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Osmotic pressure regulates alpha beta gamma -rENaC expressed in Xenopus oocytes

Hong-Long Ji, Catherine M. Fuller, and Dale J. Benos

Department of Physiology and Biophysics, University of Alabama, Birmingham, Alabama, 35294-0005

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The hypothesis that amiloride-sensitive Na+ channels (ENaC) are involved in cell volume regulation was tested. Anisosmotic ND-20 media (ranging from 70 to 450 mosM) were used to superfuse Xenopus oocytes expressing alpha beta gamma -rat ENaC (alpha beta gamma -rENaC). Whole cell currents were reversibly dependent on external osmolarity. Under conditions of swelling (70 mosM) or shrinkage (450 mosM), current amplitude decreased and increased, respectively. In contrast, there was no change in current amplitude of H2O-injected oocytes to the above osmotic insults. Currents recorded from alpha beta gamma -rENaC-injected oocytes were not sensitive to external Cl- concentration or to the K+ channel inhibitor BaCl2. They were sensitive to amiloride. The concentration of amiloride necessary to inhibit one-half of the maximal rENaC current expressed in oocytes (Ki; apparent dissociation constant) decreased in swollen cells and increased in shrunken oocytes. The osmotic pressure-induced Na+ currents showed properties similar to those of stretch-activated channels, including inhibition by Gd3+ and La3+, and decreased selectivity for Na+. alpha beta gamma -rENaC-expressing oocytes maintained a nearly constant cell volume in hypertonic ND-20. The present study is the first demonstration that alpha beta gamma -rENaC heterologously expressed in Xenopus oocytes may contribute to oocyte volume regulation following shrinkage.

rat epithelial sodium channel; mechanosensation; cell volume; amiloride; voltage clamp

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

EPITHELIA ARE ROUTINELY subjected to mechanical stimuli, most commonly changes in osmotic and hydrostatic pressure. Besides these, there are special forms of mechanical stress, such as shear stress of the endothelial cells of the vasculature, peristalsis of the intestine and renal papilla, breathing in the lungs, and air vibration on the hair cells of the inner ear, that impinge on epithelia. It is well known that all epithelia are capable of adapting to variations in osmotic microenvironments by regulating their absorbing and secreting processes for water and electrolytes. At least four classes of epithelial channels, namely, volume-sensitive Cl- channels (10, 38), mechanoactivated K+ channels (6, 24), nonselective cation channels (13, 17), and amiloride-sensitive Na+ channels (1), have been described as sensitive to membrane distension or change in cell volume.

The amiloride-sensitive epithelial Na+ channel (ENaC) is composed of alpha -, beta -, and gamma -subunits. These ENaC subunits share significant homology to the degenerins of Caenorhabditis elegans, which are sensitive to mechanical stimuli. The same apparent membrane topology and structural motifs are found in ENaC and the mec genes of C. elegans. These similarities suggest that ENaC may possess inherent mechanosensitive properties. The mechanosensation of ENaC was first reported for a cloned bovine renal ENaC (alpha -bENaC) by incorporating channel proteins into planar lipid bilayers. A hydrostatic pressure gradient across the bilayer increased channel activity but produced a concomitant decrease in amiloride affinity and monovalent cation selectivity (3). Similar observations were made in planar lipid bilayers containing alpha beta gamma -rat ENaC (alpha beta gamma -rENaC) (14), although data obtained from whole cell patch-clamp studies were less consistent (26).

Changes in cell volume mediated by changes in medium osmolarity also place a mechanical stress on membrane proteins akin to what can be induced by establishment of a hydrostatic pressure gradient across a planar lipid bilayer. However, studies examining the effects of osmolarity on Na+ channel activity have reported variable findings. Activation of a Na+ conductance has been reported in hepatocytes (36), in rat fetal distal lung epithelium (23), and in lymphocytes (1) under hypertonic stress. In contrast, amiloride-sensitive Na+ conductances were reported to be both suppressed by cell shrinkage in A6 cells (37) and increased in frog skin (7). Neither the molecular identity of the channel involved in these responses nor the intracellular signaling mechanisms involved are known.

