Tania Ferraro

Relaxin-3 in GABA projection neurons of nucleus incertus suggests widespread influence on forebrain circuits via G-protein-coupled receptor-135 in the rat

Relaxin-3 (RLX3) is a newly identified member of the relaxin/insulin peptide family that is highly conserved across a range of species from fish to mammals and is highly expressed in rat, mouse and human brain. Extensive pharmacological studies have demonstrated that RLX3 is a high affinity, selective ligand for G-protein-coupled receptor-135 (GPCR135, now classified as relaxin family peptide-3 receptor; RXFP3). In ongoing studies to understand the physiological functions of RLX3, the distribution of RLX3-containing neuronal elements in rat brain was determined by immunohistochemistry, using an affinity-purified polyclonal antiserum raised against a conserved segment of the RLX3 C-peptide (AS-R3(85-101)). Consistent with the distribution of RLX3 mRNA, neurons containing RLX3-like immunoreactivity (LI) were observed in the pontine nucleus incertus and the majority of these cells, which are known to express corticotropin-releasing factor receptor-1, were shown to express glutamic acid decarboxylase-65-immunoreactivity, suggesting a GABA phenotype. Nerve fibers and terminals containing RLX3-LI were observed adjacent to cells in the nucleus incertus and in various forebrain regions known to receive afferents from the nucleus incertus, including cortex, septum, hippocampus, thalamus, hypothalamus and midbrain. Regions that contained highest densities of RLX3-positive fibers included the medial septum, lateral preoptic area, lateral hypothalamus/medial forebrain bundle and ventral hippocampus; and additional fibers were observed in olfactory bulb and olfactory and frontal/cingulate cortices, bed nucleus of the stria terminalis, dorsal endopiriform, intergeniculate, and supramammillary nuclei, and the periaqueductal gray and dorsal raphe. The RLX3-positive network overlapped the regional distribution of GPCR135 mRNA and specific binding sites for an [125I]-GPCR135-selective, chimeric peptide. These anatomical findings further support the proposition that RLX3 is the endogenous ligand for GPCR135 in rat brain and provide evidence for broad modulatory activity of RLX3 in behavioral activation relating to autonomic and neuroendocrine control of metabolism and reproduction and higher-order processes such as stress and cognition.

The A-chain of human relaxin family peptides has distinct roles in the binding and activation of the different relaxin family peptide receptors

The relaxin peptides are a family of hormones that share a structural fold characterized by two chains, A and B, that are cross-braced by three disulfide bonds. Relaxins signal through two different classes of G-protein-coupled receptors (GPCRs), leucine-rich repeat-containing GPCRs LGR7 and LGR8 together with GPCR135 and GPCR142, now referred to as the relaxin family peptide (RXFP) receptors 1–4, respectively. Although key binding residues have been identified in the B-chain of the relaxin peptides, the role of the A-chain in their activity is currently unknown. A recent study showed that INSL3 can be truncated at the N terminus of its A-chain by up to 9 residues without affecting the binding affinity to its receptor RXFP2 while becoming a high affinity antagonist. This suggests that the N terminus of the INSL3 A-chain contains residues essential for RXFP2 activation. In this study, we have synthesized A-chain truncated human relaxin-2 and -3 (H2 and H3) relaxin peptides, characterized their structure by both CD and NMR spectroscopy, and tested their binding and cAMP activities on RXFP1, RXFP2, and RXFP3. In stark contrast to INSL3, A-chain-truncated H2 relaxin peptides lost RXFP1 and RXFP2 binding affinity and concurrently cAMP-stimulatory activity. H3 relaxin A-chain-truncated peptides displayed similar properties on RXFP1, highlighting a similar binding mechanism for H2 and H3 relaxin. In contrast, A-chain-truncated H3 relaxin peptides showed identical activity on RXFP3, highlighting that the B-chain is the sole determinant of the H3 relaxin-RXFP3 interaction. Our results provide new insights into the action of relaxins and demonstrate that the role of the A-chain for relaxin activity is both peptide- and receptor-dependent.

Thirst induced by increasing brain sodium concentration is mediated by brain angiotensin

Thirst, the longing or compelling desire to drink, arises physiologically by two main mechanisms-extracellular and cellular dehydration. The hormone angiotensin II has been implicated in the former but not in the latter brain mechanism. To test this apparent difference, experiments in 5 mammalian species examined the effect of intracerebroventricular infusion of losartan, an angiotensin II type I receptor antagonist, on the third induced by intracerebroventricular infusion of an artificial cerebrospinal fluid made hypertonic by the inclusion of 500 mM NaCl. The losartan infusion reduced the water intake due to increased brain sodium concentration in all 5 species, cattle, sheep, rabbits, rats and mice. Thus, the thirst evoked by cellular dehydration, as well as the thirst evoked by extracellular dehydration, may be mediated by angiotensin II.

