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Enaocrinol. Japon. 1989, 36(3),409-415

Calcitonin -Related in the Human

KAZUHIRO TAKAHASHI, TORAICHI MOURI, MASAHIKO SONE, OSAMU MURAKAMI, KEIICHI ITOI, YUTAKA IMAI, MAKOTO OHNEDA, KAORU YOSHINAGA AND NOBUAKI SASANO*

The Second Department of Internal Medicine and *the Department of Pathology, Tohoku University School of Medicine, Sendai, Miyagi, Japan

Abstract

Calcitonin gene-related peptide (CGRP)-like immunoreactivity (LI) in the human hypothalamus was investigated by radioimmunoassay and by im- munocytochemistry. CGRP-LI was detected from two hypothalami obtained at autopsy (2.1 and 7.0ng/g wet ) by radioimmunossay. Reverse phase high performance liquid chromatography revealed that most of the CGRP-LI in the human hypothalamus was eluted in an identical position with synthetic human CGRP. For immunocytochemistry, human hypothalami obtained at autopsy were fixed and cryostat-sectioned at 40ƒÊm. Free floating sections were immunostained with antibody to CGRP. CGRP-immunoreactive cell bodies were found in the , paraventricular nucleus and in- fundibular nucleus. These findings indicate that CGRP exists in the cell bodies of the supraoptic nucleus, paraventricular nucleus and infundibular nucleus in the human hypothalamus and CGRP may play some roles in the endocrine and other functions of the human hypothalamus.

Calcitonin gene-related peptide (CGRP) al., 1985; Skofitsch and Jacobowitz 1985; is a 37 peptide, which is encoded Mulderry et al., 1985; Okimura et al., 1987; by the same gene as calcitonin but is pro- Tiller-Borcich et al., 1988). In rat , duced by of the RNA high concentrations of CGRP-LI were re- transcript of the calcitonin gene (Amara et ported in the medulla oblongata (Mulderry al., 1982; Rosenfeld et al., 1983). et al., 1985), and immunoreactive CGRP By radioimmunoassay and immunocyto- was found in several nuclei of the brain chemistry, CGRP-like immunoreactivity (LI) stem and in their fibers projecting to the was shown to exist both in the central and hypothalamus (Rosenfeld et al., 1983). In peripheral nervous system (Rosenfeld et al., the rat hypothalamus, CGRP positive cell 1983; Wiesenfeld-Hallin et al., 1984; bodies were also observed in the paraventri- Tschopp et al., 1984 and 1985; Kawai et cular nucleus, , anterior nucleus, perifornical area and lateral area Received February 21, 1989 (Kawai et al., 1985; Skofitsch and Jacobo- Address all correspondence and reprint requests to, TORAICHI MOURI, M. D. The Second witz 1985). CGRP may act as a neuro- Department of Internal Medicine, 1-1 Seiryo-cho, transmitter or neuromodulator. Sendai, Miyagi, Japan 980 CGRP-LI was also determined in the Endocrinol.Japon. 410 TAKAHASHI et al. June 1989

including the hypothalamus creatic polypeptide, peptide YY, , , , vasoactive intestinal poly- and the by radioimmunoas- peptide or I (these were say (Tschopp et al., 1985; Tiller-Borcich obtained from Peninsula laboratories, USA). The et al., 1988). However, no report is avai- sensitivity of this assay was 2.9•}0.5pg/tube lable on the localization of immunoreactive (n=4, mean•}SD). Intra- and interassay coe- CGRP cells in the human hypothalamus. fficientsof variation were 6.2% (n=10)and In the present study, we demonstrate the 11.3% (n=8), respectively.

presence of CGRP-LI in the human hy- Fractionations of CGRP-LI in the human hy- pothalamus by radioimmunoassay and by pothalamus were carried out by reverse phase high immunocytochemistry. performance liquid chromatography (HPLC) on a Waters i Bondapak C18, 3.9X300mm column with a linear gradient from 30-60% solvent B

