Phosphatidylinositol 3-kinase activation is required for
insulin-stimulated sodium transport in A6 cells
Rae D.
Record1,
Larry L.
Froelich2,
Chris J.
Vlahos2, and
Bonnie L.
Blazer-Yost1
1 Roudebush Veterans Medical
Center and Biology Department, Indiana University-Purdue University
at Indianapolis, Indianapolis, 46202; and
2 Lilly Research Laboratories, Eli
Lilly, Indianapolis, Indiana 46285
 |
ABSTRACT |
Insulin stimulates amiloride-sensitive sodium
transport in models of the distal nephron. Here we demonstrate that, in
the A6 cell line, this action is mediated by the insulin receptor tyrosine kinase and that activation of phosphatidylinositol 3-kinase (PI 3-kinase) lies downstream of the receptor tyrosine kinase. Functionally, a specific inhibitor of PI 3-kinase, LY-294002, blocks
basal as well as insulin-stimulated sodium transport in a
dose-dependent manner (IC50
6 µM). Biochemically, PI 3-kinase is present in A6 cells and is
inhibited both in vivo and in vitro by LY-294002. Furthermore, a
subsequent potential downstream signaling element, pp70 S6 kinase, is
activated in response to insulin but does not appear to be part of the
pathway involved in insulin-stimulated sodium transport. Together with
previous reports, these results suggest that insulin may induce the
exocytotic insertion of sodium channels into the apical membrane of A6
cells in a PI 3-kinase-mediated manner.
amiloride-sensitive sodium channel; insulin signaling; receptor
tyrosine kinase; renal epithelia
 |
INTRODUCTION |
INSULIN INCREASES SODIUM REABSORPTION in dogs and
humans, and this effect appears to be manifested predominantly on the
renal distal nephron (21, 10). Models of the distal nephron, toad urinary bladder (Bufo marinus) and
the A6 cell line (derived from Xenopus
laevis kidney), have been used to demonstrate a direct effect of insulin on amiloride-sensitive sodium transport (12, 14).
Patch-clamp electrophysiology has indicated that insulin causes an
increase in the open probability
(PO) of the
apical sodium channel (20). However, blocker-induced noise analysis demonstrated that insulin induces an increase in active sodium channel
density in the apical membrane (2, 4, 11). In addition, insulin
increases the apical membrane area (11). These results suggest that
insulin may induce the insertion of sodium channels into the apical
membrane, a hypothesis supported by our study demonstrating that
brefeldin A, an inhibitor of secretion, partially inhibits
insulin-stimulated sodium transport (9).
The first step in insulin signaling is the binding of the hormone to
its receptor, followed by autophosphorylation of the receptor and
subsequent activation of the receptor tyrosine kinase (IRTK). IRTK has
several substrates, including the insulin receptor substrate (IRS)
proteins (33). The IRS proteins mediate some of the pleiotropic effects
of insulin through interactions with proteins containing SH2 domains,
including phosphatidylinositol 3-kinase (PI 3-kinase). This enzyme, a
heterodimer, phosphorylates the D-3 position of the
myo-inositol ring of
phosphatidylinositols (PtdIns; 17). PI 3-kinase is required for several
insulin-mediated effects, including the translocation of the
insulin-responsive glucose transporter (GLUT-4) to the plasma membrane
of adipocytes (7) and skeletal muscle (34), and the activation of pp70 S6 kinase, which is necessary for protein synthesis (24).
We have previously demonstrated that insulin binds to the basolateral
membrane of amphibian cells (5) and, along with others, that genistein,
a tyrosine kinase inhibitor, decreases insulin-stimulated sodium
transport (25, 27). Here we demonstrate that, in A6 cells, an IRTK
inhibitor blocks insulin-stimulated sodium transport and that a
specific PI 3-kinase inhibitor blocks insulin-stimulated sodium
transport in a dose-dependent manner. We also show that, in the A6 cell
line, insulin stimulates PI 3-kinase in vivo. In addition, our
functional and biochemical studies indicate that pp70 S6 kinase is
present in these cells and is activated by insulin but is not involved
in insulin-stimulated sodium transport. These results suggest that PI
3-kinase is necessary for insulin-stimulated sodium transport, perhaps
by mediating the insertion of sodium channels into the apical membrane
in a manner analogous to the insulin-stimulated insertion of GLUT-4.
 |
EXPERIMENTAL PROCEDURES |
Materials.
