Proenkephalin, [Metlenkephalin, and Oxytocin Immunoreactivities

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 byJ. Brachet, May 16, 1983 ABSTRACT The distribution of proenkephalin and [Met]en- opioid peptide and is highly specific for the 72-amino-acid NH2- kephalin immunoreactivities in the bovine hypothalamo-neuro- terminal portion of proenkephalin. This region of the precursor hypophyseal system was studied by use of specific antisera. Pro- does not contain the sequence of [Met]- or [Leu]enkephalin. enkephalin and [Met]enkephalin immunoreactivities were found We have previously reported that this NH2-terminal fragment in magnocellular neuronal cell bodies in the dorsal part of the su- of proenkephalin, which we have called synenkephalin, is found praoptic nuclei and in the peripheral part of the paraventricular in enkephalinergic cell bodies and terminals in various areas of nuclei. A densely staining network of nerve terminals was found the bovine brain (16). We describe here that antiserum against in the external part of the median eminence and in the posterior proenkephalin labels magnocellular neurons in the bovine hy- hypophysis. This general distribution is identical to that of the pothalamus, which nearly always contain oxytocin IR. None of neurohypophyseal hormone oxytocin. The precise localization of the vasopressin-immunoreactive magnocellular neurons were proenkephalin and [Metlenkephalin immunoreactivities was com- stained with this antiserum. pared 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 MATERIALS AND METHODS with proenkephalin and [Met]enkephalin antisera. The vasopressin-immunoreactive cells were never stained with either the pro- Bovine brains and pituitaries were obtained fresh from a local enkephalin or the antisera. slaughterhouse. Tissues were cut into slices 1 cm thick within [Met]enkephalin 1 hr after death and fixed in "Bouin Hollande sublime"' for 2 Since the discovery of the enkephalin pentapeptides, the list days (17). Tissues were then embedded in paraffin, and sec- of opioid peptides has grown rapidly. These peptides can be tions 5 ,um thick were prepared for immunohistochemical stain- divided into three families, each stemming from a distinct bio- ing. Peptides were visualized by use of the unlabeled perox- synthetic precursor (1-5). Each of these precursors is polyva- idase-antiperoxidase technique of Sternberger et al. (18) as lent, containing multiple copies of active peptides. Proenkeph- modified by Vandesande et aL (19). alincontains [Met]-and[Leu]enkephalin, theoctapeptide [Met]- Antiserum directed against [Met]enkephalin was obtained enkephalin-Arg6-Gly7-Leu8, and the heptapeptide [Metlen- from a young Dendermonde rabbit after five subcutaneous in- kephalin-Arg6-Phe7 (6, 7). Proopiomelanocortin gives rise to (- jections of the peptide, which had been conjugated to thyro- endorphin and several nonopiate peptide hormones. The prod- globulin (20) and mixed with Freund's adjuvant. In radioim- ucts of the recently characterized prodynorphin are not yet well munoassay this serum used at a final dilution of 1:6,000 has a defined, but the precursor contains the sequences of dynor- sensitivity of 35 pmol of [Met]enkephalin per liter and displays phin A, a-neoendorphin, and dynorphin B (rimorphin) (5). a 0.5% cross-reactivity with [Leu]enkephalin and 0.05% cross- Neurons containing many of these opioid peptides have been reactivity with dynorphin A. found in the hypothalamus. Cell bodies containing 1-endor- Adsorbed antisera against oxytocin and vasopressin were phin immunoreactivity (IR) are restricted to the arcuate nucleus generous gifts from F. Vandesande. Briefly, anti-vasopressin (8). Dynorphin A and other peptides derived from prodynor- antiserum was adsorbed by treatment with oxytocin coupled to phin have been found in magnocellular neurons (9-13). The Sepharose 4B beads. Anti-oxytocin--antiserum was similarly presence in magnocellular neurons of peptides derived from treated with vasopressin-Sepharose 4B. Complete details on proenkephalin is still uncertain. Watson et aL (11) and Weber the preparation and specificity of these antisera have been re- et al. (10) have proposed that proenkephalin-derived peptides ported (19). are not present in magnocellular~neurons of the rat. However, For the production of the anti-proenkephalin antiserum, a Martin and Voigt (14) and Martin et aL (15) have described the protein composed of the first 77-amino-acid residues of adrenal coexistence of [Met]enkephalin and oxytocin in nerve termi- proenkephalin