Difference in regulation mechanisms of ENaC by aldosterone and glucocorticords

doi:10.4236/health.2009.13025

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Vol.1, No.3, 152-158 (2009)

doi:10.4236/health.2009.13025

Health

Difference in regulation mechanisms of ENaC by

aldosterone and glucocorticords

Chengchun Tang1, Hao Zhang2, Su Wang3, Juyou Wu3, Yuchun Gu3, Jeng Wei3

1Department of Cardiology, The 1st Affiliated Hospital of Southeast University, Nanjing, China

2General Surgery, Hua Shan Hospital, China

3Department of Physiology, University of Birmingham, Edgbaston, UK; Corresponding author: y.gu@bham.ac.uk

Received 11 October 2009; revised 15 September 2009; accepted 22 September 2009.

ABSTRACT

Na+ transport occurs across many epithelial

surfaces and plays a key role in regulating salt

and water absorption. The molecular pathway

underlying this Na+ transport is the epithelial Na

channel (ENaC), which is strictly determined by a

variety of hormones like aldosterone, ADH and

glucocorticoids. In this study, we found that

stimulation of either aldosterone or dexameth-

asone (Dex) distributed ENaC channel on the

apical membrane of mouse cortical collecting

duct cells (M1). In the single channel recordings

from excised membrane, high density ENaC was

found in the cell with a dome shape by the

treatment of either dex or aldosterone. However,

low active ENaC was revealed in intact cells

treated with dex, when compared to cells treated

with aldosterone. Only 5.84% of cells treated with

dex containing ENaC exhibited ENaC current

transition in the cell-attach recording, whereas

40% of cells treated with aldosterone containing

ENaC exhibited ENaC current transition. ENaC

currents appeared rapid rundown within 5 min-

utes since formation of inside-out configuration

in cells treated with aldosterone but not with dex.

SKF-525A, a general antagonist of CYP, failed to

significantly enhance ENaC activity in intact cells

treated with dex, but EGTA, which deforming the

cells, increased the ENaC activity in the cells

treated with dex. PTX, an antagonist of G-protein,

reversed the effect of aldosterone on number of

active ENaC in intact cells. Based on our obser-

vation, we concluded that there are different

mechanisms in regulation of ENaC activity be-

tween stimulation of aldosterone and glucocor-

ticoids. The activation of G-protein is required to

maintain the activity of ENaC in the collecting

ducts.

Keywords: ENaC; Aldosterone; Glucocorticoids;

Single Channel Recording

1. INTRODUCTION

Active transport of Na+ across epithelial cells plays an

important role in maintenance of homeostasis of electro-

lytes and water in the body. Hormone sensitive Na+ re-

absorption is mainly mediated by epithelial Na+ channels

(ENaC), which are distributed in distal and collecting

nephron ducts, and alveolar epithelial cells. Disorder of

ENaC could lead to Na+ and water retention [1], ‘dry

lung’ and even death due to pulmonary edema [2]. Nev-

ertheless, ENaC activities are strictly under the control of

a variety of hormones like aldosterone, ADH, and glu-

cocorticoids.

Aldosterone is known to enhance the density of ENaC

on apical membrane of epithelial cells by modifying gene

expression. Several aldosterone sensitive genes including

serum and glucocorticoid (GC) inducible kinase (SGK-1)

[3,4], PI3-K [5] have implicated the action mechanism of

GC. Previously in vivo and in vitro studies in a number of

models show that endogenous and exogenous GC could

enhance expression of ENaC and increase the Na+ uptake

in airway [6-13], gut [14] and kidney [6,15]. The regula-

tion of ENaC by GC determines alveolar fluid clearance

and lung development [9]. However, it still remains un-

clear of the mechanism in regulation of ENaC by GC,

even by aldosterone. A slight difference in the expression

of ENaC subunits has been observed in some studies. The

expression of α subunit of ENaC is the most pronounced

in lung, correlating with the high GC level, whereas ex-

pression of α, β and γ subunits of ENaC is gradually in-

creased in kidney and colon [9]. Aldosterone is the major

regulator in kidney, although GC could also exert the

effects on ENaC expression in collecting ducts [6,15]. Till

today it is still less clear whether GC and aldosterone

exert the same effects on ENaC. In this study, we em-

ployed the electrophysiology methods to examine ENaC

activity in cells stimulated with either dexamethasone

(dex) or aldosterone.