Hydroxy-2-naphthalenylmethylphosphonic acid tris acetoxymethyl ester
[HNMPA-(AM)3] and
2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY-294002) were
purchased from BIOMOL Research Laboratories. Wortmannin, amiloride,
PtdIns, and PBS were purchased from Sigma Chemical. The wortmannin used
has been shown to be active against mammalian PI 3-kinase.
[32P]H3PO4
was purchased from Du Pont NEN.
[
-32P]ATP was
purchased from Amersham Life Science. The anti-PI 3-kinase 85-kDa
subunit, anti-pp70 S6 kinase, S6 kinase substrate, and an inhibitor
cocktail for S6 kinase assay were purchased from Upstate Biotechnology.
Porcine insulin was a generous gift from Eli Lilly. Rapamycin was
purchased from ICN Pharmaceuticals. Transwell inserts were purchased
from Costar. GammaBind Plus Sepharose beads were purchased from
Pharmacia Biotech. Silica gel thin-layer chromatography plates, 60A,
were purchased through Curtin Matheson Scientific. Media were purchased
from GIBCO-BRL Life Technologies. 4-(2-Aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF) was obtained from Boehringer Mannheim.
Cell culture.
The A6 cell line was obtained from American Type Tissue Culture
Collection and grown at 27°C in a humidified incubator gassed with
5% CO2 in
O2, as previously described (3).
For electrophysiology, cells were subcultured onto 24-mm Transwell
inserts; for biochemistry, cells were subcultured onto 24- or 100-mm
Transwell inserts. Cells were used 14-21 days after seeding.
Electrophysiology.
Short-circuit current (SCC) techniques were used to determine net ion
flux (19, 22). Cells grown on Transwell inserts were placed in a
modified Ussing chamber and monitored as previously described (3).
During the electrophysiological studies, cells were bathed in
serum-free media maintained at 27°C, with gentle circulation
provided by a 5% CO2-95%
O2 gas lift. Four chambers allowed
simultaneous monitoring of cells grown in tandem. The monolayers were
monitored until a stable baseline SCC was obtained (typically
0.5-2 h). The average starting currents (after baseline stabilization) in these experiments varied from 2.7 to 18.9 µA/cm2. There was some variation
between the starting currents in the different sets of experiments;
however, within each experimental protocol, the average starting
currents of the cells in all conditions were statistically the same
value. The addition of amiloride at the end of each experiment verified
that the majority of the SCC was due to net apical-to-serosal
Na+ flux, as previously reported
for A6 cells. Transcellular resistance was determined every 2 min via a
brief 2-mV pulse across the cells. The average transcellular resistance
varied from 1,372 to 3,075
· cm2. None
of the inhibitors caused a significant decrease in the transepithelial
resistance over the time course of the experimental protocols.
Porcine insulin was added to the serosal bathing medium.
HNMPA-(AM)3, wortmannin,
LY-294002, and rapamycin were dissolved in DMSO and added bilaterally.
Amiloride, dissolved in methanol, was added to the apical bathing
medium. Equal volumes of DMSO were added to the control tissues to
detect any carrier effects; none were detected.
The change in SCC (
SCC) in experimental (e) relative to control (c)
samples was calculated using the following formula:
SCC = [SCC(t)
SCC(0)]e
[SCC(t)
SCC(0)]c, where
SCC(t) is SCC at time
t after insulin addition (with or
without inhibitor preincubation), and SCC(0) is the SCC at the time of
insulin addition (with or without inhibitor pretreatment).
