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Query: UNIPROT:P06889 (Mol)
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We have used the polymerase chain reaction technique to selectively amplify guanine nucleotide-binding regulatory protein (G protein)-coupled receptor cDNA sequences from rat striatal mRNA, using sets of highly degenerate primers derived from transmembrane sequences of previously cloned G protein-coupled receptors. A novel cDNA fragment was identified, which exhibits considerable homology to various members of the G protein-coupled receptor family. This fragment was used to isolate a full-length cDNA from a rat striatal library. A 2.2-kilobase clone was obtained that encodes a protein of 326 amino acids with seven transmembrane domains, as predicted by hydropathy analysis. Stably transfected mouse A9-L cells and Chinese hamster ovary cells that expressed mRNA for this clone were screened with putative receptor ligands. Saturable and specific binding sites for the A1 adenosine antagonist [3H]-1,3-dipropyl-8-cyclopentylxanthine were identified on membranes from transfected cells. The rank order of potency and affinities of various adenosine agonist and antagonist ligands confirmed the identity of this cDNA clone as an A1 adenosine receptor. The high affinity binding of A1 adenosine agonists was shown to be sensitive to the nonhydrolyzable GTP analog guanylyl-5'-imidodiphosphate. In adenylyl cyclase assays, adenosine agonists inhibited forskolin-stimulated cAMP production by greater than 50%, in a pharmacologically specific fashion. Northern blot and in situ hybridization analyses of receptor mRNA in brain tissues revealed two transcripts of 5.6 and 3.1 kilobases, both of which were abundant in cortex, cerebellum, hippocampus, and thalamus, with lower levels in olfactory bulb, striatum, mesencephalon, and retina. These regional distribution data are in good agreement with previous receptor autoradiographic studies involving the A1 adenosine receptor. We conclude that we have cloned a cDNA encoding an A1 adenosine receptor linked to the inhibition of adenylyl cyclase activity.
Mol Pharmacol 1991 Jul
PMID:Cloning and expression of an A1 adenosine receptor from rat brain. 185 34

The DNA fragment encoding the third intracellular loop of rat m2 muscarinic receptor was fused to the gene for staphylococcal Protein A. The resultant fusion protein, expressed in bacteria, was purified via IgG affinity chromatography and used as an antigen to raise a polyclonal antiserum. Chinese hamster ovary cells transfected with cDNA coding for a single muscarinic receptor subtype were used as tissue sources to screen antisera. The antiserum was shown to immunoprecipitate quantitatively (greater than 90%) m2 receptors but not to precipitate m1, m3, m4, or m5 receptors. Additionally, immunoprecipitation by m2 antiserum could be inhibited by protein containing the third intracellular loop of the m2 receptor. This selective m2 antiserum was then used to study the distribution and density of m2 receptors in rat brain and heart. In agreement with previous studies, m2 receptors were found to be abundant in heart and comprise at least 92% of the total muscarinic receptor density. Hindbrain, brain stem, and midbrain regions such as cerebellum (75%), pons/medulla (70%), and thalamus/hypothalamus (43%) are also enriched in m2 receptors. In contrast, forebrain regions contain markedly lower percentages of m2 receptors, with cortex expressing 20%, hippocampus 19%, striatum 12%, and olfactory tubercle 20% of the total receptor density. Although the density of m2 receptors expressed as a percentage of total varied considerably from brain region to brain region, the absolute density of these receptors appeared relatively uniform throughout the brain. This study demonstrates that a gene fusion system can be used for efficient antibody production. The use of similar fusion protein antisera directed against other subtypes of muscarinic receptors should prove useful in future studies on regulation, function, and structure of muscarinic receptors.
Mol Pharmacol 1991 Jul
PMID:Distribution of m2 muscarinic receptors in rat brain using antisera selective for m2 receptors. 185 38

Nerve cells that express luteinizing hormone-releasing hormone (LHRH), essential for reproductive functions, originate in the epithelium of the medial olfactory placode. While the peripheral origin of this physiologically important brain peptide is surprising, associations between olfactory and reproductive systems are well documented in behavioral studies of pheromones and in clinical studies of disorders including hypogonadotropic hypogonadism with anosmia or olfactory-genital dysplasia. Mechanisms underlying this migration include a close association with neural cell adhesion molecules (NCAM), but are likely also to involve other physical and chemical factors.
J Steroid Biochem Mol Biol 1991 Oct
PMID:Migration of LHRH-immunoreactive neurons from the olfactory placode rationalizes olfacto-hormonal relationships. 189 87

The summated receptor potential was recorded from the vomeronasal organ (VNO) and olfactory epithelium (OE) of 49 human subjects of both sexes (18 to 55 years old) using surface non-polarizable silver-silver chloride electrodes. 15-25 pg of human putative pheromones, clove oil and a diluent were administered to the VNO or the OE in 0.3-1 s pulses from a 0.05 mm dia cannula connected to a multichannel delivery system. Local stimulation of the VNO produces negative potentials of 1.8-11.6 mV showing adaptation. Responses are not obtained when the recording electrode is placed in the nasal respiratory mucosa. Pheromone ER-830 significantly stimulates the male VNO (P less than 0.01; n = 20), while ER-670 produces a significant effect on female subjects (P less than 0.001; n = 20). The other pheromones tested do not show significantly different effects in both male and female (P greater than 0.1). Similar quantities of odorant or diluent produce an insignificant effect on the VNO. Stimulation of the OE with clove oil produces depolarization of 12.3 +/- 3.9 mV, while pheromones do not show a significant effect. Our results show that the VNO is a functional organ in adult humans having receptor sites for human putative pheromones.
J Steroid Biochem Mol Biol 1991 Oct
PMID:Effect of putative pheromones on the electrical activity of the human vomeronasal organ and olfactory epithelium. 189 88