C. C. Tang et al. / HEALTH 1 (2009) 152-158

153

2. MATERIALS AND METHODS

2.1. Cell Culture

M1 cells (mouse kidney cortical collecting duct cells)

were purchased from European Collection of Cell Cul-

tures at 21st passages. Cells were grown in the medium

containing DMEM: Ham’s F12 medium (1;1) (Sigma), 2

mM glutamine (Gibco), 5 µM dexamethasone (Sigma),

5% FBS (Sigma) in a 5% CO2 and 37 ºC incubator. 1.5

µM aldosterone (Sigma) was added into the culture me-

dium instead of 5 µM dexamethasone. When cells in the

culture flasks reached 70% confluence, cells were seeded

in low density to either coverslips or culture inserts (BD).

SKF-525A was added in the culture medium containing

dex 8 or 24 hours prior to experiments. Pertussis toxin

(PTX) was prepared according to previous description

[16] and manufacture instruction. Pertussis toxin was

added in the culture medium containing aldosterone in

final concentration 300 ng/ml 18 hours prior to experi-

ments.

2.2. Single Channel Patch Clamp Recording

Cell-attached single channel recording was performed as

previously described [17]. Briefly, M1 cells on the cov-

erslip or insert were transferred into the recording

chamber mounted on a Nikon inverted microscope

(Nikon TE 2000U). Patch pipettes with a tip resistance of

7 MΩ were fabricated from borosilicate glass capillary

(1.5 od, 0.86 id) (Warner) on a Sutter Puller (P97). Bath

solutions contained (in mM): 110 NaCl, 4.5 KCl, 1 MgCl2,

1 CaCl2, 5 HEPEs, 5 Na-HEPEs, pH 7.2. Pipette solution

contained (in mM): 110 NaCl, 4.5 KCl, 0.1 EGTA, 5

HEPEs, 5 Na-HEPEs, pH 7.2. Single channel currents

were recorded with an Axon 1D amplifier and Axon

clampex 9.0. The recording was analyzed by axon

clampfit 9.0. Due to the variance of channel open prob-

ability, the first 2-5 minute single channel recording in

normal bath medium was used as the control and NPo of

ENaC during the period of applying chemicals was di-

rectly compared with NPo of the control. Data is pre-

sented as means ± S.E.M., and statistical differences were

compared using ANOVA, taking P < 0.05 as significant

and represented as *.

2.3. Chemicals

The following chemicals were used: dexamethasone(Sigma),

aldosterone (Sigma), SKF-525A (Biomol), pertussis toxin

(PTX) (Sigma). Most chemicals which dissolved in ethanol

were made up as 1000 to 5000 times stock. Preparation of

PTX was following the previous description and manufac-

ture instruction. All chemical solutions were made as re-

quired on the day of experiments. The solvent, ethanol and

DMSO at the same dilution, was tested alone in controls and

had no effect.

Figure 1. ENaC currents obtained in single channel recordings

from excised membrane. a. In an inside-out recording, small

conductance inward currents were recorded, when pipette

voltage was held at +100 mV and +60 mV, respectively. b. In an

outside-out recording, small conductance inward currents were

monitored, when pipette voltage was held at -60 mV. Bath

application of 5 µM amiloride almost abolished these currents.

The currents were reversed, when bath amiloride was washed

off. c. Currents corresponding to the voltages from inside-out

and outside-out recordings were plotted. The points were fitted

by a linear line.

Figure 2. ENaC currents obtained in cell-attach recordings. a. In

a cell-attach recording, inward currents were recorded, when

pipette voltage was held at 0 mV and +40 mV, respectively. b.

The figure represented multiple step currents recorded in cell-

attach recording. c. The conventional histogram shows the

current events against the current size. d. The I-V curve is ob-

tained from currents in cell-attach recordings.

3. RESULTS

3.1. Identification of ENaC Currents

ENaC currents were identified in either inside-out or

outside-out recording by the unique ENaC channel con-

ductance, sensitivity to amiloride and channel kinetics.

Figure 1a represented the example currents recorded in

C. C. Tang et al. / HEALTH 1 (2009) 152-158

154

an inside-out recording. Currents were elicited when

pipette voltages (Vp) were held at +100 mV and +60 mV,

respectively. In an outside-out recording, the small con-

ductance currents possessed conductance similar to those

obtained in inside-out recording. These currents were

reversely sensitive to amiloride in the range of micro-

molar (Figure 1b). I-V curves constructed by currents

obtained in either inside-out recording (n=50) or out-

side-out recording (n=20) were almost overlapped (Fig-

ure 1c) and possessed the same conductance (5.1 pS,

fitted by linear line, r2=0.99). Figure 2a and Figure 2b

represented the currents obtained in the cell-attach re-

cordings. Currents in Figure 2b contained a multiple

transition status. I-V curve (Figure 2d) constructed by

currents in cell-attach recordings possessed the similar

conductance (5.0 pS, fitted by linear line, r2=0.99).

