Dehydroepiandrosterone Sulfate (DHEAS) Is an Endogenous Kv7 Channel Modulator That Reduces Kv7/M-Current Suppression and Inflammatory Pain.

Neuronal Kv7 voltage-gated potassium channels generate the M-current and regulate neuronal excitability. Here, we report that dehydroepiandrosterone sulfate (DHEAS) is an endogenous Kv7 channel modulator that attenuates Gq-coupled receptor-induced M-current suppression. DHEAS reduced muscarinic agonist-induced Kv7-current suppression of Kv7.1, Kv7.2, Kv7.4, or Kv7.5 homomeric currents and endogenous M-currents in rat sympathetic ganglion neurons. However, DHEAS per se did not alter the voltage dependence of these Kv7 homomeric channels or the m1 receptor-induced activation of phospholipase C or protein kinase C. DHEAS-treated Kv7.2 homomeric currents became resistant to depletion of phosphatidylinositol 4,5-bisphosphate (PIP2) induced by voltage-activated phosphatase, Ci-VSP or eVSP. Our computational models predicted a novel binding site for DHEAS in the cytoplasmic domain of Kv7 subunits. A single-point mutation of the predicted key histidine into cysteine in the rat Kv7.2 subunit, rKv7.2(H558C), resulted in a loss of effects of DHEAS on muscarinic Kv7 current suppression. Furthermore, in vivo administration of DHEAS in mice of both sexes reduced late phase pain responses in the formalin paw test. However, it did not have effects on early phase responses in the formalin paw test or responses in the hot plate test. Coadministration of a selective Kv7 inhibitor, XE991, and DHEAS eliminated analgesic effects of DHEAS in late phase responses in the formalin paw test. Collectively, these results suggest that DHEAS attenuates M-current suppression by stabilizing PIP2-Kv7 subunit interaction and can mitigate inflammatory pain.SIGNIFICANCE STATEMENT M-current suppression induced by stimulation of Gq-coupled receptors is a form of Kv7 current modulation that can reversibly increase neuronal excitability. This study demonstrates that DHEAS, an endogenous steroid hormone, is a novel Kv7 channel modulator that can attenuate M-current suppression without affecting basal Kv7 channel kinetics. Administration of DHEAS in vivo alleviated inflammatory pain in rodents. These results suggest that the degree of M-current suppression can be dynamically regulated by small molecules. Therefore, this novel form of Kv7 channel regulation holds promising potential as a therapeutic target for sensitized nervous activities, such as inflammatory pain.

Receptor-specific crosstalk between prostanoid E receptor 3 and bombesin receptor subtype 3.

Bombesin receptor subtype 3 (BRS-3) is a GPCR that is expressed in the CNS, peripheral tissues, and tumors. Our understanding of BRS-3's role in physiology and pathophysiology is limited because its natural ligand is unknown. In an attempt to identify this ligand, we screened toad skin ( Bufo bufo gargarizans Cantor) extracts and identified prostaglandins as putative ligands. In BRS-3-transfected human embryonic kidney (HEK) cells, we found that prostaglandins, with prostaglandin E2 (PGE2) being the most potent, fulfill the pharmacologic criteria of affinity, selectivity, and specificity to be considered as agonists to the BRS-3 receptor. However, PGE2 is unable to activate BRS-3 in different cellular environments. We speculated that EP receptors might be the cause of this cellular selectivity, and we found that EP3 is the receptor primarily responsible for the differential PGE2 effect. Consequently, we reconstituted the HEK environment in Chinese hamster ovary (CHO) cells and found that BRS-3 and EP3 interact to potentiate PGE2 signaling. This potentiating effect is receptor specific, and it occurs only when BRS-3 is paired to EP3. Our study represents an example of functional crosstalk between two distantly related GPCRs and may be of clinical importance for BRS-3-targeted therapies.-Zhang, Y., Liu, Y., Wu, L., Fan, C., Wang, Z., Zhang, X., Alachkar, A., Liang, X., Civelli, O. Receptor-specific crosstalk between prostanoid E receptor 3 and bombesin receptor subtype 3.

Inactivation of the melanin concentrating hormone system impairs maternal behavior.

