Arginine vasopressin stimulates phosphorylation of aquaporin-2
in rat renal tissue
Goro
Nishimoto1,
Marina
Zelenina1,
Dailin
Li1,
Masato
Yasui1,
Anita
Aperia1,
Søren
Nielsen2, and
Angus C.
Nairn3
1 Department of Woman and Child
Health, Karolinska Institute, St. Göran's Children's Hospital,
112 81 Stockholm, Sweden;
2 Department of Cell Biology,
Institute of Anatomy, University of Aarhus, DK-8000 Aarhus, Denmark;
and 3 Laboratory of Molecular and
Cellular Neuroscience, The Rockefeller University, New York, New
York 10021-6399
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ABSTRACT |
Aquaporin-2 (AQP2), the protein that mediates arginine
vasopressin (AVP)-regulated apical water transport in the renal
collecting duct, possesses a single consensus phosphorylation site for
cAMP-dependent protein kinase A (PKA) at
Ser256. The aim of this study was
to examine whether AVP, and other agents that increase cAMP levels,
could stimulate the phosphorylation of AQP2 in intact rat renal tissue.
Rat renal papillae were prelabeled with
32P and incubated with vehicle or
drugs, and then AQP2 was immunoprecipitated. Two polypeptides
corresponding to nonglycosylated (29 kDa) and glycosylated (35-48
kDa) AQP2 were identified by SDS-PAGE. AVP caused a time- and
dose-dependent increase in phosphorylation of both glycosylated and
nonglycosylated AQP2. The threshold dose for a significant increase in
phosphorylation was 10 pM, which corresponds to a physiological serum
concentration of AVP. Maximal phosphorylation was reached within 1 min
of AVP incubation. This effect on AQP2 phosphorylation was mimicked by
the vasopressin (V2) agonist,
1-desamino-[8-D-arginine]vasopressin
(DDAVP), or forskolin. Two-dimensional phosphopeptide mapping indicated
that AVP and forskolin stimulated the phosphorylation of the same site in AQP2. Immunoblot analysis using a phosphorylation state-specific antiserum revealed an increase in phosphorylation of
Ser256 after incubation of
papillae with AVP. The results indicate that AVP stimulates
phosphorylation of AQP2 at Ser256
via activation of PKA, supporting the idea that this is one of the
first steps leading to increased water permeability in collecting duct cells.
adenosine 3',5'-cyclic monophosphate; collecting duct
cells; protein kinase A; vasopressin receptor; water permeability
 |
INTRODUCTION |
ALL HEALTHY ANIMALS CAN tolerate severe losses
and/or depletion of water thanks to their capacity to
concentrate urine. This is accomplished by high water absorption in the
collecting duct mediated by an arginine vasopressin (AVP)-induced
increase in water permeability in the apical plasma membrane (3).
Discovery of the water channel proteins known as aquaporins (AQP) has
led to a better understanding of the extraordinary capacity of renal tubules to adsorb water (17, 22, 25, 32). Several members of the AQP family are expressed in the kidney (7, 8, 11, 12, 30). One
of these, aquaporin-2 (AQP2), is exclusively expressed in the renal
collecting duct and appears to play a major role in
vasopressin-regulated water transport across collecting duct cells (6,
7, 26, 27).
The precise mechanisms by which water permeability is regulated in the
renal collecting duct are not yet known. However, protein phosphorylation is thought to be of importance (5, 14, 18, 19). In the
collecting duct, AVP binds to adenylyl cyclase-coupled vasopressin
(V2) receptors, leading to
activation of cAMP-dependent protein kinase A (PKA) (9, 29). AQP2
contains a single consensus PKA phosphorylation site at
Ser256 (Arg-Arg-Gln-Ser) (6, 7).
Moreover, it has recently been reported that, in vitro and in model
cell lines, AQP2 is phosphorylated at this site by PKA and that this
may lead directly or indirectly to increased water permeability (5, 14,
15, 18, 19). We have examined the phosphorylation of AQP2
in intact rat renal tissue. The results obtained indicate that
phosphorylation of Ser256 of AQP2
is modulated by AVP or by other agents that activate the PKA pathway,
suggesting that water permeability may be regulated by this process in
collecting duct cells.
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MATERIALS AND METHODS |
Animals.
