Gordon Y. K. Ng

IDENTIFICATION OF A GABAB RECEPTOR SUBUNIT, GB2, REQUIRED FOR FUNCTIONAL GABAB RECEPTOR ACTIVITY

G protein-coupled receptors are commonly thought to bind their cognate ligands and elicit functional responses primarily as monomeric receptors. In studying the recombinant γ-aminobutyric acid, type B (GABAB) receptor (gb1a) and a GABAB-like orphan receptor (gb2), we observed that both receptors are functionally inactive when expressed individually in multiple heterologous systems. Characterization of the tissue distribution of each of the receptors by in situhybridization histochemistry in rat brain revealed co-localization of gb1 and gb2 transcripts in many brain regions, suggesting the hypothesis that gb1 and gb2 may interact in vivo. In three established functional systems (inwardly rectifying K+channel currents in Xenopus oocytes, melanophore pigment aggregation, and direct cAMP measurements in HEK-293 cells), GABA mediated a functional response in cells coexpressing gb1a and gb2 but not in cells expressing either receptor individually. This GABA activity could be blocked with the GABAB receptor antagonist CGP71872. In COS-7 cells coexpressing gb1a and gb2 receptors, co-immunoprecipitation of gb1a and gb2 receptors was demonstrated, indicating that gb1a and gb2 act as subunits in the formation of a functional GABAB receptor.

Molecular characterization and expression of cloned human galanin receptors GALR2 and GALR3.

Abstract: Galanin is a 29‐ or 30‐amino acid peptide with wide‐ranging effects on hormone release, feeding behavior, smooth muscle contractility, and somatosensory neuronal function. Three distinct galanin receptor (GALR) subtypes, designated GALR1, 2, and 3, have been cloned from the rat. We report here the cloning of the human GALR2 and GALR3 genes, an initial characterization of their pharmacology with respect to radioligand binding and signal transduction pathways, and a profile of their expression in brain and peripheral tissues. Human GALR2 and GALR3 show, respectively, 92 and 89% amino acid sequence identity with their rat homologues. Radioligand binding studies with 125I‐galanin show that recombinant human GALR2 binds with high affinity to human galanin (KD = 0.3 nM). Human GALR3 binds galanin with less affinity (IC50 of 12 nM for porcine galanin and 75 nM for human galanin). Human GALR2 was shown to couple to phospholipase C and elevation of intracellular calcium levels as assessed by aequorin luminescence in HEK‐293 cells and by Xenopus melanophore pigment aggregation and dispersion assays, in contrast to human GALR1 and human GALR3, which signal predominantly through inhibition of adenylate cyclase. GALR2 mRNA shows a wide distribution in the brain (mammillary nuclei, dentate gyrus, cingulate gyrus, and posterior hypothalamic, supraoptic, and arcuate nuclei), and restricted peripheral tissue distribution with highest mRNA levels detected in human small intestine. In comparison, whereas GALR3 mRNA was expressed in many areas of the rat brain, there was abundant expression in the primary olfactory cortex, olfactory tubercle, the islands of Calleja, the hippocampal CA regions of Ammons horn, and the dentate gyrus. GALR3 mRNA was highly expressed in human testis and was detectable in adrenal gland and pancreas. The genes for human GALR2 and 3 were localized to chromosomes 17q25 and 22q12.2–13.1, respectively.

A Transmembrane Domain-derived Peptide Inhibits D1 Dopamine Receptor Function without Affecting Receptor Oligomerization

