Neutral Ph ◽  
Rat Distal Colon ◽  
22Na Uptake ◽  
Methyl Benzene

The effects on the amiloride-blockable Na+ channel of a family of recently synthesized isothiouronium derivatives were measured in plasma membrane vesicles from rat distal colon. Some of these derivatives act as high-affinity Na(+)-like antagonists on the Na(+)-K(+)-adenosinetriphosphatase. One of the reagents tested, 1-bromo-2,4,6-tris(isothiouronium methyl)-benzene tribromide (Br-TITU), was found to be a potent blocker of the Na+ channel. At neutral pH, Br-TITU rapidly inhibits the channel mediated 22Na+ uptake, with an inhibition constant of 94 +/- 39 nM. The inhibition observed is specific and reversible. 1,3-Dibromo-2,4,6-tris(isothiouronium methyl)benzene tribromide and Br-TITU derivatives with methyl and phenyl substitutions on the isothiouronium moiety were much less effective blockers. Incubation of cells with Br-TITU at alkaline (but not neutral) pH produces irreversible inactivation of channels, possibly due ot covalent modification of a lysine residue. This inactivation can be attenuated by amiloride but not by Na+. Thus Br-TITU may be a useful reagent in identifying essential residues of the channel protein.

Amiloride-inhibitable Na+ conductive pathways in alveolar type II pneumocytes

1991 ◽  
Vol 260 (2) ◽  
pp. L90-L96 ◽  
Author(s):  
S. Matalon ◽  
R. J. Bridges ◽  
D. J. Benos
Keyword(s):  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Na Channel ◽  
Plasma Membrane Vesicle ◽  
Alveolar Type ◽  
Type Ii ◽  
Type Ii Pneumocytes ◽  
The Difference ◽  
22Na Uptake ◽  
Na Uptake

The purpose of these studies was to document the existence of electrogenic Na+ uptake by membrane vesicles of rabbit alveolar type II (ATII) cells and the extent to which this process was inhibited by amiloride. ATII cells (greater than 85% pure) were obtained by elastase digestion of lung tissue followed by Percoll centrifugation, and an enriched plasma membrane vesicle fraction was obtained by differential centrifugation. 22Na+ uptake into these vesicles was measured in the presence of a negative inside membrane potential, produced by the addition of the K+ ionophore valinomycin (10 microM) after all external K+ was removed. Electrogenic (valinomycin-sensitive) Na+ uptake (ELNa) was defined as the difference in uptake in the presence and absence of valinomycin. ELNa, normalized per milligram protein, was twice as high across ATII cells than alveolar macrophage membrane vesicles, was inhibited by amiloride (50% inhibitory concentration = 10 microM), and was decreased in the presence of an outwardly directed proton gradient (pHin 6.8; pHout 7.8), suggesting that it was not mediated by Na(+)-H+ antiport. Furthermore, ELNa was equally inhibited by increasing concentrations of amiloride and benzamil but was more sensitive to 5-(N-ethyl-N-isopropyl)-2'-4'-amiloride in concentrations of 10–1,000 microM. These findings indicate that a fraction of Na+ transport across ATII membrane vesicles occurs through a conductive pathway, probably a channel, that has different sensitivity to amiloride and its analogues than the previously described epithelial high amiloride-affinity Na+ channel.

Diabetes enhances activity of alanine transport in liver plasma membrane vesicles

1985 ◽  
Vol 248 (5) ◽  
pp. E581-E587 ◽  
Author(s):  
N. R. Rosenthal ◽  
R. Jacob ◽  
E. Barrett
Keyword(s):  
Plasma Membrane ◽  
Membrane Vesicles ◽  
Diabetic Rats ◽  
Control Group ◽  
Similar Degree ◽  
Plasma Membrane Vesicles ◽  
Fourfold Increase ◽  
Alanine Transport ◽  
Sodium Dependent ◽  
22Na Uptake

