Vasopressin stimulates sodium transport in A6 cells via a
phosphatidylinositide 3-kinase-dependent pathway
R. S.
Edinger,
M. D.
Rokaw, and
J. P.
Johnson
Renal-Electrolyte Division, University of Pittsburgh, School of
Medicine, Pittsburgh, Pennsylvania 15213
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ABSTRACT |
The enzyme
phosphatidylinositide 3-kinase (PI3K) phosphorylates the D-3 position
of the inositol ring of inositol phospholipids and produces
3-phosphorylated inositides. These novel second messengers are thought
to mediate diverse cellular signaling functions. The fungal metabolite
wortmannin covalently binds to PI3K and selectively inhibits its
activity. The role of PI3K in basal and hormone-stimulated transepithelial sodium transport was examined using this specific inhibitor. Wortmannin, 50 nM, did not affect basal,
aldosterone-stimulated, or insulin-stimulated transport in A6 cells.
Wortmannin completely inhibits vasopressin stimulation of transport in
these cells. Vasopressin stimulates PI3K activity in A6 cells.
Vasopressin stimulation of transport is also blocked by 5 µM
LY-294002, a second inhibitor of PI3K. One-hour preincubation with
wortmannin blocked vasopressin stimulation of protein kinase A activity
in the cells. Sodium transport responses to exogenous cAMP and
forskolin, which directly activates adenylate cyclase, were not
affected by wortmannin. These results indicate that wortmannin inhibits vasopressin stimulation of Na+
transport at a site proximal to activation of adenylate cyclase. The
results suggest that PI3K may be involved in receptor activation by vasopressin.
wortmannin; protein kinase A; signal transduction
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INTRODUCTION |
PHOSPHATIDYLINOSITIDE 3-kinase (PI3K) is a member of
the family of phosphoinositide kinases that acts to catalyze the
phosphorylation of inositol phospholipids on the 3 position of the
inositol ring (2, 7, 25, 31). The resulting 3-phosphoinositides are biologically active lipids, which appear to play a broad and complex role in eukaryotic cell regulation at a number of sites. These lipids
have been observed to transduce both tyrosine kinase receptor signals
(2, 19) and G protein-coupled receptor signals (27, 28). They have been
implicated in the regulation of protein secretion (8, 33) and vesicle
trafficking (11, 14). These lipids have also been observed to
participate in regulation of the actin cytoskeleton (10, 12). Many of
these observations have been made with the use of the fungal metabolite
wortmannin, which specifically inhibits PI3K activity and has provided
a powerful and selective tool with which to study the role of PI3K in
diverse cellular functions (1).
Transepithelial sodium transport mediated by the amiloride-sensitive
Na+ channel is regulated by a
number of hormones, including aldosterone, insulin, and vasopressin (4,
9). Cellular mechanisms involved with regulating sodium transport
include methylation of the channel (23), activation of either protein
kinase A or C (PKA and PKC) (4, 21, 24, 32), activation of G proteins
(6, 18, 22), and activation of tyrosine kinases (9, 16, 17). Activation
of the Na+ channel may also be
dependent on the actin cytoskeleton (4, 5). Since PI3K potentially
interacts with many of these pathways, the role of this enzyme in
hormonally regulated sodium transport was investigated by use of the
PI3K inhibitor, wortmannin.
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METHODS |
A6 cells. All studies were performed
on A6 cells grown on semipermeable supports. Cells were grown as
described (22) in amphibian media (BioWhittaker, Walkersville, MD) with
10% fetal bovine serum (Sigma, St. Louis, MO) in an atmosphere of
humidified air-4% CO2 at
28°C. Cells were grown on Millicell-HA inserts (Millipore, Bedford,
MA). Transepithelial potential difference and short-circuit current
(Isc) were
measured using a sterile in-hood short-circuiting chamber as previously
described (22). During incubations, wortmannin or LY-294002 was added
to both sides of the semipermeable supports. Hormones (aldosterone,
insulin, vasopressin) were added to the basolateral medium.
