Proenkephalin, [Metlenkephalin, and oxytocin immunoreactivities are ...

Proc. NatL Acad. Sci. USA Vol. 80, pp. 5139-5143, August 1983 Neurobiology

Proenkephalin, [Metlenkephalin, and oxytocin immunoreactivities are colocalized in bovine hypothalamic magnocellular neurons (neurosecretion/supraoptic nucleus/paraventricular nucleus/hypophysis/median eminence)

J. J. VANDERHAEGHEN*, F. LOTSTRA*, D. R. LISTONt, AND J. ROSSIERt *Neuropathology Clinic, Neuropathology Neuropeptide Research Laboratory, Brugmann University Hospital, Free University of Brussels, Brussels, Belgium; and tLaboratoire de Physiologie Nerveuse, Departement de Neurophysiologie appliquee, Centre National de la Recherche Scientifique, 91190, Cif-sur-Yvette, France

Communicated by J. Brachet, May 16, 1983 ABSTRACT The distribution of proenkephalin and [Met]enkephalin immunoreactivities in the bovine hypothalamo-neurohypophyseal system was studied by use of specific antisera. Proenkephalin and [Met]enkephalin immunoreactivities were found in magnocellular neuronal cell bodies in the dorsal part of the supraoptic nuclei and in the peripheral part of the paraventricular nuclei. A densely staining network of nerve terminals was found in the external part of the median eminence and in the posterior hypophysis. This general distribution is identical to that of the neurohypophyseal hormone oxytocin. The precise localization of proenkephalin and [Metlenkephalin immunoreactivities was compared to the distribution of oxytocin and vasopressin in serial 5,um sections through the magnocellular nuclei. Oxytocin immunoreactivity was nearly always present in cells that were stained with proenkephalin and [Met]enkephalin antisera. The vasopressin-immunoreactive cells were never stained with either the proenkephalin or the [Met]enkephalin antisera.

Since the discovery of the enkephalin pentapeptides, the list of opioid peptides has grown rapidly. These peptides can be divided into three families, each stemming from a distinct biosynthetic precursor (1-5). Each of these precursors is polyvalent, containing multiple copies of active peptides. Proenkephalincontains [Met]-and [Leu]enkephalin, the octapeptide [Met]enkephalin-Arg6-Gly7-Leu8, and the heptapeptide [Metlenkephalin-Arg6-Phe7 (6, 7). Proopiomelanocortin gives rise to (endorphin and several nonopiate peptide horm ones. The products of the recently characterized prodynorphin are not yet well defined, but the precursor contains the sequences of dynorphin A, a-neoendorphin, and dynorphin B (rimorphin) (5). Neurons containing many of these opioid peptides have been found in the hypothalamus. Cell bodies containing 1-endorphin immunoreactivity (IR) are restricted to the arcuate nucleus (8). Dynorphin A and other peptides derived from prodynorphin have been found in magnocellular neurons (9-13). The presence in magnocellular neurons of peptides derived from proenkephalin is still uncertain. Watson et aL (11) and Weber et al. (10) have proposed that proenkephalin-derived peptides are not present in magnocellular~neurons of the rat. However, Martin and Voigt (14) and Martin et aL (15) have described the coexistence of [Met]enkephalin and oxytocin in nerve terminals in the rat neurohypophysis. To clarify this issue we have used a different approach. Rather than use antisera against a restricted portion of the opioid peptides, we prepared an antiserum against the NH2-terminal portion of proenkephalin. This antiserum does not recognize [Met]enkephalin, [Leu]enkephalin, dynorphin, or any other known

opioid peptide and is highly specific for the 72-amino-acid NH2terminal portion of proenkephalin. This region of the precursor does not contain the sequence of [Met]- or [Leu]enkephalin. We have previously reported that this NH2-terminal fragment of proenkephalin, which we have called synenkephalin, is found in enkephalinergic cell bodies and terminals in various areas of the bovine brain (16). We describe here that antiserum against proenkephalin labels magnocellular neurons in the bovine hypothalamus, which nearly always contain oxytocin IR. None of the vasopressin-immunoreactive magnocellular neurons were stained with this antiserum. MATERIALS AND METHODS Bovine brains and pituitaries were obtained fresh from a local slaughterhouse. Tissues were cut into slices 1 cm thick within 1 hr after death and fixed in "Bouin Hollande sublime"' for 2 days (17). Tissues were then embedded in paraffin, and sections 5 ,um thick were prepared for immunohistochemical staining. Peptides were visualized by use of the unlabeled peroxidase-antiperoxidase technique of Sternberger et al. (18) as modified by Vandesande et aL (19). Antiserum directed against [Met]enkephalin was obtained from a young Dendermonde rabbit after five subcutaneous injections of the peptide, which had been conjugated to thyroglobulin (20) and mixed with Freund's adjuvant. In radioimmunoassay this serum used at a final dilution of 1:6,000 has a sensitivity of 35 pmol of [Met]enkephalin per liter and displays a 0.5% cross-reactivity with [Leu]enkephalin and 0.05% crossreactivity with dynorphin A. Adsorbed antisera against oxytocin and vasopressin were generous gifts from F. Vandesande. Briefly, anti-vasopressin antiserum was adsorbed by treatment with oxytocin coupled to Sepharose 4B beads. Anti-oxytocin--antiserum was similarly treated with vasopressin-Sepharose 4B. Complete details on the preparation and specificity of these antisera have been reported (19). For the production of the anti-proenkephalin antiserum, a protein composed of the first 77-amino-acid residues of adrenal proenkephalin was purified from the adrenal medulla (16). The purified protein was injected into the popliteal lymph nodes of adult male Bourgogne rabbits. Antiserum obtained 2 wk after the third injection was found to be suitable for this study. Crossreactivity in a radioimmunoassay using this antiserum was less than 0.001% for the following peptides: [Met]enkephalin, [Met]enkephalin-Lys6, [Met]enkephain-Arg6, [Met]enkephalin-Arg6-

