Involvement of the V2 receptor in vasopressin-stimulatedtranslocation of placental leucine aminopeptidase/oxytocinase in renal cells Shinako Masuda1, Akira Hattori1, Hideko Matsumoto1,*,
Trang 1Involvement of the V2 receptor in vasopressin-stimulated
translocation of placental leucine aminopeptidase/oxytocinase
in renal cells
Shinako Masuda1, Akira Hattori1, Hideko Matsumoto1,*, Shinobu Miyazawa2, Yasuhiro Natori2,
Shigehiko Mizutani3and Masafumi Tsujimoto1
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 V2receptor agonist, increased
the surface level of P-LAP, while [adamantaneacetyl1,
O-Et-D-Tyr2, Val4, aminobutyryl6, Arg8,9]-vasopressin
(AEAVP), a potent V2 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 V2receptor 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)18E 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: tsujimot@postman.riken.go.jp
Abbreviations
1 : AEAVP, [adamantaneacetyl1, O-Et-D-Tyr2, Val4,
aminobutyryl6, Arg8,9]-vasopressin; AQP, aquaporin; AVP,
arginine-vasopressin; 2MAVP,
cyclopentamethy-lenepropionyl1,O-me-Tyr2,Arg8]-vasopressin; DBcAMP,
N6,2¢-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)
Trang 2endothelial 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), N6, 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)1rabbit 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 mM 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 mMTris pH 7.8, 1% Nonidet P-40, 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 10 lgÆmL)1aprotinin) 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 4C.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)1of 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/Piand fixed with 2% paraformaldehyde in NaCl/Pi for 20 min at room temperature.Cells were permeabilized in NaCl/Pisolution containing 0.3% Triton X-100 for 5 min.Coverslips were blocked for 1 h with NaCl/Picontaining 2% horse serum (blocking buffer), and incubated for 1.5 h at 27C with 5 lgÆmL)1 affinity-purified anti-(P-LAP) Ig in blocking buffer.The cells were then washed three times with NaCl/Piand incubated with 0.5 lgÆmL)1AlexaFluor-488-labeled goat anti-(rabbit IgG)
Ig in blocking buffer for 1 h.After washing with NaCl/Pi 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 4C.NRK52E cells were grown in 100-mm-diameter dishes and washed three times
in ice-cold Krebs-Ringer phosphate Hepes (KRPH,
128 mM NaCl, 4.7 mM KCl, 1.25 mM CaCl2, 1 25 mM MgSO4, 5 mMNaPO4, 20 mMHepes, pH 7.4) and treated with 2 5 mL of 0 5 mgÆmL)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 mMTris, pH 7.8, 1% Nonidet P-40, 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100,
10 lgÆmL)1aprotinin).After determination of the protein concentration, cell lysate containing 100 lg of protein was diluted to 300 lL with lysis buffer, added 15 lL of 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
Trang 3washed 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 usingIMAGE GAUGEsoftware (Fuji
Photo Film Co., Tokyo, Japan) Results are expressed as the
means ± SE (n¼ 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 lL of 20 mM Tris/
HCl buffer (pH 7.5) containing 1 mMEDTA was incubated
at 37C.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 4C with NaCl/Pi.Cells were then scraped into 400 lL
of buffer A (150 mMNaCl, 10 mMHepes, pH 7.4, 1 mM
EGTA, 0.1 mM MgCl2) in the presence of protease
inhibitors (1 mM 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 lL of
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
4C.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)1of 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.
Trang 4prepared 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 lM, a 2.6-fold increase was observed Dose-response experiments indicate that the effect of AVP is first detectable at a concentration of 10 nM(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 (V1a, V1band V2) [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 V2receptor agonist, DDAVP.On the other hand, the potent V2receptor antagonist, AEAVP, completely suppressed the vasopressin-induced translocation of the enzyme, while the V1areceptor antagonist, 2MAVP, had no effect.These results suggest that the V2receptor is involved in mediating the hormone-induced translocation of P-LAP.Because a specific anta-gonist to the V1b 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 V2 receptors increases the intracellular cAMP, while activation
of V1receptors 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 V2receptors 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.
Trang 5translocation 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)
DBcAMP 100 227.4 ± 33.7 Forskolin 100 229.1 ± 17.2
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 C and then biotinylated.
Stimulus
Concentration (l M )
Cell surface level of P-LAP(% control ± SE)
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
and then biotinylated.
Trang 6In 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 V2 receptor agonist, DDAVP, fully
stimulated the translocation of P-LAP, while the potent V2
receptor antagonist, AEAVP, completely suppressed the
hormone-induced stimulation.These results clearly indicate
that V2 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 V2 receptors in these
regions of the kidney
RT-PCR analysis indicated the expression of mRNAs for
both V1a and V1b receptors in NRK52E cells.As PMA
could stimulate the translocation in NRK52E cells, it is
possible that V1 receptors are also involved in the
trans-location.However the possibility is unlikely, as V2receptor
antagonist could completely suppress the translocation
Further studies are required to elucidate the role of V1
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 V2
receptors are expressed in the basolateral plasma
mem-branes.These results indicate that the binding of the
hormone to V2receptors 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 V2 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 V2receptor 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 Chemical Biology Research Program from RIKEN. References
1.Mizutani, S.& Tomoda, Y.(1992) Oxytocinase: cystine amino-peptidase or placental leucine aminoamino-peptidase (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.
Trang 73 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)
ERAAP customizes peptides for MHC class I molecules in the
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) Complete amino 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.