Hormonal regulation of ENaCs: insulin and aldosterone

Bonnie L. Blazer-Yost1, Xuehong Liu2, and Sandy I. Helman2

1 Biology Department, Indiana University-Purdue University at Indianapolis, Indianapolis, Indiana 46202; and 2 Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Although a variety of hormones and other agents modulate renal Na+ transport acting by way of the epithelial Na+ channel (ENaC), the mode(s), pathways, and their interrelationships in regulation of the channel remain largely unknown. It is likely that several hormones may be present concurrently in vivo, and it is, therefore, important to understand potential interactions among the various regulatory factors as they interact with the Na+ transport pathway to effect modulation of Na+ reabsorption in distal tubules and other native tissues. This study represents specifically a determination of the interaction between two hormones, namely, aldosterone and insulin, which stimulate Na+ transport by entirely different mechanisms. We have used a noninvasive pulse protocol of blocker-induced noise analysis to determine changes in single-channel current (iNa), channel open probability (Po), and functional channel density (NT) of amiloride-sensitive ENaCs at various time points following treatment with insulin for 3 h of unstimulated control and aldosterone-pretreated A6 epithelia. Independent of threefold differences of baseline values of transport caused by aldosterone, 20 nM insulin increased by threefold and within 10-30 min the density of the pool of apical membrane ENaCs (NT) involved in transport. The very early (10 min) increases of channel density were accompanied by relatively small decreases of iNa (10-20%) and decreases of Po (28%) in the aldosterone-pretreated tissues but not the control unstimulated tissues. The early changes of iNa, Po, and NT were transient, returning very slowly over 3 h toward their respective control values at the time of addition of insulin. We conclude that aldosterone and insulin act independently to stimulate apical Na+ entry into the cells of A6 epithelia by increase of channel density.

epithelia; epithelial sodium channels; tissue culture; cortical collecting ducts; kidney; noise analysis; A6 epithelia

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

REGULATION OF RENAL Na+ reabsorption is crucial for maintaining fluid and electrolyte homeostasis. A variety of hormones and other effectors modulate Na+ absorption in the principal cells of the distal nephron and in particular in the cortical collecting duct. The A6 cell line derived from the kidney of the amphibian Xenopus laevis is a convenient tissue culture model of the Na+ transport properties of the principal cell and is responsive to the hormones that regulate mammalian Na+ reabsorptive processes, including insulin and aldosterone (1, 5, 12, 13, 17, 20, 23, 24).

Aldosterone is a steroid hormone that manifests its long-term effects via new protein synthesis; insulin is a peptide hormone that manifests immediate effects on transport via binding to basolateral plasma membranes in polarized epithelia (2, 3, 6, 13). The initial interactions of hormone-receptor binding are well characterized, and the final effect, increased transport through the apical membrane amiloride-sensitive Na+ channel, is well established. Less well understood, however, are the pathways linking receptor binding to final natriferic effects, and very little is known about the mechanisms by which multiple hormones can concurrently modulate Na+ channel function.

Using Ussing-style electrophysiological techniques, we have previously shown that the acute natriferic effects of insulin and aldosterone are additive (24). In addition, we have used the noninvasive technique of blocker-induced noise analysis to demonstrate that aldosterone increases the number of active Na+ channels in the apical membranes of A6 cells (17). Insulin also appears to increase the apical membrane Na+ influx through apical Na+ channels, but the mechanism of this increase is less well defined. Marunaka et al. (20) reported an increase in open-channel density (NPo) within 5-10 min after the application of insulin to aldosterone-pretreated A6 epithelia in cell-attached apical membrane patches. The increase at this early time of the insulin response was found to be predominantly due to an increase in channel open probability (Po). Erlij et al. (12) used 6-chloro-3,5-diaminopyrazine-2-carboxamide (CDPC) blocker-induced noise analysis to show that insulin increased the density of open channels (No = NPo) in the apical membranes of non-aldosterone-stimulated A6 cells 2 h after addition of the hormone. These investigators found no change in Po at this later time point of the insulin response. Hormonal interactions acting through common or different pathways may modify the response to other effectors or hormones. In addition, channel densities and Po may be modulated with very different time courses by various effectors. Therefore, it was of particular interest to know how insulin stimulated Na+ transport in control untreated and aldosterone-prestimulated A6 epithelia.