Male Sprague-Dawley rats weighing 200-250 g (B&K Universal,
Sollentuna, Sweden) were used. The rats were fed a standard rat chow
and had free access to normal drinking water. Rats were anesthetized with thiobutabarbital (8 mg/100 g body wt). Kidneys were rapidly removed and the papillae were excised immediately. All studies were
started between 9 and 10 a.m.
32P prelabeling.
The preweighted papillae were incubated at 30°C in 2 ml of Krebs
bicarbonate buffer containing (in mM) 124 NaCl, 4 KCl, 26 NaHCO3, 10 glucose, 1.5 MgSO4, 1.5 CaCl2, 0.25 KH2PO4,
and 1 sodium butyrate and oxygenated with 95%
O2-5%
CO2 (vol/vol). After 15 min, the
medium was replaced with the same amount of fresh buffer containing 0.5 mCi
[32P]orthophosphoric
acid (NEN Life Science Products, Boston, MA) and the tissue was
incubated for 60 min. The papillae were washed twice with 2 ml of fresh
buffer and incubated for an additional 1-10 min either with
vehicle or in the presence of
[Arg8]vasopressin
(AVP),
1-desamino-[8-D-arginine]vasopressin
(DDAVP), or forskolin plus 3-isobutyl-1-methylxanthine (IBMX)
(Sigma-Aldrich Sweden, Stockholm, Sweden). After drug treatment, buffer
was removed and tissue was rapidly frozen in dry ice and stored at
80°C.
Immunoprecipitation of AQP2.
Preweighted papillae were sonicated in 1 ml of lysis buffer (20 mM
Tris · HCl, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100,
and 0.2% BSA, pH 8.0) containing 1 mM EGTA, 50 mM NaF, and protease inhibitors (25 mM benzamidine, 100 µM phenylmethylsulfonyl fluoride, 5 µg/ml chymostatin, 5 µg/ml pepstatin A, 20 µg/ml leupeptin, and
20 µg/ml antipain). Peptides were from the Peptide Institute, Japan. For each time point, aliquots of the homogenates (2 µl) from both vehicle- and drug-treated samples were used for
determination of total 32P
incorporation into trichloroacetic acid-precipitated proteins. The
32P incorporation into the intact
renal papilla was constant among the samples (data not shown). Five
milligrams of preswollen Protein A-Sepharose CL-4B beads (Pharmacia
Biotech, Uppsala, Sweden) were added to each tube and the samples were
mixed for 20 h at 4°C. The sepharose beads and
nonspecifically adsorbed proteins were removed by centrifugation for 15 s at 10,000 g. The supernatant was
mixed for 90 min at 4°C with 8 µl of rabbit anti-serum LL126 (final dilution 1:400) raised against the final 22 amino acids of AQP2
(27). Samples were transferred to Eppendorf tubes containing 5 mg of
preswollen Protein A-Sepharose beads and incubated for 1 h at 4°C.
The beads were collected by centrifugation and washed once with 1 ml of
lysis buffer; once with 1 ml of a buffer containing 20 mM
Tris · HCl, 150 mM NaCl, 5 mM EDTA, 0.5% Triton
X-100, 0.1% SDS, and 0.2% BSA (pH 8.0); once with 1 ml of a buffer
containing 20 mM Tris · HCl, 500 mM NaCl, 0.5%
Triton X-100, and 0.2% BSA (pH 8.0); and once with 1 ml of a buffer
containing 50 mM Tris · HCl (pH 8.0). After the final
wash, the beads were resuspended in 25 µl of SDS-PAGE sample buffer
(50 mM Tris · HCl, 10% glycerol, 2% SDS, 10%
2-mercaptoethanol, and 0.01% bromphenol blue, pH 6.8), vortexed, and
centrifuged. The recovered proteins were separated by SDS-PAGE on 12%
acrylamide gels. Gels were dried, and
32P incorporation into AQP2 was
analyzed by autoradiography and by using a GS-250 Molecular Imager
(Bio-Rad Laboratories, Sundbyberg, Sweden).
Two-dimensional phosphopeptide mapping.
Gel pieces containing 32P-labeled
AQP2 were excised from dried gels, washed, and incubated with trypsin
as described (2). Aliquots were spotted on a thin-layer cellulose plate
(20 × 20 cm; in the middle and 4 cm from the bottom) and
initially separated by electrophoresis at pH 3.5 in 10% acetic
acid/1% pyridine until the dye front migrated 7 cm. Ascending
chromatography was performed in 1-butanol/acetic
acid/water/pyridine (15:3:12:10, vol/vol). Phosphopeptides were visualized by autoradiography.
