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Animal Science Journal (2015) 86, 634–640
doi: 10.1111/asj.12333
ORIGINAL ARTICLE Effects of hypothalamic dopamine on growth hormone-releasing hormone-induced growth hormone secretion and thyrotropin-releasing hormone-induced prolactin secretion in goats Jin JIN and Tsutomu HASHIZUME Faculty of Agriculture, Iwate University, Morioka, Japan
ABSTRACT The aim of the present study was to clarify the effects of hypothalamic dopamine (DA) on the secretion of growth hormone (GH) in goats. The GH-releasing response to an intravenous (i.v.) injection of GH-releasing hormone (GHRH, 0.25 μg/kg body weight (BW)) was examined after treatments to augment central DA using carbidopa (carbi, 1 mg/kg BW) and L-dopa (1 mg/kg BW) in male and female goats under a 16-h photoperiod (16 h light, 8 h dark) condition. GHRH significantly and rapidly stimulated the release of GH after its i.v. administration to goats (P < 0.05). The carbi and L-dopa treatments completely suppressed GH-releasing responses to GHRH in both male and female goats (P < 0.05). The prolactin (PRL)releasing response to an i.v. injection of thyrotropin-releasing hormone (TRH, 1 μg/kg BW) was additionally examined in male goats in this study to confirm modifications to central DA concentrations. The treatments with carbi and L-dopa significantly reduced TRH-induced PRL release in goats (P < 0.05). These results demonstrated that hypothalamic DA was involved in the regulatory mechanisms of GH, as well as PRL secretion in goats.
Key words: dopamine, GH, GHRH, goat, PRL.
INTRODUCTION Dopamine (DA) is a predominant catecholamine neurotransmitter that is located in neuronal populations in the hypothalamus and brainstem of ruminants (Tillet & Thibault 1989; Leshin et al. 1995; Tillet 1995). DA may modulate the secretion of growth hormone (GH) in sheep and cattle. DA or a DA receptor agonist (bromocriptine) was shown to inhibit basal (Soyoola et al. 1994) and GH-releasing hormone (GHRH)induced GH release (Greg et al. 1984; Soyoola et al. 1994) from cultured ovine pituitary cells. A D1 DA receptor agonist (SKF38393) stimulated the release of somatostatin (SS, a GH inhibitory factor) and inhibited the release of GHRH (a GH stimulator factor) from perifused bovine hypothalamic tissues (West et al. 1997). A subcutaneous (s.c.) injection of SKF38393 suppressed the basal and GHRH-induced release of GH in steers (McMahon et al. 1998). Although these findings were obtained in sheep and cattle, most studies have been performed in vitro (Greg et al. 1984; Soyoola et al. 1994; West et al. 1997); therefore, further in vivo studies are needed to confirm in vitro findings in rumi© 2014 Japanese Society of Animal Science
nants. Furthermore, the effects of hypothalamic DA on the secretion of GH have not yet been determined in goats. L-dopa (a precursor of DA) is often used in in vivo studies on humans in order to clarify the effects of hypothalamic DA on the secretion of GH (Mars & Genuth 1973; Camanni et al. 1978; Bansal et al. 1981; Hanew et al. 1986, 1996; Jaffe et al. 1996) because DA cannot directly cross the blood-brain barrier (BBB) (Yen 1991). L-dopa can cross the BBB and convert DA in the hypothalamus by aromatic L-amino acid decarboxylase (AADC) (Yen 1991; Tóth et al. 2002). An AADC inhibitor, such as carbidopa (carbi), is typically administered with L-dopa to humans (Mars & Genuth 1973; Camanni et al. 1978; Bansal et al. 1981; Robertson et al. 1989) because it can block the
Correspondence: Tsutomu Hashizume, Faculty of Agriculture, Iwate University, Morioka 020-8550, Japan. (Email: [email protected]) Received 27 May 2014; accepted for publication 7 August 2014.
DOPAMINE AND GHRH-INDUCED GH SECRETION
peripheral conversion of L-dopa to DA without affecting brain decarboxylation (Pinder et al. 1976). Treatments with carbi alone have neither modified basal DA levels nor induced significant changes in basal GH, prolactin (PRL) or thyroid-stimulating hormone levels in humans (Camanni et al. 1978). When carbi was administered with L-dopa to humans, higher circulating L-dopa levels were observed than after L-dopa alone (Mars & Genuth 1973; Pinder et al. 1976; Bansal et al. 1981). Previous studies demonstrated that when an AADC inhibitor was administered with L-dopa to rats, greater brain DA concentrations were present than after L-dopa alone (Pletscher 1973; Pinder et al. 1976). In ruminants, a recent study demonstrated that an intravenous (i.v.) injection of L-dopa immediately increased DA concentrations in the cerebrospinal fluid collected from the third ventricle in steers (Kasuya et al. 2013). The content of DA in the median eminence was shown to be higher under long photoperiods than short photoperiods in sheep (Thiery 1991). Increases have also been reported in DA and noradrenaline turnover in the median eminence and mediobasal hypothalamus/arcuate nucleus with a long daylength (Curlewis 1992). These findings suggest that a more active DAergic system may exist in the brain under long photoperiods. We recently demonstrated that a long photoperiod enhanced the secretion of GH in goats (Jin et al. 2012, 2013a, b), suggesting that a long photoperiod may influence the hypothalamic DAergic system to modulate the secretion of GH in goats. Therefore, the relationship between GH secretion and the hypothalamic DAergic system in goats under a long photoperiod should be examined in more detail. The secretion of PRL is generally considered to be under the dominant and tonic inhibitory control of hypothalamic DA (Neil & Nagy 1994; Freeman et al. 2000). On the other hand, the secretion of PRL is known to be stimulated by thyrotropin-releasing hormone (TRH) (Yen 1991; Neil & Nagy 1994; Freeman et al. 2000). We previously showed that TRHinduced PRL secretion was suppressed by DA both in vivo and in vitro in ruminants (Hashizume et al. 2009, 2012). Therefore, elucidating the PRL-releasing response to TRH after treatments with carbi plus L-dopa may confirm enhancements in the hypothalamic DAergic system in the present study. The aim of the present study was to clarify the effects of hypothalamic DA on GHRH-induced GH secretion in vivo in goats. The GH-releasing response to an i.v. injection of GHRH was examined after treatments with carbi plus L-dopa in both female and male goats under a long photoperiod. The PRL-releasing response to TRH was also examined in male goats in the present study to confirm modifications to central DA concentrations. Animal Science Journal (2015) 86, 634–640
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MATERIALS AND METHODS The Animal Care and Use Committee of Iwate University approved the experimental and animal care protocols (A201309).
