Báo cáo khoa học: Involvement of the V2 receptor in vasopressin-stimulated translocation of placental leucine aminopeptidase/oxytocinase in renal cells pdf

Involvement of the V
2
receptor in vasopressin-stimulated
translocation of placental leucine aminopeptidase/oxytocinase
in renal cells
Shinako Masuda
1
, Akira Hattori
1
, Hideko Matsumoto
1,
*, Shinobu Miyazawa
2
, Yasuhiro Natori
2
,
Shigehiko Mizutani
3
and Masafumi Tsujimoto
1
1
Laboratory of Cellular Biochemistry, RIKEN (The Institute of Physical and Chemical Research), Wako, Saitama, Japan;
2
Department of Clinical Pharmacology, Research Institute, International Medical Center of Japan, Shinjuku, Tokyo, Japan;
3
Department of Obstetrics and Gynecology, Nagoya University School of Medicine, Showa, Nagoya, Japan
The placental leucine aminopeptidase (P-LAP)/oxytocinase
is a membrane-bound enzyme thought to play an important
role during pregnancy. In this study, we identified the pres-
ence of P-LAP protein in the renal distal tubules and col-
lecting ducts. In rat NRK52E cells derived from renal

tubules, P-LAP was localized mainly in the intracellular
compartment. Upon the treatment of cells with 8-arginine-
vasopressin (AVP), a significant increase in the surface level
of P-LAP was observed. [deamino-Cys1,
D
-Arg8]-vaso-
pressin (DDAVP), a specific V
2
receptor agonist, increased
the surface level of P-LAP, while [adamantaneacetyl1,
O-Et-
D
-Tyr2, Val4, aminobutyryl6, Arg8,9]-vasopressin
(AEAVP), a potent V
2
receptor antagonist, blocked the
AVP-stimulated enhancement. Moreover, reagents known
to enhance the intracellular level of cAMP have also been
shown to increase the surface level of P-LAP. When we
examined the colocalization of P-LAP with the cell surface
water channel aquaporin-2 (AQP-2) that is regulated by
AVP, the P-LAP-containing vesicles had a relatively higher
density than the AQP-2-containing vesicles, suggesting that
P-LAP and AQP-2 are differently distributed in NRK52E
cells. These results suggest that AVP induces the transloca-
tion of P-LAP via V
2
receptor and P-LAP plays a role in
the regulation of excessive AVP level in the renal collecting
duct, acting as a negative feedback mechanism for the AVP

action of regulating water reabsorption.
Keywords: aquaporin-2; oxytocinase; placental leucine
aminopeptidase; vasopressin.
The serum level of placental leucine aminopeptidase
(P-LAP)/oxytocinase increases during pregnancy [1]. It is
generally believed that P-LAP plays an important role in the
maintenance of normal pregnancy, by degrading peptide
hormones, such as oxytocin, vasopressin and angiotensin
III, which may have a significant effect on uterine tonus and
utero–placental blood flow [2]. In an effort to elucidate the
structural features of P-LAP, we purified serum P-LAP
from retroplacental serum and cloned the cDNA encoding
human P-LAP [3]. Analysis of the cDNA indicates that the
enzyme is a type II membrane-spanning protein, which
belongs to the M1 family of zinc-metallopeptidases that
share the consensus HEXXH(X)
18
E motif. Our results
suggest that P-LAP is first synthesized in the placenta as a
membrane-bound protein and then secreted into the
maternal serum. A number of properties of the processing
enzyme in the placenta have been characterized recently
[4,5]. On the other hand, Keller et al. independently cloned
the rat homologue of P-LAP and designated it as the
insulin-regulated membrane aminopeptidase (IRAP) that
associates with the glucose transporter (GLUT)-4-contain-
ing vesicle [6]. This indicates the vesicular distribution of
the enzyme and that its expression is not restricted to the
placenta. It is now recognized that the association of
P-LAP/IRAP with certain intracellular vesicles is the most

characteristic feature of this enzyme.
The stimulus-induced translocation of P-LAP/IRAP
from the intracellular compartment to the plasma mem-
brane has been observed in several biological systems. It has
been well established that insulin stimulates the transloca-
tion of P-LAP/IRAP in 3T3-L1 adipocytes [7–9]. More
recently, Nakamura et al. reported that oxytocin stimulated
the translocation of P-LAP in human umbilical vascular
Correspondence to M. Tsujimoto, Laboratory of Cellular Biochemis-
try, RIKEN (The Institute of Physical and Chemical Research),
2–1 Hirosawa, Wako-shi, Saitama 351–0198 Japan,
Fax: + 81 48 462 4670, Tel.: + 81 48 467 9370,
E-mail:
Abbreviations
1
: AEAVP, [adamantaneacetyl1, O-Et-D-Tyr2, Val4,
aminobutyryl6, Arg8,9]-vasopressin; AQP, aquaporin; AVP,
arginine-vasopressin; 2MAVP, [2-mercapto-2,2-
2
cyclopentamethy-
lenepropionyl1,O-me-Tyr2,Arg8]-vasopressin; DBcAMP,
N
6
,2¢-O-dibutyryladenosine 3¢,5¢-cAMP; DDAVP, [deamino-Cys1,
D
-Arg8]-vasopressin; GLUT, glucose transporter; HUVEC, human
umbilical vascular endothelial cells; IRAP, insulin-regulated
membrane aminopeptidase; PKC, protein kinase C; P-LAP,
placental leucine aminopeptidase.
Enzymes: placental leucine aminopeptidase/oxytocinase (EC.3.4.11.3).

