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Regional distribution of immunoreactive prolactin-releasing peptide in the human brain
Peptides 21 (2000) 1551–1555
Regional distribution of immunoreactive prolactin-releasing peptide in the human brain Kazuhiro Takahashia,*, Ayako Yoshinoyaa, Zenei Ariharab, Osamu Murakamib, Kazuhito Totsuneb, Masahiko Sonea,c, Hironobu Sasanod, Shigeki Shibaharaa a
Department of Molecular Biology and Applied Physiology, Tohoku University School of Medicine, 2–1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan b Second Department of Internal Medicine, Tohoku University School of Medicine, Sendai, Miyagi 980-8574, Japan c Department of Internal Medicine, National Iwate Hospital, Ichinoseki, Iwate 021-0015, Japan d Department of Pathology, Tohoku University School of Medicine, Sendai, Miyagi 980-8575, Japan Received 26 April 2000; accepted 20 July 2000
Abstract Regional distribution of prolactin-releasing peptide (PrRP) in the human brain was studied by radioimmunoassay. The antiserum raised against human PrRP-31 in a rabbit was used in the assay, which showed 100% cross reaction with PrRP-20 and no significant cross reaction with other peptides. The highest concentrations of immunoreactive-PrRP were found in hypothalamus (912 ⫾ 519 fmol/g wet weight, n ⫽ 6, mean ⫾ SEM), followed by medulla oblongata (496 ⫾ 136 fmol/g wet weight) and thalamus (307 ⫾ 117 fmol/g wet weight). On the other hand, immunoreactive-PrRP was not detected in frontal lobe or temporal lobe (⬍50 fmol/g wet weight). Sephadex G50 column chromatography of the immunoreactive-PrRP in the hypothalamus and medulla oblongata showed three immunoreactive peaks; one peak eluting in the position of PrRP-20, one eluting in the position of PrRP-31 and one eluting earlier. Reverse phase high-performance liquid chromatography (HPLC) of these brain tissue extracts showed a peak eluting in the position of PrRP-20 and PrRP-31. The present study has shown for the first time the presence of immunoreactive-PrRP in the human brain. The immunoreactive-PrRP levels in the human hypothalamus were, however, lower than the levels of other neuropeptides with prolactin-releasing activity, such as thyrotropin-releasing hormone and vasoactive intestinal polypeptide. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Prolactin-releasing peptide; Hypothalamus; Brain; Radioimmunoassay
1. Introduction Prolactin-releasing peptide (PrRP) is a novel bioactive peptide identified from bovine hypothalamic tissues as a ligand of an orphan seven transmembrane domain receptor, hGR3 [4]. PrRP specifically stimulates prolactin secretion both in vitro and in vivo, and does not affect the secretion of other anterior pituitary hormones [4,18]. This peptide has two molecular forms, a 31-amino-acid peptide (PrRP-31) and a peptide corresponding to the C-terminal 20 amino acids of PrRP-31 (PrRP-20). Immunoreactive (IR)-PrRP was widely distributed in the central nervous system and pituitary gland of the rat, with
* Corresponding author. Tel.: ⫹81-22-717-8116; fax: ⫹81-22-7178118. E-mail address: [email protected] (K. Takahashi).
the highest concentrations found in hypothalamus [10]. Studies using immunocytochemistry and in situ hybridization showed that PrRP was expressed in some nuclei of hypothalamus (the caudal dorsomedial nucleus) and medulla oblongata (the nucleus of the solitary tract and the ventrolateral reticular nucleus) [1,7,8,12,19]. PrRP mRNA was expressed in the normal human pituitary gland and in approximately half cases of pituitary adenomas [20]. Regional distribution of IR-PrRP in the human brain, however, has not been studied. Samson et al. have reported that PrRP-20 and PrRP-31 released prolactin from dispersed anterior pituitary cells harvested from female rats only at the high doses (10-7 and 10-6 M), but TRH stimulated prolactin release at the lower dose (10-8 M) [13], raising the question as to whether PrRP functions as a physiological prolactin releasing factor. It is therefore important to quantitate the amount of PrRP-20 and PrRP-31 present in the hypothalamus. In the present study, we therefore studied the regional
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distribution of IR-PrRP in the human brain obtained at autopsy by radioimmunoassay.