Animals Five native Japanese adult male goats (Shiba goats) (age 1–2 years; mean body weight (BW) 23.1 kg) and five adult female goats (age 1–2 years; mean BW 16.4 kg) were used. Animals were housed in a room in which temperature, humidity, and lighting were strictly controlled. They were fed mixed hay of orchardgrass and reed canarygrass (1.5 kg/ head) and concentrate (50 g/head) at 09.00 hours daily. Water was continuously available. The residuum of the hay was removed before the start of blood collections, and the goats were not fed until after the experiment. Metabolizable energy (ME)/day was assumed to be 2500 Kcal. Experiments were performed in Morioka (39°42′N, 141°09′E), Japan.
Experimental design Experiment 1. Effects of hypothalamic DA on GHRH-induced GH secretion Five male goats were each housed in a separate pen in an environmentally controlled room in which room temperature, humidity and lighting were maintained at 20°C, 60% and 16 h of light (08.00 to 24.00 hours): 8 h of darkness (24.00 to 08.00 hours) (16:8 L : D), respectively. Goats were habituated for 3 weeks to all experimental conditions, and blood was then collected. Blood was drawn from an indwelling catheter at 15-min intervals for 3 h. Animals were given i.v. injections of GHRH (0.25 μg/kg BW in 2 mL of saline; hGHRH (1–29)-NH2, ANA Spec, Fremont, CA, USA) (GHRH alone) or 2 mL of saline as a control (CTL). GHRH was injected 1 h after the commencement of blood sampling. GHRH was also injected after the carbi (1 mg/kg BW in 5 mL of saline; Tokyo Chemical Industry Co. Ltd, Tokyo, Japan) and L-dopa (1 mg/kg BW in 5 mL of saline; Sigma Aldrich Co., St. Louis, MO, USA) treatments (carbi + Ldopa + GHRH). The doses of GHRH, carbi and L-dopa were selected based on the experimental protocols of our previous studies (Hashizume et al. 1997, 2005; Jin et al. 2014). Carbi and L-dopa were intravenously administered 30 min and 15 min, respectively, before the GHRH injection. Experiments were carried out at 2–3-day intervals according to the experimental protocol described in our previous study (Jin et al. 2014). Blood was collected between 11.00 and 14.00 hours. The order in which each goat received the treatments was determined at random. Blood samples were collected into centrifuge tubes containing heparin and immediately chilled with ice. Individual plasma samples were obtained after centrifugation and stored at −30°C until assayed. The experiments described for the male goats were also performed in the female goats. In the present study, five female goats were used. The goats did not exhibit estrous under the present photic condition.
Experiment 2. Effects of hypothalamic DA on TRH-induced PRL secretion This experiment was designed to confirm whether the carbi and L-dopa treatments could augment hypothalamic DA levels. Four male goats were habituated for 3 weeks to the 16:8 L : D photoperiod, and the effects of the carbi and © 2014 Japanese Society of Animal Science
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GH and PRL concentrations in the plasma were measured using a double-antibody radioimmunoassay (Hashizume et al. 1999). The standard and hormone for iodination was USDA-bGH-B-1 for GH and NIDDK oPRL-1–3 for PRL. The intra- and inter-assay coefficients of variation (CV) for GH were 10.1% and 3.2%, respectively. PRL samples were assayed in a single run and the intra-assay CV for PRL was 8.5%. The assay sensitivities for GH and PRL were 0.30 ng/mL and 0.13 ng/mL, respectively.
Statistical analysis All data from the experiments are presented as the mean ± SEM. The significance of differences in plasma GH and PRL concentrations at each sampling time between the saline-treated control (CTL) and each treated group were evaluated by a two-way repeated measures analysis of variance (ANOVA), and the Bonferroni test was used as a post hoc test. The significance of differences in plasma GH and PRL concentrations at each sampling time between GHRH alone and carbi plus L-dopa plus GHRH (carbi + L-dopa + GHRH) or TRH alone and carbi plus L-dopa plus TRH (carbi + Ldopa + TRH) were also evaluated by a two-way repeated measures ANOVA. The significance of differences in the area under the response curve (AUC) of GH for 60 min after the injection of GHRH in the male and female groups was evaluated using the Student’s t–test. All data were analyzed using GraphPad Prism (GraphPad Software, San Diego, CA, USA). Results were considered significant at P < 0.05.
