Immunocytochemical localization of endopeptidase-24.11 in the pig brain

Abstract

Endopeptidase-24.11 is an ectoenzyme found in the plasma membrane of a variety of cell types. It is most abundant in the brush-borders of kidney and intestine (Fulcher & Kenny, 1983; Fulcher et af. , 1983; Gee et af . , 1983). It is also present in pig brain membranes from which it has been purified by immuno-affinity chromatography (Relton et af . , 1983). In synaptic membranes prepared from pig striatum, endopeptidase-24.11 is the only enzyme capable of initiating the hydrolysis of substance P and [D-Ala’ ,Leu5]enkephalin (Matsas et al., 1983). In the period before the unequivocal demonstration of endopeptidase-24.11 in the central nervous system (CNS), the activity hydrolysing the Gly3-Phe4 bond of enkephalin had been referred to as ‘enkephalinase’ (Malfroy et af. , 1978), a wholly inappropriate term since the enzyme is not peptide-specific. Although the enkephalins are efficiently hydrolysed, the tachykinins, including substance P, exhibit lower K,,, and higher k,, values (Matsas et al., 1984). The distribution of endopeptidase-24.11 has been determined by two approaches. The substrate [~-Ala’,Leu]enkephalin provides a convenient assay for purified membrane fractions, the specificity of which can be established by using a specific inhibitor such as phophoramidon. Alternatively, an immunoradiometric assay (i.r.m.a.) using a monoclonal antibody generated to endopeptidase-24.11 (Gee et al., 1985) provides a specific detection system applicable also to crude extracts. Both assays, when applied to the CNS, have shown that the highest concentrations of the enzyme are present in the caudate-putamen, globus pallidus and the spinal cord throughout its length (Matsas et af . , 1985). An insight into the more precise distribution of endopeptidase-24.11 requires an immunocytochemical approach, but early attempts using a monoclonal antibody, that had revealed the brush-border localization in kidney and intestine (Gee et al., 1983), were frustrated because of the relatively small amounts in CNS membranes compared with microvilli. This obstacle has now been overcome by the use of an affinity-purified rabbit polyclonal antibody, the application of which, employing a biotinylated second antibody and a biotin-streptavidinhorseradish peroxidase detection system, is presented here. The distribution was mapped by cutting 40 pm cryostat sections of one hemisphere of a piglet brain. Macrophotography confirmed that endopeptidase-24.11 had a remarkably discrete localization. In the forebrain it was concentrated in the basal ganglia, caudateputamen, globus pallidus and olfactory tubercle and was also present in a well-defined area within the diencephalon. In the mid-brain it was identified in the substantia nigra and interpedunclar nucleus. Pial membranes covering both brain and spinal cord also stained positively. At a microscopic level the immuno-staining was confined to the neuropil; myelinated tracts were not stained. Within the neuropil of the striatum, local areas called striosomes (Graybiel, 1984), exhibiting very intense