The purposes of the present study were to characterize the responses of a cloned amiloride-sensitive Na+ channel, alpha beta gamma -rENaC heterologously expressed in Xenopus oocytes, to changes in cell volume induced by altering external osmolarity. Xenopus oocytes provide an ideal model system, since they promiscuously translate exogenous cRNAs. ENaC is a widely distributed ion channel, being present in epithelial and some endothelial tissues. Furthermore, its relationship to the mechanosensitive degenerins of C. elegans is suggestive of a role for this channel in the regulation of cell volume. The results show that expressed alpha beta gamma -rENaC in oocytes is sensitive to hypotonicity (70 mosM) and hypertonicity (450 mosM). Cell swelling decreased rENaC-associated currents, whereas cell shrinkage increased the activity of rENaC. Under anisosmotic conditions, the apparent affinity (Ki) of rENaC to amiloride and the ionic permeability ratios (PNa/PX) were shifted markedly. Measurements of relative oocyte volume in hyperosmotic solutions by digital imaging were consistent with recordings of amiloride-sensitive Na+ current changes produced by hypertonic signals in shrunken rENaC-expressing oocytes, indicating that expressed rENaC may participate in regulatory volume increase (RVI) of oocytes.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Expression of alpha beta gamma -rENaC. The vectors containing alpha -, beta -, or gamma -rENaC subunit inserts were linearized with Not I, and the insert was in vitro transcribed with T7 polymerase (Ambion) to prepare cRNA. The integrity of the cRNA was verified by denaturing gel electrophoresis through 1% agarose-formaldehyde gels. The methods for isolation of Xenopus oocytes and expression of rENaC cRNA were as previously described (16). Briefly, the ovarian tissue was removed from frogs under hypothermal anesthesia through a small incision in the lower abdomen. Oocytes at maturation stage V and VI were carefully defolliculated by hand in Ca2+-free OR-2 solution (in mM: 85.2 NaCl, 2.5 KCl, 10 HEPES, 1.0 Na2HPO4, and 0.5% streptomycin, pH 7.5), and maintained in L-15 medium (one-half strength L-15, with 15 mM HEPES, 5% heat-inactivated horse serum, and 1% antibiotic-antimycotic, pH 7.5; GIBCO) at 18°C. Oocytes were incubated in L-15 medium overnight before cRNA injection. A Nanoject (Drummond Scientific, Broomall, PA) microinjector was used for injection of 1:1:1 alpha beta gamma -rENaC cRNA (5 ng/50 nl) or the same volume of RNase-free water (control).

Electrophysiological recording. Whole cell currents were sampled from the oocytes at room temperature 24 h after cRNA injection, using pCLAMP version 5.1, and analyzed using pCLAMP 6.0 software (Axon Instruments). Voltage-clamp potentials were evoked using a TEA-200 voltage clamp (Dagan) controlled by a personal computer connected via a TL-1 interface (Axon Instruments). The injected oocyte was placed in a small chamber (1 ml) and initially perfused with ND-96 medium (in mM: 96 NaCl, 1 MgCl2, 2 KCl, 1.8 CaCl2, and 5 HEPES, pH 7.4), followed by Cl--free ND-20 (in mM: 20 sodium gluconate, 1 MgCl2, 2.0 KCl, 1.8 CaCl2, and 5.0 HEPES, pH 7.4) at a flow rate of 1.5-2 ml/min for at least 5 min before recording. Microelectrodes filled with 3 M KCl had a resistance of 0.5-3.0 MOmega . The bath was clamped by two chlorided silver wires through 3% agar bridges in 3 M KCl. The oocytes were clamped at a holding potential of 0 mV. Data were filtered at 0.5-1 kHz, digitized, and stored on hardware for offline analysis. For step current-voltage (I-V) recordings, test voltages were stepped from the holding potential to -100 mV through +100 mV in 20-mV increments for 500 ms. The currents at -80 mV and +80 mV were monitored at 20-s intervals. Samples at 200 and 450 ms of each episode were used as the steady-state currents to average data, and the sample at 490 ms was plotted in the I-V curve. Linear components of capacitance and leak currents were not subtracted. The resting membrane potentials (Vm) were read directly from the monitor window of the voltage clamp before and after application of amiloride.

