Nongenomic regulation of ENaC by aldosterone

Zhen-Hong Zhou and James K. Bubien

Department of Physiology & Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Aldosterone is involved in salt and water homeostasis. The main effect is thought to involve genomic mechanisms. However, the existence of plasma membrane steroid receptors has been postulated. We used whole cell patch clamp to test the hypothesis that epithelial sodium channels (ENaC) expressed by renal collecting duct principal cells can be regulated nongenomically by aldosterone. In freshly isolated principal cells from rabbit, aldosterone (100 nM) rapidly (<2 min) increased ENaC sodium current specifically. The aldosterone-activated current was completely inhibited by amiloride. Aldosterone also activated ENaC in cells treated with the mineralocorticoid receptor blocker spiranolactone. Nongenomic activation was inhibited by inclusion of S-adenosyl-L-homocysteine in the pipette solution, which inhibits methylation reactions. Also, the nongenomic activation required 2 mM ATP supplementation in the pipette solution. Aldosterone did not activate any ENaC current in whole cell clamped rat collecting duct principal cells. These functional studies are consistent with aldosterone membrane binding studies, suggesting the presence of a plasma membrane steroid receptor that affects cellular processes by mechanisms unrelated to altered gene expression.

epithelial sodium channels; principal cells


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE MINERALOCORTICOID HORMONE ALDOSTERONE has the ability to increase the reabsorption of salt and water. A part of this process appears to involve the stimulation of amiloride-sensitive sodium channels in the cortical collecting duct of the kidney. The classic mechanism for this regulation is that aldosterone activates a cytosolic mineralocorticoid receptor (20), which in turn has genomic effects resulting in increased transcription of the genes that produce serum glucocorticoid-regulated kinase and Na+-K+-ATPase and epithelial sodium channel (ENaC) subunits (1, 8, 10, 15, 29). A compelling reason for suspecting a genomic mechanism of regulation is that in vitro, the effects of aldosterone are not acute but, rather, take as long as 4 h to develop and can be blocked by inhibitors of transcription and translation (7, 23, 26, 34). It is important to consider that the vast majority of experiments to elucidate the mechanism of action of aldosterone have been carried out on model systems such as rat tissue and cells and A6 epithelial cells derived from Xenopus laevis. It is possible that the mechanism of action of aldosterone in other species, including humans, may be more complex than in these model systems.

Recent evidence suggests that in species other than the rat, aldosterone may produce acute effects at picomolar concentrations. Half-maximal effects have been observed at a concentration of 100 pM (13, 14). For example, rapid aldosterone-mediated effects on cellular processes in human smooth muscle cells and human lymphocytes have been observed, in the presence of spiranolactone, to inhibit the mineralocorticoid receptor (9, 32). These effects have also been observed in the presence of actinomycin D, precluding the possibility of genomic effects (13, 14). The direct demonstration of these aldosterone-induced effects led us to the hypothesis that acute aldosterone-mediated ENaC activation by nongenomic mechanisms may be present in renal principal cells. To test this hypothesis, we used whole cell patch-clamp analysis of freshly isolated renal principal cells from rat and rabbit kidneys. Identical experiments were also carried out on lymphocytes from humans, dogs, rats, mice, and rabbits.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Principal cell preparation. Collecting ducts were manually dissected from sagittal slices obtained from 50-g Sprague-Dawley rats and 0.5-kg New Zealand White rabbits. The dissected collecting ducts were suspended in RPMI 1640 supplemented with 1.5 mg/ml collagenase A (Boehringer Mannheim, Mannheim, Germany). The collecting ducts were enzymatically digested for 1.5 h to isolate individual cells. The digested cells were washed in serum-free RPMI and placed in a perfusion chamber mounted on the stage of an inverted microscope. Once the whole cell configuration was established, the capacitances were balanced (rabbit principal cell average capacitance = 12.1 ± 0.76, n = 37; rat principal cell capacitance = 10.9 ± 0.68, n = 29), and initial current measurements were made in unsupplemented RPMI ([Na+] = 133 mM; [K+] = 5.3 mM; [Cl-] = 108.3 mM). The bath solution was changed (by perfusion of the entire bath chamber) to RPMI supplemented with aldosterone (100 nM), vasopressin (250 nM), amiloride (2 µM), spiranolactone (1 µM), or various combinations of these compounds. To block the cytosolic mineralocorticoid receptor, we suspended some cells in RPMI supplemented with spiranolactone for 1 h before whole cell clamping was performed.

