scholarly journals Expression of epithelial Na channels in Xenopus oocytes.

1990 ◽  
Vol 96 (1) ◽  
pp. 23-46 ◽  
Author(s):  
L G Palmer ◽  
I Corthesy-Theulaz ◽  
H P Gaeggeler ◽  
J P Kraehenbuhl ◽  
B Rossier
Keyword(s):  
Sedimentation Coefficient ◽  
High Sensitivity ◽  
Sucrose Density Gradient ◽  
Na Channel ◽  
Channel Activity ◽  
Membrane Expression ◽  
Cell Monolayers ◽  
A6 Cells ◽  
Epithelial Na Channels ◽  
Confluent Cell

Epithelial Na channel activity was expressed in oocytes from Xenopus laevis after injection of mRNA from A6 cells, derived from Xenopus kidney. Poly A(+) RNA was extracted from confluent cell monolayers grown on either plastic or permeable supports. 1-50 ng RNA was injected into stage 5-6 oocytes. Na channel activity was assayed as amiloride-sensitive current (INa) under voltage-clamp conditions 1-3 d after injection. INa was not detectable in noninjected or water-injected oocytes. This amiloride-sensitive pathway induced by the mRNA had a number of characteristics in common with that in epithelial cells, including (a) high selectivity for Na over K, (b) high sensitivity to amiloride with an apparent K1 of approximately 100 nM, (c) saturation with respect to external Na with an apparent Km of approximately 10 mM, and (d) a time-dependent activation of current with hyperpolarization of the oocyte membrane. Expression of channel activity was temperature dependent, being slow at 19 degrees C but much more rapid at 25 degrees C. Fractionation of mRNA on a sucrose density gradient revealed that the species of RNA inducing channel activity had a sedimentation coefficient of approximately 17 S. Treatment of filter-grown cells with 300 nM aldosterone for 24 h increased Na transport in the A6 cells by up to fivefold but did not increase the ability of mRNA isolated from those cells to induce channel activity in oocytes. The apparent abundance of mRNA coding for channel activity was 10-fold less in cells grown on plastic than in those grown on filters, but was increased two- to threefold by aldosterone.

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.

Effects of prostaglandin E2 on amiloride-blockable Na+ channels in a distal nephron cell line (A6)

1994 ◽  
Vol 267 (5) ◽  
pp. C1414-C1425 ◽  
Author(s):  
K. E. Kokko ◽  
P. S. Matsumoto ◽  
B. N. Ling ◽  
D. C. Eaton
Keyword(s):  
Prostaglandin E2 ◽  
Apical Cell ◽  
Na Channel ◽  
Na Channels ◽  
Channel Activity ◽  
Distal Nephron ◽  
Open Probability ◽  
A6 Cells ◽  
Channel Inhibition ◽  
Camp Levels

We studied the mechanisms by which prostaglandin E2 (PGE2) regulates amiloride-blockable 4-pS Na+ channels in A6 distal nephron cells. With each apical cell-attached patch acting as its own control, acute (3-6 min) basolateral, but not apical, exposure to 1 microM PGE2 inhibited Na+ channel activity by decreasing the open probability (Po). This PGE2-induced inhibition was attenuated by 30 min pretreatment with the protein kinase C (PKC) antagonists 1 microM staurosporine or 100 microM D-sphingosine but was insensitive to pertussis toxin (PTX). Furthermore, the time course for channel inhibition by acute PGE2 correlated with a transient increase in intracellular inositol 1,4,5-trisphosphate (IP3) levels. In contrast, after chronic (10-50 min) exposure of A6 cells to 1 microM basolateral PGE2, channel activity was stimulated compared with controls. This stimulation was due to an increase in the number of apical Na+ channels, similar to the effect of maneuvers that increase intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels in A6 cells (22). Indeed, chronic exposure to basolateral PGE2 correlated with a sustained increase in cAMP levels. In conclusion, 1) the regulation of apical 4-pS highly selective Na+ channel activity by basolateral PGE2 is a complicated biphasic process, which includes inhibition by acute PGE2 and stimulation by chronic PGE2 exposure; 2) acute PGE2 promotes a transient generation of IP3 which activates Ca(2+)-dependent PKC and promotes a decrease in Po; 3) chronic PGE2 promotes a sustained generation of cAMP that leads to an increase in channel density; and 4) both the acute and chronic effects of PGE2 on Na+ channels are PTX-insensitive processes.