Measurement of oocyte volume. The methods for digital imaging of oocytes have been described elsewhere (40). Briefly, osmotic cell volume changes at room temperature (22°C) were monitored with an Olympus phase-contrast microscope equipped with a video camera connected to a Macintosh computer. Oocytes were transferred from 200 to 450 mosM ND-20 media with or without 10 µM amiloride. Oocytes were equilibrated in isotonic ND-20 medium for >10 min before being transferred to anisotonic ND-20 media. An oocyte image was digitized by IP-Lab Spectrum software (Signal Analytics) and stored at 30-s intervals for a total of 5 min. The imaging at time zero was taken as control. Imaging data were stored on a Zip disk (Iomega). The surface area of the sequential images was measured using the same software, assuming that the oocytes were spheres without microvilli. The oocyte volume was calculated from surface area (A) using the formula V = (4/3) × A × (A/pi )0.5, and the relative cell volume (V/Vo, where Vo is volume at time zero and V is volume at time t) was calculated from the oocyte surface area at time zero (Ao) and at time t (A): V/Vo = (A/Ao)1.5.

Statistics. Student's t-test was used to analyze the differences among groups. The results are presented as means ± SE; n is the number of oocytes. Data from rENaC-expressing oocytes for quantification of the inhibitory effect of amiloride were normalized to the maximum current in the absence of amiloride. Plots of normalized currents as a function of amiloride were fitted with the following multisite inhibition equation, using a nonlinear least-squares method: I Imax/{1 + ([Amil]/Ki)n}, where I is the steady-state current for a given amiloride concentration ([Amil]), Imax is the current amplitude in the absence of amiloride (normalized to 1.0), and n is the slope of the fitting curve.

PNa/PX were computed from a modified Goldman-Hodgkin-Katz equation (12): PNa/PX = exp [F(Er,Na - Er,X)/RT], where Er,X and Er,Na are the reversal potentials for the tested cation (X) and Na+, respectively, and F, R, and T are Faraday's constant, the gas constant, and absolute temperature in kelvin, respectively.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Basal current of expressed rENaC. The resting Vm of alpha beta gamma -rENaC-expressing oocytes were depolarized to varying degrees, from -20 to +20 mV, and were generally around 0 mV (-5.5 ± 0.3 mV, n = 20), indicating that expression of rENaC increased Na+ permeability and depolarized the Vm. In the presence of 10 µM amiloride or in Na+-free superfusate (equimolar N-methyl-D-glucamine substituted for Na+), the resting Vm in oocytes expressing rENaC were hyperpolarized compared with those of H2O-injected oocytes (-38.1 ± 1.6 mV, n = 11). The resting Vm of H2O-injected oocytes or uninjected oocytes was always less than -30 mV (-31.8 ± 0.9 mV, n = 16). The average current at +80 mV was 4,545.2 ± 205 nA (n = 9) for alpha beta gamma -rENaC-expressing oocytes perfused with ND-96 medium, whereas that for the water-injected oocytes was 124.2 ± 33.7 nA (n = 15). Amiloride had an effect on the whole cell current in rENaC-expressing oocytes that was similar to that of lower external Na+ concentration (>80% of the current was inhibited in 30 s). Additionally, 5 mM BaCl2 (a K+ channel inhibitor) or 200 µM DIDS (an anion channel inhibitor) had no effect on the current in oocytes expressing rENaC. The I-V curve was linear in the range -100 to +100 mV, consistent with the electrophysiological properties of the native and cloned ENaCs.

Cloned rENaC responds to osmotic pressure. To inhibit the endogenous, swelling-activated Cl- channel in Xenopus oocytes, Cl--free hypotonic ND-20 (70 mosM medium; see MATERIALS AND METHODS) was used in the extracellular solution, and isotonic ND-20 (200 mosM) or hyperosmotic ND-20 (450 mosM) solutions were produced by adding mannitol. The Na+ concentration in each medium was constant at 20 mM (pH 7.5). Under these experimental conditions, endogenous swelling-activated Cl- channels were not observed (2, 39). Basal currents in rENaC-expressing oocytes were activated by switching the superfusion of isotonic ND-20 to hypertonic ND-20 medium, whereas the rENaC-induced basal currents were depressed in swollen oocytes perfused with hypotonic ND-20 (Fig. 1). As shown in Fig. 2, these osmotic effects on rENaC currents were observed in most oocytes exposed to hypotonic ND-20 (22 of 24 oocytes) or hypertonic ND-20 (18 of 20 oocytes).