This study was designed to determine the time course of change of single-channel currents (iNa), total channel densities (NT), and Po of the amiloride-sensitive epithelial Na+ channels (ENaCs) that underlie the macroscopic changes of transport measured as short-circuit currents (Isc) during the response to insulin in both control untreated and aldosterone-pretreated tissues. Our recently described pulse protocol of blocker-induced noise analysis provides a noninvasive technique that can be applied in short time intervals to follow changes at multiple points during early and late transient responses (17).

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell culture. The A6 cells used in these studies were obtained from the American Type Culture Collection (Rockville, MD) at passage 69 and used at passage 84. The cells were grown at 28°C in a humidified incubator gassed with 5% CO2. The culture medium was a modified DMEM (91-5055EC; GIBCO BRL, Grand Island, NY) supplemented with penicillin (25 U/ml)-streptomycin (25 µg/ml) (GIBCO BRL) and 10% calf serum (CELLect, iron supplement calf serum; ICN Biomedicals, Aurora, OH). For analysis, cells were subcultured onto 24-mm Transwell tissue culture-treated inserts (Costar, Cambridge, MA) and used 14-21 days after seeding.

The data were obtained using blocker-induced noise analysis following a pulse protocol that we have recently described in detail (17). Briefly, the permeable supports containing confluent cell monolayers were removed from the Transwell inserts and transferred to a continuous flow perfusion chamber. The perfusion medium was the growth medium minus the calf serum and antibiotics. The tissues were short circuited for at least 1 h to allow the macroscopic Isc to stabilize. About 30 min before the control period, 10 µM CDPC (Aldrich Chemical, Milwaukee, WI) was added to the apical perfusion solution. During control and experimental periods, the apical perfusion solution was switched at intervals of 20 min to the same solution containing 30 µM CDPC for pulse intervals of 3 min and returned thereafter to the 10 µM CDPC-containing solution (see Fig. 1).

All monolayers were incubated overnight in serum-free medium; 20 nM porcine insulin (a generous gift from Eli Lilly, Indianapolis, IN) was added to the serosal perfusion medium. In the aldosterone-pretreated tissues, the cultures were incubated overnight with 2.7 µM aldosterone, and the aldosterone was added also to the serosal perfusion medium during the course of the experiments.

Analysis by noise. Current noise of short-circuited tissues was amplified after being filtered at the Nyquist frequency, digitized (4,096 points/s: 16-bit analog-to-digital convertor), and Fourier transformed to yield power density spectra (PDS). Low-frequency plateaus (So) and corner frequencies ( fc) of the blocker-induced Lorentzians were determined by nonlinear curve fitting of the spectra to the CDPC-induced Lorentzian (1/f) noise at the lower frequencies and amplifier noise at the higher frequencies <2 kHz (see Ref. 17 for details and typical PDS). The average of 60 2-s sweeps of current noise yielded Lorentzians in which the uncertainty of fc was ±1-2 Hz. PDS were measured at 10 µM CDPC just before pulse inhibition of the Isc and during exposure of the tissues to 30 µM CDPC yielding the respective f 10c and f 30c.

The sequential measurements of f 10c and f 30c were fit by nonlinear regression (TableCurve; Jandel Scientific, San Rafael, CA) to smooth curves, thereby filtering the small uncertainties in estimation of the blocker rate coefficients (see Ref. 17). Blocker on rate (kob) and blocker off rate (kbo) coefficients were calculated from the slopes and intercepts of the filtered f 10c and f 30c, at 10 and 30 µM CDPC, respectively, yielding the CDPC blocker equilibrium constant KB = kbo /kob as a function of time during the control and experimental periods of each experiment.