Therefore, we concluded that currents detected in single

channel recording in M1 cells were mediated by ENaC

channel.

Figure 3. The rundown of ENaC currents in cells treated with

either dex or aldosterone. a represented single channel currents

of ENaC in an inside-out recording within 10 minutes since

formation of inside-out configuration in a cell treated with dex.

b represented single channel currents of ENaC in an inside-out

recording in a cell treated with aldosterone. c. Summary plots of

NPo (+40 mV in a cell-attach recording) showing a sudden

rundown during the period from 3rd to 5th minute in cells treated

with aldosterone and a constant NPo in cells treated with dex.

3.2. Different ENaC Activities in M1 Cells

Treated with Either Dexmethasone (Dex)

or Aldosterone

There was no significant difference in cell morphology of

M1 cells by different treatments with either dex or al-

dosterone. Electrophysiology experiments were per-

formed, when cells formed a monolayer. As a standard

protocol, cell-attach recording was carried out for 5-10

minutes in each cell prior to single channel recording

from the excised membrane including inside-out re-

cording and outside-out recording. In cells treated with

dex, 325 cells exhibited ENaC currents in either in-

side-out or outside-out recording, but only 19 of 325 cells

possessed the detectable current transition of ENaC

channel during 5-minute of cell-attach recording (Table

1). However, in cells treated with aldosterone, 150 cells

exhibited ENaC currents in either inside-out or out-

side-out recording, and 60 of 150 cells possessed de-

tectable current transition of ENaC channel during

5-minute of cell-attach recording. Based on our observa-

tion, a large number of cells contained ENaC channels but

they did not mediate Na+ currents, when cells were in the

intact condition. About 5.84% of cells treated with dex

could mediate Na+ currents, whereas 40% of cells treated

with aldosterone could mediate Na+ currents, although

these cells contained the ENaC in apical side of mem-

brane. However, cells possessed low active ENaC

(p<0.05, in comparing with aldosterone treatment group)

on the apical membrane when cells were incubated with

aldosterone and PTX (300 ng/ml) 18 hours prior to ex-

periments.

Figure 4. The morphology of M1 cells in monolayer selected for

the patch clamping study. a, b, c, d represented the morphology

of M1 cells in monolayer. Numbers 1 to 7 represented the cell to

be studied. e and f represented the cell morphology after 10

minutes of treatment of EGTA.

Figure 5. M1 cells with certain patterns possessed high density of

ENaC currents.

3.3. Rundown of ENaC Currents in Cells

Treated with Either Dex or Aldosterone A6 [18,19] were stable for the initial 4 minutes and ex

hibited a sudden rundown during the period from the 5th

to 10th minute. In cells treated with dex, ENaC currents

Previous studies have demonstrated that ENaC currents in

Openly accessible at

C. C. Tang et al. / HEALTH 1 (2009) 152-158

155

Table 1. Active ENaC detected in different single channel configurations and cells with different treatment. In the cells treated with dex,

325 cells possessed ENaC activity in inside-out recordings but only 19 out of 325 cells possessed ENaC activity in cell-attach re-

cordings. In cells treated with aldosterone, 150 cells possessed ENaC activity in inside-out recordings and 60 out of 150 cells possessed

ENaC activity in cell-attach recordings. Deforming cells by EGTA enhanced the ENaC activity in cells treated with dex. SKF-525A did

not significantly affect ENaC activity in cells treated with dex. PTX significantly reduced the number of active ENaC in intact cells

treated with aldosterone.

Number of cells detected ENaC Dex Aldosterone Dex+EGTA Dex+SKF

525A

Aldosterone

+PTX

Inside out recording Or outside recording

(EC) 325 150 80 62 75

Cell-attach recording (CA) 19 60 15 4 1

Percentage % (CA/EC) 5.84% 40% 18.8% 6.45% 1.3%

from most cells maintained fairly constant open prob-

ability (Po) for over 10 minutes, since formation of in-

side-out configuration (Figure 3a). However, in cells

treated with aldosterone, ENaC currents were stable for

the initial 3-5 minutes and possessed a sudden rundown

from then on (Figure 3b and Figure 3c).

