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Study of the effect of 26RF- and 43RF-amides on Testosterone and Prolactin secretion in the adult male rhesus monkey (Macaca mulatta)
Peptides 36 (2012) 23–28
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Study of the effect of 26RF- and 43RF-amides on Testosterone and Prolactin secretion in the adult male rhesus monkey (Macaca mulatta) Fazal Wahab a,b,1 , Hina Salahuddin a,1 , Mariam Anees a , Jerome Leprince c , Hubert Vaudry c , Manuel Tena-Sempere d , Muhammad Shahab a,∗ a
Laboratory of Reproductive Neuroendocrinology, Department of Animal Sciences, Faculty of Biological Sciences, Quaid-i-Azam University, 45320 Islamabad, Pakistan Department of Physiology, Institute of Basic Medical Sciences, Khyber Medical University, Peshawar, Khyber Pukhtunkhawa, Pakistan c INSERM U413, Laboratory of Cellular and Molecular Neuroendocrinology, European Institute for Peptide Research (IFRMP 23), University of Rouen, 76821 Mont-Saint-Aignan, France d Department of Cell Biology, Physiology, and Immunology, University of Cordoba, 14004 Cordoba, Spain b
a r t i c l e
i n f o
Article history: Received 2 March 2012 Received in revised form 10 April 2012 Accepted 10 April 2012 Available online 17 April 2012 Keyword: 26RF amide 43RF amide GnRH Testosterone Prolactin
a b s t r a c t RF-amides (RFa), a superfamily of evolutionary-conserved neuropeptides, are expressed in both invertebrates and vertebrates. While some endocrine functions have been attributed to these peptides in lower vertebrates and few mammalian models, not much is known about their actions in primates. Therefore, the present study was designed to examine the effects of peripheral administration of two recently cloned human RFa peptides, 26RFa and 43RFa, on testosterone and prolactin secretion in the adult male adult male rhesus monkey (Macaca mulatta). For control purposes, a scrambled sequence of 26RFa (Sc-26RFa) and normal saline (1 ml) were injected. Three different doses of 26RFa and 43RFa (19-nmol, 38-nmol and 76-nmol) and a single dose (38-nmol) of Sc-26RFa were tested. A set of four chair-restraint habituated monkeys was used. Comparison of post-treatment T levels with respective pre levels showed that none of the doses of both 26RFa and 43RFa changed T release. Similarly, Sc-26RFa and saline administration also did not affect T levels. In contrast, all doses of 26RFa and 43RFa significantly (P < 0.05) stimulated prolactin secretion. 43RFa dose dependently increased prolactin secretion while dose dependency was not observed for 26RFa. Saline and Sc-26RFa injection had no effect on prolactin concentrations. Thus, present study demonstrated that peripheral administration of 26RFa and 43RFa, in the doses tested, have no effect on T secretion, suggesting possible selective lack of their neuroendocrine role in controlling hypothalamic–pituitary–gonadal axis in the adult male primates. The prominent stimulation of prolactin suggests a neuroendocrine role of RFa peptides in regulation of prolactin release in primates. © 2012 Elsevier Inc. All rights reserved.
1. Introduction Adenohypophysis serves as a relay center between the brain and peripheral endocrine organs, and hence it play critical role in the homeostatic regulation of such vital processes as metabolism, growth, reproduction and behavior [29]. The adenohypophysis is functionally linked to the hypothalamus, a critical neuroendocrine regulator area of the brain [4,23]. The hypothalamus secretes a number of peptides that reach the adenohypophysis via hypophysial portal vein and affect its functioning [9,23]. In addition
∗ Corresponding author at: Reproductive Neuroendocrinology Laboratory, Department of Animal Sciences, Faculty of Biological Sciences, Quaid-i-Azam University, 45320 Islamabad, Pakistan. Tel.: +92 51 90643014; fax: +92 51 2601176. E-mail address: [email protected] (M. Shahab). 1 These authors are contributed equally to this work. 0196-9781/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.peptides.2012.04.009
to classical hypothalamic releasing and inhibiting factors, adenohypophysis function is also regulated, directly or indirectly, by a number of other hypothalamic peptides. In recent decades, a large series of peptides with a distinctive Arg–Phe–NH2 motif at the C-terminus has been identified, and are grouped as the RF-amide (RFa) peptide superfamily [5]. In mammals, major RFa peptides are the neuropeptides FF and AF, prolactin-releasing peptide (PrRP), metastin and other kisspeptins encoded by the Kiss1 gene, as well as RF-related peptides, RFRP-1 and RFRP-3 [10–12,14,19,30,37]. More recently, a 26-amino acids RFa peptide, first isolated in the frog and named 26RFa, has also been cloned in some mammalian species [6]. This peptide is the ligand for a previously orphan G protein coupled receptor, GPR103. The GPR103 couples to Gi/o and Gq signaling pathway and it leads to rise in intracellular calcium concentrations and decrease of cAMP production in CHO-GPR103 transfected cells [8]. The Nelongated form of 26RFa, 43RFa, has the same efficacy for binding
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and activation of GPR103 [8]. Within the brain, 26RFa appears to be mainly expressed in different hypothalamic nuclei, such as the ventromedial hypothalamic nucleus and lateral hypothalamus [8]. However, 26RFa and GPR103 genes expression has also been shown in other brain areas and peripheral tissues [13]. Various physiological roles have been assigned to these peptides in various species [18,20,36]. Recently, some preliminary evidences showed the effect of 26RFa and 43RFa on gonadotropin and prolactin (PRL) secretion in rats [18]. In adult cyclic female rat, both central and peripheral administration of 26RFa and 43RFa stimulated gonadotropin secretion. In adult male rats, central as well as peripheral administration of 26RFa inhibited PRL secretion while had no effect on the gonadotropin release [18]. Later, these original findings were confirmed by the study of Patel and colleagues [20], who observed that central injection of 43RFa stimulated LH release, while peripheral administration had no effect. Until now, to the best of our knowledge, in non-rodent mammals especially in primates, the neuroendocrine role of 26RFa and 43RFa is not known at all. Therefore, we considered it relevant to explore the potential role of 26RFa and 43RFa in the regulation of the HPG axis and PRL secretion in the adult male rhesus monkey, a representative higher primate. Due to unavailability of proper facilities for measurements of the rhesus monkey LH, plasma testosterone concentration was measured as terminal marker of the HPG axis.