Single-channel currents and channel densities. Blocker-insensitive Na+ transport was measured at the end of each experiment by addition of 100 µM amiloride to the apical solution (IAmilsc). Defining the blocker-sensitive macroscopic currents IBNa = IBsc - IAmilsc, the iNa through blocker-sensitive channels at a B1 blocker concentration of 10 µM CDPC is
<IT>i</IT><SUB>Na</SUB> = <IT>i</IT><SUP>10</SUP><SUB>Na</SUB> = <FR><NU><IT>S</IT><SUP>10</SUP><SUB>o</SUB>(2&pgr;<IT>f</IT> <SUP>10</SUP><SUB>c</SUB>)<SUP>2</SUP></NU><DE>4 <IT>I</IT><SUP>10</SUP><SUB>Na</SUB><IT>k</IT><SUB>ob</SUB> <IT>B</IT><SUB>1</SUB></DE></FR> (1)
since the iNa in the absence of blocker is not significantly different from the i10Na (17). Blocker-sensitive open-channel density of functional channels at 10 µM CDPC is N10o I10Na/i10Na. In the complete absence of CDPC, open-channel density (No), expressed in units of open channels per planar centimeter squared or per 100 µm2, where the latter approximates the area per cell, is
<IT>N</IT><SUB>o</SUB> = <IT>N</IT><SUP>10</SUP><SUB>o</SUB> <FENCE>1 + <IT>P</IT><SUB>o</SUB> <FENCE><FR><NU><IT>B</IT><SUB>1</SUB></NU><DE><IT>K</IT><SUB><IT>B</IT></SUB></DE></FR></FENCE></FENCE> (2)
These measurements of No are considered to be highly reliable because the Isc are measured with high precision to within 0.01 µA/cm2 or higher when necessary. The iNa measured by noise analysis are also considered to be highly reliable because they not only give single-channel conductances in the range of the 5-pS ENaC but change predictably in accordance with the known electrophysiology of Na+-transporting epithelia like those of frog skin (16). Other channels have been reported to exist in the apical membranes of Na+-transporting epithelia (18, 22), but such channels, even if amiloride or blocker sensitive, cannot contribute appreciably to transport as measured by Isc (1, 14).

Channel Po were calculated with the values of KB and the fractional inhibition of the blocker-sensitive Na+ transport, I 30/10Na, caused by increasing the CDPC concentration from 10 to 30 µM
<IT>P</IT><SUB>o</SUB> = <FENCE><FR><NU>1 − <IT>I</IT> <SUP>30/10</SUP><SUB>Na</SUB></NU><DE>30 <IT>I</IT> <SUP>30/10</SUP><SUB>Na</SUB> − 10</DE></FR></FENCE> <IT>K</IT><SUB><IT>B</IT></SUB> (3)
Unlike determination of Po from patch-clamp data in which values of Po depend on observation of the maximum levels of current to know the channel density in the patch, the determination of Po by noise analysis is completely independent of predetermination of NT, as indicated by Eq. 3. NT was calculated from the quotient No /Po.

Experiments were carried out at room temperature. Summary data are reported as means ± SE.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have previously shown that the "acute" natriferic effects of insulin and aldosterone are additive (24). Specifically, if aldosterone was added 30 min after the initiation of an insulin response or if insulin was added 2 h after the addition of aldosterone, the amiloride-sensitive Isc 5 h after the addition of the first hormone indicated additive effects of the two hormones on the apical membrane Na+ influx. The hormone concentrations were chosen to elicit maximal responses. Therefore, these data suggest that the intracellular pathways stimulated by the two agents are largely independent.

We have also previously used the noninvasive technique of blocker-induced noise analysis to show that the response to either acute or chronic aldosterone treatment is predominantly due to an increase in the total number of active channels (NT) in the apical membrane (1, 17).

In the current experiments, we examined the response to insulin stimulation in both control and aldosterone-pretreated tissues. In Fig. 1, representative strip-chart recordings are shown to illustrate typical amiloride-sensitive Isc responses to insulin and to indicate the times at which CDPC was increased from 10 to 30 µM and returned to 10 µM. It should be noted that the first pulse after the addition of insulin was initiated at 10 min to monitor regulation during the early phase of the response. Also indicated on these tracings is the consistent finding that the Isc in these studies is amiloride sensitive, as shown by the addition of 100 µM amiloride 3 h after the addition of insulin. Amiloride-insensitive Isc averaged -0.06 ± 0.15 and 0.51 ± 0.14 in control and aldosterone-pretreated tissues, respectively.


