1 Department of Cell Biology, Institute of Anatomy, University of Aarhus, DK-8000 Aarhus, Denmark; and 2 Department of Woman and Child Health, Karolinska Institute, Astrid Lindgren Children's Hospital, 171 76 Stockholm, Sweden
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ABSTRACT |
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Phosphorylation of Ser256, in a PKA consensus site, in AQP2 (p-AQP2) appears to be critically involved in the vasopressin-induced trafficking of AQP2. In the present study, affinity-purified antibodies that selectively recognize AQP2 phosphorylated at Ser256 were developed. These antibodies were used to determine 1) the subcellular localization of p-AQP2 in rat kidney and 2) changes in distribution and/or levels of p-AQP2 in response to [desamino-Cys1,D-Arg8]vasopressin (DDAVP) treatment or V2-receptor blockade. Immunoelectron microscopy revealed that p-AQP2 was localized in both the apical plasma membrane and in intracellular vesicles of collecting duct principal cells. Treatment of rats with V2-receptor antagonist for 30 min resulted in almost complete disappearance of p-AQP2 labeling of the apical plasma membrane with only marginal labeling of intracellular vesicles remaining. Immunoblotting confirmed a marked decrease in p-AQP2 levels. In control Brattleboro rats (BB), lacking vasopressin secretion, p-AQP2 labeling was almost exclusively present in intracellular vesicles. Treatment of BB rats with DDAVP for 2 h induced a 10-fold increase in p-AQP2 labeling of the apical plasma membrane. The overall abundance of p-AQP2, however, was not increased, as determined both by immunoelectron microscopy and immunoblotting. Consistent with this, 2 h of DDAVP treatment of normal rats also resulted in unchanged p-AQP2 levels. Thus the results demonstrate that AQP2 phosphorylated in Ser256 is present in the apical plasma membrane and in intracellular vesicles and that both the intracellular distribution/trafficking, as well as the abundance of p-AQP2, are regulated via V2 receptors by altering phosphorylation and/or dephosphorylation of Ser256 in AQP2.
aquaporin-2; phosphorylation; vasopressin
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INTRODUCTION |
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VASOPRESSIN REGULATES collecting duct water reabsorption predominantly, if not exclusively, through regulated trafficking of aquaporin-2 (AQP2) from intracellular vesicles to the apical plasma membrane (35, 42, 48, 54). This acute response occurs within a few minutes and is mediated by binding of vasopressin to V2 receptors situated in the basolateral membrane of collecting duct principal cells. The synthesis of cAMP is subsequently stimulated via a V2-receptor-coupled G protein, which activates adenylate cyclase. Protein kinase A (PKA) is a multimeric protein which is activated by cAMP, and early studies have demonstrated that PKA induces phosphorylation of various membrane proteins in bovine kidney (10), and in saponin-permeabilized outer medullary collecting duct segments phosphorylation of at least two unidentified 45- and 66-kDa proteins have been demonstrated (17). Following PKA phosphorylation, intracellular vesicles containing AQP2 are transferred to the membrane possible by the involvement of dynein and dynactin, which have been shown to colocalize with AQP2-bearing vesicles in collecting duct principal cells (36). Ultimately, the vesicles fuse with the apical plasma membrane by exocytosis, which is speculated to be mediated by interaction of vesicle-targeting receptor proteins present in the vesicles (v-SNAREs) with vesicle-targeting proteins in the target membrane (t-SNAREs), e.g., VAMP-2, syntaxin-4, and SNAP-23 (20, 21, 32, 44).
The exact role of phosphorylation of AQP2 in the trafficking of AQP2 is presently not known. AQP2 contains a consensus site (Arg-Arg-Gln-Ser) for PKA phosphorylation in the cytoplasmic COOH terminus at serine-256. Recent studies have shown a very rapid phosphorylation of this serine (within 1 min) in response to vasopressin treatment of slices of kidney papilla (46). This is in agreement with the time course of vasopressin-stimulated water permeability of kidney collecting ducts (27, 53).
The water permeability of the plant water channels PM28A and
-TIP appears to be regulated by in situ phosphorylation
presumably by altering the conductance (22, 38). However, PKA
phosphorylation of AQP2 does not seem to have any significant effect on
the osmotic water permeability. In Xenopus oocytes,
cAMP-induced PKA phosphorylation of AQP2 only caused a small increase
in water permeability (28), and in AQP2-containing vesicles purified
from kidney inner medulla no increase in water permeability was seen
upon PKA stimulation (30). PKA-induced phosphorylation of AQP2 may
instead be required to modulate the distribution of AQP2 between plasma
membrane and intracellular vesicle compartments as recently indicated.
