Deparments of Medicine and Physiology and Biophysics, University of Texas Medical Branch, Galveston, Texas 77555
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ABSTRACT |
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The signal transduction mechanisms that mediate
osmotic regulation of Na+/H+ exchange are not
understood. Recently we demonstrated that hyposmolality increases
HCO3 absorption in the renal medullary thick
ascending limb (MTAL) through stimulation of the apical membrane
Na+/H+ exchanger NHE3. To investigate the
mechanism of this stimulation, MTALs from rats were isolated and
perfused in vitro with 25 mM HCO3
-containing
solutions. The phosphatidylinositol 3-kinase (PI 3-K) inhibitors
wortmannin (100 nM) and LY-294002 (20 µM) blocked completely the
stimulation of HCO3
absorption by hyposmolality. In
tissue strips dissected from the inner stripe of the outer medulla, the
region of the kidney highly enriched in MTALs, hyposmolality increased
PI 3-K activity 2.2-fold. Wortmannin blocked the hyposmolality-induced
PI 3-K activation. Further studies examined the interaction between
hyposmolality and vasopressin, which inhibits HCO3
absorption in the MTAL via cAMP and often is involved in the development of plasma hyposmolality in clinical disorders. Pretreatment with arginine vasopressin, forskolin, or 8-bromo-cAMP abolished hyposmotic stimulation of HCO3
absorption, due to an
effect of cAMP to inhibit hyposmolality- induced activation of PI 3-K.
In contrast to their effects to block stimulation by hyposmolality, PI
3-K inhibitors and vasopressin have no effect on inhibition of apical
Na+/H+ exchange (NHE3) and
HCO3
absorption by hyperosmolality. These results
indicate that hyposmolality increases NHE3 activity and
HCO3
absorption in the MTAL through activation of a
PI 3-K-dependent pathway that is inhibited by vasopressin and cAMP.
Hyposmotic stimulation and hyperosmotic inhibition of NHE3 are mediated
through different signal transduction mechanisms.
signal transduction; adenosine 3',5'-cyclic monophosphate; phosphatidylinositol 3-kinase; hyperosmolality
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INTRODUCTION |
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NA+/h+ exchangers are present in the plasma membrane of virtually all mammalian cells and participate in a variety of vital cell functions (20, 50, 57). At least five mammalian exchanger isoforms (NHE1-5) have been identified (38, 50). These isoforms differ in their tissue distribution, kinetic properties, and responses to physiological stimuli (38, 50, 57). NHE1 is expressed ubiquitously in nonpolar cells and on the basolateral membrane of epithelial cells, where it is involved in the regulation of cell volume and intracellular pH (20, 50, 57). NHE2, NHE3, and NHE4 exhibit a more restricted tissue distribution, with preferential localization in the kidney and gastrointestinal tract (38, 50, 57). NHE3 is expressed in the apical membrane of certain renal tubule and intestinal epithelial cells, where it mediates the transepithelial absorption of NaCl and NaHCO3 (2, 5, 15, 32, 38, 50, 54, 55). The activity of Na+/H+ exchangers is markedly influenced by changes in the extracellular osmolality. Hyperosmolality differentially regulates exchanger isoforms: it stimulates NHE1, NHE2, and NHE4 but inhibits NHE3 (7, 15, 22, 35, 44, 45, 52). Hyposmolality has been shown to inhibit NHE1 in several systems (19, 22, 30); however, the physiological responses of other exchanger isoforms to hyposmotic stress are poorly defined. In a study of epithelial isoforms expressed in Chinese hamster ovary (AP-1) cells, hyposmolality inhibited NHE2 but had no effect on NHE3 (22).
Transepithelial absorption of HCO3 by the
medullary thick ascending limb (MTAL) of the mammalian kidney is
mediated via apical membrane Na+/H+ exchange
(14, 18, 54). Evidence from immunocytochemical, pharmacological, and functional studies indicates that this apical exchange activity is mediated by NHE3 (2, 5, 15, 18, 28, 52,
54). Recently we demonstrated that peritubular hyposmolality increases HCO3
absorption in the MTAL through
stimulation of apical membrane Na+/H+ exchange
(54). These studies provided the first evidence that NHE3
is regulated by hyposmotic stress. We also found that hyposmolality stimulates apical Na+/H+ exchange activity
through an increase in maximal velocity
(Vmax) (54), whereas
hyperosmolality inhibits the apical exchanger by decreasing its
apparent affinity for intracellular H+ (52).
