Receptor-specific ligands distinguish natriuretic peptide receptors-A and -C in primate tissues

Systemic clearance of atrial natriuretic peptide (ANP) is in part due to neutral endopeptidase (NEP) proteolysis and natriuretic peptide receptor-C (NPR-C) mediated endocytosis. Biological responses to ANP are primarily mediated by the membrane guanylyl cyclase-A/natriuretic peptide receptor-A (NPR-A). Analogs of ANP selective for NPR-A and/or resistant to NEP may have increased activity in those tissues where NPR-C and NEP are coexpressed with NPR-A. The analog of ANP termed vANP; [(R3D, G9T, R11S, M12L, G16R)ANP] is selective for human NPR-A with at least 10,000 fold reduction in affinity for human NPR-C. We report that rat NPR-A is insensitive to 10 nM vANP, demonstrating the limitations of this species in evaluating human therapeutic candidates. As an alternative approach we tested the binding and potency of receptor-selective and NEP-resistant ANP analogs in rhesus monkey tissues. Competition binding studies with a simplified version of vANP, sANP [(G9T, R11S, G16R)rANP], in rhesus monkey kidney and lung membrane preparations shows displacement of ¹²⁵I-ANP from only a fraction of the total ANP receptor population, 30 and 85%, respectively. The remaining ANP binding sites can be occupied with the NPR-C selective ligand cANP(4-23). These data strongly suggest that only two classes of ANP receptor are present in these membrane preparations, NPR-A and NPR-C. The NEP resistant sANP derivative called sANP(TAPR) was 8 fold more potent (ED₅₀ = 0.6 nM) than rANP (ED₅₀ = SnM) in stimulating cGMP production in the lung membrane preparation. Our results demonstrate that the rhesus monkey natriuretic peptide receptors reflect the pharmacology of the human receptors, and that this species may be suitable to determine the role of NPR-C and NEP in peptide clearance and attenuating functional responses.     

Natriuretic peptides in cardiovascular diseases

The natriuretic peptide family comprises atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), dendroaspis natriuretic peptide (DNP), and urodilatin. The activities of natriuretic peptides and endothelins are strictly associated with each other. ANP and BNP inhibit endothelin-1 (ET-1) production. ET-1 stimulates natriuretic peptide synthesis. All natriuretic peptides are synthesized from polypeptide precursors. Changes in natriuretic peptides and endothelin release were observed in many cardiovascular diseases: e.g. chronic heart failure, left ventricular dysfunction and coronary artery disease.     

Biochemical analysis of neutral endopeptidase activity reveals independent catabolism of atrial and brain natriuretic peptide

Recent reports presented contradictory results regarding the catabolism of mature atrial (ANP) and brain (BNP) natriuretic peptides in circulation. Especially the role of neutral endopeptidase (NEP) in BNP degradation was conversely discussed. Our present in vitro-studies characterize the NEP-dependent metabolism of ANP and BNP in different tissues via HPLC-analysis using NEP-deficient mice and specific NEP inhibitors. Our results show a strong tissue-dependent degradation pattern of both peptides, which are not only due to the different NEP activities in these tissues. Whereas NEP rapidly degraded ANP, it had no influence in BNP-metabolism. Additional experiments with purified NEP confirmed this result. Moreover, we describe a degradation of ANP and BNP in NEP-deficient- and NEP-inhibited membranes. Consequently, we postulate the existence of at least one further natriuretic peptide (NP) degrading enzyme, which has not been characterized yet. Thus, the commonly accepted model of the natriuretic peptide system with NEP as the central degrading peptidase has to be partly revised. Moreover, the NEP-independent BNP degradation provides an effective means for achieving a beneficial BNP increase in cardiovascular pathology by inhibiting the assumed novel NP-degrading peptidase(s).     

