Margaret M. Durkin

GABA B receptors function as a heteromeric assembly of the subunits GABA B R1 and GABA B R2

The principal inhibitory neurotransmitter GABA (γ-aminobutyric acid) exerts its effects through two ligand-gated channels, GABAA and GABAC receptors, and a third receptor, GABAB (ref. 1), which acts through G proteins to regulate potassium and calcium channels. Cells heterologously expressing the cloned DNA encoding the GABABR1 protein exhibit high-affinity antagonist-binding sites, but they produce little of the functional activity expected from studies of endogenous GABAB receptors in the brain. Here we describe a new member of the GABAB polypeptide family, GABABR2, that shows sequence homology to GABABR1. Neither GABABR1 nor GABABR2, when expressed individually, activates GIRK-type potassium channels; however, the combination of GABABR1 and GABABR2 confers robust stimulation of channel activity. Both genes are co-expressed in individual neurons, and both proteins co-localize in transfected cells. Moreover, immunoprecipitation experiments indicate that the two polypeptides associate with each other, probably as heterodimers. Several G-protein-coupled receptors (GPCRs) exist as high-molecular-weight species, consistent with the formation of dimers by these receptors, but the relevance of these species for the functioning of GPCRs has not been established. We have now shown that co-expression of two GPCR structures, GABABR1 and GABABR2, belonging to the samesubfamily is essential for signal transduction by GABAB receptors.

Trace amines: Identification of a family of mammalian G protein-coupled receptors

Tyramine, β-phenylethylamine, tryptamine, and octopamine are biogenic amines present in trace levels in mammalian nervous systems. Although some “trace amines” have clearly defined roles as neurotransmitters in invertebrates, the extent to which they function as true neurotransmitters in vertebrates has remained speculative. Using a degenerate PCR approach, we have identified 15 G protein-coupled receptors (GPCR) from human and rodent tissues. Together with the orphan receptor PNR, these receptors form a subfamily of rhodopsin GPCRs distinct from, but related to the classical biogenic amine receptors. We have demonstrated that two of these receptors bind and/or are activated by trace amines. The cloning of mammalian GPCRs for trace amines supports a role for trace amines as neurotransmitters in vertebrates. Three of the four human receptors from this family are present in the amygdala, possibly linking trace amine receptors to affective disorders. The identification of this family of receptors should rekindle the investigation of the roles of trace amines in mammalian nervous systems and may potentially lead to the development of novel therapeutics for a variety of indications.

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.

A receptor autoradiographic and in situ hybridization analysis of the distribution of the 5-ht7 receptor in rat brain.

1 Receptor autoradiography and in situ hybridization histochemistry have been used to delineate the distribution of the 5‐ht7 receptor and its mRNA in rat brain. Receptor autoradiographic studies were performed using [3H]‐5‐carboxamidotryptamine (5‐CT) as the radioligand. The binding characteristics of the masking compounds were determined in Cos‐7 cells transfected with a panel of 5‐HT receptor subtype cDNAs, including the rat 5‐ht7 cDNA. In situ hybridization studies were carried out with 35S‐labelled oligonucleotide probes to the rat 5‐ht7 mRNA. 2 Specific binding of [3H]‐5‐CT was observed in many areas of the rat brain. Following co‐incubation with 1 μm ergotamine, this binding was completely eliminated. After addition of the masking ligands, [3H]‐5‐CT binding remained in layers 1–3 of cortex, septum, globus pallidus, thalamus, hypothalamus, centromedial amygdala, substantia nigra, periaquaductal gray, and superior colliculus. Addition of the antagonist, methiothepin, to the incubation regimen eliminated most of the remaining [3H]‐5‐CT binding in the brain, with the exception of the globus pallidus and substantia nigra. 3 The 5‐ht7 mRNA was discretely localized in rat brain. The most intense hybridization signals were observed over the thalamus, the anterior hippocampal rudiment, and over the CA3 region of the hippocampus. Other regions containing hybridization signals included the septum, the hypothalamus, the centromedial amygdala and the periaquaductal gray. The regions exhibiting a modest receptor binding signal after methiothepin incubation, the globus pallidus and the substantia nigra, contained no 5‐ht7 hybridization signals, suggesting a non‐5‐ht7 subtype in these two related structures. 4 The distribution of the 5‐ht7 receptor and its mRNA is suggestive of multiple roles for this novel 5‐HT receptor, within several brain systems. The limbic system (centromedial amygdala, anterior hippocampal rudiment, hypothalamus) is particularly well‐represented, indicating a potential role for the 5‐ht7 receptor in affective processes.

Localization of messenger RNAs encoding three GABA transporters in rat brain : an in situ hybridization study

Localization of the messenger RNAs encoding three gamma-aminobutyric acid (GABA) transporters, termed GAT-1, GAT-2, and GAT-3, has been carried out in rat brain using radiolabeled oligonucleotide probes and in situ hybridization histochemistry. Hybridization signals for GAT-1 mRNA were observed over many regions of the rat brain, including the retina, olfactory bulb, neocortex, ventral pallidum, hippocampus, and cerebellum. At the microscopic level, this signal appeared to be restricted to neuronal profiles, and the overall distribution of GAT-1 mRNA closely paralleled that seen in other studies with antibodies to GABA. Areas containing hybridization signals for GAT-3 mRNA included the retina, olfactory bulb, subfornical organ, hypothalamus, midline thalamus, and brainstem. In some regions, the hybridization signal for GAT-3 seemed to be preferentially distributed over glial cells, although hybridization signals were also observed over neurons, particularly in the retina and olfactory bulb. Notably, hybridization signal for GAT-3 mRNA was absent from the neocortex and cerebellar cortex, and was very weak in the hippocampus. In contrast to the parenchymal localization obtained for GAT-1 and GAT-3 mRNAs, hybridization signals for GAT-2 mRNA were found only over the leptomeninges (pia and arachnoid). The differential distribution of the three GABA transporters described here suggests that while each plays a role in GABA uptake, they do so via distinct cellular populations.

