From the Department of Ophthalmology and Visual Sciences,
Washington University School of Medicine,
St. Louis, Missouri 63110
The molecular mechanisms for regulating water
balance in many tissues are unknown. Like the kidney, the eye contains
multiple water channel proteins (aquaporins) that transport water
through membranes, including two (AQP1 and AQP4) in the ciliary body, the site of aqueous humor production. However, because humans with
defective AQP1 are phenotypically normal and because the ocular
application of phorbol esters reduce intraocular pressure, we
postulated that the water channel activity of AQP4 may be regulated by
these agents. We now report that protein kinase C activators, phorbol
12,13-dibutyrate, and phorbol 12-myristate 13-acetate strongly
stimulate the phosphorylation of AQP4 and inhibit its activity in a
dose-dependent manner. Phorbol 12,13-dibutyrate (10 µM) and phorbol 12-myristate 13-acetate (10 nM) reduced the rate of AQP4-expressing oocyte swelling by
87 and 92%, respectively. Further, phorbol 12,13-dibutyrate
significantly increased the amount of phosphorylated AQP4. These
results demonstrate that protein kinase C can regulate the activity of
AQP4 through a mechanism involving protein phosphorylation. Moreover,
they suggest important potential roles for AQP4 in several clinical
disorders involving rapid water transport such as glaucoma, brain
edema, and swelling of premature infant lungs.
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INTRODUCTION |
Aquaporins are a rapidly growing family of water channel proteins
found in animals, plant, and microorganisms (1, 2). At least eight
different aquaporins have been identified and cloned from mammals,
including AQP1 from erythrocytes (3, 4), AQP2, AQP3, and AQP6 from
kidney (5-7), AQP4 from brain (8, 9), AQP5 from salivary gland, (10),
AQP7 from testis (11), and AQP8 from testis and liver (12, 13). AQP1,
AQP2, AQP3, and AQP7 have been shown to transport the nonionic small
solutes such as urea and glycerol in addition to water (6, 11, 14), whereas AQP4, AQP5, AQP6, and AQP8 are highly selective to water permeation and exclude small solutes (8, 9, 12). With the exception of
kidney-specific, vasopressin-regulated AQP2, the aquaporins are thought
to be constitutively active (15-18). The regulation of other
aquaporins is controversial (see "Discussion") and is not well
understood.
The ciliary body expresses only two aquaporins (AQP1 and AQP4). Because
humans with mutation defects in AQP1 are phenotypically normal (19) and
because the application of phorbol esters to the eye reduces
intraocular pressure (20), we postulated that phorbol ester regulation
of AQP4 water channel activity may account for the observed reduction
of intraocular pressure using these agents. AQP4 is unique because it
encodes a water-selective channel that is not inhibited by high
concentrations of mercurial compounds such as HgCl2 (9).
Previous studies using immunocytochemistry, reverse transcription
polymerase chain reaction, and Northern blotting with AQP4 confirmed
its expression in kidney, brain, lung, and eye including retina and
ciliary body (21-23). Using an oocyte swelling assay and protein
phosphorylation studies, we demonstrate here that water channel
activity of AQP4 is regulated by phorbol ester-dependent
protein phosphorylation via protein kinase C
(PKC)1 pathway. AQP4
regulation by PKC suggests an important potential role for this
aquaporin in several clinical disorders involving rapid water transport
such as glaucoma, brain edema following stroke, and uncontrollable
swelling of premature infant lungs.
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EXPERIMENTAL PROCEDURES |
In Vitro cRNA Synthesis of AQP4--
The plasmid containing rat
AQP4 cDNA (8) was purchased from ATCC (Rockville, MD). The
EcoRI fragment of the plasmid containing entire AQP4 open
reading frame was blunt-ligated into the BglII site of the
Xenopus expression construct pXbG (3). Confirmation of the
recombinant plasmid was made by nucleotide sequencing. Sense and
antisense capped RNA transcripts of AQP4 were synthesized in
vitro with T3 RNA polymerase using two recombinant plasmids with
the AQP4 cDNA cloned in sense and antisense direction.