The NMR solution structure of the relaxin (RXFP1) receptor lipoprotein receptor class a module and identification of key residues in the N-terminal region of the module that mediate receptor activation

The receptors for the peptide hormones relaxin and insulin-like peptide 3 (INSL3) are the leucine-rich repeat-containing G-protein-coupled receptors LGR7 and LGR8 recently renamed as the relaxin family peptide (RXFP) receptors, RXFP1 and RXFP2, respectively. These receptors differ from other LGRs by the addition of an N-terminal low density lipoprotein receptor class A (LDLa) module and are the only human G-protein-coupled receptors to contain such a domain. Recently it was shown that the LDLa module of the RXFP1 and RXFP2 receptors is essential for ligand-stimulated cAMP signaling. The mechanism by which the LDLa module modulates receptor signaling is unknown; however, it represents a unique paradigm in understanding G-protein-coupled receptor signaling. Here we present the structure of the RXFP1 receptor LDLa module determined by solution NMR spectroscopy. The structure is similar to other LDLa modules but shows small differences in side chain orientations and inter-residue packing. Interchange of the module with the second ligand binding domain of the LDL receptor, LB2, results in a receptor that binds relaxin with full affinity but is unable to signal. Furthermore, we demonstrate via structural studies on mutated LDLa modules and functional studies on mutated full-length receptors that a hydrophobic surface within the N-terminal region of the module is essential for activation of RXFP1 receptor signal in response to relaxin stimulation. This study has highlighted the necessity to understand the structural effects of single amino acid mutations on the LDLa module to fully interpret the effects of these mutations on receptor activity.

Adenovirus-mediated delivery of relaxin reverses cardiac fibrosis

We have evaluated the effectiveness of systemic adenovirally delivered mouse relaxin on reversing fibrosis in a transgenic murine model of fibrotic cardiomyopathy due to beta(2)-adrenergic receptor (beta(2)AR) overexpression. Recombinant adenoviruses expressing green fluorescent protein (Ad-GFP), rat relaxin (Ad-rRLN) and mouse relaxin (Ad-mRLN) were generated and Ad-rRLN and Ad-mRLN were demonstrated to direct the expression of bioactive relaxin peptides in vitro. A single systemic injection of Ad-mRLN resulted in transgene expression in the liver and bioactive relaxin peptide in the plasma. Ad-mRLN, but not Ad-GFP, treatment reversed the increased left ventricular collagen content in beta(2)AR mice to control levels without affecting collagen levels in other heart chambers or in the lung and kidney. Hence a single systemic injection of adenovirus producing mouse relaxin reverses cardiac fibrosis without adversely affecting normal collagen levels in other organs and establishes the potential for the use of relaxin gene therapy for the treatment of cardiac fibrosis.

Studies on Soluble Ectodomain Proteins of Relaxin (LGR7) and Insulin 3 (LGR8) Receptors

Abstract: The ectodomains of both the relaxin (LGR7) and the INSL3 (LGR8) receptors can be expressed on the cell surface using only a single transmembrane domain. These membrane‐anchored proteins retain the ability to bind relaxin and can be cleaved from the cell surface. The subsequent LGR7 protein, 7BP, binds relaxin and can act as a functional relaxin antagonist. By contrast, the equivalent LGR8 protein 8BP does not bind relaxin or antagonize LGR8 activity. The 7BP protein has been successfully immobilized onto chemically derivatized surfaces for the capture of relaxin peptides and subsequent identification via SELDI‐MS analysis.

Resolving the Unconventional Mechanisms Underlying RXFP1 and RXFP2 Receptor Function

The receptors for relaxin and insulin‐like peptide 3 (INSL3) are now well‐characterized as the relaxin family peptide (RXFP) receptors RXFP1 and RXFP2, respectively. They are G‐protein‐coupled receptors (GPCRs) with closest similarity to the glycoprotein hormone receptors, with both containing large ectodomains with 10 leucine‐rich repeats (LRRs). Additionally, RXFP1 and RXFP2 are unique in the LGR family in that they contain a low‐density lipoprotein class A (LDL‐A) module at their N‐terminus. Ligand‐mediated activation of RXFP1 and RXFP2 is a complex process involving various domains of the receptors. Primary ligand binding occurs via interactions between B‐chain residues of the peptides with specific residues in the LRRs of the ectodomain. There is a secondary binding site in the transmembrane exoloops which may interact with the A chain of the peptides. Receptor signaling through cAMP then requires the unique LDL‐A module, as receptors without this domain bind ligand normally but do not signal. This is an unconventional mode of activation for a GPCR, and the precise mode of action of the LDL‐A module is currently unknown. The specific understanding of the mechanisms underlying ligand‐mediated activation of RXFP1 and RXFP2 is crucial in terms of targeting these receptors for future drug development.

