Philip J. Ryan

Relaxin-3 systems in the brain--the first 10 years.

The relaxin-3 gene was identified in 2001 by searching the human genome database for homologues of the relaxin hormone, and was subsequently discovered to encode a highly conserved neuropeptide in mammals and lower species. In the decade since its discovery there have been significant advances in our knowledge of the peptide, including the identification of its cognate receptor (a type 1 G-protein coupled receptor, GPCR135 or RXFP3), an understanding of its structure-activity and associated cellular signalling, and the elucidation of key neuroanatomical aspects of relaxin-3/RXFP3 networks in mammalian brain. The latter studies revealed that relaxin-3 is expressed within GABA neurons of the brainstem including an area known as the nucleus incertus, and that ascending relaxin-3 projections innervate a broad range of RXFP3-rich forebrain areas. These maps provided a foundation for pharmacological and physiological studies to elucidate the neurobiological nature of relaxin-3/RXFP3 signalling in vivo. Recent findings from our laboratory and others suggest the relaxin-3 neural network represents a newly identified ascending arousal system, able to modulate a range of interrelated functions including responses to stress, spatial and emotional memory, feeding and metabolism, motivation and reward, and circadian rhythm and sleep/wake states. More research is now required to discover further important facts about relaxin-3 neurons, such as their various regulatory inputs, and to characterise populations of RXFP3-positive neurons and determine their influence on particular neural circuits, physiology and complex behaviour.

Design, Synthesis, and Characterization of a Single-Chain Peptide Antagonist for the Relaxin-3 Receptor RXFP3

Relaxin-3 is a two-chain disulfide-rich peptide that is the ancestral member of the relaxin peptide family and, together with its G protein-coupled receptor RXFP3, is highly expressed in the brain. Strong evolutionary conservation of relaxin-3 suggests a critical biological function and recent studies have demonstrated modulation of sensory, neuroendocrine, metabolic, and cognitive systems. However, detailed studies of central relaxin-3-RXFP3 signaling have until now been severely hampered by the lack of a readily available high-affinity antagonist for RXFP3. Previous studies have utilized a complex two-chain chimeric relaxin peptide, R3(BΔ23-27)R/I5, in which a truncated relaxin-3 B-chain carrying an additional C-terminal Arg residue was combined with the insulin-like peptide 5 (INSL5) A-chain. In this study we demonstrate that, by replacing the native Cys in this truncated relaxin-3 B-chain with Ser, a single-chain linear peptide of 23 amino acids that retains high-affinity antagonism for RXFP3 can be achieved. In vivo studies demonstrate that this peptide, R3 B1-22R, antagonized relaxin-3/RXFP3 induced increases in feeding in rats after intracerebroventricular injection. Thus, R3 B1-22R represents an excellent tool for biological studies probing relaxin pharmacology and a lead molecule for the development of synthetically tractable, single-chain RXFP3 modulators for clinical use.

Minimization of Human Relaxin-3 Leading to High-Affinity Analogues with Increased Selectivity for Relaxin-Family Peptide 3 Receptor (RXFP3) over RXFP1

Relaxin-3 is a neuropeptide that is implicated in the regulation of stress responses and memory. The elucidation of its precise physiological role(s) has, however, been hampered by cross-activation of the relaxin-2 receptor, RXFP1, in the brain. The current study undertook to develop analogues of human relaxin-3 (H3 relaxin) that can selectively bind and activate its receptor, RXFP3. We developed a high-affinity selective agonist (analogue 2) by removal of the intra-A chain disulfide bond and deletion of 10 residues from the N terminus of the A chain. Further truncation of this analogue from the C terminus of the B chain to Cys(B22) and addition of an Arg(B23) led to a high-affinity, RXFP3-selective, competitive antagonist (analogue 3). Central administration of analogue 2 in rats increased food intake, which was blocked by prior coadministration of analogue 3. These novel RXFP3-selective peptides represent valuable pharmacological tools to study the physiological roles of H3 relaxin/RXFP3 systems in the brain and important leads for the development of novel compounds for the treatment of affective and cognitive disorders.