Phosphatidylinositol 3,4,5-trisphosphate: an early mediator of insulin-stimulated sodium transport in A6 cells

2004 ◽  
Vol 287 (2) ◽  
pp. F319-F328 ◽  
Author(s):  
Nicolas Markadieu ◽  
Daniel Blero ◽  
Alain Boom ◽  
Christophe Erneux ◽  
Renaud Beauwens
Keyword(s):  
Sodium Transport ◽  
Cell Monolayer ◽  
Sodium Reabsorption ◽  
Na Channel ◽  
Insulin Stimulation ◽  
Cell Monolayers ◽  
A6 Cells ◽  
Cell Extracts ◽  
Pkb Phosphorylation ◽  
Stimulation Of

Insulin stimulates sodium transport across A6 epithelial cell monolayers. Activation of phosphatidylinositol 3-kinase (PI 3-kinase) was suggested as an early step in the insulin-stimulated sodium reabsorption (Ref. 35). To establish that the stimulation of the PI 3-kinase signaling cascade is causing stimulation of apical epithelial Na channel, we added permeant forms of phosphatidylinositol (PI) phosphate (P) derivatives complexed with a histone carrier to A6 epithelium. Only PIP3 and PI( 3 , 4 )P2 but not PI( 4 , 5 )P2 stimulated sodium transport, although each of them penetrated into A6 cell monolayers as assessed using fluorescent permeant phosphoinositides derivatives. By Western blot analysis of A6 cell extracts, the inositol 3-phosphatase PTEN and the protein kinase B PKB were both detected. To further establish that the stimulation of sodium transport induced by insulin is related to PIP3 levels, we transfected A6 cells with human PTEN cDNA and observed a 30% decrease in the natriferic effect of insulin. Similarly, the increase in sodium transport observed by addition of permeant PIP3 was also reduced by 30% in PTEN-overexpressing cells. PKB, a main downstream effector of PI 3-kinase, was phosphorylated at both Thr 308 and Ser 473 residues upon insulin stimulation of the A6 cell monolayer. PKB phosphorylation in response to insulin stimulation was reduced in PTEN-overexpressing cells. Permeant PIP3 also increased PKB phosphorylation. Taken together, the present results establish that the d-3-phosphorylated phosphoinositides PIP3 and PI( 3 , 4 )P2 mediate the effect of insulin on sodium transport across A6 cell monolayers.

NaCl-dependent expression of amiloride-blockable Na+ channel in Xenopus oocytes

1992 ◽  
Vol 262 (2) ◽  
pp. G244-G248 ◽  
Author(s):  
C. Asher ◽  
D. Singer ◽  
R. Eren ◽  
O. Yeger ◽  
N. Dascal ◽  
...  
Keyword(s):  
Xenopus Oocytes ◽  
The Other ◽  
Na Channel ◽  
Channel Activity ◽  
High Affinity ◽  
Other Hand ◽  
Exogenous Rna ◽  
Lower Intestine ◽  
Regulation Of Transport

RNA was isolated from chicken lower intestine (both colon and coprodeum) and injected into Xenopus oocytes. 22Na+ fluxes measured after 1-4 days demonstrated the induction of an amiloride-blockable pathway. The Na+ transporter expressed by the exogenous RNA had a high affinity to amiloride (inhibitory constant less than 0.1 microM), but was insensitive to ethylisopropyl amiloride, i.e., it is likely to be the apical Na+ channel. Functional channels were readily expressed in oocytes injected with RNA derived from chickens fed a low-NaCl diet. On the other hand, no channel activity was detected in oocytes injected with RNA isolated from chickens fed a high-NaCl diet. Thus the previously reported regulation of transport by the dietary NaCl intake involves modulations in the level of mRNA that codes either for the Na+ channel or a posttranscriptional regulator of the channel.

Actin filaments regulate epithelial Na+ channel activity

1991 ◽  
Vol 261 (5) ◽  
pp. C882-C888 ◽  
Author(s):  
H. F. Cantiello ◽  
J. L. Stow ◽  
A. G. Prat ◽  
D. A. Ausiello
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Functional Role ◽  
Cytochalasin D ◽  
Actin Binding ◽  
Na Channel ◽  
Na Channels ◽  
Channel Activity ◽  
Cortical Actin ◽  
Excised Patches

