1 Biology Department, Aldosterone, a steroid hormone, regulates renal
Na+ reabsorption and, therefore,
plays an important role in the maintenance of salt and water balance.
In a model renal epithelial cell line (A6) we have found that
phosphoinositide 3-kinase (PI 3-kinase) activity is required for
aldosterone-stimulated Na+
reabsorption. Inhibition of PI 3-kinase by the specific inhibitor LY-294002 markedly reduces both basal and aldosterone-stimulated Na+ transport. Further, one of the
products of PI 3-kinase, phosphatidylinositol 3,4,5-trisphosphate, is
increased in response to aldosterone in intact A6 monolayers. This
increase occurs just before the manifestation of the functional effect
of the hormone and is also inhibited by LY-294002. With the use of
blocker-induced noise analysis, it has been demonstrated that
inhibition of phosphoinositide formation causes an inhibition of
Na+ entry in both control and
aldosterone-pretreated cultures by reducing the number of open
functional epithelial Na+ channels
(ENaCs) in the apical membrane of the A6 cells. These novel
observations indicate that phosphoinositides are required for ENaC
expression and suggest a mechanism for aldosterone regulation of
channel function.
epithelial sodium channels; noise analysis; electrophysiology; kidney; cortical collecting ducts; A6 cell line
SEVERAL HORMONES MODULATE the activity of the renal
epithelial Na+ channel (ENaC).
This regulation is critical to maintain normal salt and fluid
homeostasis, and slight alterations in these hormone-stimulated pathways may contribute to such common diseases as essential
hypertension. Despite the importance of these processes, very little is
known about the intracellular signaling pathways involved in hormonal regulation of epithelial Na+ reabsorption.
Aldosterone, a steroid hormone, mediates relatively long-term
natriferic responses. Hormone binding to the mineralocorticoid receptor
in responsive tissues is well documented. Likewise, it is known that
the final outcome of this interaction is stimulation of
Na+ transport via the
amiloride-sensitive ENaCs (1-4, 8). However, the pathway(s)
linking the aldosterone-receptor binding to activation of transport
through functional channels within the apical membrane of the cells is unknown.
Phosphoinositide 3-kinases (PI 3-kinases) are enzymes that
phosphorylate position 3 of the head group of the membrane lipid phosphatidylinositol (20). PI 3-kinases are a family of enzymes that
can be distinguished by molecular characteristics, substrate specificity, and inhibitor sensitivities (13, 20, 22-24). Classic inhibitors of these enzymes are wortmannin, a fungal metabolite, which
is an irreversible inhibitor effective in the nanomolar concentration
range, and LY-294002, a structurally unrelated compound with a very
high specificity for PI 3-kinase, which is a reversible inhibitor
effective in the micromolar concentration range (23).
These enzymes have been implicated in a variety of diverse biochemical
processes including membrane transport phenomena. Notable in this
regard is the importance of the activation of PI 3-kinase for the
insertion of the glucose transporters (GLUT-4) into the plasma membrane
of adipocytes and skeletal muscle in response to insulin (6, 24).
However, other peptide hormone-mediated transport events including
insulin-stimulated Na+ transport
via ENaC (14) and K+ uptake into
fibroblasts via the
Na+-K+-2Cl We have previously shown that the increase in aldosterone-stimulated
Na+ transport is paralleled by an
increase in the number of functional channels in the apical membrane
(8). In this regard, the steroid hormone action is similar to the
natriferic action of insulin, although the time course of the two
hormones is very different. Insulin causes an increase in the number of
functional channels within minutes, whereas aldosterone's action
requires a longer time, consistent with the requirement for new protein
synthesis (4). We have also previously demonstrated that insulin's
action is dependent on the presence of PI 3-kinase (14). Given the importance of PI 3-kinase for peptide-mediated transport, we have investigated the role of this enzyme in steroid-mediated
Na+ absorption.
Cell culture. For measurement of
macroscopic rates of transport as well as lipid profile analysis, the
A6 cells were grown at 27°C in DMEM (#91-5055EC;
GIBCO BRL, Grand Island, NY) supplemented with 25 U/ml penicillin, 25 µg/ml streptomycin, and 10% calf serum in a humidified incubator
gassed with 5% CO2. The cells
were subcultured onto 24-mm Transwell tissue-culture-treated inserts
(Costar, Cambridge, MA) for at least 14 days to achieve confluence and
used 14-21 days after seeding. For noise analysis, with the
exception of three control tissues, A6 epithelia were grown on
Transwell clear inserts at 28°C in a humidified incubator
containing 1% CO2. The growth
medium was either the modified DMEM (as above) or a mixture of Ham's
F-12 medium (N-6760; Sigma Chemical, St. Louis, MO) and L-15 Leibovitz
medium (L-4386; Sigma Chemical) supplemented with 10% defined fetal
bovine serum (FBS), 2.57 mM sodium bicarbonate, 3.84 mM
L-glutamine, 96 U/ml penicillin,
and 96 µg/ml streptomycin. Tissues were studied while continuously
perfused with growth medium without FBS and glutamine.
