The PHI (PHI-27)

Proc. NatL Acad. Sci. USA Vol. 80, pp. 895-898, February 1983 Neurobiology

The PHI (PHI-27)/corticotropin-releasing factor/enkephalin immunoreactive hypothalamic neuron: Possible morphological basis for integrated control of prolactin, corticotropin, and growth hormone secretion (hypothalamic-pituitary hormones/release regulation/peptides/coexistence) T. HOKFELT*, J. FAHRENKRUGt, K. TATEMOTOt, V. MUTTS, S. WERNERO, A.-L. HULTINGO, L. TERENIUS1, AND K. J. CHANGII Departments of *Histology and *Biochemistry, Karolinska Institutet, Stockholm, Sweden; IDepartment of Endocrinology, Karolinska Hospital, Stockholm, Sweden; Department of Pharmacology, Uppsala University, Uppsala, Sweden; tDepartment of Clinical Chemistry, Fredrikborgs Central Hospital, Hiller6d, Denmark; and flWellcome Research Laboratories, Research Triangle Park, North Carolina 27709 Communicated by U. S. von Euler, November 1, 1982

ABSTRACT By using the indirect immunofluorescence tech- MATERIAL AND METHODS nique, one and the same neuron in the parvocellular part of the Male albino rats of the Sprague-Dawley strain (body weight, paraventricular nucleus has been shown to stain with antisera 150 g) were used. Untreated rats as well as colchicine-treated against three different peptides: PHI (PHI-27), corticotropin-re- rats were studied. The latter rats received an injection into the leasing factor (CRF), and enkephalin. This could explain the well- lateral ventricle of 120 ,ug of colchicine dissolved in 20 p1 of known parallel increase in plasma prolactin, corticotropin, and physiological saline. The rats were perfused with ice-cold for- growth hormone levels-for example, under certain types of malin, and the brains were dissected out, rinsed in 0.1 M phos- stress-as being due to a concomitant release of PHI-like, CRF- phate buffer with 5% sucrose added, and cut on a cryostat. The like, and enkephalin-like peptides from the same nerve endings sections were processed for the indirect immunofluorescence in the median eminence. A hypothetical mechanism for the co- technique (17). Briefly, series of adjacent sections (thickness, ordinated release of these three anterior pituitary hormones is 3, 6, 8, 10, or 14 1m) were incubated at 40C in a humid at- discussed. mosphere with antiserum to PHI, CRF, or [Met]enkephalin for 18-24 hr. rinsed, incubated with fluorescein isothiocyanate- or It has been shown that vasoactive intestinal polypeptide (VIP) tetramethyl rhodamine-conjugated sheep anti-rabbit antibod- (1, 2) causes release ofprolactin from the anterior pituitary gland ies, rinsed, mounted, and examined in a fluorescence micro- in vitro (3-5) and in vivo (6). Therefore, this peptide may rep- scope. The production of the enkephalin antisera has been de- resent a candidate for the prolactin-releasing factor (7, 8). How- scribed (18, 19). The CRF antiserum was purchased from Penin- ever, immunohistochemical studies have so far revealed only sula Laboratories (San Carlos, CA). The PHI antiserum was single VIP-positive nerve fibers in the medial basal hypothal- raised against PHI conjugated to bovine serum albumin. It amus, including the median eminence (9-12), from which the crossreacts with VIP and secretin to a very limited extent but hypothalamic-releasing hormone and inhibitory hormone are does not display crossreactivity with a number of other pep- released into the hypophysial portal blood vessels for transport tides, including [Leu]enkephalin and [Met]enkephalin and glu- to the anterior pituitary. More recently it has been demon- cagon (unpublished data). strated that PHI-like immunoreactivity [PHI-27; the peptide In some cases the restaining technique of Tramu et aL (20) (P) having NH2-terminal histidine (H) and COOH-terminal iso- was used to investigate the presence of more than one com- leucine (I) amide and 27 amino acid residues] (13) is present in pound in the same neuron. Briefly, after photography of the a parvocellular paraventriculo-infundibular system, forming a PHI staining patterns, removal ofthe coverslip, and rinsing in dense fiber network around the portal capillaries (14). PHI has phosphate-buffered saline, the sections were immersed in acid considerable structural similarities to VIP (13) and causes pro- potassium permanganate, rinsed, incubated with fluorescein lactin release, although it is less potent than VIP in this respect isothiocyanate-conjugated antiserum (as control), and, if nega- (unpublished data). tive, incubated with enkephalin or CRF antiserum. This pro- In the present communication we report that at least part of cedure was repeated and the final incubation was made with the PHI immunoreactive neurons in the parvocellular periven- CRF or, alternatively, enkephalin antiserum. For control pur- tricular hypothalamic nuclei contain two other peptides, one poses the antisera were pretreated with an excess of the re- enkephalin-like peptide (15) and one corticotropin-releasing spective peptide (50 pug ofpeptide per ml ofantiserum diluted factor (CRF)-like peptide (16). These PHI/CRF/enkephalin 1:10 or 1:100). This abolished all staining patterns described immunoreactive neurons may be involved in the integration of below. Furthermore, each antiserum was pretreated with an control ofprolactin, corticotropin (ACTH), and growth hormone excess ofeither ofthe other peptides (concentrations as above), (GH) secretion. Abbreviations: PHI, PHI-27, the peptide (P) having NH2-terminal his- The publication costs ofthis article were defrayed in part by page charge tidine (H) and COOH-terminal isoleucine (I) amide and 27 amino acid payment. This article must therefore be hereby marked "advertise- residues; VIP, vasoactive intestinal polypeptide; CRF, corticotropin- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. releasing factor; ACTH, corticotropin; GH, growth hormone. 895 Downloaded by guest on September 25, 2021 896 Neurobiology: Hokfelt et al. Proc. Nad Acad. Sci. USA 80 (1983) and this treatment did not change the staining patterns described below. RESULTS Numerous PHI and CRF immunoreactive cell bodies were seen mainly in the parvocellular part ofthe paraventricular nucleus. On adjacent sections enkephalin immunoreactive perikarya were seen with a similar distribution, but many cells containing this immunoreactivity were seen also in other hypothalamic areas. In the median eminence a dense network of PHI, CRF, and enkephalin immunoreactive fibers was observed in the ex- ternal layer extending along the entire length of this structure into the stalk. Again, enkephalin immunoreactive fibers were seen in many other structures, whereas PHI and CRF had a much more restricted distribution and occurred only in a few other areas in the hypothalamus. Analysis ofthin adjacent sections revealed that in many cases all three immunoreactivities were present in the same neuronal cell bodies. This was confirmed by the elution-restaining experiments, in which certain cells in the parvocellular paraventricular nucleus were first demonstrated to contain PHI-like (Fig. la), then enkephalin-like (Fig. lb), and finally also CRF- like (Fig. 1c) immunoreactivities. There were also cells that seemed to contain either only enkephalin-like or only PHI-like and CRF-like or only enkephalin-like and CRF-like immunoreactivities (Fig. 1 a-c). The staining patterns ofthe three peptides in the median eminence were similar, but due to the small size of the fibers, identity could not be established. DISCUSSION Itwas demonstrated earlier that the parvocellular paraventricular nucleus ofthe rat contains enkephalin (21-23), CRF (24, 25), and PHI (14) immunoreactive neurons, but these studies dealt with only one peptide at the time and thus the question of oc- currence in one and the same neuron was not addressed. The present findings demonstrate that one and the same neuron in the parvocellular part ofthe paraventricular nucleus can contain at least three compounds-a PHI-like, a CRF-like, and an enkephalin-like peptide. However, some cells seemed to contain only one or, alternatively, two of these peptides. Whether this is true or whether the apparent absence of peptide is due to insufficient sensitivity of our technique remains to be eluci- dated. According to the classical concept ofHarris (26), the hormone secretion from the anterior pituitary is controlled by hypothalamic factors released into the hypophysial portal system and transported to the gland. Each pituitary hormone may be controlled by releasing or inhibitory factor(s), or both (27, 28). It is well known that under some conditions, there is a parallel increase in release of several pituitary hormones. Thus, it has been reported that certain types of stress cause release ofboth ACTH and prolactin (29-32) and of ACTH and GH (33). The