PI 3-kinase assay, in vitro.
PI 3-kinase activity was determined by measuring the transfer of
32P from
[
-32P]ATP to PI to
form [32P]PI-3, which was detected by thin-layer
chromatography. A6 cells cultured on Transwell inserts were incubated
in serum-free media for 16 h. The cells were incubated with or without
100 nM insulin in serum-free media for 5 min followed by one wash with
ice-cold PBS. Ice-cold immunoprecipitation buffer (10 mM Tris, pH 7.4, 1% Triton X-100, 150 mM NaCl, 0.5% nonidet P-40, 1 mM EDTA, 1 mM
EGTA, 0.2 mM sodium vanadate, 0.2 mM AEBSF, 100 mM sodium fluoride) was
added to the filters, and the cells were scraped off with a rubber
policeman. The cells were transferred to microtubes and incubated for
30 min at 4°C with occasional vortexing. The samples were
centrifuged (16,000 gmax) for 15 min in a cold room. The supernatant (containing soluble proteins) was
incubated with anti-PI 3-kinase (against the 85-kDa subunit) and
GammaBind beads for 4 h at 4°C with constant rotation. The beads
were washed once in ice-cold PBS; twice in 0.5 M LiCl, 0.1 M Tris, pH
7.5; and once in PI 3-kinase assay buffer (10 mM
MgCl2 and 20 mM HEPES, pH 7.4).
The amount of PI 3-kinase activity in the immunoprecipitations was
determined as previously described (23). The reaction was stopped with
HCl, and the lipids were extracted with methanol-chloroform (1:1 by
volume). The lipids were applied to a silica gel thin-layer chromatography plate; the residue was developed with
chloroform-methanol-water-ammonium hydroxide (45:35:8.5:1.5 by volume).
The plate was dried and autoradiographed.
PI 3-kinase activity, in vivo.
PI 3-kinase activity was determined by directly measuring the
insulin-stimulated products of PI 3-kinase
{[32P]I-3P,
[32P]I-(3,4)P2,
and [32P]I-(3,4,5)P} by use of
anion exchange chromatography and an on-line radiochemical detector. A6
cells subcultured onto 100-mm Transwell inserts and grown to confluency
were incubated in serum-free media for 16 h. Subsequently, the cells
were incubated in phosphate-free media containing 0.5 mCi/plate
[32P]H3PO4
for 2 h. The cells were incubated with or without 25 nM wortmannin or
50 µM LY-294002 for 20 min. The medium was removed, and the cells
were washed twice with phosphate-free medium with or without inhibitor.
The cells were incubated for 1 min in 100 nM insulin, serosally, with
or without inhibitor, followed by the addition of 1 N HCl-methanol (1:1
by volume). The cells were scraped and the lipids extracted as reported
(13). After thin-layer chromatography and autoradiography, the lipids
were deacylated by treatment with methylamine (8) and separated by
anion-exchange HPLC.
pp70 S6 kinase assay.
A6 cells, serum starved for 16 h, were treated for 30 min with or
without 100 nM rapamycin followed by 10 min in the presence or absence
of 100 nM insulin. Immunoprecipitations were performed as above, with
the inclusion of 10 mM
-glycerophosphate in the immunoprecipitation
buffer. A polyclonal antibody raised against a peptide corresponding to
a post-NH2-terminal region of the
human pp70 S6 kinase was used for enzyme isolation. The
immunoprecipitates were washed three times in immunoprecipitation
buffer followed by two washes in S6 kinase assay buffer [(in mM)
20 MOPS, pH 7.2, 25
-glycerophosphate, 5 EGTA, 1 orthovanadate, 1 dithiothreitol]. The washed immunoprecipitates were incubated in
10 µl of S6 kinase assay buffer, 10 µl inhibitor cocktail, 10 µl
S6 kinase assay buffer with or without substrate (11-amino acid
peptide), and 10 µl of
[
-32P]ATP mixture
(0.2 mCi/ml, 112.5 µM ATP, 16.9 mM
MgCl2, final concentrations) for
20 min at 30°C. An aliquot of the reaction mixture was placed on
phosphocellulose paper followed by three washes in 0.7% phosphoric
acid and one wash in acetone. The radioactivity was determined with a
scintillation counter. Samples without immunoprecipitates (blanks) were
run for background determinations. All samples were run in duplicate
and averaged. The blanks were subtracted from the samples, and the
endogenous substrate activity (samples without added substrate) was
subtracted from the specific substrate activity.
