Melanin-concentrating hormone in the human brain

Peptides,Vol. 14, pp. 643-646, 1993

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Melanin-Concentrating Hormone in the Human Brain T O R A I C H I M O U R I , t K A Z U H I R O T A K A H A S H I , *l H I R O S H I K A W A U C H I , ~ : M A S A H I K O SONE,* K A Z U H I T O T O T S U N E , * O S A M U M U R A K A M I , * K E I I C H I ITOI,* M A K O T O O H N E D A , * H|RONOBU SASANO,t AND NOBUAKI SASANOt

*The Second Department of Internal Medicine and ¢'The Second Department of Pathology, Tohoku University School of Medicine, Sendai, Miyagi, Japan and ~:School of Fisheries Science, Kitasato University, Sanriku, Iwate, Japan Received 22 O c t o b e r 1992

MOURI, T., K. TAKAHASHI, H. KAWAUCHI, M. SONE, K. TOTSUNE, O. MURAKAMI, K. ITOI, M. OHNEDA, H. SASANO AND N. SASANO. Melanin-concentrating hormone in the human brain. PEPTIDES 14(3) 643-646, 1993.--The presence of human melanin-concentrating hormone (MCH) was studied in the human brain by radioimmunoassay and immunocytochemistry. Immunoreactive MCH concentrations in the human brain ranged from 0.07 to 19.7 pmol/g wet weight. High performance liquid chromatography of the hypothalamus showed a large immunoreactive peak in the position of human/rat MCH, which was eluted 9 min later than that of salmon MCH. Free-floatingsections (40 urn) of the hypothalamus were immunostained. Positive MCH immunostaining was found in perifornical, tuberomammillary, and posterior nuclei. Numerous MCH-immunoreactive nerve fibers were observed throughout the hypothalamus. The presence of high concentrations of MCH in the human brain, in particular in the hypothalamus, suggests that MCH is a neurotransmitter, a neuromodulator, or a neurohormone in man. Melanin-concentrating hormone Hypothalamus Radioimmunoassay Chromatography

Brain

Human

Immunocytochemistry

METHOD

MELANIN-CONCENTRATING hormone (MCH) is a 17 amino acid peptide that was originally isolated from chum salmon pituitaries (5). Salmon MCH-like immunoreactivity (LI) has been identified not only in salmon (7) but also in the rat (10,11) and human brain (2,6,8,10). The structure of the human MCH precursor mRNA was determined using a human hypothalamic cDNA library (9), and the amino acid sequence of putative human MCH, a 19 amino acid peptide, is identical to that of rat MCH (14), but differs from salmon MCH by an N-terminal extension of two amino acids and four additional substitutions. It still remains to be determined whether the salmon MCH-LI previously shown in the human brain by other authors and us (2,6,8,10) is human MCH itself or other materials with structural similarity to salmon MCH.

Human Brain This study has been approved by the ethical committee of Tohoku University School of Medicine. Human brain tissues were obtained at autopsy within 3 h postmortem from 12 patients (seven male and five female, 52-79 years old) without neurological and endocrinological disorders. Six regions of brain were collected from five patients for radioimmunoassay and stored at - 8 0 ° C until extraction. The whole brains from seven patients were fixed in 10% formalin for 7-13 days at 4°C for immunocytochemistry. The hypothalamic tissues were dissected out and refixed in 4% paraformaldehyde in 0.01 M phosphate-buffered saline, pH 7.4, containing 0.5% glutaraldehyde and 1% saturated picric acid for 48 h at 4°C.

This study was presented partly at the 8th International Congressof Endocrinology, 1988, Kyoto (abstract 08-18-004) and the New York Academy of Sciences Conference on the Melanotropic Peptides, 1992, Rouen. Requests for reprints should be addressed to Kazuhiro Takahashi, MD, The Second Department of Internal Medicine, Tohoku University School of Medicine, 1-1 Seiryo-cho,Sendai, Miyagi 980, Japan.

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Radioirnmunoassay and Chromatography Salmon MCH (Peninsula Laboratory, Belmont, CA) was used as standard and for iodination with ~25INa by the chloramineT method (3). The antiserum against salmon MCH (No. 6461) was raised in a rabbit by injecting salmon MCH (Peninsula Laboratory) conjugated with bovine thyroblobulin (Sigma Chemical Co., St. Louis, MO) by carbodiimide. The antiserum was used at a final dilution of 1:8000. The assay could detect changes of 1.2 fmol/tube from zero with duplicate tubes at 95% confidence. The assay showed 100% cross-reactivity with human/rat MCH (Peninsula Laboratory), and twofold dilution curves of the human brain extracts were parallel to a standard curve (data not shown). The assay showed no significant cross-reaction (less than 0.001%) with other peptides such as corticotropin-releasing hormone (CRH), somatostatin, growth hormone-releasing hormone (GHRH), and a MSH. Intra- and interassay coefficients of variation were 5. I% (n = 10) and 13.2% (n = 5), respectively. Human brain tissues were extracted as previously reported (13) and assayed. The IR-MCH in the human hypothalamus was characterized by reverse-phase high performance liquid chromatography (HPLC) using a u Bondapak C18 column (3.9 × 300 mm).