*Present address: Experimental Diabetes, Metabolism and Nutrition
Section, Diabetes Branch, National Institute of Diabetes and Digestive
and Kidney Diseases, National Institutes of Health,
Bethesda MD 20892–0842, USA.
(Received 28 January 2003, revised 10 March 2003,
accepted 14 March 2003)
Eur. J. Biochem. 270, 1988–1994 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03570.x
endothelial cells (HUVEC), in a protein kinase C (PKC)-
dependent manner [10]. We have also observed the fors-
kolin-mediated translocation in PC12 cells [11]. It is
generally believed that the physiological significance of
P-LAP translocation is to enhance the cleavage of peptide
hormone substrates at the cell surface.
Aminopeptidases belonging to the M1 family of zinc-
metallopeptidases have been shown to play an important
role in the regulation of physiological and/or pathological
functions, such as blood pressure, angiogenesis and antigen
presentation [12–18]. As for P-LAP/IRAP, it was reported
recently that the enzyme is a receptor for angiotensin IV and
may play a role in the maintenance of memory [19].
In the current study, we detected the P-LAP protein in the
kidney and examined its potential role. We have observed
that 8-arginine-vasopressin (AVP) stimulated the transloca-
tion of P-LAP from the intracellular compartment to the
plasma membrane in rat renal NRK52E cells.
Materials and methods
Materials
AVP was purchased from the Peptide Institute (Osaka,
Japan). [deamino-Cys1,
D

-Arg8]-vasopressin (DDAVP),
[adamantaneacetyl1, O-Et-
D
-Tyr2, Val4, aminobutyryl6,
Arg8,9]-vasopressin (AEAVP), [2-mercapto-2,2-cyclo-
pentamethylenepropionyl1, O-me-Tyr2, Arg8]-vasopressin
(2MAVP), N
6
,2¢-O-dibutyryladenosine 3¢,5¢-cAMP
(DBcAMP), forskolin and anti-[rat aquaporin-2 (AQP-2)]
peptide Ig were purchased from Sigma (St. Louis, MO,
USA).
Western blot analysis
Test samples were separated by SDS/PAGE on a 8%
separating gel and transferred to poly(vinylidene difluoride)
membranes (Pall Corp, East Hills, NY, USA). The mem-
branes were blocked with NaCl/Tris pH 7.4, containing
0.1% (v/v) Tween-20 (NaCl/Tris/Tween) and 5% (w/v)
skimmed milk for 1 h at room temperature, then incubated
in NaCl/Tris/Tween, 5% (w/v) skimmed milk, and
1 lgÆmL
)1
rabbit anti-(P-LAP) Ig for 2 h at room tempera-
ture. The filter was washed three times with NaCl/Tris/
Tween and incubated for 1 h with horseradish peroxidase
(HRP)-conjugated goat anti-(rabbit IgG) Ig (Promega,
Madison, WI, USA), diluted to 1 : 20 000 in NaCl/Tris/
Tween containing 5% (w/v) skimmed milk. After washing
the filter three times with NaCl/Tris/Tween, the blots were
detected by an enhanced chemiluminescence method using

an ECL plus Western blotting kit obtained from Amer-
sham-Pharmacia. The results were visualized by fluoro-
graphy using RX-U Fuji medical X-ray film.
Preparation of membrane fractions
NRK52E cells derived from rat renal tubule epithelium
were grown in Dulbecco’s modified Eagle’s medium
containing 5% fetal bovine serum and 1% nonessential
amino acids and suspended in 50 m
M
Tris/HCl pH 7.5,
containing 10 lgÆmL
)1
aprotinin. The cells were homo-
genized with a Dounce homogenizer, the homogenate was
centrifuged at 700 g for 5 min and the resultant postnuclear
supernatant was centrifuged for 1 h at 100 000 g to prepare
soluble and membrane protein fractions. The membrane
fraction was resuspended in lysis buffer (10 m
M
Tris pH 7.8,
1% Nonidet P-40, 150 m
M
NaCl, 1 m
M
EDTA, 0.5%
Triton X-100, 10 lgÆmL
)1
aprotinin) and then subjected to
SDS/PAGE.
Immunohistochemical analysis

