AN IMMUNOELECTRON MICROSCOPIC STUDY OF PIG SUBSTANTIA-NIGRA SHOWS COLOCALIZATION OF ENDOPEPTIDASE-24.11 WITH SUBSTANCE-P

Endopeptidase-24.11 is a widely distributed cell surface enzyme with a role in inactivating some neuropeptides and peptide hormones. In the central nervous system it has been implicated in the metabolism of enk ephalins and tachykinins, neuropeptides which are expressed by neurons projecting to the substantia nigra. Two immunochemical methods have b een used to reveal the ultrastructural localization of endopeptidase-2 4.11 and substance P in the substantia nigra of piglets. In the first approach, substance P was revealed by immunoperoxidase staining using the rat monoclonal antibody, NCl, and endopeptidase-24.11 by 1 nm coll oidal gold using an affinity-purified rabbit polyclonal antibody, both being applied at the pre-embedding stage. NCl was shown to be highly specific for substance P, with negligible cross-reactivity with neurok inins A and B. The specificity of the immunostaining was confirmed by processing all sections for both markers, even when only one primary a ntibody was applied. In the second approach, ultrathin cryosections we re immunostained using gold particles of different diameters. In a sur vey of electron micrographs, 80% of the silver-enhanced gold particles were touching neuronal membranes, consistent with the known topology of endopeptidase-24.11. Endopeptidase-24.11 immunoreactivity was obser ved both on membranes of axons and on pre- and postsynaptic elements. Substance P immunoreactivity was seen within some boutons, apparently associated with vesicles, and in axons. In doubly stained sections, ma ny examples of immunopositive gold-labelled boutons (i.e. endopeptidas e-24.11-immunoreactive-positive) containing immunoperoxidase reaction product (i.e. substance P-immunoreactive-positive) were recorded. In u ltrathin cryosections, substance P immunoreactivity was mainly observe d in dense-core vesicles within boutons, some of which also showed mem brane-associated gold particles marking endopeptidase-24.11 immunoreac tivity. This is the first demonstration of endopeptidase-24.11 by immu nogold at the electron microscopic level and the first demonstration o f the ultrastructural co-localization of a membrane peptidase and its putative neuropeptide target. The results lend strong support to the v iew that endopeptidase-24.11 has a physiological role in the metabolis m of substance P, but do not exclude a role in the inactivation of oth er neuropeptides.