Both cAMP (14) and vasopressin or forskolin treatment (25) failed to
induce translocation of AQP2 in LLC-PK1 cells when
Ser256 was substituted by an alanine in contrast to normal
trafficking of wild-type AQP2, highlighting the importance of the PKA
consensus site for regulated and/or constitutive trafficking.
To investigate the role of phosphorylation of AQP2 in Ser256, we have used antibodies that selectively recognize AQP2 phosphorylated in this PKA consensus site and antibodies that recognize both phosphorylated and nonphosphorylated AQP2. The antibodies were used to determine 1) the subcellular localization of phosphorylated AQP2 in normal rats and in vasopressin-deficient Brattleboro rats, 2) the effect of V2-receptor antagonist treatment on phosphorylated AQP2 protein abundance and distribution in normal rats, and 3) the effect of DDAVP treatment on the abundance and distribution of phosphorylated AQP2 in Brattleboro rat kidneys.
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MATERIALS AND METHODS |
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Experimental Animals
Wistar rats were obtained from Møllegaard Breeding Center (Ejby, Denmark), and homozygous Brattleboro rats were from Harlan Nederland (Horst, The Netherlands). During experiments rats, were placed in metabolic cages with free access to standard rat diet and water. The following protocols were used.Experimental Protocols
Protocol 1. Normal rats were treated intravenously with V2-receptor antagonist 5-dimethylamino-1-(4-(2-methylbenzoylamino)benzoyl)-2,3,4,5-tetrahydro-1H-benzazepine (OPC-31260; Otsuka, Tokyo, Japan) through the femoral vein (1 mg in 0.2 ml vehicle/animal, n = 4 rats) (7). Control rats received intravenous saline (n = 4 rats). During the procedure, the animals were briefly anesthetized with isofluran. After 30 min of the injection, anesthesia was repeated and kidneys were either removed for preparation of membrane fractions or perfusion fixed for immunocytochemistry.Protocol 2. Normal rats were treated intravenously with OPC-31260 (1 mg in 0.2 ml vehicle/animal, n = 4 rats) (7). Control rats received intravenous saline (n = 4 rats). During the procedure, the animals were briefly anesthetized with isofluran. After 60 min of the injection, anesthesia was repeated and kidneys were removed for preparation of membrane fractions.
Protocol 3. Brattleboro rats were treated subcutaneously with DDAVP ([desamino-Cys1,D-Arg8]vasopressin, 50-1,000 ng in 500 µl saline/animal, n = 8 rats; Sigma-Aldrich, Vallensbaek Strand, Denmark). Control rats received subcutaneous saline (n = 7 rats). After 2 h of the injection, anesthesia was repeated and kidneys were either removed for preparation of membrane fractions (4 control rats and 4 DDAVP-treated rats) or perfusion fixed for immunocytochemistry (3 control rats and 4 DDAVP-treated rats).
Protocol 4. Normal rats were treated subcutaneously with DDAVP (500 ng in 500 µl saline/animal, n = 4 rats). Control rats received subcutaneous saline (n = 3 rats). After 2 h of the injection, anesthesia was repeated and kidneys were removed for preparation of membrane fractions.
Antibodies
For preparation of anti-phosphorylated AQP2 antibodies (anti-p-AQP2), two peptides corresponding to amino acids 253-262 of rat AQP2 were synthesized with addition of a cysteine residue at the carboxyl terminus and a glycine residue at the amino terminus (peptides 3 and 4, Table 1). Both peptides were phosphorylated at Ser256 in the PKA phosphorylation site (Arg-Arg-Gln-Ser). In one of the peptides, amino acids 259 (leucine) and 260 (histidine) were switched to reduce the antigenicity outside the PKA site (peptide 4, Table 1); this antibody has been described previously (46). The peptides were used to immunize rabbits. To remove potential clones recognizing nonphosphorylated AQP2, 1 ml of antiserum was applied three times to a column to which a nonphosphorylated peptide (amino acids 250-271 of rat AQP2 with a cysteine residue at the amino terminus, peptide 1, Table 1) was conjugated. The anti-AQP2 antibody-depleted serum was then applied to a second column containing the phosphorylated AQP2 peptide for affinity purification. The specificity of the affinity-purified anti-p-AQP2 antibodies was ensured by immunoblotting of membrane fractions from kidney inner medulla, the nonphosphorylated peptide, and the phosphorylated peptides.
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Affinity purification of anti-AQP2 antibody (LL127) (raised against a
peptide corresponding to amino acids 250-271 of rat AQP2,
peptide 1; Table 1) was performed as previously described (43).