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Fig. 1.   Analysis of amiloride-sensitive Na+ channels using 6-chloro-3,5-diaminopyrazine-2-carboxamide (CDPC) blocker pulse inhibition of blocker-sensitive short-circuit current (INa). Strip-chart recordings of INa (amiloride sensitive) during a control period (-120 to 0 min) and after treatment of the tissues with 20 nM insulin (0-180 min) are shown. Monolayer in A was pretreated overnight with 2.7 µM aldosterone; 100 µM amiloride was added at the end (180 min) of the experiments. CDPC was increased from 10 to 30 µM for 3 min at intervals of 20 min, causing pulse inhibition of the current.

The mean blocker-sensitive short-circuit current (INa) for a series of insulin responses in control and aldosterone-pretreated tissues are shown in Fig. 2. In both cases there is an almost immediate increase in apical membrane Na+ influx in response to insulin addition. The quantitative magnitude of the insulin response is greater in aldosterone-pretreated tissues. Typically, insulin-stimulated epithelia reach a maximal level of transport in 30-60 min and, thereafter, transport slowly decreases (2, 5, 6, 24). This response is seen also in the insulin-treated control tissues depicted in Fig. 2. However, in the aldosterone-pretreated tissues, the rate of decline in insulin-stimulated transport rate is much slower.


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Fig. 2.   Summary of INa responses to insulin in control and aldosterone-pretreated tissues. Insulin (20 nM) was added at time 0 to control and aldosterone-pretreated (2.7 µM, overnight) A6 monolayers. Results are presented as means ± SE.

We used noise analysis to dissect the contributions of iNa, Po, and NT to the Isc that determine the macroscopic rates of Na+ influx. Current noise PDS were measured immediately before (10 µM) and following the increase of CDPC concentration to 30 µM, providing the So and fc of the blocker-induced Lorentzians. The fractional inhibition of the macroscopic rates of Na+ transport, I 30/10Na, was measured immediately following inhibition of transport but before the onset of the autoregulatory increases of Na+ transport (17). The fc of induced current noise Lorentzians vary linearly with blocker concentration. Therefore, kob and kbo can be determined from a two-point analysis in which 2pi fc = kobB kbo. Because fc are independent of Isc magnitudes, pairs of f 10c and f 30c at each pulse interval could be used to assess the time-dependent changes of the blocker rate coefficients and thus KB. Figure 3 shows the results of a typical experiment in which fc at 10 and 30 µM CDPC are plotted as a function of time during control and experimental periods. Insulin caused no change of blocker interactions with the Na+ channels. This relationship has also been shown to be true for aldosterone. Therefore, the channels recruited by either hormone possess CDPC blocker kinetics that are the same as those present in the apical membrane before effector stimulation of transport. Zero time control values for kob (rad · s-1 · µM-1, where rad indicates radians), kbo (rad/s), and KB (µM) averaged in control tissues 7.52 ± 0.25, 245 ± 5.4, and 32.7 ± 1.4, respectively, and in aldosterone-pretreated tissues 6.61 ± 0.23, 250 ± 4.8, and 37.9 ± 0.8, respectively.


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Fig. 3.   Determination of CDPC blocker rate coefficients. A: pairs of corner frequencies ( fc) from a single insulin experiment (not aldosterone pretreated) measured at 10 and 30 µM CDPC as a function of time during control and experimental periods. Smooth curves were fit to the data, and projected values were used to calculate the blocker on rates (kob) and off rates (kbo) (B). C: time-dependent changes of the blocker equilibrium constant (KB) = kbo /kob.

Figures 4 and 5 summarize iNa (Fig. 4A), No (Fig. 4B), Po (Fig. 5A), and NT (Fig. 5B) as a function of time during an insulin response in control and aldosterone-pretreated tissues. Under both conditions, the iNa drops transiently in the first 10 min after the addition of insulin and then returns slowly to the prestimulated value. This likely represents the immediate response to depolarization across the apical membrane as Na+ enters the cell and is more pronounced, as expected, in aldosterone-pretreated tissues where the overall magnitude of the inward Na+ flux is greater. Likewise, the Po decreases in response to insulin but only in the aldosterone-pretreated cells where the starting currents are considerably higher than in the control cells. The relatively small changes in iNa and Po are accompanied by large increases in the total number of open channels in both the control and aldosterone-pretreated epithelia.


