Light and Electron Microscopic Localization of the Neutral Metalloendopeptidase EC 3.4.24.16 in the Mesencephalon of the Rat

Abstract

The topographic and cellular distribution of the neurotensin-hydrolysing neutral metalloendopeptidase 24.16 (EC 3.4.24.16) was examined by light and electron microscopic immunohistochemistry in adult rat mesencephalon. Light microscopic immunoradioautography revealed a ubiquitous distribution of the enzyme throughout the midbrain with a relative enrichment of grey matter areas including the substantia nigra, ventral tegmental area, interfascicular nucleus, interpeduncular nucleus, rostral and caudal linear raphe nuclei, central grey and superficial grey of the superior colliculus. Peroxidase - antiperoxidase immunocytochemistry revealed two distinct cellular patterns of immunostaining: (1) weakly labelled neuronal perikarya more or less uniformly distributed throughout the grey matter, and (2) intensely immunoreactive glial cells heterogeneously distributed across the mesencephalon. Areas exhibiting dense concentrations of endopeptidase 24.16-containing glial cells corresponded to those displaying enhanced immunoreactivity in immunoradioautographs, suggesting that a major proportion of brain endopeptidase 24.16 is associated with glia. Electron microscopic examination of the substantia nigra and ventral tegmental area confirmed the association of the enzyme with a subpopulation of neurons and allowed identification of labelled glial cells as protoplasmic astrocytes. In neurons, endopeptidase 24.16 immunoreactivity was distributed heterogeneously within the cytoplasm of perikarya, dendrites and axons. Reaction product was also characteristically associated with restricted zones of the plasma membrane and underlying neuroplasm. In astrocytes, endopeptidase 24.16 immunostaining was densely and uniformly distributed throughout the cytoplasm of cell bodies and processes. Many of these processes were in direct contact with endopeptidase 24.16-immunopositive neuronal elements. The present results demonstrate that within the midbrain, endopeptidase 24.16 is both intracytoplasmic and membrane-associated in neurons and predominantly intracytoplasmic in glia. The presence of a large number of immunostained elements within areas of the midbrain known to display high levels of neurotensin and/or neurotensin receptors, together with the demonstrated catabolic activity of the enzyme on neurotensin in vitro, is consistent with a role of endopeptidase 24.16 in the functional inactivation of endogenous neurotensin in this region of the brain.

Distinct Properties of Neuronal and Astrocytic Endopeptidase 3.4.24.16: A Study on Differentiation, Subcellular Distribution, and Secretion Processes

“…Our previous electron microscopic study revealed the occurrence, within the midbrain tegmentum, of endocytic invaginations at the level of appositions between endopeptidase 3.4.24.16-immunoreactive neurons and astrocytes, suggesting a possible translocation of the enzyme between these two elements (Woulfe et al, 1992). This finding prompted us to investigate the possibility of a secretion of endopeptidase 3.4.24.16 by either astrocytes or neurons.…”

“…Whether these differences reflect complementary roles of the enzyme on peptide inactivation remains to be established. In this context, it is interesting to note that, at the level of neurotensinergic pathways, endopeptidase 3.4.24.16-immunopositive astrocytic elements are often directly apposed to endopeptidase 3.4.24.16-immunoreactive nerve cell bodies and/or dendrites (Woulfe et al, 1992). It is therefore tempting to speculate that although the endopeptidase 3.4.24.16 secreted form of astrocytes would act in the extracellular space, thereby restricting diffusion of released neurotensin, the neuronal membraneassociated activity would be responsible for the physiological inactivation of the peptide either in the synaptic cleft, beside neurotensin receptors, or inside early endosomal compartments in which receptor-ligand complexes would have been internalized (Mazella et al, 1991).…”

“…Sections, 35 m thick, were cut on a Vibratome (Lancer) and collected in 0.1 M phosphate buffer. The peroxidase-antiperoxidase immunocytochemical procedure used for the visualization of endopeptidase 3.4.24.16 immunoreactivity was identical to that described previously (Woulfe et al, 1992). Visualization of bound peroxidase was achieved by reaction with a solution of 0.1 M TBS containing 0.05% DAB and 0.01% H 2 O 2 .…”

Neurotensin Activates GABAergic Interneurons in the Prefrontal Cortex

“…We found that neurotensin evoked a significant increase in extracellular GABA levels, which was comparable in magnitude to neurotensin-induced changes in GABA levels in subcortical sites. The small magnitude of neurotensinevoked GABA increases probably reflects metabolism of exogenously administered neurotensin by metalloendopeptidases (Woulfe et al, 1992), which play an important role in regulating neurotensin activity in vivo (Vincent et al, 1997a,b;O'Connor, 2001).…”

Endopeptidases 3.4.24.15 and 24.16 in endothelial cells: potential role in vasoactive peptide metabolism

“…BK is also efficiently cleaved in vitro by the closely related peptidases EP24.15 (also known as thimet oligopeptidase) and EP24.16 (also known as neurolysin) (21). We have recently shown that hypotensive responses to BK are markedly potentiated by the administration of a stable inhibitor of these enzymes N- [1- 16 has been localized to the plasma membrane in neurons, both in vivo (9,31) and in vitro (28), as well as in human kidney cells transfected with the peptidase (29). Given that neither enzyme possesses a transmembrane domain, the exact mechanism of association with the membrane is not understood.…”