Expression of placental leucine aminopeptidase/oxytocinase in neuronal cells and its action on neuronal peptides.

The placental leucine aminopeptidase (P-LAP)/oxytocinase whose serum level increases with gestation is thought to contribute to the maintenance of normal pregnancy. P-LAP mRNAs are expressed in various tissues other than the placenta. In this study, we identified P-LAP protein in the brain. In contrast with the placenta where a significant portion of P-LAP is released, the enzyme was localized in the membrane fraction in brain and PC12 cells and no soluble form of the enzyme was detected. When PC12 cells were differentiated into neuronal cells by nerve growth factor (NGF), a significant increase in the expression level of P-LAP in the cell was observed. As in the case of insulin treatment of 3T3-L1 adipocytes, treatment of PC12 cells with forskolin caused the translocation of the enzyme from intracellular vesicle to the cell surface plasma membrane. In addition, P-LAP was shown to degrade several bioactive neuropeptides such as Met-enkephalin and dynorphin A (1-8). These results suggest that P-LAP plays an important role in the regulation of neuronal cell function in the brain.     

Tissue distribution of placental leucine aminopeptidase/oxytocinase during mouse pregnancy.

Placental leucine aminopeptidase (P-LAP), also called oxytocinase, is an enzyme responsible for hydrolyzing oxytocin. This enzyme is identical to cystine aminopeptidase. We examined the tissue distribution of P-LAP in normal adult mice and also in mothers and fetuses during mouse pregnancy using immunohistochemical (IHC) analysis. P-LAP-immunoreactive protein was expressed in various organs in a cell- and gestational stage-dependent manner. In the kidney, P-LAP was located in distal and collecting tubules but not in proximal tubules. The islet of Langerhans in the maternal pancreas stained positively for P-LAP in the periphery in early gestation. This staining pattern changed so that both the periphery and inner cells were positive during mid-gestation and finally only inner cells were positive in late gestation. Among the hematopoietic cells in the fetal liver, only megakaryocytes showed strong expression of P-LAP. The staining intensity increased with gestation on the apical surface of trophoblasts in the placental labyrinth. These data demonstrate that P-LAP is present in a variety of tissues, and its presence is affected by pregnancy and fetal development. Therefore, P-LAP may play a significant role in various physiological processes in non-pregnant, pregnant, and fetal mice.     

Characterization of a recombinant soluble form of human placental leucine aminopeptidase/oxytocinase expressed in Chinese hamster ovary cells.

Serum levels of human placental leucine aminopeptidase/oxytocinase (P-LAP) increase with gestation. cDNA cloning of P-LAP revealed that the enzyme is a type II membrane-bound protein containing the consensus HEXXH(X)18E motif found in the M1 family of zinc-metallopeptidase proteins. In this study, a recombinant soluble form of P-LAP found in maternal serum was expressed in Chinese hamster ovary cells, purified to homogeneity and then characterized. Although N-terminal sequencing revealed a four-amino-acid deletion, the purified enzyme was active and was shown to be a zinc-containing homodimeric protein with molecular mass of 280 kDa in solution. Using artificial substrates, it was shown that the enzyme has broad specificity and is inhibited by several compounds known as aminopeptidase inhibitors. Subsequently, sequential N-terminal amino-acid liberation of several peptide hormones by the enzyme was monitored and structures of the products were determined. Among the hormones having a cysteine residue at their N-terminal end and intramolecular disulfide bonds, it was found that vasopressin and oxytocin, but not calcitonin and endothelins, were cleaved by the enzyme. Because the molecular properties of oxytocinase so far reported often conflict, our results provide an initial biochemical and enzymatic characterization of moleculary defined P-LAP/oxytocinase.     

Expression of glucose transporter 4 in the human pancreatic islet of Langerhans

Glucose transporter 4 (GLUT4) is the main insulin-responsive glucose transporter in skeletal muscle and adipose tissue of human and rodent, and is translocated to the plasma membrane in response to insulin. GLUT2 is well known as the main glucose transporter in pancreatic islets and could highly regulate glucose-stimulated insulin secretion by B-cells as a glucose sensor. We confirmed the presence of GLUT4 mRNA and GLUT4 protein in pancreas in the human. Indirect immunohistochemistry showed that the pancreatic islets of human and rat were conspicuously labeled by anti-GLUT4 antibody. The presence of placental leucine aminopeptidase (P-LAP), a homologue of insulin-regulated aminopeptidase (IRAP), was also shown in the human pancreatic islet. IRAP/P-LAP is thought to be involved in glucose metabolism. This study provides the first evidence that GLUT4 is present in human and rat pancreatic islets and may suggest its specific role in glucose homeostasis in conjunction with IRAP/P-LAP.     