 |
RESULTS |
We previously demonstrated that insulin binds to the basolateral
membrane and that the insulin receptor is predominantly localized to
the same membrane of amphibian cells (5). To demonstrate that
insulin-stimulated sodium transport requires the IRTK, we added
HNMPA-(AM)3 (5 µg/ml), a
specific inhibitor of IRTK (1), 60 min before the addition of 20 nM
insulin (Fig. 1).
HNMPA-(AM)3 inhibited
insulin-stimulated sodium transport ~50%. This suggests that insulin
stimulation of sodium transport is mediated through IRTK.

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Fig. 1.
Hydroxy-2-naphthalenylmethylphosphonic acid tris acetoxymethyl ester
[HNMPA-(AM)3] inhibits
insulin-stimulated sodium transport. A6 cells were preincubated with 5 µg/ml HNMPA-(AM)3 bilaterally
for 60 min. Insulin (20 nM) was added serosally at
time 0. After 120 min of insulin, 10 µM amiloride was added apically. Results are means ± SE;
n = 6. A: actual data for all conditions.
B: increase in short-circuit current
(SCC) in response to insulin and relative to the appropriate control.
SCC is calculated as described in Experimental
Procedures.
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Earlier studies by us and others (2, 4, 11) have demonstrated that
insulin stimulates an increase in the number of active sodium channels
into the apical membrane. Insulin induces the insertion of GLUT-4,
another transporter, into the plasma membrane in a PI
3-kinase-dependent manner (7, 34). To investigate whether PI 3-kinase
was necessary for insulin-stimulated sodium transport, we initially
used wortmannin, a fungal metabolite, which inhibits PI 3-kinase with
an IC50 of 1.8-4.0 nM in 3T3
cells (23). However, 25 nM wortmannin did not inhibit
insulin-stimulated sodium transport (Fig.
2). At a concentration of 100 nM,
wortmannin had an effect on basal transport and partially inhibited
only the initial (30-min) response to insulin (Fig. 2).

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Fig. 2.
Wortmannin effects on insulin-stimulated sodium transport. A6 cells
were preincubated with wortmannin (25 nM,
A; 100 nM,
B) bilaterally for 30 min. Insulin
(20 nM) was added serosally at time 0.
After 120 min of insulin, 10 µM amiloride was added apically. Results
are means ± SE; n = 4 in
A; n = 2, B.
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A more specific inhibitor of PI 3-kinase, LY-294002 (32), blocked both
basal and insulin-stimulated sodium transport in a dose-dependent
manner (Fig. 3). The lowest concentration,
2 µM, had no effect, whereas 50 µM inhibited ~90% of
insulin-stimulated sodium transport. Graphing this limited
dose-response curve yielded an
IC50 of ~6 µM (Fig.
4), which is comparable to the reported IC50 of 1.4 µM (32).

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Fig. 3.
2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyren-4-one (LY-294002)
inhibits insulin-stimulated sodium transport in a
concentration-dependent manner. A6 cells were preincubated in LY-294002
bilaterally for 30 min. LY-294002 was added at final concentrations of
2 (A), 10 (B), and 50 (C) µM, as indicated. Insulin (20 nM) was added serosally at time 0.
Results are means ± SE; n = 4.
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Fig. 4.
