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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
Neuroscience Letters 276 (1999) 103±106 www.elsevier.com/locate/neulet
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 Reiko Tokita a, Tomoko Nakata a, Harumi Katsumata a, Shunichiro Konishi a, Hidetaka Onodera b, Junko Imaki b, Shiro Minami a,* a
Department of Bioregulation, Institute of Gerontology, Nippon Medical School, Nakahara-ku, Kawasaki 211-8533, Japan b Department of Anatomy, Nippon Medical School, Bunkyo-ku, Tokyo 133-8602, Japan Received 2 August 1999; received in revised form 27 September 1999; accepted 28 September 1999
Abstract Prolactin-releasing peptide (PrRP), recently isolated from bovine hypothalamus as an endogenous ligand to a seven transmembrane-domain orphan receptor, is a candidate speci®c prolactin-releasing factor. The prolactin-releasing activity of the peptide and the expression of the PrRP gene were examined in vivo in relation to estrogen status. Plasma prolactin levels increased signi®cantly with a peak at 5 min after the administration of 50 mg/kg PrRP in female rats in estrus under urethane anesthesia as compared with those in vehicle-treated control rats, but not in female rats in diestrus or proestrus or in male rats. In ovariectomized rats treated with supraphysiological concentration of estrogen, a dose-dependent increase of prolactin secretion in response to 2±50 mg/kg PrRP was observed. However, the peak values induced by 50 mg/kg PrRP were much less than those induced by 2 mg/kg thyrotropin-releasing hormone (TRH). PrRP mRNA levels in the medulla oblongata were decreased by ovariectomy and increased by estrogen treatment. The data indicate that estrogen is prerequisite to the stimulatory effect of PrRP on the secretion of prolactin and to the increase of PrRP mRNA levels in the medulla oblongata. The weak in vivo potency of PrRP on prolactin secretion relative to TRH suggests that PrRP differs from the classical hypophysiotropic hypothalamic releasing hormones. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Prolactin; Estrogen; Prolactin-releasing peptide; Secretion; Hypothalamus; Medulla oblongata; Messenger ribonucleic acid
The secretion of prolactin from the pituitary is controlled mainly by the inhibitory neurotransmitter dopamine [6]. The speci®c hypophysiotropic stimulatory factor for prolactin secretion has not yet been identi®ed. Early studies showed that crude extracts of hypothalamic tissue contain factors that stimulate prolactin secretion [1]. Recently, a novel peptide, prolactin-releasing peptide (PrRP), was isolated from extracts of bovine hypothalamic tissue [4] as an endogenous ligand to a seven transmembrane-domain orphan receptor, hGR3 or UHR-1 [14]. The mature peptide containing 31 amino acid residues with a C-terminal amide group and the truncated C-terminal peptide containing 20 amino acids have been shown to be posttranslational products of the same preproproteins. The peptide sequences are highly conserved among the species such as bovine, rat and human. * Corresponding author. Tel.: 181-44-733-1821 ext. 865; fax: 181-44-733-1877. E-mail address: [email protected] (S. Minami)
Both peptides have been shown to possess prolactin-releasing activity from pituitary cells obtained from lactating rats and the rat pituitary adenoma-derived cell line RC-4B/C and the potency is comparable to that of thyrotropin-releasing hormone (TRH) on a molar basis [4]. These peptides do not in¯uence the secretion of other pituitary hormones. It has also been demonstrated that PrRP is less potent than TRH in releasing prolactin from dispersed pituitary cells of nonlactating random-cycle female rats and is inef®cient in releasing prolactin from dispersed pituitary cells of male rats [10]. Thus, the physiologic relevance of the peptide remains to be established. In the present study, we examined the in vivo potency of PrRP as a prolactin-releasing peptide. We examined the in¯uence of the estrous cycle, ovariectomy and an excess estrogen condition on PrRP-induced prolactin secretion under urethane anesthesia. In addition, the in¯uence of estrogen on PrRP mRNA levels in the hypothalamus and