Oxytocinase/insulin-regulated aminopeptidase is distributed throughout the sheep, female reproductive tract and is regulated by oestrogen in the uterus

Using [(125)I]Angiotensin IV (Ang IV) for the autoradiographic localisation of oxytocinase/insulin-regulated aminopeptidase (IRAP), we demonstrate for the first time that IRAP is distributed throughout the female reproductive tract. The highest concentration of IRAP was detected in the outer myometrial layer of the uterus with lower levels in the inner myometrial layer and in the luminal epithelium. High levels of the enzyme was also detected in the inner mucosal lining of the ampulla segment of the fallopian tubes with lower levels in the interstitial and isthmus. In the ovary, a high level of IRAP was found in the corpus albicans with lower levels throughout the ovarian cortex and the surrounding connective tissue. In the uterine body of ovariectomised (OVX) ewes, oestrogen treatment resulted in a significant decrease (P<0.05) in the level of IRAP in the outer myometrium. These findings indicate an important role for IRAP in reproductive physiology in regulating the action of peptide hormones.     

ADAMs, a disintegrin and metalloproteinases, mediate shedding of oxytocinase

Placental leucine aminopeptidase (P-LAP), a type-II transmembrane protease responsible for oxytocin degradation during pregnancy, is converted to a soluble form through proteolytic cleavage. The goal of this study was to determine the nature of the P-LAP secretase activity. The hydroxamic acid-based metalloprotease inhibitors GM6001 and ONO-4817 as well as the TNF-alpha protease inhibitor-2 (TAPI-2) reduced P-LAP release, while tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2, which are matrix metalloproteinase inhibitors, had no effect on P-LAP release in Chinese hamster ovary (CHO) cells stably overexpressing P-LAP, thus indicating possible involvement of ADAM (a disintegrin and metalloproteinase) members in P-LAP shedding. Furthermore, overexpression of ADAM9 and ADAM12 increased P-LAP release in P-LAP-CHO transfectants. Immunohistochemical analysis in human placenta demonstrated strong expression of ADAM12 in syncytiotrophoblasts, while little expression of ADAM9 was detected throughout the placenta. Our results suggest ADAM members, at least including ADAM12, are involved in P-LAP shedding in human placenta.     

Vasopressin is a physiological substrate for the insulin-regulated aminopeptidase IRAP

Insulin-regulated aminopeptidase (IRAP) is a membrane aminopeptidase and is homologous to the placental leucine aminopeptidase, P-LAP. IRAP has a wide distribution but has been best characterized in adipocytes and myocytes. In these cells, IRAP colocalizes with the glucose transporter GLUT4 to intracellular vesicles and, like GLUT4, translocates from these vesicles to the cell surface in response to insulin. Earlier studies demonstrated that purified IRAP cleaves several peptide hormones and that, concomitant with the appearance of IRAP at the surface of insulin-stimulated adipocytes, aminopeptidase activity toward extracellular substrates increases. In the present study, to identify in vivo substrates for IRAP, we tested potential substrates for cleavage by IRAP-deficient (IRAP(-/-)) and control mice. We found that vasopressin and oxytocin were not processed from the NH(2) terminus by isolated IRAP(-/-) adipocytes and skeletal muscles. Vasopressin was not cleaved from the NH(2) terminus after injection into IRAP(-/-) mice and exhibited a threefold increased half-life in the circulation of IRAP(-/-) mice. Consistent with this finding, endogenous plasma vasopressin levels were elevated twofold in IRAP(-/-) mice, and vasopressin levels in IRAP(-/-) brains, where plasma vasopressin originates, showed a compensatory decrease. We further established that insulin increased the clearance of vasopressin from control but not from IRAP(-/-) mice. In conclusion, we have identified vasopressin as the first physiological substrate for IRAP. Changes in plasma and brain vasopressin levels in IRAP(-/-) mice suggest a significant role for IRAP in regulating vasopressin. We have also uncovered a novel IRAP-dependent insulin effect: to acutely modify vasopressin.     

Enhanced recombinant expression and purification of human IRAP for biochemical and crystallography studies

Insulin-regulated aminopeptidase (IRAP) in humans is a membrane bound enzyme that has multiple functions. It was first described as a companion protein of the insulin-responsive glucose transporter, Glut4, in specialized vesicles. The protein has subsequently been shown to be identical to the oxytocinase/aminopeptidase or the angiotensin IV (Ang IV) receptor (AT₄ receptor). Some AT₄ ligand peptides, such as Ang IV and LVV-hemorphin-7, have been shown to act as IRAP inhibitors that exert memory-enhancing properties. As such IRAP has been a target for developing cognitive enhancers. To facilitate detailed mechanistic studies of IRAP catalysis and inhibition, and to pave the way for biophysical and structural studies of IRAP in complex with peptide inhibitors, we report here an optimized expression and purification system using High Five insect cells. We also report biochemical characterizations of the purified recombinant IRAP with a standard aminopeptidase substrate and an optimized IRAP peptide inhibitor with a Ki of 98 nM.     