Materials and Methods (solvent A=0.1% trifluoracetic acid/water; sol- vent B =0.1% trifluoracetic acid/acetonitrile) at a flow rate of 1ml/min. One ml fractions were Radioimmunoassay collected for radioimmunoassay. The hypothalami and the cerebral cortex (temporal lobe or occipital lobe) were obtained Immunocytoch ernistry at autopsy from 2 females aged 69 and 78 years Four human brain tissues were obtained at within 2 postmortem hours, and stored at -80•Ž autopsy from 2 males and 2 females aged 63-75 until extracted. Neither case had any endo- crinological or neurological disorder before death. years within 3 postmortem hours. They were immersed immediately in 10% formalin for 7-13 Each of the specimens was boiled in 1M acetic days. After macroscopical pathological examina- acid for 10 minutes, homogenized and centri- tions, the hypothalami were dissected out and fuged by 24000 Xg for 60 minutes. The super- refixed in a mixture containing 4% paraformal- natants were dried with air. Brain tissue ex- dehyde, 0.5% glutaraldehyde and 0.1% saturated tracts were reconstituted in assay buffer (0.1M picric acid in 0.01M phosphate-buffered saline phosphate buffer, pH7.4 containing 0.1% human serum albumin, 0.2% Triton X-100 and 0.1% (PBS) (pH 7.4) for 48 hours at 4•Ž. These 4 cases had no endocrinological or neurological sodium azide) and subjected to radioimmunossay. disorders. The hypothalamic tissues were rinsed The radioimmunoassay procedures for CGRP and immersed in 20% sucrose in 0.01M PBS were previously reported (Mouri et al., 1989). containing 0.05% dimethylsulfoxide and cut into The antibody to human CGRP was raised in a 40ƒÊm coronal frozen sections with a cryostat. rabbit. Synthetic human CGRP (Peninsula Sections were stored in 0.01M PBS containing Laboratories, USA) was conjugated with bovine 0.3% Triton X-100. (Sigma Chemicals Co., USA) by carbodiimide. Conjugated CGRP was injected Free floating sections were immunostained into a rabbit with complete Freund's adjuvant. with antibody to human CGRP by the Vector The final dilution of the antibody was 1:80000. ABC system. Triton X-100 diluted in 0.01 M Synthetic human CGRP was used as the standard PBS to 0.3% was used for dilution and washing and synthetic human [Tyr0]-CGRP (Peninsula in the following procedures. The sections were Laboratories, USA) was used for iodination. incubated in 1% hydrogen peroxide for 60 1251-[Tyro]-CGRP was prepared by the modifica. minutes at room temperature, washed and then tion of the chloramine-T method (Hunter and incubated in 1% normal gout serum for 60 Greenwood, 1962) without the addition of sodium minutes at room temperature. The sections were metabisulfite. The radioimmunoassay showed incubated with the antibody to human CGRP 100% cross reaction with CGRP II (Steenbergh (1:10000) for 7 days at 4•Ž, then washed and et al., 1985) (Peninsula Laboratories, USA) and incubated in biotinylated secondary antibody to less than 0.001% with calcitonin, parathyroid rabbit IgG (1:400) (Vector, USA) for 16-20 (1-84), human corticotropin-releasing hours at 4•Ž. The sections were again washed hormone, human releasing and incubated in avidin-biotinated horseradish hormone, , , peroxidase (Vector, USA) for 4 hours at room , human Y, human pan- temperature, then washed and made to react 411 HYPOTHALAMUS Vol.36, No.3 CGRP IN HUMAN with 3,3'-diaminobenzidine tetrahydrochloride (DAB). The DAB reaction product was inten- si.(ed by cobalt chloride and nickel ammonium following the procedure described by Adams, J. C. (1981). The sections were mounted on glass slides coated with gelatin.

Table 1. CGRP-LI content in the human hypothalamus and cerebral cortex of two cases.

Fig.1. Reverse phase high performance liquid chromatography of CGRP-LI in a human hypothalamic extract. CGRP: the elution n.d.: Not detectable (<0.07ng/g wet tissue) *: Occipital lobe,**: Temporal lobe position of synthetic human CGRP. CGRP II: that of CGRP II.

Results

By radioimmunoassay, CGRP-LI was detected in 2 human hypothalami (2.1 and 7.0ng/g wet tissue), the amounts being greater than those in the cerebral cortex (Table 1). Serial twofold dilution curves of tissue ex- tracts were parallel' with a standard curve of CGRP. Reverse phase HPLC revealed that most of the CGRP-LI in the human hypothalamus was eluted in an identical position with synthetic human CGRP, which was later than that of CGRP II, while a small amount of CGRP-LI was eluted in a more hydrophilic position than the elution positions of CGRP and CGRP II (Fig. 1). By immunocytochemistry, Fig.2. CGRP immunoreactive cell bodies in the supra- moderate numbers of CGRP optic nucleus (X20). OT: optic tract, DL: pars immunoreactive cell bodies dorsolateralis, DM: pars dorsomedialis, VM: pars were found in the supraoptic ventromedialis. Endocrinol. Japon. 412 TAKAHASHI et al. June 1989

Fig.3. CGRP immunoreactive cell bodies in the pars dorsolateralis of the supraoptic

nucleus (•~40).