Formalin-fixed and paraffin-embedded biopsy specimens of
human kidney remaining after diagnostic evaluation show-
ing minimal histological abnormalities were obtained from
the International Medical Center of Japan and used for
P-LAP immunohistochemical staining. Paraffin sections,
2 lm-thick, were incubated with 20 lgÆmL
)1
of affinity-
purified anti-(P-LAP) Ig overnight at 4 °C. HRP-conjugated
goat anti-(rabbit IgG) Ig diluted 1 : 500 was used as
the secondary antibody and 3,3¢-diaminobenzidine
(0.1 mgÆmL
)1
) as HRP substrate. The sections were then
counter-stained with methyl green. For negative controls,
20 lgÆmL
)1
of immunoglobulin purified from normal rabbit
serum was used instead of affinity-purified anti-(P-LAP) Ig
[11,13].
Immunocytochemical analysis of NRK52E cells
NRK52E cells grown on a cover glass were washed three
times with NaCl/P
i
and fixed with 2% paraformaldehyde in
NaCl/P
i
for 20 min at room temperature. Cells were
permeabilized in NaCl/P
i

solution containing 0.3% Triton
X-100 for 5 min. Coverslips were blocked for 1 h with
NaCl/P
i
containing 2% horse serum (blocking buffer), and
incubated for 1.5 h at 27 °Cwith5lgÆmL
)1
affinity-
purified anti-(P-LAP) Ig in blocking buffer. The cells were
then washed three times with NaCl/P
i
and incubated with
0.5 lgÆmL
)1
AlexaFluor-488-labeled goat anti-(rabbit IgG)
Ig in blocking buffer for 1 h. After washing with NaCl/P
i
three times, cells were mounted in a drop of PermaFluor
Aqueous Mounting Medium (Immunon, Pittsburgh, PA,
USA) and viewed with a Leica TCS NT laser scanning
microscope (Leica, Wetzlar, Germany) [11].
Cell surface biotinylation assay
All steps were performed at 4 °C. NRK52E cells were
grown in 100-mm-diameter dishes and washed three times
in ice-cold Krebs-Ringer phosphate Hepes (KRPH,
128 m
M
NaCl, 4.7 m
M
KCl, 1.25 m

M
CaCl
2
,1.25m
M
MgSO
4
,5m
M
NaPO
4
,20m
M
Hepes, pH 7.4) and treated
with 2.5mL of 0.5mgÆmL
)1
sulfo-NHS-SS-biotin in
KRPH for 30 min with shaking. The cells were then
washed three times with KRPH, collected and lysed
in 300 lL of lysis buffer (10 m
M
Tris, pH 7.8, 1% Nonidet
P-40, 150 m
M
NaCl, 1 m
M
EDTA, 0.5% Triton X-100,
10 lgÆmL
)1
aprotinin). After determination of the protein

concentration, cell lysate containing 100 lgofproteinwas
diluted to 300 lL with lysis buffer, added 15 lLof
immobilized streptavidin (6% cross-linked agarose, Pierce,
Rockford, IL, USA), and then allowed to bind for 2 h while
rotating. After binding, immobilized streptavidin was
recovered by centrifugation (17 400 g for 1 min) and
Ó FEBS 2003 Vasopressin-stimulated translocation of P-LAP (Eur. J. Biochem. 270) 1989
washed five times with lysis buffer. Bound proteins were
eluted by the addition of 40 lL Laemmli sample buffer, and
then subjected to Western blot analysis. Densitometric
analyses were performed using
IMAGE GAUGE
software (Fuji
Photo Film Co., Tokyo, Japan). Results are expressed as the
means ± SE (n ¼ 3) [11].
Measurement of cell surface aminopeptidase activity
Aminopeptidase activity was determined with the fluoro-
genic substrate, S-benzyl-
L
-Cys-MCA (Bachem AG, Swit-
zerland). The reaction mixture in 350 lLof20m
M
Tris/
HCl buffer (pH 7.5) containing 1 m
M
EDTA was incubated
at 37 °C. The released 7-amino-4-methyl-coumarin was
measured by spectrofluorophotometry (F-2000, Hitachi) at
an excitation wavelength of 370 nm and an emission
wavelength of 460 nm [20].

Sucrose gradient centrifugation
NRK52E cells grown to confluence were rinsed three times
at 4 °C with NaCl/P
i
. Cells were then scraped into 400 lL
of buffer A (150 m
M
NaCl, 10 m
M
Hepes, pH 7.4, 1 m
M
EGTA, 0.1 m
M
MgCl
2
) in the presence of protease
inhibitors (1 m
M
phenylmethanesulfonyl fluoride,
10 ngÆmL
)1
each of aprotinin, pepstatin and leupeptin)
and homogenized with a Dounce homogenizer. The homo-
genate was centrifuged at 700 g for 5 min, and 200 lLof
the resultant postnuclear supernatant was fractionated by
gradient centrifugation.
Samples were loaded on a 10–50% sucrose density
gradient prepared in buffer A and centrifuged at
48 000 r.p.m. in a Hitachi P50S2 rotor for 50 min at
4 °C. The samples were then collected from the top of the

