Functional Analyses of the Neuropeptide FF Receptor System Using GJ14, a Potent and Selective Antagonist
|dc.identifier.citation||Kim, J. (2016). Functional Analyses of the Neuropeptide FF Receptor System Using GJ14, a Potent and Selective Antagonist (Thesis, Doctor of Philosophy). University of Otago. Retrieved from http://hdl.handle.net/10523/6610||en|
|dc.description.abstract||The G-protein coupled neuropeptide FF receptors (NPFFR1 and NPFFR2) belong to the RFamide receptor subfamily. Of these, NPFFR1 is the most widespread and highly expressed receptor in the brain. The endogenous agonist RFamide-related peptides (RFRP) are the most potent activators of NPFFR1. This PhD reports the development and pharmacological characterisation of a novel NPFFR1 antagonist called GJ14. Using RFRP-3 and GJ14 to selectively activate or antagonise NPFFR1 respectively, the physiological functions of the NPFFR1 could be unmasked for the first time, in vivo. These functions included the neuroendocrine control of reproduction, stress and anxiety, and regulation of opioid function. Eight compounds were designed to antagonise the NPFFRs were screened by radioligand binding and cyclic adenosine monophosphate (cAMP) assays using cells stably transfected with NPFFR1 or R2. As a result of these pharmacological experiments, GJ14 was identified as the most potent NPFFR antagonist with good selectivity for NPFFR1. Any off-target effects at the structurally related neuropeptide-Y receptor-Y1 (NPY-Y1) and kisspeptin receptor (KISS1R) were also detected. This revealed an off-target KISS1R agonist action of RF9, a previously published NPFFR antagonist. RFRP is best known for its inhibitory effects in the neuroendocrine control of reproduction. The central control of fertility is driven by the gonadotropin-releasing hormone (GnRH) neurons, which drive the hypothalamic pituitary-gonadal (HPG) axis. Pulsatile GnRH release from its nerve terminals into the portal vasculature stimulates pulses of luteinising hormone (LH) secretion. The activation of KISS1R, via its endogenous agonist kisspeptin, is the most potent stimulator of gonadotropin release. Coincidently, RF9 also elicits potent LH release, which has been attributed to antagonism of a potent tonic inhibitory effect of RFRP-3 on GnRH neurons. The effects of activation and antagonism of NPFFR1 on the HPG axis were therefore tested. The potent stimulatory effect of RF9 on LH release was not observed with GJ14 injections or in KISS1R knockout mice. This, combined with the pharmacological data, provided strong evidence that the hypergonadotropic effects of RF9 are mediated by its off-target KISS1R agonism. In contrast, true blockade of the NPFFR system did not affect basal circulating LH concentrations, indicating that the RFRP peptides may not hold such an important role in the control of GnRH neuronal fuction. In addition to reproduction, there has been recent evidence indicating possible effects of RFRP and NPFFR1 on stress and anxiety. Corticosterone-releasing hormone (CRH) neurons in the paraventricular nucleus (PVN) control the neuroendocrine stress response. CRH neurons drive the release of adrenocorticotropic hormone (ACTH) from the anterior pituitary and subsequent release of the stress hormones, corticosterone (cortisol in man) from the adrenal cortex. The activation of the stress axis, if prolonged, is associated with an increased risk for psychological disorders such as anxiety. The dense expression of NPFFR1 in the PVN combined with the previously reported effects of RFRP and NPFF peptides on the stress response, suggested that activation of the NPFFR1 may have a role in controlling CRH neuron excitability. Consistent with this, central injections of RFRP-3 potently elevated circulating corticosterone levels and cFOS expression (a marker of neuronal activation) in CRH neurons in mice. These effects were all reversed with GJ14. Chronic infusions of RFRP-3 and GJ14 also respectively increased and decreased anxiety-like behaviour on the elevated plus maze, open field, and light dark box tests. RFRP-3 infused mice also exhibited elevated basal corticosterone levels. Surprisingly however, RFRP-3 had little effect on CRH neuron excitability on brain slices, suggesting that its effects are likely to be indirectly mediated. The NPFFRs collectively are best known in the literature for their putative ability to block opioid function. However in vivo evidence for this has been limited to nociceptive tests, which are confounded by the pronociceptive effects of the NPFFR agonists. To elucidate anti-opioid function of NPFFR1, magnocellular neurons of the supraoptic nucleus were used as in vivo models. In extracellular single-unit recordings from urethane anesthetised rats, the spontaneous firing rates of vasopressin and oxytocin neurons were significantly inhibited by morphine, which could be abolished with RFRP-3 pretreatment. A challenging notion is that chronic opiate treatment triggers the upregulation of the anti-opioid system, which in turn attenuates the effect of opioids, producing tolerance. Tolerance to morphine was induced by chronic morphine infusion. Vasopressin neurons from infused rats had virtually no responses to morphine, confirming morphine tolerance. Pretreatment with GJ14 reversed morphine tolerance, indicating that the reduced opioid sensitivity during tolerance is mediated at least in part, by the anti-opioid function of NPFFR1. This is the first evidence demonstrating an anti-opioid function at the cellular level, in vivo. In summary, this thesis describes the development and characterisation of GJ14, a new potent and selective antagonist. Using GJ14, the misconception that RFRP peptides hold a potent tonic restraint on the HPG axis is now cleared. The blockade of NPFFR1 has revealed its role in regulating anxiety-like behaviour. Finally, using GJ14, this thesis provides convincing evidence that the NPFFR system is an important part of a genuine anti-opioid sytem that regulates opioid sensitivity.|
|dc.publisher||University of Otago|
|dc.rights||All items in OUR Archive are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.|
|dc.title||Functional Analyses of the Neuropeptide FF Receptor System Using GJ14, a Potent and Selective Antagonist|
|thesis.degree.name||Doctor of Philosophy|
|thesis.degree.grantor||University of Otago|
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