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Involvement of the Y2 receptor subtype in the regulation of prolactin gene expression by neuropeptide Y in the male rat
ELSEVIER
Neuroscience Letters 190 (1995)77-80
UW[lg[ tm[B8
Involvement of the Y2 receptor subtype in the regulation of prolactin gene expression by neuropeptide Y in the male rat Ester Garcia de Yebenes a, Songyun Li a, Alain Fournier b, Serge St-Pierre b, Georges Pelletiera,* aMRC Group in Molecular Endocrinology, CHUL Research Center and Laval University, 2705 Laurier Boulevard, Quebec, GI V 4G2, Canada blNRS-Sant#, Montreal, Canada Received 20 February 1995; revised version received 22 March 1995; accepted 22 March 1995
Abstract
In order to determine the influence of neuropeptide Y (NPY) on the biosynthesis of prolactin (PRL), we have studied the effects of NPY and some NPY analogs on PRL gene expression in the male rat anterior pituitary gland. The following peptides (4/zg/100 g body w0: NPY, peptide YY (PYY), NPYI3_36 (a Y2 receptor agnnist) and [Leu31,Pro34]NPY (a YI receptor agonis0 were injected into the left lateral ventricle of adult male rats. Control animals received only the vehicle (0.9% NaCI). All the animals were perfused with 4% paraformaldehyde 4 h after injection and processed for in situ hybridization. The intracerebroventricular injection of NPY, PYY, and NPYI3_36 induced a significant increase in the hybridization signal (22--40% over control). On the other hand, the Yl receptor agonist [Leu31, Pro34]NPY did not influence PRL mRNA levels. These data then suggest that activation of the Y2 NPY receptor subtype at the central level can positively regulate PRL gene expression. Keywords: Prolactin; Neuropeptide Y; Y2 receptor; In situ hybridization; Anterior pituitary
There is now compelling evidence that neuropeptide Y (NPY) is involved in the regulation of reproductive function (for review see Ref. [12]). For example, it has been clearly shown that NPY plays a significant role in the control of gonadotropin secretion [12]. So far, there have been very few studies about the effect of NPY on prolactin (PRL) secretion. Harfstrand et al. [6,7] have shown that intracerebroventricular (ICV) injection of NPY increased serum plasma levels in the male rat. We have also reported that in male rats intravenous injection of NPY induced a fourfold elevation in plasma levels of prolactin [9]. Immunoneutralization of brain NPY was shown to decrease prolactin plasma levels [11], suggesting physiological involvement of the NPYergic system in the regulation of prolactin secretion. In order to improve our knowledge on the role of NPY in the regulation of PRL secretion, we have studied the effect of NPY ICV administration on PRL gene expression. Different NPY analogs were also used to identify which NPY receptor subtype(s) might be involved in the control of PRL secretion. * Corresponding author, Tel.: +1 418 6542296; Fax: +1 418 6542761.
Groups (6 animals/group) of adult male rats weighing 200-225 g received into the left lateral ventricle a 10/zl injection of one of the following peptides (4/zg/100 g body wt): PYY, NPY, the preferential agonist at the Y1 receptor [Leu31, Pro34]NpY and the preferential agonist at the Y2 receptor NPY13_36 [2,5,15]. One group of animals also received a 10/zl injection of the vehicle (0.9% NaC1). The injection was performed over a period of 5 min. All the peptides were synthesized by methods described elsewhere [4]. The animals were perfused for histological procedures as described below 4 h after the ICV injection of peptide or vehicle. The plasmid containing the rat PRL cDNA cloned in the EcoRI site of pPS64 [14] was generously provided by D.R.A. Maurer. The 717 bp fragment was purified on a 5% polyacrylamide gel and electro-eluted with an electroeluter (model 422; Bio-Rad, Richmond, CA, USA). This fragment was then labeled with [a-35S]dCTP as (1000 Ci/ mmol) (Amersham, IL, USA) by the random primer method [3] to a specific activity ranging between 0.8 and 1.2 X 109 dpm//~g. Pituitaries were fixed by vascular perfusion with 200 ml of 4% (w/v) paraformaldehyde in 0.1 M phos-
0304-3940/95/$09.50 © 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0 3 0 4 - 3 9 4 0 ( 9 5 ) 1 1505-N
78
E. Garcia de Yebenes et al. / Neuroscience Letters 190 (1995) 77-80
A
B
C
D
Fig. 1. Typical X-ray autoradiographs showing the hybridization signal obtained in pituitary sections following in situ hybridization with the 35Slabeled PRL probe. The reaction is restricted to the anterior lobe (A). Vehicle: (B) NPY; (C) NPY13_36; (D) [Leu31, Pro34]NPY. NIL, neurointermediate lobe.