Electrical measurements/noise
analysis. Macroscopic rates of transcellular
Na+ transport were measured as
short-circuit currents
(Isc) as
previously described (2, 8). After mounting in the chambers, the cells were incubated in serum-free media for 1-4 h to assure that a stable baseline was achieved before the addition of inhibitors and/or hormone.
In the noise experiments, blocker-sensitive
Na+ currents
(INa) were
determined from the difference between the
Isc and the respective 100 µM amiloride-insensitive
Na+ currents that average near 0.1 µA/cm2 before and after
treatment of the tissues with LY-294002. A pulse method of
blocker-induced noise analysis was carried out as previously described
(8), using the weak ENaC blocker
6-chloro-3,5-diaminopyrazine-2-carboxamide (CDPC; 27,788-6;
Aldrich Chemical, Milwaukee, WI). Corner frequencies (fc) and
low-frequency plateaus
(So) of the
CDPC Lorentzians characterizing the blocker-induced current noise in
power density spectra were determined by nonlinear curve fitting.
Blocker on
(kob) and off (kbo) rate
coefficients were calculated from the slopes and intercepts of
rate-concentration plots using the time-filtered
fc at the respective 10 and 30 µM concentrations of CDPC. Single-channel currents (iNa)
were calculated in the usual way from the quotient [So(2 Lipid analysis. To determine the
phosphorylation of the inositol lipids, A6 cells were incubated for 16 h in serum-free media followed by a 2-h incubation in a nominally
phosphate-free media containing
H332PO4
before any experimental manipulations were performed. After hormone
and/or inhibitor treatments, the lipids were extracted, separated, and
analyzed by anion exchange chromatography as reported previously (14,
23).
When A6 epithelia are challenged with LY-294002, a specific and
reversible inhibitor of PI 3-kinase (23), inhibition of PI 3-kinase
results in a marked decrease in basal
Na+ transport and a complete
abolition of aldosterone-stimulated Na+ transport as measured by
amiloride-sensitive
Isc. To
illustrate the time course of the basal inhibition, the data are
plotted at 5-min intervals in the 30 min following the addition of
LY-294002 (Fig.
1A).
Such observations suggest that the products of the PI 3-kinase
reaction, phosphoinositides, may be crucial intermediates in the
natriferic pathway.
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
cotransporter (19) also require PI 3-kinase activation. In addition,
these enzymes also appear to have a role in platelet-derived growth
factor activation of the
Na+/H+
exchanger (11), epidermal growth factor (EGF)-stimulated intestinal Na+ absorption (9), and
EGF-mediated inhibition of
Ca2+-dependent
Cl
secretion (21).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
fc)2]/[4INa
kobB],
where So were
corrected for the shunting of current noise (~12%) through the
apical membrane capacitance. Functional open-channel densities
(No) were
calculated as
INa/iNa.
The tissues were studied in both their unstimulated and
aldosterone-prestimulated states (0.27 µM, overnight). All
experiments were carried out at ambient room temperature. Data are
expressed as means ± SE.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effect of inhibitors of phosphoinositide 3-kinase (PI 3-kinase) on
basal and aldosterone-stimulated
Na+ transport. Macroscopic rates
of ion transport were measured as short-circuit currents
(Isc). A6
tissues were preincubated with 50 µM LY-294002
(A) or 100 nM wortmannin
(B) for 30 min. Aldosterone (2.7 µM) was added to basolateral solution at time
zero. After 5 h of aldosterone incubation, 10 µM
amiloride was added to apical media to inhibit portion of current due
to Na+ transport by epithelial
Na+ channels (ENaCs). Results are
means ± SE. For clarity, error bars are shown in one direction
only.
Interestingly, wortmannin, a structurally unrelated inhibitor of PI 3-kinase (13), also inhibited basal current but did not affect the aldosterone-stimulated portion of Na+ transport (Fig. 1B). Thus the data suggest that the baseline rate of Na+ transport requires the constitutive activation of a wortmannin- and LY-294002-sensitive PI 3-kinase, whereas aldosterone's action involves an LY-294002-sensitive but wortmannin-insensitive form of the kinase.
