Calcitonin gene-related peptide-induced suppression of luteinizing hormone pulses in the rat: the role of endogenous opioid peptides
1 Division of Reproductive Health, Endocrinology and Development
2 Cardiovascular Division
3 Department of Pharmacology and Therapeutics, New Hunt's House, King's College London, Guy's Campus, London SE1 1UL, UK
4 Henry Wellcome Laboratory for Integrative Neuroscience and Endocrinology, Dorothy Hodgkin Building, University of Bristol, Bristol BS1 3NY, UK
Abstract
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Calcitonin gene-related peptide (CGRP) is involved in a variety of stress responses in the rat. Central administration of CGRP activates the hypothalamo–pituitary–adrenal axis resulting in increased corticosterone secretion. We have previously shown that central CGRP suppresses the gonadotrophin-releasing hormone (GnRH) pulse generator, specifically LH pulses. Endogenous opioid peptides (EOPs) have been shown to play an important role in stress-induced suppression of the reproductive axis. The aim of the present study was to test the hypothesis that EOPs mediate CGRP-induced suppression of pulsatile LH secretion. Ovariectomized rats were implanted with intracerebroventricular (I.C.V.) and I.V. cannulae. Intravenous administration of the opioid antagonist naloxone (250 μg) completely blocked the suppression of LH pulses induced by 1.5 μg I.C.V. CGRP and significantly attenuated the suppression of pulsatile LH secretion induced by 5 μg I.C.V. CGRP. Furthermore, intravenous administration of naloxone was found to immediately restore normal LH pulse frequency in animals treated 90 min earlier with 1.5 μg I.C.V. CGRP. Co-administration (I.C.V.) of CGRP (1.5 μg) with the μ and opioid receptor-specific antagonists naloxone (10 μg) and norbinaltorphimine (5 μg), respectively, blocked the CGRP-induced suppression of LH pulses, whilst I.C.V. co-administration of CGRP (1.5 μg) with the opioid receptor-specific antagonist naltrindole (5 μg) did not. These data provide evidence that EOPs play a pivotal role in mediating the inhibitory effects of CGRP on pulsatile LH secretion in the rat. They also suggest that the μ and , but not the , opioid receptors may be responsible for mediating the effects of CGRP on LH pulses.
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Introduction
Calcitonin gene-related peptide (CGRP), a 37-amino-acid neuropeptide best known for its potent vasodilatory properties (Brain et al. 1985), has been shown to evoke a classic stress response by stimulating the hypothalamo–pituitary–adrenocortical (HPA) axis (Kovacs et al. 1995; Dhillo et al. 2003) and sympathetic outflow (Fisher et al. 1983). CGRP has also been implicated in anorectic (Lutz et al. 1998), addictive (Ehlers et al. 1999) and fear-related behaviours (Poore & Helmstetter, 1996). Additionally, CGRP has recently been shown to have a profound and dose dependent inhibitory action on the hypothalamic gonadotrophin-releasing hormone (GnRH) pulse generator, decreasing luteinizing hormone (LH) pulse frequency in the rat (Li et al. 2004). A pivotal role in stress-induced suppression of the GnRH pulse generator is also evident since the CGRP receptor antagonist, CGRP8–37, completely blocked hypoglycaemic stress-induced suppression of LH pulses (Li et al. 2004). The mechanisms underlying this central inhibitory action of CGRP on the reproductive neuroendocrine axis remain unknown. Corticotrophin releasing-hormone (CRH), a crucial mediator of stress-induced activation of the HPA axis, and inhibition of the GnRH pulse generator may mediate, in part, the CGRP-induced suppression of LH pulses (Li et al. 2004). There is substantial evidence for a pivotal role for endogenous opioid peptides (EOPs) in the regulation of the GnRH pulse generator. In a variety of species, including rats and primates, the suppression of LH release induced by central administration of CRH can be either attenuated or completely blocked by the opioid antagonist naloxone (Gindoff & Ferin, 1987; Almeida et al. 1988; Rivest et al. 1993). Furthermore, EOPs mediate suppression of pulsatile LH secretion in response to a variety of stressors from metabolic (Cagampang & Maeda, 1991; Bonavera et al. 1993; Cagampang et al. 1997) to behavioural (O'Byrne et al. 1989). The present study was designed to investigate whether EOPs are involved in CGRP-induced suppression of pulsatile LH secretion and to establish the opioid receptor subtypes mediating the response in the rat.
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Methods
Animals and surgical procedures
Adult female Wistar rats, weighing 230–280 g, obtained from B & K Suppliers, Ltd (Hull, UK), were housed under controlled conditions (12: 12 h light–dark; lights on at 07.00 h; temperature at 22 ± 2°C) and provided with food and water ad libitum. All animal procedures were undertaken in accordance with the UK Home Office regulations. All surgical procedures were carried out under ketamine (100 mg kg–1 I.P.; Pharmacia and Upjohn Ltd, Crawley, UK) and Rompun (10 mg kg–1 I.P.; Bayer, Leverkusen, Germany) anaesthesia. Rats were bilaterally ovariectomised. At the time of ovariectomy, all rats were also fitted with an intracerebroventricular (I.C.V.) guide cannula (22 gauge; Plastics One, Roanoke, VA, USA) directed towards the left lateral cerebral ventricle, the co-ordinates for implantation being 1.5 mm lateral, 0.6 mm posterior to Bregma, and 3.5 mm below the surface of the dura (Paxinos & Watson, 1986). The guide cannula was secured using dental cement (Dental Filling Ltd, Swindon, UK), and fitted with a dummy cannula (Plastics One) to maintain patency (Cates et al. 1999). Following a 10-day recovery period, the rats were fitted with two indwelling cardiac catheters via the jugular veins (Li et al. 2003). The catheters were exteriorized at the back of the head and secured to a cranial attachment: the rats were fitted with a 30-cm long metal spring tether (Instec Laboratories Inc., Boulder, CO, USA). The distal end of the tether was attached to a fluid swivel (Instec Laboratories), which allowed the rat freedom to move around the enclosure. Experimentation commenced 3 days later.
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Effect of CGRP on LH pulses
On the morning of experimentation, an I.C.V. injection cannula (Plastics One) with extension tubing, preloaded with drug or vehicle, was inserted into the guide cannula. The distal end of the tubing was extended outside of the animal cage to allow remote infusion without disturbing the rat during the experiment. The injection cannulae, which extended 1.0 mm beyond the tip of the guide cannula, reached the injection site, the lateral cerebral ventricle. Rats were then attached via one of the two cardiac catheters to a computed-controlled automated blood sampling system, which allows for the intermittent withdrawal of small blood samples (25 μl) without disturbing the rats (Cates et al. 1999). Experimentation commenced between 09.00 h and 11.00 h when blood samples were taken every 5 min for 6 h. After removal of each 25-μl blood sample, an equal volume of heparinized saline (10 U ml–1 normal saline; CP Pharmaceuticals Ltd, Wrexham, UK) was automatically infused into the animals to maintain patency of the catheter and blood volume. Blood samples were frozen at –20°C for later assay to determine LH concentrations. After 2 h of sampling the animals were injected I.V. with 250 μg naloxone in 200 μl saline, followed 10 min later by the substance to be tested, injected I.C.V. in 4 μl artificial cerebrospinal fluid vehicle (1.5 μg CGRP, 5 μg CGRP or aCSF alone; n = 8, 8 and 7, respectively). Automated sampling continued for 4 h after the I.C.V. injection. In separate groups of animals CGRP (1.5 μg) was administered I.C.V. after 2 h of baseline blood sampling, followed 90 min later by I.V. injection of either 250 μg/200 μl naloxone (n = 7) or 200 μl saline (n = 4). Blood sampling was continued for a further 4 h after the CGRP injection.