phate buffer (pH 7.4). They were serially cut with a cryostat into 6/zm sections. In situ hybridization was performed as described previously [14]. After hybridization, the sections were exposed for autoradiography with Kodak X-Omat films for 2 days before being coated with photographic emulsion (Kodak NTB-2). After 10 days of exposure, sections were processed and stained with hematoxylin and eosin. As control, the sections from each group were treated with pancreatic RNase A (20/~g/ml) (Boehringer Mannheim) for 30 min at room temperature before prehybridization. Densitometric measurements of autoradiographs of the whole anterior pituitary tissue were obtained with a digitized Amersham's RAS image analysis system as previously described [14]. Twenty-five pituitary sections per animal were analyzed. Data are expressed as means _+ SEM. Statistical significance was determined according to the multiple-range test of Duncan-Kramer [ 10]. The autoradiographic signal obtained following hybridization with the PRL probe was rather uniform throughout the anterior lobe of the pituitary gland (Fig. 1A). The neurointermediate lobe remained completely unlabeled. Pretreatment of sections with RNase completely prevented labeling with the [35S]PRL cDNA probe (data not shown). Moreover, we have previously shown that the signal was completely abolished when the hybridization was performed in the presence of an excess of unlabeled probe [14]. The effects of the ICV acute administration of NPY, PYY, NPY13_36 and [Leu31, Pro34]NPY are presented in Figs. 1 and 2. NPY, PYY, and NPY13_36 induced an increase of 40, 40.5, and 21.7%, respectively, in the hybridization signal. The administration of the Y1 NPY re-
ceptor agonist [Leu31, Pro34]NPY did not modify PRL mRNA levels. Observation of the sections at the light microscopic level also revealed that NPY, PYY, and NPYI3-36 increased the number of silver grains overlying mammotrophs, while [Leu31, Pro34]NPY had no effect (Fig. 3). In the present study, quantitative in situ hybridization was used to evaluate the effects of NPY and related peptides on the levels of PRL mRNA in the male rat pituitary. The results obtained clearly establish for the first time that NPY is involved in the positive regulation of
-T-
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I / / / /
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40
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20
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Fig. 2. Effect of the administration of NPY, PYY, NPY13_36 and [Leu31, Pro34]NPY on PRL mRNA levels. ***P < 0.001, vehicletreated versus all the other experimental groups.
E. Garcia de Yebenes et al. I Neuroscience Letters 190 (1995) 77-80
79
Fig. 3. Typical light microscope autoradiographs illustrating the distribution of silver grains overlying lactotrophs. (A) Vehicle; (B) NPY; (C) NPY13_ 36; (D) [Leu31,Pro341NPY. X800.