RESULTS Experiment 1. Effects of hypothalamic DA on GHRH-induced GH secretion GH plasma levels in response to an i.v. injection of GHRH with or without the carbi and L-dopa treatments in male and female goats are shown in Figures 1 and 2, respectively. Plasma GH concentrations increased immediately after the injection of GHRH in the carbi and L-dopa non-treated groups (GHRH alone) in both male and female goats (P < 0.05). Maximum values were observed 15 min after the injection of GHRH in male (40 ± 8 ng/mL) and female (48 ± 10 ng/mL) goats, respectively. The AUCs of GH for 60 min after the injection of GHRH were 1132 ± 289 ng/min/mL and 1198 ± 352 ng/min/mL, respectively, in the male and female goats. GHRH failed to stimulate the release of GH in the carbi and L-dopa-treated groups (carbi + L-dopa + GHRH), and the AUC values were 257 ± 106 ng/min/mL and 228 ± 64 ng/min/mL, respectively, in the male and female goats. No significant differences were observed © 2014 Japanese Society of Animal Science
CTL
Male
GHRH alone
* Plasma GH (ng/mL)
Hormone assay
60
GHRH 40
Carbi+L-dopa+GHRH
a
L-dopa Carbi
* a
20
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-30
b
0
30
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Time in minutes Figure 1 Plasma concentrations of growth hormone (GH) in response to the intravenous injection of GH-releasing hormone (GHRH, 0.25 μg/kg body weight (BW)) with or without the treatments with carbidopa (Carbi, 1 mg/kg BW) plus L-dopa (1 mg/kg BW) in male goats. Saline was injected as a control (CTL). Goats were kept under the photoperiod condition of 16 h of light: 8 h of dark (16:8 L : D). Each value represents the mean ± SEM of five animals. Arrows indicate the time of the injection. The different letters (a, b) at each time period denote significant differences (P < 0.05). *P < 0.05 significantly different from the corresponding values from CTL.
60
*
Female
CTL GHRH alone
Plasma GH (ng/mL)
L-dopa treatments on TRH-induced PRL secretion were examined using a similar experimental protocol to that described in experiment 1. TRH (1 μg/kg BW in 2 mL of saline; Peptide Institute Inc., Osaka, Japan) was intravenously injected 1 h after the commencement of blood sampling. The dose of TRH was chosen according to the experimental protocols of our previous studies (Hashizume et al. 2009, 2012; Yaegashi et al. 2012).
a
Carbi+L-dopa+GHRH
GHRH
40
L-dopa Carbi 20
a
b
0 -60
-30
0
b 30
60
90
120
Time in minutes Figure 2 Plasma concentrations of growth hormone (GH) in response to the intravenous injection of GH-releasing hormone (GHRH, 0.25 μg/kg body weight (BW)) with or without the treatments with carbidopa (Carbi, 1 mg/kg BW) plus L-dopa (1 mg/kg BW) in female goats. Saline was injected as a control (CTL). Goats were kept under the photoperiod condition of 16 h of light: 8 h of dark (16:8 L : D). Each value represents the mean ± SEM of five animals. Arrows indicate the time of the injection. The different letters (a, b) at each time period denote significant differences (P < 0.05). *P < 0.05 significantly different from the corresponding values from CTL.
Animal Science Journal (2015) 86, 634–640
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in AUC values between the male and female goats. The concentrations of GH 15 and 30 min after the injection of GHRH in the carbi and L-dopa-treated groups (carbi + L-dopa + GHRH) were also significantly lower than those in the non-treated groups (GHRH alone) in both the male and female goats (P < 0.05).
Experiment 2. Effects of hypothalamic DA on TRH-induced PRL secretion PRL plasma levels in response to an i.v. injection of TRH with or without the carbi and L-dopa treatments in male goats are shown in Figure 3. Plasma PRL concentrations increased immediately after the injection of TRH in the carbi and L-dopa non-treated groups (TRH alone), and reached a maximum value (543 ± 100 ng/ mL) 30 min after the injection (P < 0.05). PRL concentrations remained higher than those in the salineinjected controls (CTL) during the 105-min period (P < 0.05). Plasma PRL concentrations also increased immediately after the injection of TRH in the carbi and L-dopa-treated groups (carbi + L-dopa + TRH), and PRL concentrations 15 min after the injection of TRH (66 ± 22 ng/mL) were significantly higher than the CTL treatment (P < 0.05). The concentrations of PRL 15-105 min after the injection of TRH in the carbi and L-dopa-treated groups (carbi + L-dopa + TRH) were significantly lower than those in the non-treated group (TRH alone) (P < 0.05).
800
Male
CTL TRH alone
Plasma PRL (ng/mL)
* 600
Carbi+L-dopa+TRH
* TRH L-dopa Carbi
400
a a
*
*
a
* a a
200
*b
b
b
b
b
*
*
a
a
b
b
0 -60
-30
0
30
60
90
120
Time in minutes Figure 3 Plasma concentrations of prolactin (PRL) in response to the intravenous injection of thyrotropin-releasing hormone (TRH, 1 μg/kg body weight (BW)) with or without the treatments with carbidopa (Carbi, 1 mg/kg BW) plus L-dopa (1 mg/kg BW) in male goats. Saline was injected as a control (CTL). Goats were kept under the photoperiod condition of 16 h of light: 8 h of dark (16:8 L : D). Each value represents the mean ± SEM of four animals. Arrows indicate the time of the injection. The different letters (a, b) at each time period denote significant differences (P < 0.05). *P < 0.05 significantly different from the corresponding values from CTL.