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To test the specific opioid receptors involved in the response to CGRP, animals were co-administered 1.5 μg CGRP along with the specific opioid antagonist to be tested. These were naloxone (10 μg, a μ-receptor specific antagonist at this dose, n = 7), naltrindole (5 μg, a -receptor specific antagonist, n = 8) or norbinaltorphimine (nor-BNI, 5 μg, a -receptor specific antagonist, n = 8). Both CGRP and the specific opioid antagonists were administered I.C.V. due to the inability of naltrindole to cross the blood–brain barrier. All I.C.V. injections were given in 4 μl aCSF vehicle. To test whether -receptor activation resulted in suppression of LH pulses, the -receptor-specific agonist U69593 (100 μg, n = 9) was administered I.C.V. after 2 h of baseline blood sampling, and sampling then continued for a further 4 h. U69593 was dissolved in 1 M hydrochloric acid and then diluted in aCSF. Sodium hydroxide (1 M) was then used to neutralize this mixture before injection (total volume injected: 4 μl). Control animals received (4 μl, I.C.V.) the neutralized HCl/NaOH vehicle (n = 5). Different groups of animals were used for each individual treatment group.
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Radioimmunoassay for LH
A double antibody radioimmunoassay supplied by the National Institute of Diabetes, Digestion and Kidney Disease (NIDDK) (Monroe et al. 1968) was used to determine LH concentration in the 25 μl whole blood sample. Each blood sample was measured as a singleton. The sensitivity of the assay was 0.093 ng ml–1. The intra-assay variation was 5.8% and the interassay variation was 5.0%.
Statistical analysis
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Detection of LH pulses was established by use of the algorithm ULTRA (Van Cauter, 1988). Two intra-assay coefficients of variation of the assay were used as the reference threshold for the pulse detection. The effect of treatment on pulsatile LH secretion was calculated by comparing the mean LH pulse interval before and after drug administration and expressed as ‘prolongation of LH pulse interval’ as a percentage of the pretreatment control value. In the case of animals in which no LH pulses were observed during the post-treatment period, these were assigned a value of 4 h for the post-treatment LH pulse interval for the purposes of analysis. Statistical significance was tested on raw data using one-way ANOVA and Dunnett's test. P < 0.05 was considered statistically significant.
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Results
Intracerebroventricular administration of CGRP resulted in a suppression of pulsatile LH secretion as previously demonstrated (Li et al. 2004). Intravenous administration of naloxone (250 μg; 10 min prior to CGRP) significantly attenuated the suppression of pulsatile LH release in response to the high dose of CGRP (5 μg, I.C.V.) and completely blocked the inhibitory effects of the lower dose of CGRP (1.5 μg, I.C.V.) on LH pulses (Fig. 1C–G). Administration of either the aCSF (4 μl, I.C.V.) or naloxone (250 μg, I.V.) alone had no effect on LH pulse frequency (Fig. 1A, B and G). To determine whether EOPs are involved in merely initiating the LH response to central CGRP or play a role in maintaining LH suppression over several hours, naloxone (250 μg, I.V.) was administered 90 min after the onset of I.C.V. CGRP (1.5 μg, I.C.V.) induced suppression of pulsatile LH release. Administration of naloxone (250 μg, I.V.) 90 min after the injection of CGRP (1.5 μg, I.C.V.) immediately restored LH pulse frequency to control values whilst administration of saline had no effect (Fig. 2A–C). Given that saline administration had no effect, results from CGRP (1.5 μg, I.C.V.) followed by saline (200 μl, I.V.) treated rats were combined with results from animals treated with CGRP (1.5 μg, I.C.V.) alone for the purposes of analysis.
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Representative examples demonstrating the effects of 4 μl aCSF I.C.V. (A), 250 μg naloxone I.V. (B), 1.5 μg CGRP I.C.V. (C), 1.5 μg CGRP I.C.V. + 250 μg naloxone I.V. (D), 5 μg CGRP I.C.V. (E), and 5 μg CGRP I.C.V. + 250 μg naloxone I.V. (F) on pulsatile LH secretion in ovx rats. Asterisks denote LH pulses. G, summary of the effects of treatments with various doses of CGRP, and naloxone on pulsatile LH secretion in ovx rats. Note the dose-dependant inhibitory effect of CGRP on LH pulses. P < 0.05 versus aCSF control. P < 0.05 versus 5 μg CGRP I.C.V.n = 6–12.
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Representative examples showing the effects of 1.5 μg CGRP injected I.C.V. after 2 h of control baseline blood sampling and 200 μl saline injected I.V. 90 min later (A) and 1.5 μg CGRP injected I.C.V. after 2 h of blood sampling and 250 μg naloxone injected I.V. 90 min later (B) on pulsatile LH secretion in ovariectomized rats. C, summary of the effect of naloxone when given 90 min after CGRP administration on pulsatile LH secretion in ovx rats. Note that naloxone immediately restored LH pulse interval to pre-CGRP treatment values. Asterisks denote LH pulses. P < 0.05 versus aCSF control. P < 0.05 versus 1.5 μg CGRP I.C.V. and 200 μl saline I.V.n = 7–12.
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To investigate the specific opioid receptors involved in the inhibitory effect of CGRP on pulsatile LH secretion, we co-administered, by I.C.V. injection, CGRP (1.5 μg) with the specific opioid antagonists naloxone, naltrindole or nor-BNI. It was found that both naloxone (10 μg, I.C.V.) and nor-BNI (5 μg, I.C.V.) were effective in blocking the CGRP-induced suppression of LH pulses (Fig. 3B, C, E and H). Naltrindole (5 μg, I.C.V.) had no effect on the CGRP-induced suppression of LH pulses (Fig. 3G and H). Neither naloxone or nor-BNI had any significant effect on LH pulse frequency when given alone (Fig. 3D, F and H). Additionally, it was shown that I.C.V. administration of U69593 (100 μg), a -specific opioid receptor agonist, is capable of suppressing pulsatile LH release (Fig. 4).
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Representative examples demonstrating the effects of I.C.V. administration of 4 μl aCSF (A), 1.5 μg CGRP (B), 1.5 μg CGRP + 10 μg naloxone (C), 10 μg naloxone (D), 1.5 μg CGRP + 5 μg nor-BNI (E), 5 μg nor-BNI (F), and 1.5 μg CGRP + 5 μg naltrindole (G) on pulsatile LH secretion in ovx rats. Asterisks denote LH pulses. H, summary of the effects of treatments with 1.5 μg CGRP and specific opioid receptor antagonists on pulsatile LH secretion in ovx rats. P < 0.05 versus aCSF control. P < 0.05 versus 1.5 μg CGRP I.C.V.. n = 6–12.
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Representative examples demonstrating the effects of I.C.V. administration of 4 μl neutralized HCl/NaOH vehicle (A) and 100 μg U69593, a opioid receptor agonist (B) on pulsatile LH secretion in ovx rats. Asterisks denote LH pulses. C, the opioid receptor agonist prolonged LH interpulse interval. P < 0.05 versus vehicle control. n = 5–9.
Discussion
The data from the present study provide the first evidence that EOPs are involved in the CGRP-induced suppression of the GnRH pulse generator, since the opioid antagonist naloxone completely blocks the inhibitory effect of the lower dose of CGRP on pulsatile LH secretion. Although naloxone profoundly attenuates the inhibitory response to the higher dose of CGRP, the absence of a complete blockage may be related to an inadequate dose of naloxone or a combination of opioid and non-opioid mechanisms activated. The ability of naloxone, when given 90 min after CGRP administration (lower dose), to immediately restore a normal LH pulse frequency suggests that opioid involvement is not only important for the onset of CGRP-induced effects on LH secretion, but also for the continued maintenance of CGRP-induced suppression of the hypothalamic GnRH pulse generator.
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There is an extensive literature demonstrating that EOPs inhibit GnRH pulse generator activity. Naloxone has been shown to stimulate the release of LH in the rat (Pfeiffer & Herz, 1984) and to increase the frequency of multiunit activity volleys recorded from the mediobasal hypothalamus, an electrophysiological correlate of GnRH pulse generator activity in both the ovariectomized rat (Sano et al. 1999) and the rhesus monkey, although the presence of oestradiol is required in the latter species (Grosser et al. 1993). The absence of an effect of naloxone per se on LH pulse frequency in the present study may be related to the lower dose used compared with previous studies (Sano et al. 1999). The administration of opiate agonists such as morphine has also been demonstrated to suppress LH release (Pfeiffer & Herz, 1984; Kesner et al. 1986). Furthermore, in vitro studies show that naloxone induces GnRH release from hypothalamic explants (Rubin, 1993), whilst morphine suppresses GnRH release from isolated medio-basal hypothalamic fragments (Giri & Kaufman, 1994). It is well established that EOPs are physiologically relevant in mediating stress-induced suppression of LH pulses. In the monkey the inhibitory effects of behavioural stressors on LH pulses can be blocked by naloxone administration (O'Byrne et al. 1989), whilst in the rat naloxone administration blocks the LH pulse-suppressing effects of a variety of stressors, including fasting, interleukin-1 and insulin-induced hypoglycaemia (Cagampang & Maeda, 1991; Bonavera et al. 1993; Cagampang et al. 1997). We have previously demonstrated that endogenous CGRP is involved in hypoglycaemic stress-induced suppression of LH pulses (Li et al. 2004) and therefore our current data are consistent with the literature describing a role for EOPs in the hypoglycaemic stress-induced suppression of the GnRH pulse generator.