2. Materials and methods 2.1. Animals Four adult intact male rhesus monkeys (Macaca mulatta) were used in this study. Monkeys ranged in age from 7–8 years and weighed between 6.0 and 9.0 kg. The animals were housed in individual cages, under semi controlled colony conditions (lights on, 0700–1900 h; temperature, 22 ◦ C). The animals were fed with monkey food at 1300–1330 h daily and supplemented with fresh fruits and vegetables in the morning (0900–0930 h). Water was available ad libitum. These animals were chair-restrained habituated and housed in Department’s primate facility as described earlier [31,33–35]. All experimental protocols of the present study were approved by the Departmental Committee for Care and Use of Laboratory Animals. 2.2. Catheterization Catheterization of experimental animals was carried out as documented previously [31,33–35]. Briefly the animals were anesthetized with ketamine hydrochloride (10 mg/kg BW, i.m.), and a teflon cannula (Vasocan Branule, B. Braun Melsungen AG, Belgium; 0.8 mm/22 G O.D) was inserted in the saphenous vein. The open end of the cannula was attached to a syringe via a butterfly tubing (20 G diameter and 300 mm length). Cannulation was carried out 30 min before initiation of blood sampling and then animals were restrained to the chair. This route was used for both blood samples collection and i.v. injection of RFa and vehicle (normal saline). Blood sampling was carried out when the animals had fully regained consciousness. 2.3. Blood sampling Blood samples (2 ml) were taken in heparinized syringes and immediately transferred to culture tubes kept on ice. After completion of sampling, tubes were centrifuged at 3000 rpm at 4 ◦ C, plasma was extracted and stored at −15 ◦ C until assayed for hormones analysis. After each blood sample, equal volume of normal
saline, containing 5 IU of heparin, was injected to compensate the lost blood volume and to prevent blood clotting in the cannula. 2.4. Reagents Heparin (Rotexmedica, Trittau, Germany), GnRH (Sigma Chemical Company, St. Louis, MO, USA) and ketamine were purchased locally. Human RFa peptides (26RFa, 43RFa and Sc-26RFa, a scrambled amino acid sequence of 26RFa) were synthesized in the lab of one of the authors (HV) as described before [6]. Working solutions of RFa were made in normal saline. 2.5. General experimental design Actual experiment comprised of 8 non-consecutive days of blood sampling. All four animals were used on each day of sampling. A total of 20 blood samples were taken from each animal on each day of sampling. Sampling duration was about 5 h (1100–1800 h). Samples were obtained at 15-min intervals for 30 min before injections (−30, −15 and 0 min) and for 240 min thereafter. A bolus i.v. injection of GnRH (1 g) was given after 240 min to assess responsiveness of pituitary–testicular axis. RF amides or normal saline as vehicle was administered as i.v. bolus immediately after taking 0 min sample. A scrambled sequence of 26RFa was used as a specific control to 26RFa. Samplings were carried out from 14th December 2006 to 1st February 2007. During this period all 4 animals were taken for 8 sampling occasions with a gap of 4 days. Each animal received treatments in following order: 1. 2. 3. 4. 5. 6. 7. 8.
1 ml normal saline (0.9% NaCl) 38-nmol Sc 26RFa 19-nmol of 26RFa 38-nmol of 26RFa 76-nmol of 26RFa 19-nmol of 43RFa 38-nmol of 43RFa 76-nmol of 43RFa
Doses of amides were estimated from the neuroendocrine effective doses of kisspeptin in monkeys [26]. 2.6. Hormones assays Plasma testosterone (T) concentrations were determined by using solid phase competitive RIAs. The T RIA kits were purchased from Immunotech Marselle Cedex 9, France. The RIAs were performed as per the manufacturer’s instructions. The sensitivity of the T assay was 0.025 ng/ml and intra- and inter-assay coefficients of variation (CV) were both below 10%. Changes in plasma PRL levels were monitored using a human PRL ELISA kit (Accu Bind ELISA Microwells, Monobind Inc, Lake Forest, CA, USA) following the instructions of the manufacturer. The analytical sensitivity of the kit was 0.01 ng/ml or 0.026 mIIU/L. Intra- and inter-assay % CV were 5.96% and 2.56%, respectively. 2.7. Statistical analyses In view of individual variations in hormone levels, percent changes with respect to time 0 min were calculated and such values at different time points were analyzed by repeated measure one-way ANOVA followed by Dennett’s test. Mean % changes after administration of RFa was also compared by repeated measure ANOVA. Statistical comparisons for the mean pre- and posttreatment hormones changes were also made by paired Student’s
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Fig. 1. (A) % change in mean plasma T concentration in a 4 h period (15–240 min) after i.v. administration of vehicle, Sc-26RFa and various doses of 26RFa in the adult male rhesus monkey (n = 4). GnRH administration at 240 min significantly stimulated (P < 0.05) plasma T levels in all animals. (B) Comparison of mean % change in plasma T concentration after injection of various doses of 26RFa, Sc 26RFa and vehicle (15–240 min) in the adult male monkeys (n = 4). All doses of 26RFa like vehicle and Sc-26RFa have no effect on the plasma T values.
Fig. 2. (A) % change in mean plasma T concentration in a 4 h period (15–240 min) after i.v. administration of vehicle and various doses of 43RFa in the adult male rhesus monkey. GnRH administration at 240 min significantly stimulated (P < 0.05) plasma T levels in all animals. (B) Comparison of mean % change in plasma T concentration after injection of various doses of 43RFa and vehicle (15–240 min) in the adult male monkeys. All doses of 26RFa like vehicle and Sc-26RFa have no effect on the plasma T values.
t tests. Based upon a shorter response of PRL secretion to administration of the RFa, changes in a selected window of 0–120 min were analyzed. All data are presented as mean (±SEM). Results were considered statistically significant at P < 0.05.
of vehicle, Sc-26RFa and 26RFa is shown in Fig. 3B. All doses of 26RFa significantly increased PRL levels as compared to vehicle and Sc-26RFa levels. Comparison of % change in mean plasma PRL levels after i.v. administration of vehicle and 43RFa is shown in Fig. 4B. 43RFa administration also significantly increased mean plasma PRL level. However, PRL secretion was not affected dose dependently by 26RFa as all doses increased PRL concentrations equally. In contrast, a dose dependency was evident with 43RFa where % changes in plasma PRL concentrations increased with dose.
3. Results 3.1. Effects of 26RFa and 43RFa on T secretion The pattern of % change in plasma T secretion before and after administration of 19-, 38- and 76-nmol doses of 26RFa and 43RFa in adult male monkeys is shown in Figs. 1A and 2A, respectively. Comparison of mean pre- and post-treatment T levels or with respect to vehicle and Sc-26RFa treatments levels revealed that no dose of 26RFa altered T secretion (Fig. 1B). Likewise, no dose of 43RFa changed T secretion (Fig. 2B). In contrast, GnRH administration at end of sampling period significantly stimulated T secretion. 3.2. Effects of 26RFa and 43RFa on PRL secretion The pattern of % change in plasma PRL secretion before and after administration of 19-, 38-, 76-nmol doses 26RFa and 43RFa is shown in Figs. 3A and 4A, respectively. Comparison of % change in mean plasma PRL concentration after peripheral administration
4. Discussion In the present study, we examined the effect of peripheral administration of two human RFa peptides, 26RFa and its elongated form, 43RFa, on the T and PRL secretion in the adult male rhesus monkey. The signal finding of the present study was that i.v. administration of both the 26RFa and 43RFa in doses utilized did not affect plasma T levels. Although due to lack of unavailability of monkey LH in our lab, we could not measure LH, it was likely that absent T response to RFa in present study was due to unresponsiveness of the GnRH-LH to RFa. GnRH administration at the end of the sampling caused a robust stimulation of plasma T secretion, suggesting that the HPG axis was responsive. However, injections of both the
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Fig. 3. (A) % change in mean plasma PRL concentration in a 2 h period (15–120 min) after i.v. administration of vehicle, Sc-26RFa and various doses of 26RFa in the adult male rhesus monkey. Repeated measured ANOVA showed that administration of 38 and 76 nmol doses significantly increased % change in PRL secretion at 30, 105, and 120 min, and at 60–120 min, respectively. (B) Comparison of mean % change in plasma PRL concentration after injection of various doses of 26RFa, Sc 26RFa and Saline (15–120 min) in the adult male monkeys. Post-26RFa mean change was greater to respective pre (a P < 0.05–005) values. All doses of 26RFa significantly stimulated % PRL change as compared to vehicle- and Sc-26RFa (* P < 0.05) values.