2. Materials and methods 2.1. Tissues This study has been approved by the Ethics Committee of Tohoku University School of Medicine. Human brain tissues were obtained at autopsy performed at the Department of Pathology, Tohoku University Hospital within 4 h postmortem from 7 patients (4 male and 3 female, 31–70 years old). These patients had no neurologic or endocrinological diseases. The tissues were immediately frozen and stored at ⫺80°C prior to extraction of peptides. 2.2. Peptide extraction and radioimmunoassay Tissues were extracted, as reported previously [16]. Briefly, the tissue (approximately 750 mg) was boiled in 2 ml of 1 mol/liter acetic acid for ten minutes. Eight ml of 50% methanol in 0.5 mol/liter acetic acid was added to each sample and the tissue was homogenized. The homogenate was centrifuged by 15,000⫻ g for 30 min. The supernatant was separated, dried by air, reconstituted in assay buffer [0.1 mol/liter phosphate buffer, pH 7.5 containing 0.1% (w/v) bovine serum albumin (BSA), 0.2% (v/v) Triton X-100 and 0.1% (w/v) sodium azide] and assayed. The antiserum against human PrRP-31 (No 1118) was raised in a rabbit. Synthetic human PrRP-31 (Peptide Institute Inc., Minoh-shi, Japan) was conjugated to BSA (Sigma Chemical Co., St Louis, MO, USA) by carbodiimide (Peptide Institute Inc.), and injected into the rabbit with complete Freund adjuvant (Difco Laboratories, Detroit, Michigan, USA). The antiserum was used at a final dilution of 1:100,000. Human PrRP-31 (Peptide Institute, Inc.) was used as a standard. Human PrRP-31 was iodinated with 125 INa by the chloramine T method [5]. The assay was performed in 400 l of assay buffer. The sample or the standard peptide (200 l) was incubated with antiserum (100 l) at 4°C for 48 h and then 100 l of 125 I-PrRP-31 was added to each sample. After further 48 h incubation at 4°C, 100 l of 5% anti-rabbit IgG raised in goat (ICN Biomedicals Inc., Costa Mesa, CA, USA) and 500 l of 10% (w/v) polyethylene glycol in water were added. After 5 h incubation, the sample was centrifuged at 3000⫻ g for 30 min and the supernatant was separated. The pellets were counted by the ␥-counter. The assay could detect changes of 1.5 ⫾ 0.4 fmol/tube (mean ⫾ SD, n ⫽ 8) from zero at 95% confidence with duplicate tubes. The cross reactivity was 100% with human PrRP-20 (Phoenix Pharmaceuticals Inc., Mountain View, CA, USA), rat PrRP-20 (Phoenix Pharmaceuticals Inc.) and rat PrRP-31 (Peptide Institute, Inc.), respectively. There was no significant cross reaction (less than 0.001%) with other
peptides, including vasoactive intestinal polypeptide (VIP), substance P, pituitary adenylate cyclase activating polypeptide-38 (PACAP), neuropeptide Y (NPY), oxytocin, C-type natriuretic peptide, corticotropin releasing hormone (CRH), and growth hormone-releasing hormone (GH-RH). Intraand interassay coefficients of variation were 9.9% and 10.2%, respectively. To examine the stability of IR-PrRP in the brain during the postmortem hours, we measured IR-PrRP concentrations in the rat brains obtained immediately after death and 4 h after death. The experiment was done following the Ethics Guidelines of the Animal Experiments of Tohoku University School of Medicine. Eight adult SD rats were killed under ether anesthesia. Hypothalamus and brain stem were dissected out for peptide extraction immediately after death from the 4 rats and 4 h after death from the other 4 rats which were kept at room temperature. There were no significant decreases in the IR-PrRP concentrations in the hypothalamus and the brain stem of the rats kept at room temperature for 4 h after death [F(1,12) ⫽ 0.596, P ⬎ 0.45], suggesting that the postmortem hours at least up to 4 h had no noticeable effects on IR-PrRP levels in the brain. 2.3. Chromatography Chromatographic characterization of the tissue extracts was performed by Sephadex G-50 (superfine) column chromatography and reverse phase HPLC using a Bondapak C18 column (3.9 mm ⫻ 300 mm, Waters, Milford, MA, USA). Brain tissue extracts (hypothalamus and medulla oblongata) obtained from three cases (one male and two female) were pooled and re-extracted by a Sep-Pak C18 cartridge (Waters). The extracts were reconstituted in 1 mol/liter acetic acid containing 0.5% (w/v) BSA and loaded onto the Sephadex G-50 column (10 ⫻ 560 mm). Peptides on the column were eluted with 1 mol/liter acetic acid containing 0.5% (w/v) BSA at a flow rate of 6 ml/h. Fractions (0.8 ml/fraction) were collected, dried by air, reconstituted in assay buffer and assayed. For the HPLC analysis, the pooled tissue extracts were re-extracted with a Sep-Pak C18 cartridge (Waters), reconstituted in 0.1% (v/v) trifluoroacetic acid (TFA) and loaded onto the column. The HPLC analysis was performed with a linear gradient of acetonitrile containing 0.1% TFA from 10% to 60% at a flow rate of 1 ml/min per fraction over 50 min. Each fraction (1 ml) was collected, dried by the air, reconstituted with assay buffer and assayed.