Chemicals. Aldosterone, arginine
vasopressin, insulin, rapamycin, 8-bromo-cAMP, wortmannin, and
forskolin were purchased from Sigma, St. Louis, MO.
[
-32P]ATP was
purchased from ICN. Anti-PI3K p85 antibody was obtained from Upstate Biotechnology. LY-294002 was obtained from Calbiochem, San
Diego, CA. All other reagents were purchased from Sigma.
Protein kinase A activity. PKA
activity was measured using a colorimetric assay for kemptide
phosphorylation (Pierce). A6 cells were disrupted by
passage through a small-gauge needle in a buffer containing 100 mM
Tris · HCl, 1 mM EDTA, 1 mM dithiothreitol, 0.1 mM
Na3VO4,
0.5 µM okadaic acid, and protease inhibitors (pepstatin A, leupeptin,
antipain, and phenylmethylsulfonyl fluoride, each at 5 µg/ml) and
10% glycerol. The resulting suspension was subjected to centrifugation
for 1 h at 100,000 g at 4°C. The
resulting supernatant (crude cytosolic fraction) was protein matched,
and PKA activity was measured.
PI3K activity. PI3K activity was
measured on the immunoprecipitated enzyme as previously described (1).
A6 cells on filters were exposed to 100 mU/ml arginine vasopressin or
diluent for 10 min, and the cells were scraped from the filters. Cells
were suspended in 10 mM Tris · HCl, 1% Triton X-100,
150 mM NaCl, 0.5% Nonidet P-40, 1 mM EDTA, 0.2 mM
Na3VO4,
100 mM NaF, and protease inhibitors, pH 7.4. Cells were incubated in
Eppendorf tubes at 4°C for 30 min, vortexing every 10 min. The
samples were centrifuged at 14,000 g,
and the supernatant containing PI3K was transferred to a fresh tube.
PI3K was immunoprecipitated using the anti-PI3K antibody and GammaBind
beads for 4 h at 4°C with rotation. The beads containing the PI3K
subunit were then washed once in PBS, twice in 0.5 M LiCl, 0.1 M
Tris-Cl, pH 7.4, and once in phosphatidylinositide kinase buffer (10 mM
MgCl2, 20 mM HEPES, pH 7.4).
Beads were collected by centrifugation and resuspended in
40 µl of phosphatidylinositide kinase buffer containing 200 µg/ml
phosphatidylinositol/phosphatidylserine each. The reaction was
initiated with the addition of 10 µl of [
-32P]ATP (4,000 Ci/mmol). The samples were mixed every 2 min for 10 min at room
temperature, and the reaction was terminated by the addition of 40 µl
of 1 M HCl. Lipids were extracted from the organic phase after the
addition of 80 µl chloroform/methanol (1:1). Samples were spotted on
thin-layer chromatography plates and developed in
chloroform/methanol/water/NH3
(90:70:17:3). Radioactivity was quantified using Bio-Rad Molecular
Analysis software.
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RESULTS |
We first examined the effect of wortmannin on basal
Isc. When cells
were incubated with 50 nM wortmannin for 3 h, there was no effect on
Isc (not shown).
The effect of wortmannin on hormonally stimulated
Na+ transport was then examined.
Preincubation with wortmannin for 1 h and subsequent coincubation with
aldosterone had no effect on the
Isc response to
aldosterone (Fig. 1). Similarly, there was
no effect of wortmannin on the
Isc response to
insulin (Fig. 2). In contrast,
preincubation with 50 nM wortmannin completely inhibited the
Isc response to a
maximal concentration of vasopressin in these cells (Fig.
3). Similar results were obtained with
simultaneous addition of wortmannin and vasopressin (not shown). To
ensure that wortmannin was not acting nonspecifically, we employed a second inhibitor of PI3K, LY-294002 (29, 30). When LY-294002 was added
to A6 cells at a concentration of 5 µM (three times its
Ki of PI3K),
there was complete inhibition of vasopressin-stimulated Isc (Fig.