Fig. 4. (A) % change in mean plasma PRL concentration in a 2 h period (15–120 min) after i.v. administration of vehicle and various doses of 43RFa in the adult male rhesus monkey. % change in PRL secretion was significantly increased by 19 nmol (at 75–120 min), 38 nmol (at 60–120 min) and 76 nmol (at 45–120 min) doses of 43RFa. (B) Comparison of mean % change in plasma PRL concentration after injection of various doses of 43RFa and vehicle (15–120 min) in the adult male monkeys. Post-26RFa mean change was greater than the respective pre (a P < 0.05–005) values. All doses of 43RFa significantly stimulated % PRL change as compared to vehicle (b P < 0.05–005) values.
peptides caused an acute rise in plasma PRL levels. Together, these findings suggest that peripheral administration of both the 26RFa and 43RFa, unlike kisspeptins, had no effect on the T release in male rhesus monkeys. Our findings are consistent with a previous study in rodents which demonstrated that both the central and systemic injections of the RFa had no effect on the gonadotropin secretion in the adult male rat [18]. Similarly, another recent study showed that peripheral administration of 43RFa was unable to stimulate LH secretion but central administration of relatively high doses of 43RFa did significantly stimulate LH secretion in the adult male rat [20]. Therefore, our findings in the male rhesus monkey regarding effect of systemic RFa on T secretion are consistent with the observations in the male rat. The exact reasons for lack of the effect of the RFa on the T secretion in monkeys are not clear. It is possible that the doses of the RFa employed were low but as same doses modulated PRL secretion, the notion is less likely. Other possibility is that the primate HPG axis may be less sensitive to 26RFa and 43RFa than the hypothalamic–lactotroph axis. Therefore, the current findings did not rule out possibility of the effect of higher doses. This idea may not be convincing in view of the fact that the doses of RFa were
equimolar with effective doses of kisspeptin [26,31]. There is also possibility that RFa may act only through the hypothalamic receptors located proximal to the blood–brain-barrier and the systemic administration may not be able to reach there. This possibility is supported by some recent observations in the rats that central but not peripheral administration of 43RFa stimulated LH secretion [20]. However, other members of the RFa family especially kisspeptin is able to cross the blood–brain-barrier and activate the hypothalamic GPR54, receptor of kisspeptin [26]. In summary, we conclude with caution that in adult male non-human primates, systemic RF amides may not affect the HPG axis. Recently, 26RFa have been implicated in the regulation of PRL in rat. It led to decrease in the secretion of PRL [18]. In order to check whether lack of the effect of 26RFa and 43RFA was specific to the HPG axis in primates or to other hypothalamic–pituitary axes also, we monitored the effect of the 26- and 43-RFa on the PRL release. Systemic administration of both 26RFa and 43RFa led to an increase in the release of PRL. Our present study reports, for the first time, specific stimulatory action of RF amides on PRL secretion in higher primates. However, PRL secretion was not affected
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dose dependently by 26RFa as all doses increased PRL concentrations equally. In contrast, a dose dependency was evident with 43RFa where mean post-treatment PRL concentration appeared to rise with increase in the dose. The observed PRL stimulatory effect of 26RFa and 43RFa are in line with established PRL augmenting actions of PrRP [16], which is also a RF amide peptide. Our findings in the monkeys, however, are contradictory to observations recently made in rats. Central administration of 26RFa was observed to cause a rapid decrease in PRL levels in adult male rats and diestrous female rats [18]. Besides species differences, this discrepancy appears to be related to different route of administration of RFa utilized in our study and the rat study [18]. Our results are similar to observations made in fish. PRLreleasing peptide (PrRP) seems to be an essential stimulator of PRL transcription and secretion in teleost pituitary and peripheral organs [22]. Homologue of mammalian PrRP in salmon has also been shown to stimulate PRL release from pituitary both in salmon and tilapia [17,21,25]. Interestingly, other RFa peptides such as kisspeptins and RFRP1/GnIH have been demonstrated as important regulators of the secretion of different pituitary hormones, such as gonadotropins and/or PRL [7,10,11,24,32]. Our results, therefore, advance understanding of RFa peptides actions in primates. However, design of the current study did not allow inferring much whether our results indicated pharmacological action or physiological action of 26RFa/43RFa. Further experiments are required to asses if these peptides are involved in the physiological regulation of PRL secretion in the primates. The mechanism underlying the observed stimulatory effect of 26RFa and 43RFa on PRL secretion in monkeys remains to be determined. There are 3 possible ways regarding the mechanism of action of 26RFa in monkeys. One possibility is that peripherally administered RFa cross blood–brain-barrier and activate GPR103 receptors in periventricular nucleus leading to TRH release which would then entrain PRL secretion. Similarly, second possibility is that peripheral RFa cause activation of GPR103 in paraventricular nucleus (PVN) leading to inhibition of dopamine neurons, which would then cause increase in PRL secretion. Regarding hypothalamic site of action of 26RFa, GPR103 mRNA has been detected in PVN of human and in periventricular nucleus of mouse [27]. Recently, novel binding sites for 26RFa have been demonstrated in the PVN of rats [3]. It is relevant to mention here that TRH, which is a known PRL secretagogue, neurons are also present in the periventricular nucleus [15]. Furthermore, tuberoinfundibular dopaminergic neurons, which are primary regulators of PRL secretion, are present in the neighboring PVN [1]. It is, therefore, apparent that GPR103 expression is present in vicinity of dopamine neurons as well as in areas adjoining the location where TRH neurons are present. Parenthetically, 26RFa immunoreactive fibers have also been demonstrated in human PVN [2]. Moreover, immunohistochemical labeling using specific antibodies against human 26RFa and in situ hybridization histochemistry revealed that in the human hypothalamus 26RFa-expressing neurons are located in the PVN and ventromedial nuclei [2]. These observations support the assumption that RFa can act on dopamine and TRH neurons. It can be speculated that 26RFa causes inhibition of dopamine neurons or stimulation of TRH neurons in order to induce release of PRL. Third possibility is the direct action of the amides on pituitary GPR103 to increase PRL release. Recently, GPR103 expression has been shown in rodent’s pituitary throughout the postnatal development though the cellular localization of the receptor was not identified [18]. Furthermore, a mammalian homologue of PrRP has been shown to stimulate PRL release from fish pituitary [17,21,25,28]. Together, latter observations support the possibility of the direct action of RFa in increasing PRL release in monkeys.