Immunoblotting.
The excised rat papillae were incubated at 30°C in saline solution
containing (in mM) 137 NaCl, 5 KCl, 0.8 MgSO4, 0.33 Na2HPO4, 0.44 KH2PO4,
and 1 MgCl2, with constant
oxygenation with 95% O2-5% CO2 (vol/vol). After a 30-min
preincubation, 10
7 M AVP or
vehicle was added and the tissue was incubated for an additional 5 min,
then frozen in dry ice and stored at
80°C.
Both control and AVP-incubated papillae were homogenized in buffer
containing 0.3 M sucrose, 25 mM imidazole, and 1 mM EDTA, pH 7.2, containing protease inhibitors (5 µg/ml leupeptin and 1 mM
phenylmethylsulfonyl fluoride) and protein phosphatase inhibitors (25 mM NaF, 1 mM sodium orthovanadate, and
10
7 M okadaic acid). The
homogenate was suspended in Laemmli sample buffer, analyzed for protein
concentration by the Lowry method, and fractionated by SDS-PAGE (10 µg protein per lane). The proteins were transferred to Hybond-P
polyvinylidene difluoride (PVDF) membrane (Amersham Sweden) by
electroelution. A total membrane fraction from kidney inner medulla was
also prepared in the same manner as the papillae and used in some
immunoblots. Phosphorylated AQP2 was detected with an affinity-purified
(see below) phosphorylation state-specific rabbit antibody (AN83-2)
raised against a synthetic peptide corresponding to amino acids
253-262 of AQP2 with two amino acids changed to reduce
antigenicity, a glycine residue added at the NH2 terminus,
and a cysteine added at the COOH terminus (SRRQSVEHLSPC). This was
chemically phosphorylated at
Ser256 (prepared by The
Rockefeller University Biotechnology Facility). AQP2 phosphorylated at
Ser256 was visualized using an
ECL-Plus Western blotting analysis system (Amersham Sweden) and
autoradiography. The obtained films were subjected to densitometry
using NIH Image 1.57 software.
As a control for the total amount of AQP2 in each sample, the PVDF
membrane was washed with 62.5 mM Tris · HCl, pH 6.7, 2% SDS, and 100 mM 2-mercaptoethanol for 30 min at 50°C, then with 80 mM
Na2HPO4,
20 mM
NaH2PO4,
100 mM NaCl, and 0.1% Tween 20 (pH 7.5) for two 10-min washes at room
temperature to remove the
phospho-Ser256 antibodies, and
immunodetection was repeated using rabbit antiserum LL126 raised
against the COOH terminus of AQP2 (27). In some experiments, membranes were incubated first with LL126 antiserum, then,
after washing, with antiserum against phosphorylated
Ser256.
Double affinity purification of the
anti-phospho-AQP2 antibody.
To obtain antibody clones that exclusively recognize phosphorylated
AQP2, we subjected the antisera raised against the phospho-AQP2 peptide
to affinity purification using a two-step sequential
affinity-purification protocol. For this purpose, both the phospho-AQP2
peptide mentioned above and a non-phospho-AQP2 peptide (1, 27) were
used. The peptides (2 mg) were immobilized via covalent sulfhydryl
linkage to activated agarose beads in columns (Sulfolink
Immunobilization Kit No. 2; Pierce, Rockford, IL). As the first step of
purification, 1 ml of the antiserum (anti-phospho-AQP2 serum) was
applied to the column on which the non-phospho peptide was immobilized.
The 1 ml antiserum preparation was applied on the same column three times (to obtain maximal extraction of potential clones that may recognize nonphosphorylated AQP2 and therefore need to be removed). The
final eluate was then subjected to the second step of affinity purification. The preparation was applied to the column on which the
phospho-AQP2 peptide was immobilized and recycled several times (to
maximize extraction of antibody clones recognizing the phospho-AQP2
peptide and thus phosphorylated AQP2). Finally, the column was washed
extensively and the column-bound antibody was eluted at low pH (1).
This preparation was used for immunoblotting using the phospho-AQP2
peptide and the non-phospho-AQP2 peptide. Immunoblotting indicated that
the final preparation of the affinity-purified antibody exclusively
recognized the phospho-AQP2 peptide and did not cross-react with the
non-phospho-AQP2 peptide (see Fig. 5).
Statistics.