Distribution of a rat galanin receptor mRNA in rat brain

In situ hybridization histochemistry has been employed to determine the distribution of the mRNA encoding a recently cloned rat galanin receptor (rGalR1). The galanin receptor mRNA has been found to be discretely localized in rat brain. The most intense hybridization signals were found over neurons in the nucleus of the lateral olfactory tract, in the ventral posterior hippocampus, and in the lateral external subdivision of the parabrachial nucleus. A number of other brain regions also contain significant hybridization signals, including the hypothalamus, brain stem and spinal cord. The localization of rGalR1mRNA indicates that this receptor may play a role in the varied functions ascribed to GAL, among them feeding, cognition and modulation of sensory information.

5-HT1F receptor agonists inhibit neurogenic dural inflammation in guinea pigs.

THE serotonin (5-HT) receptor subtype mediating inhibition of neurogenic dural inflammation in guinea pigs was investigated using a series of serotonin agonists with differing affinities for the 5-HT1B, 5-HT1D and 5-HT1F receptors. When agonist potencies for inhibiting neurogenic inflammation were compared with affinities for these receptor subtypes, a significant positive correlation was seen only with the 5-HT1F receptor. The potency of agonists in inhibiting adenylate cyclase in cells transfected with human 5-HT1F receptor was also highly correlated with their potency in the animal model of migraine. In situ hybridization demonstrated 5-HT1F receptor mRNA in guinea pig trigeminal ganglion neurons. These data suggest that the 5-HT1F receptor is a rational target for migraine therapeutics.

Distribution of the neuropeptide Y Y2 receptor mRNA in rat central nervous system

Our group has recently reported the expression cloning of the human neuropeptide Y Y2 receptor DNA and subsequently the cloning of the rat homologue. These studies have made it possible to localize the mRNA encoding this NPY receptor subtype in rat tissues. We have, thus, carried out in situ hybridization studies, using radiolabeled oligonucleotide probes to the rat Y2 receptor mRNA, to determine the distribution of Y2 mRNA in rat brain and limited peripheral ganglia. Probe specificity was confirmed by testing antisense and sense probes in transfected cells. In rat brain, hybridization signals obtained with the antisense probes were discrete and were restricted to neuronal profiles in specific subregions of the cortex, hippocampus, amygdala, thalamus, hypothalamus, mesencephalon and pons. Among the regions exhibiting the most intense labeling were the CA3 region of the hippocampus, the arcuate nucleus of the hypothalamus and layer 3 of the piriform cortex. Other regions containing labeled neurons included the medial amygdala, the centromedial thalamic nucleus, the dorsal raphe, the dorsal motor nucleus of the vagus and the trigeminal ganglion. The present results indicate that the mRNA encoding the Y2 receptor is discretely localized in the rat brain and that the distribution is generally consistent with previous radioligand-binding studies. This study should help clarify the relationship between the Y2 receptor distribution and functional studies of NPY receptor subtype classification and provides further evidence for the involvement of the Y2 receptor in multiple physiological processes.

Localization of mRNA and Receptor Binding Sites for the alpha sub 1a-Adrenoceptor Subtype in the Rat, Monkey and Human Urinary Bladder and Prostate

PURPOSE To localize the mRNAs and receptor binding sites for the alpha 1a/A, alpha 1b/B and alpha 1d/D- adrenoceptor (AR) subtypes in the rat, monkey and human urinary bladder and prostate. MATERIALS AND METHODS alpha 1-AR mRNAs were localized on slide mounted tissue sections by in situ hybridization using [35S]-labeled subtype specific oligonucleotide probes. alpha 1-AR receptor binding sites were localized on slide mounted tissue sections by competitive displacement of [3H]-prazosin using subtype selective ligands. RESULTS Only the alpha 1a-AR subtype mRNA was discernible by in situ hybridization. The alpha 1a-AR mRNA was localized in all smooth muscle areas of the rat, monkey and human urinary bladder and prostate. High levels of alpha 1a mRNA were detected in bladder dome and bladder base urothelium. Competitive displacement studies using the alpha 1A-AR selective ligand SNAP 5272 revealed that the alpha 1A-AR represented over 80% of the total alpha 1-AR in monkey bladder and prostate. In general, localization of the alpha 1A-AR corresponded to the alpha 1a-AR mRNA localization, that is, receptor protein was localized to smooth muscle areas of the bladder dome, trigone and base and prostate. One notable exception was the bladder urothelium, which contained high levels of alpha 1a-AR mRNA, but undetectable levels of alpha 1A-AR protein. The alpha 1a-AR mRNA appeared to be transcribed but not translated in bladder urothelium. CONCLUSIONS The alpha 1A-AR represents the major subtype in the smooth muscle of rat, monkey and human urinary systems. Selective alpha 1A-AR agents are therefore potentially useful in the treatment of multiple urinary smooth muscle related disorders.