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Fig. 1.   Regulation of osmotic pressures on alpha beta gamma -rat amiloride-sensitive epithelial Na+ channels (alpha beta gamma -rENaC) expressed in Xenopus oocytes. A: 1 representative recording (of 7) demonstrates that changes in current evoked by changes in external osmolarity are reversible. Differing osmolarity was produced by addition of mannitol to hypotonic ND-20 medium (20 mM Na+, 70 mosM). Arrows, beginning of application of 10 µM amiloride (up arrows) and of washout (down arrows). B: stepping currents of rENaC treated with 200 mosM (isotonicity) and 450 mosM (hypertonicity). C: rENaC-associated currents recorded from an oocyte under unstretched (200 mosM) or swollen (70 mosM) conditions.


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Fig. 2.   Summarized data comparing expressed rENaC currents with those of H2O-injected oocytes. A: currents recorded at holding potential of -80 mV in swollen oocytes (n = 18) are decreased significantly. B: currents recorded at -80 mV in shrunken cells are stimulated markedly (n = 24). For comparison of effects of anisosmolarity on rENaC, scale of y-axis is same in A and B.

The sensitivity of the cloned rENaC expressed in oocytes to changes in external osmolarity was reversible. Figure 1A shows a typical recording (repeated in 7 oocytes) for osmolarity-induced changes in total rENaC currents. Currents recorded in both hypotonic and hypertonic ND-20 were sensitive to nanomolar concentrations of amiloride. Application of amiloride directly to the chamber or to the chamber perfusate immediately blocked the currents in rENaC-expressing oocytes (>90% of the total current). Both the increase and decrease in current induced by changes in solution osmolarity were reversible. The current was restored after washing out the applied amiloride, and application of amiloride could be repeated several times on the same oocytes with identical results.

The osmolarity dependence of expressed rENaC currents was investigated by superfusing oocytes with ND-20 media of variable osmolarities (from 70 to 490 mosM; Fig. 3). In H2O-injected oocytes, no significant changes in the current magnitude were found (Fig. 3A). However, in rENaC-expressing oocytes, osmolarities as high as 490 mosM increased current, whereas perfusing the cells with hypotonic ND-20 decreased currents (Fig. 3B). Figure 3C shows that the relationship between current and osmolarity was almost linear over the range of osmolarities studied. Osmolarities >500 mosM caused an irreversible increase in current, and the electrodes were easily dislodged from the oocytes due to the large decrease in cell volume.


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Fig. 3.   Osmotic pressure dependence of rENaC. Oocytes were perfused with ND-20 media, ranging from 70 to 490 mosM. A: overlapped current traces from a water-injected oocyte evoked at clamping potential of -80 mV. B: inward currents of rENaC-expressing oocyte at -80 mV are sensitive to external osmolarity. C: currents in B normalized to that obtained in isotonic ND-20 medium (1.0) and replotted as a function of extracellular osmolarity. Data in A and B are representative of at least 3 similar experiments.

Inhibition by amiloride. Amiloride is not only a specific inhibitor of ENaC (at the nanomolar level) but also an inhibitor (at the micromolar level) of stretch-activated channels in hair cells and in Xenopus oocytes (for review, see Ref. 11). Observations from lipid bilayer experiments incorporating alpha -bENaC or alpha beta gamma -rENaC (3, 14) and whole cell patch-clamp studies on human B lymphocytes (1) showed that the amiloride sensitivity of ENaC tended to decrease with stretch. Our results using Xenopus oocytes expressing rENaC showed that hypertonic solutions (450 mosM) caused a leftward shift in the dose-inhibition curve of amiloride, and that hypotonic solutions (70 mosM) shifted the dose-response curve of amiloride to the right (Figs. 4 and 5). In rENaC-expressing oocytes exposed to hypotonic ND-48 medium (containing 48 mM Na+; 100 mosM), the Ki of amiloride increased to 800 nM from 176 nM (recorded in isotonic ND-48 medium), consistent with the observations for the shift in amiloride Ki recorded in ND-20 medium and the results of planar lipid bilayer experiments (14).