gradient in 22 fractions.
Results
Expression of P-LAP protein in the kidney
In our previous work, we examined the expression of
P-LAP in various human tissues by Western blot analysis
and found that the kidney is one of the major tissues
expressing the enzyme [11]. Therefore, in the initial experi-
ments of this study, we analyzed the immunohistochemical
localization of P-LAP in the kidney. To detect immuno-
reactivity, we employed the affinity-purified anti-(P-LAP).
This antibody does not recogize adipocyte-derived leucine
aminopeptidase (A-LAP), which is highly related to P-LAP,
confirming its specificity. Figure 1 shows the localization of
P-LAP in the cortex of the human kidney. In this
preparation, P-LAP immunoreactivity was detected in the
distal tubules. Control immunoglobulin showed no
immunoreactivity on the sample (data not shown). We also
analyzed the immunohistochemical localization of the
enzyme in the kidney of Wistar Kyoto rats with immuno-
reactivity detected in the distal tubules of the cortex and the
collecting ducts of the cortex and medulla (data not shown).
Vesicular distribution of P-LAP protein in NRK52E cells
The subcellular localization of P-LAP in rat renal tubule-
derived NRK52E cells was then analyzed. To examine
whether the enzyme is localized in the membrane, we
Fig. 1. Detection of the immunoreactivity of
P-LAP in the human renal cortex. After incu-
bation with 20 lgÆmL
)1
of affinity-purified

anti-(P-LAP) Ig, bound antibody was detected
using HRP-conjugated goat anti-(rabbit IgG)
Ig and 3,3¢-diaminobenzidine. The section was
counterstained with methyl green. Arrow-
heads indicate P-LAP immunoreactivity
found in the distal tubules. Bar ¼ 50 lm.
1990 S. Masuda et al.(Eur. J. Biochem. 270) Ó FEBS 2003
prepared soluble and membrane fractions from the
NRK52E cells. We detected by Western blot analysis that
P-LAP was mainly associated with the membrane (parti-
culate) fraction as a  170 kDa protein (Fig. 2A). When the
culture medium was analyzed, a band with molecular mass
of  150 kDa was detected after extensive concentration,
indicating that a small portion of the enzyme was secreted
by the cell (data not shown) [5].
Figure 2B shows the immunocytochemical localization
of the P-LAP in the cell. Immunoreactivity was observed in
the cytoplasm, as a punctate pattern around the perinuclear
region. These results suggest that a major part of the enzyme
is associated with intracellular vesicles in NRK52E cells.
AVP-stimulated enhancement of cell surface P-LAP
It has been shown that insulin stimulates the translocation
of IRAP/P-LAP-associated GLUT-4 vesicles to the cell
surface in adipocytes [8,9]. Oxytocin, a substrate of P-LAP,
was also shown to stimulate the translocation of P-LAP
in HUVEC [10]. Therefore, we examined the stimulus-
mediated translocation of P-LAP in NRK52E cells. We
used AVP to stimulate the cells because it is cleaved by
P-LAP and plays an important role in the regulation of
water reabsorption in the renal collecting ducts, where

P-LAP is expressed [21].
Figure 3 shows the data from a representative experiment
on the AVP-induced translocation of P-LAP to plasma
membrane. The NRK52E cells treated with the hormone
and then surface-labeled with sulfo-NHS-SS-biotin were
analyzed by Western blot analysis. As shown in Fig. 3A,
AVP induced a significant increase in the cell surface P-LAP
immunoreactivity. Densitometric analysis indicates that at
0.1 and 1 l
M
, a 2.6-fold increase was observed. Dose-
response experiments indicate that the effect of AVP is first
detectable at a concentration of 10 n
M
(Fig. 3B), suggesting
that the hormone can mediate its effect at physiological
concentrations. In repeated experiments, AVP induced a
two- to threefold increase in the cell surface levels of P-LAP
in a dose-dependent manner. Kinetic analysis revealed that
the increase in the surface level of P-LAP was detectable at
30 min after AVP addition (Fig. 3C). We also examined the
AVP-mediated increase in the cell surface P-LAP activity
and found that AVP treatment caused a 1.8-fold increase in
the rate of hydrolysis of S-benzyl-Cys-MCA, a preferential
synthetic substrate for the enzyme (data not shown).
The role of vasopressin receptors in AVP-stimulated
translocation of P-LAP
AVP exerts its effects through three principal cell surface
receptors (V
1a

,V
1b
and V
2
) [22]. To elucidate the role of
vasopressin receptors, we examined the effects of vasopressin
agonists and antagonists on the hormone-induced translo-
cation of P-LAP in NRK52E cells (Table 1). The level of
P-LAP at the plasma membrane was about 2.1-fold greater
in response to the hormone. A comparable increase was also
observed when cells were treated with the specific V
2
receptor
agonist, DDAVP. On the other hand, the potent V
2
receptor
antagonist, AEAVP, completely suppressed the vasopressin-
induced translocation of the enzyme, while the V
1a
receptor
antagonist, 2MAVP, had no effect. These results suggest
that the V
2
receptor is involved in mediating the hormone-
induced translocation of P-LAP. Because a specific anta-
gonist to the V
1b
receptor is not available, we could not
estimate the contribution of this receptor at present.
In order to elucidate the mechanism of action further,