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In summary, present data indicate, for the first time, that 26RFa and 43RFa administration does not affect HPG axis but stimulate PRL secretion in adult male rhesus monkey, suggesting a potential role of the evolutionary conserved RFa in the central or pituitary mechanisms controlling PRL release in higher primates. Disclosure Authors have nothing to disclose. Acknowledgements The work presented here was funded by the Higher Education Commission (HEC), Islamabad, Pakistan. References [1] Ben-Jonathan N. Dopamine: a prolactin-inhibiting hormone. Endocr Rev 1985;6:564–89. [2] Bruzzone F, Lectez B, Tollemer HL, Leprince JR, Dujardin C, Rachidi W, et al. Anatomical distribution and biochemical characterization of the novel RFamide peptide 26RFa in the human hypothalamus and spinal cord. J Neurochem 2006;99:616–27. [3] Bruzzone F, Lectez B, Alexandre D, Jegou S, Mounien L, Tollemer H, et al. Distribution of 26RFa binding sites and GPR103 mRNA in the central nervous system of the rat. J Comp Neurol 2007;503:573–91. [4] Charlton H. Hypothalamic control of anterior pituitary function: a history. J Neuroendocrinol 2008;20:641–6. [5] Chartrel N, Dujardin C, Leprince J, Desrues L, Tonon MC, Cellier E, et al. Isolation, characterization, and distribution of a novel neuropeptide, Rana RFamide (RRFa), in the brain of the European green frog Rana esculenta. J Comp Neurol 2002;448:111–27. [6] Chartrel N, Dujardin C, Anouar Y, Leprince J, Decker A, Clerens S, et al. Identification of 26RFa, a hypothalamic neuropeptide of the RFamide peptide family with orexigenic activity. Proc Natl Acad Sci USA 2003;100:15247–52. [7] Colledge WH. GPR54 and puberty. Trends Endocrinol Metab 2004;15:448–53. [8] Fukusumi S, Yoshida H, Fujii R, Maruyama M, Komatsu H, Habata Y, et al. A new peptidic ligand and its receptor regulating adrenal function in rats. J Biol Chem 2003;278:46387–95. [9] Guillemin R. Hypothalamic hormones a.k.a. hypothalamic releasing factors. J Endocrinol 2005;184:11–28. [10] Hinuma S, Habata Y, Fujii R, Kawamata Y, Hosoya M, Fukusumi S. A prolactinreleasing peptide in the brain. Nature 1998;393:272–6. [11] Hinuma S, Onda H, Fujino M. The quest for novel biological peptides utilizing orphan seven-transmembrane-domain receptors. J Mol Med 1999;777:495–504. [12] Hinuma S, Shintani Y, Fukusumi S, Iijima N, Matsumoto Y, Hosoya M, et al. New neuropeptides containing carboxy-terminal RFamide and their receptor in mammals. Nat Cell Biol 2000;2:703–8. [13] Jiang Y, Luo L, Gustafson EL, Yadav D, Laverty M, Murgolo N. Identification and characterization of a novel RF-amide peptide ligand for orphan G-proteincoupled receptor SP9155. J Biol Chem 2003;278:27652–7. [14] Kotani M, Detheux M, Vandenbogaerde A, Communi D, Vanderwinden JM, Le Poul Brézillon S, et al. The metastasis suppressor gene KiSS-1 encodes kisspeptins, the natural ligands of the orphan G protein-coupled receptor GPR54. J Biol Chem 2001;276:34631–43436. [15] Liposits ZS, Paull WK, Wu P, Jackson IMD, Lechan RM. Hypophysiotrophic thyrotropin releasing hormone (TRH) synthesizing neurons Ultrastructure, adrenergic innervation and putative transmitter action. Histochem Cell Biol 1987;88:1–10. [16] Matsumoto H, Noguchi J, Horikoshi Y, Kawamata Y, Kitada C, Hinuma S, et al. Stimulation of prolactin release by prolactin-releasing peptide in rats. Biochem Biophys Res Commun 1999;259:321–4. [17] Moriyama S, Ito T, Takahashi A, Amano M, Sower SA, Hirano T, et al. A homolog of mammalian PRL-releasing peptide (fish arginyl-phenylalanyl-amide peptide) is a major hypothalamic peptide of PRL release in teleost fish. Endocrinology 2002;143:2071–9. [18] Navarro VM, Fernández-Fernández R, Nogueiras R, Vigo E, Tovar S, Chartrel N, et al. Novel role of 26RFa, a hypothalamic RFamide orexigenic peptide, as putative regulator of the gonadotropic axis. J Physiol 2006;573:237–49. [19] Ohtaki T, Shintani Y, Honda S, Matsumoto H, Hori A, Kanehashi K, et al. Metastasis suppressor gene KiSS-1 encodes peptide ligand of a G protein-coupled receptor. Nature 2001;411:613–7. [20] Patel SR, Murphy KG, Thompson EL, Patterson M, Curtis AE, Ghatei MA, et al. Pyroglutamylated RFamide peptide 43 stimulates the hypothalamic–pituitary–gonadal axis via gonadotropin-releasing hormone in rats. Endocrinology 2008;149:4747–54. [21] Sakamoto T, Agustsson T, Moriyama S, Itoh T, Takahashi A, Kawauchi H, et al. Intra-arterial injection of prolactin-releasing peptide elevates prolactin gene expression and plasma prolactin levels in rainbow trout. J Comp Physiol 2003;173:333–7.
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[22] Sakamoto T, Oda A, Narita K, Takahashi H, Oda T, Fujiwara J, et al. Prolactin: fishy tales of its primary regulator and function. Ann N Y Acad Sci 2005;1040:184–8. [23] Schindler WJ. Hypothalamic neurohumoral control of pituitary function. Proc R Soc Med 1962;55:125–30. [24] Seal LJ, Small CJ, Kim MS, Stanley SA, Taheri S, Ghatei MA, et al. Prolactinreleasing peptide (PrRP) stimulates luteinizing hormone (LH) and follicle stimulating hormone (FSH) via a hypothalamic mechanism in male rats. Endocrinology 2000;141:1909–12. [25] Seale AP, Itoh T, Moriyama S, Takahashi A, Kawauchi H, Sakamoto T, et al. Isolation and characterization of a homologue of mammalian prolactin-releasing peptide from the tilapia brain and its effect on prolactin release from the tilapia pituitary. Gen Comp Endocrinol 2002;125:328–39. [26] Shahab M, Mastronardi C, Seminara SB, Crowley WF, Ojeda SR, Plant TM. Increased hypothalamic GPR54 signaling a potential mechanism for initiation of puberty in primates. Proc Natl Acad Sci USA 2005;102:2129–34. [27] Takayasu S, Sakurai T, Iwasaki S, Teranishi H, Yamanaka A, Williams SC, et al. A neuropeptide ligand of the G protein-coupled receptor GPR103 regulates feeding, behavioral arousal, and blood pressure in mice. Proc Natl Acad Sci U S A 2006;103:7438–43. [28] Tokita R, Nakata T, Kutsumata H, Konishi S, Onodera H, Imaki J, et al. Prolactin secretion in response to prolactin-releasing peptide and the expression of prolactin-releasing peptide gene in the medulla oblongata are estrogen dependent in rats. Neurosci Lett 1999;276:103–6. [29] Treier M, Rosenfeld MG. The hypothalamic–pituitary axis: co-development of two organs. Curr Opin Cell Biol 1996;8:833–43. [30] Tsutsui K, Saigoh E, Ukena K, Teranishi H, Fujisawa Y, Kikuchi M, et al. A novel avian hypothalamic peptide inhibiting gonadotropin release. Biochem Biophys Res Commun 2000;275:661–7.
[31] Wahab F, Aziz F, Irfan S, Zaman W, Shahab M. Short-term fasting attenuates the response of the HPG axis to kisspeptin challenge in the adult male rhesus monkey (Macaca mulatta). Life Sci 2008;83:633–7. [32] Wahab F, Quinton R, Seminara SB. The kisspeptin signaling pathway and its role in human isolated GnRH deficiency. Mol Cell Endocrinol 2011;346: 29–36. [33] Wahab F, Bano R, Jabeen S, Irfan S, Shahab M. Effect of peripheral kisspeptin administration on adiponectin, leptin, and resistin secretion under fed and fasting condition in the adult male rhesus monkey (Macaca mulatta). Horm Metab Res 2010;42:570–4. [34] Wahab F, Tanzeela R, Shahab M. Study of the effect of peripheral kisspeptin administration on basal and glucose-induced insulin secretion under fed and fasting conditions in the adult male rhesus monkey (Macaca mulatta). Horm Metab Res 2011;43:37–42. [35] Wahab F, Ullah F, Chan YM, Seminara SB, Shahab M. Decrease in hypothalamic Kiss1 and Kiss1r expression: a potential mechanism for fasting-induced suppression of the HPG axis in the adult male rhesus monkey (Macaca mulatta). Horm Metab Res 2011;43:81–5. [36] Wahab F, Riaz T, Khan LA, Leprince J, Vaudry H, Tena-Sempere M, et al. Peripheral administration of the human kisspeptin-10 and 26RF-aimde inhibit plasma testosterone levels in the adult male broiler breeder birds (Gallus domesticus). Pak J Zool 2012;44:7–14. [37] Yang H-YT, Fratta W, Majane EA, Costa E. Isolation, sequencing, synthesis and pharmacological characterization of two brain neuropeptides that modulate the action of morphine. Proc Natl Acad Sci USA 1985;82: 7757–61.