3.4. Enhancement of Na+ Currents in

Dex Treated Cells

Although the apical side membrane of M1 cells contained

ENaC channels, only 5.84% of cells treated with Dex

could mediate Na+ currents. SKF-525A, a general an-

tagonist of CYP, was then used to test whether the low

activity of ENaC was due to 11,12-EET [20,21]. M1 cells

were incubated with SKF-525A (60 µM) for 8 hours and

even 24 hours. Activity of ENaC was then determined in

the inside-out recording from 62 cells. About 4 out of 62

cells possessed the ENaC currents in the cell-attach re-

cording (Table 1). There was no significant difference,

when compared to cells treated with dex. Bath EGTA was

then used to stretch the membrane and rearrange the cy-

toskeleton (Figure 4e and Figure 4f), which may affect

ENaC activities. After the treatment of EGTA for 10

minutes, the cell morphology displayed change within

monolayer. The change always occurred in a patch. The

patch was in the shape of a circle. Cells in the patch lost

the flat structure and became swollen (Figure 4e) with

dome. Cells in the right side of Figure 4f were still flat

and tight, but cells on the left side were pulled by EGTA.

After the wash off EGTA, single channel recordings were

then performed. ENaC currents were detected in the in-

side-out or outside-out recordings from 80 cells. About 15

out of 80 cells possessed the ENaC currents in the

cell-attach recording.

3.5. The Linkage Between Cell Morphology

and Enac Detection

The common morphology of M1 cells from a monolayer

could be summarized into 7 shapes as shown in Figures

4a-4d. Type 1 was flat and could be found in a very flat

part of the monolayer. Types 2, 3, 5 and 6 had recogniz-

able surface domes. Type 4 also had a reasonable dome

and formed a circle with other similar shape cells. Type 7

was constituted by round cells found on the joint of flat

patches. The percentage to detect ENaC currents in Type

1 was 3% (3 out of 100), as compared to over 60% (90 out

of 150) in Type 4. In addition, before cells formed the

monolayer, there were the patterns as shown in Figure 5a

and Figure 5b with high probability to detect ENaC

currents over 60% (12 out of 20).

4. DISCUSSION

Dex and aldosterone were suggested to stimulate the

activity and expression of ENaC in renal and pulmonary

systems. In this study, we showed that both dex and al-

dosterone could allocate ENaC on the apical membrane.

However, these ENaC channels mediating Na+ entry

depended on the treatment on the cells. Only 5.84% of

them in cells treated with dex could permeate Na+ entry,

as compared to over 40% of them in cells treated with

aldosterone. ENaC in cells treated with aldosterone pos-

sessed a quick rundown. The low percentage of ENaC

permeating Na+ entry in cells treated by dex was not

related to inhibition by 11,12-EET and likely due to ac-

tivation of G-protein and how channels were anchored on

the membrane. Deformation of cytoskeleton of cells

could enhance the percentage of ENaC to permeate Na+

entry in intact cells.

In addition to the regulation on the ENaC transcription,

hormones like dex, aldosterone and epidermal growth

factor [22] exert the acute mediation on ENaC activity.

These acute effects were revealed to link with the cellular

level of PIP2. Anionic phosphilipids such as PIP2

[18,19,23] and PIP3 [24-27], located in the inner leaflet of

plasma membrane, were suggested to regulate the activity

of ENaC [26]. The negatively charged head group of PIP2

or PIP3 could directly interact with the positive charged

cytoplasmic parts in β and/or γ subunits [19,24,26,28,29]

of ENaC to exert the regulation. Evidences further indi-

C. C. Tang et al. / HEALTH 1 (2009) 152-158

156

cated that aldosterone elevates the cellular concentration

of PIP3 by activating PI-3-kinase to lock the cytoplasmic

termini of ENaC to the inner surface of plasma membrane

[3] and/or pull the channel to activate ENaC [25]. Al-

dosterone-induced protein K-Ras localizes PI3-K near

ENaC. The spatial organization of phosphatidylinositides

and specific phospholipid precursors [30,31] within cel-

lular-membrane might determine the turnover of ENaC to

allow Na+ currents [32]. Therefore, the activation of

PI3-K and spatial organization of phosphatidylinositides

on the apical membrane by aldosterone might explain the

active ENaC in the cells treated with aldosterone, but not

dex, suggesting the presence of another mechanism.

Degeneration of PIP2 by PLC [33] could lead to

unlocking of cytoplasmic termini of ENaC [25,33], re-

sulting in decreased ENaC activity. It was manifested as

the rundown of ENaC in excise membrane recording.