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medulla oblongata, where PrRP-containing cells are located [3,7,9] was observed. Adult Sprague±Dawley rats were used for this study. Female rats weighing 200±220 g and male rats weighing 250±280 g were housed in air-conditioned animal quarters with lights on between 08:00 and 20:00 h. Food and water were available ad libitum. Vaginal smears were taken and inspected daily and rats with 4-day estrous cycles were used for this study. To examine the effect of estrogen, ovariectomy with (OVX 1 E2 group) or without (OVX group) a subcutaneously implanted silastic capsule (inner diameter 0.5 mm, outer diameter 1.0 mm, length 10 mm) containing b-estradiol 3-bezoate (E2, SIGMA Chemical Co., St. Louis, MO) was performed 2 weeks before the experiment. On the day of the experiment, blood samples were taken and assayed for estradiol by using the double antibody estradiol radioimmunoassay kit (Diagnostic Products Corporation, CA). The plasma concentration of estradiol was 25.0 ^ 1.0 pg/ml (n 6) in normal female rats during proestrus, 109.8 ^ 6.8 pg/ml (n 30, P , 0:01 vs. proestrus females by Student's t-test) in OVX 1 E2 rats and not detectable (,1.4 pg/ml) in all OVX rats. A two-week treatment with an implanted estrogen capsule causes the state of excess estrogen (supraphysiological) in the rat. All studies were performed with the approval of the Experimental Animal Ethics Committee of Nippon Medical School. Six rats were used for each treatment group. In the ®rst experiment, the effect of intravenous (i.v.) administration of PrRP (prolactin-releasing peptide-31, Peptide Institute, Inc., Osaka, Japan) on prolactin secretion was examined. Rats were anesthetized with an intraperitoneal injection of urethane (1.5 g/kg body weight) and a silastic cannula was inserted via the jugular vein to the right atrium for blood collection and peptide administration. Thirty minutes after anesthesia, PrRP dissolved in 0.9% NaCl was administered through the cannula and blood samples were collected serially. Two micrograms of TRH (thyrotropin-releasing hormone, Peptide Institute, Inc.) was administered to some rats with OVX 1 E2 for comparison with PrRP to stimulate prolactin secretion. The plasma was separated and stored at 2408C until assayed. All experiments were performed between 09:00 and 12:00 h to avoid spontaneous prolactin surges that occur in the afternoon. The plasma concentration of prolactin was determined by radioimmunoassay using materials supplied by NHPP and by using rPRL-RP-3 as a standard. To measure the mRNA levels of PrRP in the hypothalamus and medulla oblongata, RNase protection assay was performed using female rats in diestrus, OVX rats, OVX 1 E2 rats and male rats. The rats were killed by decapitation and the hypothalamus and medulla oblongata were taken and frozen immediately in liquid nitrogen. Total RNA was isolated by the guanidine thiocyanate method using Isogen (Nippon Gene, Tokyo, Japan). A plasmid containing the PrRP cDNA was obtained by a previously described method [9]. To produce the antisense RNA probe, the plasmid was
linearized with EcoRI. The pTRI-b-Actin 125-Rat plasmid (Ambion, Austin, TX) which encodes rat b-actin was used as a control. Using T7 RNA polymerase (Boeringer Mannheim Gmbh, Germany) for PrRP and T3 RNA polymerase (Boeringer Mannheim Gmbh) for b-actin, radioactive RNA copies were synthesized from the linearized plasmids with [ 32P]CTP. Radiolabeled probes for PrRP and b-actin mRNAs were annealed with the total RNA (5 mg) and digested using the Ribonuclease Protection Assay Kit, RPA II e (Ambion). Digested RNA was analyzed on 5% polyacrylamide, 8 M urea denaturing gels. The gels were exposed to X-ray ®lm for 5 days (PrRP mRNA) or 8 h (bactin mRNA). Protected fragments of PrRP mRNA in 2.5, 5.0 and 10 mg total RNA, isolated from the medulla oblongata of diestrus female rat were shown in Fig. 3A. Autoradiographic exposures were quanti®ed by scanning densitometry using NIH image (NIH, Washington D.C.). The densitometric values for PrRP mRNA were calculated relative to those for the b-actin mRNA in the same sample and expressed as arbitrary units. In each study, data from six rats for each treatment group were used for analysis. Statistical analysis was carried out by one-way ANOVA followed by Dunn's multiple comparison procedure and P , 0:05 was considered to be signi®cant. Intravenous administration of 50 mg/kg PrRP increased plasma prolactin levels signi®cantly during estrus compared with vehicle from 2.5 to 10 min after administration and with a peak at 5 min (Fig. 1C). However, the administration of 50 mg/kg PrRP did not in¯uence plasma prolactin levels in female rats during diestrus or proestrus (Fig. 1A,B) or in male rats (Fig. 1D). Baseline levels of plasma prolactin were increased by
Fig. 1. Prolactin (PRL) secretion in response to 50 mg/kg PrRP in urethane anesthetized female rats during the estrous cycle (A±C) and in male rats (D). Normal cycling female rats were examined in the morning of diestrus (A), proestrus (B) and estrus (C). Values are expressed as the mean ^ SEM. The number of rats in each treatment group was six. *P , 0:05 compared with vehicle-treated rats.