cells were treated with reagents known to increase intracel-
lular cAMP levels or PKC activity. The activation of V
2
receptors increases the intracellular cAMP, while activation
of V
1
receptors stimulates PKC [22]. As shown in Table 2,
DBcAMP and forskolin, which are known to induce the
increase in intracellular cAMP, caused an increase in the
surface level of P-LAP comparable to the level induced by
AVP, strongly suggesting that cAMP mediates the translo-
cation of P-LAP. From these results, it is plausible that in
NRK52E cells the activation of V
2
receptors by AVP causes
an increase in intracellular cAMP, which in turn mediates
the translocation of P-LAP. It is noteworthy to state that,
similar to the oxytocin-induced translocation of P-LAP in
HUVEC, PMA stimulation of PKC also caused an increase
in the cell surface P-LAP.
Different distribution of P-LAP and AQP-2
in NRK52E cells
It is well known that AVP regulates water reabsorption in
renal collecting duct principal cells by the cAMP-dependent
Fig. 2. Vesicular distribution of P-LAP in NRK52E cells. (A) Cells
were first lysed in a Dounce homogenizer and used as a total fraction
(T). The supernatant (S) and membrane (P) fractions were collected by
centrifugation. Samples were then subjected to Western blot analysis
employing affinity-purified anti-(P-LAP) Ig. In each fraction 25 lg
of protein was separated by SDS/PAGE. (B) Immunocytochemical

analysis was performed as described in Materials and methods
employing affinity-purified anti-(P-LAP) Ig. Bar ¼ 20 lm.
Ó FEBS 2003 Vasopressin-stimulated translocation of P-LAP (Eur. J. Biochem. 270) 1991
translocation of the water channel AQP-2 [21,23,24]. Similar
to P-LAP, translocation of AQP-2 is detected within 30 min
after the addition of vasopressin. Moreover, it has been
reported that as in GLUT-4-containing vesicles, leucine
aminopeptidase activity is associated with AQP-2-contain-
ing vesicles [25]. As we detected AQP-2 in NRK52E cells
by Western blot analysis, we examined whether AQP-2 and
P-LAP are colocalized in the same vesicle. For this purpose,
we conducted density gradient centrifugation. When cell
lysate was loaded on a sucrose gradient and centrifuged,
AQP-2 fractionated close to the top of the gradient,
suggesting the low-density nature of the AQP-2-containing
vesicles. On the other hand, P-LAP fractionated differently
and was recovered mainly in the high-density fractions
(Fig. 4). These data suggest that the majority of the intra-
cellular P-LAP is not associated with the AQP-2-containing
vesicles.
Discussion
Four mammalian membrane-bound aminopeptidases
belonging to the M1 family have been identified and
characterized [3,6,26–28]. Among them, P-LAP is unique in
its vesicular distribution and stimulus-dependent transloca-
tion from its intracellular compartment to the plasma
membrane [7–9,20,29], while the other three aminopeptid-
ases are localized primarily at the plasma membrane. Two
di-leucine motifs in the relatively long cytoplasmic domain
have been attributed to the vesicular distribution of the

enzyme [30].
Table 2. Intracellular cAMP enhances the surface level of P-LAP in
NRK52E cells. Cells were incubated with various compounds for
30 min at 37 °C and then biotinylated and analyzed as described in
Materials and methods.
Stimulus
Concentration
(l
M
)
Cell surface level of
P-LAP(% control ± SE)
None – 100
AVP 0.1 244.1 ± 30.0
DBcAMP 100 227.4 ± 33.7
Forskolin 100 229.1 ± 17.2
PMA 0.1 247.1 ± 38.9
Table 1. Effects of vasopressin V
2
receptor agonist and antagonist on the
surface level of P-LAP in NRK52E cells. Cells were treated with
agonists (AVP or DDAVP) for 30 min at 37 °C and then biotinylated
and analyzed as described in Materials and methods. Cells were pre-
treated with antagonists (2MAVP or AEAVP) 30 min before the
addition of AVP and further incubated for 30 min at 37 °Candthen
biotinylated.
Stimulus
Concentration
(l
M

)
Cell surface level of
P-LAP(% control ± SE)
None – 100
AVP 0.1 209.3 ± 41.4
DDAVP 0.1 210.0 ± 9.7
2MAVP + AVP 1.0 + 0.1 231.6 ± 7.9
AEAVP + AVP 1.0 + 0.1 66.1 ± 5.4
Fig. 3. AVP-stimulated enhancement of the surface level of P-LAP in
NRK52E cells. (A) NRK52E cells incubated with AVP for 30 min at
37 °C were biotinylated and analyzed as described in Materials and
methods. (B) Dose-response experiment of AVP-stimulated enhance-
ment of the surface level of P-LAP. Cells were treated with various
concentrations of AVP for 30 min at 37 °C and then biotinylated. (C)
Kinetic analysis of AVP-stimulated enhancement of the surface level of
P-LAP. Cells were treated with 100 n
M
of AVP for the times indicated
andthenbiotinylated.
1992 S. Masuda et al.(Eur. J. Biochem. 270) Ó FEBS 2003
In the current study we found that AVP stimulated the
translocation of P-LAP from the intracellular compartment
to the plasma membrane in NRK52E cells. This transloca-
tion was observed within 30 min, suggesting that the
reaction is independent from de novo synthesis of new
proteins. The specific V
2
receptor agonist, DDAVP, fully
stimulated the translocation of P-LAP, while the potent V
2