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DISCUSSION The present study attempted to clarify the relationship between the hypothalamic DA and the GHRH-induced secretion of GH in vivo in goats. The results obtained clearly demonstrated that the treatments with carbi and L-dopa suppressed GHRH-induced GH release in both male and female goats under the long photoperiod condition. This is the first study to demonstrate that hypothalamic DA may alter GHRH-induced GH release in goats. The present study was performed using a long photoperiod, which was previously shown to enhance the secretion of GH in male and female goats (Jin et al. 2012, 2013a, b). We previously reported that the GH pulse amplitude was slightly bigger and the mean GH concentrations were significantly higher in the long photoperiod (16:8 L : D) than in the short photoperiod (8:16 L : D) (Jin et al. 2012, 2013a, b). The present study was conducted under such enhanced GH secretory conditions. Previous studies demonstrated that the secretory pattern of GH was sexually dimorphic in rats (Tannenbaum & Martin 1976; Jansson et al. 1985; Veldhuis 1998) and humans (Stolar & Baumann 1986; Veldhuis 1996, 1998). In ruminants, sexual steroid hormones were suggested to be involved in modifications to pulsatile GH secretion in female goats (Yonezawa et al. 2005); however, we did not observed any significant differences in the mean concentration, pulse amplitude or pulse frequency of GH between the estrous and luteal phases in goats (Hashizume et al. 2000). Furthermore, testicular androgen was suggested to modify the secretory pattern of GH pulses in male goats (Mogi et al. 2002). However, enhancing hypothalamic DA had no significant effect on the GHRH-induced release of GH between the sexes in the present study. The oral administration of L-dopa alone (Mars & Genuth 1973; Hanew et al. 1986), an AADC inhibitor (MK-486) plus L-dopa (Mars & Genuth 1973) and carbi plus L-dopa (Bansal et al. 1981) was shown to increase basal plasma GH concentrations in humans. In these studies, the GH peaks induced by L-dopa appeared 60–90 min after its administration. L-dopainduced basal GH secretion was suppressed by a GHRH antagonist in humans (Hanew et al. 1996; Jaffe et al. 1996); therefore, the stimulation of basal GH secretion by dopaminergic pathways in humans appears to require endogenous GHRH. Although these findings were reported in humans, the pretreatment with carbi plus L-dopa completely suppressed GHRH-induced GH release in the present study. The present results in vivo in goats support a selective DA D1 receptor agonist inhibiting the GHRH-induced release of GH in vivo in steers (McMahon et al. 1998). However, the effects of the treatment with carbi plus L-dopa on basal GH © 2014 Japanese Society of Animal Science
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secretion in goats also need to be examined in future studies. We did not examine the effects of carbi alone on basal and GHRH-induced GH secretion in the present study because it was reported that the treatment with carbi alone did not modify basal GH levels in humans (Camanni et al. 1978). However, the effects of the treatment with carbi alone on basal and GHRHinduced GH secretion in goats may also need to be examined in future studies. The secretion of GH is mainly regulated by GHRH neurons in the arcuate nucleus (ARC) and somatostatin (SS) neurons in the periventricular nucleus (PeVN) (Leshin et al. 1994; McMahon et al. 2001). DA receptors have been separated into two categories: D1-like receptors, which can increase intracellular levels of cyclic adenosine 3′,5′-monophosphate (cAMP) by activating adenylate cyclase (AC), and D2-like receptors, which can inhibit the activation of AC (Missale et al. 1998). The stimulation of D1 receptors by subcutaneous injections of a DA1 receptor agonist has been shown to activate SS neurons in the PeVN and suppress basal and GHRH-induced GH secretion in steers (McMahon et al. 1998). The stimulation of D1 receptors was shown to increase the release of SS, but decreased the release of GHRH from bovine hypothalamic tissues in vitro (West et al. 1997). Furthermore, DA stimulated the release of SS from isolated hypothalamic synaptosomes in rats (Wakabayashi et al. 1977). Therefore, in the present study, enhancing hypothalamic DA may have stimulated the secretion of SS via D1 receptors in PeVN while SS suppressed GHRH-induced GH secretion. Increased SS levels may act directly on somatotrophs to inhibit GHRH-induced GH secretion. SS has been shown to suppress GHRHinduced GH secretion from bovine anterior pituitary cells (Hashizume et al. 1994). In addition, enhanced hypothalamic DA may also have directly inhibited the release of GH from somatotrophs in the pituitary gland via D2 receptors. A treatment with a D2 receptor agonist reduced the secretion of GH by 30% in human pituitary tumors (Gruszka et al. 2012). DA inhibited GHRH-elevated GH secretion by 50% and lowered cAMP levels by 30% in ovine pituitary cells in vitro (Law et al. 1984). McMahon et al. (2001) also reported that DA could inhibit the secretion of GH from somatotrophs via D2 receptors in domestic animals. PRL-releasing responses to TRH were blunted after the treatments with carbi and L-dopa in goats. This result was consistent with previous findings in humans (Carlson 1986). The present PRL results suggest that the carbi and L-dopa treatments could effectively enhance central DA-derived L-dopa in the goat brain, and also that enhanced hypothalamic DA could reach the pituitary lactotrophs through the pituitary portal circulation. DA has been shown to inhibit the synthesis and release of PRL by acting directly on lactotrophs (Yen 1991; Freeman et al. © 2014 Japanese Society of Animal Science
2000). After binding DA, the D2 receptor complex activates a membrane inhibitory Gi-protein, that is negatively coupled to the AC and phospholipase C (PLC) systems (Yen 1991; Missale et al. 1998). TRH stimulates the release of PRL from lactotrophs by activating the PLC system (Yen 1991); therefore, the PLC system associated with PRL secretion may be blocked by elevated central DA levels. In conclusion, hypothalamic DA was found to be involved in the regulatory mechanisms underlying GH secretion, as well as PRL secretion in goats.