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levels in female rats in diestrus and in male rats were not different. Our results show that prolactin secretion in response to PrRP in normal female rats is dependent on the estrous cycle. The stimulatory response was seen only during estrus. Plasma levels of estradiol are highest during the afternoon of proestrus and decrease toward the day of estrus [2]. A two-week treatment with supraphysiological concentration of estrogen in OVX rats enhanced the prolactin response as well as baseline prolactin levels. These data indicate that exposure to estrogen is prerequisite to prolactin release induced by PrRP. Matsumoto et al. showed that iv administration of PrRP induced prolactin release not only during estrus, but during proestrus and metestrus under urethane anesthesia [8]. They used larger dose of PrRP, 50 nmol (180 mg)/kg, than that used in the present study and also reported that a huge dose of PrRP, 500 nmol (1.8 mg)/kg, was effective in male rats. Their experiments were performed in the afternoon of each estrous period, whereas ours were done in the morning. These differences of the experimental conditions may explain the discrepancy of the results. Our data in estrogen-treated rats indicate that PrRP is much less potent than TRH in its ability to stimulate prolacFig. 2. Effect of estrogen on prolactin (PRL) secretion induced by PrRP in urethane anesthetized rats. (A) Prolactin secretion in response to 50 mg/kg PrRP in ovariectomized rats with (OVX 1 E2) and without (OVX) estrogen treatment. The prolactin response to 2 mg/kg TRH in OVX 1 E2 rats is shown in the inset for comparison. Note the difference of the scale in the inset. (B) Prolactin secretion in response to increasing doses of PrRP. Values are expressed as the mean ^ SEM. The number of rats in each treatment group was six. *P , 0:05 compared with vehicle-treated rats.
estrogen treatment for 2 weeks (Fig. 2A). In OVX 1 E2 rats, iv administration of 50 mg/kg PrRP increased the levels of plasma prolactin signi®cantly compared with vehicle administration and the time course of the response was same as that observed in normal estrus rats. The administration of PrRP was not effective in OVX rats (Fig. 2A). The peak value of the prolactin response in OVX 1 E2 rats was larger than that seen in normal estrus rats (53.8 ^ 5.6 vs. 21.2 ^ 8.3 ng/ml, P , 0:05), as expected from the higher baseline levels. As shown in Fig. 2B, responses of prolactin secretion induced by PrRP were dose-dependent from 2.0 to 50 mg/kg. However, the peak value induced by 50 mg (13.6 nmol)/kg PrRP was smaller than that induced by i.v. administration of 2 mg (5.5 nmol)/kg TRH (53.8 ^ 5.6 ng/ml vs. 345 ^ 44.9 ng/ml, P , 0:01) (Fig. 2A; inset). PrRP mRNA levels in the hypothalamus did not vary signi®cantly between females in diestrus, OVX, OVX 1 E2 and male rats (Fig. 3B). In the medulla oblongata, PrRP mRNA levels in OVX rats were signi®cantly lower than those in diestrus rats (Fig. 3C). Estrogen treatment increased PrRP mRNA levels in OVX rats. PrRP mRNA
Fig. 3. Expression of PrRP mRNA by RNase protection. (A) Protected fragments of PrRP mRNA in 2.5, 5.0 and 10 mg total RNA, isolated from the medulla oblongata (arrow). (B,C) Expression of PrRP mRNA in the hypothalamus and medulla oblongata of normal female rats in diestrus, OVX rats, OVX 1 E2 rats and male rats. PrRP mRNA levels were examined by RNase protection assay and expressed as the ratio to b-actin mRNA (arbitrary units). Values are expressed as the mean ^ SEM. The number of rats in each treatment group was six. *P , 0:05.