Values are presented as means ± SE. Comparisons between data were
made by unpaired t-test. Values of
P < 0.05 were considered significant.
 |
RESULTS |
The phosphorylation of AQP2 was initially analyzed in
32P-prelabeled rat renal papilla
by immunoprecipitation followed by SDS-PAGE electrophoresis and
autoradiography. Under control conditions, two phosphorylated
polypeptides were detected that corresponded, respectively, to the
nonglycosylated (29 kDa) and glycosylated (35-48 kDa) forms of
AQP2, indicating that there was basal phosphorylation of AQP2 in intact
rat renal tissue. The basal phosphorylation of AQP2 found in our
experiments may be attributed to the presence of circulating AVP in the
anesthetized rats or to the constitutive activation of adenylyl cyclase
in the kidney papilla preparation. Addition of either AVP
(10
7 M) or DDAVP
(10
7 M) increased
phosphorylation of both nonglycosylated and glycosylated forms of AQP2
(Fig. 1). In the presence of AVP, the
increase in phosphorylation was rapid, reaching a maximal level within
1 min and remaining elevated throughout the time course of the
incubation (Fig. 2). The effect of AVP was
dose-dependent (Fig. 3), with a significant
increase in phosphorylation being observed with 10 pM AVP compared with
vehicle. A significant increase in phosphorylation of AQP2 was also
observed after incubation with forskolin
(10
5 M) plus IBMX (5 × 10
4 M),
concentrations of which would increase cAMP levels (Fig. 4A).

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Fig. 1.
Effect of arginine vasopressin (AVP) and
1-desamino-[8-D-arginine]vasopressin
(DDAVP) on phosphorylation of aquaporin-2 (AQP2) in rat renal papillae.
32P-prelabeled rat renal papillae
were incubated with vehicle (lane
1), 10 7 M
AVP (lane 2), or
10 7 M DDAVP
(lane 3) for 5 min at 30°C. An
antibody specific for the 22 COOH-terminal amino acids of rat AQP2 was
used for immunoprecipitation. Samples were separated by SDS-PAGE and
analyzed by autoradiography. The 2 bands with apparent
Mr values of 29 kDa and 35-48 kDa correspond to nonglycosylated and glycosylated
AQP2, respectively.
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Fig. 2.
Time course of AVP-stimulated AQP2 phosphorylation. Tissue was
incubated with vehicle or
10 7 M AVP for various
periods of time and samples were processed as described in legend to
Fig. 1. 32P incorporation into
AQP2 was analyzed by autoradiography and quantitation was carried out
using a GS-250 Molecular Imager (Bio-Rad Laboratories). Data shown
represent only glycosylated form of AQP2. Relative increase in
32P incorporation was same for
nonglycosylated polypeptide. Data are expressed as percentage of
32P incorporated into AQP2 under
control conditions for each respective time point. Values are means ± SE of 4-6 determinations.
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Fig. 3.
AVP stimulates phosphorylation of AQP2 in a concentration-dependent
manner. Tissue was incubated with vehicle or various concentrations of
AVP for 5 min and samples were processed as described in legend to Fig.
1. For each concentration of AVP, data are expressed as percentage of
32P incorporated into AQP2 under
control conditions. Values are means ± SE of 3-6
determinations. At 10 pM AVP, 32P
incorporation was increased 1.63-fold compared with vehicle
(P < 0.05).
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Fig. 4.
AVP and forskolin increase phosphorylation of the same site in AQP2.
A:
32P-labeled papillae were
incubated with vehicle (lane 1) or
10 5 M forskolin plus 0.5 mM
3-isobutyl-1-methyxanthine (IBMX) (lane
2) for 5 min. Samples were processed as described in
legend to Fig. 1. B: two-dimensional
tryptic phosphopeptide maps of phosphorylated AQP2.
32P-labeled AQP2 samples, prepared
as shown in Fig. 4A, were excised from
dried gels, washed, and digested with trypsin. Tryptic digests were
separated by electrophoresis in first dimension and by chromatography
in second dimension. O, origin; positive electrode is on left, negative
electrode is on right. Phosphopeptides were visualized by
autoradiography. Results shown are representative of 2 independent
experiments. In other experiments, both peptides 1 and 2 were
identified, but peptide 1 was present at higher level than peptide 2. This supports idea that peptides 1 and 2 are derived from alternative
tryptic cleavage of a single phosphorylation site. A minor peptide
could be detected in maps from highly phosphorylated samples that ran
just below peptide 2. Level of this peptide varied in different samples
and is likely to be a derivative of peptide 1 or 2.