PRL gene expression. They are in agreement with our previous data showing that the intravenous injection of NPY produced within 60 min a fourfold increase in PRL plasma levels [9] and also with previous reports [6,7] demonstrating that ICV injection of NPY induced a stimulatory action on PRL release in the male rat. With the intravenous method, it was not possible to determine whether NPY was acting at the pituitary or hypothalamic level such as the median eminence which lacks a bloodbrain barrier. In the present work and in previous studies [6,7], since NPY was injected into the lateral ventricle, it is very likely that the peptide exerts its action on PRL at a central level, although it cannot be totally excluded that small amounts of peptides injected could eventually reach the anterior pituitary gland. It has been reported by few groups which used hemipituitary fragments or long-term cultures that NPY did not modify PRL release [1,9,13]. On the other hand, there is one report indicating a stimulatory effect of NPY on PRL release in pituitary cells in culture [1]. Although this discrepancy cannot be ex-
plained, the absence of NPY receptors in the anterior pituitary [2] makes it unlikely that NPY could exert a direct effect on mammotrophs. The results obtained with the different agonists used strongly suggest that the Y2 NPY receptor subtype mediates the stimulatory effect of NPY on PRL gene expression. This inference is on the basis that the positive influence of NPY was mimicked by PYY and NPY13_36, and not by [LeUal, Pro34]NPY1, a preferential agonist at Y1 receptor. The high degree of activity of PYY eliminates the possibility that the Y3 NPY receptor subtype might be involved in the regulation of PRL gene expression by NPY. The present studies do not provide precise information on the mechanism of action of NPY on PRL secretion. The presence of Y2 NPY receptor subtypes in the hypothalamus [2] suggests that NPY might exert some influence on hypothalamic nuclei. Since NPY has been shown to positively regulate tuberoinfundibular dopaminergic (TIDA) neurons by activating the Yl NPY receptor sub-
80
E. Garcia de Yebenes et al. / Neuroscience Letters 190 (1995) 77-80
type [8], it is u n l i k e l y that the action of N Y P on PRL secretion could be m e d i a t e d b y an action of the peptide on T I D A neurons. O n the other hand, it c a n n o t be excluded that N P Y could regulate the release o f d o p a m i n e by free nerve e n d i n g s in the m e d i a n e m i n e n c e . In fact, it has been s h o w n that NPYt3_36 could reduce d o p a m i n e utilization in the external zone o f the m e d i a n e m i n e n c e [6,7]. In conclusion, it appears that activation o f the Y2 receptor s u b t y p e c a n stimulate not only the release [6,7,9] but also the biosynthesis o f P R L as evaluated by m R N A level m e a s u r e m e n t s . T h e precise m e c h a n i s m o f action of N P Y r e m a i n s to be elucidated. [1] Cmwley, W.R. and Kalra, S.P., Neuropeptide Y stimulates the release of luteinizing hormone-releasing hormone from medial basal hypothalamus in vitro: modulation by ovarian hormones, Neuroendocrinology, 46 (1987) 97-103. [2] Dumont, Y., Foumier, A., St-Pierre, S., Schwartz, T.W. and Quirion, R., Differential distribution of neuropeptide Y1 and Y2 receptors in the rat brain, Eur. J. Pharmacol., 191 0990) 501-503. [3] Feinberg, A.P. and Vogelstein, B., A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity, Anal. Biochem., 132 (1983) 6-13. [4] Forest, M. and Fournier, A., BOP reagent for the coupling of pGlu and Boc-His(Tos) in solid phase peptide synthesis, Int. J. Peptide Protein Res., 35 (1990) 89-94. [5] Fuhlendofff, J., Gether, U., Aakerlund, L., Langeland-Johansen, N., Thogersen, H., Melberg, S., Olsen, U.B., Thastrup, O. and Schwartz, T.W., [Leu31Pro34] neuropeptide Y: a specific YI receptor agonist, Proc. Natl. Acad. Sci. USA, 87 (1990) 182186. [6] Hiirfstrand, A., Eneroth, P., Agnati, L.F. and Fuxe, K., Further studies on the effects of central administration of neuropeptide Y on neuroendocrine function in the male rat: relationship to hypothalamic catecholamines, Regul. Pept., 17 (1987) 167-169.