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Fig. 4.   Summary of time-dependent changes of single-channel currents (iNa) and density of open channels (No) in response to insulin in control and aldosterone-pretreated tissues. Values are means ± SE at each time point.


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Fig. 5.   Summary of time-dependent changes of open probability (Po) and total number of channels (NT) in response to insulin in control and aldosterone-pretreated cells. Values are means ± SE at each time point.

It is evident from the data presented in Figs. 1 and 2 that the quantitative response to insulin is dependent on the transport rate at the time of hormone addition. To better compare the contribution of each of the components to the changes in macroscopic current, the data from Figs. 2, 4, and 5 have been normalized to the zero time values of insulin addition (Fig. 6). From these graphs, it is clear that the insulin-induced changes of NT are proportional to the initial values before hormone addition. Insulin stimulates a nearly threefold increase in NT regardless of the initial density of channels. In contrast, when insulin is added to aldosterone-pretreated tissues that are already transporting Na+ at a relatively high rate, the Po shows a marked but transient decrease in the first 10 min. Accordingly, the apparent relative stability of the Isc following insulin in aldosterone-prestimulated tissues appears to result from transient compensatory decreases of NT, like those in control tissues, and secondary increases of Po, unlike those that occur in control tissues.


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Fig. 6.   Summary of zero time normalized values of INa, iNa, Po, and NT in response to insulin in control (solid circles) and aldosterone-pretreated (gray circles) tissues. A-D: time-dependent changes of control (n = 5) and aldosterone-pretreated (n = 6) groups of tissues expressed as experimental/zero time control values. Values are means ± SE at each time point.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Aldosterone is a major regulator of fluid and electrolyte balance. The interaction of this steroid hormone with the mineralocorticoid receptor initiates a classic genetic derepression pathway leading to the synthesis of aldosterone-induced proteins that mediate the final effect: an increase in Na+ reabsorption via the amiloride-sensitive Na+ channel. The nature and mode of action of the induced proteins remain unknown.

Insulin stimulates transcellular Na+ flux in a variety of transporting epithelia, and this effect is manifested as an increase in Na+ reabsorption in animal models (7, 21), normal and diabetic humans (7, 8), and models of the distal nephron (2, 3, 5, 6, 12, 13, 20). It is clear that the effect of insulin in both native and model epithelia is initiated by insulin binding to a cell surface receptor located on the basolateral membrane. The location and hormone binding affinities of the amphibian receptor have been shown to be virtually identical to the mammalian counterpart (3). Postreceptor pathways involve insulin receptor phosphorylation (23, 26), and our recent data have implicated the enzyme phosphatidylinositol 3-kinase as a component of this pathway (23) in a manner analogous to the insulin regulation of glucose uptake into cells (4).

Thus, whereas the intracellular pathways initiated by hormone-receptor binding are quite different, the final effect of both hormones is manifested at the level of the amiloride-sensitive Na+ channel. We have recently demonstrated that both acute and chronic natriferic effects of aldosterone involve an increase in the number of active Na+ channels in the apical membrane with no increases in iNa or Po (1, 17). Interestingly, the results of our present experiments indicate that the major effect responsible for the increase in insulin-stimulated Na+ transport is also an increase in the number of active channels in the membrane. We had previously shown that the acute effects of insulin and aldosterone were additive, and here we show that insulin causes a rather rapid threefold increase in the number of channels regardless of whether the basal rate of transport was relatively low (control) or comparatively higher (aldosterone pretreated). It was also of interest to note that, despite differences of basal rates of transport and despite differences in responses of Po in control and aldosterone-pretreated tissues, the secondary falloff of NT was quite similar (Fig. 6D) in control and aldosterone-pretreated tissues.