利钠肽家族基因与心血管疾病研究新进展

利钠肽家族是一组由心肌细胞分泌的激素,主要包括A型、B型和C型利钠肽,具有相似的基因结构和生理学效应,可对心血管系统产生血压调节、抗心肌肥厚、抗心肌纤维化和抗心肌弛缓等保护作用。利钠肽受体A、B和C亦介导多种生理活性,调节心血管稳态。利钠肽受体A选择性结合A型、B型利钠肽。利钠肽受体B结合C型利钠肽。利钠肽受体C结合各型利钠肽,通过受体介导的内化和退化作用清除血液循环中利钠肽。对利钠肽家族及其受体基因单核甘酸多态性及功能研究显示,其与多种心血管疾病(房颤、高血压、心力衰竭等)的易感性相关。利钠肽家族及其受体基因缺失的转基因小鼠表现为心肌肥厚、心肌纤维化,与高血压、心肌病及心力衰竭的发生发展相关。各种导致心肌肥厚和缺血性损伤的刺激均参与利钠肽及其受体基因的表达调控。临床将脑钠肽作为左室功能障碍和心力衰竭失代偿的一个预测指标。静脉注射重组脑钠肽已经成为治疗急性心力衰竭的有效手段。深入了解利钠肽家族基因变异及其信号调控有助于探索心血管疾病的病理生理机制,为临床诊疗开辟新思路。     

利钠肽家族基因与心血管疾病研究新进展

利钠肽家族是一组由心肌细胞分泌的激素, 主要包括A型、B型和C型利钠肽, 具有相似的基因结构和生理学效应, 可对心血管系统产生血压调节、抗心肌肥厚、抗心肌纤维化和抗心肌弛缓等保护作用。利钠肽受体A、B和C亦介导多种生理活性, 调节心血管稳态。利钠肽受体A选择性结合A型、B型利钠肽。利钠肽受体B结合C型利钠肽。利钠肽受体C结合各型利钠肽, 通过受体介导的内化和退化作用清除血液循环中利钠肽。对利钠肽家族及其受体基因单核甘酸多态性及功能研究显示, 其与多种心血管疾病(房颤、高血压、心力衰竭等)的易感性相关。利钠肽家族及其受体基因缺失的转基因小鼠表现为心肌肥厚、心肌纤维化, 与高血压、心肌病及心力衰竭的发生发展相关。各种导致心肌肥厚和缺血性损伤的刺激均参与利钠肽及其受体基因的表达调控。临床将脑钠肽作为左室功能障碍和心力衰竭失代偿的一个预测指标。静脉注射重组脑钠肽已经成为治疗急性心力衰竭的有效手段。深入了解利钠肽家族基因变异及其信号调控有助于探索心血管疾病的病理生理机制, 为临床诊疗开辟新思路。     

三种钠尿肽抑制大鼠肺动脉平滑肌细胞增殖效应的比较

本文比较了心房钠尿肽（ＡＮＰ）、Ｃ－型钠尿肽（ＣＮＰ）、血管钠肽（ＶＮＰ）抑制肺动脉平滑肌细胞（ＰＡＳＭＣｓ）增殖的效应。用蛋白激酶Ｃ激动剂佛波酯（ＰＭＡ）刺激体外培养大鼠ＰＡＳＭＣｓ的增殖,以总蛋白含量和ＭＴＴ比色ＯＤ值为指标,观察三种钠尿肽对ＰＭＡ刺激大鼠ＰＡＳＭＣｓ增殖的影响。结果表明,ＰＭＡ（１０＾－９－１０＾－７ｍｏｌ／Ｌ）显著升高（Ｐ＜０．０５）ＰＡＳＭＣｓ的总蛋白含量和ＭＴＴＯＤ值,     

The Role of the Circulation in Processing pro-Brain Natriuretic Peptide (proBNP) to Amino-Terminal BNP and BNP-32

Hunt, P. J., E. A. Espiner, G. M. Nicholls, A. M. Richards and T. G. Yandle. The role of the circulation in processing pro-brain natriuretic peptide (proBNP) to amino-terminal BNP and BNP-32. Peptides 18(10) 1475–1481, 1997.—Human proBNP (purified from cardiac tissue) was incubated at 37°C in whole blood, serum and plasma and the products analyzed by size exclusion high pressure liquid chromatography and radioimmunoassay (RIA). In addition to RIAs for BNP-32 and NT-proBNP(1-13), a newly developed RIA for proBNP(62-76) was also used to identify the peptides. Incubation with serum resulted in the formation of a 9 kDa and a 3 kDa peptide, consistent with the N-terminal and the C-terminal peptides of the propeptide. Minimal processing of proBNP was seen in blood or plasma, suggesting that the circulation does not play a major role in the activation of proBNP. Analysis of degradation products of human proBNP using site directed specific antisera indicates that removal of N-terminal amino acids from proBNP occurs in serum. These findings support the view that the “high molecular weight BNP-32” previously identified in human plasma comprises amino-terminal deleted forms, and is unlikely to be intact proBNP(1-108).     