[7] H~'fstrand, A., Fuxe, K., Agnati, L.F., Eneroth, P., Zini, I., Zoli, M., Andersson, K., Euler, G.v., Terenius, L., Mutt, V. and Goldstein, M., Studies on neuropeptide Y-catecholamine interactions in the hypothalamus and in the forebrain of the male rat. Relationship to neuroendocrine function, Neurochem. Int., 8 (1986) 355376. [8] Hong, M., Li, S. and Pelletier, G., Role of neuropeptide Y in the regulation of tyrosine hydroxylase ribonucleic acid levels in the male rat areuate nucleus as evaluated by in situ hybridization, J. Neuroendocrinol., 7 (1995) 25-28. [9] Kerkedan, L., Guy, J., Lef~vre, G. and Pelletier, G., Effects of neuropeptide Y (NPY) on the release of anterior pituitary hormones in the rat, Peptides, 6 (1985) 1201-1204. [10] Kramer, C.Y., Extension of multiple range tests to group means with unique numbers of replications, Biometrics, 12 (1956) 307310. [11] McCann, S.M., Rettoni, V., Milenkovic, L., Riedel, M., Aguila, C. and McDonald, LK., The role of neuropeptide Y (NPY) in the control of anterior pituitary hormone release in the rat. In K. Mutt, K. Fuxe, A. Hokfelt and J.M. Lundberg (Eds.), Neuropepfide Y. Karolinska Institute Nobel Conference Series, Raven Press, New York, 1989, pp. 215-220. [12] McDonald, J.K. and Koenig, J.I., Neuropeptide Y actions on reproductive and endocrine functions. In W.F. Colmers and C. Wahlestedt (Eds.), The Biology of Neuropeptide Y and Related Peptides, Humana Press, Totowa, NJ, 1993, pp. 419-456. [13] Rodriguez-Sierra, J.F., Jacobowitz, D.M. and Bloke, C.A., Effects of neuropeptide Y on LH, FSH, and TSH release in male rats, Peptides, 8 (1987) 539-542. [14] Tong, Y., Simard, J., Labrie, C., Zhao, H.F., Labrie, F. and Pelletier, G., Inhibitory effect of androgen on estrogen-induced prolactin messenger ribonucleic acid accumulation in the male rat anterior pituitary gland, Endocrinology, 125 (1989) 1821-1828. [15] Wahlestedt, C., Grundemar, L., Hakanson, R., Heilig, M., Sben, G.H., Zukowska-Grojec, Z. and Reis, D.L, Neuropeptide Y receptor subtypes Yl and Y2, Ann. N. Y. Acad. Sci., 611 (1990) 7-26.
Neuroscience Letters 190 (1995)77-80
UW[lg[ tm[B8
Involvement of the Y2 receptor subtype in the regulation of prolactin gene expression by neuropeptide Y in the male rat Ester Garcia de Yebenes a, Songyun Li a, Alain Fournier b, Serge St-Pierre b, Georges Pelletiera,* aMRC Group in Molecular Endocrinology, CHUL Research Center and Laval University, 2705 Laurier Boulevard, Quebec, GI V 4G2, Canada blNRS-Sant#, Montreal, Canada Received 20 February 1995; revised version received 22 March 1995; accepted 22 March 1995
Abstract
In order to determine the influence of neuropeptide Y (NPY) on the biosynthesis of prolactin (PRL), we have studied the effects of NPY and some NPY analogs on PRL gene expression in the male rat anterior pituitary gland. The following peptides (4/zg/100 g body w0: NPY, peptide YY (PYY), NPYI3_36 (a Y2 receptor agnnist) and [Leu31,Pro34]NPY (a YI receptor agonis0 were injected into the left lateral ventricle of adult male rats. Control animals received only the vehicle (0.9% NaCI). All the animals were perfused with 4% paraformaldehyde 4 h after injection and processed for in situ hybridization. The intracerebroventricular injection of NPY, PYY, and NPYI3_36 induced a significant increase in the hybridization signal (22--40% over control). On the other hand, the Yl receptor agonist [Leu31, Pro34]NPY did not influence PRL mRNA levels. These data then suggest that activation of the Y2 NPY receptor subtype at the central level can positively regulate PRL gene expression. Keywords: Prolactin; Neuropeptide Y; Y2 receptor; In situ hybridization; Anterior pituitary
There is now compelling evidence that neuropeptide Y (NPY) is involved in the regulation of reproductive function (for review see Ref. [12]). For example, it has been clearly shown that NPY plays a significant role in the control of gonadotropin secretion [12]. So far, there have been very few studies about the effect of NPY on prolactin (PRL) secretion. Harfstrand et al. [6,7] have shown that intracerebroventricular (ICV) injection of NPY increased serum plasma levels in the male rat. We have also reported that in male rats intravenous injection of NPY induced a fourfold elevation in plasma levels of prolactin [9]. Immunoneutralization of brain NPY was shown to decrease prolactin plasma levels [11], suggesting physiological involvement of the NPYergic system in the regulation of prolactin secretion. In order to improve our knowledge on the role of NPY in the regulation of PRL secretion, we have studied the effect of NPY ICV administration on PRL gene expression. Different NPY analogs were also used to identify which NPY receptor subtype(s) might be involved in the control of PRL secretion. * Corresponding author, Tel.: +1 418 6542296; Fax: +1 418 6542761.