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Fig. 4.   Inhibition of current by amiloride in rENaC-expressing Xenopus oocytes. Top: currents in ND-20 perfusing media (control). Middle: currents following application of amiloride (100 nM). Bottom: amiloride-sensitive currents.


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Fig. 5.   Osmotic pressures shift amiloride affinity of rENaC. Apparent dissociation constant (Ki) of amiloride for rENaC-expressing oocytes in 200 mosM (), 450 mosM (bullet ), and 70 mosM (open circle ) superfusates are 114 ± 0.03 nM (n = 5), 55 ± 0.001 nM (n = 3-5), and 4,199 ± 1.1 nM (n = 4 or 5), respectively. Remaining percentages of total current (100%) are plotted against concentration of applied amiloride.

To determine whether the osmotic pressure sensitivity of the currents recorded from rENaC-expressing oocytes was a specific characteristic of Na+ channel expression, amiloride (10 µM) was added to the chamber containing isotonic ND-20 medium before the chamber was switched to hypotonic or hypertonic ND-20 medium with an equal concentration of amiloride. Amiloride inhibited >90% of the total current in oocytes injected with rENaC cRNA. As expected, application of osmotic pressure (70 or 450 mosM) did not modify the amiloride-insensitive currents in rENaC-expressing oocytes, identical to the results obtained from uninjected or H2O-injected oocytes studied under the same conditions (Fig. 6).


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Fig. 6.   Amiloride pretreatment blocks response to mechanical signals of rENaC. Left: currents. Right: corresponding current-voltage curves. A: control; basal current recorded from an rENaC-expressing oocyte in isotonic ND-20 medium. Vm, membrane potential. B: inhibition of current in isotonic ND-20 medium in presence of 10 µM amiloride. C: in continual presence of 10 µM amiloride, there is no change in current following superfusion with hypertonic medium (450 mosM ND-20). Data are representative recordings of 3 oocytes.

Effect of osmotic pressure on ion selectivity. ENaC are highly selective for Na+ and Li+ and much less permeable to K+ and other monovalent cations (4, 8, 9). The application of hydrostatic or osmotic pressure on human B lymphocytes (1) and hydrostatic pressure across planar lipid bilayers containing ENaC (3, 14) showed that mechanical stress on ENaCs may modify the ionic selectivity of the pore. The ionic selectivity of rENaC was calculated according to the reversal potentials of amiloride-sensitive currents (Table 1) recorded from the same oocyte with different extracellular monovalent cations.

                              
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Table 1.   Reversal potentials of amiloride-sensitive rENaC currents expressed in Xenopus oocytes

Oocytes were superfused in ND-20 with variable osmolarity or media in which Na+ was replaced with the equimolar test cation (K+, Li+, or Cs+). An I-V curve was then run from -100 to +80 mV in the presence or absence of 10 µM amiloride. As summarized in Table 2, the relative permeability of Na+ vs. K+ or Cs+ was higher in ND-20 media than in ND-96; the PNa/PK was 20.0 ± 0.9 (n = 7) in ND-20 vs. 5.0 ± 0.8 in ND-96, and PNa/PCs was 33.3 ± 1.1 (n = 8) in ND-20 vs. 8.3 ± 1.8 in ND-96. However, under conditions of identical ionic strength, osmotic pressure resulted in changes in ion selectivity. The PNa/PK decreased to 6.3 and 11.0 for rENaC stressed by hypotonicity and hypertonicity, respectively, compared with a selectivity of 20 in isotonic ND-20 medium. The selectivity of the channel for Cs+ (PNa/PCs) similarly changed to 5.0 and 12.5 in 70 and 450 mosM ND-20 solutions, respectively, from a ratio of 33 in isotonic ND-20.