Immunoblotting (Fig. 1) demonstrated that
this AQP2 antibody recognizes both nonphosphorylated and phosphorylated
AQP2 peptides (amino acids 250-271). There are no significant
changes in the affinity for the two peptides.
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Immunocytochemistry
Immunocytochemistry was performed essentially as previously described (7, 41, 42). Kidneys were perfusion fixed with either 1) 2% paraformaldehyde in PLP (0.01 M NaIO4, 0.075 M L-lysine, and 0.0375 M Na2HPO4, pH 6.2); or 2) 0.1% glutaraldehyde plus 2% paraformaldehyde in 0.1 M sodium cacodylate, pH 7.4; or 3) 0.2% glutaraldehyde plus 2% paraformaldehyde in 0.1 M sodium cacodylate, pH 7.4, via the abdominal aorta. Tissue blocks were prepared from kidney inner medulla or inner stripe outer medulla. The blocks were infiltrated with 2.3 M sucrose/2% paraformaldehyde for 30 min, mounted on holders, and rapidly frozen in liquid nitrogen. Frozen tissue blocks were either directly used for cryosectioning or subjected to a cryosubstitution and Lowicryl HM20 embedding prior to ultramicrotomy. Cryosubstitution was performed as described previously (37, 45). The frozen samples were freeze-substituted in a Reichert AFS (Reichert, Vienna, Austria). Samples were sequentially equilibrated over 3 days in 0.5% uranyl acetate in methanol at temperatures gradually increasing fromSemithin (0.85 µm) cryosections and ultrathin (80 nm) Lowicryl sections, cut on a Reichert Ultracut FCS, were preincubated with PBS containing 0.1% skimmed milk and 0.05 M glycine (for light microscopy) or with TBST (0.05 M Tris, pH 7.4, 0.1% Triton X-100) containing 0.1% sodium borohydride and 0.05 M glycine followed by incubation with TBST containing 0.2% skimmed milk (for electron microscopy). The preincubation was followed by incubation with affinity-purified anti-p-AQP2 or anti-AQP2 antibody. For light microscopy, labeling was visualized by use of peroxidase-conjugated secondary antibody (P448; Dako, Copenhagen, Denmark). For immunoelectron microscopy, the labeling was visualized with goat anti-rabbit IgG conjugated to 10-nm colloidal gold particles (GAR.EM10 or GAFR.EM10, 1:50; Biocell Research Laboratories, Cardiff, UK), and grids were stained with uranyl acetate for 10 min and with lead citrate for 5 s. Immunolabeling controls using anti-p-AQP2 preabsorbed with the phosphorylated peptide (peptides 3 and 4, respectively; Table 1) revealed a complete absence of labeling, whereas preabsorption of anti-p-AQP2 with the nonphosphorylated peptide (peptide 1, Table 1) did not ablate the labeling. Light microscopy was performed using a Leica Laborlux S microscope, and electron microscopy was performed using a Philips CM100 electron microscope.
Semi-Quantitation Of Phosphorylated AQP2 Immunogold Labeling
Electron micrographs were taken of the apical part of inner medullary collecting duct principal cells from Brattleboro rats treated with DDAVP (sc) for 2 h (n = 4) and control Brattleboro rats receiving saline treatment (sc) for 2 h (n = 3). Electron micrographs were printed at a final magnification of ×63,000. The total area of the cytoplasm was determined using a lattice square test system with a size of the squares of 30 mm × 30 mm. The total labeling density was calculated as the total number of gold particles per cytoplasm area. To assure that the immunogold quantitation was performed on comparable tissue sampling, the length of the apical plasma membrane and the area of the cell (excluding nuclei) were determined. The lengths of the apical plasma membrane as a fraction of the area of cytoplasm in untreated and DDAVP-treated Brattleboro rats were 0.21 ± 0.04 and 0.23 ± 0.01, respectively. In untreated and in OPC-31260-treated rats, the values were 0.17 ± 0.01 and 0.16 ± 0.01, respectively. This indicates equal sampling, i.e., same ratio of apical surface to cytoplasmic area for both set of animals. The length of the apical plasma membrane was determined by a manual tracing device, and the number of gold particles per length apical plasma membrane was calculated. The number of gold particles associated with apical plasma membranes, intracellular vesicles, and multivesicular bodies was determined. Gold particles in structures that could not be identified distinctly as vesicles or multivesicular bodies or apical plasma membrane were counted separately. This is likely to represent labeling of tangentially sectioned vesicles or parts of the rough endoplasmic reticulum. The total labeling density was also determined in the apical part of inner medullary collecting duct principal cells in normal rats treated with OPC-31260 for 30 min (iv) (n = 4) and corresponding control animals receiving saline treatment (iv) for 30 min (n = 4).Membrane Fractionation for Immunoblotting