Converting enzyme inhibition in rabbits : effects on sodium and water intake/excretion and blood pressure

Earlier studies in rabbits revealed that in this species, in contrast to most other species studied, water intake was not influenced by injection or infusion of angiotensin II (ANG II). In order to establish whether ANG II has any role in the regulation of water intake of rabbits, a comprehensive study of the effect of converting enzyme inhibition was undertaken. Enalaprilat was given systemically in various doses to sodium- and water-replete, sodium-deplete, and water-deprived rabbits, and the intake and excretion of water and sodium was measured. In replete rabbits systemic injection of enalaprilat, 8 mg/kg and 8 micrograms/kg, but not 0.8 mg/kg, was followed by increased daily water intake. In sodium-deplete rabbits injection of enalaprilat, 80 mg/kg, was followed by water drinking within 1 h, and daily sodium intake was reduced. Systemic administration of ANG II increased, but did not restore to control level the sodium appetite of sodium-deplete rabbits attenuated by 80 mg/kg enalaprilat. Rabbits deprived of water for 24 h, however, drank the same amount of water after injection of vehicle or enalaprilat, 80 and 8 mg/kg. The efficacy of converting enzyme inhibition was also tested by measuring the blood pressure response to ANG I. Blood pressure responses revealed that in replete animals converting enzyme activity was depressed below control levels for 30 h after injection of 80 mg/kg enalaprilat. In sodium-deplete rabbits blood pressure fell following injection of 80 mg/kg enalaprilat and did not return to control level until 48 h after the injection.(ABSTRACT TRUNCATED AT 250 WORDS)

Design and development of analogues of dimers of insulin-like peptide 3 B-chain as high-affinity antagonists of the RXFP2 receptor†

Insulin‐like peptide 3 (INSL3) is one of 10 members of the human relaxin–insulin superfamily of peptides. It is a peptide hormone that is expressed by fetal and postnatal testicular Leydig cells and postnatal ovarian thecal cells. It mediates testicular descent during fetal life and suppresses sperm apoptosis in adult males, whereas, in females, it causes oocyte maturation. INSL3 has also been shown to promote thyroid tumor growth and angiogenesis in human. These actions of INSL3 are mediated through its G protein‐coupled receptor, RXFP2. INSL3, a two‐chained peptide, binds to its receptor primarily via its B‐chain, whereas elements of the A‐chain are essential for receptor activation. In an attempt to design a high‐affinity antagonist with potential clinical application as an anticancer agent as well as a contraceptive, we have previously prepared a synthetic parallel dimer of INSL3 B‐chain and demonstrated that it binds to RXFP2 with high affinity. In this work, we undertook full pharmacological characterization of this peptide and show that it can antaogonize INSL3‐mediated cAMP signaling through RXFP2. Further refinement by truncation of 18 residues yielded a minimized analogue that retained full binding affinity and INSL3 antagonism. It is an attractive lead peptide for in vivo evaluation as an inhibitor of male and female fertility and of INSL3‐mediated carcinogenesis.

Effect of ICV infusion of CRF on blood pressure and adrenal steroids in rabbits

The effect of prolonged, 22 h long, intracerebroventricular (i.c.v.) infusion of corticotropin-releasing hormone (CRF) on plasma cortisol, corticosterone and electrolyte concentrations, mean arterial blood pressure (MAP) and heart rate (HR) were investigated in conscious rabbits. During i.c.v. infusion of CRF, 1 and 3 micrograms/h, at a rate of 17 microliters/h, plasma cortisol and corticosterone concentrations rose to the level noted after ACTH stimulation in rabbits. Plasma [Na] did not change, but plasma [K] was reduced and plasma osmolality increased during the infusion of CRF, 3 micrograms/h. MAP and HR, recorded continuously during i.c.v. infusion of CRF, changed only with the higher dose of CRF: MAP was elevated during the first 5 h of infusion, and then returned to the control level. HR was lower than control at the end of the first hour of infusion and again between 9 and 15 h of infusion. The prolonged rise of CRF concentration in the brain induced a sustained rise in circulating adrenal steroid hormones. MAP did not increase to the level noted after bolus i.c.v. injection of CRF and the rise in MAP was not sustained.