Pierre J.-J. Vaysse

Antidepressant, anxiolytic and anorectic effects of a melanin-concentrating hormone-1 receptor antagonist.

Melanin concentrating hormone (MCH) is an orexigenic hypothalamic neuropeptide, which plays an important role in the complex regulation of energy balance and body weight. Here we show that SNAP-7941, a selective, high-affinity MCH1 receptor (MCH1-R) antagonist, inhibited food intake stimulated by central administration of MCH, reduced consumption of palatable food, and, after chronic administration to rats with diet-induced obesity, resulted in a marked, sustained decrease in body weight. In addition, after mapping the binding sites for [3H]SNAP-7941 in rat brain, we evaluated its effects in a series of behavioral models. SNAP-7941 produced effects similar to clinically used antidepressants and anxiolytics in three animal models of depression/anxiety: the rat forced-swim test, rat social interaction and guinea pig maternal-separation vocalization tests. Given these observations, an MCH1-R antagonist may be useful not only in the management of obesity but also as a treatment for depression and/or anxiety.

Identification and Characterization of Two G Protein-coupled Receptors for Neuropeptide FF

The central nervous system octapeptide, neuropeptide FF (NPFF), is believed to play a role in pain modulation and opiate tolerance. Two G protein-coupled receptors, NPFF1 and NPFF2, were isolated from human and rat central nervous system tissues. NPFF specifically bound to NPFF1 (K d = 1.13 nm) and NPFF2 (K d = 0.37 nm), and both receptors were activated by NPFF in a variety of heterologous expression systems. The localization of mRNA and binding sites of these receptors in the dorsal horn of the spinal cord, the lateral hypothalamus, the spinal trigeminal nuclei, and the thalamic nuclei supports a role for NPFF in pain modulation. Among the receptors with the highest amino acid sequence homology to NPFF1 and NPFF2 are members of the orexin, NPY, and cholecystokinin families, which have been implicated in feeding. These similarities together with the finding that BIBP3226, an anorexigenic Y1 receptor ligand, also binds to NPFF1 suggest a potential role for NPFF1 in feeding. The identification of NPFF1 and NPFF2 will help delineate their roles in these and other physiological functions.

Expression Cloning and Pharmacological Characterization of a Human Hippocampal Neuropeptide Y/Peptide YY Y2 Receptor Subtype

The pancreatic polypeptide family includes neuropeptide Y (NPY), one of the most abundant neuropeptides in the mammalian nervous system, as well as peptide YY (PYY) and pancreatic polypeptide (PP). This peptide family is involved in numerous physiological processes such as memory, pain, blood pressure, appetite, anxiety, and circadian rhythm. Of the multiple Y-type receptors proposed for PP family members, only the Y1 subtype was cloned previously. We now report the isolation of a human Y2 (hhY2) receptor cDNA by expression cloning from a human hippocampal cDNA library, using a 125I-PYY binding assay. hhY2 cDNA encodes a predicted protein of 381 amino acids with low amino acid identity to the human Y1 receptor (31% overall; 41% transmembrane). 125I-PYY binding to transiently expressed hY2 receptors was saturable (pKd = 10.17) and displaceable by human PP family members in rank order: PYY (pKi = 9.47) ∼ NPY (pKi = 9.27) PP (pKi < 6) and by peptide analogs: NPY2-36 (pKi = 8.80) ∼ NPY13-36 (pKi = 8.55) ∼ C2-NPY (pKi = 8.54) > NPY26-36 (pKi = 6.51) ∼ [Leu31,Pro34]NPY (pKi = 6.23). Human PYY decreased [cAMP] and increased intracellular [Ca2+] in hY2-transfected 293 cells.

CLONED HUMAN AND RAT GALANIN GALR3 RECEPTORS: PHARMACOLOGY AND ACTIVATION OF G-PROTEIN INWARDLY RECTIFYING K+ CHANNELS

The neuropeptide galanin has been implicated in the regulation of processes such as nociception, cognition, feeding behavior, and hormone secretion. Multiple galanin receptors are predicted to mediate its effects, but only two functionally coupled receptors have been reported. We now report the cloning of a third galanin receptor distinct from GALR1 and GALR2. The receptor, termed GALR3, was isolated from a rat hypothalamus cDNA library by both expression and homology cloning approaches. The rat GALR3 receptor cDNA can encode a protein of 370 amino acids with 35% and 52% identity to GALR1 and GALR2, respectively. Localization of mRNA by solution hybridization/RNase protection demonstrates that the GALR3 transcript is widely distributed, but expressed at low abundance, with the highest levels in the hypothalamus and pituitary. We also isolated the gene encoding the human homologue of GALR3. The human GALR3 receptor is 90% identical to rat GALR3 and contains 368 amino acids. Binding of porcine 125I-galanin to stably expressed rat and human GALR3 receptors is saturable (rat K D = 0.98 nm and human K D = 2.23 nm) and displaceable by galanin peptides and analogues in the following rank order: rat galanin, porcine galanin ≃ M32, M35 ≃ porcine galanin-(−7 to +29), galantide, human galanin > M40, galanin-(1–16) > [d-Trp2]galanin-(1–29), galanin-(3–29). This profile resembles that of the rat GALR1 and GALR2 receptors with the notable exception that human galanin, galanin-(1–16), and M40 show lower affinity at GALR3. InXenopus oocytes, activation of rat and human GALR3 receptors co-expressed with potassium channel subunits GIRK1 and GIRK4 resulted in inward K+ currents characteristic of Gi/Go-coupled receptors. These data confirm the functional efficacy of GALR3 receptors and further suggest that GALR3 signaling pathways resemble those of GALR1 in that both can activate potassium channels linked to the regulation of neurotransmitter release.