Although increase of NT is the major factor determining stimulation of the macroscopic rate of transport (Isc), transient changes of both iNa and Po play contributing roles that yield somewhat different Isc responses to insulin in the control and aldosterone-pretreated epithelia. The decrease of iNa that was measured immediately after the addition of insulin is predictably due to electrical depolarization across the apical membrane, decreasing the driving force for Na+ entry and arising from changes of fractional transcellular resistance. Within 2-3 h, the iNa returned slowly toward the zero time control values in both groups of tissues, thereby partially offsetting the relatively slow decreases of NT. The immediate decrease in Po following insulin in the aldosterone-pretreated tissues was unanticipated and stands in contrast to patch-clamp experiments in which hormones and effectors have, in general, been reported to increase Po (9, 18, 20). These data are, however, consistent with our previous observations (17). In a series of experiments examining iNa, Po, and NT in control and aldosterone-pretreated A6 cells grown under a variety of culture conditions and having a wide range of basal transport rates, we observed that Po was inversely related to the basal current, and this relationship was independent of hormone treatment. Our current findings support and extend these observations, noting, however, that the immediate but relatively small decreases of Po (compared quantitatively to those of NT) were not sustained. Po in the aldosterone-pretreated tissues increased slowly toward the zero time control values within 2 h, whereas, in control tissues, the Po remained essentially unchanged for 3 h following treatment of the epithelia with insulin.

Our findings, particularly with regard to insulin regulation of Po and NT, are opposite to the findings of Marunaka et al. (20) who used cell-attached patch-clamp methodology to examine the effect of insulin on single channels in aldosterone-pretreated epithelia (control tissues were not studied). It was reported that increases of the average NPo per patch (where N is number of channels per patch and Po is average value of open probability in individual channels in the patch) were due principally to an increase in the Po. There are differences in growth media, serum, substrate on which the A6 cells were grown, source of insulin, passage of cells, and other differences that cannot be ruled out completely to explain the disparity of observation. However, we believe that the origin of this disparity may also be related to the way in which channels respond to hormonal regulation in preformed patches of plasma membranes.

It is, for example, interesting to note that, while examining the role of antidiuretic hormone (ADH) and cAMP in regulation of ENaCs, Marunaka and Eaton (19) found that patches formed after stimulation of transport exhibited large (5-fold) increases of N with no significant change of Po. This result is in complete agreement with noise experiments (10, 11). In both the noise and patch-clamp experiments, Po averaged near 0.4. Interestingly, in these experiments, Marunaka and Eaton (19) also demonstrated that patches formed before hormonal treatment with ADH or cAMP did not respond to hormonal regulation. The authors speculated that mechanical formation of the patch disrupts the ability of the patched membrane to respond to regulation by cAMP. Other investigators have noted similar problems when studying regulation of channels in membrane patches. Gray et al. (15) reported that the results of their experiments were different with stimulation (secretin, forskolin, dibutyryl cAMP) of pancreatic duct cell Cl- channels, depending on patch formation either before or after hormonal stimulation of the cells (15). Their speculation included the possibility of mechanical damage to the apical plasma membrane or the cytoskeleton during gigaseal formation. In patch-clamp experiments by Strong et al. (25) of Aplysia neurons, phorbol ester stimulation of Ca2+ channels was prevented completely by the prior formation of cell-attached patches. Taken collectively, it appears that channels observed in patches after hormonal treatment can retain the changes in activity caused by the hormone (15, 19); preformed patches either change the response to the hormone (15) or, in the cases of cAMP stimulation of A6 and phorbol ester treatment of Aplysia neurons, obliterate the response completely (19, 25). Because the effects of insulin have not been evaluated in patches following hormonal stimulation, it remains unknown whether the results from noise and patch are irreconcilable.

Our data support and extend the findings of Erlij et al. (12), who found an increase in the number of open channels ~2 h after the addition of 100 mU/ml insulin. These investigators found no changes in iNa or Po at this late time point, consistent with our own results. The changes of iNa that we observed as an immediate response to insulin were anticipated by Erlij et al. (see discussion in Ref. 12) but not demonstrated, likely due to the relatively late time point of analysis and the small transient behavior of the iNa.