was purified from the adrenal medulla (16). The nals in the rat neurohypophysis. purified protein was injected into the popliteal lymph nodes of To clarify this issue we have used a different approach. Rather adult male Bourgogne rabbits. Antiserum obtained 2 wk after than use antisera against a restricted portion of the opioid pep- the third injection was found to be suitable for this study. Cross- tides, we prepared an antiserum against the NH2-terminal por- reactivity in a radioimmunoassay using this antiserum was less tion ofproenkephalin. This antiserum does not recognize [Met]- than 0.001% forthefollowingpeptides: [Met]enkephalin, [Met]- enkephalin, [Leu]enkephalin, dynorphin, or any other known enkephalin-Lys6, [Met]enkephain-Arg6, [Met]enkephalin-Arg6- Arg7, [Met]enkephalin-Arg6-Phe7, [Met]enkephalin-Arg6-Gly7- The publication costs of this article were defrayed in part by page charge Leu8, [Met]enkephalin-Arg6-Gly7-Leu8-Lys9, Arg°-[Met]en- payment. This article must therefore be hereby marked "advertise- ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: IR, immunoreactivity.

5139 Downloaded by guest on September 30, 2021 5140 Neurobiology: Vanderhaeghen et aLPProc. Natl. Acad. Sci. USA 80 (1983) kephalin, [Leu]enkephalin, [Leu]enkephalin-Lys6, [Leu]en- tem and found to be superimposable (Fig. 2 E and F). In serial kephalin sulfate, a-endorphin, /B3endorphin, y-endorphin, a- 5-,um sections through the supraoptic nucleus stained alter- neoendorphin, ,3neoendorphin, dynorphin A18, BAM22P (21), nativelywithproenkephalinantiserum (Fig. 2E) andwith [Met]- peptide E (22), peptide F (23). To minimize nonspecific stain- enkephalin antiserum (Fig. 2F), nearly all cell bodies contained ing, antisera were always used at a dilution greater than 1:5,000. both IRs. Further specificity controls were performed routinely on ad- The distribution of proenkephalin IR was also compared with jacent sections with antisera in the presence of an excess of the the distribution of oxytocin IR and vasopressin IR. Only mag- relevant antigen. nocellular neurons in the dorsal part of the supraoptic nucleus were stained with the oxytocin antiserum (Fig. 3B). Magno- cellular neurons in the ventral part showed positive staining RESULTS with the vasopressin antiserum (not shown). The same differ- The general distribution of proenkephalin IR in the bovine hy- ential labeling was found in the peripheral and central parts of pothalamo-neurohypophyseal system is given in Fig. 1. In the the paraventricular nuclei (Fig. 1C). supraoptic nuclei (Fig. 1A), staining was restricted to magno- By use of serial sections (Fig. 3) it was possible to show that cellular neurons located in the dorsal part of these nuclei. No proenkephalin IR and oxytocin IR were often located in the same IR was found in the ventral part just above the tractus opticus magnocellular neurons. Because of the large size of the mag- (T.O.). In the paraventricular nuclei (Fig. 1B) proenkephalin IR nocellular neurons (25 ,um), when the nucleus of a particular was restricted to magnocellular neurons located mostly in the cell is present in one of the 5-,um sections, at least some part lateral periphery of these nuclei. No IR was found in the vi- of the same cell is present in the adjacent section. By using this cinity of the third ventricle (3V). For comparison, Fig. 1C shows criterion, nearly all of the cells that were stained with the pro- that the oxytocin-immunoreactive magnocellular neurons also enkephalin antiserum also were stained with the oxytocin anti- are restricted to the lateral periphery of the paraventricular nu- serum. Several oxytocin-immunoreactive cells remained un- cleus. A dense layer of fibers was found in the external zone of stained with the proenkephalin antiserum. In contrast, cells the median eminence (Fig. 1D). In the hypophysis, proen- visualized with the vasopressin antiserum were never stained kephalin-immunoreactive fibers were present only in the neural in adjacent sections with the proenkephalin antiserum. lobe and not in the anterior or intermediate lobes (Fig. 1E). In [Met]enkephalin IR and oxytocin IR also were found fre- the neural lobe, dense layers of fibers were concentrated in the quently in the same magnocellular neurons (Fig. 2 A-D). The periphery of the gland. intensity of the staining in consecutive sections with the [Met]- The distributions of [Met]enkephalin IR and proenkephalin enkephalin antiserum always appeared weaker than that with IR were compared in the hypothalamo-neurohypophyseal sys- the oxytocin antiserum.