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tin secretion. This is consistent with in vitro data showing that the magnitude of prolactin release from pituitary cells of normal cycling female rats in the presence of 1 mM PrRP was much less than that obtained by a similar dose of TRH [10]. We have not examined lactating rats, whose pituitary cells are reported to secrete prolactin in response to the same potency of PrRP as TRH [4]. PrRP may therefore have some physiological roles in lactation. In situ hybridization and immunocytochemistry studies have shown that the perikarya producing PrRP are located in discrete regions in the rat brain, namely, the hypothalamic dorsomedial nucleus, the solitary tract nucleus and the caudal ventrolateral medulla [3,7,9]. No other hypophysiotropic-releasing or -inhibiting factors are known to be derived from neurons in these regions. An immunocytochemical study of PrRP using a monoclonal antibody demonstrated a wide distribution of immunoreactive nerve ®ber networks in the rat brain. However, no immunopositive ®bers were observed in the external layer of the median eminence [7]. These histological data indicate that PrRP is not transported to the anterior pituitary via the ordinary mechanism through hypophyseal portal blood, even if PrRP acts on lactotrophs in certain situations. In the hypothalamic paraventricular nucleus, some PrRP-positive neurons showed synaptic contact with oxytocin-positive cell bodies [7]. It is possible that PrRP acts in the hypothalamus or other brain regions to provoke prolactin secretion secondarily. Our data also demonstrate that PrRP gene expression in the medulla oblongata is in¯uenced by estrogen. The physiological signi®cance of this phenomenon is not clear. The distribution of PrRP neurons in the brain-stem are compatible to areas A1 and A2 where noradrenergic neurons exist [5,11] and PrRP-like immunoreactivity is expressed in a population of tyrosine hydroxylase-positive cells [3]. A1 noradrenergic neurons contain neuropeptide Y, an orexigenic peptide and send nerve ®bers to the hypothalamus [11,12]. Noradrenergic input to the preoptic area has important roles in the regulation of gonadotropin-releasing hormone secretion [13]. PrRP may have some functional roles in hypothalamic neuroendocrine control mechanism, such as in energy balance or gonadal function. In conclusion, stimulation of prolactin secretion in response to PrRP and expression of the PrRP gene in the medulla oblongata depend on the estrogen status in female rats. The potency of PrRP is relatively weak in vivo compared with TRH under excess estrogen conditions. Together with histological observations [7,9], it is conceivable that PrRP differs from the classical hypophysiotropic hypothalamic releasing hormones. The central effects of PrRP will be investigated in the future. We thank NIDDK-NIH for providing the rPRL RIA kit. This work was supported in part by Grants-in-Aid for Scien-
ti®c Research from the Japanese Ministry of Education, Science, Sports and Culture.
[1] Boyd, A.E., Spencer, E., Jackson, I.M.D. and Reichlin, S., Prolactin-releasing factor (PRF) in porcine hypothalamic extract distinct from TRH. Endocrinology, 99 (1976) 861±871. [2] Butcher, R.L., Collins, W.E. and Fugo, N.W., Plasma concentration of LH, FSH, prolactin, progesterone and estradiol-17b throughout the 4-day estrous cycle of the rat. Endocrinology, 94 (1974) 1704±1708. [3] Chen, C.-T., Dun, S.L., Dun, N.L. and Chang, J.-K., Prolactinreleasing peptide-immunoreactivity in A1 and A2 noradrenergic neurons of the rat medulla. Brain Res., 822 (1999) 276±279. [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. and Fujino, M., A prolactin-releasing peptide in the brain. Nature, 393 (1998) 272±276. [5] Hokfelt, T., Martensson, R., Bjorklund, A., Kleinau, S. and Goldstein, M., Distribution maps of tyrosine-hydroxylaseimmunoreactive neurons in the rat brain. In A. Bjorklund and T. Hokfelt (Eds.), Handbook of Chemical Neuroanatomy, Elsevier, Amsterdam, 1984, pp. 277±379. [6] Lamberts, S.W.L. and Macleod, R.M.M., Regulation of prolactin secretion at the level of the lactotroph. Physiol. Rev., 70 (1990) 279±318. [7] Maruyama, M., Matsumoto, H., Fujiwara, K., Kitada, C., Hinuma, S., Onda, H., Fujino, M. and Inoue, K., Immunocytochemical localization of prolactin-releasing peptide in the rat brain. Endocrinology, 140 (1999) 2326±2333. [8] Matsumoto, H., Noguchi, J., Horikoshi, Y., Kawamata, Y., Kitada, C., Hinuma, S., Onda, H., Nishimura, O. and Fujino, M., Stimulation of prolactin release by prolactin-releasing peptide in rats. Biochem. Biophys. Res. Commun., 259 (1999) 321±324. [9] Minami, S., Nakata, T., Tokita, R., Onodera, H. and Imaki, J., Cellular localization of prolactin-releasing peptide messenger RNA in the rat brain. Neurosci. Lett., 266 (1999) 73±75. [10] Samson, W.K., Resch, Z.T., Murphy, T.C. and Chang, J.