receptor antagonist, AEAVP, completely suppressed the
hormone-induced stimulation. These results clearly indicate
that V
2
receptors are involved in the signal transduction
pathway for P-LAP translocation. Moreover, reagents that
increase intracellular cAMP also activate the translocation
of P-LAP, indicating that AVP stimulates the transloca-
tion of P-LAP from the intracellular compartment to the
plasma membrane via a cAMP-dependent mechanism. As
immunohistochemical analysis revealed that P-LAP is
expressed in the renal distal tubules and collecting ducts, it
may be speculated that there is a vasopressin-mediated
translocation of P-LAP through V
2
receptors in these
regions of the kidney.
RT-PCR analysis indicated the expression of mRNAs for
both V
1a
and V
1b
receptors in NRK52E cells. As PMA
could stimulate the translocation in NRK52E cells, it is
possible that V
1
receptors are also involved in the trans-
location. However the possibility is unlikely, as V
2
receptor

antagonist could completely suppress the translocation.
Further studies are required to elucidate the role of V
1
receptors in the PKC-dependent translocation of P-LAP
employing other cell lines.
It is well known that AVP regulates water reabsorption in
renal collecting duct principal cells by activating the cAMP-
dependent translocation of AQP-2 from an intracellular
compartment to the apical plasma membranes [21,23,24].
Moreover, in the principal cells, it is also known that V
2
receptors are expressed in the basolateral plasma mem-
branes. These results indicate that the binding of the
hormone to V
2
receptors on the basolateral membrane of
principal cells stimulates cAMP synthesis, which leads to the
translocation of AQP-2 to the apical membrane [31].
Both P-LAP and AQP-2 are expressed in NRK52E
cells and translocated from the intracellular compartment
to the plasma membrane by AVP. Because it has been
reported that aminopeptidase activity is associated with
AQP-2-containing vesicles [25], we examined whether these
two proteins are colocalized in the same vesicles in NRK52E
cells. Our data shown in Fig. 4 indicates that the majority of
P-LAP-containing vesicles have a relatively higher density
when compared to AQP-2-containing vesicles, suggesting
that these two proteins are differently distributed in the
intracellular compartment of NRK52E cells. However, we
cannot rule out the possibility that these two proteins are

colocalized in some minor vesicle population, because it is
possible that the P-LAP-containing vesicles might be
heterogeneous. It has been reported that there are two
types of GLUT-4-containing vesicles in adipocytes, one that
is rich in P-LAP/IRAP and the other that is not [32]. It is
known that although AQP-2 is abundant in the apical
plasma membrane and apical vesicles in the collecting duct
principal cells, some AQP-2 is associated with the baso-
lateral plasma membrane [21]. These results also suggest the
presence of a minor population of AQP-2-containing
vesicles.
Taken together, we suggest the biological relevance of
our findings as follows. The binding of AVP to V
2
receptors on the renal collecting duct principal cells
stimulates cAMP-dependent translocation of P-LAP to
the basolateral plasma membrane. Translocated P-LAP
might then facilitate the degradation of excessive vaso-
pressin, acting as a negative feedback mechanism of AVP
action on the regulation of water reabsorption. As P-LAP
is the receptor for angiotensin IV [18], it is also possible
that P-LAP facilitates the enhancement of superficial
cortical blood flow in renal tubules after translocation to
the plasma membrane [33,34]. To understand the role of
P-LAP in the kidney, further studies are required to
determine the actual movement of the vesicles containing
P-LAP employing more distinctive renal cells such as the
primary cell culture model [35].
In summary, we have shown that AVP stimulates the
translocation of P-LAP from the intracellular compartment

to the plasma membrane in NRK52E cells. The effect is
mainly due to V
2
receptor activation and is mediated by
cAMP-dependent signaling. As both AQP-2 and P-LAP are
expressed in renal collecting ducts and both are translocated
by AVP, our findings should have some physiological
relevance.
Acknowledgements
We are grateful to Dr D. G. Hunt of the Experimental Diabetes,
Metabolism and Nutrition Section, DB/NIDDK/NIH for careful
reading of the manuscript and helpful suggestions. We also thank Prof
N. Yamanaka and Dr A. Shimizu of the Department of Pathology,
Nippon Medical School, for advice concerning the immunohisto-
chemical analyses.
This work was supported in part by Grants-in-Aid from the
Ministry of Education, Science, Sports and Culture of Japan and a
grant for ÔChemical Biology Research ProgramÕ from RIKEN.
References
1. Mizutani, S. & Tomoda, Y. (1992) Oxytocinase: cystine amino-
peptidase or placental leucine aminopeptidase (P-LAP). Semin.
Reprod. Endocrinol. 10, 146–153.
2. Mizutani, S. & Tomoda, Y. (1996) Effects of placental proteases
on maternal and fetal blood pressure in normal pregnancy and
preeclampsia. Am. J. Hypertens. 9, 591–597.
Fig. 4. Sucrose density gradient analysis of P-LAP- and AQP-2-con-
taining vesicles. A postnuclear supernatant of NRK52E cells was
prepared as described in Materials and methods. The sample was
fractionated in a 10–50% sucrose gradient at 48 000 r.p.m. in a
Hitachi P50S2 rotor for 50 min. Fractions were collected starting from