4).

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Fig. 1.
Wortmannin has no effect on aldosterone-stimulated short-circuit
current (Isc).
A6 cells were preincubated in wortmannin (50 nM) or diluent for 1 h.
Aldosterone (1 µM) was added at time
0, and
Isc was measured
sequentially; , aldosterone alone; , aldosterone + wortmannin.
Results are means ± SE; n = 6.
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Fig. 2.
Wortmannin has no effect on insulin-stimulated
Isc. Cells were
preincubated with wortmannin (50 nM) or diluent for 1 h, and then 100 mU/ml insulin was added to serosal solution.
Isc is shown from
time of addition of insulin (time
0); , insulin alone; , insulin + wortmannin;
n = 6.
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Fig. 3.
Wortmannin inhibits vasopressin-stimulated
Isc. A6 cells
were preincubated with wortmannin or diluent for 1 h. Arginine
vasopressin (100 mU/ml) was added to serosal solution; , vasopressin
alone; , vasopressin + wortmannin. Inhibition of vasopressin current
is significant at each time point; n = 6.
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Fig. 4.
LY-294002 inhibits vasopressin-stimulated
Isc. A6 cells
were incubated with vasopressin (100 mU/ml) or vasopressin and 5 µM
LY-294002, added simultaneously; , vasopressin alone; ,
vasopressin + LY-294002. Inhibition of vasopressin current is
significant at each time point; n = 6.
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Inhibition of vasopressin action by wortmannin suggested that this
hormone might act, in part, through stimulation of PI3K. To examine
this possibility, we measured the effect of vasopressin on PI3K
activity in A6 cells. PI3K was immunoprecipitated from A6 cells
following exposure to vasopressin (100 mU/ml for 10 min) and compared
with activity from control cells as described in METHODS. Phosphorylation of
phosphatidylinositol was quantified by autoradiography and
densitometry. Activity from control cells was 240 ± 10.9 U and from
cells exposed to vasopressin was 408 ± 10.8 U
(n = 3, P < 0.01). These results demonstrate
that vasopressin stimulated PI3K activity in A6 cells. The next
experiments sought to determine the possible site of wortmannin
inhibition of vasopressin-stimulated Na+ transport. Vasopressin acts to
increase apical Na+ channel number
and activity through activation of adenylate cyclase and subsequent
activation of PKA (16). Wortmannin (50 nM) has no effect on
unstimulated PKA activity in A6 cells (wortmannin/control PKA activity, 1.04 ± 0.08; n = 4, P = not significant). Figure 5 demonstrates that wortmannin inhibits
vasopressin activation of PKA activity in A6 cells. If PI3K is acting
to couple receptor activation by vasopressin to enzyme stimulation,
then agents that bypass these steps, such as cAMP or forskolin, might
still stimulate transport even in the presence of PI3K inhibition. We
therefore examined the effect of wortmannin on the transport response
to cAMP and forskolin in A6 cells. Figure 6
shows the effects of a permeable cAMP analog, 8-bromo-cAMP on
Isc in A6 cells.
Wortmannin does not inhibit the
Isc response to
this agent. Figure 7 demonstrates that
wortmannin does not inhibit the stimulation of
Isc mediated by
forskolin, an agent known to stimulate adenylate cyclase independent of
vasopressin receptor activation. There is an early (5 min) apparent
enhancement of forskolin-stimulated
Isc by
wortmannin, which is not sustained at 15 min.

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Fig. 5.
Wortmannin inhibits vasopressin-induced increase in protein kinase A
(PKA) activity. A6 cells were preincubated with wortmannin or diluent
for 1 h and exposed to vasopressin (100 mU/ml) or diluent for 1 h.
Cells were then scraped from filters, and PKA activity was measured as
described in METHODS. Units for PKA
activity are determined from a standard curve. Column
1, control PKA activity; column
2, PKA activity after vasopressin addition;
column 3, PKA activity following
vasopressin in presence of wortmannin;
n = 4.