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t4> E N K .RO- 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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FIG. 2. Frontal sections through the bovine supraoptic nucleus showing colocalization of[Metlenkephalin (MET-ENK) IR with oxytocin (OXY) IR and proenkephalin (PRO-ENK) IR. In serial 5-gm sections incubated with anti-[Metlenkephalin (A and B) or anti-oxytocin antiserum (C and D), there was nearly a 1:1 correspondence between positively staining cells. B and D are at higher magnification. Proenkephalin IR (E) was also found in cells containing [Metlenkephalin IR (F) in adjacent sections. (Bar = 100 jim.) -1*,t DISCUSSION as are found oxytocinergic cell bodies (19, 25). Proenkephalin- The magnocellular neurons of the hypothalamo-neurohypo- and [Met]enkephalin-immunoreactive fibers and terminals were physeal system contain a wide variety of peptide hormones, found in the external median eminence and were concentrated including vasopressin, oxytocin, cholecystokinin, and cortico- in the periphery of the neurohypophysis. We found a similar tropin-releasing factor (19, 24-26). The hypothalamus is also distribution for oxytocin in these tissues as has been reported rich in opiate peptides (8-13). We have shown here that proen- by others (19, 25). kephalin and [Met]enkephalin IRs are present in oxytocin-con- The staining of cell bodies in the magnocellular nuclei with taining magnocellular neurons of the bovine hypothalamus. the [Met]enkephalin and proenkephalin antisera was restricted There is considerable evidence that the biosynthetic path- nearly always to neurons that also stained for oxytocin, dem- way of [Met]enkephalin in the brain is similar to that found in onstrating that [Met]enkephalin coexists with oxytocin in these the adrenal gland (16, 27-29). IRs for the heptapeptide [Met]- cells. In some preparations there was nearly a 1:1 correspon- enkephalin-Arg6-Phe7, the octapeptide [Met]enkephalin-Arg - dence between oxytocin IR and proenkephalin IR in adjacent Gly7-Leu8, and BAM22P have been shown to be present in en- sections. These results extend the observations of Martin and kephalinergic neurons (28, 29). All of these peptides are de- Voigt (14) and Martin et a. (15), who have shown that, in the rived from a common precursor, proenkephalin, so it is not sur- rat neurohypophysis, [Met]enkephalin IR coincides with oxy- prising that proenkephalin IR and [Met]enkephalin IR are found tocin IR in neuronal terminals. By use of antisera against the in the same neurons. The degree of staining with the anti-pro- biosynthetic precursor and the daughter peptide, we have shown enkephalin antiserum was always more intense than that with here that [Met]enkephalin is colocalized with oxytocin in the the anti-[Met]enkephalin antiserum. This may be due to a greater magnocellular neuronal perikarya of the bovine supraoptic and efficiency of fixation or to a greater availability of antigenic de- paraventricular nuclei. terminants obtained with larger proteins. It has been reported that another opioid peptide, dynorphin, The general distribution of proenkephalin IR and [Met]en- is present in vasopressin-containing magnocellular neurons (9, kephalin IR in the hypothalamo-neurohypophyseal system was 13). The dynorphins and neoendorphins arise from a precursor superimposable on the distribution of