In summary, we have used a pulse protocol of CDPC-induced noise analysis to examine the time course of stimulation of Na+ transport by insulin in the presence and absence of exogenous aldosterone stimulation of Na+ transport in A6 epithelia. The major contributing factor to the increased transport is an increase in the number of active or functional channels in the apical membrane. Although our data do not allow us to postulate the exact mechanism (e.g., activation of preexisting apical membrane channels or insertion of the new channels from an intracellular pool), several other studies from our own and other laboratories support the contention that insulin causes the insertion of channels from an intracellular pool. Erlij et al. (12) observed that insulin increased apical membrane capacitance in A6 cells and hence membrane area (assuming constancy of the dielectric constant at audio frequencies), suggesting that insulin's action may be associated with the exocytotic delivery of channels to the apical membrane. Our previous data showing that brefeldin A, an inhibitor of exocytotic events, partially inhibits both basal and insulin-stimulated Na+ transport are consistent with these findings (5). Also in agreement are our recent studies showing that the enzyme phosphatidylinositol 3-kinase is involved in the intracellular signaling pathway of insulin's natriferic effect (23). This enzyme has been shown to be one of the prime components involved in the insulin-stimulated insertion of the glucose transporter (GLUT-4) into the plasma membrane (4). Full elucidation of this thesis will, however, require further investigation.

    ACKNOWLEDGEMENTS

We are grateful to A. L. Helman for assistance in preparation of this manuscript.

    FOOTNOTES

This work was supported by a Veterans Affairs Merit Review Grant and a Grant-In-Aid from the American Heart Association, Indiana Affiliate, to B. L. Blazer-Yost and by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-30824 to S. I. Helman.

X. Liu is a doctoral student in the Dept. of Molecular and Integrative Physiology (Univ. of Illinois at Urbana).

Address for reprint requests: B. L. Blazer-Yost, Biology Dept., Indiana University-Purdue University at Indianapolis., Rm. SL358, 723 West Michigan St., Indianapolis, IN 46202.

Received 15 September 1997; accepted in final form 4 February 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Baxendale-Cox, L. M., R. L. Duncan, X. Liu, K. Baldwin, W. J. Els, and S. I. Helman. Steroid hormone-dependent expression of blocker-sensitive ENaCs in apical membranes of A6 epithelia. Am. J. Physiol. 273 (Cell Physiol. 42): C1650-C1656, 1997[Abstract/Free Full Text].

2.   Blazer-Yost, B. L., M. Cox, and R. Furlanetto. Insulin and IGF I receptor-mediated Na+ transport in toad urinary bladders. Am. J. Physiol. 257 (Cell Physiol. 26): C612-C620, 1989[Abstract/Free Full Text].

3.   Blazer-Yost, B. L., N. Shah, L. Jarett, M. Cox, and R. M. Smith. Insulin and IGF1 receptors in a model renal epithelium: receptor localization and characterization. Biochem. Int. 28: 143-153, 1992[Medline].

4.   Cheatham, B., C. J. Vlahos, L. Cheatham, L. Wang, J. Blenis, and C. R. Kahn. Phosphatidylinositol 3-kinase activation is required for insulin stimulation of pp70 S6 kinase, DNA synthesis, and glucose transporter translocation. Mol. Cell. Biol. 14: 4902-4911, 1994[Abstract].

5.   Coupaye-Gerard, B., H. J. Kim, A. Singh, and B. L. Blazer-Yost. Differential effects of brefeldin A on hormonally regulated Na+ transport in a model renal epithelial cell line. Biochim. Biophys. Acta 1190: 449-456, 1994[Medline].

6.   Cox, M., and I. Singer. Insulin-mediated Na+ transport in the toad urinary bladder. Am. J. Physiol. 232 (Renal Fluid Electrolyte Physiol. 1): F270-F277, 1977[Medline].

7.   DeFronzo, R. A. The effect of insulin on renal sodium metabolism. Diabetologia 21: 165-171, 1981[Medline].

8.   DeFronzo, R. A., C. R. Cooke, R. Andres, G. R. Faloona, and P. J. Davis. The effect of insulin on renal handling of sodium, potassium, calcium, and phosphate in man. J. Clin. Invest. 55: 845-855, 1975[Medline].

9.   Eaton, D. C., A. Becchetti, H. P. Ma, and B. N. Ling. Renal sodium channels: regulation and single channel properties. Kidney Int. 48: 941-949, 1995[Medline].

10.   Els, W. J., and S. I. Helman. Activation of epithelial Na channels by hormonal and autoregulatory mechanisms of action. J. Gen. Physiol. 98: 1197-1220, 1991[Abstract].