Groups (6 animals/group) of adult male rats weighing 200-225 g received into the left lateral ventricle a 10/zl injection of one of the following peptides (4/zg/100 g body wt): PYY, NPY, the preferential agonist at the Y1 receptor [Leu31, Pro34]NpY and the preferential agonist at the Y2 receptor NPY13_36 [2,5,15]. One group of animals also received a 10/zl injection of the vehicle (0.9% NaC1). The injection was performed over a period of 5 min. All the peptides were synthesized by methods described elsewhere [4]. The animals were perfused for histological procedures as described below 4 h after the ICV injection of peptide or vehicle. The plasmid containing the rat PRL cDNA cloned in the EcoRI site of pPS64 [14] was generously provided by D.R.A. Maurer. The 717 bp fragment was purified on a 5% polyacrylamide gel and electro-eluted with an electroeluter (model 422; Bio-Rad, Richmond, CA, USA). This fragment was then labeled with [a-35S]dCTP as (1000 Ci/ mmol) (Amersham, IL, USA) by the random primer method [3] to a specific activity ranging between 0.8 and 1.2 X 109 dpm//~g. Pituitaries were fixed by vascular perfusion with 200 ml of 4% (w/v) paraformaldehyde in 0.1 M phos-
0304-3940/95/$09.50 © 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0 3 0 4 - 3 9 4 0 ( 9 5 ) 1 1505-N
78
E. Garcia de Yebenes et al. / Neuroscience Letters 190 (1995) 77-80
A
B
C
D
Fig. 1. Typical X-ray autoradiographs showing the hybridization signal obtained in pituitary sections following in situ hybridization with the 35Slabeled PRL probe. The reaction is restricted to the anterior lobe (A). Vehicle: (B) NPY; (C) NPY13_36; (D) [Leu31, Pro34]NPY. NIL, neurointermediate lobe.
phate buffer (pH 7.4). They were serially cut with a cryostat into 6/zm sections. In situ hybridization was performed as described previously [14]. After hybridization, the sections were exposed for autoradiography with Kodak X-Omat films for 2 days before being coated with photographic emulsion (Kodak NTB-2). After 10 days of exposure, sections were processed and stained with hematoxylin and eosin. As control, the sections from each group were treated with pancreatic RNase A (20/~g/ml) (Boehringer Mannheim) for 30 min at room temperature before prehybridization. Densitometric measurements of autoradiographs of the whole anterior pituitary tissue were obtained with a digitized Amersham's RAS image analysis system as previously described [14]. Twenty-five pituitary sections per animal were analyzed. Data are expressed as means _+ SEM. Statistical significance was determined according to the multiple-range test of Duncan-Kramer [ 10]. The autoradiographic signal obtained following hybridization with the PRL probe was rather uniform throughout the anterior lobe of the pituitary gland (Fig. 1A). The neurointermediate lobe remained completely unlabeled. Pretreatment of sections with RNase completely prevented labeling with the [35S]PRL cDNA probe (data not shown). Moreover, we have previously shown that the signal was completely abolished when the hybridization was performed in the presence of an excess of unlabeled probe [14]. The effects of the ICV acute administration of NPY, PYY, NPY13_36 and [Leu31, Pro34]NPY are presented in Figs. 1 and 2. NPY, PYY, and NPY13_36 induced an increase of 40, 40.5, and 21.7%, respectively, in the hybridization signal. The administration of the Y1 NPY re-
ceptor agonist [Leu31, Pro34]NPY did not modify PRL mRNA levels. Observation of the sections at the light microscopic level also revealed that NPY, PYY, and NPYI3-36 increased the number of silver grains overlying mammotrophs, while [Leu31, Pro34]NPY had no effect (Fig. 3). In the present study, quantitative in situ hybridization was used to evaluate the effects of NPY and related peptides on the levels of PRL mRNA in the male rat pituitary. The results obtained clearly establish for the first time that NPY is involved in the positive regulation of
-T-
////~
IO0
/ / / / /
/////
- 801 -7--
i!,o
I / / / /
/////
/ / / / I
..,la
1
i i l l l i i i i i
40
i l l l i
\ k ~ k ~
//i/~
20
0
_u
I I I 1 ~ I I I I I
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IIIII
\\\\~
I I I I I
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Fig. 2. Effect of the administration of NPY, PYY, NPY13_36 and [Leu31, Pro34]NPY on PRL mRNA levels. ***P < 0.001, vehicletreated versus all the other experimental groups.