FIG. 1. Immunofluorescence micrographs of the paraventricular nucleus of a colchicine-treated rat after incubation with antiserum to PHI (a), enkephalin (ENK) (b), and CRF (c). All micrographs show the same section, which, afterphotography of thefirstdistribution pattern (PHI), has been subjected to elution and restaining with antiserum to the next antigen (CRF) and then to the third one (ENK). Note that many cells (single arrowhead) are immunoreactive to all three peptides but that some exhibit only PHI-like and CRF-like immunoreactivity (double arrowheads), some only ENK-like immunoreactivity (triple arrowheads), and some ENK-like and CRF-like immunoreactivity (quadruple arrowheads). The apparent presence of only one or two peptides must be looked upon with caution, because negative immunohistochemical results are difficult to interpret. X denotes the same blood vessel. Bar indicates 50 ,um. Downloaded by guest on September 25, 2021 USA 80 897 Neurobiology: H~kfelt et aL Proc. Nati. Acad. Sci. (1983) three peptides (immunoreactive PHI, CRF, and enkephalin) from the same nerve endings in the median eminence, can cause a coordinated release of three pituitary hormones (prolactin, ACTH, and GH). The present findings represent a further example of coexistence of multiple messengers in one and the same neuron (cf. refs. 42 and 43) and suggest that such a coexistence can explain a well-known physiological event, as, for example, parallel release of several pituitary hormones upon stress. ~~~~~~~~~~~~~~~Portal We thank Prof. B. Hokfelt, Malmo General Hospital, for constructive discussions. We thank Miss A. Edin, W. Hiort, and A. Peters for ex- for secretarial DA PHI CRF cellent technical assistance and Miss H. Olofsson expert help. This study was supported by the Swedish Medical Research Coun- cil (04X-2887, 04X-3766, 13X-01010), The NOVO Foundation, Alice och Knut Wallenbergs Stiftelse, Magnus Bergvalls Stiftelse, and Ollie och Elof Erikssons Stiftelse.