Distribution of adipocyte-derived leucine aminopeptidase (A-LAP)/ER-aminopeptidase (ERAP)-1 in human uterine endometrium.

Adipocyte-derived leucine aminopeptidase (A-LAP, endoplasmic reticulum aminopeptidase ERAP1) is specialized to produce peptides presented on the class I major histocompatibility complex (MHC) by trimming epitopes to eight or nine residues, in addition to its enzymatic activity to degrade angiotensin II. Previously we identified placental leucine aminopeptidase (P-LAP), another member of the oxytocinase subfamily of aminopeptidases, in human uterine endometrial epithelial cells. Here we analyzed the distribution of A-LAP in human cyclic endometrium. Western blotting analysis showed that A-LAP was present in the endometrial tissue throughout the menstrual cycle. Immunohistochemical (IHC) analysis of A-LAP showed a similar distribution to that of P-LAP. A-LAP was localized predominantly in the endometrial glands and the luminal surface epithelium. However, the intracellular localization change that accompanied apocrine secretion, which was observed in P-LAP, was not shown in A-LAP. Subcellular localization of A-LAP, demonstrated by immunofluorescence, was ER in the cultured glandular epithelial cells. Our results indicate that A-LAP may fit the endometrial localization as an antigen-presenting ER aminopeptidase. Further understanding of the function(s) of A-LAP in the endometrium appear likely to yield insights into the cyclic changes during the normal endometrial cycle.     

Involvement of the V2 receptor in vasopressin-stimulated translocation of placental leucine aminopeptidase/oxytocinase in renal cells.

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 presence of P-LAP protein in the renal distal tubules and collecting 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]-vasopressin (DDAVP), a specific V2 receptor 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 translocation of P-LAP via V2 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.     

Relationship between the changes in placental blood flow resistance assessed by Doppler technique and maternal serum placental aminopeptidases, which degrade vaso-active peptides, in pre-eclampsia.

Our study showed that there were statistically significant correlations between the systolic and diastolic ratio (S/D) of maternal uterine or umbilical artery and the levels of maternal serum aminopeptidase activities in pre-eclampsia. Kininase I was positively correlated with the S/D ratios, whereas placental leucine aminopeptidase (P-LAP) and aminopeptidase A were negatively correlated with the S/D ratios. It is known that the increased S/D ratios reflect the increased utero-placental blood flow resistance. Since our previous study showed that placental aminopeptidases degrade vasoactive peptides such as oxytocin, angiotensin and bradykinin, which the fetus actively produces, our present study suggests that the increased vascular resistance in feto-placental circulation in pre-eclampsia is partly controlled by changes in vaso-active peptides, via degradation by placental aminopeptidases.     

Adhesive and degradative properties of human placental cytotrophoblast cells in vitro

Human fetal development depends on the embryo rapidly gaining access to the maternal circulation. The trophoblast cells that form the fetal portion of the human placenta have solved this problem by transiently exhibiting certain tumor-like properties. Thus, during early pregnancy fetal cytotrophoblast cells invade the uterus and its arterial network. This process peaks during the twelfth week of pregnancy and declines rapidly thereafter, suggesting that the highly specialized, invasive behavior of the cytotrophoblast cells is closely regulated. Since little is known about the actual mechanisms involved, we developed an isolation procedure for cytotrophoblasts from placentas of different gestational ages to study their adhesive and invasive properties in vitro. Cytotrophoblasts isolated from first, second, and third trimester human placentas were plated on the basement membrane-like extracellular matrix produced by the PF HR9 teratocarcinoma cell line. Cells from all trimesters expressed the calcium-dependent cell adhesion molecule cell-CAM 120/80 (E-cadherin) which, in the placenta, is specific for cytotrophoblasts. However, only the first trimester cytotrophoblast cells degraded the matrices on which they were cultured, leaving large gaps in the basement membrane substrates and releasing low molecular mass 3H-labeled matrix components into the medium. No similar degradative activity was observed when second or third trimester cytotrophoblast cells, first trimester human placental fibroblasts, or the human choriocarcinoma cell lines BeWo and JAR were cultured on radiolabeled matrices. To begin to understand the biochemical basis of this degradative behavior, the substrate gel technique was used to analyze the cell-associated and secreted proteinase activities expressed by early, mid, and late gestation cytotrophoblasts. Several gelatin-degrading proteinases were uniquely expressed by early gestation, invasive cytotrophoblasts, and all these activities could be abolished by inhibitors of metalloproteinases. By early second trimester, the time when cytotrophoblast invasion rapidly diminishes in vivo, the proteinase pattern of the cytotrophoblasts was identical to that of term, noninvasive cells. These results are the first evidence suggesting that specialized, temporally regulated metalloproteinases are involved in trophoblast invasion of the uterus. Since the cytotrophoblasts from first trimester and later gestation placentas maintain for several days the temporally regulated degradative behavior displayed in vivo, the short-term cytotrophoblast outgrowth culture system described here should be useful in studying some of the early events in human placen     