                              
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Table 2.   Effect of osmotic pressure on PNa/PX of expressed rENaC

Changes in the external osmotic pressure did not alter the relative permeability sequence for monovalent cations. The sequence of ionic permeability was the same in isotonic ND-96, isotonic ND-20, hypotonic ND-20, and hypertonic ND-20 media, namely, Li+ > Na+ > K+ > Cs+. The PNa/PLi was also less sensitive to changes in osmotic pressure.

Mechanogated channel inhibitors. Although La3+ and Gd3+ inhibit several ion channels (19, 35), they have been applied to stretch-activated and volume-sensitive cation or anion channels in a variety of cell species (11). To test the hypothesis that the response of expressed rENaC to osmolarity represents its potential mechanosensitivity, the effects of GdCl3 (50 µM) and LaCl3 (2 mM) were studied on the volume-sensitive currents in shrunken or swollen oocytes expressing rENaC (Fig. 7). The currents of rENaC under isotonic conditions were -1,560.2 ± 201 and -1,511.3 ± 115 nA in the absence and presence of 2 mM La3+, respectively. Similarly, no significant change in current amplitude was found with 50 µM Gd3+ (-1,747.8 ± 201 nA for control vs. -1,750.1 ± 58 nA for Gd3+). However, in both shrunken and swollen cells, the rENaC-associated currents became sensitive to 2 mM La3+. The currents decreased to 45.9 ± 7.3% (n = 4, P < 0.01) and 80.3 ± 14.2% (n = 4, P < 0.05) of control in shrunken and swollen oocytes, respectively. Inhibition of volume-sensitive currents by 50 µM Gd3+ was similar to that by La3+. Under conditions of hypertonicity and hypotonicity, currents reduced to 75.3 ± 3.0% (n = 4, P < 0.05) and 61.9 ± 10% (n = 3, P < 0.05) of controls, respectively. Pretreatment of rENaC cRNA-injected oocytes with Gd3+ or La3+ prevented the response of rENaC-injected oocytes to changes in bath osmolarity (data not shown) without affecting the basal currents. The inhibitory effects of these mechanogated channel inhibitors were concentration dependent (data not shown). In H2O-injected oocytes, neither Gd3+ nor La3+ had any effect on basal current under conditions of swelling or shrinkage, consistent with the response of uninjected oocytes to changes in solution osmolarity.


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Fig. 7.   Inhibition by La3+ and Gd3+ of osmotic pressure-sensitive rENaC currents. Left: LaCl3 (2 mM) inhibition. rENaC-associated currents (-80 mV) in swollen (n = 4) or shrunken (n = 4) oocytes were sensitive to LaCl3. Currents were normalized to maximal amplitudes without LaCl3. Right: GdCl3 (50 µM) decreased currents in swollen (n = 9) or shrunken (n = 4) oocytes injected with rENaC cRNA.

Relative oocyte volume. It has been proposed that stretch-activated epithelial ion channels can function as a sensor of cell volume changes (30). We therefore hypothesized that rENaC may be involved in regulation of cell volume. As shown in Fig. 8A, oocytes expressing rENaC kept the cell volume constant following a slight decrease in the first 1 min of exposure to hypertonic ND-20 medium; in contrast, the volume of the H2O-injected oocyte was much smaller after 5-min incubation in hypertonic ND-20 medium. The ability of rENaC-expressing oocytes to regulate their volume in hypertonic ND-20 medium was diminished with amiloride (10 µM). The absolute oocyte volumes at time zero and 5 min in 450 mosM ND-20 medium are summarized in Table 3. The normal absolute volume of Xenopus oocytes (H2O-injected oocytes) was 9.4 × 10-4 cm3 (n = 12), similar to that reported previously (40). In oocytes incubated for 2 h in hypertonic media, 63.1 ± 7.0% of the eggs injected with H2O shrank. In contrast, rENaC-expressing oocytes were able to regulate their volume such that only 18.4 ± 3.7% of the eggs shrank, consistent with activation of rENaC conductance by an increase in external osmotic pressure (Fig. 8B). Digital imaging of both water-injected and alpha beta gamma -rENaC-injected oocytes showed that, although water-injected eggs shrank on exposure to hypertonic ND-20 medium, rENaC-expressing oocytes maintained their volume over a 5-min time course (Fig. 8C)