New aspects of the interactions between the cardiovascular nitric oxide system and natriuretic peptides

Arterial blood pressure is regulated by a variety of endocrine, autocrine and neuronal systems. Natriuretic peptides and nitric oxide are important factors that exert synergistic vascular and cardiac actions and their activities are closely linked. The existence of a novel signal transduction mechanism involved in activation of nitric oxide synthase via natriuretic peptides is currently being explored. Since several cardiovascular disorders are associated with dysfunction of natriuretic peptides activity, selective modulation of the natriuretic peptides pathway represents an important therapeutic target. This review article highlights the current findings on cross-talk between natriuretic peptides and the nitric oxide system.     

Neutral endopeptidase and natriuretic peptide receptors participate in the regulation of C-type natriuretic peptide expression in renal interstitial fibrosis

The initiation and progression of renal interstitial fibrosis (RIF) is a complicated process in which many factors may play an activate role. Among these factors, C-type natriuretic peptide (CNP) is an endothelium-derived hormone and acts in a local, paracrine fashion to regulate vascular smooth muscle tone and proliferation. In this study, we established a rat model of unilateral ureteral obstruction (UUO). CNP expression tends to be higher immediately after ligation and declined at later time points, occurring predominantly in tubular epithelial cells. A high-level CNP may contribute to the elevated expression of natriuretic peptide receptor (NPR)-B in the early phase of UUO. However, the sustained expression of NPR-C and neutral endopeptidase (NEP) observed throughout the study period (that is up to 3 months) helps to, at least partly, explain the subsequent decline of CNP. Thus, NEP and NPRs participate in the regulation of CNP expression in RIF.     

Identification and gene expression analyses of natriuretic peptide system in the ovary of goat (Capra hircus)

Natriuretic peptides (NPs) are involved in maintaining cardiovascular and fluid homeostasis, regulating reproductive processes and bone growth, and other numerous functions. To better understand the role of NPs in goat (Capra hircus), in the present study, full-length cDNAs of goat Nppa (natriuretic peptide precursor A), Nppb (natriuretic peptide precursor B) and Nppc (natriuretic peptide precursor C), respectively encoding ANP, BNP and CNP, were cloned from adult goat heart and ovary. The putative prepropeptide ANP (prepro-ANP) and prepro-CNP share a high amino acid sequence identity with other species. Real-time PCR showed that Nppa, Nppb and Nppc were widely expressed in adult goat tissues. The mRNA expression of Nppa and Nppb in the heart was extremely higher compared with other tissues. Nppc mRNA expression in the lung and uterus was also higher than in other tissues. The expression of Nppa, Nppb and Nppc genes was examined at different ovarian follicle stages using RT-PCR. The mRNAs of Nppa and Nppb were detected in secondary follicles as well as in COCs (cumulus–oocyte-complexes) and granulosa cells of antral follicles. However, the mRNA expression of Nppc was observed throughout ovarian follicle development, and it was especially higher in granulosa cells of antral follicles. In vitro, stimulating goat granulosa cells with FSH led to an increase in the expression of Nppc by dose- and time-dependent manners and a rapid decline was induced by LH stimulation, but the expression of Nppa and Nppb did not change after FSH or LH treatment. These results suggest that Nppc is a gonadotropin-induced gene in granulosa cells of goat ovary and CNP may be involved in the regulation of ovarian follicle development and oocyte maturation.     

Structural determinants of natriuretic peptide receptor specificity and degeneracy

Cardiovascular homeostasis and blood pressure regulation are reliant, in part, on interactions between natriuretic peptide (NP) hormones and natriuretic peptide receptors (NPR). The C-type NPR (NPR-C) is responsible for clearance of NP hormones from the circulation, and displays a cross-reactivity for all NP hormones (ANP, BNP, and CNP), in contrast to other NPRs, which are more restricted in their specificity. In order to elucidate the structural determinants for the binding specificity and cross-reactivity of NPR-C with NP hormones, we have determined the crystal structures of the complexes of NPR-C with atrial natriuretic peptide (ANP), and with brain natriuretic peptide (BNP). A structural comparison of these complexes, with the previous structure of the NPR-C/CNP complex, reveals that NPR-C uses a conformationally inflexible surface to bind three different, highly flexible, NP ligands. The complex structures support a mechanism of rigid promiscuity rather than conformational plasticity by the receptor. While ANP and BNP appear to adopt similar receptor-bound conformations, the CNP structure diverges, yet shares sets of common receptor contacts with the other ligands. The degenerate versus selective hormone recognition properties of different NPRs appears to derive largely from two cavities on the receptor surfaces, pocket I and pocket II, that serve as anchoring sites for hormone side-chains and modulate receptor selectivity.     