Preparation of lymphocytes. Human and canine lymphocytes were isolated from peripheral blood samples by differential centrifugation over Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden). The cells were washed and resuspended in serum-free RPMI. Subsequently, suspended cells were placed in the perfusion chamber. Whole cell patch clamp and testing of aldosterone were carried out with techniques and protocols identical to those described for principal cells. Rabbit, rat, and mouse lymphocytes were obtained by mincing sections of spleen and manually freeing the cells by agitation. The cell suspensions were then centrifuged over Ficoll-Paque and treated identically to the lymphocytes obtained from peripheral blood. All procedures were carried out under the guidelines for animal use and with Institutional Board Approval for Human Use.

Whole cell patch clamp. Micropipettes were constructed using a Narashigi pp-83 two-stage micropipette puller. The tips of these pipettes had an inner diameter of ~0.3-0.5 µm and an outer diameter of 0.7-0.9 µm. When filled with an electrolyte solution containing (in mM) 100 K-gluconate, 30 KCl, 10 NaCl, 20 HEPES, 0.5 EGTA, and 4 ATP as well as <10 nM free Ca2+, pH 7.2, the electrical resistance of the tip was 1-3 MOmega . The bath solution was serum-free RPMI-1640 cell culture medium. The solutions accurately approximate the ionic gradients across the cell membrane in vivo. Pipettes were mounted in a holder and connected to the head stage of an Axon 200A patch-clamp amplifier affixed to a three-dimensional micromanipulator system attached to the microscope. The pipettes were abutted to the cells, and slight suction was applied. Seal resistance was continuously monitored (model 300 Nicolet oscilloscope) by using 0.1-mV electrical pulses from an electrical pulse generator. After seals were formed with resistances in excess of 1 GOmega , another suction pulse was applied to form the whole cell configuration by rupturing the membrane within the seal but leaving the seal intact. Successful completion of this procedure produced a sudden increase in capacitance with no change in seal resistance. The magnitude of the capacitance increase is a direct function of the membrane available to be voltage clamped (i.e., the membrane area, and hence cell size). Typically, this capacitance was between 5 and 10 pF for activated peripheral blood lymphocytes.

Previous measurements of transmembrane voltage showed that once the whole cell configuration was obtained, the pipette solution and the cellular interior equilibrated within 30 s. The cells were then held at a membrane potential of -60 mV and clamped sequentially for 0.8 s each to membrane potentials of -160, -140, -120, -100, -80, -60, -40, -20, 0, 20, and 40 mV, returning to the holding potential of -60 mV for 0.8 s between each test voltage. This procedure provided voltages sufficient to measure inward sodium (at more hyperpolarized potentials) and outward potassium (at more depolarized potentials) currents. The currents were recorded digitally and filed in real time. The entire procedure was performed with a DOS Pentium computer modified for analog-to-digital (A/D) signals with pCLAMP 6 software, with an A/D interface controlled by pCLAMP (Axon Instruments, Sunnyvale, CA).