Because intracellular signaling processes must occur before the final
functional effect manifested by the cell, the phosphatidylinositol lipid profile was examined just before the onset of
aldosterone-stimulated transport (20 min after hormone addition) and
just after an aldosterone-induced increase in transport was clearly
measurable (60 min). The lipid profiles of stimulated tissues were
compared with control (non-hormone-treated) tissues that had been grown
in parallel cultures (Fig. 2). Twenty minutes after the addition of aldosterone, there was an increase in the phosphoinositides phosphatidylinositol 3-phosphate
[PtdIns(3)P] and
phosphatidylinositol 3,4,5-trisphosphate
[PtdIns(3,4,5)P3]. Accurate
quantitation of PtdIns(3)P as well as phosphatidylinositol 3,4-bisphosphate
[PtdIns(3,4)P2]
was not possible in all experiments due to the proximity of these
compounds to unidentified peaks that have a similar elution time. For
this reason, the
PtdIns(3,4,5)P3 peak, which can be accurately quantitated, was used as the measure of
PI 3-kinase activity in a more extensive series of experiments (Fig. 3).
|
|
To normalize for experimental variability due to sample recovery, the PtdIns(3,4,5)P3 peak was compared with the PtdIns(4,5)P2 peak in each sample. The amount of PtdIns(4,5)P2 is not likely to change under these experimental conditions. The PtdIns(3,4,5)P3/PtdIns(4,5)P2 ratio from each sample was compared with the same ratio in a control sample that was grown, labeled, and processed in tandem (Fig. 3).
An increased level of
PtdIns(3,4,5)P3
is detectable before a measurable increase in aldosterone-stimulated
Na+ transport (20 min) in each
sample analyzed. After 60 min of hormone stimulation,
PtdIns(3,4,5)P3
remains elevated over control in all samples. With one exception,
LY-294002 inhibits the production of
PtdIns(3,4,5)P3
below control levels even in the presence of aldosterone. Thus
LY-294002 inhibited not only the aldosterone-mediated increase in the
production of the phosphoinositides (Fig. 2 and 3) but also the
Na+ transport response (Figs. 1
and 4).
|
Finally, we determined the underlying time-dependent changes of amiloride-sensitive Na+ transport (INa) in response to LY-294002 at the single-channel level. Noninvasive methods of blocker-induced noise analysis were used to monitor the changes of the single-channel currents (iNa) and open-channel densities (No) in confluent short-circuited monolayers of A6 epithelia. For the purpose of these studies, the effects of the inhibitor on both unstimulated and aldosterone-pretreated tissues were examined (Fig. 4). Zero time values shown in Fig. 4 are typical of those reported for unstimulated and aldosterone-pretreated tissues where aldosterone causes a twofold or larger stimulation of transport by increase of No with no significant change of iNa (8). From zero time values of INa that averaged 4.0 ± 0.1 µA/cm2 in unstimulated tissues and 9.1 ± 0.9 µA/cm2 in aldosterone-pretreated tissues, the amiloride-sensitive Isc were inhibited by LY-294002, on average, to 33% and 38% of the zero time values, respectively, within 90 min (Fig. 4A), following, however, an initial small transient increase in transport (<5 min, data not shown). The inhibitory effect was relatively slow, with the mean INa response to 10 µM LY-294002 exhibiting quasiexponential decays with time constants of 22.4 and 24.4 min in control and aldosterone-pretreated tissues, respectively.
The changes of single-channel current caused by the inhibitor in the two groups of tissues are summarized in Fig. 4B. From zero time values that averaged ~0.39 pA, LY-294002 caused similar small time-dependent increases of iNa. Consequently, the inhibitory effect of LY-294002 on Na+ transport is due to a decrease in the number of functionally open channels within the apical membrane of the cells. Underlying the trend in INa, No fell on average to 30% and 34% of the respective zero time values in control and aldosterone-pretreated tissues, respectively, after 90 min of exposure to LY-294002. Inhibition of the macroscopic rates of transport and open-channel densities is fully reversible on removal of the LY-294002 (Fig. 4).
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DISCUSSION |
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Tight epithelia of the A6 cell line, derived from the kidney of Xenopus laevis, are commonly used as a model for the transport properties of the principal cells of the distal nephron. A6 cells contain ENaCs and respond to hormones known to regulate Na+ reabsorption in the mammalian distal nephron (12). In these cells, aldosterone stimulates an increase in transepithelial Na+ transport with a slow time course that is consistent with its action through new protein synthesis (3). The nature of the induced proteins as well as the identity of other intracellular regulators of the hormone-stimulated transport process are unknown.