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Study of the effect of 26RF- and 43RF-amides on Testosterone and Prolactin secretion in the adult male rhesus monkey (Macaca mulatta) Fazal Wahab a,b,1 , Hina Salahuddin a,1 , Mariam Anees a , Jerome Leprince c , Hubert Vaudry c , Manuel Tena-Sempere d , Muhammad Shahab a,∗ a
Laboratory of Reproductive Neuroendocrinology, Department of Animal Sciences, Faculty of Biological Sciences, Quaid-i-Azam University, 45320 Islamabad, Pakistan Department of Physiology, Institute of Basic Medical Sciences, Khyber Medical University, Peshawar, Khyber Pukhtunkhawa, Pakistan c INSERM U413, Laboratory of Cellular and Molecular Neuroendocrinology, European Institute for Peptide Research (IFRMP 23), University of Rouen, 76821 Mont-Saint-Aignan, France d Department of Cell Biology, Physiology, and Immunology, University of Cordoba, 14004 Cordoba, Spain b
a r t i c l e
i n f o
Article history: Received 2 March 2012 Received in revised form 10 April 2012 Accepted 10 April 2012 Available online 17 April 2012 Keyword: 26RF amide 43RF amide GnRH Testosterone Prolactin
a b s t r a c t RF-amides (RFa), a superfamily of evolutionary-conserved neuropeptides, are expressed in both invertebrates and vertebrates. While some endocrine functions have been attributed to these peptides in lower vertebrates and few mammalian models, not much is known about their actions in primates. Therefore, the present study was designed to examine the effects of peripheral administration of two recently cloned human RFa peptides, 26RFa and 43RFa, on testosterone and prolactin secretion in the adult male adult male rhesus monkey (Macaca mulatta). For control purposes, a scrambled sequence of 26RFa (Sc-26RFa) and normal saline (1 ml) were injected. Three different doses of 26RFa and 43RFa (19-nmol, 38-nmol and 76-nmol) and a single dose (38-nmol) of Sc-26RFa were tested. A set of four chair-restraint habituated monkeys was used. Comparison of post-treatment T levels with respective pre levels showed that none of the doses of both 26RFa and 43RFa changed T release. Similarly, Sc-26RFa and saline administration also did not affect T levels. In contrast, all doses of 26RFa and 43RFa significantly (P < 0.05) stimulated prolactin secretion. 43RFa dose dependently increased prolactin secretion while dose dependency was not observed for 26RFa. Saline and Sc-26RFa injection had no effect on prolactin concentrations. Thus, present study demonstrated that peripheral administration of 26RFa and 43RFa, in the doses tested, have no effect on T secretion, suggesting possible selective lack of their neuroendocrine role in controlling hypothalamic–pituitary–gonadal axis in the adult male primates. The prominent stimulation of prolactin suggests a neuroendocrine role of RFa peptides in regulation of prolactin release in primates. © 2012 Elsevier Inc. All rights reserved.
1. Introduction Adenohypophysis serves as a relay center between the brain and peripheral endocrine organs, and hence it play critical role in the homeostatic regulation of such vital processes as metabolism, growth, reproduction and behavior [29]. The adenohypophysis is functionally linked to the hypothalamus, a critical neuroendocrine regulator area of the brain [4,23]. The hypothalamus secretes a number of peptides that reach the adenohypophysis via hypophysial portal vein and affect its functioning [9,23]. In addition
∗ Corresponding author at: Reproductive Neuroendocrinology Laboratory, Department of Animal Sciences, Faculty of Biological Sciences, Quaid-i-Azam University, 45320 Islamabad, Pakistan. Tel.: +92 51 90643014; fax: +92 51 2601176. E-mail address: [email protected] (M. Shahab). 1 These authors are contributed equally to this work. 0196-9781/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.peptides.2012.04.009
to classical hypothalamic releasing and inhibiting factors, adenohypophysis function is also regulated, directly or indirectly, by a number of other hypothalamic peptides. In recent decades, a large series of peptides with a distinctive Arg–Phe–NH2 motif at the C-terminus has been identified, and are grouped as the RF-amide (RFa) peptide superfamily [5]. In mammals, major RFa peptides are the neuropeptides FF and AF, prolactin-releasing peptide (PrRP), metastin and other kisspeptins encoded by the Kiss1 gene, as well as RF-related peptides, RFRP-1 and RFRP-3 [10–12,14,19,30,37]. More recently, a 26-amino acids RFa peptide, first isolated in the frog and named 26RFa, has also been cloned in some mammalian species [6]. This peptide is the ligand for a previously orphan G protein coupled receptor, GPR103. The GPR103 couples to Gi/o and Gq signaling pathway and it leads to rise in intracellular calcium concentrations and decrease of cAMP production in CHO-GPR103 transfected cells [8]. The Nelongated form of 26RFa, 43RFa, has the same efficacy for binding
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and activation of GPR103 [8]. Within the brain, 26RFa appears to be mainly expressed in different hypothalamic nuclei, such as the ventromedial hypothalamic nucleus and lateral hypothalamus [8]. However, 26RFa and GPR103 genes expression has also been shown in other brain areas and peripheral tissues [13]. Various physiological roles have been assigned to these peptides in various species [18,20,36]. Recently, some preliminary evidences showed the effect of 26RFa and 43RFa on gonadotropin and prolactin (PRL) secretion in rats [18]. In adult cyclic female rat, both central and peripheral administration of 26RFa and 43RFa stimulated gonadotropin secretion. In adult male rats, central as well as peripheral administration of 26RFa inhibited PRL secretion while had no effect on the gonadotropin release [18]. Later, these original findings were confirmed by the study of Patel and colleagues [20], who observed that central injection of 43RFa stimulated LH release, while peripheral administration had no effect. Until now, to the best of our knowledge, in non-rodent mammals especially in primates, the neuroendocrine role of 26RFa and 43RFa is not known at all. Therefore, we considered it relevant to explore the potential role of 26RFa and 43RFa in the regulation of the HPG axis and PRL secretion in the adult male rhesus monkey, a representative higher primate. Due to unavailability of proper facilities for measurements of the rhesus monkey LH, plasma testosterone concentration was measured as terminal marker of the HPG axis.