The inner medulla was dissected from each kidney, minced finely, and homogenized in 2 ml of dissecting buffer (0.3 M sucrose, 25 mM imidazole, and 1 mM EDTA, pH 7.2, and containing the protease and phosphatase inhibitors 8.5 µM leupeptin, 1 mM phenylmethylsulfonyl fluoride, 100 nM okadaic acid, 1 mM sodium orthovanadate, and 25 mM sodium fluoride) (34, 35, 46). This homogenate was centrifuged in a Beckman L8M centrifuge at 4,000 g for 15 min at 4°C. The supernatant was centrifuged at 200,000 g for 1 h, and the resultant pellet, containing a mixture of plasma membranes and intracellular vesicles, was resuspended in dissecting buffer.Electrophoresis and Immunoblotting
Samples of membrane fractions from inner medulla were subjected to SDS-PAGE (29) using 12% polyacrylamide minigels (Bio-Rad Mini Protean II) and transferred to nitrocellulose paper by electroelution. Blots were blocked with 5% milk in PBS-T (80 mM Na2HPO4, 20 mM NaH2PO4, 100 mM NaCl, and 0.1% Tween 20, pH 7.5) for 1 h, and incubated overnight at 4°C with anti-p-AQP2 or anti-AQP2 antibody. After washing in PBS-T, the blots were incubated for 1 h with horseradish peroxidase-conjugated secondary antibody (P448; Dako). After final washing in PBS-T, p-AQP2 or total AQP2 was visualized using the ECL enhanced chemiluminescence system (Amersham International, Buckinghamshire, UK). Immunolabeling controls using preabsorbed anti-p-AQP2 antibody revealed no labeling (data not shown).Statistical Analysis
For densitometry of immunoblots, samples from OPC-31260- and DDAVP-treated animals were run on gels with corresponding samples from control animals. Films were scanned using a AGFA ARCUS II scanner and Adobe Photoshop Software. The scanning was performed using ECL exposures that gave bands in lower gray scale where there is a linear correlation between signal and protein levels (34). The labeling density was quantitated using specially written software (35). p-AQP2 labeling in the samples from the experimental animals was calculated as a fraction of the mean control value for that film. Both the 29-kDa and the 35- to 50-kDa band (corresponding to nonglycosylated and the glycosylated AQP2) were scanned (34, 42, 43). Values were corrected by densitometry of Coomassie-stained preliminary gels. Values are presented in the text as means ± SE. Comparisons between groups were made by unpaired t-test. P < 0.05 was considered significant. ![]() |
RESULTS |
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Selectivity of Anti-p-AQP2 Antibody
As shown in Fig. 1, the nonselective anti-AQP2 antibody (LL127) recognized both the phosphorylated and the nonphosphorylated peptides with similar efficiency. In contrast, the affinity-purified anti-phosphorylated AQP2 antibodies selectively recognized p-AQP2 as determined by immunoblotting. Figure 2 shows the selectivity of the antibody produced from peptide 3 (Table 1). In Fig. 2A immune serum was used, and in Fig. 2B a double affinity-purified preparation was used. Both antibody preparations recognized 29- and 35- to 50-kDa bands corresponding to nonglycosylated and glycosylated AQP2 (43) and selectively recognized the phosphorylated peptide corresponding to amino acids 253-262 of rat AQP2 but not the nonphosphorylated peptide corresponding to amino acids 250-271 of rat AQP2. The same results were obtained with the antibody produced from peptide 4 (Table 1) (46). Absorption controls using preabsorbed anti-p-AQP2 antibody showed no labeling (data not shown).
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Cellular and Subcellular Distribution of p-AQP2
Our previously described AQP2 antibody (9, 43) (LL127, recognizing both phosphorylated and nonphosphorylated AQP2, Fig. 1) labeled the apical plasma membrane and intracellular domains in collecting duct principal cells (Fig. 3A) consistent with previous investigations (43).
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Figure 3B shows a cryosection from inner stripe of the outer
medulla from a normal rat labeled with the anti-p-AQP2 antibody. Phosphorylated AQP2 was present in apical plasma membrane domains in
collecting duct principal cells significantly. Labeling of intracellular domains was more pronounced using a higher concentration of the antibody (inset, Fig. 3B). A similar cellular
location of p-AQP2 was found in cryosections from inner medulla from a normal rat (Fig. 3, C and D). In kidney inner medulla,
p-AQP2 was located in apical plasma membrane domains (Fig. 3C),
and labeling with a higher concentration of the antibody resulted in
abundant labeling of intracellular domains (Fig. 3D).