Reciprocal regulation of natriuretic peptide receptors by insulin in adipose cells

Atrial- and brain-type natriuretic peptides (ANP and BNP, respectively) have been shown to exert potent lipolytic action in adipocytes. A family of natriuretic peptide receptors (NPRs), NPR-1, NPR-2, and NPR-3, mediates their physiologic effects. NPR-1 and NPR-2 are receptor guanylyl cyclases, while NPR-3 lacks enzymatic activity and functions primarily as a clearance receptor for natriuretic peptides. ANP has a high affinity for NPR-1 and NPR-3 than other natriuretic peptides. There is a possibility that ANP may exhibit its lipolytic effect through the balance of NPR-1 and NPR-3 expressions in adipocytes. However, the regulation of adipose NPRs has not been fully elucidated. We here examined the regulation of mouse adipose NPRs by insulin, an anti-lipolytic hormone. Among the insulin target organs, NPR-1 mRNA levels were higher in white adipose tissue (WAT) than in liver and skeletal muscle. NPR-3 mRNA was expressed most abundantly in WAT. Fasting condition induced NPR-1 mRNA level while suppressed NPR-3 mRNA level in WAT. Administration of streptozotocin resulted in the increase of NPR-1 mRNA level while the decrease of NPR-3 mRNA level in WAT. In ob/ob mice, hyperinsulinemic model, NPR-1 mRNA level was lower whereas NPR-3 mRNA level was higher compared to lean control mice. In 3T3-L1 adipocytes, insulin significantly reduced NPR-1 mRNA level while increased NPR-3 mRNA levels both through phosphatidylinositol 3-kinase (PI3-kinase) pathway. In summary, NPR-1 and NPR-3 were highly expressed in WAT and adipose NPR-1 and NPR-3 were reciprocally regulated by insulin. This study suggests that insulin may efficiently promote lipogenesis partly by reducing the lipolytic action of ANP through the opposite regulation of NPR-1 and NPR-3.     

Dendroaspis natriuretic peptide binds to the natriuretic peptide clearance receptor

Dendroaspis natriuretic peptide (DNP) is a newly-described natriuretic peptide which lowers blood pressure via vasodilation. The natriuretic peptide clearance receptor (NPR-C) removes natriuretic peptides from the circulation, but whether DNP interacts with human NPR-C directly is unknown. The purpose of this study was to test the hypothesis that DNP binds to NPR-C. ANP, BNP, CNP, and the NPR-C ligands AP-811 and cANP(4-23) displaced [(125)I]-ANP from NPR-C with pM-to-nM K(i) values. DNP displaced [(125)I]-ANP from NPR-C with nM potency, which represents the first direct demonstration of binding of DNP to human NPR-C. DNP showed high pM affinity for the GC-A receptor and no affinity for GC-B (K(i)>1000 nM). DNP was nearly 10-fold more potent than ANP at stimulating cGMP production in GC-A expressing cells. Blockade of NPR-C might represent a novel therapeutic approach in augmenting the known beneficial actions of DNP in cardiovascular diseases such as hypertension and heart failure.     

Intracellular trafficking and metabolic turnover ofligand-bound guanylyl cyclase/atrial natriuretic peptide receptor-A into subcellular compartments