Single-channel patch clamp. For inside-out patch recordings, the pipette and bath solution were 140 mM Na-gluconate and 20 mM HEPES, pH 7.4. For outside-out patches, the solution was supplemented with 5 mM vanadate and 0.1 mM fluoride to inhibit lipid peroxidation and reduce channel rundown. Single-channel activity was filtered at 50 Hz and recorded on videotape. For amplitude histogram analysis, the taped records were digitized (Fetchex, pCLAMP), measured (Fetchan, pCLAMP), and analyzed (pSTAT, pCLAMP) by computer. The current-voltage (I-V) relation was constructed from the average currents obtained at a variety of positive and negative voltages applied to patches from six different cells.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Nongenomic ENaC activation in rabbit principal cells. Enzymatic dispersion of collecting ducts resulted in two morphologically distinct cell types, large oval cells and smaller round cells. The large oval cells have been shown previously to be principal cells, whereas the smaller round cells were most likely intercalated cells (2). Only the large round cells were whole cell clamped. These cells were exposed to 100 nM aldosterone. Positive identification of principal cells was determined by positive response either to aldosterone or, if the cells failed to respond to aldosterone, to vasopressin. No cells failed both tests. Upon entering the bath, the aldosterone induced a specific activation of the inward currents at hyperpolarized membrane potential clamp voltages. The activated currents were subsequently inhibited completely with 2 µM amiloride (in the continued presence of aldosterone), confirming that the activated currents were amiloride sensitive. A typical experiment is shown in Fig. 1. Figure 2 shows the average I-V relations for the current activated by aldosterone and for the current inhibited by amiloride after aldosterone activation. These I-V relations show inward current up to +40 mV, the most positive membrane potential tested. This I-V relation is expected for a highly selective sodium conductance since, under our experimental conditions, the equilibrium potential for sodium was +67 mV. Thus the activated current is carried primarily by sodium. In a separate set of experiments, rabbit principal cells were resuspended in RPMI supplemented with 1 µM spiranolactone for 1 h before use for whole cell patch clamp. Figure 3 shows that spiranolactone had no effect on the inward currents. However, even with mineralocorticoid receptor inhibition, 100 nM aldosterone specifically activated the inward currents. These experiments indicate that the acute activation of ENaC by aldosterone did not utilize the mineralocorticoid receptor and was therefore nongenomic. Aldosterone also acutely activates ENaC current in human lymphocytes. These cells were used to confirm that the effect of increased inward current was not caused by translation of ENaC. To inhibit protein translation, we incubated human lymphocytes in 10 µg/ml cyclohexamide (33) and subsequently whole cell clamped them. In cyclohexamide-treated cells, aldosterone acutely activated ENaC current within seconds of exposure to the cells (Fig. 4), supporting the hypothesis that acute ENaC activation by aldosterone did not involve genomic mechanisms.


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Fig. 1.   Whole cell current records from a freshly isolated rabbit renal principal cell. Top: basal current record. The increased inward currents in response to aldosterone (middle) were completely inhibited by amiloride in the continued presence of aldosterone (bottom). Typically, activation of the inward sodium currents occurred within 1 min after aldosterone entered the bath.



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Fig. 2.   Average current-voltage relations for the whole cell current activated by aldosterone and inhibited by amiloride. These current-voltage relations go to zero current at an equilibrium potential for sodium that is determined by the sodium gradient across the membrane, indicating the high sodium selectivity of the activated and inhibited current.



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Fig. 3.   Preincubation of rabbit principal cells with the mineralocorticoid receptor inhibitor spiranolactone failed completely to inhibit the aldosterone-induced activation of the inward sodium currents. Thus the acute epithelial sodium channel (ENaC) activation observed in these experiments was independent of the mineralocorticoid receptor.



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Fig. 4.   Whole cell currents obtained from a human lymphocyte pretreated with 10 µg/ml cyclohexamide for 90 min to inhibit protein transcription. These records show that inhibition of protein transcription has no effect on the acute activation of inward current by 100 nM aldosterone. This finding supports the hypothesis that the acute activation of these currents does not involve genomic mechanisms.

Aldosterone does not acutely activate ENaC in rat principal cells. In contrast to the findings in the rabbit principal cells, aldosterone had no acute effect on principal cells isolated from the collecting ducts of rats. Figure 5 shows typical records from a whole cell- clamped rat principal cell, where procedures used were identical to those used on the rabbit principal cells. Because aldosterone had no effect, the expression of ENaC and the cell type (principal cell) were confirmed in every experiment by subsequent acute, specific activation of the amiloride-sensitive inward currents by 250 nM vasopressin (Fig. 5, bottom). The same protocol was carried out on rabbit principal cells (Fig. 6). Once ENaC was activated by aldosterone, subsequent stimulation with vasopressin did not activate the inward currents any further, i.e., there was no synergistic effect of the two ENaC agonists, implying that they activated the same set of channels. For most experiments, vasopressin was added after aldosterone was washed out of the bath. During short-term experiments (<10 min), currents did not inactivate upon washout of aldosterone. Thus, to test for synergy, vasopressin was superfused after aldosterone washout. Some experiments were performed with both ENaC agonists present (not shown), with identical results.


