Electron microscopic localization of immunoreactive enkephalinase (EC 3.4.24.11) in the neostriatum of the rat

Abstract

The fine structural distribution of the enzyme-neutral endopeptidase EC 3.4.24.11 (enkephalinase) was examined by immunoradioautography (using an iodinated monoclonal antibody) and peroxidase immunocytochemistry (using the same probe in nonradioactive form) in the neostriatum of the rat. At the light microscopic level, both techniques revealed a heterogeneous distribution of immunoreactive enkephalinase in the caudoputamen, characterized by the presence of patches of intense immunolabeling prominent against a relatively strong immunoreactive matrix, a pattern reminiscent of mu opioid receptors radioautographically labeled in the same region. Pilot experiments indicated that fixation of the brain with a mixture of 4% paraformaldehyde, 0.05% glutaraldehyde, and 0.2% picric acid did not modify the distribution and only slightly reduced the intensity of striatal enkephalinase antigenicity, provided that the post-fixation period did not exceed 1 hr. In the neostriatum of animals fixed according to this protocol, enkephalinase immunoreactivity was found by electron microscopic immunoradioautography to be exclusively confined to neuronal and glial membrane interfaces. Immunoperoxidase cytochemistry confirmed the association of immunoreactive enkephalinase with the plasma membrane of neurons and, to a lesser extent, of astrocytes and oligodendrocytes. Both immunoradioautographic and immunoperoxidase techniques revealed a predominant association of the enzyme with neuronal perikarya and dendrites. The morphological features of the labeled perikarya, together with the presence of immunoreactive dendritic spines, suggested that some of these neurons corresponded to striatofugal medium spiny neurons. Immunoreactive enkephalinase was also detected at the level of myelinated and unmyelinated axons and axon terminals. These axons could potentially have originated from intrinsic striatal neurons or from the substantia nigra. Statistical analysis of silver grain distribution in electron microscopic immunoradioautographs indicated that immunoreactive enkephalinase was not preferentially concentrated at the level of specific membrane interfaces, but rather, was more or less uniformly distributed on the surface of neurons and/or glial cells. A similarly diffuse localization of the enzyme was apparent in peroxidase-reacted material, though the latter technique also revealed a microheterogeneity in the deposition of the reaction product along the labeled membranes. Finally, quantitative analysis of immunoradioautographs clearly indicated an absence of enkephalinase enrichment at the level of synaptic junctions. The similarity between the light and electron microscopic distribution of enkephalinase observed in the present study, and that previously reported for mu opioid receptors, lends support to the concept that this ectoenzyme may be involved in the inactivation of endogenous opioids in the mammalian neostriatum.

Synaptic localization of kappa opioid receptors in guinea pig neostriatum.

“…Given that -10-15% of these exclusive grains may be accounted for by cross-fire from radioactive sources associated with nearby plasma membranes (23,32), these results suggest that only a small proportion of the bound radioactivity was actually intraneuronal. This fraction was comparable to that of tL opioid-binding sites but markedly lower than that of 8 sites labeled in rat neostriatum (14,15).…”

“…Although this type of labeling may originate from the membrane of abutting elements as well as from that of the dendrites themselves (23,32), several lines of evidence favored a predominant association of tracer with the dendritic side: (i) high labeling incidence of dendritic appositions, (ii) marked enrichment of dendrodendritic interfaces, and (iii) occurrence of dendritic profiles labeled at several contact points along their plasma membrane. This interpretation also conforms with the internal labeling of striatal perikarya and dendrites, as well as with the results of cytotoxic lesion studies that favor association of K receptors with striatal neurons (33).…”

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

“…, 1988a.b; Back and Gorenstein. 1989;Marcel et a / . , 1990), both enkephalinase and angiotensin converting enzyme have also been demonstrated in glial cells (Lentzen et itl.. 1983(Lentzen et itl.. .…”

Inhibition of anandamide hydrolysis in rat brain tissue by (E)‐6‐(bromomethylene) tetrahydro‐3‐(1‐naphthalenyl)‐2H‐pyran‐2‐one

“…These fall into two main categories: reuptake into cells mediated by transporter proteins terminates the synaptic effects of biogenic amines, glutamate and y-aminobutyrate [1,2], while breakdown by extracellular hydrolyzing enzymes terminates the effects of acetylcholine [3], and participates in the inactivation of many neuropeptides [3][4][5]. Because of these essential regulatory functions, neurotransmitter-inactivating processes have provided targets for drugs of both experimental and clinical significance, ranging from amine reuptake blockers to acetylcholinesterase inhibitors.…”