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The mechanism through which EOPs affect the GnRH pulse generator is currently unknown. Whilst in both the rat and the monkey direct synaptic contacts have been found between opioidergic and GnRH neurones (Thind & Goldsmith, 1988; Chen et al. 1989), GnRH neuronal cell bodies do not appear to express μ, , or opioid receptor mRNA (Sannella & Petersen, 1997; Mitchell et al. 1997). Additionally, studies in GT1–7 cells, a GnRH neuronal cell line, indicate that these cells do not express μ opioid receptors (Maggi et al. 1995a), nor is GnRH release from the GT1–7 cells affected by treatment with μ opioid agonists (Maggi et al. 1995b). However, it has recently been shown that GnRH neurones in vivo are not as poorly innervated as previously thought, and in fact receive abundant synaptic inputs onto dendritic processes, which were hitherto not realized to have such far reaching projections (Campbell et al. 2005). This raises the exciting possibility that despite a lack of opioid receptors on the cell body, direct interactions of EOPs with the GnRH neurones may still be possible through synaptic inputs onto their dendritic processes, particularly since opioid receptors have been found on neuronal dendrites in other regions of the rat brain (Wang et al. 2003; Pickel et al. 2004).
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Nevertheless, an intermediary role for other neurotransmitter systems, such as -aminobutyric acid (GABA), in EOP effects on the GnRH neural network has to be considered. Indeed, GABA neurones within the preoptic area have been shown to contain abundant opioid receptors and also form extensive synapses onto GnRH neurones (Leranth et al. 1985). However, whilst it has been shown that GABA receptor stimulation in the preoptic area attenuates GnRH release (Tomaszewska-Zaremba et al. 2002), functional studies indicate that the inhibitory effects of EOP on LH release do not involve the preoptic GABAergic system (Jarry et al. 1995).
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Our results with the selective opioid receptor antagonists indicate a role for both the μ and opioid receptors in the CGRP-induced suppression of LH pulses, but not for the opioid receptors. Naltrindole and norbinaltorphimine are highly selective and antagonists, respectively, whereas naloxone displays much less selectivity for the μ receptor (Corbett et al. 1993). In in vitro assays naltrindole has Ke values at the , μ and receptors of 0.3, 22 and 100 nM, respectively, norbinaltorphimine has Ke values at the , μ and receptors of 16, 25 and 0.05 nM, respectively, and naloxone has Ke values at the , μ and receptors of 46, 2 and 16 nM respectively. The concentrations of the antagonists used in the present study are similar to those used in investigations of the role of the different opioid receptors in a number of different paradigms (Kalra et al. 2001; Ishihara et al. 2001; Silva et al. 2001). It is well established that μ opioid receptor activation results in a suppression of LH levels (Marko & Romer, 1983). There is, however, contradictory evidence over the ability of or opioid receptor agonists to inhibit LH. Several studies have demonstrated that and opioid receptor agonists suppress LH levels (Leadem & Yagenova, 1987; Pfeiffer et al. 1987) whilst others have failed to show an effect on LH secretion (Pfeiffer et al. 1983; Mallory & Gallo, 1990). Our data indicate that the opioid receptor agonist U69593 is able to decrease LH pulse frequency. In binding assays, U69593 has Ki values of 1.4, 2350 and 20000 nM at the , μ and receptors, respectively (Corbett et al. 1993). The analgesic effects of 25 μg U69693 I.C.V. are blocked by norbinaltorphimine 30 μg I.C.V., a dose of norbinaltorphimine which does not affect analgesia produced by the selective agonists given I.C.V. [D-Ala2,MePhe4,Gly-ol5]enkephalin (μ) or [D-Pen2,D-Pen5]enkephalin () (Spanagel et al. 1994). This supports the suggestion that 100 μg U-69593 I.C.V. will selectively activate receptors. Thus the inhibitory effects of CGRP on LH pulses may be mediated through the opioid receptors in addition to the μ opioid receptor.
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The results of the current study are in keeping with our previously published data demonstrating a pivotal role of CRH in mediating, in part, the inhibitory effects of central CGRP on the GnRH pulse generator (Li et al. 2004). Given that in both the monkey and the rat the suppression of LH release induced by central CRH can be blocked or attenuated by opioid antagonists (Gindoff & Ferin, 1987; Almeida et al. 1988; Rivest et al. 1993), it seems likely that EOPs are involved downstream of CRH in the mechanism relaying the inhibitory effects of central CGRP on pulsatile LH secretion. We are currently working towards further characterizing the neural circuitry involved.
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References
Almeida OF, Nikolarakis KE & Herz A (1988). Evidence for the involvement of endogenous opioids in the inhibition of luteinizing hormone by corticotropin-releasing factor. Endocrinology 122, 1034–1041.
Bonavera JJ, Kalra SP & Kalra PS (1993). Mode of action of interleukin-1 in suppression of pituitary LH release in castrated male rats. Brain Res 612, 1–8.
Brain SD, Williams TJ, Tippins JR, Morris HR & MacIntyre I (1985). Calcitonin gene-related peptide is a potent vasodilator. Nature 313, 54–56.
, 百拇医药
Cagampang FR, Cates PS, Sandhu S, Strutton PH, McGarvey C, Coen CW & O'Byrne KT (1997). Hypoglycaemia-induced inhibition of pulsatile luteinizing hormone secretion in female rats: role of oestradiol, endogenous opioids and the adrenal medulla. J Neuroendocrinol 9, 867–872.
Cagampang FR & Maeda K (1991). Effects of intracerebroventricular administration of opiate receptor antagonists on the suppressed pulsatile LH release during acute fasting in ovariectomized estradiol-treated rats. Life Sci 49, 1823–1828.
, 百拇医药
Campbell RE, Han SK & Herbison AE (2005). Biocytin filling of adult GnRH neurons in situ reveals extensive, spiny, dendritic processes. Endocrinology 146, 1163–1169.
Cates PS, Forsling ML & O'Byrne KT (1999). Stress-induced suppression of pulsatile luteinising hormone release in the female rat: role of vasopressin. J Neuroendocrinol 11, 677–683.
Chen M, Lee J, Huang BS, Grekin RJ & Malvin RL (1989). Clonidine and morphine increase atrial natriuretic peptide secretion in anesthetized rats. Proc Soc Exp Biol Medical 191, 299–303.
, 百拇医药
Corbett AD, Paterson SJ & Kosterlitz HW (1993). Selectivity of ligands for opioid receptors. Handbook Exp Pharmacol 104, 645–679.
Dhillo WS, Small CJ, Jethwa PH, Russell SH, Gardiner JV, Bewick GA, Seth A, Murphy KG, Ghatei MA & Bloom SR (2003). Paraventricular nucleus administration of calcitonin gene-related peptide inhibits food intake and stimulates the hypothalamo-pituitary-adrenal axis. Endocrinology 144, 1420–1425.
, http://www.100md.com Ehlers CL, Somes C, Li TK, Lumeng L, Hwang BH, Jimenez P & Mathe AA (1999). Calcitonin gene-related peptide (CGRP) levels and alcohol. Int J Neuropsychopharmacol 2, 173–179.
Fisher LA, Kikkawa DO, Rivier JE, Amara SG, Evans RM, Rosenfeld MG, Vale WW & Brown MR (1983). Stimulation of noradrenergic sympathetic outflow by calcitonin gene-related peptide. Nature 305, 534–536.
Gindoff PR & Ferin M (1987). Endogenous opioid peptides modulate the effect of corticotropin-releasing factor on gonadotropin release in the primate. Endocrinology 121, 837–842.