the top of the gradient. Equal-volume of the fractions was subjected to
SDS/PAGE and P-LAP and AQP-2 proteins were detected by West-
ern blot analysis.
Ó FEBS 2003 Vasopressin-stimulated translocation of P-LAP (Eur. J. Biochem. 270) 1993
3. Rogi, T., Tsujimoto, M., Nakazato, H., Mizutani, S. & Tomoda,
Y. (1996) Human placental leucine aminopeptidase/oxytocinase: a
new member of type II membrane-spanning zinc metallopeptidase
family. J. Biol. Chem. 271, 56–61.
4. Iwase, A., Nomura, S. & Mizutani, S. (2001) Characterization of a
secretase activity for placental leucine aminopeptidase. Arch.
Biochem. Biophys. 393, 163–169.
5. Ofner, L.D. & Hooper, N.M. (2002) Ectodomain shedding
of cystinyl aminopeptidase from human placental membranes.
Placenta 23, 65–70.
6. Keller,S.R.,Scott,H.M.,Mastick,C.C.,Aebersold,R.&Lien-
hard, G. (1995) Cloning and characterization of a novel insulin-
regulated membrane aminopeptidase from GLUT-4 vesicles.
J. Biol. Chem. 270, 23612–23618.
7. Ross,S.A.,Scott,H.M.,Morris,N.J.,Leung,W Y.,Mao,F.,
Lienhard, G.E. & Keller, S. (1996) Characterization of the insulin-
regulated membrane aminopeptidase in 3T3-L1 adipocytes. J. Biol.
Chem. 271, 3328–3332.
8. Waters, S.B., D’Auria, M., Martin, S.S., Nguyen, C.M., Kozuma,
L.M. & Luskey, K.L. (1997) The aminoterminus of insulin-
responsive aminopeptidase causes GLUT-4 translocation in
3T3-L1 adipocytes. J. Biol. Chem. 272, 23323–23327.
9. Kandror, K.V. (1999) Insulin regulation of protein traffic in rat
adipose cells. J. Biol. Chem. 274, 25210–25217.
10.Nakamura,H.,Itakura,A.,Okamura,M.,Ito,M.,Iwase,A.,
Nakanishi, Y., Okada, M., Nagasaka, T. & Mizutani, S. (2000)

Oxytocin stimulates the translocation of oxytocinase of human
vascular endothelial cells via activation of oxytocin receptors.
Endocrinology 141, 4481–4485.
11. Matsumoto, H., Nagasaka, T., Hattori, A., Rogi, T., Tsuruoka,
N., Mizutani, S. & Tsujimoto, M. (2001) Expression of placental
leucine aminopeptidase/oxytocinase in neuronal cells and its
action on neuronal peptides. Eur. J. Biochem. 268, 3259–3266.
12. Reaux,A.,Fournie-Zaluski,M.C.,David,C.,Zini,S.,Roques,B.,
Corvol, P. & Llorens-Cortes, C. (1999) Aminopeptidase A
inhibitors as potential central antihypertensive agents. Proc. Natl
Acad. Sci. USA 96, 13415–13420.
13. Hattori, A., Kitatani, K., Matsumoto, H., Miyazawa, S., Rogi, T.,
Tsuruoka, N., Mizutani, S., Natori, Y. & Tsujimoto, M. (2000)
Characterization of recombinant human adipocyte-derived leu-
cine aminopeptidase expressed in Chinese hamster ovary cells.
J. Biochem. (Tokyo) 128, 755–762.
14. Yamamoto, N., Nakayama, J., Kobayashi-Yamakawa, K.,
Hamaguchi, H., Miyazaki, R. & Arinami, T. (2002) Identification
of 33 polymorphism in the adipocyte-derived leucine amino-
peptidase (ALAP) gene and possible association with hyperten-
sion. Hum. Mutat. 19, 251–257.
15. Pasquqlini,R.,Koivunen,E.,Kain,R.,Lahdenranta,J.,Saka-
moto,M.,Stryhn,A.,Ashmun,R.A.,Sapiro,L.,Arap,W.&
Ruoslahti, E. (2000) Aminopeptidase N is a receptor for tumor-
homing peptides and a target for inhibiting angiogenesis. Cancer
Res. 60, 722–727.
16. Miyashita, H., Yamazaki, T., Akada, T., Niizeki, O., Ogata, M.,
Nishikawa, S. & Sato, Y. (2002) A mouse orthologue of puro-
mycin-insensitive leucyl-specific aminopeptidase is expressed in
endothelial cells and plays an important role in angiogenesis.