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Fig. 5.   Current records from a whole cell-clamped rat renal principal cell. In 6 of 6 rat principal cell whole cell preparations, aldosterone failed completely to activate any current. However, in each cell, vasopressin activated the inward sodium currents, and these currents were completely inhibited by amiloride.



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Fig. 6.   In rabbit principal cells, aldosterone activated the inward current. When activation was followed by vasopressin, no additional current was activated. All of the activated current was inhibited by amiloride. Thus there was no apparent synergy between these sodium channel agonists, suggesting the same channels could be activated independently by either aldosterone or vasopressin.

Figure 7 shows the average current activated by these ENaC agonists measured at the equilibrium potential for potassium. At this potential (-80 mV) there can be no potassium current; thus all of the current activated by aldosterone or vasopressin must be sodium current. In the absence of aldosterone, or if aldosterone activation was inhibited, vasopressin activated the inward currents. Comparison of the average currents resulting from each treatment was assessed statistically with an unpaired Student's t-test. The mean current levels showed that for rat principal cells, aldosterone did not significantly alter the current, but vasopressin increased it significantly, while amiloride restored the current to the basal level. For the rabbit principal cells, aldosterone significantly increased the current, vasopressin had no additional effect, and amiloride returned the conductance to the basal level. Also, the basal conductance and the activated conductance in rat and rabbit principal cells were not significantly different between the species.


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Fig. 7.   Average whole cell current at the equilibrium potential for potassium (EK; -80 mV) in rabbit and rat cells. At this potential there can be no potassium current; thus the current activated by each ENaC agonist can only be carried by sodium. Also, the current recorded at -80 mV is within the physiological range. Values represent means from 6 different cells that received all of the treatments, i.e., aldosterone (Aldo), vasopressin (Vaso), and amiloride (Amil). Current amplitudes are reported in pA.

Nongenomic ENaC activation utilizes methylation. We next tested the hypothesis that aldosterone-mediated nongenomic activation of ENaC utilizes methylation rather than protein kinase A-mediated phosphorylation to induce channel activation. For these experiments, pipette solutions were supplemented with 100 µM S-adenosyl-L-homocysteine (SAH). Once the whole cell configuration was formed, the cells were given 5 min to ensure that the SAH equilibrated with the cytosol. The compound is an end-product inhibitor of methyl esterification and has been shown previously to inhibit Na+ transport (23). When SAH was included in the pipette solutions, acute ENaC activation by aldosterone was completely prevented in rabbit principal cells. However, in the same cells, vasopressin activated the ENaC Na+ currents normally (Fig. 8). The average sodium conductance in these cells after each treatment is shown in Fig. 9. This finding implies that the vasopressin response was independent of methyl esterification.


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Fig. 8.   Whole cell currents showing that inclusion of S-adenosyl-L-homocysteine (SAH; 100 µM) in the pipette solution completely prevents ENaC activation by aldosterone in rabbit principal cells. However, in the same cell, vasopressin produced ENaC activation. SAH inhibits methylation. Thus these experiments suggest that methylation contributes to aldosterone-mediated nongenomic activation of ENaC. The cells were allowed to equilibrate with the pipette solution for 5 min before the cells were exposed to aldosterone and the voltage-clamp protocols were run.



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Fig. 9.   There was no change in the average inward sodium conductance in response to aldosterone when SAH was included in the pipette solution (NSD, no significant difference; n = no. of cell preparations). However, in the same cells, vasopressin induced a significant specific inward conductance increase (P < 0.05). Conductance was calculated as the chord between the average current measured between a clamp potential of -140 mV and the reversal potential for each cell.