-K., Gender-biased activity of the novel prolactin releasing peptides: comparison with thyrotropin releasing hormone reveals only pharmacologic effects. Endocrine, 9 (1999) 289±291. [11] Sawchenko, P. and Swanson, L.W., The organization of noradrenergic pathways from the brain-stem to the paraventricular and supraoptic nuclei in the rat. Brain Res. Rev., 4 (1982) 275±325. [12] Sawchenko, P.E., Swanson, L.W., Grzanna, R., Howe, P.R.C., Bloom, S.R. and Polak, J.M., Colocalization of neuropeptide Y immunoreactivity in brain-stem catecholaminergic neurons that project to the paraventricular nucleus of the hypothalamus. J. Comp. Neurol., 241 (1985) 138±153. [13] Terasawa, E., Krook, C., Hei, D.L., Gearing, M., Schultz, N.J. and Davis, G.A., Norepinephrine is a possible neurotransmitter stimulating pulsatile release of luteinizing hormone releasing hormone in the rhesus monkey. Endocrinology, 123 (1988) 1808±1816. [14] Welch, S.K., O'Hara, B.F., Kilduff, T.S. and Heller, H.C., Sequence and tissue distribution of a candidate G-coupled receptor cloned from rat hypothalamus. Biochem. Biophys. Res. Commun., 209 (1995) 606±613.
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 Reiko Tokita a, Tomoko Nakata a, Harumi Katsumata a, Shunichiro Konishi a, Hidetaka Onodera b, Junko Imaki b, Shiro Minami a,* a
Department of Bioregulation, Institute of Gerontology, Nippon Medical School, Nakahara-ku, Kawasaki 211-8533, Japan b Department of Anatomy, Nippon Medical School, Bunkyo-ku, Tokyo 133-8602, Japan Received 2 August 1999; received in revised form 27 September 1999; accepted 28 September 1999
Abstract Prolactin-releasing peptide (PrRP), recently isolated from bovine hypothalamus as an endogenous ligand to a seven transmembrane-domain orphan receptor, is a candidate speci®c prolactin-releasing factor. The prolactin-releasing activity of the peptide and the expression of the PrRP gene were examined in vivo in relation to estrogen status. Plasma prolactin levels increased signi®cantly with a peak at 5 min after the administration of 50 mg/kg PrRP in female rats in estrus under urethane anesthesia as compared with those in vehicle-treated control rats, but not in female rats in diestrus or proestrus or in male rats. In ovariectomized rats treated with supraphysiological concentration of estrogen, a dose-dependent increase of prolactin secretion in response to 2±50 mg/kg PrRP was observed. However, the peak values induced by 50 mg/kg PrRP were much less than those induced by 2 mg/kg thyrotropin-releasing hormone (TRH). PrRP mRNA levels in the medulla oblongata were decreased by ovariectomy and increased by estrogen treatment. The data indicate that estrogen is prerequisite to the stimulatory effect of PrRP on the secretion of prolactin and to the increase of PrRP mRNA levels in the medulla oblongata. The weak in vivo potency of PrRP on prolactin secretion relative to TRH suggests that PrRP differs from the classical hypophysiotropic hypothalamic releasing hormones. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Prolactin; Estrogen; Prolactin-releasing peptide; Secretion; Hypothalamus; Medulla oblongata; Messenger ribonucleic acid
The secretion of prolactin from the pituitary is controlled mainly by the inhibitory neurotransmitter dopamine [6]. The speci®c hypophysiotropic stimulatory factor for prolactin secretion has not yet been identi®ed. Early studies showed that crude extracts of hypothalamic tissue contain factors that stimulate prolactin secretion [1]. Recently, a novel peptide, prolactin-releasing peptide (PrRP), was isolated from extracts of bovine hypothalamic tissue [4] as an endogenous ligand to a seven transmembrane-domain orphan receptor, hGR3 or UHR-1 [14]. The mature peptide containing 31 amino acid residues with a C-terminal amide group and the truncated C-terminal peptide containing 20 amino acids have been shown to be posttranslational products of the same preproproteins. The peptide sequences are highly conserved among the species such as bovine, rat and human. * Corresponding author. Tel.: 181-44-733-1821 ext. 865; fax: 181-44-733-1877. E-mail address: [email protected] (S. Minami)