In this study, we show that a peptide based on the sequence of transmembrane domain 6 of the D1 dopamine receptor (D1DR) specifically inhibited D1DR binding and function, without affecting receptor oligomerization. It has been shown that an analogous peptide from the β2-adrenergic receptor disrupted dimerization and adenylyl cyclase activation in the β2-adrenergic receptor (Hebert, T. E., Moffett, S., Morello, J. P., Loisel, T. P., Bichet, D. G., Barret, C., and Bouvier, M. (1996) J. Biol. Chem. 271, 16384–16392). Treatment of D1DR with the D1DR transmembrane 6 peptide resulted in a dose-dependent, irreversible inhibition of D1DR antagonist binding, an effect not seen in D1DR with peptides based on transmembrane domains of other G protein-coupled receptors. Incubation with the D1DR transmembrane 6 peptide also resulted in a dose-dependent attenuation of both dopamine-induced [35S]guanosine 5′-3-O-(thio)triphosphate (GTPγS) binding and receptor-mediated dopamine stimulation of adenylyl cyclase activity. Notably, GTPγS binding and cAMP production were reduced to levels below baseline, indicating blockade of ligand-independent, intrinsic receptor activity. Immunoblot analyses of the D1DR revealed the receptor existed as monomers, dimers, and higher order oligomers and that these oligomeric states were unaffected after incubation with the D1DR transmembrane 6 peptide. These findings represent the first demonstration that a peptide based on the transmembrane 6 of the D1DR may represent a novel category of noncompetitive D1DR antagonists.

Phosphorylation and palmitoylation of the human D2L dopamine receptor in Sf9 cells.

Abstract: We have expressed and biochemically characterized the human D2long (D2L) dopamine receptor isoform using the baculovirus/Sf9 cell system. The expressed receptor bound ligands with a pharmacological profile similar to that reported for neuronal and cloned D2L receptors expressed in mammalian cell lines. Dopamine binding to D2L receptor was sensitive to guanine nucleotides, indicating receptor coupling to endogenous G proteins. A D2L receptor‐specific antibody identified two major protein species at ∼44 kDa and at ∼93 kDa in immunoblots, suggesting the presence of D2L receptor monomers and dimers. Both species were purified by immunoprecipitation from digitonin‐solubilized preparation of cells expressing D2L receptor prelabeled with 32Pi or [3H]‐palmitate. These results constitute the first direct evidence for D2L receptor phosphorylation and palmitoylation.

Genotypic differences in brain dopamine receptor function in the DBA/2J and C57BL/6J inbred mouse strains

The propensity for high ethanol preference and high ethanol consumption (herein referred to as ethanol abuse) may be a consequence of a congenital deficit in central dopaminergic activity. This hypothesis was examined in the ethanol-avoiding DBA/2J (DBA) and ethanol-preferring C57BL/6J (C57) inbred mouse strains. Endogenous dopamine D1 and D2 receptor functions differed between strains in the nigrostriatal/mesolimbic dopamine system. At the level of the forebrain, the C57 mouse exhibited higher dopamine D1 and D2 receptor mRNA abundance and elevated dopamine D1 and D2 receptor densities in the striatum compared to DBA mouse. A likely explanation for these observations might be that higher dopamine receptor gene expression could be a consequence of low synaptic dopamine activity. Accordingly, we found higher striatal dopamine-sensitive adenylyl cyclase activity in the C57 mouse. The C57 mouse exhibited an enhanced dopamine D1-D2 receptor link as suggested by an enhanced up-regulation of striatal dopamine D2 receptor mRNA following dopamine D1 receptor blockade with SCH-23390 compared to DBA mouse. At the level of the mesencephalon and hind brain, the C57 mouse had lower dopamine D2 receptor mRNA in the medulla pons, and correspondingly lower midbrain and medulla pons dopamine D2 receptor densities. Adenylyl cyclase activities in these regions were similar to the DBA mouse suggesting that the coupling of these dopamine D2 receptors could be a factor regulating their function. Strain differences in dopamine D2 receptor function were also observed in the diencephalic dopamine system. The C57 mouse exhibited lower dopamine D2 receptor density in the hippocampus and lower dopamine D2 receptor mRNA abundance and lower adenylyl cyclase activity in the hypothalamus. Changes in brain dopamine receptor gene expression following ethanol intake inferred an increase in the activities of central dopamine pathways in both the DBA and C57 mouse supporting an association between dopamine receptor function and ethanol drinking. These lines of evidence provide a basis for the hypothesis that a genetically determined brain dopaminergic deficit mediated by dopamine D1-D2 receptor mechanisms may be involved in at least a part of the risk for ethanol abuse in the C57 inbred mouse strain.