Immunoelectron microscopy showed prominent labeling of the apical
plasma membrane (Fig. 4). In addition,
there was a significant labeling associated with intracellular
vesicles. Thus, in normal rats p-AQP2 is located both in the apical
plasma membrane and in intracellular vesicles in collecting duct
principal cells.
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Effects of V2-Receptor Antagonist Treatment on p-AQP2 Levels and Intracellular Distribution in Normal Rats
To assess the effect of V2-receptor antagonist treatment on p-AQP2 protein levels in normal rats, rats were treated intravenously with OPC-31260 (1 mg) for 30 or 60 min using protocols previously described (7). OPC-31260 treatment caused a marked increase in urine production (7). Immunoblots of membrane fractions from kidney inner medulla of saline-injected control rats and OPC-31260-treated animals (Fig. 5) showed a marked reduction in p-AQP2 protein levels after 30 and 60 min of V2-receptor antagonist treatment (n = 4, P < 0.005). The reduced expression of p-AQP2 was confirmed by immunocytochemistry using thin cryosections from kidney inner medulla labeled with anti-p-AQP2 antibody (Fig. 6). A marked reduction in the overall p-AQP2 labeling was seen in rats treated with OPC-31260 for 30 min (Fig. 6B) compared with control rats (Fig. 6A). This was further evidenced by immunoelectron microscopy showing only sparse labeling of intracellular vesicles in collecting duct principal cells in rats exposed to V2-receptor antagonist for 30 min (Fig. 7B) compared with controls (Fig. 7A). The total labeling density was only 11.0 ± 1.3 particles/µm2 (n = 4) in OPC-31260-treated animals compared with 56.6 ± 1.9 particles/µm2 (n = 4) in control animals.1
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Effects of DDAVP Treatment on p-AQP2 Levels and Intracellular Distribution in Brattleboro Rats
Prior to investigation of the effect of DDAVP treatment on p-AQP2 levels and intracellular distribution in Brattleboro rats, the urine production was determined. Brattleboro rats were kept in metabolic cages for 2 h after exposure to DDAVP (protocol 3). DDAVP treatment had a marked antidiuretic effect resulting in a 93% reduction in urine output (19.3 ± 1.1 vs. 1.4 ± 0.5 ml/2 h, n = 20). To investigate the subcellular distribution of p-AQP2 in Brattleboro rats and to see whether DDAVP treatment changes the subcellular distribution, immunocytochemistry was performed. In untreated Brattleboro rats, a distinct labeling of p-AQP2 was seen (Fig. 8A). Thus in vasopressin-deficient Brattleboro rats, significant levels of p-AQP2 are present. In untreated Brattleboro rats, p-AQP2 was almost exclusively found in intracellular domains and not in plasma membrane domains (Fig. 8A). In contrast, DDAVP treatment caused a marked increase in the labeling of apical plasma membrane domains (Fig. 8B). To further examine the subcellular distribution of p-AQP2, immunoelectron microscopy was carried out. A distinct p-AQP2 labeling of intracellular vesicles was observed in untreated Brattleboro rats, whereas low labeling was associated with the apical plasma membrane(Fig. 9A). In contrast, there was an extensive labeling of the apical plasma membrane in response to DDAVP treatment (Fig. 9B). The changes in the subcellular distribution of p-AQP2 in response to DDAVP treatment were additionally confirmed by semi-quantitation of the immunogold labeling in the apical part of collecting duct principal cells (Table 2). The fraction of total p-AQP2 labeling in the apical plasma membrane was only 0.022 ± 0.007 (n = 3) in control rats. After DDAVP exposure, the fraction of p-AQP2 in the apical plasma membrane was increased to 0.21 ± 0.02 (n = 4).
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The immunocytochemistry (Fig. 8) indicated no major changes in the
levels of overall labeling. To confirm this, semi-quantitative immunoblotting and semi-quantitative immunogold labeling was
undertaken. Immunoblotting showed no significant changes in p-AQP2
levels after DDAVP treatment of Brattleboro rats (0.91 ± 0.21 vs.
1.00 ± 0.18 in controls, n = 4) (Fig.