E. Garcia de Yebenes et al. I Neuroscience Letters 190 (1995) 77-80
79
Fig. 3. Typical light microscope autoradiographs illustrating the distribution of silver grains overlying lactotrophs. (A) Vehicle; (B) NPY; (C) NPY13_ 36; (D) [Leu31,Pro341NPY. X800.
PRL gene expression. They are in agreement with our previous data showing that the intravenous injection of NPY produced within 60 min a fourfold increase in PRL plasma levels [9] and also with previous reports [6,7] demonstrating that ICV injection of NPY induced a stimulatory action on PRL release in the male rat. With the intravenous method, it was not possible to determine whether NPY was acting at the pituitary or hypothalamic level such as the median eminence which lacks a bloodbrain barrier. In the present work and in previous studies [6,7], since NPY was injected into the lateral ventricle, it is very likely that the peptide exerts its action on PRL at a central level, although it cannot be totally excluded that small amounts of peptides injected could eventually reach the anterior pituitary gland. It has been reported by few groups which used hemipituitary fragments or long-term cultures that NPY did not modify PRL release [1,9,13]. On the other hand, there is one report indicating a stimulatory effect of NPY on PRL release in pituitary cells in culture [1]. Although this discrepancy cannot be ex-
plained, the absence of NPY receptors in the anterior pituitary [2] makes it unlikely that NPY could exert a direct effect on mammotrophs. The results obtained with the different agonists used strongly suggest that the Y2 NPY receptor subtype mediates the stimulatory effect of NPY on PRL gene expression. This inference is on the basis that the positive influence of NPY was mimicked by PYY and NPY13_36, and not by [LeUal, Pro34]NPY1, a preferential agonist at Y1 receptor. The high degree of activity of PYY eliminates the possibility that the Y3 NPY receptor subtype might be involved in the regulation of PRL gene expression by NPY. The present studies do not provide precise information on the mechanism of action of NPY on PRL secretion. The presence of Y2 NPY receptor subtypes in the hypothalamus [2] suggests that NPY might exert some influence on hypothalamic nuclei. Since NPY has been shown to positively regulate tuberoinfundibular dopaminergic (TIDA) neurons by activating the Yl NPY receptor sub-
80
E. Garcia de Yebenes et al. / Neuroscience Letters 190 (1995) 77-80
type [8], it is u n l i k e l y that the action of N Y P on PRL secretion could be m e d i a t e d b y an action of the peptide on T I D A neurons. O n the other hand, it c a n n o t be excluded that N P Y could regulate the release o f d o p a m i n e by free nerve e n d i n g s in the m e d i a n e m i n e n c e . In fact, it has been s h o w n that NPYt3_36 could reduce d o p a m i n e utilization in the external zone o f the m e d i a n e m i n e n c e [6,7]. In conclusion, it appears that activation o f the Y2 receptor s u b t y p e c a n stimulate not only the release [6,7,9] but also the biosynthesis o f P R L as evaluated by m R N A level m e a s u r e m e n t s . T h e precise m e c h a n i s m o f action of N P Y r e m a i n s to be elucidated. [1] Cmwley, W.R. and Kalra, S.P., Neuropeptide