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Fig. 6.
Wortmannin has no effect on 8-bromo-cAMP stimulation of
Isc. Following
1-h preincubation with ( ) or without ( ) wortmannin, 2 mM
8-bromo-cAMP was added to basolateral surface at time
60 min; n = 12.
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Fig. 7.
Wortmannin has no effect on forskolin-stimulated
Isc. Effect of 2 µM forskolin on
Isc in presence
( ) or absence ( ) of wortmannin (50 nM). Wortmannin or diluent was
present for 1 h prior to addition of forskolin;
n = 12.
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DISCUSSION |
PI3K has been implicated in signal transduction of a number of cellular
processes including G protein-coupled receptor signaling, activation of
the actin cytoskeleton, tyrosine kinase-activated receptor signals, and
secretion or targeting of proteins. Many of these processes have also
been implicated in activation of the amiloride-sensitive epithelial
Na+ channel by hormones.
Aldosterone stimulation of Na+
channel activity is dependent on transcription and translation of new
proteins and occurs in several phases. The early phase, from 1-4 h
involves activation of preexisting channels without significant
synthesis of new channels, whereas the late phase, from 4-12 h,
may involve transcription of new channel subunits as well as
basolateral membrane increase in
Na+-K+-ATPase
activity (4, 9, 13). The activation of channels occurs by one of
several proposed mechanisms including G protein activation (9, 13, 22),
channel subunit carboxylmethylation (23), or possibly phosphorylation
(26). Insulin activation of channel activity is dependent on tyrosine
kinase activity and appears to be primarily due to an increase in open
probability of existing channels (9, 16, 17), although some studies have suggested an increase in total number of active channels (3). Finally, vasopressin activation of channel activity
appears to be dependent on PKA activity, which appears to induce the
insertion of new channels into the apical membrane of responsive
epithelia (4, 9, 15). This study sought to examine the possible participation of PI3K in Na+
transport activation by use of the specific inhibitor of PI3K, wortmannin.
The results demonstrate that wortmannin has no effect on basal
transport or transport stimulated by either aldosterone or insulin. The
lack of an effect on insulin-stimulated
Na+ transport is somewhat
surprising, since insulin has been described as stimulating PI3K in
other systems and this has been linked to membrane insertion of glucose
transporters and stimulation of enzyme activity (8, 19). Although
insulin activity in A6 cells is dependent on tyrosine kinase activity
(17), subsequent PI3K activation does not appear to be involved in
Na+ channel activation by insulin
as judged by sensitivity to wortmannin. In contrast to the lack of
effect of wortmannin on basal transport or transport stimulated by
either aldosterone or insulin, transport stimulation by vasopressin is
completely inhibited by wortmannin and by a second inhibitor of PI3K,
LY-294002. This suggests that vasopressin might stimulate PI3K activity
in A6 cells, and current results indicate that it does. Moreover, the
PI3K inhibitor, wortmannin, blocks vasopressin stimulation of PKA. This
result suggests that vasopressin stimulation of PKA activity is
dependent on activation of PI3K as a signal transduction pathway
linking receptor activation to enzyme stimulation as has been seen in
other systems (27). Stimulation of transport by forskolin, which
activates adenylate cyclase, and exogenous cAMP are both unaffected by
wortmannin. It appears that channel activation or
insertion into the apical membrane is not dependent on
wortmannin-sensitive PI3K activity. These results indicate that
wortmannin inhibits vasopressin action in A6 cells at a site prior to
activation of adenylate cyclase and suggest that PI3K activity is
required for vasopressin receptor activation of that enzyme in these cells.
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FOOTNOTES |
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: J. P. Johnson, 935 Scaife Hall, 3550 Terrace St., Pittsburgh, PA 15213 (E-mail: johnson{at}med1.dept-med.pitt.edu).
Received 18 September 1998; accepted in final form 8 June 1999.
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