Nongenomic aldosterone-mediated ENaC activation requires ATP. The present experiments extend those obtained from intact rabbit cortical collecting ducts and help to resolve an apparent contradiction. A considerable delay (2-4 h) was observed when the in vitro effect of aldosterone was measured using intact collecting duct segments (21, 26, 34). In the present study, aldosterone-stimulated ENaC activation was observed within seconds. The only obvious difference between the cited studies and the present study was that we were able to control the cytosolic ATP concentration, whereas in intact collecting duct segments, the cellular ATP is controlled by cellular metabolism. Once these segments are dissected, it is possible that ATP becomes depleted. It has been shown previously that reduced ATP induces a loss of structural support for the plasma membrane (11) and reduces phosphorylation in renal epithelia (19). Thus we hypothesized that the difference between the two preparations might be a difference in cytosolic ATP. With a conventional whole cell configuration, the pipette solution is contiguous with the cytosol and, in our experiments, routinely supplemented with 4 mM ATP. With intact collecting ducts, it may not be possible to maintain such a high level of cytosolic ATP concentration. If the thermodynamics of aldosterone-mediated signal transduction require high levels of ATP, these reactions may be lost if there is even a slight reduction of ATP. To test this hypothesis, we examined nongenomic ENaC activation by aldosterone, using four concentrations of ATP in the pipette solutions (0, 0.5, 1, and 2 mM ATP). We found that in the complete absence of ATP in the pipette solutions, aldosterone failed completely to activate ENaC. The same result was observed when the pipette solution contained 0.5 mM ATP. With 1 mM ATP, we observed incomplete and intermittent ENaC activation by aldosterone. Full ENaC activation was observed in rabbit principal cells only when the pipette solutions were supplemented with a minimum of 2 mM ATP. Figure 10 shows representative whole cell currents for three principal cells with different concentrations of ATP in the pipette solutions. Responses of similar magnitude were obtained when the pipette solutions contained 4 mM ATP (Figs. 1, 3, and 5), implying that a saturating concentration of ATP for this signal transduction was ~2 mM.


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Fig. 10.   Whole cell records from 3 rabbit principal cells showing that aldosterone fails to activate the inward sodium current in the absence of ATP. These titrations of the pipette ATP concentration suggest that 2 mM ATP is required for full activation of ENaC by aldosterone.

To test the hypothesis that ATP hydrolysis was required in this signal transduction pathway, we supplemented the pipette solution with 10 mM 5'-adenylylimidodiphosphate (AMP-PNP, a nonhydrolyzable ATP analog). With AMP-PNP in the pipette solution, aldosterone (100 nM) failed to activate any inward current (Fig. 11). To confirm that the aldosterone maintained its potency, we whole cell clamped an additional three cells using a pipette solution supplemented with 4 mM ATP, and the cells were treated with the same 100 nM aldosterone solution. With 4 mM ATP in the pipette solution, inward currents were activated within seconds. These findings indicate that hydrolysis, and not simple occupation of ATP binding sites, is required for aldosterone stimulated activation of ENaC.


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Fig. 11.   Whole cell records showing that when the pipette solution was supplemented with 10 mM 5'-adenylylimidodiphosphate (AMP-PNP, a nonhydrolyzable ATP analog), aldosterone failed completely to activate acutely any inward current. Subsequent control experiments with an ATP-supplemented pipette solution were performed to ensure the potency of the aldosterone solution and to eliminate the possibility that impotent aldosterone resulted in the inability to activate the currents.

Aldosterone-activated lymphocyte sodium currents. We have previously demonstrated that lymphocytes express an amiloride-sensitive sodium conductance that is abnormal in lymphocytes from individuals with Liddle's disease (4, 5, 17). Lymphocytes can be easily isolated from a small blood sample from any mammalian species. Aldosterone has been shown to bind to the plasma membrane of lymphocytes with high affinity (Ks = 0.1 nM) (13, 14, 32). Also, since aldosterone failed to activate any sodium current in rat principal cells, lymphocytes provided a second cell type to confirm this negative observation. For these reasons we tested the hypothesis that aldosterone was able to activate specifically lymphocyte amiloride-sensitive sodium currents in peripheral blood lymphocytes from five species (human, dog, rabbit, rat, and mouse). Lymphocytes from each of these species were whole cell clamped under conditions identical to those used to whole cell clamp renal principal cells. After the basal current levels were established, the cells were superfused with 100 nM aldosterone. Subsequently, the cells were superfused with 40 µM 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (8-CPT-cAMP). Figure 12 shows the results obtained from lymphocytes of each species. We found that 100 nM aldosterone activated specifically the inward sodium conductance in lymphocytes from humans, dogs, and rabbits. These currents were inhibited completely by 2 µM amiloride (not shown), the same as when they are activated by 8-CPT-cAMP, cholera toxin, or pertussis toxin (3-6). However, just as was found in rat principal cells, aldosterone failed completely to activate any currents in rat and mouse lymphocytes. This finding was not due to the lack of sodium channel expression, because 8-CPT-cAMP activated sodium currents in each rat and mouse whole cell-clamped lymphocyte. This observation confirmed the negative effect of aldosterone on rat principal cells. In human, dog, and rabbit lymphocytes, after sodium current activation by aldosterone, 8-CPT-cAMP inhibited the activated currents. This finding was similar to findings previously reported showing that pertussis toxin and cholera toxin each activated the sodium conductance alone but that, when one was followed by the other, the conductance was inhibited (5).