3. Results Fig. 1 shows a standard curve of human PrRP-31 and a dilution curve of a human brain extract (thalamus). The dilution curve was parallel with the standard curve of PrRP31. The highest concentrations of IR-PrRP were found in
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Fig. 1. A standard curve of prolactin-releasing peptide-31 (PrRP-31) (F) and a two-fold dilution curve of a brain tissue extract (thalamus) (E). The tissue extract was two-fold diluted serially and assayed. B/Bo (%), a percentage of bound counts in the presence of the sample/counts bounds in the absence of added peptide ⫻100.
hypothalamus (912 ⫾ 519 fmol/g wet weight, n ⫽ 6, mean ⫾ SEM), followed by medulla oblongata (496 ⫾ 136 fmol/g wet weight, n ⫽ 6) and thalamus (307 ⫾ 117 fmol/g wet weight, n ⫽ 6) (Fig. 2). Lower concentrations of IRPrRP were detected in occipital lobe, pons and cerebellum. On the other hand, IR-PrRP was not detected in frontal lobe and temporal lobe (⬍50 fmol/g wet weight, n ⫽ 5). IRPrRP levels in the hypothalamus ranged from 555 to 3467 fmol/g wet weight in three female subjects, and from 49 to 421 fmol/g wet weight in three male subjects, although there was no significant difference in the IR-PrRP levels between male and female subjects [F(1,4) ⫽ 1.941, P ⬎ 0.2].
Fig. 3. Sephadex G50 (superfine) column chromatography of the human brain. A, hypothalamus. B, medulla oblongata. Vo, void volume. The arrows indicate the elution positions of PrRP-20 and PrRP-31, respectively.
Sephadex G50 column chromatography of the IR-PrRP in the hypothalamus and medulla oblongata showed three immunoreactive peaks; one peak eluting in the position of PrRP-20, one eluting in the position of PrRP-31 and one eluting earlier (Fig. 3). Reverse phase HPLC of these brain tissue extracts showed a peak eluting in the position of PrRP-20 and PrRP-31 (Fig. 4). PrRP-20 and PrRP-31 were eluted in the same position on HPLC.
4. Discussion
Fig. 2. Regional distribution of immunoreactive prolactin-releasing peptide (IR-PrRP) in the human brain. Data are shown as mean ⫾ SEM. Cerebellum was dissected into the hemisphere (h) and vermis (v).
We have shown for the first time the presence of IR-PrRP in the human brain, with the highest concentrations found in the hypothalamus. High concentrations of IR-PrRP in human medulla oblongata are in accord with the previous reports on the expression of PrRP in the nucleus of the solitary tract and the ventrolateral reticular nucleus in rats [1,7,8,12,19]. The chromatographic study showed that both PrRP-20 and PrRP-31 were present in the human brain.
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Fig. 4. Reverse phase HPLC of the human brain. A, hypothalamus. B, medulla oblongata. The arrow indicates the elution position of PrRP-20 and PrRP-31, which were eluted in the same position. The dotted line indicates a gradient of acetonitrile.