10). Consistent with this,
semi-quantitation of the immunogold labeling of p-AQP2 revealed no
significant changes in total labeling density between untreated and
DDAVP-treated Brattleboro rats (Table 2). In normal rats, 2 h of DDAVP
treatment also failed to increase p-AQP2 levels (0.79 ± 0.18 vs. 1.00 ± 0.15 in controls, n = 4). Thus 2 h of DDAVP treatment
causes a marked increase in apical plasma membrane labeling of
phosphorylated AQP2 in Brattleboro rats, but the overall abundance of
p-AQP2 is not changed.
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DISCUSSION |
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Considerable evidence has been provided to support the view that phosphorylation of AQP2 in Ser256 (a PKA consensus site) is critically involved in regulation of AQP2 trafficking (14, 25). However, the exact role of phosphorylation of AQP2 with respect to the onset and offset response to vasopressin remains to be identified. To further investigate the role of PKA phosphorylation of AQP2 in regulated trafficking, we have produced and characterized antibodies that either selectively recognize phosphorylated AQP2 or recognize both nonphosphorylated and phosphorylated AQP2 (with respect to the PKA consensus site). Immunocytochemistry at the light microscopic and electron microscopic level showed that p-AQP2 was present in both the apical plasma membrane and in subapical vesicles in collecting duct principal cells in normal rats. V2-receptor antagonist treatment of normal rats markedly reduced p-AQP2 levels as determined by semi-quantitative immunoblotting, and immunocytochemistry confirmed marked reduction both in intracellular vesicles and the apical plasma membrane. In vasopressin-deficient Brattleboro rats, p-AQP2 was almost exclusively present in intracellular vesicles. Two hours of DDAVP treatment induced a dramatic increase in the apical plasma membrane labeling of p-AQP2, whereas total p-AQP2 levels were not increased. Taken together, the results support the view that AQP2 is phosphorylated in intracellular vesicles and that p-AQP2 is subjected to trafficking to the apical plasma membrane in response to DDAVP treatment. Furthermore, the results demonstrate that phosphorylation and/or dephosphorylation of AQP2 is regulated via V2 receptors. The results are consistent with the hypothesis that phosphorylation of AQP2 at Ser256 is involved in regulated trafficking of AQP2 to control collecting duct water permeability but that other mechanisms may be also required to regulate the trafficking/docking/fusion of AQP2 bearing vesicles with the apical plasma membrane.
p-AQP2 Trafficking
In vasopressin-deficient Brattleboro rats, p-AQP2 was almost exclusively present in intracellular vesicles. Two hours of DDAVP treatment induced a 10-fold increase in the apical plasma membrane content of p-AQP2. This dramatic change in p-AQP2 labeling is consistent with previous studies on Brattleboro rats, where AQP2 has been shown to be translocated to the apical plasma membrane in response to acute or chronic vasopressin treatment (9, 48, 54). The phosphorylation of AQP2 at Ser256 and the trafficking of AQP2 can in principle take place in three ways: 1) phosphorylation of AQP2 in an intracellular compartment with subsequent vasopressin-mediated trafficking induced via other/additional signaling mechanisms; 2) vasopressin-induced phosphorylation of AQP2 in vesicles directly causing exocytosis of p-AQP2; 3) vasopressin-induced trafficking of unphosphorylated AQP2 followed by phosphorylation in the apical plasma membrane and subsequent internalization of p-AQP2. These possibilities will be discussed in the following.One mechanism could potentially involve trafficking of AQP2 residing in a phosphorylated state in intracellular vesicles. Vasopressin stimulation would then induce insertion of p-AQP2 into the apical plasma membrane. Phosphorylation in the PKA site may not necessarily be involved in the last step of regulated targeting/docking of AQP2 bearing vesicles. It is likely that phosphorylation in this site plays a permissive or facilatory role in trafficking but does not itself induce exocytosis. This mechanism would be consistent with the absence of an overall change in total abundance of p-AQP2 in response to 2 h of DDAVP treatment (both in Brattleboro and normal rats) and the relative high levels of p-AQP2 in vasopressin-deficient Brattleboro rats. The observation that phosphorylation of AQP2 in the PKA consensus site is regulated via V2 receptors may not necessarily undermine the hypothesis, since vasopressin-stimulation/phosphorylation may indeed assure that AQP2 enter a compartment from where it can be recruited for exocytosis.