11.   Els, W. J., and S. I. Helman. Dual role of prostaglandins (PGE2) in regulation of channel density and open probability of epithelial Na+ channels in frog skin (R. pipiens). J. Membr. Biol. 155: 75-87, 1997[Medline].

12.   Erlij, D., P. De Smet, and W. Van Driessche. Effect of insulin on area and Na+ channel density of apical membrane of cultured toad kidney cells. J. Physiol. (Lond.) 481: 533-542, 1994[Abstract].

13.   Fidelman, M. L., J. M. May, T. U. L. Biber, and C. O. Watlington. Insulin stimulation of Na+ transport and glucose metabolism in cultured kidney cells. Am. J. Physiol. 242 (Cell Physiol. 11): C121-C123, 1982[Abstract/Free Full Text].

14.   Fisher, R. S., F. G. Grillo, and S. Sariban-Sohraby. Brefeldin A inhibition of apical Na+ channels in epithelia. Am. J. Physiol. 270 (Cell Physiol. 39): C138-C147, 1996[Abstract/Free Full Text].

15.   Gray, M. A., J. R. Greenwell, and B. E. Argent. Secretin-regulated chloride channel on the apical plasma membrane of pancreatic duct cells. J. Membr. Biol. 105: 131-142, 1988[Medline].

16.   Helman, S. I., and N. L. Kizer. Apical sodium ion channels of tight epithelia as viewed from the perspective of noise analysis. In: Current Topics in Membranes and Transport, edited by S. I. Helman, and W. Van Driessche. New York: Academic, 1990, vol. 37, p. 117-155.

17.   Helman, S. I., X. Liu, K. Baldwin, B. Blazer-Yost, and W. J. Els. Time-dependent stimulation by aldosterone of blocker-sensitive ENaCs in A6 epithelia. Am. J. Physiol. 274 (Cell Physiol. 43): C947-C957, 1998[Abstract/Free Full Text].

18.   Kemendy, A. E., T. R. Kleyman, and D. C. Eaton. Aldosterone alters the open probability of amiloride-blockable sodium channels in A6 epithelia. Am. J. Physiol. 263 (Cell Physiol. 32): C825-C837, 1992[Abstract/Free Full Text].

19.   Marunaka, Y., and D. C. Eaton. Effects of vasopressin and cAMP on single amiloride-blockable Na channels. Am. J. Physiol. 260 (Cell Physiol. 29): C1071-C1084, 1991[Abstract/Free Full Text].

20.   Marunaka, Y., N. Hagiwara, and H. Tohda. Insulin activates single amiloride-blockable Na channels in a distal nephron cell line (A6). Am. J. Physiol. 263 (Renal Fluid Electrolyte Physiol. 32): F392-F400, 1992[Abstract/Free Full Text].

21.   Nizet, A., P. Lefebvre, and J. Crabbé. Control by insulin of sodium, potassium and water excretion by the isolated dog kidney. Pflügers Arch. 323: 11-20, 1971[Medline].

22.   Palmer, L. G. Epithelial Na channels: function and diversity. Annu. Rev. Physiol. 54: 51-66, 1992[Medline].

23.   Record, R. D., L. Froelich, C. J. Vlahos, and B. L. Blazer-Yost. Phosphatidylinositol 3-kinase activation is required for insulin-stimulated sodium transport in A6 cells. Am. J. Physiol. 274 (Endocrinol. Metab. 37): E611-E617, 1998[Abstract/Free Full Text].

24.   Record, R. D., M. Johnson, S. Y. Lee, and B. L. Blazer-Yost. Aldosterone and insulin stimulate amiloride-sensitive sodium transport in A6 cells by additive mechanisms. Am. J. Physiol. 271 (Cell Physiol. 40): C1079-C1084, 1996[Abstract/Free Full Text].

25.   Strong, J. A., A. P. Fox, R. W. Tsien, and L. K. Kaczmarek. Stimulation of protein kinase C recruits covert calcium channels in Aplysia bag cell neurons. Nature 325: 714-717, 1987[Medline].

26.   White, M. F., and C. R. Kahn. The insulin signaling system. J. Biol. Chem. 269: 1-4, 1994[Free Full Text].


AJP Cell Physiol 274(5):C1373-C1379
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