Materials eluting in the earlier peak in Sephadex G50 column chromatography may represent IR-PrRP with a larger molecular weight, possibly the PrRP precursor or its processed form. IR-PrRP levels in the human brain including the hypothalamus are compatible with the levels in the rat brain reported by Matsumoto et al. [10]. The secretion of prolactin from the anterior pituitary is controlled mainly by the inhibitory neurotransmitter, dopamine [6]. Several peptides possessing prolactin-releasing activity, such as thyrotropin-releasing hormone (TRH), VIP, substance P, neurotensin, galanin and PACAP, were identified, but none of them has been demonstrated to be a specific hypophysiotropic stimulatory factor for prolactin secretion. PrRP-31 and PrRP-20 have been proposed to be promising candidates for a specific prolactin-releasing hormone by Hinuma et al. [4]. On the other hand, Samson et al. reported that PrRP-20 and PrRP-31 released prolactin from dispersed anterior pituitary cells harvested from random
cycle female rats only at the high doses (10-7 and 10-6 M), but TRH stimulated prolactin release at the lower dose (10-8 M) [13]. Such lower potency of PrRPs in prolactin release was also observed in dispersed anterior pituitary cells harvested from lactating rats [14]. In the present study, IR-PrRP levels in the human hypothalamus were much lower than the levels of other neuropeptides with prolactin-releasing activity, such as TRH [11], VIP [2], substance P [2], neurotensin [2] and PACAP [3,17]. Postmortem degradation is unlikely to explain these low values of PrRP, because IR-PrRP levels in the rat brain were not decreased at least up to 4 h after death. The immunocytochemical studies by others showed that PrRP nerve fibers were scarcely observed in the external layer of the median eminence in which hypothalamic hormones were abundantly detected [8,19]. Taken together, PrRP may not act as a hypophysiotropic factor like classic hypothalamic hormones, such as CRH and GH-RH. Recently, biologic actions of PrRP other than prolactin release have been suggested [9,12,14,15]. Central administration of PrRP stimulated oxytocin release [9] and increased blood pressure in rats [14]. Intracerebroventricular injection of PrRP-31 stimulated the secretion of LH and FSH via the hypothalamic release of LH-RH in male rats [15]. On the other hand, it is noteworthy that IR-PrRP levels in the hypothalamus in female subjects were in the higher range than in male subjects. In 9-week old rats, IR-PrRP levels in the hypothalamus were about two times higher in female rats than in male rats (Takahashi K, unpublished observations). The present study, however, studied a limited number of human samples and could not show the statistical difference in the IR-PrRP levels in the hypothalamus between male and female subjects. There is a possibility that hypothalami of lactating women have much higher levels of PrRP. The present study has shown the presence of PrRP in the human brain, with the highest concentrations found in hypothalamus, followed by medulla oblongata and thalamus. These findings are in accord with the possibility that PrRP-20 and PrRP-31 act as neuromodulators in the human brain.
Acknowledgments This work was supported in part by Grants-in-aid for Scientific Research (B) and (C), and on Priority Areas (A) from the Ministry of Education, Science, Sports and Culture of Japan and by the Mochida Memorial Foundation for Medical and Pharmaceutical Research.
References [1] Chen C-T, Dun SL, Dun NJ, Chang J-K. Prolactin-releasing peptideimmunoreactivity in A1 and A2 noradrenergic neurons of the rat medulla. Brain Res 1999;822:276 –9.
K. Takahashi et al. / Peptides 21 (2000) 1551–1555 [2] Ghatei MA, Bloom SR, Langevin H, McGregor GP, Lee YC, Adrian TE, O’Shaughnessy DJ, Blank MA, Ottenthal LO. Regional distribution of bombesin and seven other regulatory peptides in the human brain. Brain Res 1984;293:101–9. [3] Ghatei MA, Takahashi K, Suzuki Y, Gardiner J, Jones PM, Bloom SR. Distribution, molecular characterization of pituitary adenylate cyclase-activating polypeptide and its precursor encoding messenger RNA in human and rat tissues. J Endocrinol 1993;136:159 – 66. [4] Hinuma S, Habata Y, Fujii R, Kawamata Y, Hosoya M, Fukusumi S, Kitada C, Masuo Y, Asano T, Matsumoto H, Sekiguchi M, Kurokawa T, Nishimura O, Onda H, Fujino M. A prolactin-releasing peptide in the brain. Nature 1998;393:272– 6. [5] Hunter WM, Greenwood FC. Preparation of iodine-131-labelled human growth hormone of high specific activity. Nature 1962;194: 495– 6. [6] Lamberts SWL, Macleod RMM. Regulation of prolactin secretion at the level of the lactotroph. Physiol Rev 1990;70:279 –318. [7] Lee Y, Yang S-P, Soares MJ, Voogt JL. Distribution of prolactinreleasing peptide mRNA in the rat brain. Brain Res Bull 2000;51: 171– 6. [8] Maruyama M, Matsumoto H, Fujiwara K, Kitada C, Hinuma S, Onda H, Fujino M, Inoue K. Immunocytochemical localization of prolactinreleasing peptide in the rat brain. Endocrinology 1999;140:2326 –33. [9] Maruyama M, Matsumoto H, Fujiwara K, Noguchi J, Kitada C, Hinuma S, Onda H, Nishimura O, Fujino M, Higuchi T, Inoue K. Central administration of prolactin-releasing peptide stimulates oxytocin release in rats. Neurosci Lett 1999;276:193– 6. [10] Matsumoto H, Murakami Y, Horikoshi Y, Noguchi J, Habata Y, Kitada C, Hinuma S, Onda H, Fujino M. Distribution and characterization of immunoreactive prolactin-releasing peptide (PrRP) in rat tissue and plasma. Biochem Biophys Res Commun 1999;257:264 – 8. [11] Okon E, Koch Y. Localisation of gonadotropin-releasing and thyrotropin-releasing hormones in human brain by radioimmunoassay. Nature 1976;263:345–7.