Relaxin-3/RXFP3 system regulates alcohol-seeking

Significance Relapse and hazardous drinking represent the most difficult clinical problems in treating patients with alcohol use disorders. Increasing our understanding of the brain circuits and chemicals that regulate alcohol intake and relapse offers the potential for more targeted therapeutic approaches to assist in relapse prevention. Using a rat model of alcohol use and alcohol-seeking, we provide the first evidence that a neuropeptide, namely relaxin-3, acts upon specific receptors (relaxin family peptide 3) within the brain to regulate alcohol self-administration and relapse-like behavior. In the case of relapse-like alcohol-seeking, this system appears particularly involved in stress-mediated relapse via actions within a brain region called the bed nucleus of the stria terminalis. Relapse and hazardous drinking represent the most difficult clinical problems in treating patients with alcohol use disorders. Using a rat model of alcohol use and alcohol-seeking, we demonstrated that central administration of peptide antagonists for relaxin family peptide 3 receptor (RXFP3), the cognate receptor for the highly conserved neuropeptide, relaxin-3, decreased self-administration of alcohol in a dose-related manner and attenuated cue- and stress-induced reinstatement following extinction. By comparison, RXFP3 antagonist treatment did not significantly attenuate self-administration or reinstatement of sucrose-seeking, suggesting a selective effect for alcohol. RXFP3 is densely expressed in the stress-responsive bed nucleus of the stria terminalis, and bilateral injections of RXFP3 antagonist into the bed nucleus of the stria terminalis significantly decreased self-administration and stress-induced reinstatement of alcohol, suggesting that this brain region may, at least in part, mediate the effects of RXFP3 antagonism. RXFP3 antagonist treatment had no effect on general ingestive behavior, activity, or procedural memory for lever pressing in the paradigms assessed. These data suggest that relaxin-3/RXFP3 signaling regulates alcohol intake and relapse-like behavior, adding to current knowledge of the brain chemistry of reward-seeking.

Increased feeding and body weight gain in rats after acute and chronic activation of RXFP3 by relaxin-3 and receptor-selective peptides: functional and therapeutic implications.

This paper provides a review of the effects of relaxin-3 and structurally related analogues on food intake and related behaviours, in relation to hypothalamic neural networks and chemical messengers known to control feeding, metabolism and body weight, including other neuropeptides and hormones. Soon after relaxin-3 was discovered, pharmacological studies identified the ability of the native peptide to stimulate feeding acutely in adult rats. Although interpretation of these data was confounded by ligand cross-reactivity at relaxin-family peptide (RXFP) receptors, studies with relaxin-3 analogues selective for the native relaxin-3 receptor, RXFP3, confirmed that acute and chronic activation of RXFP3 increased feeding and weight gain, and produced changes in plasma leptin and insulin. These studies also identified the hypothalamus as a locus of action. Studies are now required to identify RXFP3-positive neuron populations involved in the effects of relaxin-3/RXFP3 signalling on metabolic and neuroendocrine homeostasis, and to determine whether peptide-based, nonpeptide-based or gene-based RXFP3 treatments can alter food intake and body weight in animal models of obesity and eating disorders, as a reflection of the therapeutic potential of this newly identified transmitter system.

Relaxin-3 mRNA levels in nucleus incertus correlate with alcohol and sucrose intake in rats

BACKGROUND Chronic alcohol intake produces multiple neuroadaptive changes, including up- and down-regulation of neuropeptides and receptors. There are widespread projections of relaxin-3 containing neurons to, and abundant relaxin family peptide 3 receptor (RXFP3) expression within, brain regions involved in modulating alcohol intake. Recently we demonstrated the involvement of relaxin-3/RXFP3 signalling in alcohol-seeking in rats; therefore in this study we examined whether relaxin-3 and/or RXFP3 expression were altered by chronic alcohol intake in alcohol-preferring iP rats. METHODS Expression of relaxin-3 mRNA in the hindbrain nucleus incertus and RXFP3 radioligand binding levels in discrete forebrain regions were investigated following voluntary intake of alcohol or sucrose for 12 weeks, with a 2 day washout, using quantitative in situ hybridisation histochemistry and in vitro receptor autoradiography, respectively, in cohorts of adult, male iP rats. RESULTS Levels of relaxin-3 mRNA in the hindbrain nucleus incertus were positively correlated with the level of intake of both alcohol (r(12)=0.59, p=0.03) and sucrose (r(7)=0.70, p=0.04) in iP rats. Dense binding of the RXFP3-selective radioligand, [(125)]-R3/I5, was detected in hypothalamic and extrahypothalamic sites, but no significant changes in the density of RXFP3 were observed in the brain regions quantified following chronic sucrose or ethanol intake. CONCLUSIONS Our findings suggest high endogenous relaxin-3 expression may be associated with higher intake of rewarding substances, rather than its expression being regulated in response to their intake, consistent with an active role for the relaxin-3/RXFP3 system in modulating ingestive and alcohol-related behaviours.