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Fig. 12.   Whole cell clamps identical to those performed on renal principal cells were performed on lymphocytes from 5 species. These records show that aldosterone activated the inward sodium currents in lymphocytes from humans, rabbits, and dogs but failed to activate these currents in lymphocytes from rats and mice. 8-CPT-cAMP, 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate.

Aldosterone modifies single-channel characteristics. The "ragged" kinetics of the whole cell currents in principal cells and lymphocytes has generated some concern about the identity of the channels that are activated by aldosterone. To address this concern, we examined single-channel characteristics of the sodium conductors expressed by human lymphocytes. We have recently demonstrated the expression of human ENaC (hENaC) mRNA by RT-PCR and direct sequencing of the PCR products by human lymphocytes. To determine the single-channel conductance, we formed inside-out patches (Fig. 13, top) were formed in symmetrical Na-gluconate solutions (140 mM). The patches were clamped to potentials ranging from -80 to +80 mV. The current amplitudes were determined by either direct measurement of individual openings or amplitude histogram analysis (pCLAMP) (Fig. 13, middle right). From the slope of the I-V relation (Fig. 13, middle left), it was found that the single-channel conductance was 8.4 ± 1.1 pS. This conductance is the same as the single-channel conductance reported for hENaC (alpha ,beta ,gamma -subunits) expressed in Xenopus oocytes (16, 28).


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Fig. 13.   Top: records showing single-channel activity in an inside-out patch from an untreated lymphocyte plasma membrane. Middle left: average currents from similar records observed in patches from 6 cells were used to estimate the slope conductance (G) from the current-voltage relation. I/O, inside-out. Middle right: amplitude histograms constructed from all-points histograms were used to confirm the accuracy of measurements made on individual channel transitions. Bottom: records obtained from an outside-out patch showing that a submaximal concentration of amiloride (100 nM) shortens the transition times but does not alter the single-channel conductance.

To test the amiloride sensitivity of the single channels, we formed outside-out patches, because amiloride interacts with the outside surface of the channels. We experienced the "rundown" phenomena typical of ENaC in the inside-out patch experiments. To inhibit lipid peroxidation and delay channel rundown, we added vanadate (5 mM) and fluoride (0.1 mM) to the Na-gluconate pipette and bath solutions. Outside-out patches had channels with the same conductance and with the long open and closed times observed in inside-out patches, typical of ENaC. When the bath solution was supplemented with 100 nM amiloride, the open-closed transition times were shortened (Fig. 13, bottom) and channel open probability (NPo) was reduced by 60% from 1.97 to 0.77. This finding shows the incomplete inhibitory action of amiloride on these channels at an amiloride concentration close to the IC50 for amiloride and ENaC (75 nM). When the amiloride concentration was increased to 2 µM, single-channel activity was completely abolished.