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[12] Roland BL, Sutton SW, Wilson SJ, Luo L, Pyati J, Huvar R, Erlander MG, Lovenberg TW. Anatomical distribution of prolactin-releasing peptide and its receptor suggests additional functions in the central nervous system and periphery. Endocrinology 1999;140:5736 – 45. [13] Samson WK, Resch ZT, Murphy TC, Chang J-K. Gender-biased activity of the novel prolactin releasing peptides. Endocrine 1998;9: 289 –91. [14] Samson WK, Resch ZT, Murphy TC. A novel action of the newly described prolactin-releasing peptides: cardiovascular regulation. Brain Res 2000;858:19 –25. [15] Seal LJ, Small CJ, Kim MS, Stanley SA, Taheri S, Ghatei MA, Bloom SR. Prolactin releasing peptide (PrRP) stimulates luteinizing hormone (LH) and follicle stimulating hormone (FSH) via a hypothalamic mechanism in male rats. Endocrinology 2000;141:1909 –12. [16] Takahashi K, Totsune K, Sone M, Ohneda M, Murakami O, Itoi K, Mouri T. Human brain natriuretic peptide-like immunoreactivity in human brain. Peptides 1992;13:121–3. [17] Takahashi K, Totsune K, Murakami O, Satoh F, Sone M, Ohneda M, Sasano H, Mouri T. Pituitary adenylate cyclase activating polypeptide (PACAP)-like immunoreactivity in human hypothalamus: co-localization with arginine vasopressin. Regul Pept 1994;50:267–75. [18] Tokita R, Nakata T, Katsumata H, Konishi S, Onodera H, Imaki J, Minami S. Prolactin secretion in response to prolactin-releasing peptide and the expression of the prolactin-releasing peptide gene in the medulla oblongata are estrogen dependent in rats. Neurosci Lett 1999;276:103– 6. [19] Yamakawa K, Kudo K, Kanba S, Arita J. Distribution of prolactinreleasing peptide-immunoreactive neurons in the rat hypothalamus. Neurosci Lett 1999;267:113– 6. [20] Zhang X, Danila DC, Katai M, Swearingen B, Klibanski A. Expression of prolactin-releasing peptide and its receptor messenger ribonucleic acid in normal human pituitary and pituitary adenomas. J Clin Endocrinol Metab 1999;84:4652–5.
Regional distribution of immunoreactive prolactin-releasing peptide in the human brain Kazuhiro Takahashia,*, Ayako Yoshinoyaa, Zenei Ariharab, Osamu Murakamib, Kazuhito Totsuneb, Masahiko Sonea,c, Hironobu Sasanod, Shigeki Shibaharaa a
Department of Molecular Biology and Applied Physiology, Tohoku University School of Medicine, 2–1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan b Second Department of Internal Medicine, Tohoku University School of Medicine, Sendai, Miyagi 980-8574, Japan c Department of Internal Medicine, National Iwate Hospital, Ichinoseki, Iwate 021-0015, Japan d Department of Pathology, Tohoku University School of Medicine, Sendai, Miyagi 980-8575, Japan Received 26 April 2000; accepted 20 July 2000
Abstract Regional distribution of prolactin-releasing peptide (PrRP) in the human brain was studied by radioimmunoassay. The antiserum raised against human PrRP-31 in a rabbit was used in the assay, which showed 100% cross reaction with PrRP-20 and no significant cross reaction with other peptides. The highest concentrations of immunoreactive-PrRP were found in hypothalamus (912 ⫾ 519 fmol/g wet weight, n ⫽ 6, mean ⫾ SEM), followed by medulla oblongata (496 ⫾ 136 fmol/g wet weight) and thalamus (307 ⫾ 117 fmol/g wet weight). On the other hand, immunoreactive-PrRP was not detected in frontal lobe or temporal lobe (⬍50 fmol/g wet weight). Sephadex G50 column chromatography of the immunoreactive-PrRP in the hypothalamus and medulla oblongata showed three immunoreactive peaks; one peak eluting in the position of PrRP-20, one eluting in the position of PrRP-31 and one eluting earlier. Reverse phase high-performance liquid chromatography (HPLC) of these brain tissue extracts showed a peak eluting in the position of PrRP-20 and PrRP-31. The present study has shown for the first time the presence of immunoreactive-PrRP in the human brain. The immunoreactive-PrRP levels in the human hypothalamus were, however, lower than the levels of other neuropeptides with prolactin-releasing activity, such as thyrotropin-releasing hormone and vasoactive intestinal polypeptide. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Prolactin-releasing peptide; Hypothalamus; Brain; Radioimmunoassay
1. Introduction Prolactin-releasing peptide (PrRP) is a novel bioactive peptide identified from bovine hypothalamic tissues as a ligand of an orphan seven transmembrane domain receptor, hGR3 [4]. PrRP specifically stimulates prolactin secretion both in vitro and in vivo, and does not affect the secretion of other anterior pituitary hormones [4,18]. This peptide has two molecular forms, a 31-amino-acid peptide (PrRP-31) and a peptide corresponding to the C-terminal 20 amino acids of PrRP-31 (PrRP-20). Immunoreactive (IR)-PrRP was widely distributed in the central nervous system and pituitary gland of the rat, with
* Corresponding author. Tel.: ⫹81-22-717-8116; fax: ⫹81-22-7178118. E-mail address: [email protected] (K. Takahashi).