oxytocin. Proenkephalin- protein, prodynorphin, which does not contain the sequence of and [Met]enkephalin-immunostained cell bodies were visual- [Met]enkephalin (5). In the present study, proenkephalin IR ized in the dorsal part of the supraoptic nuclei and in the lateral and [Metlenkephalin IR were never found in cells that stained peripheral part of the paraventricular nuclei, in the same areas positively for vasopressin. In contrast, by use of two different Downloaded by guest on September 30, 2021 5142 Neurobiology: Vanderhaeghen et aL Proc. NatL Acad. Sci. USA 80 (1983)

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anti-[Leu]enkephalin antisera, we have observed positive staining Further study with semithin sections is needed to determine of vasopressin-containing magnocellular neurons in the central whether proenkephalin IR, [Met]enkephalin IR, and chole- parts of the bovine supraoptic and paraventricular nuclei. This cystokinin IR are all present in the same oxytocin-containing staining pattern is similar to that reported in the rat brain with magnocellular neurons of the bovine brain. antisera against dynorphin (9). Thus, it appears that the proen- and are in different This work was supported by the Belgian Medical Research Fund kephalin prodynorphin systems present (3.45.21.82), the Queen Elisabeth Medical Research Foundation, the subpopulations of magnocellular neurons, the enkephalin fam- Esther A. and Joseph Klingenstein Fund (New York), Centre National ily being present in most oxytocin-immunoreactive cells and de la Recherche Scientifique (Action Thematique Programmee Inter- the dynorphin family, in some vasopressin-immunoreactive cells. nationale and Centre National de la Recherche Scientifique-National The physiological significance of this anatomical separation Science Foundation Exchange Program), and Institut National de la Sante of opiate peptides may be related to the differential potency et de la Recherche Medicale (CRL 81 6025). that they exhibit at the various subclasses of opiate receptors. 1. Noda, M., Furitani, Y., Takahashi, H., Toyosata, M., Hirose, T., Peptides derived from proenkephalin are highly potent ligands Inayama, S., Nakanishi, S. & Numa, S. (1982) Nature (London) for the ,u and 8 receptors (30), and dynorphin has been shown 295, 202-206. to be much more potent at the K receptor (31). These peptides 2. Gubler, U., Seeburg, P., Hoffman, B. J., Gage, L. P. & Uden- are likely candidates as modulators of oxytocin and vasopressin friend, S. (1982) Nature (London) 295, 206-208. release at the neurohypophyseal level (32, 33). A distinction 3. Comb, M., Seeburg, P., Adelman, J., Eiden, L. & Herbert, E. between the receptors activated concurrent release of oxy- (1982) Nature (London) 295, 663-666. by 4. Roberts, J. L., Seeburg, P. H., Shine, J., Herbert, E., Baxter, J. tocin/enkephalin or vasopressin/dynorphin would minimize D. & Goodman, H. M. (1979) Proc. NatL Acad. Sci. USA 76, 2153- "crosstalk" between the two systems, allowing independent 2157. regulation of oxytocin and vasopressin release. This is remi- 5. Kakidani, H., Furutani, Y., Takahashi, H., Noda, M., Morimoto, niscent of the monoaminergic system, where closely related Y., Hirose, T., Asai, M., Inayama, S., Nakanishi, S. & Numa, S. compounds, through selective activation of different receptors, (1982) Nature (London) 298, 245-249. & distinct responses. 