Vasopressin stimulation may induce phosphorylation of AQP2 residing in intracellular vesicles. This alone or together with other regulatory steps induces exocytosis of p-AQP2 to the apical plasma membrane. This is not immediately consistent with the absence of an overall increase in p-AQP2 levels in response to 2 h of DDAVP treatment. Intuitively, we would have expected p-AQP2 levels to increase in response to DDAVP treatment. There may be several explanations for the absence of the increase. It is likely that there is a temporary increase in p-AQP2 levels in response to acute vasopressin or DDAVP treatment. That vasopressin treatment indeed can induce an increase in p-AQP2 levels was demonstrated in a parallel study using slices of kidney inner medulla. Interestingly, the level of p-AQP2 returned toward baseline in response to continued treatment for 1 h (unpublished results). These observations in in vitro systems are consistent with the present results. Both vasopressin-induced phosphorylation and vasopressin-regulated changes in the osmotic water permeability have been shown to be rapid processes (46, 53). Thus it is possible that levels of p-AQP2 return to the steady state due to dephosphorylation of AQP2 still residing in the apical plasma membrane. Whether this means that phosphorylation of AQP2 is only needed for the trafficking to the membrane but not for AQP2 to reside in the apical plasma membrane remains to be tested in future studies.
The observation that there is a substantial level of phosphorylated AQP2 in intracellular vesicles in untreated normal rats together with the observed marked reduction in p-AQP2 levels in response to vasopressin receptor antagonist treatment supports the view that in collecting duct principal cells of normal untreated rats there is a considerable constitutive activation of PKA (or other kinases) which induces phosphorylation of AQP2 in the vesicular reservoir. AQP1 is structurally organized as homotetramers in the membrane (1), and recent studies indicate that AQP2 also exists as homotetramers in the membrane (23). Thus it may be speculated that one or more monomers may be phosphorylated in the unstimulated state (basal state), whereas after vasopressin stimulation more monomers may be phosphorylated resulting in exocytosis. Thus phosphorylation may be required of three or more monomers to induce trafficking.
A third possibility would be vasopressin-regulated trafficking of AQP2 in a nonphosphorylated state. After insertion of nonphosphorylated AQP2 into the apical plasma membrane, AQP2 then becomes phosphorylated by PKA. For this to be the case, it would require that p-AQP2 can be internalized in a phosphorylated state (resulting in the observed p-AQP2 in intracellular vesicles). This hypothesis remains unlikely, mainly because it is inconsistent with the data described above demonstrating that Ser256 is essential for translocation of AQP2 to the membrane (14, 25).
In conclusion, the most likely mechanism involved is vasopressin-induced phosphorylation of AQP2 in an intracellular compartment, and this together with additional steps results in exocytosis of p-AQP2 to the apical plasma membrane. The fraction of p-AQP2 inserted in the membrane is likely to be dephosphorylated, thereby returning total levels of p-AQP2 back to the steady state. The additional steps triggering exocytosis as well as endocytosis of AQP2 remain to be identified.
High Levels of p-AQP2 in Kidneys from Vasopressin-Deficient Brattleboro Rats
There is a high level of p-AQP2 in intracellular vesicles in kidneys from vasopressin-deficient Brattleboro rats. It remains to be identified what stimulates PKA-dependent AQP2 phosphorylation in the absence of vasopressin, but the results suggest an upregulation of other signaling pathways, i.e., PKA may be activated by cAMP through other signal transduction pathways than via V2 receptors. Other ligands using the vasopressin signaling cascade may also be upregulated, e.g., oxytocin, which has been shown to have antidiuretic effects (5). Similar to high levels of p-AQP2, high expression levels of total AQP2 protein have previously been demonstrated in untreated Brattleboro rats (26, 47). There is a cAMP-responsive element in the 5'-flanking region of the AQP2 gene, and several studies have indicated an importance of this in regulating AQP2 gene expression (19, 52, 55). Thus, high activity of cAMP/PKA (or perhaps other kinases) in Brattleboro rat kidney collecting duct might therefore induce both AQP2 protein synthesis and phosphorylation of AQP2 without triggering of the water channel exocytosis.Regulatory Step(s) Inducing Exocytosis of AQP2
As discussed above, one or more additional steps appear to be necessary in addition to phosphorylation of AQP2 in the PKA consensus site to induce docking and fusion (exocytosis). These factors may include phosphorylation or chemical modification of AQP2 or other proteins participating in the exocytosis.Other kinases may potentially phosphorylate AQP2 and be involved in regulating AQP2 trafficking (offset or onset response). In addition to the PKA consensus site, AQP2 contains potential phosphorylation sites for protein kinase C (PKC) and casein kinase II (CKII) (15). These enzymes are both present in the kidney. Several PKC isoforms were identified in rat medullary thick ascending limb and inner medullary collecting duct, and recent studies using renal epithelial cells have demonstrated that PKC is activated by vasopressin (2, 3, 6). CKII activity was observed in bovine kidney and in chicken kidney CKII mRNA has been shown to be expressed (12, 33). cGMP-dependent protein kinase (PKG), Ca2+-, calmodulin-dependent protein kinase (PK-CaM) and protein kinase B (PKB) have been shown to phosphorylate the same serine residues as PKA in different proteins (8, 11, 49), and isoforms of these three kinases are expressed in the kidney (16, 39, 50). It is possible that one of these kinases may also phosphorylate Ser256 in AQP2.