ACKNOWLEDGMENTS The authors wish to thank Dr. T. Johke, National Institute of Animal Industry, Tsukuba, Japan, for providing monkey anti-bGH, and the USDA Animal Hormone Program, Beltsville, MD, USA, for providing USDAbGH-B-1. They also wish to thank Dr. A. F. Parlow, National Hormone and Peptide Program, Harbor-UCLA Medical Center, Torrance, CA, USA for providing oPRL (NIDDK oPRL-1-3) and oPRL antiserum (AFP-C358106). This research was supported in part by a Grant-in-Aid for Scientific Research (No.24380149) provided by the Japan Society for the Promotion of Science (JSPS).
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Animal Science Journal (2015) 86, 634–640
Animal Science Journal (2015) 86, 634–640
doi: 10.1111/asj.12333
ORIGINAL ARTICLE Effects of hypothalamic dopamine on growth hormone-releasing hormone-induced growth hormone secretion and thyrotropin-releasing hormone-induced prolactin secretion in goats Jin JIN and Tsutomu HASHIZUME Faculty of Agriculture, Iwate University, Morioka, Japan
ABSTRACT The aim of the present study was to clarify the effects of hypothalamic dopamine (DA) on the secretion of growth hormone (GH) in goats. The GH-releasing response to an intravenous (i.v.) injection of GH-releasing hormone (GHRH, 0.25 μg/kg body weight (BW)) was examined after treatments to augment central DA using carbidopa (carbi, 1 mg/kg BW) and L-dopa (1 mg/kg BW) in male and female goats under a 16-h photoperiod (16 h light, 8 h dark) condition. GHRH significantly and rapidly stimulated the release of GH after its i.v. administration to goats (P < 0.05). The carbi and L-dopa treatments completely suppressed GH-releasing responses to GHRH in both male and female goats (P < 0.05). The prolactin (PRL)releasing response to an i.v. injection of thyrotropin-releasing hormone (TRH, 1 μg/kg BW) was additionally examined in male goats in this study to confirm modifications to central DA concentrations. The treatments with carbi and L-dopa significantly reduced TRH-induced PRL release in goats (P < 0.05). These results demonstrated that hypothalamic DA was involved in the regulatory mechanisms of GH, as well as PRL secretion in goats.
Key words: dopamine, GH, GHRH, goat, PRL.
INTRODUCTION Dopamine (DA) is a predominant catecholamine neurotransmitter that is located in neuronal populations in the hypothalamus and brainstem of ruminants (Tillet & Thibault 1989; Leshin et al. 1995; Tillet 1995). DA may modulate the secretion of growth hormone (GH) in sheep and cattle. DA or a DA receptor agonist (bromocriptine) was shown to inhibit basal (Soyoola et al. 1994) and GH-releasing hormone (GHRH)induced GH release (Greg et al. 1984; Soyoola et al. 1994) from cultured ovine pituitary cells. A D1 DA receptor agonist (SKF38393) stimulated the release of somatostatin (SS, a GH inhibitory factor) and inhibited the release of GHRH (a GH stimulator factor) from perifused bovine hypothalamic tissues (West et al. 1997). A subcutaneous (s.c.) injection of SKF38393 suppressed the basal and GHRH-induced release of GH in steers (McMahon et al. 1998). Although these findings were obtained in sheep and cattle, most studies have been performed in vitro (Greg et al. 1984; Soyoola et al. 1994; West et al. 1997); therefore, further in vivo studies are needed to confirm in vitro findings in rumi© 2014 Japanese Society of Animal Science
nants. Furthermore, the effects of hypothalamic DA on the secretion of GH have not yet been determined in goats. L-dopa (a precursor of DA) is often used in in vivo studies on humans in order to clarify the effects of hypothalamic DA on the secretion of GH (Mars & Genuth 1973; Camanni et al. 1978; Bansal et al. 1981; Hanew et al. 1986, 1996; Jaffe et al. 1996) because DA cannot directly cross the blood-brain barrier (BBB) (Yen 1991). L-dopa can cross the BBB and convert DA in the hypothalamus by aromatic L-amino acid decarboxylase (AADC) (Yen 1991; Tóth et al. 2002). An AADC inhibitor, such as carbidopa (carbi), is typically administered with L-dopa to humans (Mars & Genuth 1973; Camanni et al. 1978; Bansal et al. 1981; Robertson et al. 1989) because it can block the
Correspondence: Tsutomu Hashizume, Faculty of Agriculture, Iwate University, Morioka 020-8550, Japan. (Email: [email protected]) Received 27 May 2014; accepted for publication 7 August 2014.
DOPAMINE AND GHRH-INDUCED GH SECRETION
peripheral conversion of L-dopa to DA without affecting brain decarboxylation (Pinder et al. 1976). Treatments with carbi alone have neither modified basal DA levels nor induced significant changes in basal GH, prolactin (PRL) or thyroid-stimulating hormone levels in humans (Camanni et al. 1978). When carbi was administered with L-dopa to humans, higher circulating L-dopa levels were observed than after L-dopa alone (Mars & Genuth 1973; Pinder et al. 1976; Bansal et al. 1981). Previous studies demonstrated that when an AADC inhibitor was administered with L-dopa to rats, greater brain DA concentrations were present than after L-dopa alone (Pletscher 1973; Pinder et al. 1976). In ruminants, a recent study demonstrated that an intravenous (i.v.) injection of L-dopa immediately increased DA concentrations in the cerebrospinal fluid collected from the third ventricle in steers (Kasuya et al. 2013). The content of DA in the median eminence was shown to be higher under long photoperiods than short photoperiods in sheep (Thiery 1991). Increases have also been reported in DA and noradrenaline turnover in the median eminence and mediobasal hypothalamus/arcuate nucleus with a long daylength (Curlewis 1992). These findings suggest that a more active DAergic system may exist in the brain under long photoperiods. We recently demonstrated that a long photoperiod enhanced the secretion of GH in goats (Jin et al. 2012, 2013a, b), suggesting that a long photoperiod may influence the hypothalamic DAergic system to modulate the secretion of GH in goats. Therefore, the relationship between GH secretion and the hypothalamic DAergic system in goats under a long photoperiod should be examined in more detail. The secretion of PRL is generally considered to be under the dominant and tonic inhibitory control of hypothalamic DA (Neil & Nagy 1994; Freeman et al. 2000). On the other hand, the secretion of PRL is known to be stimulated by thyrotropin-releasing hormone (TRH) (Yen 1991; Neil & Nagy 1994; Freeman et al. 2000). We previously showed that TRHinduced PRL secretion was suppressed by DA both in vivo and in vitro in ruminants (Hashizume et al. 2009, 2012). Therefore, elucidating the PRL-releasing response to TRH after treatments with carbi plus L-dopa may confirm enhancements in the hypothalamic DAergic system in the present study. The aim of the present study was to clarify the effects of hypothalamic DA on GHRH-induced GH secretion in vivo in goats. The GH-releasing response to an i.v. injection of GHRH was examined after treatments with carbi plus L-dopa in both female and male goats under a long photoperiod. The PRL-releasing response to TRH was also examined in male goats in the present study to confirm modifications to central DA concentrations. Animal Science Journal (2015) 86, 634–640
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MATERIALS AND METHODS The Animal Care and Use Committee of Iwate University approved the experimental and animal care protocols (A201309).