Identification and Characterization of Two Neuromedin U Receptors Differentially Expressed in Peripheral Tissues and the Central Nervous System

Two structurally related, G-protein-coupled receptors were identified as receptors for the neuropeptide, neuromedin U. This peptide is found in highest levels in the gut and genitourinary system where it potently contracts smooth muscle but is also expressed in the spinal cord and discrete regions of the brain. Binding sites for neuromedin U have been characterized in rat uterus, however, little is known about the activity of this peptide in the regions of the central nervous system where it is expressed. The receptors characterized in this report are activated by neuromedin U at nanomolar potency in heterologous expression systems and bind radiolabeled neuromedin U with high affinity. Localization of the receptor RNA by quantitative reverse transcription-polymerase chain reaction in a variety of human tissues shows distinct expression patterns for the two receptors. NMU1 is expressed predominantly in peripheral tissues, whereas NMU2 is more highly expressed in the central nervous system. Identification of neuromedin U receptor subtypes will greatly aid in the determination of the physiological roles of this peptide.

Molecular biology and pharmacology of multiple NPY Y5 receptor species homologs

NPY is a 36-amino acid peptide which exerts its physiological effects through the activation of a family of G-protein coupled receptors. In vivo and in vitro characterization of the recently cloned rat Y5 receptor suggests that it is a primary mediator of NPY-induced feeding (Gerald et al., Nature 1996;382:168-171). We now report the molecular cloning and pharmacological characterization of the human, dog and mouse homologs of the Y5 receptor. With the exception of a 21 amino acid repeat in the amino terminus of the mouse Y5 receptor, the sequence of the four species homologs appear to be highly conserved, with 88% to 97% amino acid identities between any two species. Similarly, the pharmacological profiles of the four species homologs as determined in porcine 125I-PYY binding assays show a great deal of conservation, with the following rank order of affinity: human or porcine NPY, PYY, [Leu31,Pro34]NPY, NPY(2-36), human PP > human [D-Trp32]NPY > rat PP, C2-NPY. Northern blot analysis reveals that the Y5 receptor is widely distributed in the human brain, with the strongest signals detected in the cortex, putamen and caudate nucleus. The chromosomal localization of the human Y5 receptor, previously shown to be overlapping and in the opposite orientation to the Y1 receptor, is determined to be 4q31, the same locus as previously demonstrated for the human Y1 receptor (Herzog et al., J Biol Chem 1993;268:6703-6707), suggesting that these receptors may be coregulated. These Y5 species homologs along with corresponding animal models may be useful in the search for novel therapeutics in the treatment of obesity and related feeding disorders.

A structure-activity analysis of the cloned rat and human Y4 receptors for pancreatic polypeptide

We cloned and expressed the rat Y4 receptor for pancreatic polypeptide (PP). Structure-activity profiles derived from 125I-PP binding assays and [cAMP] radioimmunoassays reveal a selective receptor interaction with rat PP vs. neuropeptide Y (NPY) or peptide YY (PYY). Rat and human Y4 receptor clones share 75% amino acid identity. Based on [cAMP] radioimmunoassay, the human Y4 receptor exhibits a less selective interaction with rat PP vs. NPY or PYY and a greater dependence on N-terminal PP residues, relative to rat Y4. Differences in sequence and structure-activity profiles suggest the rat be used with caution to model human Y4 receptor function.

[3H]5-hydroxytryptamine labels the agonist high affinity state of the cloned rat 5-HT4 receptor.

We have used the cloned rat 5-HT4 receptor, and determined that the single protein product produced is able to bind both [3H]5-HT and [3H]GR113808 ([1-[(2-methyl sulphonyl) amino] ethyl-4-piperidinyl] methyl-1-methyl-1H-indole-3-carboxylate) with high affinity. The affinities of agonists for the [3H]5-HT (agonist)-labelled receptor were significantly higher than for the [3H]GR113808 (antagonist)-labelled receptor. Furthermore, [3H]5-HT binding was reduced by addition of guanyl nucleotides. These results strongly support the hypothesis that the 5-HT4 receptor displays two interconvertible affinity states (high and low) for agonists, characteristic of many G protein coupled receptors. [3H]5-HT, at the concentration employed, therefore labels the agonist high affinity state of the 5-HT4 receptor in systems in which high densities of this receptor are found.

A convenient synthesis of 2-amino-2-oxazolines and their pharmacological evaluation at cloned human α adrenergic receptors

Abstract A number of 2-amino-2-oxazolines including rilmenidine ( 7 ) were synthesized in good yield by thermal cyclization of 2-chloroethylurea 2 in the presence of potassium fluoride on alumina. These compounds were assayed for their binding affinity and efficacy at cloned human α adrenergic receptors.

Pharmacological evaluation of UK-14,304 analogs at cloned human α adrenergic receptors

As a part of a program directed at the identification of subtype selective α2 agonists, a series of analogs of UK-14,304 (1) were synthesized. Binding affinities and functional efficacies were measured at cloned human α adrenergic receptors. A number of analogs showed high binding affinity and good selectivity for the human α2A receptor subtype.