Preparation of Oocytes and Measurement of
Pf--
Defolliculated stage V and VI
oocytes from female Xenopus laevis (24) were injected with
20 nl of water or cRNAs (1 mg/ml). After incubation in 200 mosmol
modified Barth's buffer at 18 °C for 72 h, oocytes were
transferred to 70 mosmol Barth's buffer diluted with distilled water,
and the time course of osmotic volume increase was monitored at
20 °C. Because the time course of cell swelling was principally
linear during the initial 40 s, osmotic water permeability
(Pf) of oocytes was calculated from this 40-s response as described previously (25). The effects of phorbol 12,13-dibutyrate (PDBu), phorbol 12-myristate 13-acetate (PMA), 4
-phorbol, 4
-phorbol 12,13-didecanoate (4
-PDD), (all from
Calibiochem), and HgCl2 were examined by incubating oocytes
in Barth's buffer containing appropriate concentrations of the reagent
for 15 min prior to Pf measurements.
In Vitro Phosphorylation--
Rat brain homogenate (75 µg) was
incubated at 25 °C for 30 min in the presence of 50 µM
{
-32P}ATP; 0.045 µg of PKC (Calibiochem) and PKC
activators in phosphorylation buffer containing 20 mM
Tris-HCl (pH 7.4); 100 mM NaCl; 5 mM
MgCl2; 5 mM NaH2PO4;
1.5 mM CaCl2; 0.2% (v/v) Triton X-100; 1 mM EDTA; 1 mM dithiothreitol; 1 mM
phenylmethylsulfonyl fluoride; 5 µg/ml each of leupeptin, pepstatin,
and antipain. At the end of incubation period the phosphorylation
reaction was stopped by immunoprecipitation with AQP4 antibody as
described below.
Immunoprecipitation of Phosphorylated AQP4--
Phosphorylated
homogenate was incubated with 10 mg of preswollen protein A-Sepharose
beads and incubated for 1 h at 4 °C. The Sepharose
bead-associated, nonspecifically adsorbed proteins were removed by
centrifugation for 10 s at 15,000 rpm in microcentrifuge. The
supernatant was then mixed with 2 µl of AQP4 antibody (kindly provided by Dr. Verkman, University of California San Francisco), and
the mixture was incubated for 12 h at 4 °C. The samples were then transferred to Eppendorf tube containing 10 mg of preswollen protein A-Sepharose beads and incubated for 1-2 h at 4 °C. The beads were collected by centrifugation and washed once with lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.2% bovine serum albumin (pH
8.0) containing 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml each of leupeptin, pepstatin, and antipain); three times with 1 ml buffer containing 20 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, 0.1% SDS, 0.2%
bovine serum albumin (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 50 µl of SDS-PAGE sample buffer (50 mM Tris-HCl, 10% glycerol, 2% SDS, 10%
2-mercaptoethanol, 0.01% bromphenol blue (pH 6.8)), vortexed, and
centrifuged. The recovered proteins were separated by SDS-PAGE (7.5%).
Gels were dried and subjected to autoradiography.
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RESULTS AND DISCUSSION |
Effect of Phorbol Esters on AQP4 Water Channel
Activity--
An expression construct was prepared by inserting
the AQP4 coding sequences between 5'- and 3'-untranslated sequences of
the Xenopus
-globin cDNA as described under
"Experimental Procedures." Defolliculated oocytes were
microinjected with 20 ng of in vitro transcribed AQP4 cRNA.
Osmotic water permeability after transfer of oocytes from a 200 to a 70 mosmol solution was determined by monitoring changes in cell volume as
described under "Experimental Procedures." Fig.
1 shows the effect of PDBu (PKC
activator) on the osmotic water permeability of oocytes expressing
AQP4. Oocytes incubated for 15 min in 200 mosmol Barth's buffer
containing PDBu showed considerably decreased subsequent rate of
swelling in 70 mosmol buffer compared with control oocytes, whereas
oocytes incubated with 4
-phorbol (inactive phorbol) for 15 min in
200 mosmol Barth's buffer showed no effect on subsequent rate of
swelling in 70 mosmol buffer. The effect of PDBu was
dose-dependent; at 10 µM PDBu reduced the
rate of oocyte swelling by 87% in 70 mosmol buffer. Similarly, oocytes
incubated for 15 min in 200 mosmol Barth's buffer containing PMA
showed a significantly lower subsequent rate of swelling in 70 mosmol
buffer (Fig. 2) versus control
oocytes. Oocytes incubated for 15 min in 200 mosmol Barth's buffer
containing 4
-PDD (inactive PMA) showed no effect on subsequent rate
of swelling in 70 mosmol buffer. The effect of PMA was also
dose-dependent; at 10 nM PMA reduced the rate
of oocyte swelling by 92% in 70 mosmol buffer. Ethanol (0.1%) was
used to make 1 mM stock solutions of PDBu, 4
-phorbol,
PMA, and 4
-PDD and had no effect alone on swelling. Swelling of
oocytes expressing AQP4 was not blocked by 1 mM
HgCl2 (data not shown), because AQP4 is a
mercury-insensitive water channel (8, 9). Oocytes injected with water
or antisense cRNA showed a very low swelling rate that was unaffected
by either PDBu or PMA treatment.