Animals Five native Japanese adult male goats (Shiba goats) (age 1–2 years; mean body weight (BW) 23.1 kg) and five adult female goats (age 1–2 years; mean BW 16.4 kg) were used. Animals were housed in a room in which temperature, humidity, and lighting were strictly controlled. They were fed mixed hay of orchardgrass and reed canarygrass (1.5 kg/ head) and concentrate (50 g/head) at 09.00 hours daily. Water was continuously available. The residuum of the hay was removed before the start of blood collections, and the goats were not fed until after the experiment. Metabolizable energy (ME)/day was assumed to be 2500 Kcal. Experiments were performed in Morioka (39°42′N, 141°09′E), Japan.
Experimental design Experiment 1. Effects of hypothalamic DA on GHRH-induced GH secretion Five male goats were each housed in a separate pen in an environmentally controlled room in which room temperature, humidity and lighting were maintained at 20°C, 60% and 16 h of light (08.00 to 24.00 hours): 8 h of darkness (24.00 to 08.00 hours) (16:8 L : D), respectively. Goats were habituated for 3 weeks to all experimental conditions, and blood was then collected. Blood was drawn from an indwelling catheter at 15-min intervals for 3 h. Animals were given i.v. injections of GHRH (0.25 μg/kg BW in 2 mL of saline; hGHRH (1–29)-NH2, ANA Spec, Fremont, CA, USA) (GHRH alone) or 2 mL of saline as a control (CTL). GHRH was injected 1 h after the commencement of blood sampling. GHRH was also injected after the carbi (1 mg/kg BW in 5 mL of saline; Tokyo Chemical Industry Co. Ltd, Tokyo, Japan) and L-dopa (1 mg/kg BW in 5 mL of saline; Sigma Aldrich Co., St. Louis, MO, USA) treatments (carbi + Ldopa + GHRH). The doses of GHRH, carbi and L-dopa were selected based on the experimental protocols of our previous studies (Hashizume et al. 1997, 2005; Jin et al. 2014). Carbi and L-dopa were intravenously administered 30 min and 15 min, respectively, before the GHRH injection. Experiments were carried out at 2–3-day intervals according to the experimental protocol described in our previous study (Jin et al. 2014). Blood was collected between 11.00 and 14.00 hours. The order in which each goat received the treatments was determined at random. Blood samples were collected into centrifuge tubes containing heparin and immediately chilled with ice. Individual plasma samples were obtained after centrifugation and stored at −30°C until assayed. The experiments described for the male goats were also performed in the female goats. In the present study, five female goats were used. The goats did not exhibit estrous under the present photic condition.
Experiment 2. Effects of hypothalamic DA on TRH-induced PRL secretion This experiment was designed to confirm whether the carbi and L-dopa treatments could augment hypothalamic DA levels. Four male goats were habituated for 3 weeks to the 16:8 L : D photoperiod, and the effects of the carbi and © 2014 Japanese Society of Animal Science
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GH and PRL concentrations in the plasma were measured using a double-antibody radioimmunoassay (Hashizume et al. 1999). The standard and hormone for iodination was USDA-bGH-B-1 for GH and NIDDK oPRL-1–3 for PRL. The intra- and inter-assay coefficients of variation (CV) for GH were 10.1% and 3.2%, respectively. PRL samples were assayed in a single run and the intra-assay CV for PRL was 8.5%. The assay sensitivities for GH and PRL were 0.30 ng/mL and 0.13 ng/mL, respectively.
Statistical analysis All data from the experiments are presented as the mean ± SEM. The significance of differences in plasma GH and PRL concentrations at each sampling time between the saline-treated control (CTL) and each treated group were evaluated by a two-way repeated measures analysis of variance (ANOVA), and the Bonferroni test was used as a post hoc test. The significance of differences in plasma GH and PRL concentrations at each sampling time between GHRH alone and carbi plus L-dopa plus GHRH (carbi + L-dopa + GHRH) or TRH alone and carbi plus L-dopa plus TRH (carbi + Ldopa + TRH) were also evaluated by a two-way repeated measures ANOVA. The significance of differences in the area under the response curve (AUC) of GH for 60 min after the injection of GHRH in the male and female groups was evaluated using the Student’s t–test. All data were analyzed using GraphPad Prism (GraphPad Software, San Diego, CA, USA). Results were considered significant at P < 0.05.