In the present study plasma membrane vesicles were prepared from livers of control and alloxan-induced diabetic rats and the substrate specificity and kinetic characteristics of alanine transport determined in both groups. Sodium-dependent alanine uptake at physiological alanine concentrations (100 microM) was enhanced threefold in diabetic as compared with control animals (0.31 +/- 0.04 vs. 0.11 +/- 0.01 nmol X mg protein-1 X 10 s-1). This accelerated influx corresponded to a three- to fourfold increase in the Vmax of alanine transport in diabetic versus control group (7.1 +/- 2.1 vs. 1.6 +/- 0.2 nmol X mg protein-1 X 10 s-1, P less than 0.05), whereas the Km of alanine uptake was unchanged (2.8 +/- 1.2 vs. 1.4 +/- 0.1 mM). Other neutral amino acids (20 mM) inhibited alanine transport to a similar degree in both groups. The sodium-dependent influx of glutamine (100 microM) was similar in diabetic and control groups (0.17 +/- 0.03 and 0.14 +/- 0.02 nmol X mg protein-1 X 10 s-1, respectively). The initial velocity of 22Na uptake (80 mM) into vesicles and half-maximal stimulation of alanine transport was achieved at essentially identical sodium concentrations (approximately 40 mM) in both groups.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Interactive Actions of Aldosterone and Insulin on Epithelial Na+ Channel Trafficking

2020 ◽  
Vol 21 (10) ◽  
pp. 3407 ◽  
Author(s):  
Rie Marunaka ◽  
Yoshinori Marunaka
Keyword(s):  
Blood Pressure ◽  
Inhibitory Action ◽  
Apical Membrane ◽  
Short Circuit ◽  
Recycling Rate ◽  
Na Channel ◽  
Stimulatory Action ◽  
Channel Trafficking ◽  
Short Circuit Currents ◽  
Epithelial Na Channel

Epithelial Na+ channel (ENaC) participates in renal epithelial Na+ reabsorption, controlling blood pressure. Aldosterone and insulin elevate blood pressure by increasing the ENaC-mediated Na+ reabsorption. However, little information is available on the interactive action of aldosterone and insulin on the ENaC-mediated Na+ reabsorption. In the present study, we tried to clarify if insulin would modify the aldosterone action on the ENaC-mediated Na+ reabsorption from a viewpoint of intracellular ENaC trafficking. We measured the ENaC-mediated Na+ transport as short-circuit currents using a four-state mathematical ENaC trafficking model in renal A6 epithelial cells with or without aldosterone treatment under the insulin-stimulated and -unstimulated conditions. We found that: (A) under the insulin-stimulated condition, aldosterone treatment (1 µM for 20 h) significantly elevated the ENaC insertion rate to the apical membrane ( k I ) 3.3-fold and the ENaC recycling rate ( k R ) 2.0-fold, but diminished the ENaC degradation rate ( k D ) 0.7-fold without any significant effect on the ENaC endocytotic rate ( k E ); (B) under the insulin-unstimulated condition, aldosterone treatment decreased k E 0.5-fold and increased k R 1.4-fold, without any significant effect on k I or k D . Thus, the present study indicates that: (1) insulin masks the well-known inhibitory action of aldosterone on the ENaC endocytotic rate; (2) insulin induces a stimulatory action of aldosterone on ENaC apical insertion and an inhibitory action of aldosterone on ENaC degradation; (3) insulin enhances the aldosterone action on ENaC recycling; (4) insulin has a more effective action on diminution of ENaC endocytosis than aldosterone.

scholarly journals Differential Effects of Protein Kinase C on the Levels of Epithelial Na+Channel Subunit Proteins

2000 ◽  
Vol 275 (33) ◽  
pp. 25760-25765 ◽  
Author(s):  
James D. Stockand ◽  
Hui-Fang Bao ◽  
Julie Schenck ◽  
Bela Malik ◽  
Pam Middleton ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Na Channel ◽  
Differential Effects ◽  
Kinase C ◽  
Epithelial Na Channel

scholarly journals CRISPR/Cas9 Mediated Knock Down of δ-ENaC Blunted the TNF-Induced Activation of ENaC in A549 Cells

2021 ◽  
Vol 22 (4) ◽  
pp. 1858
Author(s):  
Waheed Shabbir ◽  
Nermina Topcagic ◽  
Mohammed Aufy ◽  
Murat Oz
Keyword(s):  
A549 Cells ◽  
Na Channel ◽  
Na Current ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Knock Down ◽  
Whole Cell Patch Clamp ◽  
Glycosylation Sites ◽  
Epithelial Na Channel

Tumor necrosis factor (TNF) is known to activate the epithelial Na+ channel (ENaC) in A549 cells. A549 cells are widely used model for ENaC research. The role of δ-ENaC subunit in TNF-induced activation has not been studied. In this study we hypothesized that δ-ENaC plays a major role in TNF-induced activation of ENaC channel in A549 cells which are widely used model for ENaC research. We used CRISPR/Cas 9 approach to knock down (KD) the δ-ENaC in A549 cells. Western blot and immunofluorescence assays were performed to analyze efficacy of δ-ENaC protein KD. Whole-cell patch clamp technique was used to analyze the TNF-induced activation of ENaC. Overexpression of wild type δ-ENaC in the δ-ENaC KD of A549 cells restored the TNF-induced activation of whole-cell Na+ current. Neither N-linked glycosylation sites nor carboxyl terminus domain of δ-ENaC was necessary for the TNF-induced activation of whole-cell Na+ current in δ-ENaC KD of A549 cells. Our data demonstrated that in A549 cells the δ-ENaC plays a major role in TNF-induced activation of ENaC.