, 百拇医药
Giri M & Kaufman JM (1994). Opioidergic modulation of in vitro pulsatile gonadotropin-releasing hormone release from the isolated medial basal hypothalamus of the male guinea pig. Endocrinology 135, 2137–2143.
Grosser PM, O'Byrne KT, Williams CL, Thalabard JC, Hotchkiss J & Knobil E (1993). Effects of naloxone on estrogen-induced changes in hypothalamic gonadotropin-releasing hormone pulse generator activity in the rhesus monkey. Neuroendocrinology 57, 115–119.
, 百拇医药
Ishihara S, Tsuchiya S, Horie S, Murayama T & Watanabe K (2001). Stimulatory effects of centrally injected kappa-opioid receptor agonists on gastric acid secretion in urethane-anesthetized rats. Eur J Pharmacol 418, 187–194.
Jarry H, Leonhardt S & Wuttke W (1995). The inhibitory effect of beta-endorphin on LH release in ovariectomized rats does not involve the preoptic GABAergic system. Exp Clin Endocrinol Diabetes 103, 317–323.
, 百拇医药
Kalra A, Urban MO & Sluka KA (2001). Blockade of opioid receptors in rostral ventral medulla prevents antihyperalgesia produced by transcutaneous electrical nerve stimulation (TENS). J Pharmacol Exp Ther 298, 257–263.
Kesner JS, Kaufman JM, Wilson RC, Kuroda G & Knobil E (1986). The effect of morphine on the electrophysiological activity of the hypothalamic luteinizing hormone-releasing hormone pulse generator in the rhesus monkey. Neuroendocrinology 43, 686–688.
, 百拇医药
Kovacs A, Biro E, Szeleczky I & Telegdy G (1995). Role of endogenous CRF in the mediation of neuroendocrine and behavioral responses to calcitonin gene-related peptide in rats. Neuroendocrinology 62, 418–424.
Leadem CA & Yagenova SV (1987). Effects of specific activation of mu-, delta- and kappa-opioid receptors on the secretion of luteinizing hormone and prolactin in the ovariectomized rat. Neuroendocrinology 45, 109–117.
, 百拇医药
Leranth C, MacLusky NJ, Sakamoto H, Shanabrough M & Naftolin F (1985). Glutamic acid decarboxylase-containing axons synapse on LHRH neurons in the rat medial preoptic area. Neuroendocrinology 40, 536–539.
Li XF, Bowe JE, Mitchell JC, Brain SD, Lightman SL & O'Byrne KT (2004). Stress-induced suppression of the gonadotropin-releasing hormone pulse generator in the female rat: a novel neural action for calcitonin gene-related peptide. Endocrinology 145, 1556–1563.
, http://www.100md.com
Li XF, Mitchell JC, Wood S, Coen CW, Lightman SL & O'Byrne KT (2003). The effect of oestradiol and progesterone on hypoglycaemic stress-induced suppression of pulsatile luteinizing hormone release and on corticotropin-releasing hormone mRNA expression in the rat. J Neuroendocrinol 15, 468–476.
Lutz TA, Rossi R, Althaus J, Del Prete E & Scharrer E (1998). Amylin reduces food intake more potently than calcitonin gene-related peptide (CGRP) when injected into the lateral brain ventricle in rats. Peptides 19, 1533–1540.
, 百拇医药
Maggi R, Pimpinelli F, Martini L & Piva F (1995a). Inhibition of luteinizing hormone-releasing hormone secretion by delta-opioid agonists in GT1-1 neuronal cells. Endocrinology 136, 5177–5181.
Maggi R, Pimpinelli F, Martini L & Piva F (1995b). Characterization of functional opioid delta receptors in a luteinizing hormone-releasing hormone-producing neuronal cell line. Endocrinology 136, 289–295.
Mallory DS & Gallo RV (1990). Medial preoptic-anterior hypothalamic area involvement in the suppression of pulsatile LH release by a mu-opioid agonist in the ovariectomized rat. Brain Res Bull 25, 251–257.
, 百拇医药
Marko M & Romer D (1983). Inhibitory effect of a new opioid agonist on reproductive endocrine activity in rats of both sexes. Life Sci 33, 233–240.
Mitchell V, Prevot V, Jennes L, Aubert JP, Croix D & Beauvillain JC (1997). Presence of mu and kappa opioid receptor mRNAs in galanin but not in GnRH neurons in the female rat. Neuroreport 8, 3167–3172.
Monroe SE, Parlow AF & Midgley AR Jr (1968). Radioimmunoassay for rat luteinizing hormone. Endocrinology 83, 1004–1012.
, 百拇医药
O'Byrne KT, Lunn SF & Dixson AF (1989). Naloxone reversal of stress-induced suppression of LH release in the common marmoset. Physiol Behav 45, 1077–1080.
Paxinos G & Watson C (1986). The Rat Brain in Stereotaxic Coordinates. Academic Press, London.
Pfeiffer A & Herz A (1984). Endocrine actions of opioids. Horm Metab Res 16, 386–397.
Pfeiffer DG, Pfeiffer A, Almeida OF & Herz A (1987). Opiate suppression of LH secretion involves central receptors different from those mediating opiate effects on prolactin secretion. J Endocrinol 114, 469–476.
, 百拇医药
Pfeiffer DG, Pfeiffer A, Shimohigashi Y, Merriam GR & Loriaux DL (1983). Predominant involvement of mu- rather than delta- or kappa-opiate receptors in LH secretion. Peptides 4, 647–649.
Pickel VM, Chan J, Kash TL, Rodriguez JJ & MacKie K (2004). Compartment-specific localization of cannabinoid 1 (CB1) and mu-opioid receptors in rat nucleus accumbens. Neuroscience 127, 101–112.
Poore LH & Helmstetter FJ (1996). The effects of central injections of calcitonin gene-related peptide on fear-related behavior. Neurobiol Learn Mem 66, 241–415.
, 百拇医药
Rivest S, Plotsky PM & Rivier C (1993). CRF alters the infundibular LHRH secretory system from the medial preoptic area of female rats: possible involvement of opioid receptors. Neuroendocrinology 57, 236–246.
Rubin BS (1993). Naloxone stimulates comparable release of luteinizing hormone-releasing hormone from tissue fragments from ovariectomized, estrogen-treated young and middle-aged female rats. Brain Res 601, 246–254.
, 百拇医药 Sannella MI & Petersen SL (1997). Dual label in situ hybridization studies provide evidence that luteinizing hormone-releasing hormone neurons do not synthesize messenger ribonucleic acid for mu, kappa, or delta opiate receptors. Endocrinology 138, 1667–1672.
Sano A, Funabashi T, Kawaguchi M, Shinohara K & Kimura F (1999). Intravenous injections of nicotine decrease the pulsatile secretion of LH by inhibiting the gonadotropin-releasing hormone (GnRH) pulse generator activity in female rats. Psychoneuroendocrinology 24, 397–407.
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Silva RM, Hadjimarkou MM, Rossi GC, Pasternak GW & Bodnar RJ (2001). endorphin-induced feeding: pharmacological characterization using selective opioid antagonists and antisense probes in rats. J Pharmacol Exp Ther 297, 590–596.
Spanagel R, Almeida OF & Shippenberg TS (1994). Evidence that nor-binaltorphimine can function as an antagonist at multiple opioid receptor subtypes. Eur J Pharmacol 264, 157–162.
Thind KK & Goldsmith PC (1988). Infundibular gonadotropin-releasing hormone neurons are inhibited by direct opioid and autoregulatory synapses in juvenile monkeys. Neuroendocrinology 47, 203–216.
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Tomaszewska-Zaremba D, Mateusiak K & Przekop F (2002). The Involvement of GABAA receptors in the control of GnRH and beta-endorphin release, and catecholaminergic activity in the preoptic area in anestrous ewes. Exp Clin Endocrinol Diabetes 110, 336–342.
Van Cauter E (1988). Estimating false-positive and false-negative errors in analyses of hormonal pulsatility. Am J Physiol 254, E786–E794.
