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.

Gene products from LUQ neurons in the abdominal ganglion are present at the renal pore of Aplysia californica.

The L2-4,6 and L5 cells located in the left upper quadrant of the abdominal ganglion of Aplysia californica express the L5-67 and LUQ-1 genes, respectively, in a nonoverlapping manner. These cells send major neurites to the kidney and at least some of them were shown to innervate the renal pore closer muscle, and thereby control its function. By using in-situ hybridization and immunofluorescence, the presence of L5-67 and LUQ-1 mRNAs and peptides was studied in the kidney, with emphasis on the region of the renal pore. We detected immunoreactive materials in many small varicose nerve fibers running along the central epithelium in the inner parts of the kidney, and in neurites located within a large nerve associated with muscles inside the renal pore. Our observations represent the first direct evidence of the presence of gene products from LUQ cells at the renal pore, suggesting that they may be responsible for mediating LUQ cell signals. Furthermore, mRNAs coding for the L5-67 and LUQ-1 peptides were also found in the nerve structure inside the renal pore. Our report documents a striking example of neuropeptide mRNA targeting nerve terminals that are very distant from their cell bodies.

The N-terminal segment of endothelin-converting enzyme (ECE)-1b contains a di-leucine motif that can redirect neprilysin to an intracellular compartment in Madin-Darby canine kidney (MDCK) cells.

Endothelin-converting enzyme (ECE)-1 is a membrane-bound metallopeptidase of the neprilysin (NEP) family. ECE-1 is responsible for the conversion of inactive big-endothelins into active endothelins. Three different isoforms of human ECE-1 (ECE-1a, ECE-1b and ECE-1c) have been identified. They differ in their N-terminal cytosolic regions, have distinct tissue distribution and intracellular localization. ECE-1a and ECE-1c are both located at the cell surface whereas ECE-1b is targeted to an intracellular compartment. To better understand the nature of the signal responsible for the targeting of ECE-1b to the intracellular compartment, we have constructed several ECE/NEP chimaeric proteins and expressed them by transfection into Madin-Darby canine kidney (MDCK) cells. This allowed us to identify a nine amino acid segment in the cytosolic tail of ECE-1b that is sufficient to relocate NEP from the cell surface to an intracellular compartment. Site-directed mutagenesis on these chimaeras led to the identification of two leucine residues as part of the intracellular retention signal.

Cloning and characterization of Aplysia neutral endopeptidase, a metallo-endopeptidase involved in the extracellular metabolism of neuropeptides in Aplysia californica.

Cell surface metallo-endopeptidases play important roles in cell communication by controlling the levels of bioactive peptides around peptide receptors. To understand the relative relevance of these enzymes in the CNS, we characterized a metallo-endopeptidase in the CNS of Aplysia californica, whose peptidergic pathways are well described at the molecular, cellular, and physiological levels. The membrane-bound activity cleaved Leu-enkephalin at the Gly3-Phe4 bond with an inhibitor profile similar to that of the mammalian neutral endopeptidase (NEP). This functional homology was supported by the molecular cloning of cDNAs from the CNS, which demonstrated that the Aplysia and mammalian NEPs share all the same amino acids that are essential for the enzymatic activity. The protein is recognized both by specific anti-Aplysia NEP (apNEP) antibodies and by the [125I]-labeled NEP-specific inhibitor RB104, demonstrating that the apNEP gene codes for the RB104-binding protein. In situ hybridization experiments on sections of the ganglia of the CNS revealed that apNEP is expressed in neurons and that the mRNA is present both in the cell bodies and in neurites that travel along the neuropil and peripheral nerves. When incubated in the presence of a specific NEP inhibitor, many neurons of the buccal ganglion showed a greatly prolonged physiological response to stimulation, suggesting that NEP-like metallo-endopeptidases may play a critical role in the regulation of the feeding behavior in Aplysia. One of the putative targets of apNEP in this behavior is the small cardioactive peptide, as suggested by RP-HPLC experiments. More generally, the presence of apNEP in the CNS and periphery may indicate that it could play a major role in the modulation of synaptic transmission in Aplysia and in the metabolism of neuropeptides close to their point of release.

Molecular cloning, sequence analysis and expression distribution of an aminopeptidase in Aplysia california.

We are investigating the role of membrane-bound peptidases in the inactivation of neuropeptides in Aplysia californica. Recently, we reported the biochemical characterization of a membrane-bound neuropeptide-degrading enzyme which has enzymatic characteristics similar to those of the mammalian aminopeptidase N (Bawab W, Querido E, Crine P, DesGroseillers L. Identification and characterization of aminopeptidases from Aplysia californica, Biochem J 1992;286:967-975). We now report the cloning and sequencing of a cDNA encoding an aminopeptidase enzyme (apAP) and the localization of the apAP transcript in Aplysia. The apAP cDNA encodes a putative protein of 1007 amino acids, which shows around 34% sequence identity to mammalian aminopeptidases A and N sequences. The deduced amino acid sequence suggests that apAP is a type II membrane-bound protein, with a long extracellular domain in which the consensus sequence of zinc-binding metallopeptidases (His-Glu-Xxx-Xxx-His) is found. RT-PCR and Northern blot experiments showed that the apAP gene is expressed as a single 6.8-kb transcript in the central nervous system, gill, heart, kidney and ovotestis.