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Fig. 1.
Effect of PDBu on the osmotic water
permeability of oocytes expressing AQP4 RNA. Oocytes were injected
with 20 ng of AQP4 cRNA 72 h prior to experiments. Oocytes were
incubated with PDBu or 4 -phorbol as described under "Experimental
Procedures." After the treatments, osmotic swelling of the oocytes
was monitored, and oocyte volume was calculated. Each point represents
the mean ± S.E. of 8-12 oocytes. Similar results were obtained
in three separate experiments with oocytes from different frogs.
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Fig. 2.
Effect of PMA on the osmotic water
permeability of oocytes expressing AQP4 RNA. Oocytes were injected
with 20 ng of AQP4 cRNA 72 h prior to experiments. Oocytes were
incubated with PMA or 4 -PDD as described under "Experimental
Procedures." After the treatments, osmotic swelling of the oocytes
was monitored, and oocyte volume was calculated. Each point represents
the mean ± S.E. of 8-12 oocytes. Similar results were obtained
in three separate experiments with oocytes from different frogs.
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The coefficients of osmotic water permeability
(Pf) at 20 °C calculated from the rates of
swelling were 32.4 ± 2 µm/s (mean ± S.E.,
n = 8) for untreated AQP4 injected oocytes and 4.3, 5.3, and 9.1 ± 1 µm/s (mean ± S.E., n = 12) for PDBu-treated oocytes at 10, 5, and 1 µM
concentrations, respectively (Fig. 3),
whereas the Pf values of oocytes incubated with
4
-phorbol were 31 ± 2 µm/s (mean ± S.E.,
n = 8), suggesting that the decrease in
Pf of AQP4 due to PDBu was specific. In another
experiment, similar results were obtained using PMA. The
Pf values of oocytes incubated with PMA at 10, 5, and 1 nM, were 2.4, 8.2, and 13 ± 2 µm/s
(mean ± S.E., n = 10), respectively, whereas the
Pf values of untreated oocytes and
4
-PDD-treated oocytes were 29 and 24.9 ± 3 µm/s (mean ± S.E., n = 10), respectively. The
Pf values of oocytes injected with water (data
not shown) were 2.2 ± 1 µm/s (mean ± S.E.,
n = 8) suggesting that microinjection itself had no
effect on the oocyte swelling. The phorbol ester-dependent
decrease in water permeability suggests that AQP4 could participate in
receptor-mediated regulation of water fluxes in a variety of tissues,
such as kidney, heart, brain, lung, and eye, in which it is widely
distributed (21, 22).

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Fig. 3.
Changes in Pf of
oocytes expressing AQP4 RNA after PDBu and PMA treatments. Oocytes
were injected with 20 ng of AQP4 cRNA 72 h prior to experiments.
Oocytes were incubated with PDBu or PMA as described under
"Experimental Procedures." After the treatments, osmotic swelling
of the oocytes was monitored, and Pf was
calculated. Pf is shown as the mean ± S.E.
of 8-12 oocytes. Similar results were obtained in three separate
experiments with different batches of oocytes.
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In Vitro Phosphorylation of AQP4--
Activation of PKC by phorbol
esters is known to stimulate the phosphorylation of several proteins,
thereby modulating their function. Therefore, we tested whether
phosphorylation of AQP4 could be achieved in vitro by PKC.
For this purpose, we incubated equal aliquots of rat brain homogenate
with or without PDBu in the presence of
-32P and PKC.