Blood 99, 3241–3249.
17. Serwold,T.,Gonzalez,F.,Kim,J.,Jacob,R.&Shastri,N.(2002)
ERAAPcustomizespeptidesforMHCclassImoleculesinthe
endoplasmic reticulum. Nature 419, 480–483.
18.Saric,T.,Chang,S C.,Hattori,A.,York,I.A.,Markant,S.,
Rock, K.L., Tsujimoto, M. & Goldberg, A.L. (2002) An
IFN-c-induced aminopeptidase in the ER, ERAP I, trims
precursors to MHC class I-presented peptides. Nature Immunol. 3,
1169–1176.
19. Albiston, A.L., McDowall, S.G., Matsacos, D., Sim, P., Clune, E.,
Mustafa, T., Lee, J., Mendelsohn, F.A.O., Simpson, R.J., Conn-
olly, L.M. & Chai, S.Y. (2001) Evidence that the angiotensin IV
(AT
4
) receptor is the enzyme insulin-regulated aminopeptidase.
J. Biol. Chem. 276, 48623–48626.
20. Matsumoto, H., Rogi, T., Yamashiro, K., Kodama, S., Tsuruoka,
N., Hattori, A., Takio, K., Mizutani, S. & Tsujimoto, M. (2000)
Characterization of a recombinant soluble form of human pla-
cental leucine aminopeptidase/oxytocinase expressed in Chinese
hamster ovary cells. Eur. J. Biochem. 267, 46–52.
21. Nielsen,S.,Frokiar,J.,Marples,D.,Kwon,T H.,Agre,P.&
Knepper, M.A. (2002) Aquaporins in the kidney: from molecules
to medicine. Physiol. Rev. 82, 205–244.
22. Birnbaumer, M. (2000) Vasopressin receptors. Trends Endocrinol.
Metab. 11, 406–410.
23. Nielsen,S.,Chou,C L.,Marples,D.,Christensen,E.I.,Kishore,
B.K. & Knepper, M.A. (1995) Vasopressin increases water per-
meability of kidney collecting duct by inducing translocation of
aquaporin-CD water channels to plasma membrane. Proc. Natl

Acad. Sci. USA 92, 1013–1017.
24. Valenti,G.,Frigeri,A.,Ronco,P.M.,D’Ettorre,C.&Svelto,M.
(1996) Expression and functional analysis of water channels in a
stably AQP2-transfected human collecting duct cell line. J. Biol.
Chem. 271, 24365–24370.
25. Harris, H.W., Zeidel, M.L., Jo, I. & Hammond, T.G. (1994)
Characterization of purified endosomes containing the antidiuretic
hormone-sensitive water channel from rat renal papilla. J. Biol.
Chem. 269, 11993–20000.
26. Wu,Q.,Lahti,J.M.,Air,G.M.,Burrows,P.D.&Cooper,M.D.
(1990) Molecular cloning of the murine BP-1/6C3 antigen: a
member of the zinc-dependent metallopeptidase family. Proc. Natl
Acad. Sci. USA 87, 993–997.
27. Olsen, J., Cowell, G.M., Konigsofer, E., Danielsen, M., Moller, J.,
Laustsen, L., Hansen, O.C., Welinder, K., Engberg, J., Hunzilker,
W.,Spiess,M.,Sjostrom,H.&Noren,O.(1988)Completeamino
acid sequence of human intestinal aminopeptidase N as deduced
from cloned cDNA. FEBS Lett. 238, 307–314.
28. Schauder, B., Schmburg, L., Kohler, J. & Bauer, K. (1994)
Cloning of a cDNA encoding an ectoenzyme that degrades
thyrotropin-releasing hormone. Proc. Natl Acad. Sci. USA 91,
9534–9538.
29. Thoidis, G. & Kandror, K.V. (2001) A Glut-4-vesicle marker
protein, insulin-responsive aminopeptidase, is localized in a novel
vesicular compartment in PC 12 cells. Traffic 2, 577–587.
30. Johnson, A.O., Lampson, M.A. & McGraw, T.E. (2001) A
di-leucine sequence and a cluster of acidic amino acids are required
for dynamic retention in the endosomal recycling compartment of
fibroblasts. Mol. Biol. Cell. 12, 367–381.
31. Laycock, J.F. & Hanoune, J. (1998) From vasopressin receptor

to water channel: intracellular traffic, constraint and by-pass.
J. Endocrinol. 159, 361–372.
32. Kupriyanova, T.A., Kandror, V. & Kandror, K.V. (2002) Iso-
lation and characterization of the two major intracellular Glut4
storage compartments. J. Biol. Chem. 277, 9133–9138.
33. Coleman,J.K.M.,Krebs,I.T.,Hamilton,T.A.,Ong,B.,Law-
rence, K.A., Sardinia, M.F., Harding, J.W. & Wright, J.W. (1998)
Autoradiographic identification of kidney angiotensin IV binding
sites and angiotensin IV-induced renal cortical blood flow changes
in rats. Peptides 19, 269–277.
34. Handa, R.K. (2001) Characterization and signaling of the AT
4
receptor in human proximal tubule epithelial (HK-2) cells. J. Am.
Soc. Nephrol. 12, 440–449.
35. Maric, K., Oksche, A. & Rosenthal, W. (1998) Aquaporin-2
expression in primary cultured rat inner medullary collecting duct
cells. Am. J. Physiol. 275, F796–F801.
1994 S. Masuda et al.(Eur. J. Biochem. 270) Ó FEBS 2003