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Two-dimensional phosphopeptide mapping of
32P-labeled AQP2 was carried out
to further examine the basal and stimulated phosphorylation of the
protein. Under control conditions, four phosphorylated peptides were
detected (Fig. 4B, labeled 1-4).
In the presence of AVP, there was a pronounced increase in
phosphorylation of peptides 1 and 2, and an apparent decrease in
phosphorylation of peptides 3 and 4. An essentially identical result
was found for the AQP2 sample obtained from tissue incubated with forskolin.
To clarify the identity of the peptides phosphorylated, we prepared a
phosphorylation state-specific antibody that specifically recognized
the phosphorylated, but not the dephosphorylated, form of
Ser256 (Fig.
5). Absorption of the double
affinity-purified antibody with the immunizing phospho-peptide revealed
a complete absence of labeling (data not shown). Immunoblotting studies
of papillary tissue incubated under control conditions indicated that
there was a measurable level of
Ser256 phosphorylation in intact
normally hydrated rats. Incubation with AVP significantly increased the
phosphorylation of Ser256 in both
the glycosylated and nonglycosylated forms of AQP2. Under AVP
treatment, the level of phosphorylation was 218 ± 18%
(n = 9) compared with a corresponding
control (Fig.
6A). The
total amount of AQP2 was not altered by AVP treatment (Fig.
6B).

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Fig. 5.
Characterization of an antibody that specifically recognizes
phosphorylated form of Ser256 in
AQP2. Nonphosphorylated AQP2 peptide (residues 250-271; 50 ng in
lane 3 and 200 ng in
lane 4) or phosphorylated AQP2
peptide (P-AQP2; residues 253-262; 50 ng in lane
5 and 200 ng in lane
6) were separated by SDS-PAGE (12% acrylamide).
Peptides were transferred to nitrocellulose membrane, and the
phosphorylated form of AQP2 was detected using a phosphorylation
state-specific antibody raised against a synthetic peptide
corresponding to amino acids 253-262 of AQP2, which was chemically
phosphorylated at Ser256. Antibody
binding was detected by enhanced chemiluminescence. As a control,
membranes from kidney inner medulla, prepared in the presence of
protein phosphatase inhibitors (1 µg in lane
1 and 5 µg in lane
2), were also analyzed. The antibody labeled 2 bands
of 28 and 35-45 kDa corresponding to the nonglycosylated and
glycosylated forms, respectively, of basally phosphorylated
AQP2.
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Fig. 6.
Ser256 of AQP2 is phosphorylated
in kidney papilla. A: papillae
incubated with 10 7 M AVP
for 5 min (lanes 2, 4,
6) or control papillae from the same
rats (lanes 1, 3,
5) were homogenized and homogenates
were suspended in Laemmli sample buffer and separated by SDS-PAGE (10 µg protein per lane). Proteins were transferred to Hybond-P PVDF
membrane, and phosphorylated form of AQP2 was detected using
phosphorylation state-specific antibody. Antibody binding was detected
by enhanced chemiluminescence. Similar results were obtained in 3 independent experiments. B: reprobing
of same membrane using antiserum that recognized both phosphorylated
and nonphosphorylated forms of AQP2. Total amount of AQP2 was not
altered by AVP treatment.
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 |
DISCUSSION |
It is generally agreed that AVP exerts its antidiuretic effect by
acting on V2 receptors in the
collecting duct and that the target protein is AQP2. The effect of AVP
on AQP2 involves several steps. Under conditions of normal hydration,
AQP2-containing vesicles are located intracellularly (23, 26, 27).
After activation of V2 receptors,
the vesicles then fuse with the apical membrane, leading to a dramatic
increase in the membrane water permeability (26).
V2 receptors are coupled to the
adenylyl cyclase-cAMP-PKA pathway. AQP2 contains a single PKA consensus
phosphorylation site (6, 7), and phosphorylation of the water channel
has been suggested to play a role in AVP-dependent increases in water permeability in kidney collecting ducts (5, 14, 18, 19).
To date, studies of AQP2 phosphorylation have been carried out either
in vitro (18, 19) or in cells expressing recombinant AQP2 (5, 14, 18).