2. Materials and methods 2.1. Animals Four adult intact male rhesus monkeys (Macaca mulatta) were used in this study. Monkeys ranged in age from 7–8 years and weighed between 6.0 and 9.0 kg. The animals were housed in individual cages, under semi controlled colony conditions (lights on, 0700–1900 h; temperature, 22 ◦ C). The animals were fed with monkey food at 1300–1330 h daily and supplemented with fresh fruits and vegetables in the morning (0900–0930 h). Water was available ad libitum. These animals were chair-restrained habituated and housed in Department’s primate facility as described earlier [31,33–35]. All experimental protocols of the present study were approved by the Departmental Committee for Care and Use of Laboratory Animals. 2.2. Catheterization Catheterization of experimental animals was carried out as documented previously [31,33–35]. Briefly the animals were anesthetized with ketamine hydrochloride (10 mg/kg BW, i.m.), and a teflon cannula (Vasocan Branule, B. Braun Melsungen AG, Belgium; 0.8 mm/22 G O.D) was inserted in the saphenous vein. The open end of the cannula was attached to a syringe via a butterfly tubing (20 G diameter and 300 mm length). Cannulation was carried out 30 min before initiation of blood sampling and then animals were restrained to the chair. This route was used for both blood samples collection and i.v. injection of RFa and vehicle (normal saline). Blood sampling was carried out when the animals had fully regained consciousness. 2.3. Blood sampling Blood samples (2 ml) were taken in heparinized syringes and immediately transferred to culture tubes kept on ice. After completion of sampling, tubes were centrifuged at 3000 rpm at 4 ◦ C, plasma was extracted and stored at −15 ◦ C until assayed for hormones analysis. After each blood sample, equal volume of normal
saline, containing 5 IU of heparin, was injected to compensate the lost blood volume and to prevent blood clotting in the cannula. 2.4. Reagents Heparin (Rotexmedica, Trittau, Germany), GnRH (Sigma Chemical Company, St. Louis, MO, USA) and ketamine were purchased locally. Human RFa peptides (26RFa, 43RFa and Sc-26RFa, a scrambled amino acid sequence of 26RFa) were synthesized in the lab of one of the authors (HV) as described before [6]. Working solutions of RFa were made in normal saline. 2.5. General experimental design Actual experiment comprised of 8 non-consecutive days of blood sampling. All four animals were used on each day of sampling. A total of 20 blood samples were taken from each animal on each day of sampling. Sampling duration was about 5 h (1100–1800 h). Samples were obtained at 15-min intervals for 30 min before injections (−30, −15 and 0 min) and for 240 min thereafter. A bolus i.v. injection of GnRH (1 g) was given after 240 min to assess responsiveness of pituitary–testicular axis. RF amides or normal saline as vehicle was administered as i.v. bolus immediately after taking 0 min sample. A scrambled sequence of 26RFa was used as a specific control to 26RFa. Samplings were carried out from 14th December 2006 to 1st February 2007. During this period all 4 animals were taken for 8 sampling occasions with a gap of 4 days. Each animal received treatments in following order: 1. 2. 3. 4. 5. 6. 7. 8.
1 ml normal saline (0.9% NaCl) 38-nmol Sc 26RFa 19-nmol of 26RFa 38-nmol of 26RFa 76-nmol of 26RFa 19-nmol of 43RFa 38-nmol of 43RFa 76-nmol of 43RFa
Doses of amides were estimated from the neuroendocrine effective doses of kisspeptin in monkeys [26]. 2.6. Hormones assays Plasma testosterone (T) concentrations were determined by using solid phase competitive RIAs. The T RIA kits were purchased from Immunotech Marselle Cedex 9, France. The RIAs were performed as per the manufacturer’s instructions. The sensitivity of the T assay was 0.025 ng/ml and intra- and inter-assay coefficients of variation (CV) were both below 10%. Changes in plasma PRL levels were monitored using a human PRL ELISA kit (Accu Bind ELISA Microwells, Monobind Inc, Lake Forest, CA, USA) following the instructions of the manufacturer. The analytical sensitivity of the kit was 0.01 ng/ml or 0.026 mIIU/L. Intra- and inter-assay % CV were 5.96% and 2.56%, respectively. 2.7. Statistical analyses In view of individual variations in hormone levels, percent changes with respect to time 0 min were calculated and such values at different time points were analyzed by repeated measure one-way ANOVA followed by Dennett’s test. Mean % changes after administration of RFa was also compared by repeated measure ANOVA. Statistical comparisons for the mean pre- and posttreatment hormones changes were also made by paired Student’s
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Fig. 1. (A) % change in mean plasma T concentration in a 4 h period (15–240 min) after i.v. administration of vehicle, Sc-26RFa and various doses of 26RFa in the adult male rhesus monkey (n = 4). GnRH administration at 240 min significantly stimulated (P < 0.05) plasma T levels in all animals. (B) Comparison of mean % change in plasma T concentration after injection of various doses of 26RFa, Sc 26RFa and vehicle (15–240 min) in the adult male monkeys (n = 4). All doses of 26RFa like vehicle and Sc-26RFa have no effect on the plasma T values.
Fig. 2. (A) % change in mean plasma T concentration in a 4 h period (15–240 min) after i.v. administration of vehicle and various doses of 43RFa in the adult male rhesus monkey. GnRH administration at 240 min significantly stimulated (P < 0.05) plasma T levels in all animals. (B) Comparison of mean % change in plasma T concentration after injection of various doses of 43RFa and vehicle (15–240 min) in the adult male monkeys. All doses of 26RFa like vehicle and Sc-26RFa have no effect on the plasma T values.
t tests. Based upon a shorter response of PRL secretion to administration of the RFa, changes in a selected window of 0–120 min were analyzed. All data are presented as mean (±SEM). Results were considered statistically significant at P < 0.05.
of vehicle, Sc-26RFa and 26RFa is shown in Fig. 3B. All doses of 26RFa significantly increased PRL levels as compared to vehicle and Sc-26RFa levels. Comparison of % change in mean plasma PRL levels after i.v. administration of vehicle and 43RFa is shown in Fig. 4B. 43RFa administration also significantly increased mean plasma PRL level. However, PRL secretion was not affected dose dependently by 26RFa as all doses increased PRL concentrations equally. In contrast, a dose dependency was evident with 43RFa where % changes in plasma PRL concentrations increased with dose.
3. Results 3.1. Effects of 26RFa and 43RFa on T secretion The pattern of % change in plasma T secretion before and after administration of 19-, 38- and 76-nmol doses of 26RFa and 43RFa in adult male monkeys is shown in Figs. 1A and 2A, respectively. Comparison of mean pre- and post-treatment T levels or with respect to vehicle and Sc-26RFa treatments levels revealed that no dose of 26RFa altered T secretion (Fig. 1B). Likewise, no dose of 43RFa changed T secretion (Fig. 2B). In contrast, GnRH administration at end of sampling period significantly stimulated T secretion. 3.2. Effects of 26RFa and 43RFa on PRL secretion The pattern of % change in plasma PRL secretion before and after administration of 19-, 38-, 76-nmol doses 26RFa and 43RFa is shown in Figs. 3A and 4A, respectively. Comparison of % change in mean plasma PRL concentration after peripheral administration
4. Discussion In the present study, we examined the effect of peripheral administration of two human RFa peptides, 26RFa and its elongated form, 43RFa, on the T and PRL secretion in the adult male rhesus monkey. The signal finding of the present study was that i.v. administration of both the 26RFa and 43RFa in doses utilized did not affect plasma T levels. Although due to lack of unavailability of monkey LH in our lab, we could not measure LH, it was likely that absent T response to RFa in present study was due to unresponsiveness of the GnRH-LH to RFa. GnRH administration at the end of the sampling caused a robust stimulation of plasma T secretion, suggesting that the HPG axis was responsive. However, injections of both the
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Fig. 3. (A) % change in mean plasma PRL concentration in a 2 h period (15–120 min) after i.v. administration of vehicle, Sc-26RFa and various doses of 26RFa in the adult male rhesus monkey. Repeated measured ANOVA showed that administration of 38 and 76 nmol doses significantly increased % change in PRL secretion at 30, 105, and 120 min, and at 60–120 min, respectively. (B) Comparison of mean % change in plasma PRL concentration after injection of various doses of 26RFa, Sc 26RFa and Saline (15–120 min) in the adult male monkeys. Post-26RFa mean change was greater to respective pre (a P < 0.05–005) values. All doses of 26RFa significantly stimulated % PRL change as compared to vehicle- and Sc-26RFa (* P < 0.05) values.