the highest concentrations found in hypothalamus [10]. Studies using immunocytochemistry and in situ hybridization showed that PrRP was expressed in some nuclei of hypothalamus (the caudal dorsomedial nucleus) and medulla oblongata (the nucleus of the solitary tract and the ventrolateral reticular nucleus) [1,7,8,12,19]. PrRP mRNA was expressed in the normal human pituitary gland and in approximately half cases of pituitary adenomas [20]. Regional distribution of IR-PrRP in the human brain, however, has not been studied. Samson et al. have reported that PrRP-20 and PrRP-31 released prolactin from dispersed anterior pituitary cells harvested from female rats only at the high doses (10-7 and 10-6 M), but TRH stimulated prolactin release at the lower dose (10-8 M) [13], raising the question as to whether PrRP functions as a physiological prolactin releasing factor. It is therefore important to quantitate the amount of PrRP-20 and PrRP-31 present in the hypothalamus. In the present study, we therefore studied the regional
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distribution of IR-PrRP in the human brain obtained at autopsy by radioimmunoassay.
2. Materials and methods 2.1. Tissues This study has been approved by the Ethics Committee of Tohoku University School of Medicine. Human brain tissues were obtained at autopsy performed at the Department of Pathology, Tohoku University Hospital within 4 h postmortem from 7 patients (4 male and 3 female, 31–70 years old). These patients had no neurologic or endocrinological diseases. The tissues were immediately frozen and stored at ⫺80°C prior to extraction of peptides. 2.2. Peptide extraction and radioimmunoassay Tissues were extracted, as reported previously [16]. Briefly, the tissue (approximately 750 mg) was boiled in 2 ml of 1 mol/liter acetic acid for ten minutes. Eight ml of 50% methanol in 0.5 mol/liter acetic acid was added to each sample and the tissue was homogenized. The homogenate was centrifuged by 15,000⫻ g for 30 min. The supernatant was separated, dried by air, reconstituted in assay buffer [0.1 mol/liter phosphate buffer, pH 7.5 containing 0.1% (w/v) bovine serum albumin (BSA), 0.2% (v/v) Triton X-100 and 0.1% (w/v) sodium azide] and assayed. The antiserum against human PrRP-31 (No 1118) was raised in a rabbit. Synthetic human PrRP-31 (Peptide Institute Inc., Minoh-shi, Japan) was conjugated to BSA (Sigma Chemical Co., St Louis, MO, USA) by carbodiimide (Peptide Institute Inc.), and injected into the rabbit with complete Freund adjuvant (Difco Laboratories, Detroit, Michigan, USA). The antiserum was used at a final dilution of 1:100,000. Human PrRP-31 (Peptide Institute, Inc.) was used as a standard. Human PrRP-31 was iodinated with 125 INa by the chloramine T method [5]. The assay was performed in 400 l of assay buffer. The sample or the standard peptide (200 l) was incubated with antiserum (100 l) at 4°C for 48 h and then 100 l of 125 I-PrRP-31 was added to each sample. After further 48 h incubation at 4°C, 100 l of 5% anti-rabbit IgG raised in goat (ICN Biomedicals Inc., Costa Mesa, CA, USA) and 500 l of 10% (w/v) polyethylene glycol in water were added. After 5 h incubation, the sample was centrifuged at 3000⫻ g for 30 min and the supernatant was separated. The pellets were counted by the ␥-counter. The assay could detect changes of 1.5 ⫾ 0.4 fmol/tube (mean ⫾ SD, n ⫽ 8) from zero at 95% confidence with duplicate tubes. The cross reactivity was 100% with human PrRP-20 (Phoenix Pharmaceuticals Inc., Mountain View, CA, USA), rat PrRP-20 (Phoenix Pharmaceuticals Inc.) and rat PrRP-31 (Peptide Institute, Inc.), respectively. There was no significant cross reaction (less than 0.001%) with other