Dose-response curve for the effect of LY-294002 on insulin-stimulated
sodium transport. Data depicted in Fig. 3 were used to construct a
limited dose-response relationship. The % SCC of insulin alone at 30 min after insulin addition are plotted vs. concentration of LY-294002
(log scale). Results are means ± SE;
n = 4.
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The effects of insulin on transcellular
Na+ transport are manifested quite
rapidly. Figure 5 illustrates the very
early time course of the insulin response. Because activation of the
signal transduction mechanism would be expected to precede the final effect on ion flux, the biochemical assays were designed to examine effects within the early phase of the insulin response.

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Fig. 5.
Initial response to insulin, in which paired data represent initial
response to insulin in A6 cells. Results are means ± SE;
n = 4.
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To determine whether the LY-294002 effect was due to PI 3-kinase
inhibition, we immunoprecipitated PI 3-kinase from A6 cells and assayed
the activity associated with the immunoprecipitates (Fig.
6). In agreement with the electrophysiology
studies, immunoprecipitated PI 3-kinase activity was inhibited by 50 µM LY-294002, whereas wortmannin (25 nM) had little or no effect. As
has been reported (31), treating the cells with insulin did not
increase the amount of PI 3-kinase activity in immunoprecipitates.

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Fig. 6.
In vitro PI 3-kinase activity. Serum-starved (16-h) A6 cells were
treated with (I) or without (CT) 100 nM insulin for 5 min and
solubilized. Solubilized proteins were immunoprecipitated with anti-PI
3-kinase 85-kDa subunit. Immunoprecipitates were assayed for activity
with or without 25 nM wortmannin (W) or 50 µM LY-294002 (LY); lipids
were extracted and separated by thin-layer chromatography (TLC). Arrow
indicates phosphatidylinositol (PtdIns) monophosphate. Data are
representative of 5 experiments.
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We also examined the ability of insulin to stimulate the formation of
D-3 phosphorylated phosphoinositides in vivo. As seen in Fig.
7, PtdIns(3)P, PtdIns(3,4)P, and
PtdIns(3,4,5)P increase with insulin treatment (100 nM; 1 min).
Wortmannin (25 nM) inhibited the insulin-stimulated formation of
PtdIns(3)P and PtdIns(3,4)P but not PtdIns(3,4,5)P. LY-294002 (50 µM)
inhibited production of all three products, preventing the formation of
any detectable PtdIns(3,4,5)P. Together, these results suggest that PI
3-kinase is present in A6 cells and can be stimulated by insulin.
Furthermore, the A6 cell PI 3-kinase is inhibited by LY-294002,
partially inhibited by wortmannin, and is critical for
insulin-stimulated sodium transport.

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Fig. 7.
In vivo PI 3-kinase activity. Serum-starved (16-h) A6 cells were
metabolically labeled with
32Pi
for 2 h, followed by 20 min with or without 25 nM wortmannin or 50 µM
LY-294002. Preliminary experiments determined that stimulation for 1 min with 100 nM insulin (data not shown) was sufficient for
production of detectable phosphatidylinositol 3,4,5-trisphosphate
[PtdIns(3,4,5)P]. After stimulation with insulin, cells
were harvested, and lipids were extracted and separated by TLC.
Phosphorylated PtdIns were extracted from TLC plates, deacylated, and
analyzed by anion-exchange HPLC.
[3H]PtdIns(4)P and
[3H]PtdIns(4,5)P were
used as internal standards (Std). Data are representative of 3 experiments.
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pp70 S6 kinase is one of the insulin-stimulated elements downstream of
PI 3-kinase necessary for several metabolic actions (7). To determine
whether pp70 S6 kinase is involved in insulin-stimulated sodium
transport, A6 cells were pretreated with rapamycin, an inhibitor of
pp70 S6 kinase. At all of the concentrations used, 10 nM, 50 nM (not
shown), or 100 nM (Fig.