Fig. 4. (A) % change in mean plasma PRL concentration in a 2 h period (15–120 min) after i.v. administration of vehicle and various doses of 43RFa in the adult male rhesus monkey. % change in PRL secretion was significantly increased by 19 nmol (at 75–120 min), 38 nmol (at 60–120 min) and 76 nmol (at 45–120 min) doses of 43RFa. (B) Comparison of mean % change in plasma PRL concentration after injection of various doses of 43RFa and vehicle (15–120 min) in the adult male monkeys. Post-26RFa mean change was greater than the respective pre (a P < 0.05–005) values. All doses of 43RFa significantly stimulated % PRL change as compared to vehicle (b P < 0.05–005) values.
peptides caused an acute rise in plasma PRL levels. Together, these findings suggest that peripheral administration of both the 26RFa and 43RFa, unlike kisspeptins, had no effect on the T release in male rhesus monkeys. Our findings are consistent with a previous study in rodents which demonstrated that both the central and systemic injections of the RFa had no effect on the gonadotropin secretion in the adult male rat [18]. Similarly, another recent study showed that peripheral administration of 43RFa was unable to stimulate LH secretion but central administration of relatively high doses of 43RFa did significantly stimulate LH secretion in the adult male rat [20]. Therefore, our findings in the male rhesus monkey regarding effect of systemic RFa on T secretion are consistent with the observations in the male rat. The exact reasons for lack of the effect of the RFa on the T secretion in monkeys are not clear. It is possible that the doses of the RFa employed were low but as same doses modulated PRL secretion, the notion is less likely. Other possibility is that the primate HPG axis may be less sensitive to 26RFa and 43RFa than the hypothalamic–lactotroph axis. Therefore, the current findings did not rule out possibility of the effect of higher doses. This idea may not be convincing in view of the fact that the doses of RFa were
equimolar with effective doses of kisspeptin [26,31]. There is also possibility that RFa may act only through the hypothalamic receptors located proximal to the blood–brain-barrier and the systemic administration may not be able to reach there. This possibility is supported by some recent observations in the rats that central but not peripheral administration of 43RFa stimulated LH secretion [20]. However, other members of the RFa family especially kisspeptin is able to cross the blood–brain-barrier and activate the hypothalamic GPR54, receptor of kisspeptin [26]. In summary, we conclude with caution that in adult male non-human primates, systemic RF amides may not affect the HPG axis. Recently, 26RFa have been implicated in the regulation of PRL in rat. It led to decrease in the secretion of PRL [18]. In order to check whether lack of the effect of 26RFa and 43RFA was specific to the HPG axis in primates or to other hypothalamic–pituitary axes also, we monitored the effect of the 26- and 43-RFa on the PRL release. Systemic administration of both 26RFa and 43RFa led to an increase in the release of PRL. Our present study reports, for the first time, specific stimulatory action of RF amides on PRL secretion in higher primates. However, PRL secretion was not affected
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dose dependently by 26RFa as all doses increased PRL concentrations equally. In contrast, a dose dependency was evident with 43RFa where mean post-treatment PRL concentration appeared to rise with increase in the dose. The observed PRL stimulatory effect of 26RFa and 43RFa are in line with established PRL augmenting actions of PrRP [16], which is also a RF amide peptide. Our findings in the monkeys, however, are contradictory to observations recently made in rats. Central administration of 26RFa was observed to cause a rapid decrease in PRL levels in adult male rats and diestrous female rats [18]. Besides species differences, this discrepancy appears to be related to different route of administration of RFa utilized in our study and the rat study [18]. Our results are similar to observations made in fish. PRLreleasing peptide (PrRP) seems to be an essential stimulator of PRL transcription and secretion in teleost pituitary and peripheral organs [22]. Homologue of mammalian PrRP in salmon has also been shown to stimulate PRL release from pituitary both in salmon and tilapia [17,21,25]. Interestingly, other RFa peptides such as kisspeptins and RFRP1/GnIH have been demonstrated as important regulators of the secretion of different pituitary hormones, such as gonadotropins and/or PRL [7,10,11,24,32]. Our results, therefore, advance understanding of RFa peptides actions in primates. However, design of the current study did not allow inferring much whether our results indicated pharmacological action or physiological action of 26RFa/43RFa. Further experiments are required to asses if these peptides are involved in the physiological regulation of PRL secretion in the primates. The mechanism underlying the observed stimulatory effect of 26RFa and 43RFa on PRL secretion in monkeys remains to be determined. There are 3 possible ways regarding the mechanism of action of 26RFa in monkeys. One possibility is that peripherally administered RFa cross blood–brain-barrier and activate GPR103 receptors in periventricular nucleus leading to TRH release which would then entrain PRL secretion. Similarly, second possibility is that peripheral RFa cause activation of GPR103 in paraventricular nucleus (PVN) leading to inhibition of dopamine neurons, which would then cause increase in PRL secretion. Regarding hypothalamic site of action of 26RFa, GPR103 mRNA has been detected in PVN of human and in periventricular nucleus of mouse [27]. Recently, novel binding sites for 26RFa have been demonstrated in the PVN of rats [3]. It is relevant to mention here that TRH, which is a known PRL secretagogue, neurons are also present in the periventricular nucleus [15]. Furthermore, tuberoinfundibular dopaminergic neurons, which are primary regulators of PRL secretion, are present in the neighboring PVN [1]. It is, therefore, apparent that GPR103 expression is present in vicinity of dopamine neurons as well as in areas adjoining the location where TRH neurons are present. Parenthetically, 26RFa immunoreactive fibers have also been demonstrated in human PVN [2]. Moreover, immunohistochemical labeling using specific antibodies against human 26RFa and in situ hybridization histochemistry revealed that in the human hypothalamus 26RFa-expressing neurons are located in the PVN and ventromedial nuclei [2]. These observations support the assumption that RFa can act on dopamine and TRH neurons. It can be speculated that 26RFa causes inhibition of dopamine neurons or stimulation of TRH neurons in order to induce release of PRL. Third possibility is the direct action of the amides on pituitary GPR103 to increase PRL release. Recently, GPR103 expression has been shown in rodent’s pituitary throughout the postnatal development though the cellular localization of the receptor was not identified [18]. Furthermore, a mammalian homologue of PrRP has been shown to stimulate PRL release from fish pituitary [17,21,25,28]. Together, latter observations support the possibility of the direct action of RFa in increasing PRL release in monkeys.