Wang H, Cuzon VC & Pickel VM (2003). Postnatal development of mu-opioid receptors in the rat caudate-putamen nucleus parallels asymmetric synapse formation. Neuroscience 118, 695–708., http://www.100md.com(J. E Bowe, X. F Li, J. S )
2 Cardiovascular Division
3 Department of Pharmacology and Therapeutics, New Hunt's House, King's College London, Guy's Campus, London SE1 1UL, UK
4 Henry Wellcome Laboratory for Integrative Neuroscience and Endocrinology, Dorothy Hodgkin Building, University of Bristol, Bristol BS1 3NY, UK
Abstract
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Calcitonin gene-related peptide (CGRP) is involved in a variety of stress responses in the rat. Central administration of CGRP activates the hypothalamo–pituitary–adrenal axis resulting in increased corticosterone secretion. We have previously shown that central CGRP suppresses the gonadotrophin-releasing hormone (GnRH) pulse generator, specifically LH pulses. Endogenous opioid peptides (EOPs) have been shown to play an important role in stress-induced suppression of the reproductive axis. The aim of the present study was to test the hypothesis that EOPs mediate CGRP-induced suppression of pulsatile LH secretion. Ovariectomized rats were implanted with intracerebroventricular (I.C.V.) and I.V. cannulae. Intravenous administration of the opioid antagonist naloxone (250 μg) completely blocked the suppression of LH pulses induced by 1.5 μg I.C.V. CGRP and significantly attenuated the suppression of pulsatile LH secretion induced by 5 μg I.C.V. CGRP. Furthermore, intravenous administration of naloxone was found to immediately restore normal LH pulse frequency in animals treated 90 min earlier with 1.5 μg I.C.V. CGRP. Co-administration (I.C.V.) of CGRP (1.5 μg) with the μ and opioid receptor-specific antagonists naloxone (10 μg) and norbinaltorphimine (5 μg), respectively, blocked the CGRP-induced suppression of LH pulses, whilst I.C.V. co-administration of CGRP (1.5 μg) with the opioid receptor-specific antagonist naltrindole (5 μg) did not. These data provide evidence that EOPs play a pivotal role in mediating the inhibitory effects of CGRP on pulsatile LH secretion in the rat. They also suggest that the μ and , but not the , opioid receptors may be responsible for mediating the effects of CGRP on LH pulses.
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Introduction
Calcitonin gene-related peptide (CGRP), a 37-amino-acid neuropeptide best known for its potent vasodilatory properties (Brain et al. 1985), has been shown to evoke a classic stress response by stimulating the hypothalamo–pituitary–adrenocortical (HPA) axis (Kovacs et al. 1995; Dhillo et al. 2003) and sympathetic outflow (Fisher et al. 1983). CGRP has also been implicated in anorectic (Lutz et al. 1998), addictive (Ehlers et al. 1999) and fear-related behaviours (Poore & Helmstetter, 1996). Additionally, CGRP has recently been shown to have a profound and dose dependent inhibitory action on the hypothalamic gonadotrophin-releasing hormone (GnRH) pulse generator, decreasing luteinizing hormone (LH) pulse frequency in the rat (Li et al. 2004). A pivotal role in stress-induced suppression of the GnRH pulse generator is also evident since the CGRP receptor antagonist, CGRP8–37, completely blocked hypoglycaemic stress-induced suppression of LH pulses (Li et al. 2004). The mechanisms underlying this central inhibitory action of CGRP on the reproductive neuroendocrine axis remain unknown. Corticotrophin releasing-hormone (CRH), a crucial mediator of stress-induced activation of the HPA axis, and inhibition of the GnRH pulse generator may mediate, in part, the CGRP-induced suppression of LH pulses (Li et al. 2004). There is substantial evidence for a pivotal role for endogenous opioid peptides (EOPs) in the regulation of the GnRH pulse generator. In a variety of species, including rats and primates, the suppression of LH release induced by central administration of CRH can be either attenuated or completely blocked by the opioid antagonist naloxone (Gindoff & Ferin, 1987; Almeida et al. 1988; Rivest et al. 1993). Furthermore, EOPs mediate suppression of pulsatile LH secretion in response to a variety of stressors from metabolic (Cagampang & Maeda, 1991; Bonavera et al. 1993; Cagampang et al. 1997) to behavioural (O'Byrne et al. 1989). The present study was designed to investigate whether EOPs are involved in CGRP-induced suppression of pulsatile LH secretion and to establish the opioid receptor subtypes mediating the response in the rat.
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Methods
Animals and surgical procedures
Adult female Wistar rats, weighing 230–280 g, obtained from B & K Suppliers, Ltd (Hull, UK), were housed under controlled conditions (12: 12 h light–dark; lights on at 07.00 h; temperature at 22 ± 2°C) and provided with food and water ad libitum. All animal procedures were undertaken in accordance with the UK Home Office regulations. All surgical procedures were carried out under ketamine (100 mg kg–1 I.P.; Pharmacia and Upjohn Ltd, Crawley, UK) and Rompun (10 mg kg–1 I.P.; Bayer, Leverkusen, Germany) anaesthesia. Rats were bilaterally ovariectomised. At the time of ovariectomy, all rats were also fitted with an intracerebroventricular (I.C.V.) guide cannula (22 gauge; Plastics One, Roanoke, VA, USA) directed towards the left lateral cerebral ventricle, the co-ordinates for implantation being 1.5 mm lateral, 0.6 mm posterior to Bregma, and 3.5 mm below the surface of the dura (Paxinos & Watson, 1986). The guide cannula was secured using dental cement (Dental Filling Ltd, Swindon, UK), and fitted with a dummy cannula (Plastics One) to maintain patency (Cates et al. 1999). Following a 10-day recovery period, the rats were fitted with two indwelling cardiac catheters via the jugular veins (Li et al. 2003). The catheters were exteriorized at the back of the head and secured to a cranial attachment: the rats were fitted with a 30-cm long metal spring tether (Instec Laboratories Inc., Boulder, CO, USA). The distal end of the tether was attached to a fluid swivel (Instec Laboratories), which allowed the rat freedom to move around the enclosure. Experimentation commenced 3 days later.
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Effect of CGRP on LH pulses
On the morning of experimentation, an I.C.V. injection cannula (Plastics One) with extension tubing, preloaded with drug or vehicle, was inserted into the guide cannula. The distal end of the tubing was extended outside of the animal cage to allow remote infusion without disturbing the rat during the experiment. The injection cannulae, which extended 1.0 mm beyond the tip of the guide cannula, reached the injection site, the lateral cerebral ventricle. Rats were then attached via one of the two cardiac catheters to a computed-controlled automated blood sampling system, which allows for the intermittent withdrawal of small blood samples (25 μl) without disturbing the rats (Cates et al. 1999). Experimentation commenced between 09.00 h and 11.00 h when blood samples were taken every 5 min for 6 h. After removal of each 25-μl blood sample, an equal volume of heparinized saline (10 U ml–1 normal saline; CP Pharmaceuticals Ltd, Wrexham, UK) was automatically infused into the animals to maintain patency of the catheter and blood volume. Blood samples were frozen at –20°C for later assay to determine LH concentrations. After 2 h of sampling the animals were injected I.V. with 250 μg naloxone in 200 μl saline, followed 10 min later by the substance to be tested, injected I.C.V. in 4 μl artificial cerebrospinal fluid vehicle (1.5 μg CGRP, 5 μg CGRP or aCSF alone; n = 8, 8 and 7, respectively). Automated sampling continued for 4 h after the I.C.V. injection. In separate groups of animals CGRP (1.5 μg) was administered I.C.V. after 2 h of baseline blood sampling, followed 90 min later by I.V. injection of either 250 μg/200 μl naloxone (n = 7) or 200 μl saline (n = 4). Blood sampling was continued for a further 4 h after the CGRP injection.
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To test the specific opioid receptors involved in the response to CGRP, animals were co-administered 1.5 μg CGRP along with the specific opioid antagonist to be tested. These were naloxone (10 μg, a μ-receptor specific antagonist at this dose, n = 7), naltrindole (5 μg, a -receptor specific antagonist, n = 8) or norbinaltorphimine (nor-BNI, 5 μg, a -receptor specific antagonist, n = 8). Both CGRP and the specific opioid antagonists were administered I.C.V. due to the inability of naltrindole to cross the blood–brain barrier. All I.C.V. injections were given in 4 μl aCSF vehicle. To test whether -receptor activation resulted in suppression of LH pulses, the -receptor-specific agonist U69593 (100 μg, n = 9) was administered I.C.V. after 2 h of baseline blood sampling, and sampling then continued for a further 4 h. U69593 was dissolved in 1 M hydrochloric acid and then diluted in aCSF. Sodium hydroxide (1 M) was then used to neutralize this mixture before injection (total volume injected: 4 μl). Control animals received (4 μl, I.C.V.) the neutralized HCl/NaOH vehicle (n = 5). Different groups of animals were used for each individual treatment group.