Chemical synthesis and orexigenic activity of rat/mouse relaxin-3

The insulin-like peptide, relaxin-3 was first identified just a decade ago via a genomic database search and is now recognized to be a key neuropeptide with several roles including the regulation of arousal, stress responses and neuroendocrine homeostasis. It also has significant potential as a drug to treat stress and obesity. Its actions are mediated via its cognate G protein-coupled receptor, RXFP3, which is found in abundant numbers in the brain. However, much remains to be determined with respect to the mechanism of neurological action of this peptide. Consequently, the chemical synthesis of the rat and mouse (which share identical primary structures) two-chain, three disulfide peptide was undertaken and the resulting peptide subjected to detailed in vitro and in vivo assay. Use of efficient solid-phase synthesis methods provided the two regioselectively S-protected A- and B-chains which were readily combined via sequential disulfide bond formation. The synthetic rat/mouse relaxin-3 was obtained in high purity and good overall yield. It demonstrated potent orexigenic activity in rats in that central intracerebroventricular infusion led to significantly increased food intake and water drinking.

Production of Colony-Stimulating Factors and IL-5 by Organs from Three Types of Mice with Inflammatory Disease Due to Loss of the Suppressor of Cytokine Signaling-1

Organs from neonatal mice dying from IFN-γ-dependent inflammatory disease initiated by loss of the gene encoding the suppressor of cytokine signaling-1 (SOCS-1) had a normal capacity to produce G-CSF in vitro but a reduced capacity to produce GM-CSF, most evident with the lung, and some reduction in the production of M-CSF by muscle tissue. In contrast, organs from mice lacking the genes for both SOCS-1 and IFN-γ had a normal capacity to produce CSFs. Organs from young adult mice dying with polymyositis and myocarditis that lacked SOCS-1 but were heterozygous for IFN-γ had a normal capacity to produce GM-CSF and M-CSF, but muscle tissue produced significantly increased amounts of G-CSF and IL-5 with IL-5 production also being elevated for the salivary gland, thymus, and heart. Loss of the IFN-γ gene alone had no impact on organ production of these cytokines in vitro. In none of the inflammatory disease models was IL-3 production detected. The SOCS-1 protein appears to have no direct influence on the cellular production of these cytokines and the abnormalities observed either depend on the coaction of IFN-γ, or more likely, are linked with the invasion and destruction of tissue by T lymphocytes, macrophages, eosinophils, and neutrophils. The ability of local organs to produce these proinflammatory cytokines could contribute to the development and progression of these inflammatory lesions.

The multi-organ origin of interleukin-5 in the mouse

Murine Ba/F3 cells were transfected with cDNA for the α-chain of the murine interleukin-5 (IL-5) receptor and cloned lines of these cells were able to proliferate in response to as little as 2.5 pg/ml of IL-5. The bioassay was demonstrated to be specific for IL-5 and was able to measure IL-5 produced in culture by organs from adult C57BL/6 and BALB/c mice. The highest levels of IL-5 were produced by lung tissue but thymus and bladder consistently produced IL-5 and more variable production was observed by the heart, spleen, muscle, bone shaft, uterus and testes. Bone marrow cells produced no detectable IL-5. Observed levels of production of IL-5 were similar when using organs from mice lacking high-affinity receptors for IL-5 and from nu/nu, RAG-1−/− and NOD/SCID mice lacking T lymphocytes. In inflammatory peritoneal exudates induced by the injection of casein plus bacteria, levels of induced IL-5 were higher if the mice lacked high-affinity receptors for IL-5. The data indicate that T lymphocytes are not the dominant cellular source of IL-5 in organ-conditioned media and that local IL-5 production can occur with a wide range of normal murine organs.

The Neurocircuitry of fluid satiation

Fluid satiation, or quenching of thirst, is a critical homeostatic signal to stop drinking; however, its underlying neurocircuitry is not well characterized. Cutting‐edge genetically encoded tools and techniques are now enabling researchers to pinpoint discrete neuronal populations that control fluid satiation, revealing that hindbrain regions, such as the nucleus of the solitary tract, area postrema, and parabrachial nucleus, primarily inhibit fluid intake. By contrast, forebrain regions such as the lamina terminalis, primarily stimulate thirst and fluid intake. One intriguing aspect of fluid satiation is that thirst is quenched tens of minutes before water reaches the circulation, and the amount of water ingested is accurately calibrated to match physiological needs. This suggests that ‘preabsorptive’ inputs from the oropharyngeal regions, esophagus or upper gastrointestinal tract anticipate the amount of fluid required to restore fluid homeostasis, and provide rapid signals to terminate drinking once this amount has been consumed. It is likely that preabsorptive signals are carried via the vagal nerve to the hindbrain. In this review, we explore our current understanding of the fluid satiation neurocircuitry, its inputs and outputs, and its interconnections within the brain, with a focus on recent studies of the hindbrain, particularly the parabrachial nucleus.