However, it was not known if the phosphorylation of AQP2 occurs in
intact renal tissue or if the phosphorylation of
Ser256 of AQP2 was regulated by
AVP. The present study demonstrates that
Ser256 of AQP2 is phosphorylated
in response to the actions of AVP acting as a first messenger in intact
rat renal tissue. DDAVP, the V2 receptor agonist, and forskolin increased phosphorylation of AQP2. The
two-dimensional phosphopeptide mapping studies indicated that AVP and
forskolin treatment led to phosphorylation of the same site, suggesting
that the effect of AVP on AQP2 phosphorylation was mediated via
adenylyl cyclase and activation of PKA. By use of a phosphorylation
state-specific antibody that specifically recognized the phosphorylated
form of Ser256, we demonstrated
that Ser256 was the major residue
phosphorylated under control conditions and in response to incubation
with AVP. Under basal conditions, AQP2 was phosphorylated to a low
level at sites distinct from Ser256. The presence of basal
phosphorylation of these sites leads to a slight underestimation of the
efficacy of the AVP action in the results shown in Figs. 1-3.
Moreover, in the immunoblotting study, where only
Ser256 phosphorylation was
detected, the calculated increase in AQP2 phosphorylation in response
to AVP was significantly higher than that obtained by
immunoprecipitation. The identity of the other phosphorylation site(s)
is not known. There are four other potential sites for phosphorylation
in AQP2, one for protein kinase C (PKC) and three for casein kinase II
(6, 7). Future studies will hopefully reveal whether the minor site(s)
phosphorylated under control conditions correspond to the PKC or casein
kinase II sites, and whether phosphorylation of these sites might
regulate AQP2.
The results obtained in this study demonstrate that the phosphorylation
of AQP2 is regulated in a physiologically relevant manner in rat renal
tissue. AQP2 is phosphorylated under basal conditions, but within 1 min
after addition of AVP, the level of AQP2 phosphorylation is
significantly increased. These results are consistent with data
obtained using perfused collecting ducts, in which water permeability
is altered within 1 min by AVP treatment (33). A significant increase
in phosphorylation of Ser256 of
AQP2 was observed at concentrations of
10
11-10
7
M AVP. The plasma concentration of AVP is about 2 pM in normal hydrated
rats, and increases up to 12 pM after 24-h dehydration (13, 34). Thus
the present results support the concept that phosphorylation of AQP2 is
one of the first steps leading to increased water permeability in the
collecting duct cells. Recent studies of
LLC-PK1 cells expressing
recombinant AQP2 (5, 14) have indicated that phosphorylation of AQP2 is
one of a series of regulatory processes involved in the exocytosis of
AQP2-containing vesicles (4, 16, 20, 28). The development of the
phosphorylation state-specific antibody will hopefully help in future
studies in defining the precise role of AVP-mediated phosphorylation of AQP2 in regulating its translocation and/or membrane insertion.
Several observations have demonstrated that sustained activation of
V2 receptors may lead to increased
AQP2 synthesis, suggesting that AVP may play a role in the long-term
regulation of AQP2 (1, 21, 22, 31). We recently reported that a single
injection of DDAVP into rats resulted in significant accumulation of
AQP2 mRNA within 6 h (35). It was also shown that AQP2 mRNA abundance is decreased in animals after treatment with a
V2-receptor antagonist (10). We
have demonstrated that human AQP2 gene promoter activity was increased
after V2 receptor stimulation and
activation of the PKA pathway in
LLC-PK1 cells (36). This effect
was mediated by cAMP-responsive element (CRE) and AP-1 consensus sites
present in the AQP2 promoter region. Vasopressin increased
CRE binding (CREB) protein phosphorylation and CREB-CRE binding, as
well as c-Fos mRNA and protein
expression and
c-Fos/c-Jun-AP-1
binding (36). Upregulation of CRE binding proteins was also
demonstrated in dehydrated rats (24). All of these
observations, together with data presented here, suggest that AVP
regulates AQP2 both on a short- and long-term basis and that cAMP plays
the central role in both processes. Such a dual effect of AVP would
guarantee a sustained increase in urinary concentrating capacity after
prolonged dehydration.
 |
ACKNOWLEDGEMENTS |
Expert technical assistance was provided by M. Vistisen and G. Christensen.
 |
FOOTNOTES |
This work was supported by grants from the Swedish Medical Research
Council (03644), the Swedish Heart-Lung Foundation, and the Märta
and Gunnar V. Philipson Foundation.
Address for reprint requests: A. C. Nairn, Laboratory of Molecular and
Cellular Neuroscience, Rockefeller Univ., New York, NY 10021-6399.
Received 22 December 1997; accepted in final form 9 October 1998.
 |
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