peptides, including vasoactive intestinal polypeptide (VIP), substance P, pituitary adenylate cyclase activating polypeptide-38 (PACAP), neuropeptide Y (NPY), oxytocin, C-type natriuretic peptide, corticotropin releasing hormone (CRH), and growth hormone-releasing hormone (GH-RH). Intraand interassay coefficients of variation were 9.9% and 10.2%, respectively. To examine the stability of IR-PrRP in the brain during the postmortem hours, we measured IR-PrRP concentrations in the rat brains obtained immediately after death and 4 h after death. The experiment was done following the Ethics Guidelines of the Animal Experiments of Tohoku University School of Medicine. Eight adult SD rats were killed under ether anesthesia. Hypothalamus and brain stem were dissected out for peptide extraction immediately after death from the 4 rats and 4 h after death from the other 4 rats which were kept at room temperature. There were no significant decreases in the IR-PrRP concentrations in the hypothalamus and the brain stem of the rats kept at room temperature for 4 h after death [F(1,12) ⫽ 0.596, P ⬎ 0.45], suggesting that the postmortem hours at least up to 4 h had no noticeable effects on IR-PrRP levels in the brain. 2.3. Chromatography Chromatographic characterization of the tissue extracts was performed by Sephadex G-50 (superfine) column chromatography and reverse phase HPLC using a Bondapak C18 column (3.9 mm ⫻ 300 mm, Waters, Milford, MA, USA). Brain tissue extracts (hypothalamus and medulla oblongata) obtained from three cases (one male and two female) were pooled and re-extracted by a Sep-Pak C18 cartridge (Waters). The extracts were reconstituted in 1 mol/liter acetic acid containing 0.5% (w/v) BSA and loaded onto the Sephadex G-50 column (10 ⫻ 560 mm). Peptides on the column were eluted with 1 mol/liter acetic acid containing 0.5% (w/v) BSA at a flow rate of 6 ml/h. Fractions (0.8 ml/fraction) were collected, dried by air, reconstituted in assay buffer and assayed. For the HPLC analysis, the pooled tissue extracts were re-extracted with a Sep-Pak C18 cartridge (Waters), reconstituted in 0.1% (v/v) trifluoroacetic acid (TFA) and loaded onto the column. The HPLC analysis was performed with a linear gradient of acetonitrile containing 0.1% TFA from 10% to 60% at a flow rate of 1 ml/min per fraction over 50 min. Each fraction (1 ml) was collected, dried by the air, reconstituted with assay buffer and assayed.
3. Results Fig. 1 shows a standard curve of human PrRP-31 and a dilution curve of a human brain extract (thalamus). The dilution curve was parallel with the standard curve of PrRP31. The highest concentrations of IR-PrRP were found in
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Fig. 1. A standard curve of prolactin-releasing peptide-31 (PrRP-31) (F) and a two-fold dilution curve of a brain tissue extract (thalamus) (E). The tissue extract was two-fold diluted serially and assayed. B/Bo (%), a percentage of bound counts in the presence of the sample/counts bounds in the absence of added peptide ⫻100.
hypothalamus (912 ⫾ 519 fmol/g wet weight, n ⫽ 6, mean ⫾ SEM), followed by medulla oblongata (496 ⫾ 136 fmol/g wet weight, n ⫽ 6) and thalamus (307 ⫾ 117 fmol/g wet weight, n ⫽ 6) (Fig. 2). Lower concentrations of IRPrRP were detected in occipital lobe, pons and cerebellum. On the other hand, IR-PrRP was not detected in frontal lobe and temporal lobe (⬍50 fmol/g wet weight, n ⫽ 5). IRPrRP levels in the hypothalamus ranged from 555 to 3467 fmol/g wet weight in three female subjects, and from 49 to 421 fmol/g wet weight in three male subjects, although there was no significant difference in the IR-PrRP levels between male and female subjects [F(1,4) ⫽ 1.941, P ⬎ 0.2].
Fig. 3. Sephadex G50 (superfine) column chromatography of the human brain. A, hypothalamus. B, medulla oblongata. Vo, void volume. The arrows indicate the elution positions of PrRP-20 and PrRP-31, respectively.