Arg7, [Met]enkephalin-Arg6-Phe7, [Met]enkephalin-Arg6-Gly7Leu8, [Met]enkephalin-Arg6-Gly7-Leu8-Lys9, Arg°-[Met]en-

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Abbreviation: IR, immunoreactivity. 5139

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kephalin, [Leu]enkephalin, [Leu]enkephalin-Lys6, [Leu]enkephalin sulfate, a-endorphin, /B3endorphin, y-endorphin, aneoendorphin, ,3neoendorphin, dynorphin A18, BAM22P (21), peptide E (22), peptide F (23). To minimize nonspecific staining, antisera were always used at a dilution greater than 1:5,000. Further specificity controls were performed routinely on adjacent sections with antisera in the presence of an excess of the relevant antigen.

tem and found to be superimposable (Fig. 2 E and F). In serial 5-,um sections through the supraoptic nucleus stained alternatively with proenkephalin antiserum (Fig. 2E) and with [Met] enkephalin antiserum (Fig. 2F), nearly all cell bodies contained both IRs. The distribution of proenkephalin IR was also compared with the distribution of oxytocin IR and vasopressin IR. Only magnocellular neurons in the dorsal part of the supraoptic nucleus were stained with the oxytocin antiserum (Fig. 3B). Magnocellular neurons in the ventral part showed positive staining with the vasopressin antiserum (not shown). The same differential labeling was found in the peripheral and central parts of the paraventricular nuclei (Fig. 1C). By use of serial sections (Fig. 3) it was possible to show that proenkephalin IR and oxytocin IR were often located in the same magnocellular neurons. Because of the large size of the magnocellular neurons (25 ,um), when the nucleus of a particular cell is present in one of the 5-,um sections, at least some part of the same cell is present in the adjacent section. By using this criterion, nearly all of the cells that were stained with the proenkephalin antiserum also were stained with the oxytocin antiserum. Several oxytocin-immunoreactive cells remained unstained with the proenkephalin antiserum. In contrast, cells visualized with the vasopressin antiserum were never stained in adjacent sections with the proenkephalin antiserum. [Met]enkephalin IR and oxytocin IR also were found frequently in the same magnocellular neurons (Fig. 2 A-D). The intensity of the staining in consecutive sections with the [Met]enkephalin antiserum always appeared weaker than that with the oxytocin antiserum.

RESULTS The general distribution of proenkephalin IR in the bovine hypothalamo-neurohypophyseal system is given in Fig. 1. In the supraoptic nuclei (Fig. 1A), staining was restricted to magnocellular neurons located in the dorsal part of these nuclei. No IR was found in the ventral part just above the tractus opticus (T.O.). In the paraventricular nuclei (Fig. 1B) proenkephalin IR was restricted to magnocellular neurons located mostly in the lateral periphery of these nuclei. No IR was found in the vicinity of the third ventricle (3V). For comparison, Fig. 1C shows that the oxytocin-immunoreactive magnocellular neurons also are restricted to the lateral periphery of the paraventricular nucleus. A dense layer of fibers was found in the external zone of the median eminence (Fig. 1D). In the hypophysis, proenkephalin-immunoreactive fibers were present only in the neural lobe and not in the anterior or intermediate lobes (Fig. 1E). In the neural lobe, dense layers of fibers were concentrated in the periphery of the gland. The distributions of [Met]enkephalin IR and proenkephalin IR were compared in the hypothalamo-neurohypophyseal sys-

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FIG. 1. General distribution of proenkephalin (PRO-ENK) ER in the bovine hypothalamo-neurohypophyseal system. (A) Proenkephalin ER in the supraoptic nucleus was restricted to cell bodies in the dorsal part ofthe nucleus. In the paraventricular nucleus, proenkephalin IR (B) and oxytocin,(OXY) IR (C) were located in the lateral periphery.. Proenkephalin-immunoreactive nerve terminals were present in the external median eminence (D) and in the neurohypophysis (E). T.O., tractus opticus; 3V, third ventricle; LA, anterior lobe; LI,.intermediate lobe; LP, posterior lobe. (Bar = 100,um.)