RESULTS Experiment 1. Effects of hypothalamic DA on GHRH-induced GH secretion GH plasma levels in response to an i.v. injection of GHRH with or without the carbi and L-dopa treatments in male and female goats are shown in Figures 1 and 2, respectively. Plasma GH concentrations increased immediately after the injection of GHRH in the carbi and L-dopa non-treated groups (GHRH alone) in both male and female goats (P < 0.05). Maximum values were observed 15 min after the injection of GHRH in male (40 ± 8 ng/mL) and female (48 ± 10 ng/mL) goats, respectively. The AUCs of GH for 60 min after the injection of GHRH were 1132 ± 289 ng/min/mL and 1198 ± 352 ng/min/mL, respectively, in the male and female goats. GHRH failed to stimulate the release of GH in the carbi and L-dopa-treated groups (carbi + L-dopa + GHRH), and the AUC values were 257 ± 106 ng/min/mL and 228 ± 64 ng/min/mL, respectively, in the male and female goats. No significant differences were observed © 2014 Japanese Society of Animal Science
CTL
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Time in minutes Figure 1 Plasma concentrations of growth hormone (GH) in response to the intravenous injection of GH-releasing hormone (GHRH, 0.25 μg/kg body weight (BW)) with or without the treatments with carbidopa (Carbi, 1 mg/kg BW) plus L-dopa (1 mg/kg BW) in male goats. Saline was injected as a control (CTL). Goats were kept under the photoperiod condition of 16 h of light: 8 h of dark (16:8 L : D). Each value represents the mean ± SEM of five animals. Arrows indicate the time of the injection. The different letters (a, b) at each time period denote significant differences (P < 0.05). *P < 0.05 significantly different from the corresponding values from CTL.
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L-dopa treatments on TRH-induced PRL secretion were examined using a similar experimental protocol to that described in experiment 1. TRH (1 μg/kg BW in 2 mL of saline; Peptide Institute Inc., Osaka, Japan) was intravenously injected 1 h after the commencement of blood sampling. The dose of TRH was chosen according to the experimental protocols of our previous studies (Hashizume et al. 2009, 2012; Yaegashi et al. 2012).
a
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Time in minutes Figure 2 Plasma concentrations of growth hormone (GH) in response to the intravenous injection of GH-releasing hormone (GHRH, 0.25 μg/kg body weight (BW)) with or without the treatments with carbidopa (Carbi, 1 mg/kg BW) plus L-dopa (1 mg/kg BW) in female goats. Saline was injected as a control (CTL). Goats were kept under the photoperiod condition of 16 h of light: 8 h of dark (16:8 L : D). Each value represents the mean ± SEM of five animals. Arrows indicate the time of the injection. The different letters (a, b) at each time period denote significant differences (P < 0.05). *P < 0.05 significantly different from the corresponding values from CTL.
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in AUC values between the male and female goats. The concentrations of GH 15 and 30 min after the injection of GHRH in the carbi and L-dopa-treated groups (carbi + L-dopa + GHRH) were also significantly lower than those in the non-treated groups (GHRH alone) in both the male and female goats (P < 0.05).
Experiment 2. Effects of hypothalamic DA on TRH-induced PRL secretion PRL plasma levels in response to an i.v. injection of TRH with or without the carbi and L-dopa treatments in male goats are shown in Figure 3. Plasma PRL concentrations increased immediately after the injection of TRH in the carbi and L-dopa non-treated groups (TRH alone), and reached a maximum value (543 ± 100 ng/ mL) 30 min after the injection (P < 0.05). PRL concentrations remained higher than those in the salineinjected controls (CTL) during the 105-min period (P < 0.05). Plasma PRL concentrations also increased immediately after the injection of TRH in the carbi and L-dopa-treated groups (carbi + L-dopa + TRH), and PRL concentrations 15 min after the injection of TRH (66 ± 22 ng/mL) were significantly higher than the CTL treatment (P < 0.05). The concentrations of PRL 15-105 min after the injection of TRH in the carbi and L-dopa-treated groups (carbi + L-dopa + TRH) were significantly lower than those in the non-treated group (TRH alone) (P < 0.05).
800
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* TRH L-dopa Carbi
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Time in minutes Figure 3 Plasma concentrations of prolactin (PRL) in response to the intravenous injection of thyrotropin-releasing hormone (TRH, 1 μg/kg body weight (BW)) with or without the treatments with carbidopa (Carbi, 1 mg/kg BW) plus L-dopa (1 mg/kg BW) in male goats. Saline was injected as a control (CTL). Goats were kept under the photoperiod condition of 16 h of light: 8 h of dark (16:8 L : D). Each value represents the mean ± SEM of four animals. Arrows indicate the time of the injection. The different letters (a, b) at each time period denote significant differences (P < 0.05). *P < 0.05 significantly different from the corresponding values from CTL.