Phosphorylation of other proteins may also be important for trafficking. Early studies have demonstrated that PKA induces phosphorylation of various membrane proteins in bovine kidney (10), and in saponin-permeabilized outer medullary collecting duct segments phosphorylation of at least two unidentified 45- and 66-kDa proteins has been demonstrated (17). Vesicle targeting receptors, also known to be present in the kidney collecting duct, may potentially also be subjected to phosphorylation. The vesicle-associated membrane protein VAMP/synaptobrevin and the vesicle-targeting protein SNAP-25 have been shown to be phosphorylated in vitro by protein kinases (18, 40). Furthermore, PKA and CKII can phosphorylate syntaxin-4 in vitro (13), whereas SNAP-23 was not phosphorylated by either PKA or CKII and only minimally by PKC (13). Thus phosphorylation of SNARE proteins may hypothetically play a role for trafficking of AQP2. CKII may also be involved in the regulation of dynein function as evidenced by in vitro studies showing phosphorylation of dynein by CKII as well as binding of CKII to dynein (24).
Regulation of p-AQP2 Levels in Response to V2-Receptor Antagonist Treatment
Phosphorylated AQP2 expression levels were markedly reduced in response to both 30 and 60 min of V2-receptor antagonist treatment. Previous studies using the same protocol showed no changes in total AQP2 (nonphosphorylated plus phosphorylated) protein abundance after 60 min of OPC-31260 treatment (7). This suggests that V2-receptor antagonist treatment modulates p-AQP2 abundance at the posttranslational level, i.e., it changes the phosphorylation state of AQP2. V2-receptor antagonist treatment may either reduce PKA activity and/or induce dephosphorylation of AQP2, e.g., by increased phosphatase activity. Several phosphatases have been identified in the kidney, e.g., protein phosphatases 1 and 2a and the calcium/calmodulin-dependent protein phosphatase calcineurin (4, 31, 51). Whether these are subject to regulation via V2 receptors is unknown. It is likely that there is a constant activity of phosphatases in collecting duct principal cells and that the absence of V2 receptor stimulation (achieved by treatment with V2-receptor antagonist) reduces the buildup of p-AQP2 resulting in a net decrease.The offset response to vasopressin could be hypothesized to take place by three mechanisms with regard to the role of AQP2 (de)phosphorylation. 1) p-AQP2 residing in the apical plasma membrane could be subjected to dephosphorylation, and dephosphorylated AQP2 might then be internalized (either directly as a consequence of dephosphorylation or due to other regulatory steps). 2) Alternatively, phosphorylated AQP2 could be internalized and be subjected to dephosphorylation after internalization. 3) A combination of the two possibilities listed above may exist. This study does not allow us to discriminate between these potential mechanisms.
Conclusion
Our studies strongly support the view that PKA-mediated phosphorylation of AQP2 at Ser256 is involved in vasopressin-regulated trafficking of AQP2 water channel protein. Moreover, the results strongly suggest that other factors are necessary and involved in regulated AQP2 bearing vesicle trafficking, docking, and fusion. These may include further chemical modification of AQP2 and/or involve other regulatory components as discussed above. Future studies will be needed to characterize this in detail. ![]() |
ACKNOWLEDGEMENTS |
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We thank Zhila Nikrozi, Mette Vistisen, Gitte Christensen, and Annette Blak Rasmussen for expert technical assistance.
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FOOTNOTES |
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Support for this study was provided by the Karen Elise Jensen Foundation; Danish Medical Research Council; The Medical Faculty; University of Aarhus; Novo Nordisk Foundation; University of Aarhus Research Foundation; Swedish Medical Research Council Grant 03644; and Märta and Gunnar Phillipson Foundation.
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.
1 Because of the low count of gold particles in V2-receptor antagonist-treated animals, it was not possible to quantify meaningfully and compare the changes in labeling of the apical plasma membrane and in intracellular vesicles.
Address for reprint requests and other correspondence: S. Nielsen, Dept. of Cell Biology, Institute of Anatomy, Univ. of Aarhus, DK-8000 Aarhus, Denmark (E-mail: SN{at}ANA.AU.DK).
Received 25 February 1999; accepted in final form 17 August 1999.
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