Y stimulates the release of luteinizing hormone-releasing hormone from medial basal hypothalamus in vitro: modulation by ovarian hormones, Neuroendocrinology, 46 (1987) 97-103. [2] Dumont, Y., Foumier, A., St-Pierre, S., Schwartz, T.W. and Quirion, R., Differential distribution of neuropeptide Y1 and Y2 receptors in the rat brain, Eur. J. Pharmacol., 191 0990) 501-503. [3] Feinberg, A.P. and Vogelstein, B., A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity, Anal. Biochem., 132 (1983) 6-13. [4] Forest, M. and Fournier, A., BOP reagent for the coupling of pGlu and Boc-His(Tos) in solid phase peptide synthesis, Int. J. Peptide Protein Res., 35 (1990) 89-94. [5] Fuhlendofff, J., Gether, U., Aakerlund, L., Langeland-Johansen, N., Thogersen, H., Melberg, S., Olsen, U.B., Thastrup, O. and Schwartz, T.W., [Leu31Pro34] neuropeptide Y: a specific YI receptor agonist, Proc. Natl. Acad. Sci. USA, 87 (1990) 182186. [6] Hiirfstrand, A., Eneroth, P., Agnati, L.F. and Fuxe, K., Further studies on the effects of central administration of neuropeptide Y on neuroendocrine function in the male rat: relationship to hypothalamic catecholamines, Regul. Pept., 17 (1987) 167-169.
[7] H~'fstrand, A., Fuxe, K., Agnati, L.F., Eneroth, P., Zini, I., Zoli, M., Andersson, K., Euler, G.v., Terenius, L., Mutt, V. and Goldstein, M., Studies on neuropeptide Y-catecholamine interactions in the hypothalamus and in the forebrain of the male rat. Relationship to neuroendocrine function, Neurochem. Int., 8 (1986) 355376. [8] Hong, M., Li, S. and Pelletier, G., Role of neuropeptide Y in the regulation of tyrosine hydroxylase ribonucleic acid levels in the male rat areuate nucleus as evaluated by in situ hybridization, J. Neuroendocrinol., 7 (1995) 25-28. [9] Kerkedan, L., Guy, J., Lef~vre, G. and Pelletier, G., Effects of neuropeptide Y (NPY) on the release of anterior pituitary hormones in the rat, Peptides, 6 (1985) 1201-1204. [10] Kramer, C.Y., Extension of multiple range tests to group means with unique numbers of replications, Biometrics, 12 (1956) 307310. [11] McCann, S.M., Rettoni, V., Milenkovic, L., Riedel, M., Aguila, C. and McDonald, LK., The role of neuropeptide Y (NPY) in the control of anterior pituitary hormone release in the rat. In K. Mutt, K. Fuxe, A. Hokfelt and J.M. Lundberg (Eds.), Neuropepfide Y. Karolinska Institute Nobel Conference Series, Raven Press, New York, 1989, pp. 215-220. [12] McDonald, J.K. and Koenig, J.I., Neuropeptide Y actions on reproductive and endocrine functions. In W.F. Colmers and C. Wahlestedt (Eds.), The Biology of Neuropeptide Y and Related Peptides, Humana Press, Totowa, NJ, 1993, pp. 419-456. [13] Rodriguez-Sierra, J.F., Jacobowitz, D.M. and Bloke, C.A., Effects of neuropeptide Y on LH, FSH, and TSH release in male rats, Peptides, 8 (1987) 539-542. [14] Tong, Y., Simard, J., Labrie, C., Zhao, H.F., Labrie, F. and Pelletier, G., Inhibitory effect of androgen on estrogen-induced prolactin messenger ribonucleic acid accumulation in the male rat anterior pituitary gland, Endocrinology, 125 (1989) 1821-1828. [15] Wahlestedt, C., Grundemar, L., Hakanson, R., Heilig, M., Sben, G.H., Zukowska-Grojec, Z. and Reis, D.L, Neuropeptide Y receptor subtypes Yl and Y2, Ann. N. Y. Acad. Sci., 611 (1990) 7-26.