In order to prepare the mother for the demands of pregnancy and lactation, the maternal brain is subjected to a number of adaptations. Maternal behaviors are regulated by complex neuronal interactions. Here, we show that the melanin concentrating hormone (MCH) system is an important regulator of maternal behaviors. First, we report that melanin concentrating hormone receptor 1 knockout (MCHR1 KO) mice display a disruption of maternal behavior. Early postpartum MCHR1 KO females exhibit poor nesting, deficits in pup retrieval and maternal aggression. In addition, ablation of MCH receptors results in decreased milk production and prolactin mRNA levels. Then we show that these results are in line with those obtained in wild type mice (WT) treated with the specific MCHR1 antagonist GW803430. Furthermore, following pups retrieval, MCHR1 KO mice display a lower level of Fos expression than WT mice in the ventral tegmental area, and nucleus accumbens. With the progression of the lactation period, however, the MCHR1 KO mice improve maternal care towards their pups. This is manifested by an increase in the pups׳ survival rate and the decrease in pups׳ retrieval time beyond the second day after parturition. In conclusion, we show that the MCH system plays a significant role in the initiation of maternal behavior. In this context, MCH may play a role in integrating information from multiple sources, and connecting brain reward, homeostatic and regulatory systems.

Anatomical characterization of bombesin receptor subtype-3 mRNA expression in the rodent central nervous system.

Bombesin receptor subtype-3 (BRS-3) is an orphan G-protein-coupled receptor (GPCR) involved in the regulation of energy homeostasis. Mice deficient in BRS-3 develop late-onset mild obesity with metabolic defects, while synthetic agonists activating BRS-3 show antiobesity profiles by inhibiting food intake and increasing metabolic rate in rodent models. The molecular mechanisms and the neural circuits responsible for these effects, however, remain elusive and demand better characterization. We report here a comprehensive mapping of BRS-3 mRNA in the rat and mouse brain through in situ hybridization. Furthermore, to investigate the neurochemical characteristics of the BRS-3-expressing neurons, double in situ hybridization was performed to determine whether BRS-3 colocalizes with other neurotransmitters or neuropeptides. Many, but not all, of the BRS-3-expressing neurons were found to be glutamatergic, while few were found to be cholinergic or GABAergic. BRS-3-containing neurons do not express some of the well-characterized neuropeptides, such as neuropeptide Y (NPY), proopiomelanocortin (POMC), orexin/hypocretin, melanin-concentrating hormone (MCH), thyrotropin-releasing hormone (TRH), gonadotropin-releasing hormone (GnRH), and kisspeptin. Interestingly, BRS-3 mRNA was found to partially colocalize with corticotropin-releasing factor (CRF) and growth hormone-releasing hormone (GHRH), suggesting novel interactions of BRS-3 with stress- and growth-related endocrine systems. Our study provides important information for evaluating BRS-3 as a potential therapeutic target for the treatment of obesity.

Disruption of the melanin-concentrating hormone receptor 1 (MCH1R) affects thyroid function.

Melanin-concentrating hormone (MCH) is a peptide produced in the hypothalamus and the zona incerta that acts on one receptor, MCH receptor 1 (MCH1R), in rodents. The MCH system has been implicated in the regulation of several centrally directed physiological responses, including the hypothalamus-pituitary-thyroid axis. Yet a possible direct effect of the MCH system on thyroid function has not been explored in detail. We now show that MCH1R mRNA is expressed in thyroid follicular cells and that mice lacking MCH1R [MCH1R-knockout (KO)] exhibit reduced circulating iodothyronine (T(4), free T(4), T(3), and rT(3)) levels and high TRH and TSH when compared with wild-type (WT) mice. Because the TSH of MCH1R-KO mice displays a normal bioactivity, we hypothesize that their hypothyroidism may be caused by defective thyroid function. Yet expression levels of the genes important for thyroid hormones synthesis or secretion are not different between the MCH1R-KO and WT mice. However, the average thyroid follicle size of the MCH1R-KO mice is larger than that of WT mice and contained more free and total T(4) and T(3) than the WT glands, suggesting that they are sequestered in the glands. Indeed, when challenged with TSH, the thyroids of MCH1R-KO mice secrete lower amounts of T(4). Similarly, secretion of iodothyronines in the plasma upon (125)I administration is significantly reduced in MCH1R-KO mice. Therefore, the absence of MCH1R affects thyroid function by disrupting thyroid hormone secretion. To our knowledge, this study is the first to link the activity of the MCH system to the thyroid function.