Anterior 1. Said, S. I. & Mutt, V. (1970) Science 169, 1217-1218. Y) K J ~~~~~~pituitary 2. Mutt, V. & Said, S. I. (1974) Eur. J. Biochem. 42, 581-584. 3. Kato, Y., Iwasaki, Y., Iwasaki, J., Abe, H., Yanaihara, N. & Imura, H. (1978) Endocrinology 103, 554-558. 4. Ruberg, M., Rotsztejn, W., Arancibia, S., Besson, J. & Enalbert, PHI! FIG. 2. Schematic illustration of a possible functional role of A. (1978) Eur. J. Pharmacol. 51, 319-320. CRF/enkephalin (ENK) neurons in the control of prolactin (PRL), 5. Shaar, C. J., Clemens, J. A. & Dininger, N. B. (1979) Life Sci. ACTH, and GH secretion (for references, see text). CRF stimulates 25, 2071-2074. ACTH secretion at the pituitary level. ENK-like and PHI-like peptides 6. Vijayan, E., Samson, W., Said, S. I. & McCann, S. M. (1979) enhance PRL secretion via two different mechanisms: direct stim- Endocrinology 104, 53-57. ulation of PRL cells in the pituitary by PHI and (ii) an inhibitory in- 7. Nicoll, C. S., Fiorinde, R. P., McKennee, C. T. & Parsons, J. A. fluence by ENK, at the median eminence level, on nerve endings con- (1970) in Hypophysiotrophic Hormones of the Hypothalamus: tainingdopamine (DA), which is aprolactin inhibitory factor (PIF) GH Assay and Chemistry, ed. Meites, J. (Williams & Wilkins, Balti- secretion is increased by depression of the GH-release inhibiting hor- more), pp. 115-150. ENK or PHI, mone, somatostatin (SOM), via an inhibitory action of 8. Valverde, R. C., Chieffo, V. & Reichlin, 5. (1972) Endocrinology eminence level. ENK, and or both, on SOM release at the median CRF, 91, 982-993. PHI denote peptides, against which the antisera were raised. For fur- 9. Fuxe, K., H~kfelt, T., Said, S. I. & Mutt, V. (1977) Neurosci. ther explanations, see text. Lett. 5, 241-246. 10. I., Emson, P. C., Fahrenkrug, J., Bjorklund, A., Alu- Lorkn, 1953- present findings that neurons contain CRF-like, enkephalin- mets, J., Hikanson, R. & Sundler, F. (1979) Science 4, mor- 1976. like, and PHI-like immunoreactivities may represent the A. several 11. Sims, K. B., Hoffman, D. L., Said, S. I. & Zimmerman, E. phological basis for such an integrated, parallel release of (1980) Brain Res. 186, 165-183. pituitary hormones, which is in harmony with the known func- 12. Hbkfelt, T., Schultzberg, M., Lundberg, J. M., Fuxe, K., Mutt, tions of these three peptides, as reported in the literature. Thus, V., Fahrenkrug, J. & Said, S. I. (1982) in Vasoactive Intestinal other opioid CRF causes release of ACTH (16). Enkephalin (and Polypeptide, Advances in Peptide Hormone Research, ed. said, peptides and morphine) has a wide spectrum of effects, includ- S. I. (Raven, New York), pp. 65-90. en- 13. Tatemoto, K. & Mutt, V. (1981) Proc. Natl. Acad. Sci. USA 78, ing actions at the neuroendocrine axis (33). For example, 6603-6607. kephalin and enkephalin analogues facilitate prolactin release 14. H6kfelt, T., Fahrenkrug, J., Tatemoto, K., Mutt, V. & Werner, (34-39) and stimulate GH secretion (34-39). In preliminary 5. (1982) Acta Physiol. Scand., in press. experiments it has been demonstrated that PHI causes release 15. Hughes, J., Smith, T. W., Kosterlitz, H. W., Fothergill, L. A., of prolactin in vitro (unpublished data). Morgan, B. A. & Morris, H. R. (1975) Nature (London) 258, 577- A hypothetical mechanism that may underlie the parallel re- 579. GH is shown in 2. CRF, 16. Vale, W., Spiess, J., Rivier, C. & Rivier, J. (1981) Science 213, lease of ACTH, prolactin, and Fig. 1394-1397. transported via the portal vessels, stimulates the pituitary cells 17. Coons, A. H. (1958) in General Cytochemical Methods, ed. Dan- to release ACTH. PHI, or arelated peptide, may, after transport ielli, J. F. (Academic, New York), pp. 399-422. in the portal vessels, act directly on the pituitary to release pro- 18. Schultzberg, M., Lundberg, J. M., H~kfelt, T., Terenius, L., lactin. 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USA 80 (1983)

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