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: tsujimot@postman.riken.go.jp 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. 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