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Fig. 8.   rENaC expression mediated cell volume regulation. A: time course of relative cell volume (RCV) of treated oocytes in hypertonic ND-20 (450 mosM). RCV of oocytes expressing rENaC was kept approximately constant, and 10 µM amiloride inhibited ~60% of regulatory volume increase, while RCV of uninjected oocytes decreased markedly. Data are representative of 8 measurements. B: average percentages of shrunken oocytes following 2-h incubation in hypertonic ND-20 medium (450 mosM) of H2O-injected oocytes and alpha beta gamma -rENaC-injected oocytes (n = 3). C: digital images of H2O-injected oocyte (top) and alpha beta gamma -rENaC-expressing oocyte (bottom) treated in hypertonic ND-20 (450 mosM). Images were taken at time 0 (control), and then 1 image/min was taken. Left to right: images from 0, 1, 2, 3, 4, and 5 min. H2O-injected oocytes shrank under these conditions, whereas rENaC-expressing oocytes maintained their volume.

                              
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Table 3.   Absolute volume of Xenopus oocytes before and after hypertonic treatment

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The purpose of the present study was to test whether wild type alpha beta gamma -rENaC expressed in Xenopus oocytes could respond to changes in osmotic pressure using the two-electrode voltage-clamp technique. The main finding of our study is that wild type alpha beta gamma -rENaC expressed in oocytes behaved as a mechanoactivated cation channel sensitive to changes in external osmotic pressure. Extracellular hypotonicity inhibited the oocyte whole cell currents, whereas extracellular hypertonicity stimulated rENaC-associated currents in a reversible fashion. Changes in osmotic pressure also modified the sensitivity of rENaC to amiloride and the ionic selectivity of the channel for monovalent cations. Under conditions of hypertonic stress, rENaC-associated currents were sensitive to the mechanogated channel antagonists La3+ and Gd3+. Moreover, rENaC in heterologously expressed oocytes was involved in regulatory cell volume restoration of oocytes.

Although Xenopus oocytes have been used as an expression model to translate a number of volume-sensitive Cl- channels (20) and ENaCs (3, 8, 9, 14, 15), an important concern of the present study was whether the current component activated by changes in external osmotic pressure was due to activation of endogenous channels. Expressed currents were not sensitive to 200 µM DIDS or 100 µM niflumic acid, whereas endogenous Cl- channels were activated by oocyte swelling rather than shrinkage (2). No shrinkage-activated anion channels have yet been identified in Xenopus oocytes. In addition, Cl--free ND-20 medium would be expected to diminish the endogenous Cl- channel activity. The endogenous volume-sensitive cation channels and the mechanogated cation channels that have been previously described in Xenopus oocytes were difficult to demonstrate under our experimental conditions in the present study (39). However, the small whole cell current magnitude (~100 nA), the poor cation selectivity (equal for Na+, K+, and Ca2+), and the rapid adaptation of their mechanosensitivity are very different from the properties associated with expression of rENaC. Although these native channels are blocked by amiloride and Gd3+ (21, 39), Ki (0.5 mM) was 120- to 625-fold higher than those of rENaC (500 vs. 0.8 µM in hypotonic ND-48 medium; 500 vs. 4.19 µM in hypotonic ND-20 medium). The presence of stretch-activated endogenous cation channels has not been verified by two-electrode voltage-clamping record, and, under our experimental conditions, the channels were not activated in H2O-injected or uninjected oocytes (Fig. 2).

The present study demonstrates that alpha beta gamma -rENaC expressed in Xenopus oocytes is sensitive to the mechanogated channel inhibitors La3+ and Gd3+. La3+ and Gd3+, as specific antagonists of stretch-activated (mechanosensitive) channels, have been used to inhibit many epithelial or nonepithelial cation channels (11, 25, 28, 31). Our observations show that Gd3+ and La3+ block rENaC-associated currents elicited by changes in osmotic pressure. The mechanisms of Gd3+ and La3+ inhibition of rENaC are not known. It is conceivable that cryptic binding sites for Gd3+ and La3+ could be revealed by membrane conformational change as a result of changes in bath solution tonicities.