When the cells were treated with 100 nM aldosterone, a somewhat different channel behavior was observed. In the absence of aldosterone, the frequency of encountering single channels was less than 1 in 20 patches. After treatment with aldosterone, the frequency increased to ~8 in 10 patches. In outside-out patches, the unitary conductance was unchanged, but the channels opened and closed in groups (Fig. 14, top). Single-channel activity was virtually abolished when amiloride (2 µM, final concentration) was added to the bath solution (Fig. 14, bottom). We have demonstrated that this single-channel behavior produces the "ragged" whole cell currents that are characteristic of the sodium conductance of principal cells and lymphocytes by reconstructing the whole cell currents from single-channel recordings (6). This behavior of opening and closing in groups should not be too surprising because it has been shown by freeze fracture that ENaC aggregates into groups when expressed in oocytes (12). Finally, it should be mentioned that the vast majority of patches (>80%) were devoid of single-channel activity in cells that were not pretreated with aldosterone. These single-channel findings are completely consistent with the whole cell findings and establish by biophysical characteristics that the channels that are activated by aldosterone are ENaC.


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Fig. 14.   Single-channel records obtained from an outside-out patch made from a lymphocyte treated with 100 nM aldosterone. Top: records showing that the single-channel conductance is not altered by aldosterone treatment; rather, the channel openings appear in groups. Bottom: records showing that 2 µM amiloride virtually eliminates channel opening. These records show that single-channel kinetics behavior accounts for the "ragged" appearance of the whole cell currents induced by aldosterone at hyperpolarized membrane potentials.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The direct electrophysiological findings from these studies address three major issues concerning the role of aldosterone in the regulation of sodium currents by principal cells. First, the findings demonstrate directly an acute, nongenomic activation of an amiloride-sensitive sodium conductance (ENaC) in renal principal cells of the rabbit. In contrast, the findings also show that aldosterone fails to activate any current in principal cells of the rat. Because the kidneys of both species are used extensively to study renal salt and water regulation, this difference has wide-ranging implications for our understanding of the role of aldosterone in controlling sodium reabsorption in the collecting duct. For example, on the basis of numerous previous studies indicating that aldosterone increases expression of sodium channels by interaction with the cytosolic mineralocorticoid receptor (1, 17, 26), inhibition of this receptor with spiranolactone has been a recognized treatment for primary aldosteronism. However, since the experiments presented here show that aldosterone has the ability to activate nascent channels in the presence of spiranolactone, a more effective therapy may be the combination of spiranolactone plus amiloride for cases of primary or secondary aldosteronism where surgery is not warranted or cannot be performed.

We are aware of previous experiments utilizing rabbit cortical collecting ducts that showed delayed effects of aldosterone on sodium conductance but that failed to show the immediate effect demonstrated in our whole cell experiments (7, 27, 34). Because some of the experiments measured transepithelial potential (TEP) and could demonstrate an acute change in TEP induced by vasopressin but not by aldosterone, the findings suggested that aldosterone did not produce an acute ENaC activation in rabbit cortical collecting ducts, contrary to the whole cell clamp findings presented in this paper. We hypothesized that by resupplying cytosolic ATP with the use of ATP-supplemented pipette solutions, we were able to replete ATP to a concentration that was apparently higher than the ATP concentration in intact cortical collecting ducts in vitro. Also, these findings support the hypothesis that the depleted ATP was essential for signal transduction between aldosterone and ENaC to produce nongenomic ENaC activation. We found that in the absence of pipette ATP, we obtained the same result as has been reported for intact collecting ducts, i.e., no effect of aldosterone but an acute increase in ENaC current by vasopressin. By titrating back the pipette ATP, we were able to establish that for full aldosterone-mediated acute ENaC activation, a minimum concentration of 2 mM ATP was required.

One question that remains is, how can vasopressin or 8-CPT-cAMP activate ENaC current in the absence of pipette solution ATP? The only established intracellular effect of cAMP that we are aware of is to bind to the regulatory subunit of protein kinase A, thereby inducing activity of the catalytic subunit. The activated catalytic subunit, in turn, catalyzes phosphorylation of a variety of substrates. This reaction requires ATP. We speculate that there must be separate pools of ATP, one that becomes depleted and one that does not. Our findings indicate that aldosterone-stimulated signal transduction for ENaC regulation requires ATP from the depletable pool but that vasopressin-stimulated signal transduction for ENaC activation utilizes ATP from a pool that is not depleted. This line of reasoning reconciles the difference between the whole cell clamp findings and the findings obtained in intact collecting ducts. In intact tubules the in vitro metabolism may not be robust (due to reduced oxygen delivery) enough to continuously provide the cellular concentrations of ATP that are required for aldosterone-mediated ENaC regulation.