These different kinetic mechanisms suggest that the opposing effects of
hyposmolality and hyperosmolality on NHE3 activity in the MTAL may be
mediated through different signaling pathways. At present, however, the
signaling mechanisms involved in osmotic regulation of
Na+/H+ exchange activity are largely unknown.
Phosphatidylinositol 3-kinase (PI 3-K) phosphorylates the 3-position of the inositol ring of phosphatidylinositides, resulting in the production of lipid second messengers that regulate a variety of cellular processes, including proliferation and survival, glucose transport and metabolism, and cytoskeletal organization (40, 48). Recent studies indicate that PI 3-K is activated by hyposmotic cell swelling and plays a role in mediating swelling-induced stimulation of glycogen synthesis in hepatocytes and skeletal muscle cells (25, 29, 47). In addition, PI 3-K was found to play a role in the stimulation of apical membrane Na+/H+ exchange activity by epidermal growth factor in intestinal epithelial cells (23) and to increase plasma membrane NHE3 activity in transfected AP-1 cells (27). These findings suggest that PI 3-K could be a component of the signaling pathway that mediates hyposmotic stimulation of apical Na+/H+ exchange activity in the MTAL. At present, however, the role of PI 3-K in osmotic regulation of Na+/H+ exchange has not been defined. Also, whether PI 3-K activity is osmotically regulated in renal tubules is not known.
In a variety of clinical disorders, the development of plasma
hyposmolality involves renal water retention mediated through elevated
levels of vasopressin (4). We have demonstrated previously that arginine vasopressin (AVP) inhibits HCO3
absorption in the MTAL via cAMP (13), an effect opposite
to the stimulation of HCO3
absorption by
hyposmolality (54). These findings raise the possibility
that AVP and hyposmolality could function in a negative feedback
system, in which inhibition of HCO3
absorption by AVP
is opposed by stimulation of HCO3
absorption by a
decrease in osmolality. The feasibility of such a counterregulatory
mechanism is supported by our previous observation that AVP and
hyperosmolality regulate MTAL HCO3
absorption through
independent pathways (15). However, despite the close
association between hyposmolality and increased vasopressin levels in
many clinical states, the interacting effects of hyposmolality and
vasopressin in the regulation of ion transport and signaling pathways
in renal tubules have not been investigated.
The aims of the present study were 1) to identify signaling pathways
involved in the hyposmotic stimulation of apical membrane Na+/H+ exchange and HCO3
absorption in the rat MTAL and 2) to examine the interacting effects of
hyposmolality and AVP in the regulation of HCO3
absorption. The results demonstrate that the effect of hyposmolality to
increase HCO3
absorption through stimulation of
apical membrane Na+/H+ exchange is mediated
through activation of PI 3-K. Vasopressin blocks the hyposmotic
stimulation of HCO3
absorption by increasing cAMP,
which inhibits the hyposmolality-induced stimulation of PI 3-K
activity. We also show that PI 3-K is not involved in the inhibition of
HCO3
absorption by hyperosmolality, indicating that
hyposmolality and hyperosmolality regulate apical
Na+/H+ exchange (NHE3) activity in the MTAL
through different signaling pathways.
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METHODS |
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Tubule Perfusion and Measurement of Net
HCO3 Absorption
The protocol for study of transepithelial HCO3
absorption was as described (13, 15, 54). The tubules were
equilibrated for 20-30 min at 37°C in the initial perfusion and
bath solutions, and the luminal flow rate (normalized per unit tubule
length) was adjusted to 1.5-2.0
nl · min
1 · mm
1. Two or
three 10-min tubule fluid samples were then collected for each period
(initial, experimental, and recovery). The tubules were allowed to
reequilibrate for 5-15 min after a change in the composition of
the lumen and/or bath solutions. The absolute rate of
HCO3
absorption (JHCO3
,
pmol · min
1 · mm
1) was
calculated from the luminal flow rate and the difference between total
CO2 concentrations measured in perfused and collected fluids (13). An average HCO3
absorption
rate was calculated for each period studied in a given tubule. When
repeat measurements were made at the beginning and end of an experiment
(initial and recovery periods), the values were averaged. Single tubule
values are presented in the figures. Mean values ± SE
(n = number of tubules) are reported in the text. In
separate experiments, epithelial cell volume was determined from
measurements of inner and outer tubule diameters as previously described (15, 52). The protocol and conditions for the
cell volume experiments were virtually identical to those used in the HCO3
transport experiments.