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Radioimmunoassay for LH
A double antibody radioimmunoassay supplied by the National Institute of Diabetes, Digestion and Kidney Disease (NIDDK) (Monroe et al. 1968) was used to determine LH concentration in the 25 μl whole blood sample. Each blood sample was measured as a singleton. The sensitivity of the assay was 0.093 ng ml–1. The intra-assay variation was 5.8% and the interassay variation was 5.0%.
Statistical analysis
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Detection of LH pulses was established by use of the algorithm ULTRA (Van Cauter, 1988). Two intra-assay coefficients of variation of the assay were used as the reference threshold for the pulse detection. The effect of treatment on pulsatile LH secretion was calculated by comparing the mean LH pulse interval before and after drug administration and expressed as ‘prolongation of LH pulse interval’ as a percentage of the pretreatment control value. In the case of animals in which no LH pulses were observed during the post-treatment period, these were assigned a value of 4 h for the post-treatment LH pulse interval for the purposes of analysis. Statistical significance was tested on raw data using one-way ANOVA and Dunnett's test. P < 0.05 was considered statistically significant.
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Results
Intracerebroventricular administration of CGRP resulted in a suppression of pulsatile LH secretion as previously demonstrated (Li et al. 2004). Intravenous administration of naloxone (250 μg; 10 min prior to CGRP) significantly attenuated the suppression of pulsatile LH release in response to the high dose of CGRP (5 μg, I.C.V.) and completely blocked the inhibitory effects of the lower dose of CGRP (1.5 μg, I.C.V.) on LH pulses (Fig. 1C–G). Administration of either the aCSF (4 μl, I.C.V.) or naloxone (250 μg, I.V.) alone had no effect on LH pulse frequency (Fig. 1A, B and G). To determine whether EOPs are involved in merely initiating the LH response to central CGRP or play a role in maintaining LH suppression over several hours, naloxone (250 μg, I.V.) was administered 90 min after the onset of I.C.V. CGRP (1.5 μg, I.C.V.) induced suppression of pulsatile LH release. Administration of naloxone (250 μg, I.V.) 90 min after the injection of CGRP (1.5 μg, I.C.V.) immediately restored LH pulse frequency to control values whilst administration of saline had no effect (Fig. 2A–C). Given that saline administration had no effect, results from CGRP (1.5 μg, I.C.V.) followed by saline (200 μl, I.V.) treated rats were combined with results from animals treated with CGRP (1.5 μg, I.C.V.) alone for the purposes of analysis.
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Representative examples demonstrating the effects of 4 μl aCSF I.C.V. (A), 250 μg naloxone I.V. (B), 1.5 μg CGRP I.C.V. (C), 1.5 μg CGRP I.C.V. + 250 μg naloxone I.V. (D), 5 μg CGRP I.C.V. (E), and 5 μg CGRP I.C.V. + 250 μg naloxone I.V. (F) on pulsatile LH secretion in ovx rats. Asterisks denote LH pulses. G, summary of the effects of treatments with various doses of CGRP, and naloxone on pulsatile LH secretion in ovx rats. Note the dose-dependant inhibitory effect of CGRP on LH pulses. P < 0.05 versus aCSF control. P < 0.05 versus 5 μg CGRP I.C.V.n = 6–12.
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Representative examples showing the effects of 1.5 μg CGRP injected I.C.V. after 2 h of control baseline blood sampling and 200 μl saline injected I.V. 90 min later (A) and 1.5 μg CGRP injected I.C.V. after 2 h of blood sampling and 250 μg naloxone injected I.V. 90 min later (B) on pulsatile LH secretion in ovariectomized rats. C, summary of the effect of naloxone when given 90 min after CGRP administration on pulsatile LH secretion in ovx rats. Note that naloxone immediately restored LH pulse interval to pre-CGRP treatment values. Asterisks denote LH pulses. P < 0.05 versus aCSF control. P < 0.05 versus 1.5 μg CGRP I.C.V. and 200 μl saline I.V.n = 7–12.
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To investigate the specific opioid receptors involved in the inhibitory effect of CGRP on pulsatile LH secretion, we co-administered, by I.C.V. injection, CGRP (1.5 μg) with the specific opioid antagonists naloxone, naltrindole or nor-BNI. It was found that both naloxone (10 μg, I.C.V.) and nor-BNI (5 μg, I.C.V.) were effective in blocking the CGRP-induced suppression of LH pulses (Fig. 3B, C, E and H). Naltrindole (5 μg, I.C.V.) had no effect on the CGRP-induced suppression of LH pulses (Fig. 3G and H). Neither naloxone or nor-BNI had any significant effect on LH pulse frequency when given alone (Fig. 3D, F and H). Additionally, it was shown that I.C.V. administration of U69593 (100 μg), a -specific opioid receptor agonist, is capable of suppressing pulsatile LH release (Fig. 4).
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Representative examples demonstrating the effects of I.C.V. administration of 4 μl aCSF (A), 1.5 μg CGRP (B), 1.5 μg CGRP + 10 μg naloxone (C), 10 μg naloxone (D), 1.5 μg CGRP + 5 μg nor-BNI (E), 5 μg nor-BNI (F), and 1.5 μg CGRP + 5 μg naltrindole (G) on pulsatile LH secretion in ovx rats. Asterisks denote LH pulses. H, summary of the effects of treatments with 1.5 μg CGRP and specific opioid receptor antagonists on pulsatile LH secretion in ovx rats. P < 0.05 versus aCSF control. P < 0.05 versus 1.5 μg CGRP I.C.V.. n = 6–12.
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Representative examples demonstrating the effects of I.C.V. administration of 4 μl neutralized HCl/NaOH vehicle (A) and 100 μg U69593, a opioid receptor agonist (B) on pulsatile LH secretion in ovx rats. Asterisks denote LH pulses. C, the opioid receptor agonist prolonged LH interpulse interval. P < 0.05 versus vehicle control. n = 5–9.
Discussion
The data from the present study provide the first evidence that EOPs are involved in the CGRP-induced suppression of the GnRH pulse generator, since the opioid antagonist naloxone completely blocks the inhibitory effect of the lower dose of CGRP on pulsatile LH secretion. Although naloxone profoundly attenuates the inhibitory response to the higher dose of CGRP, the absence of a complete blockage may be related to an inadequate dose of naloxone or a combination of opioid and non-opioid mechanisms activated. The ability of naloxone, when given 90 min after CGRP administration (lower dose), to immediately restore a normal LH pulse frequency suggests that opioid involvement is not only important for the onset of CGRP-induced effects on LH secretion, but also for the continued maintenance of CGRP-induced suppression of the hypothalamic GnRH pulse generator.
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There is an extensive literature demonstrating that EOPs inhibit GnRH pulse generator activity. Naloxone has been shown to stimulate the release of LH in the rat (Pfeiffer & Herz, 1984) and to increase the frequency of multiunit activity volleys recorded from the mediobasal hypothalamus, an electrophysiological correlate of GnRH pulse generator activity in both the ovariectomized rat (Sano et al. 1999) and the rhesus monkey, although the presence of oestradiol is required in the latter species (Grosser et al. 1993). The absence of an effect of naloxone per se on LH pulse frequency in the present study may be related to the lower dose used compared with previous studies (Sano et al. 1999). The administration of opiate agonists such as morphine has also been demonstrated to suppress LH release (Pfeiffer & Herz, 1984; Kesner et al. 1986). Furthermore, in vitro studies show that naloxone induces GnRH release from hypothalamic explants (Rubin, 1993), whilst morphine suppresses GnRH release from isolated medio-basal hypothalamic fragments (Giri & Kaufman, 1994). It is well established that EOPs are physiologically relevant in mediating stress-induced suppression of LH pulses. In the monkey the inhibitory effects of behavioural stressors on LH pulses can be blocked by naloxone administration (O'Byrne et al. 1989), whilst in the rat naloxone administration blocks the LH pulse-suppressing effects of a variety of stressors, including fasting, interleukin-1 and insulin-induced hypoglycaemia (Cagampang & Maeda, 1991; Bonavera et al. 1993; Cagampang et al. 1997). We have previously demonstrated that endogenous CGRP is involved in hypoglycaemic stress-induced suppression of LH pulses (Li et al. 2004) and therefore our current data are consistent with the literature describing a role for EOPs in the hypoglycaemic stress-induced suppression of the GnRH pulse generator.