Fig.4. CGRP immunoreactive cell bodies in the pars dorsomedialis of the supraoptic nucleus (•~200). 413 Vol.36, No.3 CGRP IN HUMAN HYPOTHALAMUS

⇔ Fig.5. CGRP immunoreactive cell bodies in the paraven- tricular nucleus (X40). V: third ventricle

nucleus (Fig.2-4). CGRP immunoreactive cell bodies were also found in the para- ventricular and infundibular nuclei, but the numbers of

positive cells in these two nuclei were less than those in the supraoptic nucleus (Fig. 5,6). The absorption of CGRP antibody with a large amount of synthetic human CGRP

(2.5,ƒÊg/l ml of 1:10000 CGRP antibody) for 3 hours at room temperature abolished im- munostaining completely. The

pretreatment of CGRP anti- body with large amounts of corticotropin-releasing hor- mone, arginine vasopressin and oxytocin (2.5ƒÊg/l ml of 1:10000 CGRP antibody) did not in- fluence the immunostaining of CGRP. Normal rabbit serum (1:10000) instead of CGRP antibody did not show any positive immunostainings.

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Fig.6. CGRP immunoreactive cell bodies in the infundibularnucleus (•~100). Endocrinol. Japon. 414 TAKAHASHI et ol. June 1989

(Brain et al., 1985; Hanko et al., 1985). Discussion The infusion of CGRP reduced gastric acid output, pepsin and plasma levels In this study, we detected CGRP-LI in of , gastric inhibitory peptide, entero- the human hypothalamus in a radioim- glucagon and in man (Kraenzlin munoassay. By reverse phase HPLC, most et al., 1985). And intracisternal injection of the CGRP-LI in the human hypothala- of CGRP inhibited gastric secretion in rats mus was eluted in an identical position (Tache et al., 1984). But the physiological with synthetic human CGRP, although a roles of CGRP in the hypothalmo-pituitary small amount of CGRP-LI was found in a system are not clear and need further in- more hydrophilic position. By immunocyto- vestigation. chemistry, CGRP-LI was found in the cell bodies of the supraoptic nucleus, paraven- tricular nucleus and infundibular nucleus. Acknowledgements These findings indicate that CGRP exists in the cell bodies of the human hypothala- We thank Assistant Professor T. Yamamoto mic nuclei mentioned above. The origin of the Department of Neuropathology, Tohoku of CGRP-LI eluted in a more hydrophilic University School of Medicine for his kind position in HPLC is not clear. Further advice on the method of immunocytochemistry studies are needed to clarify whether this and Dr. T. Masuda for his kind advice on the material is an artificial product of CGRP printing of the photomicrographs. We also in the procedures of tissue extraction and thank Ms T. Katayanagi for her secretarial assistance. HPLC, or a hydrophilic form of CGRP-like This study is partly supported by a research material. grant for Intractable Disease from the Japanese High concentrations of CGRP in the Ministry of Health and Welfare and a grant-in- pituitary glands were reported earlier aid for scientific research from the Ministry of (Tschopp et al., 1984 and 1985; Tiller- Education, Science and Culture, Japan (No. Borcich et al., 1988). From these reports 63570519). and our finding that CGRP exists in the supraoptic nucleus, paraventricular nucleus and infundibular nucleus, it is suggested References that CGRP plays a physiological role in the hypothalamo-pituitary system. Adams, J. C.(1981). Heavy metal intensifica- In the human hypothalamus, arginine tion of DAB-based HRP reaction product. J. vasopressin and oxytocin were known to Histochem. Cytochem. 29, 775. exist in the paraventricular nucleus and Amara, S. G., V. Jonas, M. G. Rosenfeld, E. S. Ong and R. M. Evans (1982). Alternative supraoptic nucleus and be transported RNA processing in calcitonin through the nerve fibers to the pituitary generates mRNAs encoding different poly- gland (Dierickx and Vandesande 1977). peptide products. Nature 298, 240-244. CGRP which exists in these nuclei may Brain, S. D., T. J. Williams, J. R. Tippins, have some modulatory effects on the actions H. R. Morris and I. Maclntyre (1985). Cal- of arginine vasopressin and oxytocin. But citonin gene-related peptide is a potent vaso- further studies are needed to clarify if dilator. Nature 313, 54-56. Dierickx, K. and F. Vandesande (1977). Im- CGRP co-exists in the supraoptic nucleus munocytochemical localization of the vaso- and/or paraventricular nucleus with arginine pressinergic and the oxytocinergic in vasopressin and/or oxytocin. the human hypothalamus. Cell Tiss. Res. CGRP has a potent vasodilator action 184, 15-27. 4151 Vol.36, No.3 CGRP IN HUMAN HYPOTHALAMUS