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Radioimmunoassay and HPLC High concentrations of IR-MCH were detected from all the regions of the human brain examined: hypothalamus, 6.38 _+ 3.87 pmol/g wet weight (mean _+ SEM), n = 4; thalamus, 3.09 _ 1.73, n = 4; pons, 1.41 + 0.47, n = 4; hemisphere of cerebellum, 0.82 + 0.47, n = 4; cortex, 0.41 + 0.14, n = 5; vermis of cerebellum, 0.26 + 0.11, n = 3. Synthetic human/rat MCH was eluted nine fractions later than salmon MCH on the HPLC. Most of(>80%) IR-MCH in the hypothalamic extracts was eluted in the position of human/ rat MCH (Fig. 1). Two small peaks were found in similar positions with salmon MCH and the oxidized form of human/rat MCH, respectively.

Immunocytochemistry Positive immunostaining was observed in all seven cases examined. Melanin-concentrating hormone-immunoreactive perikarya were present in the posterior part of human hypothalamus; MCH immunoreactive perikarya were observed in the perifornical nucleus (Fig. 2A), tuberomammillary nucleus (Fig. 2B), and posterior nucleus. Melanin-concentrating hormone-immunoreactive nerve fibers were observed throughout the hypothalamus, including the periventricular area (Fig. 2C) and the infundibulum. No M C H - i m m u n o reactive perikarya were found in the paraventricular nucleus or supraoptic nucleus. Positive immunostaining was completely abolished by the preabsorption of the antiserum with synthetic human MCH. The pretreatment of the antiserum with CRH, GHRH, and c~-MSH did not affect the i m m u n o -

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FIG. l. Reverse-phase high performance liquid chromatography of the human hypothalamic extracts of two cases, sMCH, oxi MCH and h/rMCH show the elution positions of synthetic salmon MCH, the oxidized form of human/rat MCH, and human/rat MCH, respectively. The dotted line indicates a gradient of acetonitrile. A linear gradient of acetonitrile in 0.1% trifluoroacetic acid was performed from 10% to 60% over 50 min at a flow rate of 1 ml/min/fraction. IR-MCH: immunoreactive MCH.

staining. N o n i m m u n e rabbit serum instead of the antiserum did not show any immunostaining (data not shown). DISCUSSION Sekiya et al. reported the presence of salmon MCH-LI in the h u m a n brain (10), but their chromatographical study using fast protein liquid chromatography showed that most of salmon MCH-LI in the h u m a n hypothalamus eluted in the positions different from that of the synthetic salmon MCH, suggesting the species difference of MCH molecule. In the present study, most of IR-MCH in the human hypothalamus was chromatographically identical to the recently sequenced human/rat MCH. The material eluting in the position of the oxidized form of human/rat MCH is probably generated by the oxidation of human/rat MCH during the extraction procedure.

MCH IN H U M A N BRAIN

645

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FIG. 2. MCH-immunoreactive neurons in (A) the perifornical nucleus (×100) and (B) the tuberomammillary nucleus (x 100), and (C) MCHirnmunoreactive nerve fibers in the periventricular area (X250). In (C), perikarya in the paravenlricular nucleus were not immunostained. F: fornix.

Pelletier et al. (8) reported that salmon MCH-immunoreactive perikarya were observed in the periventricular area of human hypothalamus obtained from three subjects, and Bresson et al. (2) reported that salmon M C H - i m m u n o r e a c t i v e perikarya were distributed in the posterior hypothalamus, behind the paraventricular nucleus, within areas extending from the juxtaventricular to the perifornical regions of the hypothalamus obtained from one adult subject. Our findings in immunocytochemistry were similar to those of Bresson et al. (2).

Melanin-concentrating hormone is reported to inhibit A C T H and cortisol secretion in teleost fish (1), while its intracerebroventricular injection into rat released A C T H (4). Thus, physiological roles of MCH in the mammalian, in particular in human, have not been clarified. But it is noteworthy that numerous M C H - i m m u n o r e a c t i v e nerve fibers are present throughout the hypothalamus, including the periventricular area and infundibulum, where many neurons secreting hypothalamic hormones are present.

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M O U R I E T AL. REFERENCES

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