The functional role of the cytoskeleton in the control of ion channel activity is unknown. In the present study, immunocolocalization of Na+ channels with specific antibodies and fluorescein isothiocyanate-phalloidin to stain the cortical cytoskeleton indicates that actin is always present in close proximity to apical Na+ channels in A6 cells. The patch-clamp technique was used to assess the effect of cortical actin networks on apical Na+ channels in these A6 epithelial cells. The actin filament disrupter, cytochalasin D (5 micrograms/ml), induced Na+ channel activity in cell-attached patches within 5 min of addition. Cytochalasin D also induced and/or increased Na+ channel activity in 90% of excised patches tested within 2 min. Addition of short actin filaments (greater than 5 microM) to excised patches also induced channel activity. This effect was enhanced by addition of ATP and/or cytochalasin D. The effect of actin on Na+ channel activity was reversed by addition of the G actin-binding protein DNase I or completely prevented by treatment of the excised patches with this enzyme. Addition of the actin-binding protein, filamin, reversibly inhibited both spontaneous and actin-induced Na+ channels. Thus actin filament networks, achieved by either depolymerizing endogenous actin filaments by treatment with cytochalasin D, the addition of exogenous short actin filaments plus ATP, or actin plus cytochalasin D, regulate apical Na+ channel activity. This conclusion was supported by the observation that the addition of short actin filaments in the form of actin-gelsolin complexes in molar ratios less than 8:1 was also effective in activating Na+ channels. We have thus demonstrated a functional role for the cortical actin network in the regulation of epithelial Na+ channels that may complement a structural role for membrane protein targetting and assembly.

Regulation of amiloride-sensitive Na+ channels by endothelin-1 in distal nephron cells

1996 ◽  
Vol 271 (2) ◽  
pp. F451-F460 ◽  
Author(s):  
M. S. Gallego ◽  
B. N. Ling
Keyword(s):  
Na Channel ◽  
Na Channels ◽  
Channel Activity ◽  
Distal Nephron ◽  
Endothelin 1 ◽  
Channel Inhibition ◽  
Current Voltage ◽  
Mean Time ◽  
Current Voltage Relationship

We used patch-clamp methods to investigate the effects of basolateral endothelin-1 (ET-1) on the amiloride-sensitive Na+ channel in A6 distal nephron cells. One hundred picomolar ET-1 decreased channel activity via an increase in mean time closed (P < 0.01, n = 10). Channel inhibition by pM ET-1 was mimicked by an ET-B receptor agonist (P < 0.05, n = 7) and was prevented by ET-B antagonists (P = 0.14, n = 10) but not by an ET-A antagonist (P < 0.05, n = 4). With the inhibitory ET-B receptor blocked, higher doses of ET-1 (10 nM) actually increased channel activity through an increase in mean time open (P < 0.001, n = 12). The current-voltage relationship and the number of channels were not changed by basolateral ET-1 exposure. We conclude that 1) basolateral ET-1 regulates amiloride-sensitive Na+ channels; 2) binding of picomolar ET-1 to ET-B receptors inhibits, whereas the binding of nanomolar ET-1 to a different ET receptor (likely ET-A) stimulates, channel activity; and 3) these dose-dependent, distal nephron responses provide a potential mechanism for the in vivo natriuresis and antinatriuresis observed in response to "subpressor" and "pressor" concentrations of ET-1, respectively.

DEMONSTRATION OF A CYTOPLASMIC RECEPTOR PROTEIN FOR OESTROGEN IN THE CANINE PROSTATE GLAND

1978 ◽  
Vol 78 (1) ◽  
pp. 131-139 ◽  
Author(s):  
N. CHAISIRI ◽  
Y. VALOTAIRE ◽  
BRONWEN A. J. EVANS ◽  
C. G. PIERREPOINT
Keyword(s):  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Prostate Gland ◽  
Sedimentation Coefficient ◽  
Sucrose Density Gradient ◽  
Receptor Protein ◽  
Receptor Complex ◽  
Binding Properties ◽  
Sucrose Density Gradient Centrifugation ◽  
A Value

A receptor protein that selectively binds oestrogens has been demonstrated in the cytosol of the canine prostate gland. The steroid–receptor complex was found to have a sedimentation coefficient of 4–5 S with respect to bovine serum albumin after sucrose density-gradient centrifugation. The high affinity and low capacity of the protein for oestrogens was indicated by displacement studies, which gave a value of 3·8 ± 1·53 (s.d.) × 10−10 mol/l for the dissociation constant. A metastasizing prostatic tumour was also shown to possess this receptor, with binding properties similar to those exhibited by the receptor in normal prostatic cytosol. The implications of these findings are discussed with regard to normal prostatic function in the dog and the virtually inevitable advent of prostatic hyperplasia with age in this species.

Sign in / Sign up

Export Citation Format

Share Document