A new approach regarding the treatment of preeclampsia and preterm labor

Both preeclampsia and preterm delivery are important complications in pregnancy and are leading causes for maternal and perinatal morbidity and mortality. The underlying molecular mechanisms of both diseases remain unknown, thus treatments (beta2-stimulants and magnesium sulfate) are essentially symptomatic. Both molecules have molecular weights less than 5-8 kDa and cross the placental barrier thus exerting their effects on the fetus. In addition, the fetus produces peptide hormones that are highly vasoactive and uterotonic and increase in response to maternal stress and with continued development. Fetal peptides are also small molecules that inevitably leak across into the maternal circulation. Aminopeptidases such as placental leucine aminopeptidase (P-LAP) and aminopeptidase A (APA) are large molecules that do not cross the placental barrier. We have shown that APA acts as an antihypertensive agent in the pregnant spontaneously hypertensive rat by degrading vasoactive peptides and as a result returns the animal to a normotensive state. We have also noted that P-LAP acts as an anti-uterotonic agent by degrading uterotonic peptides, and as a result prolongs gestation in the pregnant mouse. Thus, P-LAP and APA represent promising agents for the treatment of preeclampsia and preterm labor by degrading bioactive hormones derived from the feto-placental circulation.     

Insulin stimulates placental leucine aminopeptidase/oxytocinase/insulin-regulated membrane aminopeptidase expression in BeWo choriocarcinoma cells

Placental leucine aminopeptidase (P-LAP), a cystine aminopeptidase that is identical to insulin-regulated membrane aminopeptidase, hydrolyzes oxytocin, which results in the loss of oxytocin activity. We previously isolated genomic clones containing the human P-LAP promoter region, which included two sites homologous to the 10-bp-insulin responsive element (IRE) that was identified on the phosphoenolpyruvate carboxinase gene. We therefore postulated that insulin regulates P-LAP expression via these IREs and investigated this notion using BeWo choriocarcinoma trophoblastic cells cultured in the presence of insulin. Insulin increased P-LAP activity in a time- and dose-dependent manner. Physiological concentrations of insulin at 10(-7) M exhibited the most potent effect on P-LAP activity. Western blotting demonstrated that 10(-7) M insulin increased P-LAP protein levels. Semi-quantitative RT-PCR and Southern blotting showed that insulin also increased P-LAP mRNA, which was abrogated by prior exposure to cycloheximide. Luciferase assay did not reveal any regulatory regions within 1.1 kb upstream of the P-LAP gene that could explain the insulin-induced P-LAP mRNA accumulation. These findings indicate that insulin induces P-LAP expression in trophoblasts, and that it acts via de novo synthesis of other proteins, which partially contradicts our initial hypothesis.     

Molecular cloning of adipocyte-derived leucine aminopeptidase highly related to placental leucine aminopeptidase/oxytocinase

In the current study, we report the cloning and initial characterization of a novel human cytosolic aminopeptidase named adipocyte-derived leucine aminopeptidase (A-LAP). The sequence encodes a 941-amino acid protein with significant homology (43%) to placental leucine aminopeptidase (P-LAP)/oxytocinase. The predicted A-LAP contains the HEXXH(X)18E consensus sequence, which is characteristic of the M1 family of zinc-metallopeptidases. Although the deduced sequence contains a hydrophobic region near the N-terminus, the enzyme localized mainly in cytoplasm when expressed in COS-7 cells. Northern blot analysis revealed that A-LAP was expressed in all the tissues tested, some of which expressed at least three forms of mRNA, suggesting that the regulation of the gene expression is complex. When aminopeptidase activity of A-LAP was measured with various synthetic substrates, the enzyme revealed a preference for leucine, establishing that A-LAP is a novel leucine aminopeptidase with restricted substrate specificity. The identification of A-LAP, which reveals strong homology to P-LAP, might lead to the definition of a new subfamily of zinc-containing aminopeptidases belonging to the M1 family of metallopeptidases.