Glucocorticoid regulation of epithelial sodium channel genes in human fetal lung

1997 ◽  
Vol 273 (1) ◽  
pp. L227-L233 ◽  
Author(s):  
V. C. Venkatesh ◽  
H. D. Katzberg
Keyword(s):  
Rna Polymerase Ii ◽  
Actinomycin D ◽  
Critical Role ◽  
Fetal Lung ◽  
Explant Culture ◽  
Late Gestation ◽  
Na Channel ◽  
Second Trimester ◽  
Cyclic Monophosphate ◽  
Human Fetal Lung

Pulmonary epithelial Na+ channels (ENaC), composed of three distinct subunits (alpha, beta, and gamma), play a critical role in the regulation of fluid reabsorption from airspaces of late-gestation fetal lung. We studied the expression of ENaC subunit genes in cultured human fetal lung. All three mRNAs were expressed at low levels in second trimester lung (13-32% of adult values at 24 wk gestation). There was a spontaneous increase of approximately threefold over preculture values of all three subunits within 24 h of explant culture in serum-free Waymouth's medium. Dexamethasone (Dex) induced all three mRNAs by two- to threefold. Maximal induction was noted by 8 h with 30-100 nM Dex and half-maximal stimulation with 3-10 nM Dex. Cycloheximide decreased basal expression of all three subunits by 8 h but did not alter the response to Dex. Actinomycin D and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), inhibitors of RNA polymerase II, decreased the basal and the Dex-induced expression of all three subunits with a more marked effect on human hENaC-gamma than on hENaC-alpha or hENaC-beta. Under conditions where transcription was blocked by actinomycin D or DRB, Dex did not alter the stability of the three mRNAs. Triiodothyronine (T3) at low (2 nM) or high (100 nM) concentrations had no effect on the expression of the three subunits in the presence or absence of low (10 nM) or high (100 nM) concentrations of Dex for 8 or 24 h. Similarly, 8-bromoadenosine 3',5'-cyclic monophosphate (2 microM) had no effect on basal or Dex-induced increase in the three subunits. We conclude that the three Na+ channel subunit genes are expressed in second trimester human fetal lung and are coordinately upregulated by glucocorticoid hormones but not by T3 or adenosine 3',5'-cyclic monophosphate. Glucocorticoid induction is receptor mediated, is primarily transcriptional, and does not require the induction of an intermediate protein for transcriptional enhancement. We speculate that induction of lung ENaC may contribute to the beneficial effects of antenatal glucocorticoids in premature babies.

scholarly journals Acute effects of aldosterone on the epithelial Na channel in rat kidney

2015 ◽  
Vol 308 (6) ◽  
pp. F572-F578 ◽  
Author(s):  
Gustavo Frindt ◽  
Lawrence G. Palmer
Keyword(s):  
Rat Kidney ◽  
Surface Protein ◽  
Early Response ◽  
Surface Expression ◽  
Na Channel ◽  
Channel Activity ◽  
Acute Effects ◽  
Chronic Stimulation ◽  
Epithelial Na Channels ◽  
Epithelial Na Channel

The acute effects of aldosterone administration on epithelial Na channels (ENaC) in rat kidney were examined using electrophysiology and immunodetection. Animals received a single injection of aldosterone (20 μg/kg body wt), which reduced Na excretion over the next 3 h. Channel activity was assessed in principal cells of cortical collecting ducts as amiloride-sensitive whole cell clamp current ( INa). INa averaged 100 pA/cell, 20–30% of that reported for the same preparation under conditions of chronic stimulation. INa was negligible in control animals that did not receive hormone. The acute physiological response correlated with changes in ENaC processing and trafficking. These effects included increases in the cleaved forms of α-ENaC and γ-ENaC, assessed by Western blot, and increases in the surface expression of β-ENaC and γ-ENaC measured after surface protein biotinylation. These changes were qualitatively and quantitatively similar to those of chronic stimulation. This suggests that altered trafficking to or from the apical membrane is an early response to the hormone and that later increases in channel activity require stimulation of channels residing at the surface.

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