Biochemical roles of the oligosaccharide chains in thyrostimulin, a heterodimeric hormone of glycoprotein hormone subunits alpha 2 (GPA2) and beta 5 (GPB5).

Thyrostimulin is a heterodimeric hormone composed of GPA2 and GPB5, and shares the thyroid-stimulating hormone receptor (TSHR). Thyrostimulin has three N-linked oligosaccharide chains, two in GPA2 and one in GPB5. The roles of these N-linked oligosaccharides in secretion, heterodimer formation and signal transduction were analyzed. Recombinant GPA2s lacking either of the two oligosaccharides were obtained from conditioned medium, whereas dual site-disrupted GPA2 and the GPB5 mutant were not expressed in either the conditioned medium or cell lysate. The binding between GPA2 and GPB5 was weaker than that between TSH subunits GPA1 and TSH beta. Neither of the oligosaccharides in GPA2 had significant effects on heterodimerization. Disruption of either of the oligosaccharides in GPA2 significantly decreased receptor activation, suggesting their critical role in receptor activation.

The orphanin FQ/nociceptin (OFQ/N) system.

Orphanin FQ/nociceptin (OFQ/N) was the first novel neuropeptide discovered as the natural ligand of an orphan G protein-coupled receptor (GPCR). Orphan GPCRs are proteins classified as receptors on the basis of their sequence similarities to known GPCRs but that lack the ligands that activate them in vivo. One such orphan GPCR exhibited sequence similarities with the opioid receptors. OFQ/N was isolated as its natural ligand and shown to also share sequence similarities to the opioid peptides. This led to numerous studies attempting to find functional similarities and differences between the OFQ/N and opioid systems. This chapter will summarize our knowledge of the OFQ/N system and of its roles in the organism.

A Drosophila adenosine receptor activates cAMP and calcium signaling.

Adenosine receptors (AdoR) are members of the G protein-coupled receptor superfamily and mediate extracellular adenosine signaling, but the mechanism of adenosine signaling is still unclear. Here we report the first characterization of an insect AdoR, encoded by the Drosophila gene CG9753. Adenosine stimulation of Chinese hamster ovary cells carrying transiently expressed CG9753 led to a dose-dependent increase of intracellular cAMP and calcium, but untransfected controls showed no such response, showing that CG9753 encodes a functional AdoR. Endogenous CG9753 transcripts were detected in the brain, imaginal discs, ring gland and salivary glands of third-instar Drosophila larvae, and CG9753 overexpression in vivo caused lethality or severe developmental anomalies. These developmental defects were reduced by adenosine depletion, consistent with the proposed function of the CG9753 product as an AdoR. Overexpression of the G protein subunit Galpha(s) or of the catalytic subunit of protein kinase A (PKA) partially mimicked and enhanced the defects caused by ectopic expression of AdoR. Our results suggest that AdoR is an essential part of the adenosine signaling pathway and Drosophila offers a unique opportunity to use genetic analysis to study conserved aspects of the adenosine signaling pathway.

GPR39 receptor expression in the mouse brain.

GPR39, an orphan G protein-coupled receptor, has been recently identified as the receptor for the bioactive peptide obestatin. Obestatin is secreted from the stomach and acts as an anti-appetite hormone. This activity is induced whether obestatin is administered intraperitoneally or intracerebroventricularly. GPR39 is known to be expressed in the central nervous system but its precise localization is unknown. In view of the growing importance of this system, we decided to study the sites of GPR39 mRNA expression by in-situ hybridization. We find the highest levels of GPR39 mRNA in the amygdala, the hippocampus, and the auditory cortex and low levels in several other brain regions. Surprisingly, we find no expression of GPR39 in the hypothalamus, expected to be the site of the anorexigenic action of obestatin.

Salusin beta is a surrogate ligand of the mas-like G protein-coupled receptor MrgA1.