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The mechanism through which EOPs affect the GnRH pulse generator is currently unknown. Whilst in both the rat and the monkey direct synaptic contacts have been found between opioidergic and GnRH neurones (Thind & Goldsmith, 1988; Chen et al. 1989), GnRH neuronal cell bodies do not appear to express μ, , or opioid receptor mRNA (Sannella & Petersen, 1997; Mitchell et al. 1997). Additionally, studies in GT1–7 cells, a GnRH neuronal cell line, indicate that these cells do not express μ opioid receptors (Maggi et al. 1995a), nor is GnRH release from the GT1–7 cells affected by treatment with μ opioid agonists (Maggi et al. 1995b). However, it has recently been shown that GnRH neurones in vivo are not as poorly innervated as previously thought, and in fact receive abundant synaptic inputs onto dendritic processes, which were hitherto not realized to have such far reaching projections (Campbell et al. 2005). This raises the exciting possibility that despite a lack of opioid receptors on the cell body, direct interactions of EOPs with the GnRH neurones may still be possible through synaptic inputs onto their dendritic processes, particularly since opioid receptors have been found on neuronal dendrites in other regions of the rat brain (Wang et al. 2003; Pickel et al. 2004).
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Nevertheless, an intermediary role for other neurotransmitter systems, such as -aminobutyric acid (GABA), in EOP effects on the GnRH neural network has to be considered. Indeed, GABA neurones within the preoptic area have been shown to contain abundant opioid receptors and also form extensive synapses onto GnRH neurones (Leranth et al. 1985). However, whilst it has been shown that GABA receptor stimulation in the preoptic area attenuates GnRH release (Tomaszewska-Zaremba et al. 2002), functional studies indicate that the inhibitory effects of EOP on LH release do not involve the preoptic GABAergic system (Jarry et al. 1995).
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Our results with the selective opioid receptor antagonists indicate a role for both the μ and opioid receptors in the CGRP-induced suppression of LH pulses, but not for the opioid receptors. Naltrindole and norbinaltorphimine are highly selective and antagonists, respectively, whereas naloxone displays much less selectivity for the μ receptor (Corbett et al. 1993). In in vitro assays naltrindole has Ke values at the , μ and receptors of 0.3, 22 and 100 nM, respectively, norbinaltorphimine has Ke values at the , μ and receptors of 16, 25 and 0.05 nM, respectively, and naloxone has Ke values at the , μ and receptors of 46, 2 and 16 nM respectively. The concentrations of the antagonists used in the present study are similar to those used in investigations of the role of the different opioid receptors in a number of different paradigms (Kalra et al. 2001; Ishihara et al. 2001; Silva et al. 2001). It is well established that μ opioid receptor activation results in a suppression of LH levels (Marko & Romer, 1983). There is, however, contradictory evidence over the ability of or opioid receptor agonists to inhibit LH. Several studies have demonstrated that and opioid receptor agonists suppress LH levels (Leadem & Yagenova, 1987; Pfeiffer et al. 1987) whilst others have failed to show an effect on LH secretion (Pfeiffer et al. 1983; Mallory & Gallo, 1990). Our data indicate that the opioid receptor agonist U69593 is able to decrease LH pulse frequency. In binding assays, U69593 has Ki values of 1.4, 2350 and 20000 nM at the , μ and receptors, respectively (Corbett et al. 1993). The analgesic effects of 25 μg U69693 I.C.V. are blocked by norbinaltorphimine 30 μg I.C.V., a dose of norbinaltorphimine which does not affect analgesia produced by the selective agonists given I.C.V. [D-Ala2,MePhe4,Gly-ol5]enkephalin (μ) or [D-Pen2,D-Pen5]enkephalin () (Spanagel et al. 1994). This supports the suggestion that 100 μg U-69593 I.C.V. will selectively activate receptors. Thus the inhibitory effects of CGRP on LH pulses may be mediated through the opioid receptors in addition to the μ opioid receptor.
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The results of the current study are in keeping with our previously published data demonstrating a pivotal role of CRH in mediating, in part, the inhibitory effects of central CGRP on the GnRH pulse generator (Li et al. 2004). Given that in both the monkey and the rat the suppression of LH release induced by central CRH can be blocked or attenuated by opioid antagonists (Gindoff & Ferin, 1987; Almeida et al. 1988; Rivest et al. 1993), it seems likely that EOPs are involved downstream of CRH in the mechanism relaying the inhibitory effects of central CGRP on pulsatile LH secretion. We are currently working towards further characterizing the neural circuitry involved.
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References
Almeida OF, Nikolarakis KE & Herz A (1988). Evidence for the involvement of endogenous opioids in the inhibition of luteinizing hormone by corticotropin-releasing factor. Endocrinology 122, 1034–1041.
Bonavera JJ, Kalra SP & Kalra PS (1993). Mode of action of interleukin-1 in suppression of pituitary LH release in castrated male rats. Brain Res 612, 1–8.
Brain SD, Williams TJ, Tippins JR, Morris HR & MacIntyre I (1985). Calcitonin gene-related peptide is a potent vasodilator. Nature 313, 54–56.
, 百拇医药
Cagampang FR, Cates PS, Sandhu S, Strutton PH, McGarvey C, Coen CW & O'Byrne KT (1997). Hypoglycaemia-induced inhibition of pulsatile luteinizing hormone secretion in female rats: role of oestradiol, endogenous opioids and the adrenal medulla. J Neuroendocrinol 9, 867–872.
Cagampang FR & Maeda K (1991). Effects of intracerebroventricular administration of opiate receptor antagonists on the suppressed pulsatile LH release during acute fasting in ovariectomized estradiol-treated rats. Life Sci 49, 1823–1828.
, 百拇医药
Campbell RE, Han SK & Herbison AE (2005). Biocytin filling of adult GnRH neurons in situ reveals extensive, spiny, dendritic processes. Endocrinology 146, 1163–1169.
Cates PS, Forsling ML & O'Byrne KT (1999). Stress-induced suppression of pulsatile luteinising hormone release in the female rat: role of vasopressin. J Neuroendocrinol 11, 677–683.
Chen M, Lee J, Huang BS, Grekin RJ & Malvin RL (1989). Clonidine and morphine increase atrial natriuretic peptide secretion in anesthetized rats. Proc Soc Exp Biol Medical 191, 299–303.
, 百拇医药
Corbett AD, Paterson SJ & Kosterlitz HW (1993). Selectivity of ligands for opioid receptors. Handbook Exp Pharmacol 104, 645–679.
Dhillo WS, Small CJ, Jethwa PH, Russell SH, Gardiner JV, Bewick GA, Seth A, Murphy KG, Ghatei MA & Bloom SR (2003). Paraventricular nucleus administration of calcitonin gene-related peptide inhibits food intake and stimulates the hypothalamo-pituitary-adrenal axis. Endocrinology 144, 1420–1425.
, http://www.100md.com Ehlers CL, Somes C, Li TK, Lumeng L, Hwang BH, Jimenez P & Mathe AA (1999). Calcitonin gene-related peptide (CGRP) levels and alcohol. Int J Neuropsychopharmacol 2, 173–179.
Fisher LA, Kikkawa DO, Rivier JE, Amara SG, Evans RM, Rosenfeld MG, Vale WW & Brown MR (1983). Stimulation of noradrenergic sympathetic outflow by calcitonin gene-related peptide. Nature 305, 534–536.
Gindoff PR & Ferin M (1987). Endogenous opioid peptides modulate the effect of corticotropin-releasing factor on gonadotropin release in the primate. Endocrinology 121, 837–842.