Determination of PI 3-K Activity
Tissue preparation.
The tissue preparation used to study PI 3-K activity has been described
previously (3, 51). In brief, rats were anesthetized with
pentobarbital sodium (50 mg/kg ip) and both kidneys were removed and
sliced in ice-cold control solution. The inner stripe of the outer
medulla was cut from the slices and dissected into thin strips of
tissue as described (3, 51). The strips were divided into
four samples of equal amount and then incubated in vitro in the same
solutions used for HCO3 transport experiments. The
tissue samples were equilibrated at 37°C for 1 h in control
solution and then either maintained in control solution or incubated in
hyposmotic solution (50 mM mannitol removed) for an additional 15 min.
Identical samples were run in individual experiments with either 100 nM
wortmannin or 10
4 M 8-bromo-cAMP (8-BrcAMP) in control
and hyposmotic solutions. These protocols were chosen to reproduce
those used in the HCO3
transport experiments. The
tissue samples were bubbled continuously with 95% O2-5%
CO2 throughout the incubation for mixing and to maintain
the oxygen tension and pH of the solutions. After incubation, the
tissue samples were suspended immediately in ice-cold Triton lysis
buffer (20 mM Tris, pH 7.4, 137 mM NaCl, 2 mM EDTA, 1% Triton X-100,
25 mM
-glycerophosphate, 1 mM sodium orthovanadate, 2 mM sodium
pyrophosphate, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, and
10 mg/ml leupeptin), homogenized using a Dounce pestle, and lysed for
2.5 h at 4°C on an orbital shaker. The cell lysates were then
centrifuged at 3,600 g for 5 min, and the supernatants were
isolated and assayed for protein concentration (Micro BCA kit, Pierce,
Rockford, IL). The inner stripe of the outer medulla is highly enriched
in MTALs, which comprise the majority of the protein mass and cellular
volume of this region (3, 24). We demonstrated previously
that changes in protein kinase activities measured in the inner stripe
reproduce accurately changes measured in the MTAL (3, 51,
53).
Immunoprecipitation and PI 3-K assay.
PI 3-K activity was determined in a lipid kinase assay with
phosphatidylinositol as substrate, using previously described methods
(12, 43). Equal amounts of protein (200 µg) from
experimental samples were incubated for 2 h at 4°C with 10 µg
of monoclonal antibody against the p85 subunit of PI 3-K (Santa Cruz
Biotechnology, Santa Cruz, CA) and 20 µl of a protein A-agarose bead
suspension (Santa Cruz). The immunoprecipitates were washed three times
with lysis buffer and three times with 10 mM Tris · HCl, pH
7.4. To measure PI 3-K activity, the immune complexes were incubated
for 10 min at 4°C in 10 µl of 1 mg/ml sonicated
L-
-phosphatidylinositol (Avanti Polar Lipids, Alabaster,
AL) in 30 mM HEPES, pH 7.4. Forty microliters of kinase buffer (30 mM
HEPES, pH 7.4, 30 mM MgCl2, 200 µM adenosine, 50 µM
ATP, and 20 µCi [
-32P]ATP) were then added, and the
assays were carried out at room temperature for 15 min. The reactions
were stopped with 100 µl of 1N HCl, and the lipids were extracted by
the addition of 200 µl of chloroform-methanol (1:1). The organic
phase was collected, and 20-µl samples were spotted onto silica-gel
thin-layer chromatography (TLC) plates impregnated with 1% potassium
oxalate. The TLC solvent was chloroform-methanol-water-ammonium
hydroxide (18:14:3:1). The plates were dried, and phosphorylated
substrate was detected by autoradiography. Autoradiograms were
digitized, and substrate phosphorylation was quantified by densitometry
(ImageQuant; Molecular Dynamics, Sunnyvale, CA). There was no
detectable phosphorylation of substrate in negative control experiments
in which sample protein was omitted from the assay. Equal amounts of PI
3-K in immunoprecipitates were verified in parallel samples for all
protocols by immunoblotting with the same antibody used for immunoprecipitation.