The mas-like G protein-coupled receptors form a subfamily of G protein-coupled receptors that includes variable member numbers across different species and that have been shown to bind a wide variety of ligands from peptides to amino acid derivatives. While screening a library of peptides against different orphan G protein-coupled receptors, we found that human salusin beta activates the mouse mas-like G protein-coupled receptor, mMrgA1 with an EC(50) of about 300 nM. Salusin beta is a bioactive peptide recently discovered through bioinformatics analysis which stimulates arginine-vasopressin release from rat pituitary and causes rapid and profound hypotension and bradycardia. However, when we further analyzed the generality of the mMrgA1 activation, we found that human salusin beta does not activate corresponding human mas-like G protein-coupled receptors. Our results show that human salusin beta is a surrogate ligand of the mouse MrgA1 and raises a cautionary flag for experiments that analyze the pharmacological profiles of mas-like G protein-coupled receptors from different species.

Cancer metastasis-suppressing peptide metastin upregulates excitatory synaptic transmission in hippocampal dentate granule cells.

Metastin is an antimetastatic peptide encoded by the KiSS-1 gene in cancer cells. Recent studies found that metastin is a ligand for the orphan G-protein-coupled receptor GPR54, which is highly expressed in specific brain regions such as the hypothalamus and parts of the hippocampus. This study shows that activation of GPR54 by submicromolar concentrations of metastin reversibly enhances excitatory synaptic transmission in hippocampal dentate granule cells in a mitogen-activated protein (MAP) kinase-dependent manner. Synaptic enhancement by metastin was suppressed by intracellular application of the G-protein inhibitor GDP-beta-S and the calcium chelator BAPTA. Analysis of miniature excitatory postsynaptic currents (mEPSCs) revealed an increase in the mean amplitude but no change in event frequency. This indicates that GPR54 and the mechanism responsible for the increase in EPSCs are postsynaptic. Metastin-induced synaptic potentiation was abolished by 50 microM PD98059 and 20 microM U0126, two inhibitors of the MAP kinases ERK1 and ERK2. The effect was also blocked by inhibitors of calcium/calmodulin-dependent kinases and tyrosine kinases. RT-PCR experiments showed that both KiSS-1 and GPR54 are expressed in the hippocampal dentate gyrus. Metastin is thus a novel endogenous factor that modulates synaptic excitability in the dentate gyrus through mechanisms involving MAP kinases, which in turn may be controlled upstream by calcium-activated kinases and tyrosine kinases.

Pharmacological characterization of human and murine neuropeptide s receptor variants.

We have recently shown that Neuropeptide S (NPS) can promote arousal and induce anxiolytic-like effects after central administration in rodents. Another study reported a number of natural polymorphisms in the human NPS receptor gene. Some of these polymorphisms were associated with increased risk of asthma and possibly other forms of atopic diseases, but the physiological consequences of the mutations remain unclear. One of the polymorphisms produces an Asn-Ile exchange in the first extracellular loop of the receptor protein, and a C-terminal splice variant of the NPS receptor was found overexpressed in human asthmatic airway tissue. We sought to study the pharmacology of the human receptor variants in comparison with the murine receptor protein. Here, we report that the N107I polymorphism in the human NPS receptor results in a gain-of-function characterized by an increase in agonist potency without changing binding affinity in NPSR Ile107. In contrast, the C-terminal splice variant of the human NPS receptor shows a pharmacological profile similar to NPSR Asn107. The mouse NPS receptor, which also carries an Ile residue at position 107, displays an intermediate pharmacological profile. Structure-activity relationship studies show that the amino terminus of NPS is critical for receptor activation. The altered pharmacology of the Ile107 isoform of the human NPS receptor implies a mechanism of enhanced NPS signaling that might have physiological significance for brain function as well as peripheral tissues that express NPS receptors.

Orphan G protein-coupled receptors and obesity.