Immunohistochemical and biochemical assessment of caspase-3 activation and DNA fragmentation following transient focal ischemia in the rat

In the present study, we evaluated the time-course of caspase-3 activation, and the evolution of cell death following focal cerebral ischemia produced by transient middle cerebral artery occlusion in rats. Ischemia-induced active caspase-3 immunoreactivity in the striatum but not the cortex at 3 and 6 h time points post-reperfusion. Furthermore, using a novel approach to visualize enzymatic activity, deltaC-APP, a C-terminal cleavage product of APP generated by caspase-3, was found to immunolocalize to the same areas as active caspase-3. Double-labeling studies demonstrated co-localization of these two proteins at the cellular level. Further double-labeling experiments revealed that active caspase-3 was confined to neuronal cells which were still viable and thus immunoreactive for NeuN. DNA fragmentation, assessed histologically by terminal dUTP nick-end labeling (TUNEL), was observed in a small number of cells in the striatum as early as 3 h, but only began to appear in the cortex by 6 h. DNA fragmentation was progressive, and by 24 h post-reperfusion, large portions of both the striatum and cortex showed TUNEL positive cells. However, double-labeling of active caspase-3 with TUNEL showed only minimal co-localization at all time-points. Thus, caspase-3 activation is an event that appears to occur prior to DNA fragmentation. As a confirmation of the histological TUNEL data, 24 h ischemia also induced the generation of nucleosome fragments, evidenced by cell death enzyme-linked immunosorbent assay. Using a novel ischemia-induced substrate cleavage biochemical approach, spectrin P120 fragment, a caspase-specific cleavage product of alpha II spectrin, a cytoskeletal protein, was shown to be elevated by western blotting. Brain concentrations of both nucleosomes and spectrin P120 correlate with the degree of injury previously assessed by triphenyltetrazolium chloride staining and infarct volume calculation. Together, our findings suggest a possible association between caspase-3 activation and ischemic cell death following middle cerebral artery occlusion brain injury.

Dopamine receptor agonist reduces ethanol self-administration in the ethanol-preferring C57BL/6J inbred mouse

This report experimentally examined whether the genetically determined low nigrostriatal/mesolimbic dopaminergic activity in the C57BL/6J (herein referred to as C57) inbred mouse mediated the congenital high risk for ethanol abuse (ethanol consumption and ethanol preference) in this model. C57 mice pretreated with dopamine D1 receptor agonist ((+)-SKF-38393) or dopamine D2 receptor agonist (bromocriptine) to augment synaptic dopamine availability exhibited marked 76% and 38% reductions in voluntary ethanol intake in comparison to untreated controls. Dopamine receptor agonist administration resulted in changes in dopamine D1 and D2 receptor mRNA in the cell bodies and dopamine D1 and D2 receptor densities principally in the afferent targets of nigrostriatal/mesolimbic dopamine neurons. Dopamine receptor agonists promoted a decrease of striatal dopamine D1 and D2 receptor densities and corresponding down-regulation of olfactory tubercle dopamine D1 and D2 receptor mRNA abundance. Dopamine receptor agonist-induced increases in forebrain dopaminergic activity was compensated with increased dopamine D2 receptor density and correspondingly higher dopamine D2 receptor mRNA content in the brain stem. When bromocriptine was administered to ethanol-sensitized mice, it was ineffective in reducing voluntary ethanol abuse. In these mice, treatment with the dopamine D2 receptor antagonist haloperidol led to a 28% reduction in the absolute amount of ethanol consumed, but not in voluntary ethanol preference. These data indicated that nigrostriatal/mesolimbic dopamine D1-D2 receptor mechanism(s) mediating the potential for becoming high ethanol drinking on exposure to ethanol are distinct from factors mediating voluntary ethanol drinking after sensitization to ethanol. These data constitute direct evidence supporting a dopamine hypothesis for ethanol abuse in the genetically ethanol-preferring C57 mouse.