Statistical Analysis
Results are presented as means ± SE. Differences between means were evaluated using the paired Student's t-test or ANOVA with Newman-Keuls multiple-range test, as appropriate. P < 0.05 was considered statistically significant. ![]() |
RESULTS |
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Hyposmolality Stimulates HCO3
Absorption
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Role of PI 3-K in Hyposmotic Stimulation of
HCO3 Absorption
Inhibitors of PI 3-K block stimulation of HCO3
absorption by hyposmolality.
PI 3-K has been implicated in signal transduction by hyposmolality in
several cell types (25, 29, 47). To determine whether PI
3-K is involved in hyposmotic stimulation of HCO3
absorption in the MTAL, we examined the effects of wortmannin and
LY-294002, two selective inhibitors of PI 3-K activity (37, 49,
56). The results in Fig. 2 show
that in tubules bathed with either 100 nM wortmannin or 20 µM
LY-294002, decreasing osmolality by the removal of 50 mM mannitol or 25 mM NaCl had no effect on HCO3
absorption (11.7 ± 1.1, inhibitors, vs. 11.6 ± 1.1 pmol · min
1 · mm
1,
inhibitors + hyposmolality, n = 6;
P = not significant). These results suggest that PI 3-K
plays an essential role in the stimulation of HCO3
absorption by hyposmolality.
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Hyposmolality stimulates PI 3-K activity.
To confirm that hyposmolality regulates PI 3-K activity in the MTAL, we
examined PI 3-K in the inner stripe of the outer medulla, the region of
the kidney highly enriched in MTALs (3, 24). Inner stripe
tissue was incubated in control or hyposmotic (50 mM mannitol removed)
solution for 15 min in the absence and presence of 100 nM wortmannin,
and then PI 3-K activity was determined in an immune complex assay
using phosphatidylinositol as substrate (see METHODS). As
shown in Fig. 3, hyposmolality increased
PI 3-K activity 2.2-fold (P < 0.05). This increase was
blocked by pretreatment with wortmannin. In control solution,
wortmannin decreased basal PI 3-K activity by 40% (cont vs. wort, Fig.
3). These results support the view that hyposmolality increases
HCO3 absorption through stimulation of PI 3-K
activity.
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Inhibitors of PI 3-K decrease basal HCO3
absorption.
The experiments in Fig. 3 show that the MTAL expresses constitutive PI
3-K activity that is inhibited by wortmannin. We therefore tested
whether this constitutive activity influences the basal rate of
HCO3
absorption. In tubules studied in control
(isosmotic) solution, addition of wortmannin or LY-294002 to the bath
decreased HCO3
absorption from 11.8 ± 0.8 to
10.0 ± 1.0 pmol · min
1 · mm
1
(n = 6; P < 0.001) (Fig.
4). This decrease was observed within 15 min after addition of the inhibitors and was stable for up to 60 min.
Thus PI 3-K activity is a determinant of the basal rate of
HCO3
absorption.
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Inhibitors of PI 3-K do not block inhibition of
HCO3 absorption by hyperosmolality.
In contrast to the stimulation by hyposmolality, hyperosmolality
inhibits HCO3
absorption in the MTAL through
inhibition of apical membrane Na+/H+ exchange
(15, 52). The results in Fig.
5 show that in tubules bathed with
wortmannin or LY-294002, increasing osmolality in the lumen and bath by
the addition of 50 mM mannitol or 75 mM NaCl decreased
HCO3
absorption by 46%, from 10.9 ± 1.0 to
5.8 ± 0.8 pmol · min
1 · mm
1
(n = 8; P < 0.001). This decrease is
similar to that observed previously in the absence of the inhibitors
(15, 51). Thus PI 3-K is not involved in the inhibition of
HCO3
absorption by hyperosmolality.
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Role of Protein Kinase C in Hyposmotic Stimulation of
HCO3 Absorption
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Interaction Between Hyposmolality and Vasopressin
AVP blocks stimulation of HCO3 absorption by
hyposmolality.
