Ontogeny, regional and cellular distribution of the novel metalloprotease neprilysin 2 in the rat: a comparison with neprilysin and endothelin-converting enzyme-1.

The localisation of the gene transcripts of a recently discovered peptidase, neprilysin 2 (NEP2), was established by in situ hybridisation in rat tissues during development and adulthood. It was compared with those of neprilysin (NEP), a closely related enzyme in terms of sequence homology or substrate specificity, and of endothelin-converting enzyme 1 (ECE-1) which, like the other two, belongs to the M-13 sub-family of zinc-dependent metallopeptidases. The ontogeny of the three enzymes differed markedly, the expression of NEP2 being restricted to developing and differentiating fields of the CNS, whereas NEP and ECE-1 genes were broadly expressed early on in the CNS and periphery. In contrast to the wide expression of NEP and ECE-1 in peripheral adult tissues and in CNS, NEP2 was almost exclusively expressed in selected neuronal populations of the brain and spinal cord. The only exceptions were the intermediate and anterior lobes of the pituitary as well as the choroid plexuses, where NEP2 was also strongly expressed. These localisations as well as those in the hypothalamic nuclei, together with the previously established pattern of cleaved peptides, suggest the involvement of NEP2 in the metabolism of neurohormones of the hypothalamo-pituitary axis.Complementary distributions of NEP and NEP2 mRNAs were observed in a large number of brain areas with, for instance the former being highly expressed in the striatum in which NEP2 transcripts were almost undetectable. In contrast, NEP2 was highly expressed in numerous thalamic, hypothalamic and brainstem nuclei from which NEP was absent. Since both peptidases are able to cleave the same neuropeptides, this pattern may suggest a complementary role in their peptide inactivation functions in the CNS. Finally, ECE-1 mRNAs were generally observed in neuronal populations known to express the pre-proendothelin-1 gene, confirming the function of the metallopeptidase in endothelin-1 generation.

Effects of histamine H3 receptor agonist and antagonist on histamine co-transmitter expression in rat brain.

The histaminergic H3-receptor (H3R) controls histamine synthesis and release in the tuberomamillary nucleus. We evaluated the effects of stimulating or blocking of H(3)R on glutamate-decarboxylase 67 kDa (GAD-67) and galanin mRNA expression, two histamine co-transmitters.After in situ hybridization histochemistry (ISHH), we observed a colocalization of 100% between histidine decarboxylase (HDC) and GAD-67 or H3R and of 80 to 97% with galanin. Adult rats received an H3R agonist ((R)alpha-Methylhistamine) or antagonist (ciproxifan) and were sacrificed 1 or 3 hours later. Treatment effects on HDC, galanin and GAD-67 mRNA were studied by quantitative ISHH on serial sections. Treatment with the H3R agonist known to decrease histamine neuron activity initially reduced HDC and galanin gene expression but an inverse change, presumably reflecting a compensatory mechanism, was observed after 3 h on both markers. In contrast, the H3R antagonist known to activate histamine neurons, had opposite effects on the two markers, suggesting that co-transmitters are submitted to independent control mechanisms. Furthermore, GAD-67 mRNA levels were not significantly modified by these treatments.

Neprilysin II: A putative novel metalloprotease and its isoforms in CNS and testis.

Metalloproteases of the M13 subfamily, comprising namely neprylisin (NEP) and endothelin-converting enzyme (ECE), are involved in the metabolism of various neuronal and hormonal peptides, and inhibitors thereof have already led to therapeutically useful agents. Using homology cloning, we have identified a new member of this family in rat tissues. It is a glycosylated, type II integral membrane protein of 774 amino acids, containing a zinc-binding consensus motif, highly homologous to NEP and, therefore, designated NEPII. We have characterized multiple splice variants of NEPII mRNA with distinct expression patterns in brain regions, pituitary and testis. In situ hybridization of testis, where levels of the NEPII gene transcript are the highest, reveals a localization within round spermatids. In brain, NEPII is expressed heterogeneously among several neuronal populations and according to a pattern grossly complementary to that of NEP.