Rundown of ENaC currents in excise membrane re-

cordings reflected the simultaneous/continuous activation

of PLC in a platform interacting with PIP2 and ENaC.

Such platforms were revealed in G protein activated in-

ward rectifying K+ channel [34], trp channel [35] and

suggested to locate in β and γ subunits of ENaC [19].

Moreover, PLC-γ via receptor typrosine kinase [22] and

PLC-β via Gq/11 [33,36] were demonstrated to mediate the

degeneration of PIP2 in epithelial cells. However, the

effects of PIP2 on ENaC activity are suggested to be

permissive rather than regulatory [26]. Declines in PIP2

levels are parallel to the loss of ENaC activity, whereas

addition of exogenous PIP2 rarely causes ENaC open

probability to exceed the control level in excise mem-

brane recording [36]. Increase in ENaC open probability

by addition of PIP2 with GTP in inside-out recordings [19]

implicates that the activation of G-protein plays an im-

portant role in regulating ENaC activity. Decrease in

ENaC open probability by dialysis of intracellular GTP,

which is not dependent on PI-3 K [37], further supported

this hypothesis. We, therefore, proposed that activation of

G-protein in cells treated with aldosterone might be the

key to keep active ENaC on the membrane. Activation of

G-protein could activate PLC, resulting in PIP2 degen-

eration. The rapid rundown of ENaC in cells treated with

aldosterone might reflect activation of G-protein. In intact

cells, the degeneration and resynthesis of phosphatidy-

linositides reach a balance to maintain the lipid compo-

nent of the membrane structure [38,39], but the lack of

resynthesis of PIP2 pathway in excised membrane re-

cording causes the decline of PIP2, resulting in rundown

of ENaC. Like aldosterone, dex too distributes ENaC on

the apical membrane via a similar mechanism as aldos-

terone via SGK-1 [3,4,7,40,41]. It is demonstrated by the

ENaC activity detected in inside-out recordings in cells

treated with dex. The low active ENaC in intact cells

treated with dex and constant ENaC open probability in

inside-out recording might be attributed to the low activ-

ity of G-protein. The linkage between aldosterone and

activation of G-protein could be implicated by experi-

ments that prevention of ATP release due to stimulation of

aldosterone abolishes the aldosterone action [17]. Our

observation that PTX incubation significantly reverses

the effect of aldosterone on active ENaC in intact cells is

consistent to previous report [42] and further supports our

hypothesis.

11,12 - EET, a metabolite of AA via CYP epoxygenase,

was demonstrated to mediate the direct inhibition of

ENaC [20]. The evidence that EET mediates the inhibi-

tion effect of adenosine [21] on ENaC indicates a pro-

found pathway to regulate the cellular EET level. The

other rational explanation to low active ENaC in cells

treated with dex is due to presence of cellular inhibitor of

ENaC. We, therefore, applied SKF-525A, a general an-

tagonist for CYP, to reduce the cellular EET level. There

was no significant difference in the ENaC activity in

intact cells, suggesting that EET is not the case to respond

to low active ENaC.

It is widely accepted that ENaC activity is strongly

regulated by cytoskeleton elements [43-45]. Recent evi-

dence that activation of P2 receptors in epithelial baso-

lateral membrane could deform the cells and eventually

activate ENaC addressed the classic observation that

aldosterone action on Na+ reabsorption relies on ATP

production. Cell cytoskeletons could be deformed by

many methods like ATP stimulation and EGTA. Our pre-

vious work demonstrated that elevation in [Ca2+]i could

reduce ENaC open probability, but there was no signifi-

cant difference in ENaC activity, when 500 nM and 0 nM

Ca2+ were in cytoplasmic medium. EGTA was, therefore,

used to deform the cell. A significant increase in ENaC

activity in cells treated with dex demonstrated that ele-

ments of cytoskeleton regulate ENaC activity. It also

explains that ENaC activity is commonly observed in the

excised membrane recording. However, the mechanism

remains unclear. Consistent to previous observation [11],

ENaC channels are in high density and distributed in the

cells containing splitting pattern, suggesting that ENaC

might involve cell differentiation and proliferation. The

recognized dome in some cells, which contain ENaC on

apical membrane, might be formed due to Na+ entry via

ENaC with subsequent water entry.

In conclusion, aldosterone and glucocorticords could

increase the expression of ENaC and distribute ENaC on

the apical membrane of cortical collecting duct cells. Low

active ENaC is found in cells treated with dex. Activation

of G-protein is required to keep the channel active. Al-

dosterone could lead to activation of G-protein to there-

fore possess high ENaC activity in intact cells.

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