Atrial natriuretic peptide (ANP) is the first described member of the natriuretic peptide hormone family. ANP elicits natriuretic, diuretic, vasorelaxant and antiproliferative effects, important factors in the control of blood pressure homeostasis. One of the principal loci involved in the regulatory action of ANP is the guanylyl cyclase-linked ANP-receptor which has been designated as NPRA, also referred to as GC-A, whose ANP-binding efficiency and guanylyl cyclase activity vary remarkably in different target tissues. However, the cellular and molecular basis of these activities and the functional expression and regulation of NPRA are not well understood. The mature form of receptor resides in the plasma membrane and consists of an extracellular ligand-binding domain, a single transmembrane-spanning region, and intracellular protein kinase-like homology and guanylyl cyclase catalytic domains. In this review, emphasis has been placed on the interaction of ANP with NPRA, the ligand-mediated endocytosis, trafficking, and subcellular distribution of ligand-receptor complexes from cell surface to the intracellular compartments. Furthermore, it is implicated that after internalization, the ANP/NPRA complexes dissociate into the subcellular compartments and a population of receptor recycles back to the plasma membrane. This is an interesting area of research in the natriuretic peptide receptor field because there is currently debate over whether ANP/NPRA complexes internalize at all or whether cell utilizes some other mechanisms to release ANP from the bound receptor molecules. Indeed, controversy exist since it has been previously reported by default that among the three natriuretic peptide receptors only NPRC internalizes with bound ligand. Hence, from a thematic standpoint it is clearly evident that there is a current need to review this subject and provide a consensus forum that establishes the cellular trafficking, sequestration and processing of ANP/NPRA complexes in intact cells. Towards this aim the cellular life-cycle of NPRA will be described in the context of ANP-binding, internalization, metabolic processing, and/or inactivation, down-regulation, and degradation of ligand-receptor complexes in model cell systems.     

NPR-A regulates self-renewal and pluripotency of embryonic stem cells

Self-renewal and pluripotency of embryonic stem (ES) cells are maintained by several signaling cascades and by expression of intrinsic factors, such as Oct4, Nanog and Sox2. The mechanism regulating these signaling cascades in ES cells is of great interest. Recently, we have demonstrated that natriuretic peptide receptor A (NPR-A), a specific receptor for atrial and brain natriuretic peptides (ANP and BNP, respectively), is expressed in pre-implantation embryos and in ES cells. Here, we examined whether NPR-A is involved in the maintenance of ES cell pluripotency. RNA interference-mediated knockdown of NPR-A resulted in phenotypic changes, indicative of differentiation, downregulation of pluripotency factors (such as Oct4, Nanog and Sox2) and upregulation of differentiation genes. NPR-A knockdown also resulted in a marked downregulation of phosphorylated Akt. Furthermore, NPR-A knockdown induced accumulation of ES cells in the G1 phase of the cell cycle. Interestingly, we found that ANP was expressed in self-renewing ES cells, whereas its level was reduced after ES cell differentiation. Treatment of ES cells with ANP upregulated the expression of Oct4, Nanog and phosphorylated Akt, and this upregulation depended on NPR-A signaling, because it was completely reversed by pretreatment with either an NPR-A antagonist or a cGMP-dependent protein kinase inhibitor. These findings provide a novel role for NPR-A in the maintenance of self-renewal and pluripotency of ES cells.     

Immunohistochemical localisation of natriuretic peptides in the heart and brain of the gulf toadfish Opsanus beta

Summary The distribution of natriuretic peptide immunoreactivity was determined in the heart and brain of the gulf toadfish Opsanus beta using the avidin-biotin peroxidase technique. Four antisera were used: the first raised against porcine brain natriuretic peptide which cross-reacts with atrial natriuretic and C-type natriuretic peptides (termed natriuretic peptide-like immunoreactivity); the second raised against porcine brain natriuretic peptide which cross-reacts with C-type natriuretic peptide but not with atrial natriuretic peptide (termed porcine brain natriuretic peptide-like immunoreactivity); the third raised against rat atrial natriuretic peptide; and the fourth raised against eel atrial natriuretic peptide. Natriuretic peptide- and porcine brain natriuretic peptide-like immunoreactivity was observed in all cardiac muscle cells of the atrium. In the ventricle, natriuretic peptide-like immunoreactivity was found in all cardiac muscle cells, however porcine brain natriuretic peptidelike immunoreactivity was confined to muscle cells adjacent to the epicardium. There was no discernible difference in the distribution of natriuretic peptide-like immunoreactivity and porcine brain natriuretic peptide-like immunoreactivity in the brain. Immunoreactive perikarya were observed only in the preoptic region of the diencephalon, and many immunoreactive fibres were found in the telencephalon, preoptic area, and rostral hypothalamus, lateral to the thalamic region. There was no immunoreactivity in any region of the hypophysis. A pair of distinct immunoreactive fibre tracts ran caudally from the preoptic area to the thalamic region, from which fibres extended to the posterior commissure, area praetectalis, dorsolateral regions of the midbrain tegmentum, and tectum. Many immunoreactive fibres were present in the rostral regions of the inferior lobes of the hypothalamus and in the dorsolateral and ventrolateral aspects of the rhombencephalon. No immunoreactivity was observed in the heart and brain using rat atrial natriuretic and eel natriuretic peptide antisera. Although the chemical structure of natriuretic peptides in the heart and brain of toadfish is unknown, these observations show that a component of the natriuretic peptide complement is similar to porcine brain natriuretic and/or porcine C-type natriuretic peptides. The presence of natriuretic peptides in the brain suggests that they could be important neuromodulators and/or neurotransmitters.     