8A),
insulin-stimulated sodium transport was not inhibited. An
IC50 of 0.5-1.0 nM has been
reported for this inhibitor in Chinese hamster ovary cells (26).
However, rapamycin did inhibit insulin-stimulated S6 kinase activity in
pp70 S6 kinase immunoprecipitations from the A6 cell (Fig.
8B), indicating that pp70 S6 kinase,
although present in A6 cells and inhibited by rapamycin, is not
involved in insulin-stimulated sodium transport.

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Fig. 8.
Effects of 100 nM rapamycin on insulin-stimulated sodium transport and
pp70 S6 kinase. A: A6 cells were
pretreated with 100 nM rapamycin bilaterally for 30 min. Insulin (20 nM) was added serosally at time 0.
After 120 min of insulin, 10 µM amiloride was added apically. Results
are means ± SE; n = 9. B: serum-starved A6 cells were treated
with or without 100 nM rapamycin for 30 min, followed by 5 min with or
without 100 nM insulin. Solubilized proteins were immunoprecipitated
with anti-pp70 S6 kinase. Immunoprecipitates were assayed for activity.
Results are expressed as multiples of control level, means ± SE;
n = 5. * P < 0.05 vs. control;
** P < 0.01 vs. rapamycin and
insulin.
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 |
DISCUSSION |
Previous work has demonstrated that insulin stimulates the
amiloride-sensitive epithelial sodium channel in models of the distal
nephron. The mechanism of how basolaterally presented insulin can
affect the channel in the apical membrane has not been elucidated. We
have previously demonstrated that insulin specifically binds the
basolateral membrane of amphibian cells and that the insulin receptor
is immunologically localized to the same membrane (5). In this report,
we demonstrate that an inhibitor of the IRTK, HNMPA-(AM)3, can inhibit
insulin-stimulated sodium transport (Fig. 1). However, it should be
noted that HNMPA-(AM)3 also has an
effect on basal transport, so we cannot exclude effects of the
inhibitor on tyrosine kinases other than the insulin receptor.
Insulin has pleiotropic effects mediated by several postreceptor
signaling pathways. A key enzyme activated by insulin is PI 3-kinase.
Activation of this enzyme is required for the insertion of the
insulin-sensitive glucose transporter (GLUT-4) into the plasma membrane
and for activation of pp70 S6 kinase, which is involved in protein
synthesis. We used a specific inhibitor of PI 3-kinase, LY-294002, to
demonstrate that insulin-stimulated sodium transport also requires
activation of this enzyme (Fig. 3). Because intracellular pathways are
stimulated before the manifestation of a final effect on transcellular
transport, we measured PI 3-kinase activity in the very early phase of
the insulin response. PI 3-kinase is present in A6 cells and can be
inhibited both in vitro (Fig. 6) and in vivo (Fig. 7) by LY-294002. The
inability of wortmannin to completely inhibit PI 3-kinase in these
cells (Fig. 2) may be characteristic of Xenopus
laevis; others have reported a PI 3-kinase with similar
sensitivities to LY-294002 and wortmannin in Xenopus
laevis oocytes (29). Although the initial effects of
LY-294002 and wortmannin were similar, the wortmannin effect was not
sustained over time and was less complete than that of LY-294002. Our
functional assay provides a unique opportunity to monitor the inhibitor
effect continuously over long time courses and may, therefore, detect
interactions or effects that are not easily assessed in other systems.
In Chinese hamster ovary cells, activation of PI 3-kinase is necessary
for pp70 S6 kinase activation and subsequent protein synthesis (26).
Rapamycin, an immunosuppressant and inhibitor of pp70 S6 kinase, was
used to reveal that pp70 S6 kinase is not involved in
insulin-stimulated sodium transport (Fig.
8A). Although the pp70 S6 kinase is
present in A6 cells, is activated by insulin, and can be inhibited by
rapamycin (Fig. 8B), it does not
appear to be involved in the natriferic effect of insulin.