Both peptides have been shown to possess prolactin-releasing activity from pituitary cells obtained from lactating rats and the rat pituitary adenoma-derived cell line RC-4B/C and the potency is comparable to that of thyrotropin-releasing hormone (TRH) on a molar basis [4]. These peptides do not in¯uence the secretion of other pituitary hormones. It has also been demonstrated that PrRP is less potent than TRH in releasing prolactin from dispersed pituitary cells of nonlactating random-cycle female rats and is inef®cient in releasing prolactin from dispersed pituitary cells of male rats [10]. Thus, the physiologic relevance of the peptide remains to be established. In the present study, we examined the in vivo potency of PrRP as a prolactin-releasing peptide. We examined the in¯uence of the estrous cycle, ovariectomy and an excess estrogen condition on PrRP-induced prolactin secretion under urethane anesthesia. In addition, the in¯uence of estrogen on PrRP mRNA levels in the hypothalamus and
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medulla oblongata, where PrRP-containing cells are located [3,7,9] was observed. Adult Sprague±Dawley rats were used for this study. Female rats weighing 200±220 g and male rats weighing 250±280 g were housed in air-conditioned animal quarters with lights on between 08:00 and 20:00 h. Food and water were available ad libitum. Vaginal smears were taken and inspected daily and rats with 4-day estrous cycles were used for this study. To examine the effect of estrogen, ovariectomy with (OVX 1 E2 group) or without (OVX group) a subcutaneously implanted silastic capsule (inner diameter 0.5 mm, outer diameter 1.0 mm, length 10 mm) containing b-estradiol 3-bezoate (E2, SIGMA Chemical Co., St. Louis, MO) was performed 2 weeks before the experiment. On the day of the experiment, blood samples were taken and assayed for estradiol by using the double antibody estradiol radioimmunoassay kit (Diagnostic Products Corporation, CA). The plasma concentration of estradiol was 25.0 ^ 1.0 pg/ml (n 6) in normal female rats during proestrus, 109.8 ^ 6.8 pg/ml (n 30, P , 0:01 vs. proestrus females by Student's t-test) in OVX 1 E2 rats and not detectable (,1.4 pg/ml) in all OVX rats. A two-week treatment with an implanted estrogen capsule causes the state of excess estrogen (supraphysiological) in the rat. All studies were performed with the approval of the Experimental Animal Ethics Committee of Nippon Medical School. Six rats were used for each treatment group. In the ®rst experiment, the effect of intravenous (i.v.) administration of PrRP (prolactin-releasing peptide-31, Peptide Institute, Inc., Osaka, Japan) on prolactin secretion was examined. Rats were anesthetized with an intraperitoneal injection of urethane (1.5 g/kg body weight) and a silastic cannula was inserted via the jugular vein to the right atrium for blood collection and peptide administration. Thirty minutes after anesthesia, PrRP dissolved in 0.9% NaCl was administered through the cannula and blood samples were collected serially. Two micrograms of TRH (thyrotropin-releasing hormone, Peptide Institute, Inc.) was administered to some rats with OVX 1 E2 for comparison with PrRP to stimulate prolactin secretion. The plasma was separated and stored at 2408C until assayed. All experiments were performed between 09:00 and 12:00 h to avoid spontaneous prolactin surges that occur in the afternoon. The plasma concentration of prolactin was determined by radioimmunoassay using materials supplied by NHPP and by using rPRL-RP-3 as a standard. To measure the mRNA levels of PrRP in the hypothalamus and medulla oblongata, RNase protection assay was performed using female rats in diestrus, OVX rats, OVX 1 E2 rats and male rats. The rats were killed by decapitation and the hypothalamus and medulla oblongata were taken and frozen immediately in liquid nitrogen. Total RNA was isolated by the guanidine thiocyanate method using Isogen (Nippon Gene, Tokyo, Japan). A plasmid containing the PrRP cDNA was obtained by a previously described method [9]. To produce the antisense RNA probe, the plasmid was