Binding of Brain and Atrial Natriuretic Peptides to Cultured Mouse Astrocytes and Effect on Cyclic GMP

125I-Porcine brain natriuretic peptide (125I-pBNP) bound to mouse astrocytes in primary culture in a time-dependent manner (t1/2 = 4.5 min), similar to 125I-human atrial natriuretic peptide (125I-hANP) (t1/2 = 5 min). Binding was saturable and reached equilibrium after 90 min at 22 degrees C for both radioligands. Scatchard analysis suggested a single class of binding sites for pBNP with a binding affinity and capacity (KD = 0.08 nM; Bmax = 78.3 fmol/mg of protein) similar to those of hANP1-28 (KD = 0.1 nM; Bmax = 90.3 fmol/mg of protein). In competition binding studies, pBNP or human/rat atrial natriuretic peptide (ANP) analogues [hANP1-28, rat ANP1-28 (rANP1-28), and rANP5-28] displaced 125I-hANP, 125I-pBNP, and 125I-rANP1-28 completely, all with IC50 values of less than nM (0.14-0.83 nM). All four peptides maximally stimulated cyclic GMP (cGMP) production by 10 min at 22 degrees C at concentrations of 1 microM with EC50 values ranging from 50 to 100 nM. However, maximal cGMP induction by brain natriuretic peptide (BNP) (25.9 +/- 2.1 pmol/mg of protein) was significantly greater than that by hANP1-28 (11.5 +/- 2.2 pmol/mg of protein), rANP1-28 (16.5 +/- 2.0 pmol/mg of protein), and rANP5-28 (15.8 +/- 2.2 pmol/mg of protein). These studies indicate that BNP and ANPs act on the same binding sites and with similar affinities in cultured mouse astrocytes. BNP, however, exerts a greater effect on cGMP production. The difference in both affinity and selectivity between binding and cGMP production may indicate the existence of receptor subtypes that respond differentially to natriuretic peptides despite similar binding characteristics.     

The precursor to B-type natriuretic peptide is an O-linked glycoprotein

Human pro-B-type natriuretic peptide (proBNP), the precursor for B-type natriuretic peptide (BNP), was expressed in Chinese hamster ovary cells (CHO) and compared by Western blot analysis to BNP cross-reacting material immunoprecipitated from the plasma of heart failure patients. Both recombinant and native forms co-migrated as a diffuse band centered around 25 kDa and were reduced to a 12 kDa species by treatment with a mixture of O-link deglycosylation enzymes. The 108-amino acid CHO-expressed protein was examined by tryptic mapping and LC-MS and found to be an O-linked glycoprotein. Determination of the sites of O-glycosyl addition by blank cycle sequencing of tryptic and Glu-C (Staphylococcus aureus V8 protease) peptides showed that there are seven sites of glycosylation confined to a 36-amino acid residue stretch within the center of the propeptide region. This data is consistent with previous observations of higher molecular weight isoforms of BNP.     

Neutral endopeptidase is expressed on the follicular granulosa cells of rabbit ovaries

Neutral endopeptidase (NEP) is a zinc metallopeptidase ubiquitously distributed in various tissues in mammals. This peptidase is involved in the post-secretory metabolism of various neuropeptides and peptide hormones in vivo, such as enkephalins, bradykinin, atrial natriuretic peptide, substance P and endothelins. In this paper we show that NEP is expressed in ovaries as a 110-kDa glycosylated integral membrane protein with enzymatic properties similar to those of the kidney protein. Using immunohistochemistry, we localize the peptidase in the granulosa cells of follicles at all stages of maturation, with the exception of atretic follicles. We also observe immunoreactive staining in the epithelia that lines the blood vessels in the medulla and the surface of the ovary. The co-localization of NEP and bioactive peptides known to be physiological substrates of NEP in other tissues suggests an important role for this protein in processes such as follicle maturation, ovulation, and/or regulation of ovarian blood flow, by modulating the physiological function of these peptides.