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DISCUSSION The magnocellular neurons of the hypothalamo-neurohypophyseal system contain a wide variety of peptide hormones, including vasopressin, oxytocin, cholecystokinin, and corticotropin-releasing factor (19, 24-26). The hypothalamus is also rich in opiate peptides (8-13). We have shown here that proenkephalin and [Met]enkephalin IRs are present in oxytocin-containing magnocellular neurons of the bovine hypothalamus. There is considerable evidence that the biosynthetic pathway of [Met]enkephalin in the brain is similar to that found in the adrenal gland (16, 27-29). IRs for the heptapeptide [Met]enkephalin-Arg6-Phe7, the octapeptide [Met]enkephalin-Arg Gly7-Leu8, and BAM22P have been shown to be present in enkephalinergic neurons (28, 29). All of these peptides are derived from a common precursor, proenkephalin, so it is not surprising that proenkephalin IR and [Met]enkephalin IR are found in the same neurons. The degree of staining with the anti-proenkephalin antiserum was always more intense than that with the anti-[Met]enkephalin antiserum. This may be due to a greater efficiency of fixation or to a greater availability of antigenic determinants obtained with larger proteins. The general distribution of proenkephalin IR and [Met]enkephalin IR in the hypothalamo-neurohypophyseal system was superimposable on the distribution of oxytocin. Proenkephalinand [Met]enkephalin-immunostained cell bodies were visualized in the dorsal part of the supraoptic nuclei and in the lateral peripheral part of the paraventricular nuclei, in the same areas

as are found oxytocinergic cell bodies (19, 25). Proenkephalinand [Met]enkephalin-immunoreactive fibers and terminals were found in the external median eminence and were concentrated in the periphery of the neurohypophysis. We found a similar distribution for oxytocin in these tissues as has been reported by others (19, 25). The staining of cell bodies in the magnocellular nuclei with the [Met]enkephalin and proenkephalin antisera was restricted nearly always to neurons that also stained for oxytocin, demonstrating that [Met]enkephalin coexists with oxytocin in these cells. In some preparations there was nearly a 1:1 correspondence between oxytocin IR and proenkephalin IR in adjacent sections. These results extend the observations of Martin and Voigt (14) and Martin et a. (15), who have shown that, in the rat neurohypophysis, [Met]enkephalin IR coincides with oxytocin IR in neuronal terminals. By use of antisera against the biosynthetic precursor and the daughter peptide, we have shown here that [Met]enkephalin is colocalized with oxytocin in the magnocellular neuronal perikarya of the bovine supraoptic and paraventricular nuclei. It has been reported that another opioid peptide, dynorphin, is present in vasopressin-containing magnocellular neurons (9, 13). The dynorphins and neoendorphins arise from a precursor protein, prodynorphin, which does not contain the sequence of [Met]enkephalin (5). In the present study, proenkephalin IR and [Metlenkephalin IR were never found in cells that stained positively for vasopressin. In contrast, by use of two different

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bxv, FIG. 3. Serial 5-,tm sections through the bovine supraoptic nucleus showing colocalization of proenkephalin (PRO-ENK) IR andoxytocin (OXY) IR. Most cells staining positively with the anti-proenkephalin antiserum (A and C) also were stained with the anti-oxytocin antiserum (B and D). C and D are at higher magnification. (Bar = 100 am.)

anti-[Leu]enkephalin antisera, we have observed positive staining of vasopressin-containing magnocellular neurons in the central parts of the bovine supraoptic and paraventricular nuclei. This staining pattern is similar to that reported in the rat brain with antisera against dynorphin (9). Thus, it appears that the proenkephalin and prodynorphin systems are present in different subpopulations of magnocellular neurons, the enkephalin family being present in most oxytocin-immunoreactive cells and the dynorphin family, in some vasopressin-immunoreactive cells. The physiological significance of this anatomical separation of opiate peptides may be related to the differential potency that they exhibit at the various subclasses of opiate receptors. Peptides derived from proenkephalin are highly potent ligands for the ,u and 8 receptors (30), and dynorphin has been shown to be much more potent at the K receptor (31). These peptides are likely candidates as modulators of oxytocin and vasopressin release at the neurohypophyseal level (32, 33). A distinction between the receptors activated by concurrent release of oxytocin/enkephalin or vasopressin/dynorphin would minimize "crosstalk" between the two systems, allowing independent regulation of oxytocin and vasopressin release. This is reminiscent of the monoaminergic system, where closely related compounds, through selective activation of different receptors, produce distinct physiological responses. We have reported previously that some oxytocinergic magnocellular neurons also contain cholecystokinin IR (19). Martin et al. (15) have shown recently that these peptides are colocalized in neuronal terminals within the rat neurohypophysis.

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