The use of orphan G protein-coupled receptors (GPCRs) as targets to identify new transmitters has led over the last decade to the discovery of 12 novel neuropeptide families. Each one of these new neuropeptides has opened its own field of research, has brought new insights in distinct pathophysiological conditions and has offered new potentials for therapeutic applications. Interestingly, several of these novel peptides have seen their roles converge on one physiological response: the regulation of food intake and energy expenditure. In this manuscript, we discuss four deorphanized GPCR systems, the ghrelin, orexins/hypocretins, melanin-concentrating hormone (MCH) and neuropeptide B/neuropeptide W (NPB/NPW) systems, and review our knowledge of their role in the regulation of energy balance and of their potential use in therapies directed at feeding disorders.

High-throughput real-time monitoring of Gs-coupled receptor activation in intact cells using cyclic nucleotide-gated channels.

Cyclic adenosine-monophosphate (cAMP) is one of the major second messenger molecules transmitting extracellular stimuli into short- and long-term changes of intracellular homeostasis. Measurements of cellular cAMP levels are often used to quantify and characterize signaling by G protein-coupled receptors. Current assays for cAMP determination are usually end-point assays involving cell lysis. We have developed a technology to monitor real-time changes of cAMP levels in living cells. This method uses a modified cyclic nucleotide-gated (CNG) Ca(2+) channel which is opened by intracellular cAMP. Thus, changes in cAMP levels are translated into changes in free Ca(2+) which can easily be measured using fluorimetric imaging technologies compatible with high-throughput screening formats. The new assay method was used to characterize the pharmacology of various endogenously and heterologously expressed G protein-coupled receptors and allows for the simultaneous study of G(s), G(i) and G(q)-linked receptors in the same cell population.

Different structural requirements for melanin-concentrating hormone (MCH) interacting with rat MCH-R1 (SLC-1) and mouse B16 cell MCH-R.

Melanin-concentrating hormone (MCH) is a neuropeptide occurring in all vertebrates and some invertebrates and is now known to stimulate pigment aggregation in teleost melanophores and food-intake in mammals. Whereas the two MCH receptor subtypes hitherto cloned, MCH-R1 and MCH-R2, are thought to mediate mainly the central effects of MCH, the MCH-R on pigment cells has not yet been identified, although in some studies MCH-R1 was reported to be expressed by human melanocytes and melanoma cells. Here we present data of a structure-activity study in which 12 MCH peptides were tested on rat MCH-R1 and mouse B16 melanoma cell MCH-R, by comparing receptor binding affinities and biological activities. For receptor binding analysis with HEK-293 cells expressing rat MCH-R1 (SLC-1), the radioligand was [125I]-[Tyr13]-MCH with the natural sequence. For B16 cells (F1 and G4F sublines) expressing B16 MCH-R, the analog [125I]-[D-Phe13, Tyr19]-MCH served as radioligand. The bioassay used for MCH-R1 was intracellular Ca2+ mobilization quantified with the FLIPR instrument, whereas for B16 MCH-R the signal determined was MAP kinase activation. Our data show that some of the peptides displayed a similar relative increase or decrease of potency in both cell types tested. For example, linear MCH with Ser residues at positions 7 and 16 was almost inactive whereas a slight increase in side-chain hydrophilicity at residues 4 and 8, or truncation of MCH at the N-terminus by two residues hardly changed binding affinity or bioactivity. On the other hand, salmonic MCH which also lacks the first two residues of the mammalian sequence but in addition has different residues at positions 4, 5, 9, and 18 exhibited a 5- to 10-fold lower binding activity than MCH in both cell systems. A striking difference in ligand recognition between MCH-R1 and B16 MCH-R was however observed with modifications at position 13 of MCH: whereas L-Phe13 in [Phe13, Tyr19]-MCH was well tolerated by both MCH-R1 and B16 MCH-R, change of configuration to D-Phe13 in [D-Phe13, Tyr19]-MCH or [D-Phe13]-MCH led to a complete loss of biological activity and to a 5- to 10-fold lower binding activity with MCH-R1. By contrast, the D-Phe13 residue increased the affinity of [D-Phe13, Tyr19]-MCH to B16 MCH-R about 10-fold and elicited MAP kinase activation as observed with [Phe13, Tyr19]-MCH or MCH. These data demonstrate that ligand recognition by B16 MCH-R differs from that of MCH-R1 in several respects, indicating that the B16 MCH-R represents an MCH-R subtype different from MCH-R1.