Hanko, J., J.-E. Hardebo, J. Kahrstrom, C. L. W. Swanson, P. E. Sawchenko, J. Rivier,. Owman and F. Sundler (1985). Calcitonin W. W. Vale and R. M. Evans (1983). Pro- gene-related peptide is present in mammalian duction of a novel neuropeptide encoded by cerebrovascular nerve fibers and dilates pial the calcitonin gene via tissue-specific RNA. and peripheral arteries. Neurosci. Lett. 57, processing. Nature 304, 129-135. 91-95. Skofitsch, G. and D. M. Jacobowitz (1985). Hunter, W. M. and F. C. Greenwood (1962). Calcitonin gene-related peptide: detailed im- Preparation of iodine-131 labelled human munohistochemical distribution in the . Peptides 6, 721-745. growth hormone of high specific activity. Nature 4827, 495-496. Steenbergh, P. H., J. W. M. Hoppener, J. Kawai, Y., K. Takami, S. Shiosaka, P. C. Zandberg, C. J. M. Lips and H. S. Jansz. Emson, C. J. Hillyard, S. Girgis, I. Maclntyre (1985). A second human calcitonin/CGRP. and M. Tohyama (1985). Topographic locali- gene. FEBS letters 183, 403-407. zation of calcitonin gene-related peptide in the Tache, Y., M. Gunion, M. Lauffenberger and rat brain: an immunohistochemical analysis. Y. Goto (1984). Inhibition of gastric acid Neuroscience 15, 747-763. secretion by intracerebral injection of calcitonin Kraenzlin, M.E., J.L.C. Ch'ng, P.K. Mulderry, gene related peptide in rats. Life Sci. 35, M. A. Ghatei and S.R. Bloom (1985). In- 871-878. fusion of a novel peptide, calcitonin gene- Tiller-Borcich, J. K., H. Capili and G. S. Gordan. related peptide (CGRP) in man. Pharmacoki- (1988). Human brain calcitonin gene-related netics and effects on gastric acid secretion and peptide (CGRP) is concentrated in the - on gastrointestinal . Regul. Pept. caeruleus. 11, 55-61. 10, 189-197. Tschopp, F. A., P. H. Tobler and J. A. Fischer Mouri, T., K. Takahashi, M. Sone, O. Murakami, (1984). Calcitonin gene-related peptide in the M. Ohneda, K. Itoi, Y. Imai, K. Yoshinaga human , pituitary and brain. Mol. and N. Sasano (1989). Calcitonin gene-related Cell. Endocrinol. 36, 53-57. Tschopp, F. A., H. Henke, J. B. Petermann,. peptide-like immunoreactivities in pheochromo- cytomas. Peptides 10, 201-204. P. H. Tobler, R. Janzer, T. Hokfelt, J. M. Lundberg, C. Cuello and J. A. Fischer (1985). Mulderry, P. K., M. A. Ghatei, A. E. Bishop, Y. S. Allen, J. M. Polak and S. R. Bloom Calcitonin gene-related peptide and its binding: sites in the human central nervous system and (1985). Distribution and chromatographic characterization of CGRP-like immunoreac- pituitary. Proc. Natl. Acad. Sci. USA 82, 248-252. tivity in the brain and gut of the rat. Regul. Pept. 12, 133-143. Wiesenfeld-Hallin, Z., T. Hokfelt, J. M. Lundberg, W. G. Forssmann, M. Reinecke, F. A. Tschopp. Okimura, Y., K. Chihara, H. Abe, T. Kita, Y. and J. A. Fischer (1984). Immunoreactive Kashio, M. Sato and T. Fujita (1987). Cal- calcitonin gene-related peptide and substance citonin gene-related peptide-like immunoreac- P co-exist in sensory neurons to the spinal tivity in the central nervous system and peri- cord and interact in spinal behavioral res- pheral organs of rats. Regul. Pept. 17, 327- 337. ponses of the rat. Neurosci. Lett. 52, 199-204 Rosenfeld, M. G., J.-J. Mermod, S. G. Amara,