Genotypic differences in mesolimbic enkephalin gene expression in DBA/2J and C57BL/6J inbred mice

The DBA/2J and C57BL/6J (herein referred to as DBA and C57) inbred mouse strains exhibit low and high predispositions for voluntary ethanol consumption, respectively, but the neurobiological basis underlying this differential drug vulnerability remains poorly understood. Comparison of endogenous brain proenkephalin gene expression showed the C57 mouse, compared to the DBA mouse, had lower preproenkephalin mRNA abundance, proenkephalin concentration and processed [Met5]enkephalin-immunoreactive peptide levels in the mid brain. No strain differences in enkephalin gene expression was observed in the striatum, hypothalamus, or medulla pons. Neurochemical analysis of C57 mice, following high voluntary ethanol consumption (approximately 17 g/kg/day), revealed markedly higher enkephalin gene expression in the striatum and mid brain compared to ethanol-naive animals. These findings suggested that mesolimbic enkephalin is augmented following ethanol consumption, and that endogenous low enkephalin biosynthesis may be associated with an increased vulnerability for ethanol abuse. However, the neurobiological basis of this behaviour may not be quite this simple. C57 mice pretreated with the dopamine receptor agonist, bromocriptine, had reduced striatum and mid brain preproenkephalin mRNA levels, and showed a 41% lower voluntary ethanol consumption compared to controls. We conclude that functional connectivity exists between enkephalin and dopamine systems, and although low mesolimbic enkephalin may predispose to high ethanol preference, dopamine is a more important determinant than enkephalin in the hierarchy of neurotransmitter pathways that mediate the increased vulnerability for ethanol consumption in the C57 mouse.

Neuroprotective effects of M826, a reversible caspase-3 inhibitor, in the rat malonate model of Huntington's disease

Caspases, key enzymes in the apoptosis pathway, have been detected in the brain of HD patients and in animal models of the disease. In the present study, we investigated the neuroprotective properties of a new, reversible, caspase‐3‐specific inhibitor, M826 (3‐({(2S)‐2‐[5‐tert‐butyl‐3‐{[(4‐methyl‐1,2,5‐oxadiazol‐3‐yl)methyl]amino}‐2‐oxopyrazin‐1(2H)‐yl]butanoyl}amino)‐5‐[hexyl(methyl)amino]‐4‐oxopentanoic acid), in a rat malonate model of HD. Pharmacokinetic and autoradiography studies after intrastriatal (i.str.) injection of 1.5 nmol of M826 or its tritiated analogue [3H]M826 indicated that the compound diffused within the entire striatum. The elimination half‐life (T1/2) of M826 in the rat striatum was 3 h. I.str. injection of 1.5 nmol of M826 10 min after malonate infusion induced a significant reduction (66%) in the number of neurones expressing active caspase‐3 in the ipsilateral striatum. Inhibition of active caspase‐3 translated into a significant but moderate reduction (39%) of the lesion volume, and of cell death (24%), 24 h after injury. The efficacy of M826 at inhibiting cell death was comparable to that of the noncompetitive NMDA receptor antagonist MK801. These data provide in vivo proof‐of‐concept of the neuroprotective effects of reversible caspase‐3 inhibitors in a model of malonate‐induced striatal injury in the adult rat.

A novel gene codes for a putative G protein‐coupled receptor with an abundant expression in brain

Following the cloning of the dopamine receptors we continued a search of the human genome for related genes. We searched an EST data base and discovered cDNA fragments encoding novel G protein‐coupled receptor genes. The available GenBank sequence of one of these EST fragments showed that it encoded a receptor with closest similarity to the D2 dopamine and adrenergic receptors. This cDNA was used to isolate the gene (GPR19), and the encoded receptor also demonstrated similarity with the neuropeptide Y receptor. The gene was mapped to chromosome 12, in region p13.2–12.3. Northern blot analysis revealed expression of GPR19 in peripheral regions, and brain regions significantly overlapping with the D2 receptor gene expression. A sequence of the rat orthologue of GPR19 was obtained and in situ hybridization analysis demonstrated a very abundant expression in rat brain.