We showed previously that AVP inhibits HCO3
absorption in the MTAL (13); thus, in principle, the
stimulation of HCO3
absorption by hyposmolality could
counteract the inhibitory effect of AVP. To examine the interaction
between hyposmolality and AVP in the regulation of
HCO3
absorption, we tested the effect of
hyposmolality in the presence of AVP. The results in Fig.
7A show that in tubules bathed
with 10
10 M AVP, hyposmolality had no effect on
HCO3
absorption (6.3 ± 0.5 pmol · min
1 · mm
1, AVP vs.
6.4 ± 0.5 pmol · min
1 · mm
1, AVP + hyposmolality, n = 4 ; P = not
significant). Thus the stimulation of HCO3
absorption
by hyposmolality is inhibited by AVP.
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cAMP blocks stimulation of HCO3 absorption by
hyposmolality.
AVP inhibits HCO3
absorption in the MTAL by
increasing cAMP (13, 14). To determine whether cAMP
mediates the effect of AVP to block stimulation by hyposmolality,
tubules were bathed with forskolin or 8-BrcAMP, agents that induce
maximal cAMP-dependent inhibition of HCO3
absorption
(13). The results in Fig. 8
show that in the presence of either 10
6 M forskolin or
10
4 M 8-BrcAMP, hyposmolality had no effect on
HCO3
absorption (8.0 ± 0.7, agent vs. 8.0 ± 0.7, agent + hyposmolality, n = 8;
P = not significant). Thus agents that elevate cAMP
prevent stimulation by hyposmolality. These results support the view
that the effect of AVP to inhibit stimulation of HCO3
absorption by hyposmolality is mediated through cAMP.
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cAMP blocks stimulation of PI 3-K activity by hyposmolality.
To investigate the mechanism by which AVP and cAMP block hyposmotic
stimulation of HCO3 absorption, we examined the
effect of cAMP on the PI 3-K signaling pathway. Inner stripe tissue was
incubated in control or hyposmotic (50 mM mannitol removed) solution
for 15 min in the absence and presence of 10
4 M 8-BrcAMP,
and then PI 3-K activity was determined in an immune complex assay as
described in METHODS. As shown in Fig.
9, 8-BrcAMP blocked completely the
stimulation of PI 3-K activity by hyposmolality. 8-BrcAMP had no effect
on basal PI 3-K activity (control vs. 8-BrcAMP, Fig. 9). These results
support the conclusion that cAMP blocks hyposmotic stimulation of
HCO3
absorption by inhibiting the
hyposmolality-induced increase in PI 3-K activity.
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DISCUSSION |
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The molecular mechanisms that mediate osmotic regulation of
Na+/H+ exchange activity are not understood.
Recently we demonstrated that hyposmolality increases
HCO3 absorption in the MTAL through stimulation of
the apical membrane Na+/H+ exchanger NHE3
(54). The present study demonstrates that this stimulation
is mediated through activation of PI 3-K. We also show that the
hyposmotic stimulation of HCO3
absorption is blocked
by vasopressin and cAMP because of an effect of cAMP to inhibit
hyposmolality-induced stimulation of PI 3-K activity. In contrast,
inhibition of apical Na+/H+ exchange and
HCO3
absorption by hyperosmolality does not involve
PI 3-K and is not blocked by vasopressin (14, 15),
indicating that hyposmolality and hyperosmolality regulate NHE3
activity in the MTAL through different signaling mechanisms. PI 3-K
pathways are involved in the regulation of a number of cellular
processes, including growth and survival, oncogenic transformation,
vesicle trafficking, and glucose transport and metabolism (40,
48). Our studies establish a role for PI 3-K in the osmotic
regulation of Na+/H+ exchange activity and
identify an important interaction between the PI 3-K and cAMP signaling
pathways in the control of renal epithelial transport.
Hyposmolality Stimulates NHE3 and
HCO3 Absorption Through Activation of
PI 3-K
Recent studies indicate that NHE3 is present in intracellular vesicles
and that its acute regulation involves the redistribution of
transporters between the plasma membrane and one or more subapical vesicular compartments. In AP-1 cells expressing NHE3, NHE3 activity was localized both in the plasma membrane and in recycling endosomes (9). In human colon carcinoma (Caco-2) cells, inhibition
of NHE3 activity by PKC was mediated in part by subcellular relocation of NHE3 from the apical membrane to subapical cytoplasmic compartments (21). Evidence for regulation of NHE3 through protein
trafficking also has been presented in the renal proximal tubule.