, 百拇医药
Giri M & Kaufman JM (1994). Opioidergic modulation of in vitro pulsatile gonadotropin-releasing hormone release from the isolated medial basal hypothalamus of the male guinea pig. Endocrinology 135, 2137–2143.
Grosser PM, O'Byrne KT, Williams CL, Thalabard JC, Hotchkiss J & Knobil E (1993). Effects of naloxone on estrogen-induced changes in hypothalamic gonadotropin-releasing hormone pulse generator activity in the rhesus monkey. Neuroendocrinology 57, 115–119.
, 百拇医药
Ishihara S, Tsuchiya S, Horie S, Murayama T & Watanabe K (2001). Stimulatory effects of centrally injected kappa-opioid receptor agonists on gastric acid secretion in urethane-anesthetized rats. Eur J Pharmacol 418, 187–194.
Jarry H, Leonhardt S & Wuttke W (1995). The inhibitory effect of beta-endorphin on LH release in ovariectomized rats does not involve the preoptic GABAergic system. Exp Clin Endocrinol Diabetes 103, 317–323.
, 百拇医药
Kalra A, Urban MO & Sluka KA (2001). Blockade of opioid receptors in rostral ventral medulla prevents antihyperalgesia produced by transcutaneous electrical nerve stimulation (TENS). J Pharmacol Exp Ther 298, 257–263.
Kesner JS, Kaufman JM, Wilson RC, Kuroda G & Knobil E (1986). The effect of morphine on the electrophysiological activity of the hypothalamic luteinizing hormone-releasing hormone pulse generator in the rhesus monkey. Neuroendocrinology 43, 686–688.
, 百拇医药
Kovacs A, Biro E, Szeleczky I & Telegdy G (1995). Role of endogenous CRF in the mediation of neuroendocrine and behavioral responses to calcitonin gene-related peptide in rats. Neuroendocrinology 62, 418–424.
Leadem CA & Yagenova SV (1987). Effects of specific activation of mu-, delta- and kappa-opioid receptors on the secretion of luteinizing hormone and prolactin in the ovariectomized rat. Neuroendocrinology 45, 109–117.
, 百拇医药
Leranth C, MacLusky NJ, Sakamoto H, Shanabrough M & Naftolin F (1985). Glutamic acid decarboxylase-containing axons synapse on LHRH neurons in the rat medial preoptic area. Neuroendocrinology 40, 536–539.
Li XF, Bowe JE, Mitchell JC, Brain SD, Lightman SL & O'Byrne KT (2004). Stress-induced suppression of the gonadotropin-releasing hormone pulse generator in the female rat: a novel neural action for calcitonin gene-related peptide. Endocrinology 145, 1556–1563.
, http://www.100md.com
Li XF, Mitchell JC, Wood S, Coen CW, Lightman SL & O'Byrne KT (2003). The effect of oestradiol and progesterone on hypoglycaemic stress-induced suppression of pulsatile luteinizing hormone release and on corticotropin-releasing hormone mRNA expression in the rat. J Neuroendocrinol 15, 468–476.
Lutz TA, Rossi R, Althaus J, Del Prete E & Scharrer E (1998). Amylin reduces food intake more potently than calcitonin gene-related peptide (CGRP) when injected into the lateral brain ventricle in rats. Peptides 19, 1533–1540.
, 百拇医药
Maggi R, Pimpinelli F, Martini L & Piva F (1995a). Inhibition of luteinizing hormone-releasing hormone secretion by delta-opioid agonists in GT1-1 neuronal cells. Endocrinology 136, 5177–5181.
Maggi R, Pimpinelli F, Martini L & Piva F (1995b). Characterization of functional opioid delta receptors in a luteinizing hormone-releasing hormone-producing neuronal cell line. Endocrinology 136, 289–295.
Mallory DS & Gallo RV (1990). Medial preoptic-anterior hypothalamic area involvement in the suppression of pulsatile LH release by a mu-opioid agonist in the ovariectomized rat. Brain Res Bull 25, 251–257.
, 百拇医药
Marko M & Romer D (1983). Inhibitory effect of a new opioid agonist on reproductive endocrine activity in rats of both sexes. Life Sci 33, 233–240.
Mitchell V, Prevot V, Jennes L, Aubert JP, Croix D & Beauvillain JC (1997). Presence of mu and kappa opioid receptor mRNAs in galanin but not in GnRH neurons in the female rat. Neuroreport 8, 3167–3172.
Monroe SE, Parlow AF & Midgley AR Jr (1968). Radioimmunoassay for rat luteinizing hormone. Endocrinology 83, 1004–1012.
, 百拇医药
O'Byrne KT, Lunn SF & Dixson AF (1989). Naloxone reversal of stress-induced suppression of LH release in the common marmoset. Physiol Behav 45, 1077–1080.
Paxinos G & Watson C (1986). The Rat Brain in Stereotaxic Coordinates. Academic Press, London.
Pfeiffer A & Herz A (1984). Endocrine actions of opioids. Horm Metab Res 16, 386–397.
Pfeiffer DG, Pfeiffer A, Almeida OF & Herz A (1987). Opiate suppression of LH secretion involves central receptors different from those mediating opiate effects on prolactin secretion. J Endocrinol 114, 469–476.
, 百拇医药
Pfeiffer DG, Pfeiffer A, Shimohigashi Y, Merriam GR & Loriaux DL (1983). Predominant involvement of mu- rather than delta- or kappa-opiate receptors in LH secretion. Peptides 4, 647–649.
Pickel VM, Chan J, Kash TL, Rodriguez JJ & MacKie K (2004). Compartment-specific localization of cannabinoid 1 (CB1) and mu-opioid receptors in rat nucleus accumbens. Neuroscience 127, 101–112.
Poore LH & Helmstetter FJ (1996). The effects of central injections of calcitonin gene-related peptide on fear-related behavior. Neurobiol Learn Mem 66, 241–415.
, 百拇医药
Rivest S, Plotsky PM & Rivier C (1993). CRF alters the infundibular LHRH secretory system from the medial preoptic area of female rats: possible involvement of opioid receptors. Neuroendocrinology 57, 236–246.
Rubin BS (1993). Naloxone stimulates comparable release of luteinizing hormone-releasing hormone from tissue fragments from ovariectomized, estrogen-treated young and middle-aged female rats. Brain Res 601, 246–254.
, 百拇医药 Sannella MI & Petersen SL (1997). Dual label in situ hybridization studies provide evidence that luteinizing hormone-releasing hormone neurons do not synthesize messenger ribonucleic acid for mu, kappa, or delta opiate receptors. Endocrinology 138, 1667–1672.
Sano A, Funabashi T, Kawaguchi M, Shinohara K & Kimura F (1999). Intravenous injections of nicotine decrease the pulsatile secretion of LH by inhibiting the gonadotropin-releasing hormone (GnRH) pulse generator activity in female rats. Psychoneuroendocrinology 24, 397–407.
, http://www.100md.com
Silva RM, Hadjimarkou MM, Rossi GC, Pasternak GW & Bodnar RJ (2001). endorphin-induced feeding: pharmacological characterization using selective opioid antagonists and antisense probes in rats. J Pharmacol Exp Ther 297, 590–596.
Spanagel R, Almeida OF & Shippenberg TS (1994). Evidence that nor-binaltorphimine can function as an antagonist at multiple opioid receptor subtypes. Eur J Pharmacol 264, 157–162.
Thind KK & Goldsmith PC (1988). Infundibular gonadotropin-releasing hormone neurons are inhibited by direct opioid and autoregulatory synapses in juvenile monkeys. Neuroendocrinology 47, 203–216.
, 百拇医药
Tomaszewska-Zaremba D, Mateusiak K & Przekop F (2002). The Involvement of GABAA receptors in the control of GnRH and beta-endorphin release, and catecholaminergic activity in the preoptic area in anestrous ewes. Exp Clin Endocrinol Diabetes 110, 336–342.
Van Cauter E (1988). Estimating false-positive and false-negative errors in analyses of hormonal pulsatility. Am J Physiol 254, E786–E794.
Wang H, Cuzon VC & Pickel VM (2003). Postnatal development of mu-opioid receptors in the rat caudate-putamen nucleus parallels asymmetric synapse formation. Neuroscience 118, 695–708., http://www.100md.com(J. E Bowe, X. F Li, J. S )