Insulin-activated PI 3-kinase is necessary for the translocation of
GLUT-4 from a cytosolic pool of vesicles to the plasma membrane of
adipocytes and skeletal muscle. Although the steps downstream of PI
3-kinase are not known, it is thought that the activation of PI
3-kinase brings the enzyme to its substrates (PtdIns) in the membrane
and that the lipids are the mediators of insulin signaling (17). We
would suggest that this pathway may also be utilized for
insulin-stimulated sodium transport.
Several groups have demonstrated that insulin induces an increase in
the number of active sodium channels in the apical membrane. Preliminary reports indicate that insulin increases channel density in
the apical membranes of A6 cells (2, 4). Published experiments demonstrate that insulin increases the apical membrane area and the
number of open sodium channels in the apical membrane (11). These
results suggest that insulin induces the exocytotic insertion of sodium
channels into the apical membrane. This hypothesis is supported by
experiments with brefeldin A, an inhibitor of exocytosis, which also
inhibits insulin-stimulated sodium transport (9). Conversely,
patch-clamp experiments demonstrated an insulin-stimulated increase in
Po, the sodium
channel. These results could not be confirmed by using noise analysis
(4). The reasons for this discrepancy are not obvious, and we can only
suggest that blocker-induced noise analysis is a less invasive
procedure that will not perturb potential signaling pathways, such as
those involving the cellular cytoskeleton.
There is precedence for transporters to be translocated in response to
stimuli. In addition to GLUT-4, water channels (aquaporin) are inserted
into the apical membrane of amphibian skin and urinary bladder and
mammalian kidney epithelia in response to antidiuretic hormone (16); in
stimulated gastric parietal cells,
H+-K+-ATPase
is redistributed to the apical surface (15); and the number of
H+ ATPases found in turtle bladder
apical membrane increases in response to carbon dioxide (6). Thus the
hypothesis that sodium channels are exocytotically inserted into the
apical membrane in response to insulin is reasonable.
PI 3-kinase has recently been shown to mediate epidermal growth factor
(EGF) stimulation of intestinal NaCl absorption and Na+/H+
exchange (18). The ion transport occurs at the ileal brush-border membrane; the EGF receptor is located at the basolateral membrane. The
authors suggest that PI 3-kinase may be directly associating with
Na+/H+
exchangers, because the SH3 domain of the PI 3-kinase 85-kDa subunit
could interact with proline-rich sequences in the exchanger. Similarly,
the
-subunit of the sodium channel also contains proline-rich regions (28) that may directly interact with PI 3-kinase in A6 cells.
These are suggestive of interactions within the
proline-rich region of effector proteins. However, it is also possible
that the lipid products of PI 3-kinase can directly interact with
effectors and regulate activity. In this regard, it is unclear whether
PI(3,4)P2 or
PI(3,4,5)P3
(PIP3) would be the crucial
product for signal transduction in this system. Both molecules have
been shown to regulate various signaling systems (30). However, it is
interesting to note that LY-294002 inhibits
PIP3 production and (subsequently) sodium transport, whereas wortmannin has no effect on
PIP3 production and little
sustained effect on sodium transport in these cells. However, more
experiments need to be performed before it is possible to state that
PIP3 is the crucial product for
signaling in this epithelium or whether the PI 3-kinase may have a more
direct action on the channel components.
In conclusion, we demonstrate for the first time that PI 3-kinase is
necessary for insulin-stimulated sodium transport. We suggest that
insulin may induce the insertion of sodium channels into the apical
membrane of A6 cells in a manner analogous to insulin-stimulated
translocation of GLUT-4.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Simon Rhodes and Judy Boyd-White for critically
reviewing this manuscript and Dr. Ray Russo for help with the computer
graphics.
 |
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).
Address for reprint requests: B. L. Blazer-Yost, Biology Dept.,
I.U.P.U.I., 723 W. Michigan St., Indianapolis, IN 46202.
Received 15 July 1997; accepted in final form 8 January 1998.
 |
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