Taken together, the data presented here indicate that PI 3-kinases are required for transcellular Na+ transport with specific involvement of apical membrane ENaCs. Analysis of the lipid profile in A6 cells that have been treated with aldosterone indicates that this steroid hormone mediates an increase in the amount of phosphoinositides, specifically PtdIns(3,4,5)P3. This increase over the level found in control cells can be detected before the increase in the functional effect of enhanced Na+ transport, consistent with a regulatory role for the phosphoinositides in aldosterone-mediated natriferic activity. These results are similar to our previous studies that showed that insulin-mediated Na+ reabsorption via ENaC also involves the stimulation of an LY-294002-sensitive PI 3-kinase and that the PtdIns(3,4,5)P3 formed by this enzyme is increased before the functional action on the channel (14). Insulin and aldosterone exhibit additive natriferic responses, suggesting that they are mediated by at least partially independent pathways (15). However, both hormone responses are a result of an increase in the number of active ENaCs in the apical membrane (4, 8). The similarities in PI 3-kinase inhibitor effects on the two hormone responses suggest that the enzyme may be mediating an effect at or near the channel.
PI 3-kinases play pivotal roles in a wide variety of metabolic processes. Specificity in the regulation of these diverse pathways may be explained, in part, by the growing number of PI 3-kinase variants that have been described recently (7, 14, 20, 22). Multiple variants can coexist in a single cell. In fact, four different isoforms of PI 3-kinase have been identified in adipocytes. Our results indicate that both basal Na+ transport as well as the aldosterone-stimulated portion of the natriferic activity are sensitive to inhibition by LY-294002, whereas only the basal transport is inhibited by wortmannin. Again, this is consistent with our previous studies showing that the insulin-stimulated increase in Na+ transport is a wortmannin-insensitive, LY-294002-sensitive process, whereas basal transport is sensitive to both inhibitors (14). These data suggest that at least two variants of PI 3-kinase are involved in transcellular Na+ transport.
LY-294002 inhibition of transport assessed macroscopically and at the single-channel level is fully reversible after removal of LY-294002, indicating the dynamic role of PI 3-kinase in regulation of transport. Notably, inhibition of transport is essentially the same for both control and aldosterone-pretreated tissues, indicating the importance of PI 3-kinase for functional expression of ENaCs.
The time course of the inhibitory effect in both basal and aldosterone-pretreated tissues is relatively slow. These results can be compared with the data of Kurashima et al. (10) who found that inhibitors of PI 3-kinase alter the constitutive endosomal recycling of the Na+/H+ exchanger NHE3. The time course of the inhibitory effect on the functional activity of the exchanger in intact cells was remarkably similar to the inhibition of Na+ transport in our study. It is also worth noting that ENaC has been shown to be a relatively short-lived protein with an estimated half time of ~1 h (18). The data obtained in the Kurashima studies suggested that the functional effects were likely due to a decrease in the cycling of intracellular Na+/H+ exchangers to the plasma membrane (10). Although the very low number of endogenous Na+ channels in our native epithelial cells makes similar studies examining recycling very difficult, we have examined the effect of LY-294002 on the number of active channels expressed on the apical membrane of A6 cells. When the inhibitory effect was assessed at a single-channel level, we found that the effect on macroscopic transport is clearly due to a decrease in the number of active channels in the apical membrane (Fig. 4). Although this result, combined with the relatively slow time course, is certainly consistent with a role for PI 3-kinase in dynamic recycling of Na+ channels, it should be noted that these biophysical measurements of active channels cannot distinguish between insertion of new channels from an intracellular pool and activation of quiescent channels that were preexisting in the membrane.
ENaC has been shown to be involved in rare forms of blood pressure aberrations (5, 17) and has been implicated as a potential factor in many forms of essential hypertension (16). Therefore, a complete elucidation of the hormonal pathways that regulate the activity of the channel is important to understand the etiology of pathological processes that may arise from aberrant regulation of this signaling. These studies provide evidence of a crucial role for phosphoinositide formation in aldosterone-stimulated Na+ transport and represent novel data regarding the intracellular pathway linking mineralocorticoid receptor binding to the final natriferic effect in transporting epithelial cells. Although PI 3-kinases have been linked to a variety of peptide factor transport responses, this is the first demonstration of a role for these enzymes in a steroid hormone response.
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ACKNOWLEDGEMENTS |
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We thank T. Lahr and R. Yost for critically reviewing the manuscript.
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FOOTNOTES |
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This work was supported by grants from the American Heart Association, Indiana Affiliate (to B. Blazer-Yost), and the National Institutes of Health (to S. I. Helman).
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 and other correspondence: B. L. Blazer-Yost, Biology Dept., SL 358, Indiana Univ., Purdue Univ. at Indianapolis, 723 W. Michigan St., Indianapolis IN 46202 (E-mail: bblazer{at}iupui.edu).
Received 28 September 1998; accepted in final form 28 May 1999.
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