6. Rossier, J., Audigier, Y., Ling, N., Cros, J. Udenfriend, S. (1980) produce physiological Nature (London) 288, 88-90. We have reported previously that some oxytocinergic mag- 7. Ikeda, Y., Nakao, K., Yoshimasa, T., Yanaihara, N., Numa, S. & nocellular neurons also contain cholecystokinin IR (19). Martin Imura, H. (1982) Biochem. Biophys. Res. Commun. 107, 656-662. et al. (15) have shown recently that these peptides are colocal- 8. Bloom, F., Battenberg, E., Rossier, J., Ling, N. & Guillemin, R. ized in neuronal terminals within the rat neurohypophysis. (1978) Proc. Nat. Acad. Sci. USA 75, 1591-1595. Downloaded by guest on September 30, 2021 Neurobiology: Vanderhaeghen et aL Proc. Natl. Acad. Sci. USA 80 (1983) 5143 9. Watson, S. J., Akil, H., Fischli, W., Goldstein, A., Zimmerman, 22. Kilpatrick, D. L., Taniguchi, T., Jones, B. N., Stern, A. S., Shively, E., Nilaver, G. & van Wimersma Greidanus, T. B. (1982) Science J. E., Hullihan, J., Kimura, S., Stein, S. & Udenfriend, S. (1981) 216, 85-87. Proc. Natl Acad. Sci. USA 78, 3265-3268. 10. Weber, E., Roth, K. A., Evans, C. J., Chang, J.-K. & Barchas, J. 23. Jones, B. N., Stern, A. S., Lewis, R. V., Kimura, S., Stein, S. & D. (1982) Life Sci. 31, 1761-1764. Udenfriend, S. (1980) Arch. Biochem. Biophys. 204, 392-395. 11. Watson, S. J., Khachaturian, H., Akil, H., Coy, D. H. & Gold- 24. Vanderhaeghen, J. J., Lotstra, F., De Mey, J. & Gilles, C. (1980) stein, A. (1982) Science 218, 1134-1136. Proc. NatL Acad. Sci. USA 77, 1190-1194. 12. Weber, E., Roth, K. A. & Barchas, J. D. (1982) Proc. Natl Acad. 25. Vanderhaeghen, J. J., Lotstra, F., Vandesande, F. & Dierickx, K. Sci. USA 79, 3062-3066. (1981) Cell Tissue Res. 221, 227-231. 13. Weber, E., Evans, C. J. & Barchas, J. D. (1982) in Regulatory 26. Roth, K. A., Weber, E. & Barchas, J. D. (1982) Life Sci. 31, 1857- Peptides: From Molecular Biology to Function, eds. Costa, E. & 1860. Trabucchi, M. (Raven, New York), pp. 519-526. 27. Dandekar, S. & Sabal, S. L. (1982) Biochem. Biophys. Res. Com- 14. Martin, R. & Voigt, K. H. (1981) Nature (London) 289, 502-504. mun. 105, 67-74. 15. Martin, R., Geis, R., Holl, R., Schafer, M. & Voight, H. K. (1983) 28. Bloch, B., Baird, A., Ling, N., Benoit, R. & Guillemin, R. (1983) Neuroscience 8, 213-227. Brain Res. 263, 251-257. 16. Liston, D. R., Vanderhaeghen, J. J. & Rossier, J. (1983) Nature 29. Khachaturian, H., Lewis, M. E. & Watson, S. J. (1982) Life Sci. (London) 302, 62-65. 31, 1879-1882. 17. Ganter, P. & Jolles, G. (1970) Histochimie Normale et Patholo- 30. Corbett, A. D., Paterson, S. J., McKnight, A. T., Magnan, J. & gique (Gauthier-Villars, Paris), Vol. 2, p. 1395. Kosterlitz, H. (1982) Nature (London) 299, 79-81. 18. Sternberger, L. A., Hardy, P. H., Jr., Cuculis, J. J. & Meyer, H. 31. Chavkdn, C., James, I. F. & Goldstein, A. (1982) Science 215, 413- G. (1970) Histochem. Cytochem. 18, 315-333. 415. 19. Vandesande, F., Dierickx, K. & De Mey, J. (1977) Cell Tissue Res. 32. Rossier, J., Battenberg, E., Pittman, Q., Bayon, A., Koda, L., 180, 443-452. Miller, R., Guillemin, R. & Bloom, F. (1979) Nature (London) 277, 20. Skowsky, W. R. & Fisher, D. A. (1972)J. Lab. Clin. Med. 80, 134- 653-655. 144. 33. Clark, G., Wood, P., Merrick, L. & Lincoln, D. W. (1979) Nature 21. Mizuno, K., Minamino, N., Kangawer, K. & Matsuo, H. (1980) (London) 282, 746-748. Biochem. Biophys. Res. Commun. 97, 1283-1290. 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