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DISCUSSION The present study attempted to clarify the relationship between the hypothalamic DA and the GHRH-induced secretion of GH in vivo in goats. The results obtained clearly demonstrated that the treatments with carbi and L-dopa suppressed GHRH-induced GH release in both male and female goats under the long photoperiod condition. This is the first study to demonstrate that hypothalamic DA may alter GHRH-induced GH release in goats. The present study was performed using a long photoperiod, which was previously shown to enhance the secretion of GH in male and female goats (Jin et al. 2012, 2013a, b). We previously reported that the GH pulse amplitude was slightly bigger and the mean GH concentrations were significantly higher in the long photoperiod (16:8 L : D) than in the short photoperiod (8:16 L : D) (Jin et al. 2012, 2013a, b). The present study was conducted under such enhanced GH secretory conditions. Previous studies demonstrated that the secretory pattern of GH was sexually dimorphic in rats (Tannenbaum & Martin 1976; Jansson et al. 1985; Veldhuis 1998) and humans (Stolar & Baumann 1986; Veldhuis 1996, 1998). In ruminants, sexual steroid hormones were suggested to be involved in modifications to pulsatile GH secretion in female goats (Yonezawa et al. 2005); however, we did not observed any significant differences in the mean concentration, pulse amplitude or pulse frequency of GH between the estrous and luteal phases in goats (Hashizume et al. 2000). Furthermore, testicular androgen was suggested to modify the secretory pattern of GH pulses in male goats (Mogi et al. 2002). However, enhancing hypothalamic DA had no significant effect on the GHRH-induced release of GH between the sexes in the present study. The oral administration of L-dopa alone (Mars & Genuth 1973; Hanew et al. 1986), an AADC inhibitor (MK-486) plus L-dopa (Mars & Genuth 1973) and carbi plus L-dopa (Bansal et al. 1981) was shown to increase basal plasma GH concentrations in humans. In these studies, the GH peaks induced by L-dopa appeared 60–90 min after its administration. L-dopainduced basal GH secretion was suppressed by a GHRH antagonist in humans (Hanew et al. 1996; Jaffe et al. 1996); therefore, the stimulation of basal GH secretion by dopaminergic pathways in humans appears to require endogenous GHRH. Although these findings were reported in humans, the pretreatment with carbi plus L-dopa completely suppressed GHRH-induced GH release in the present study. The present results in vivo in goats support a selective DA D1 receptor agonist inhibiting the GHRH-induced release of GH in vivo in steers (McMahon et al. 1998). However, the effects of the treatment with carbi plus L-dopa on basal GH © 2014 Japanese Society of Animal Science
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secretion in goats also need to be examined in future studies. We did not examine the effects of carbi alone on basal and GHRH-induced GH secretion in the present study because it was reported that the treatment with carbi alone did not modify basal GH levels in humans (Camanni et al. 1978). However, the effects of the treatment with carbi alone on basal and GHRHinduced GH secretion in goats may also need to be examined in future studies. The secretion of GH is mainly regulated by GHRH neurons in the arcuate nucleus (ARC) and somatostatin (SS) neurons in the periventricular nucleus (PeVN) (Leshin et al. 1994; McMahon et al. 2001). DA receptors have been separated into two categories: D1-like receptors, which can increase intracellular levels of cyclic adenosine 3′,5′-monophosphate (cAMP) by activating adenylate cyclase (AC), and D2-like receptors, which can inhibit the activation of AC (Missale et al. 1998). The stimulation of D1 receptors by subcutaneous injections of a DA1 receptor agonist has been shown to activate SS neurons in the PeVN and suppress basal and GHRH-induced GH secretion in steers (McMahon et al. 1998). The stimulation of D1 receptors was shown to increase the release of SS, but decreased the release of GHRH from bovine hypothalamic tissues in vitro (West et al. 1997). Furthermore, DA stimulated the release of SS from isolated hypothalamic synaptosomes in rats (Wakabayashi et al. 1977). Therefore, in the present study, enhancing hypothalamic DA may have stimulated the secretion of SS via D1 receptors in PeVN while SS suppressed GHRH-induced GH secretion. Increased SS levels may act directly on somatotrophs to inhibit GHRH-induced GH secretion. SS has been shown to suppress GHRHinduced GH secretion from bovine anterior pituitary cells (Hashizume et al. 1994). In addition, enhanced hypothalamic DA may also have directly inhibited the release of GH from somatotrophs in the pituitary gland via D2 receptors. A treatment with a D2 receptor agonist reduced the secretion of GH by 30% in human pituitary tumors (Gruszka et al. 2012). DA inhibited GHRH-elevated GH secretion by 50% and lowered cAMP levels by 30% in ovine pituitary cells in vitro (Law et al. 1984). McMahon et al. (2001) also reported that DA could inhibit the secretion of GH from somatotrophs via D2 receptors in domestic animals. PRL-releasing responses to TRH were blunted after the treatments with carbi and L-dopa in goats. This result was consistent with previous findings in humans (Carlson 1986). The present PRL results suggest that the carbi and L-dopa treatments could effectively enhance central DA-derived L-dopa in the goat brain, and also that enhanced hypothalamic DA could reach the pituitary lactotrophs through the pituitary portal circulation. DA has been shown to inhibit the synthesis and release of PRL by acting directly on lactotrophs (Yen 1991; Freeman et al. © 2014 Japanese Society of Animal Science
2000). After binding DA, the D2 receptor complex activates a membrane inhibitory Gi-protein, that is negatively coupled to the AC and phospholipase C (PLC) systems (Yen 1991; Missale et al. 1998). TRH stimulates the release of PRL from lactotrophs by activating the PLC system (Yen 1991); therefore, the PLC system associated with PRL secretion may be blocked by elevated central DA levels. In conclusion, hypothalamic DA was found to be involved in the regulatory mechanisms underlying GH secretion, as well as PRL secretion in goats.
ACKNOWLEDGMENTS The authors wish to thank Dr. T. Johke, National Institute of Animal Industry, Tsukuba, Japan, for providing monkey anti-bGH, and the USDA Animal Hormone Program, Beltsville, MD, USA, for providing USDAbGH-B-1. They also wish to thank Dr. A. F. Parlow, National Hormone and Peptide Program, Harbor-UCLA Medical Center, Torrance, CA, USA for providing oPRL (NIDDK oPRL-1-3) and oPRL antiserum (AFP-C358106). This research was supported in part by a Grant-in-Aid for Scientific Research (No.24380149) provided by the Japan Society for the Promotion of Science (JSPS).
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