We confirmed that amiloride sensitivity (Ki) of the cloned rENaC expressed in Xenopus oocytes was altered after exposure to anisosmotic media. Similar findings have been reported for bENaC (3) and rENaC (14). These earlier studies showed that the activation of the epithelial amiloride-sensitive Na+ conductances were linearly dependent on the hydrostatic pressure across an artificial membrane. In the present study, amiloride Ki values were related to the extracellular osmolarity; as depicted in Fig. 5, Ki values estimated with 450, 200, 100, and 70 mosM were 55, 114, 800, and 4,199 nM, respectively, indicating that as osmolarity decreased, the Ki of amiloride for the channel increased in parallel.

The ionic permeability of a channel is a critical parameter for channel characterization. Ionic selectivity studies revealed that expressed rENaC was highly selective for Na+ and was dependent on the extracellular Na+ concentration. After exposure to 70 or 450 mosM ND-20 medium, the monovalent cation permeability of the channel (Na+, K+, Li+, and Cs+) was blunted. Observations from both cell-free (3, 14) and whole cell systems (1) on ENaC behavior under similar conditions are consistent with the present study. Similarly, stable expression of the alpha -subunit of ENaC in an osteoblast cell line was associated with the appearance of a stretch-activated nonselective cation channel (18).

The cellular mechanisms underlying volume regulation in oocytes expressing alpha beta gamma -rENaC are unknown. However, in the cell, ENaC is structurally connected to actin, spectrin, and their associated proteins (29, 32). Evidence that actin can regulate ENaC (5, 27) supports the idea that hypertonicity may activate rENaC through a mechanism involving G-actin. The underlying signal may involve an increase in intracellular Ca2+ concentration ([Ca2+]i). An increase in [Ca2+]i has been shown in several cell lines to be a consequence of hypotonic shock (22, 33). As rENaC is converted to a less selective or nonselective cation channel by changing osmotic pressures, an increase in the permeability of Ca2+ through this pathway could give rise to an increase in [Ca2+]i.

In the present study, we demonstrate that expressed amiloride-sensitive rENaC is involved in RVI in shrunken oocytes (Fig. 8). When the degree of amiloride sensitivity of whole cell currents (>90%) is compared with amiloride-inhibited changes in cell volume (~60%), it is clear that some apparently "amiloride-resistant" pathways were sensitive to osmotic signals outside the cell. A native Na+/H+ antiporter that was sensitive to amiloride and to hypertonicity (34) could contribute to RVI in shrunken oocytes. The relative contributions of epithelial Na+ channel, Na+/H+ exchange, and Na+-K+-Cl- symport to RVI in rat hepatocytes were reported to be in the proportions 4:1:1 (37). This observation that the Na+ channel makes a major contribution to RVI in rat hepatocytes is consistent with the findings of the present study that expression of ENaC enhances RVI in oocytes.

In conclusion, we report that the cloned ENaC isolated from the rat colon is responsive to changes in external osmotic pressure when expressed in Xenopus oocytes. The sensitivity of expressed rENaC in oocytes to osmotic pressure relates to RVI under hypertonic conditions, but the role of native alpha beta gamma -rENaC in the physiological regulation of epithelial cell volume remains to be determined.

    ACKNOWLEDGEMENTS

We acknowledge Eddie J. Walthall (Dept. of Physiology and Biophysics) for superb technical assistance, Dr. Michael DuVall (Dept. of Anesthesiology) for helpful discussions of the manuscript, Shawn Williams (Dept. of Cell Biology) for excellent assistance in imaging Xenopus oocytes, and Dr. Lan Chen (Dept. of Anesthesiology) for help in the dissection of Xenopus ovaries.

    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-37206 and National Heart, Lung, and Blood Institute Grant HL-50487.

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. §1734 solely to indicate this fact.

Address for reprint requests: D. J. Benos, Department of Physiology and Biophysics, The University of Alabama at Birmingham, BHSB 706, 1918 University Blvd., Birmingham, AL 35294-0005.

Received 6 April 1998; accepted in final form 8 July 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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Am J Physiol Cell Physiol 275(5):C1182-C1190
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