After separation of immunoprecipitated proteins using AQP4-specific
antibody, we observed two bands with an apparent molecular masses of 31 and 40-45 kDa (Fig. 4). The 31-kDa band corresponds to unglycosylated protein, and the 40-45-kDa band corresponds to glycosylated proteins. The intensity of phosphorylated bands in the presence of PDBu was significantly higher than the bands
in the absence of PDBu or in the presence of PKC inhibitor or
4
-phorbol. Analysis of Fig. 4 by densitometry showed that the
density of the 31-kDa band in the presence of PDBu was 12-12.5 times
higher than that in the absence of PDBu or in the presence of PKC
inhibitor or 4
-phorbol. These results strongly suggested that both
glycosylated and unglycosylated AQP4 peptides were phosphorylated by
PKC in the presence of PDBu.

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Fig. 4.
Effect of PDBu on the phosphorylation of rat
brain AQP4 protein. Phosphorylation of AQP4 from rat brain was
identified as the incorporation of radioactivity into the protein by
PKC in the presence of 5 µM of PDBu (with or without PKC
inhibitor) or 4 -phorbol for 30 min. Autoradiogram was obtained after
immunoprecipitation of phosphorylated AQP4 and separation of recovered
proteins by SDS-PAGE. Similar results were obtained in two separate
experiments using brain homogenate from different rats.
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Decreased oocyte Pf in AQP4 expressing oocytes
by PDBu and PMA but not by inactive phorbol esters and increased
32P incorporation into AQP4 in in vitro
phosphorylation in the presence of PDBu but not in the presence of
inactive phorbol or PKC inhibitor suggest that the phorbol
ester-dependent phosphorylation of AQP4 is involved in the
regulation of its water channel activity. Further studies are necessary
to identify the unique amino acid residue responsible for the
phosphorylation of AQP4 and determine whether phosphorylation by
phorbol ester-dependent PKC changes the activity or
distribution pattern of AQP4 in native tissue. Thr-107, Ser-111, and
Ser-180 (8) may be the potential phosphorylation sites for AQP4 because
they are contained in the recognition motifs (S*/T*)X(R/K)
or RXXS* used by serine/threonine protein kinases such as
PKC (26).
The regulation of aquaporins is the subject of major controversy.
Recently, it was reported that forskolin stimulated the water channel
activity of AQP1 (27). Although some investigators failed to reproduce
these observations (28), we were able to duplicate these results in our
laboratory (25, 29). Further, we have shown recently that AQP1 is
regulated by arginine vasopressin and atrial natriuretic peptide in
oocytes (25). Previous studies have shown that water channel activity
of AQP2 is stimulated by cAMP-dependent protein
phosphorylation (30). These results have also been controversial in
oocyte (31) as well as in phosphorylation (32) studies using AQP2. Yet
a recent report suggested that protein kinase activators such as
forskolin and PMA had no effect on the water channel activity of AQP1,
AQP2, AQP3, AQP4, or AQP5 expressing oocytes and concluded that
phosphorylation is not involved in the regulation of these proteins
(33). Some of these discrepancies may be due to variation in oocyte
batches undertaken for the studies. For example, studies with
activation of ionic currents in Xenopus oocytes by arginine
vasopressin showed that not all donor frogs are responsive to this
peptide, and the response was variable between oocytes from a single
donor (34). Furthermore, these studies indicated that there may be a
seasonal variation in expression of the receptors for these
neuropeptides.
The regulation of AQP4 is likely significant clinically because it is a
major water channel protein in brain and may therefore play an
important role in the swelling that follows stroke. In lung, where
there is a sharp increase in AQP4 expression just after birth (35) it
may play an important role in the clearance of fluid from the newborn
lungs. In eye, its presence in the ciliary body (9, 23, 36) may
contribute to aqueous humor production and elevated intraocular
pressure as occurs in glaucoma. Furthermore, its presence in retinal
Muller cells may contribute to visual function by its involvement in
the light-dependent hydration of space-surrounding
photoreceptors (see Ref. 37). In addition, recent evidence demonstrates
that AQP4 knockout mice have impaired ability to concentrate urine,
suggesting a functional role for this aquaporin in the kidney (38). At
the least, our results provide mounting evidence that in addition to
AQP2, other aquaporins such as AQP4 are likely amenable to
pharmacological regulation and furthermore such activity appears to be
of physiologic relevance.