Immunocytochemical studies using NHE-specific antibodies showed that
NHE3 was present in both the brush-border membrane and in subapical
cytoplasmic vesicles in rat proximal tubule (5).
Furthermore, the regulation of NHE3 activity in proximal tubule cells
by a variety of factors, including parathyroid hormone
(59), acute hypertension (58), metabolic
acidosis (6), and endothelin (39), involves
the redistribution of NHE3 protein between the apical membrane and intracellular vesicular compartments. Of particular relevance to the
present study, PI 3-K has been shown to be involved in vesicular
trafficking in both yeast and mammalian cells (40, 48) and
recent studies implicate PI 3-K in the regulation of NHE3 recycling in
transfected AP-1 cells (27). On the basis of these
findings and the results of our current and previous (54)
studies, we propose that hyposmolality stimulates
HCO3 absorption in the MTAL via the following
mechanism: hyposmotic cell swelling increases PI 3-K activity, which
leads to the redistribution of NHE3 from subapical intracellular
vesicles to the apical membrane. The recruitment of apical membrane
transporters results in the increased apical NHE3 activity that
mediates stimulation of HCO3
absorption. Our finding
that hyposmolality increases NHE3 activity in the MTAL through an
increase in Vmax is consistent with an increase
in the number of membrane transporters through trafficking (54). In previous studies in which NHE3 was
immunolocalized to the apical membrane of the MTAL (2, 5),
an intracellular pool of NHE3 was not evident; however, there are
reasons why NHE3 may not have been detected in subapical membrane
compartments in the MTAL in these studies, including a lack of
sufficient resolution and/or an inability of the antibodies to
recognize NHE3 in the intracellular vesicles. Further studies are
needed to define the contribution of PI 3-K-dependent trafficking to
hyposmotic stimulation of NHE3 activity in the MTAL.
PKC has been identified as a downstream target of PI 3-K in a number of
systems (40, 48) and recent studies indicate a role for
PKC in regulating NHE3 trafficking in Caco-2 cells (21). Moreover, we found that PKC can mediate stimulation of
HCO3 absorption in the MTAL under certain conditions
(16, 17). These findings suggested that PKC may be a
downstream mediator of the PI 3-K-dependent stimulation of NHE3 in the
MTAL. We found, however, that inhibitors of PKC that abolish
PKC-dependent stimulation of HCO3
absorption by other
agonists (3, 16, 17) did not prevent stimulation by
hyposmolality. Thus it is unlikely that PKC is involved in mediating
the hyposmotic stimulation of NHE3. We also found that PI 3-K
inhibitors (as well as AVP) blocked hyposmotic stimulation of
HCO3
absorption but did not prevent cell swelling.
This indicates that the mechanical stresses and/or structural changes
associated with cell swelling are not in themselves sufficient to cause
stimulation of NHE3 activity in the absence of a functioning PI 3-K
signaling pathway. Our studies do not address whether cell swelling is
necessary for PI 3-K activation. However, the observation in
hepatocytes that cell swelling induced by either hyposmotic medium or
increased amino acid uptake causes activation of PI 3-K
(25) suggests that increased cell volume is the signal
leading to kinase activation. Important goals for future work will be
to define the mechanism(s) involved in hyposmotic activation of PI 3-K
and the downstream effectors that mediate PI 3-K-dependent stimulation
of NHE3 activity in the MTAL.
There is precedent for osmotic regulation of PI 3-K in other systems.