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In summary, present data indicate, for the first time, that 26RFa and 43RFa administration does not affect HPG axis but stimulate PRL secretion in adult male rhesus monkey, suggesting a potential role of the evolutionary conserved RFa in the central or pituitary mechanisms controlling PRL release in higher primates. Disclosure Authors have nothing to disclose. Acknowledgements The work presented here was funded by the Higher Education Commission (HEC), Islamabad, Pakistan. References [1] Ben-Jonathan N. Dopamine: a prolactin-inhibiting hormone. Endocr Rev 1985;6:564–89. [2] Bruzzone F, Lectez B, Tollemer HL, Leprince JR, Dujardin C, Rachidi W, et al. Anatomical distribution and biochemical characterization of the novel RFamide peptide 26RFa in the human hypothalamus and spinal cord. J Neurochem 2006;99:616–27. [3] Bruzzone F, Lectez B, Alexandre D, Jegou S, Mounien L, Tollemer H, et al. Distribution of 26RFa binding sites and GPR103 mRNA in the central nervous system of the rat. J Comp Neurol 2007;503:573–91. [4] Charlton H. Hypothalamic control of anterior pituitary function: a history. J Neuroendocrinol 2008;20:641–6. [5] Chartrel N, Dujardin C, Leprince J, Desrues L, Tonon MC, Cellier E, et al. Isolation, characterization, and distribution of a novel neuropeptide, Rana RFamide (RRFa), in the brain of the European green frog Rana esculenta. J Comp Neurol 2002;448:111–27. [6] Chartrel N, Dujardin C, Anouar Y, Leprince J, Decker A, Clerens S, et al. Identification of 26RFa, a hypothalamic neuropeptide of the RFamide peptide family with orexigenic activity. Proc Natl Acad Sci USA 2003;100:15247–52. [7] Colledge WH. GPR54 and puberty. Trends Endocrinol Metab 2004;15:448–53. [8] Fukusumi S, Yoshida H, Fujii R, Maruyama M, Komatsu H, Habata Y, et al. A new peptidic ligand and its receptor regulating adrenal function in rats. J Biol Chem 2003;278:46387–95. [9] Guillemin R. Hypothalamic hormones a.k.a. hypothalamic releasing factors. J Endocrinol 2005;184:11–28. [10] Hinuma S, Habata Y, Fujii R, Kawamata Y, Hosoya M, Fukusumi S. A prolactinreleasing peptide in the brain. Nature 1998;393:272–6. [11] Hinuma S, Onda H, Fujino M. The quest for novel biological peptides utilizing orphan seven-transmembrane-domain receptors. J Mol Med 1999;777:495–504. [12] Hinuma S, Shintani Y, Fukusumi S, Iijima N, Matsumoto Y, Hosoya M, et al. New neuropeptides containing carboxy-terminal RFamide and their receptor in mammals. Nat Cell Biol 2000;2:703–8. [13] Jiang Y, Luo L, Gustafson EL, Yadav D, Laverty M, Murgolo N. Identification and characterization of a novel RF-amide peptide ligand for orphan G-proteincoupled receptor SP9155. J Biol Chem 2003;278:27652–7. [14] Kotani M, Detheux M, Vandenbogaerde A, Communi D, Vanderwinden JM, Le Poul Brézillon S, et al. The metastasis suppressor gene KiSS-1 encodes kisspeptins, the natural ligands of the orphan G protein-coupled receptor GPR54. J Biol Chem 2001;276:34631–43436. [15] Liposits ZS, Paull WK, Wu P, Jackson IMD, Lechan RM. Hypophysiotrophic thyrotropin releasing hormone (TRH) synthesizing neurons Ultrastructure, adrenergic innervation and putative transmitter action. Histochem Cell Biol 1987;88:1–10. [16] Matsumoto H, Noguchi J, Horikoshi Y, Kawamata Y, Kitada C, Hinuma S, et al. Stimulation of prolactin release by prolactin-releasing peptide in rats. Biochem Biophys Res Commun 1999;259:321–4. [17] Moriyama S, Ito T, Takahashi A, Amano M, Sower SA, Hirano T, et al. A homolog of mammalian PRL-releasing peptide (fish arginyl-phenylalanyl-amide peptide) is a major hypothalamic peptide of PRL release in teleost fish. Endocrinology 2002;143:2071–9. [18] Navarro VM, Fernández-Fernández R, Nogueiras R, Vigo E, Tovar S, Chartrel N, et al. Novel role of 26RFa, a hypothalamic RFamide orexigenic peptide, as putative regulator of the gonadotropic axis. J Physiol 2006;573:237–49. [19] Ohtaki T, Shintani Y, Honda S, Matsumoto H, Hori A, Kanehashi K, et al. Metastasis suppressor gene KiSS-1 encodes peptide ligand of a G protein-coupled receptor. Nature 2001;411:613–7. [20] Patel SR, Murphy KG, Thompson EL, Patterson M, Curtis AE, Ghatei MA, et al. Pyroglutamylated RFamide peptide 43 stimulates the hypothalamic–pituitary–gonadal axis via gonadotropin-releasing hormone in rats. Endocrinology 2008;149:4747–54. [21] Sakamoto T, Agustsson T, Moriyama S, Itoh T, Takahashi A, Kawauchi H, et al. Intra-arterial injection of prolactin-releasing peptide elevates prolactin gene expression and plasma prolactin levels in rainbow trout. J Comp Physiol 2003;173:333–7.
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[22] Sakamoto T, Oda A, Narita K, Takahashi H, Oda T, Fujiwara J, et al. Prolactin: fishy tales of its primary regulator and function. Ann N Y Acad Sci 2005;1040:184–8. [23] Schindler WJ. Hypothalamic neurohumoral control of pituitary function. Proc R Soc Med 1962;55:125–30. [24] Seal LJ, Small CJ, Kim MS, Stanley SA, Taheri S, Ghatei MA, et al. Prolactinreleasing peptide (PrRP) stimulates luteinizing hormone (LH) and follicle stimulating hormone (FSH) via a hypothalamic mechanism in male rats. Endocrinology 2000;141:1909–12. [25] Seale AP, Itoh T, Moriyama S, Takahashi A, Kawauchi H, Sakamoto T, et al. Isolation and characterization of a homologue of mammalian prolactin-releasing peptide from the tilapia brain and its effect on prolactin release from the tilapia pituitary. Gen Comp Endocrinol 2002;125:328–39. [26] Shahab M, Mastronardi C, Seminara SB, Crowley WF, Ojeda SR, Plant TM. Increased hypothalamic GPR54 signaling a potential mechanism for initiation of puberty in primates. Proc Natl Acad Sci USA 2005;102:2129–34. [27] Takayasu S, Sakurai T, Iwasaki S, Teranishi H, Yamanaka A, Williams SC, et al. A neuropeptide ligand of the G protein-coupled receptor GPR103 regulates feeding, behavioral arousal, and blood pressure in mice. Proc Natl Acad Sci U S A 2006;103:7438–43. [28] Tokita R, Nakata T, Kutsumata H, Konishi S, Onodera H, Imaki J, et al. Prolactin secretion in response to prolactin-releasing peptide and the expression of prolactin-releasing peptide gene in the medulla oblongata are estrogen dependent in rats. Neurosci Lett 1999;276:103–6. [29] Treier M, Rosenfeld MG. The hypothalamic–pituitary axis: co-development of two organs. Curr Opin Cell Biol 1996;8:833–43. [30] Tsutsui K, Saigoh E, Ukena K, Teranishi H, Fujisawa Y, Kikuchi M, et al. A novel avian hypothalamic peptide inhibiting gonadotropin release. Biochem Biophys Res Commun 2000;275:661–7.
[31] Wahab F, Aziz F, Irfan S, Zaman W, Shahab M. Short-term fasting attenuates the response of the HPG axis to kisspeptin challenge in the adult male rhesus monkey (Macaca mulatta). Life Sci 2008;83:633–7. [32] Wahab F, Quinton R, Seminara SB. The kisspeptin signaling pathway and its role in human isolated GnRH deficiency. Mol Cell Endocrinol 2011;346: 29–36. [33] Wahab F, Bano R, Jabeen S, Irfan S, Shahab M. Effect of peripheral kisspeptin administration on adiponectin, leptin, and resistin secretion under fed and fasting condition in the adult male rhesus monkey (Macaca mulatta). Horm Metab Res 2010;42:570–4. [34] Wahab F, Tanzeela R, Shahab M. Study of the effect of peripheral kisspeptin administration on basal and glucose-induced insulin secretion under fed and fasting conditions in the adult male rhesus monkey (Macaca mulatta). Horm Metab Res 2011;43:37–42. [35] Wahab F, Ullah F, Chan YM, Seminara SB, Shahab M. Decrease in hypothalamic Kiss1 and Kiss1r expression: a potential mechanism for fasting-induced suppression of the HPG axis in the adult male rhesus monkey (Macaca mulatta). Horm Metab Res 2011;43:81–5. [36] Wahab F, Riaz T, Khan LA, Leprince J, Vaudry H, Tena-Sempere M, et al. Peripheral administration of the human kisspeptin-10 and 26RF-aimde inhibit plasma testosterone levels in the adult male broiler breeder birds (Gallus domesticus). Pak J Zool 2012;44:7–14. [37] Yang H-YT, Fratta W, Majane EA, Costa E. Isolation, sequencing, synthesis and pharmacological characterization of two brain neuropeptides that modulate the action of morphine. Proc Natl Acad Sci USA 1985;82: 7757–61.