Previous studies have shown that SAH inhibits substrate methylation (24, 25, 30, 31). In the present study aldosterone failed to activate ENaC currents when the pipette solution was supplemented with SAH. Thus methylation appears to play a role in the signal transduction pathway between aldosterone and ENaC for nongenomic signaling. This finding is consistent with the findings of others who also suggest that methylation reactions regulate ENaC. For example, with the use of purified ENaC protein subunits, it has been demonstrated directly that carboxymethylation of the beta -subunit alters the biophysical properties of ENaC (18, 22). It also has been shown that isoprenlycysteine-O-carboxylmethyl transferase regulates aldosterone-sensitive sodium reabsorption (31). While the findings of the present study do not identify the specific components of the signal transduction pathway, they are consistent with these previous findings in suggesting that aldosterone-mediated nongenomic ENaC regulation utilizes methylation as one component of the signal transduction pathway.

Another interesting finding in the present study is that in lymphocytes, aldosterone-activated sodium currents were inhibited by subsequent exposure to 8-CPT-cAMP. The same phenomenon was observed when lymphocyte sodium currents were activated by using pertussis toxin to activate the channels initially (5). Because pertussis toxin ADP-ribosylates G proteins, the implication of the present study is that, at least in lymphocytes, a G protein may be involved in the aldosterone-mediated regulation of the sodium conductance. Others also have implicated guanine nucleotides and GTP binding proteins in aldosterone-mediated regulation of ENaC (24) and have demonstrated directly that treatment with aldosterone plus GTP induces methylation in purified ENaC polypeptides from A6 epithelia (25).

The observation that the amiloride-sensitive sodium conductance of lymphocytes can be activated by aldosterone is important for a number of reasons. First, the finding implies that lymphocytes are affected when circulating aldosterone levels rise, such as during periods of salt deprivation. Lymphocyte sodium conductance can be activated by norepinephrine via alpha 1-adrenergic receptors (3). The findings of the experiments described here suggest that another receptor (i.e., a plasma membrane steroid receptor) can also regulate ENaC by a distinct and independent signal transduction pathway. Why two independent pathways have evolved for the acute regulation of ENaC is not clear. Rats do not appear to express a signal transduction pathway for acute aldosterone-mediated ENaC activation, and yet they have no apparent deficiencies in renal function. Rats may not need acute ENaC activation to the extent that rabbits, dogs, and humans do. Alternatively, the presence of both pathways may provide more precise regulation of the sodium-retentive mechanisms in rabbits and humans. Whatever the ultimate explanation for this difference, it remains important to recognize that aldosterone induces nongenomic activation of principal cells from rabbits but does not do so in principal cells from rats.

In addition to the rat-rabbit differences and to the direct stimulation of ENaC in the absence of mineralocorticoid receptor involvement and its role in blood pressure regulation is the possibility that this mechanism may be involved in other systemic pathophysiology associated with aldosteronism, such as vascular damage, and in cardiac pathophysiology. These experiments show that lymphocytes are sensitive to aldosterone. Thus, if the plasma level of aldosterone is increased, there is a possibility that the steroid can provoke an unwarranted immunological response. It is well known that glucocorticoids are immunosuppressive. We show here that a mineralocorticoid has agonistic effects that mimic the actions of cytokines. Thus the basic findings described here may have even broader physiological implications than we currently understand.


    ACKNOWLEDGEMENTS

We thank Drs. Janos Peti-Peterdi and P. Darwin Bell for providing renal tubules and Drs. Dale J. Benos and James A Schafer for critical input into these studies.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1-DK-52789 (J. K. Bubien). J. K. Bubien is an Established Investigator of the American Heart Association.

Address for reprint requests and other correspondence: J. K. Bubien, Dept. of Physiology and Biophysics, 876 McCallum Bldg., Univ. of Alabama at Birmingham, Birmingham, Alabama 35294 (E-mail: bubien{at}uab.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 22 March 2001; accepted in final form 25 May 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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