In isolated hepatocytes and human intestine 407 cells, hyposmotic cell
swelling induced a two- to threefold increase in PI 3-K activity
(25, 47). Swelling-induced activation of PI 3-K has been
shown to play a role in mediating increased glycogen synthesis in liver
and skeletal muscle cells (25, 29) and in the regulation
of Cl channel activity and cell volume in intestine and
hepatoma cells (11, 47). On the other hand, hyperosmotic
stress decreased PI 3-K activity in a fibroblast cell line, leading to
a decrease in the activity of the PI 3-K/Akt survival pathway and an
increased susceptibility to agonist-induced apoptotic cell death
(60). The results of the present study
establish that PI 3-K is osmotically regulated in native renal
tubule epithelial cells and demonstrate a role for this pathway in the
control of MTAL ion transport. The identification of signaling pathways
regulated in response to osmotic stress is of particular physiological
relevance for cells of the renal medulla, which are routinely exposed
to large and rapid changes in extracellular osmolality due to the
normal function of the urinary concentrating mechanism
(31). In this context, the role of PI 3-K as a component
of signaling pathways that convey cell survival is noteworthy. Recent
work suggests that osmotic stress and changes in cell volume modify
cell growth and may be associated with the induction of programmed cell
death in several cell types, including renal inner medullary collecting duct cells (8, 26, 41, 60). It is conceivable, therefore, that the effect of decreasing osmolality to stimulate PI 3-K activity in the MTAL may serve the dual function of regulating transepithelial ion transport and inducing cell survival signals that preserve the
integrity of the MTAL epithelium in the constantly changing osmotic
environment of the renal medulla. Further work is needed to explore
this hypothesis.
Hyposmolality and Hyperosmolality Regulate NHE3 Activity Through Different Signaling Mechanisms
Hyperosmolality decreases HCO3Role of PI 3-K in Basal
HCO3 Absorption
Vasopressin and cAMP Inhibit Stimulation by Hyposmolality
Decreases in plasma osmolality can be both the primary cause of, and the secondary consequence of, a change in the plasma vasopressin level (4, 31). Thus identification of the interacting effects of hyposmolality and vasopressin at the cellular level is important for understanding the pathophysiology of H2O balance. In the MTAL, vasopressin inhibits HCO3Vasopressin inhibits the stimulation of HCO3
absorption by hyposmolality by increasing intracellular cAMP, which
inhibits the hyposmolality-induced stimulation of PI 3-K activity. This conclusion is based on the following observations: 1)
vasopressin increases cAMP production in the MTAL (34),
2) agents that increase cell cAMP reproduce the effect of
vasopressin to block hyposmotic stimulation of HCO3
absorption, 3) stimulation of HCO3
absorption by hyposmolality requires the activation of PI 3-K, and
4) increased cAMP blocks the hyposmolality-induced
stimulation of PI 3-K activity. These findings provide new evidence for
an interaction between the cAMP and PI 3-K pathways in the control of
renal epithelial transport and reveal a previously undescribed mechanism by which cAMP can influence Na+/H+
exchange activity, namely, through modulation of PI 3-K-dependent regulation. An effect of cAMP to inhibit agonist-induced activation of
PI 3-K has been reported previously in a number of nonepithelial systems (1, 33, 36, 42). The mechanism of this interaction has not been defined. Of relevance to the present study, cAMP was found
to inhibit insulin-induced stimulation of glucose transport in
adipocytes by inhibiting PI 3-K-mediated translocation of the glucose
transporter GLUT-4 to the plasma membrane (36). By
analogy, it is plausible that cAMP may inhibit hyposmolality-induced
stimulation of HCO3
absorption in the MTAL by
inhibiting PI 3-K-mediated translocation of NHE3 to the apical
membrane. The identification of the cAMP effect is important not only
to understand the interactions between signal transduction pathways
that control ion transport in the MTAL but also to provide insight into
a new mechanism by which vasopressin and cAMP can influence other
processes in renal cells that may be regulated through PI 3-K, such as
growth and survival and responses to osmotic stress. Further work is
needed to define the physiological roles of PI 3-K signaling pathways
in the regulation of ion transporters and other cellular processes in
the MTAL and to define the modulation of these processes by cAMP.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-38217.
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FOOTNOTES |
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Address for reprint requests and other correspondence: D. W. Good, 4.200 John Sealy Annex 0562, Univ. of Texas Medical Branch, 301 Univ. Boulevard, Galveston, TX 77555.
1
In a recent study using A6 cells, a toad
kidney-derived cell line, wortmannin blocked vasopressin stimulation of
protein kinase A activity and Na+ transport
(10). In contrast, in the MTAL we found no effect of
wortmannin on AVP-induced inhibition of HCO3
absorption, a cAMP-mediated process (Good, unpublished results). Thus
it is unlikely that PI 3-K is involved in mediating stimulation of cAMP
production and activation of protein kinase A by AVP in this nephron segment.
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. Section 1734 solely to indicate this fact.
Received 8 March 2000; accepted in final form 12 June 2000.
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