linearized with EcoRI. The pTRI-b-Actin 125-Rat plasmid (Ambion, Austin, TX) which encodes rat b-actin was used as a control. Using T7 RNA polymerase (Boeringer Mannheim Gmbh, Germany) for PrRP and T3 RNA polymerase (Boeringer Mannheim Gmbh) for b-actin, radioactive RNA copies were synthesized from the linearized plasmids with [ 32P]CTP. Radiolabeled probes for PrRP and b-actin mRNAs were annealed with the total RNA (5 mg) and digested using the Ribonuclease Protection Assay Kit, RPA II e (Ambion). Digested RNA was analyzed on 5% polyacrylamide, 8 M urea denaturing gels. The gels were exposed to X-ray ®lm for 5 days (PrRP mRNA) or 8 h (bactin mRNA). Protected fragments of PrRP mRNA in 2.5, 5.0 and 10 mg total RNA, isolated from the medulla oblongata of diestrus female rat were shown in Fig. 3A. Autoradiographic exposures were quanti®ed by scanning densitometry using NIH image (NIH, Washington D.C.). The densitometric values for PrRP mRNA were calculated relative to those for the b-actin mRNA in the same sample and expressed as arbitrary units. In each study, data from six rats for each treatment group were used for analysis. Statistical analysis was carried out by one-way ANOVA followed by Dunn's multiple comparison procedure and P , 0:05 was considered to be signi®cant. Intravenous administration of 50 mg/kg PrRP increased plasma prolactin levels signi®cantly during estrus compared with vehicle from 2.5 to 10 min after administration and with a peak at 5 min (Fig. 1C). However, the administration of 50 mg/kg PrRP did not in¯uence plasma prolactin levels in female rats during diestrus or proestrus (Fig. 1A,B) or in male rats (Fig. 1D). Baseline levels of plasma prolactin were increased by
Fig. 1. Prolactin (PRL) secretion in response to 50 mg/kg PrRP in urethane anesthetized female rats during the estrous cycle (A±C) and in male rats (D). Normal cycling female rats were examined in the morning of diestrus (A), proestrus (B) and estrus (C). Values are expressed as the mean ^ SEM. The number of rats in each treatment group was six. *P , 0:05 compared with vehicle-treated rats.
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levels in female rats in diestrus and in male rats were not different. Our results show that prolactin secretion in response to PrRP in normal female rats is dependent on the estrous cycle. The stimulatory response was seen only during estrus. Plasma levels of estradiol are highest during the afternoon of proestrus and decrease toward the day of estrus [2]. A two-week treatment with supraphysiological concentration of estrogen in OVX rats enhanced the prolactin response as well as baseline prolactin levels. These data indicate that exposure to estrogen is prerequisite to prolactin release induced by PrRP. Matsumoto et al. showed that iv administration of PrRP induced prolactin release not only during estrus, but during proestrus and metestrus under urethane anesthesia [8]. They used larger dose of PrRP, 50 nmol (180 mg)/kg, than that used in the present study and also reported that a huge dose of PrRP, 500 nmol (1.8 mg)/kg, was effective in male rats. Their experiments were performed in the afternoon of each estrous period, whereas ours were done in the morning. These differences of the experimental conditions may explain the discrepancy of the results. Our data in estrogen-treated rats indicate that PrRP is much less potent than TRH in its ability to stimulate prolacFig. 2. Effect of estrogen on prolactin (PRL) secretion induced by PrRP in urethane anesthetized rats. (A) Prolactin secretion in response to 50 mg/kg PrRP in ovariectomized rats with (OVX 1 E2) and without (OVX) estrogen treatment. The prolactin response to 2 mg/kg TRH in OVX 1 E2 rats is shown in the inset for comparison. Note the difference of the scale in the inset. (B) Prolactin secretion in response to increasing doses of PrRP. Values are expressed as the mean ^ SEM. The number of rats in each treatment group was six. *P , 0:05 compared with vehicle-treated rats.