Sephadex G50 column chromatography of the IR-PrRP in the hypothalamus and medulla oblongata showed three immunoreactive peaks; one peak eluting in the position of PrRP-20, one eluting in the position of PrRP-31 and one eluting earlier (Fig. 3). Reverse phase HPLC of these brain tissue extracts showed a peak eluting in the position of PrRP-20 and PrRP-31 (Fig. 4). PrRP-20 and PrRP-31 were eluted in the same position on HPLC.
4. Discussion
Fig. 2. Regional distribution of immunoreactive prolactin-releasing peptide (IR-PrRP) in the human brain. Data are shown as mean ⫾ SEM. Cerebellum was dissected into the hemisphere (h) and vermis (v).
We have shown for the first time the presence of IR-PrRP in the human brain, with the highest concentrations found in the hypothalamus. High concentrations of IR-PrRP in human medulla oblongata are in accord with the previous reports on the expression of PrRP in the nucleus of the solitary tract and the ventrolateral reticular nucleus in rats [1,7,8,12,19]. The chromatographic study showed that both PrRP-20 and PrRP-31 were present in the human brain.
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Fig. 4. Reverse phase HPLC of the human brain. A, hypothalamus. B, medulla oblongata. The arrow indicates the elution position of PrRP-20 and PrRP-31, which were eluted in the same position. The dotted line indicates a gradient of acetonitrile.
Materials eluting in the earlier peak in Sephadex G50 column chromatography may represent IR-PrRP with a larger molecular weight, possibly the PrRP precursor or its processed form. IR-PrRP levels in the human brain including the hypothalamus are compatible with the levels in the rat brain reported by Matsumoto et al. [10]. The secretion of prolactin from the anterior pituitary is controlled mainly by the inhibitory neurotransmitter, dopamine [6]. Several peptides possessing prolactin-releasing activity, such as thyrotropin-releasing hormone (TRH), VIP, substance P, neurotensin, galanin and PACAP, were identified, but none of them has been demonstrated to be a specific hypophysiotropic stimulatory factor for prolactin secretion. PrRP-31 and PrRP-20 have been proposed to be promising candidates for a specific prolactin-releasing hormone by Hinuma et al. [4]. On the other hand, Samson et al. reported that PrRP-20 and PrRP-31 released prolactin from dispersed anterior pituitary cells harvested from random
cycle female rats only at the high doses (10-7 and 10-6 M), but TRH stimulated prolactin release at the lower dose (10-8 M) [13]. Such lower potency of PrRPs in prolactin release was also observed in dispersed anterior pituitary cells harvested from lactating rats [14]. In the present study, IR-PrRP levels in the human hypothalamus were much lower than the levels of other neuropeptides with prolactin-releasing activity, such as TRH [11], VIP [2], substance P [2], neurotensin [2] and PACAP [3,17]. Postmortem degradation is unlikely to explain these low values of PrRP, because IR-PrRP levels in the rat brain were not decreased at least up to 4 h after death. The immunocytochemical studies by others showed that PrRP nerve fibers were scarcely observed in the external layer of the median eminence in which hypothalamic hormones were abundantly detected [8,19]. Taken together, PrRP may not act as a hypophysiotropic factor like classic hypothalamic hormones, such as CRH and GH-RH. Recently, biologic actions of PrRP other than prolactin release have been suggested [9,12,14,15]. Central administration of PrRP stimulated oxytocin release [9] and increased blood pressure in rats [14]. Intracerebroventricular injection of PrRP-31 stimulated the secretion of LH and FSH via the hypothalamic release of LH-RH in male rats [15]. On the other hand, it is noteworthy that IR-PrRP levels in the hypothalamus in female subjects were in the higher range than in male subjects. In 9-week old rats, IR-PrRP levels in the hypothalamus were about two times higher in female rats than in male rats (Takahashi K, unpublished observations). The present study, however, studied a limited number of human samples and could not show the statistical difference in the IR-PrRP levels in the hypothalamus between male and female subjects. There is a possibility that hypothalami of lactating women have much higher levels of PrRP. The present study has shown the presence of PrRP in the human brain, with the highest concentrations found in hypothalamus, followed by medulla oblongata and thalamus. These findings are in accord with the possibility that PrRP-20 and PrRP-31 act as neuromodulators in the human brain.
Acknowledgments This work was supported in part by Grants-in-aid for Scientific Research (B) and (C), and on Priority Areas (A) from the Ministry of Education, Science, Sports and Culture of Japan and by the Mochida Memorial Foundation for Medical and Pharmaceutical Research.
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