estrogen treatment for 2 weeks (Fig. 2A). In OVX 1 E2 rats, iv administration of 50 mg/kg PrRP increased the levels of plasma prolactin signi®cantly compared with vehicle administration and the time course of the response was same as that observed in normal estrus rats. The administration of PrRP was not effective in OVX rats (Fig. 2A). The peak value of the prolactin response in OVX 1 E2 rats was larger than that seen in normal estrus rats (53.8 ^ 5.6 vs. 21.2 ^ 8.3 ng/ml, P , 0:05), as expected from the higher baseline levels. As shown in Fig. 2B, responses of prolactin secretion induced by PrRP were dose-dependent from 2.0 to 50 mg/kg. However, the peak value induced by 50 mg (13.6 nmol)/kg PrRP was smaller than that induced by i.v. administration of 2 mg (5.5 nmol)/kg TRH (53.8 ^ 5.6 ng/ml vs. 345 ^ 44.9 ng/ml, P , 0:01) (Fig. 2A; inset). PrRP mRNA levels in the hypothalamus did not vary signi®cantly between females in diestrus, OVX, OVX 1 E2 and male rats (Fig. 3B). In the medulla oblongata, PrRP mRNA levels in OVX rats were signi®cantly lower than those in diestrus rats (Fig. 3C). Estrogen treatment increased PrRP mRNA levels in OVX rats. PrRP mRNA
Fig. 3. Expression of PrRP mRNA by RNase protection. (A) Protected fragments of PrRP mRNA in 2.5, 5.0 and 10 mg total RNA, isolated from the medulla oblongata (arrow). (B,C) Expression of PrRP mRNA in the hypothalamus and medulla oblongata of normal female rats in diestrus, OVX rats, OVX 1 E2 rats and male rats. PrRP mRNA levels were examined by RNase protection assay and expressed as the ratio to b-actin mRNA (arbitrary units). Values are expressed as the mean ^ SEM. The number of rats in each treatment group was six. *P , 0:05.
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tin secretion. This is consistent with in vitro data showing that the magnitude of prolactin release from pituitary cells of normal cycling female rats in the presence of 1 mM PrRP was much less than that obtained by a similar dose of TRH [10]. We have not examined lactating rats, whose pituitary cells are reported to secrete prolactin in response to the same potency of PrRP as TRH [4]. PrRP may therefore have some physiological roles in lactation. In situ hybridization and immunocytochemistry studies have shown that the perikarya producing PrRP are located in discrete regions in the rat brain, namely, the hypothalamic dorsomedial nucleus, the solitary tract nucleus and the caudal ventrolateral medulla [3,7,9]. No other hypophysiotropic-releasing or -inhibiting factors are known to be derived from neurons in these regions. An immunocytochemical study of PrRP using a monoclonal antibody demonstrated a wide distribution of immunoreactive nerve ®ber networks in the rat brain. However, no immunopositive ®bers were observed in the external layer of the median eminence [7]. These histological data indicate that PrRP is not transported to the anterior pituitary via the ordinary mechanism through hypophyseal portal blood, even if PrRP acts on lactotrophs in certain situations. In the hypothalamic paraventricular nucleus, some PrRP-positive neurons showed synaptic contact with oxytocin-positive cell bodies [7]. It is possible that PrRP acts in the hypothalamus or other brain regions to provoke prolactin secretion secondarily. Our data also demonstrate that PrRP gene expression in the medulla oblongata is in¯uenced by estrogen. The physiological signi®cance of this phenomenon is not clear. The distribution of PrRP neurons in the brain-stem are compatible to areas A1 and A2 where noradrenergic neurons exist [5,11] and PrRP-like immunoreactivity is expressed in a population of tyrosine hydroxylase-positive cells [3]. A1 noradrenergic neurons contain neuropeptide Y, an orexigenic peptide and send nerve ®bers to the hypothalamus [11,12]. Noradrenergic input to the preoptic area has important roles in the regulation of gonadotropin-releasing hormone secretion [13]. PrRP may have some functional roles in hypothalamic neuroendocrine control mechanism, such as in energy balance or gonadal function. In conclusion, stimulation of prolactin secretion in response to PrRP and expression of the PrRP gene in the medulla oblongata depend on the estrogen status in female rats. The potency of PrRP is relatively weak in vivo compared with TRH under excess estrogen conditions. Together with histological observations [7,9], it is conceivable that PrRP differs from the classical hypophysiotropic hypothalamic releasing hormones. The central effects of PrRP will be investigated in the future. We thank NIDDK-NIH for providing the rPRL RIA kit. This work was supported in part by Grants-in-Aid for Scien-
ti®c Research from the Japanese Ministry of Education, Science, Sports and Culture.
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