Odor molecules may be considered as molecular ligands which bind to receptors in the olfactory sensory neurons to give rise to the sensory response. Binding studies in whole sensory epithelia suggest that the receptors also bind muscarinic cholinergic antagonists. Preliminary electrophysiological evidence indicates that muscarinic and beta adrenergic antagonists block odor-elicited membrane currents in single isolated salamander sensory neurons. These results support the idea that models developed for analyzing ligand binding by members of the 7 transmembrane domain family of membrane receptors may apply rather closely to olfactory transduction. We suggest that sensory neurons express single receptor types with differing degrees of affinity for different ligands. We further suggest that glomeruli in the olfactory bulb function as labeled lines for particular sets of odor ligand determinants, and that interglomerular circuits bind together similar glomeruli and enhance contrast between dissimilar glomeruli. The odor image laid down in the sensory neuron population is thus subjected to abstracting and enhancement at the glomerular stage, prior to being transmitted for further processing in the deeper layers of the olfactory bulb and in the olfactory cortex.
J Steroid Biochem Mol Biol 1991 Oct
PMID:Toward a pharmacology of odor receptors and the processing of odor images. 189 89

Studies on the properties of olfactory receptors and of the olfactory glomeruli indicate that there is spatial segregation of response to particular characteristics of odorant molecules at the input level of the olfactory bulb. Existing anatomical information and studies of synaptic mechanisms in the olfactory bulb suggest that the bulb circuitry might act as a contrast detection mechanism analyzing a spatially organized input. Recent electrophysiological studies have supported this idea. Extracellular recordings have shown that the similarity between responses of cell pairs to the same stimulus odor depend upon the distance between those cells. Intracellular recordings from mitral and tufted cells have shown spatially organized excitatory and inhibitory responses to localized electrical stimulation of the input layer of the bulb. Some of the major interneurons of the olfactory bulb have also been identified during odor and localized electrical stimulation. These recordings are also consistent with a spatially based organization.
J Steroid Biochem Mol Biol 1991 Oct
PMID:Central processing of olfaction. 189 90

The vomeronasal organ (VNO) and accessory olfactory system (AOS) are present in most terrestrial vertebrates except birds and higher primates. The receptor neurons of the AOS are sequestered inside the VNO, away from the main airflow to the main olfactory receptor neurons. Mechanisms of stimulus access to the sensory neurons vary across species but in most cases there is a system for delivering stimuli faster than would be possible by diffusion. Vomeronasal (VN) receptor neurons typically lack cilia, the site of most of the transduction apparatus in the main olfactory receptors. The VN receptor neurons have a restricted but privileged pathway to the areas of the brain concerned with reproduction and social behavior. In contrast, the main olfactory neurons have a broad pathway to wide areas of the brain, including the neocortex. Experiments where the VNOs or other parts of the accessory olfactory pathway were ablated indicate that the system is important in many behavioral and physiological responses to pheromones (chemical signals carrying information about gender or reproductive or dominance status), some of which may be proteins. VN sensory neurons respond to both volatile and non-volatile stimuli. There is no evidence in the vertebrate AOS for the extreme sensitivity or selectivity characteristic of insect pheromone detectors, but this has not been adequately tested. There is some evidence for learning, possibly by synaptic modification at the second-order neuron level. Social and reproductive cues stimulating the AOS often elicit an intracerebral release of LHRH--which may act at receptors different from those of the pituitary to facilitate behavior. Whether the LHRH release is necessary for AOS-mediated behavioral response is not yet clear.
J Steroid Biochem Mol Biol 1991 Oct
PMID:Sensory processing in the main and accessory olfactory systems: comparisons and contrasts. 189 91

A synthetic oligopeptide (QCDKRKRRKQQYQQRQSV) corresponding to a carboxyl-terminal sequence of the rat m3 receptor (amino acids 561-578) was coupled to carrier proteins and used to generate a polyclonal antiserum. This serum selectively immunoprecipitates at least 90% of the m3 receptors expressed by A9 cells transfected with the cDNA encoding the m3 muscarinic receptor but does not precipitate receptors from cells transfected with cDNA encoding m1, m2, m4, or m5 receptors. Using this m3 antiserum, the density of m3 receptors in various regions of rat brain was quantified. Areas expressing the highest density of m3 receptors are the cortex, hippocampus, striatum, and olfactory tubercle, with 232 fmol/mg, 197 fmol/mg, 140 fmol/mg, and 130 fmol/mg, respectively. Hindbrain regions (i.e., cerebellum, thalamus/hypothalamus, and pons/medulla) expressed fewer m3 receptors, both as a percentage of total muscarinic receptors (5-6%) and in terms of absolute receptor density (12-70 fmol/mg). A panel of subtype-selective antisera (m1, m2, and m3) was used to determine receptor distribution in several peripheral tissues of the rat (lung, ileum, and bladder). The m2 receptor subtype constitutes the majority of total receptors in the bladder (86%), lung (91%), and ileum (69%). The m3 receptor was found at lower densities in these tissues (5-11%), whereas the m1 receptor is present in highest amounts in the ileum (17%). Human clonal cell lines, in which regulation of muscarinic receptors has been commonly studied, were also examined. The SK-N-SH neuroblastoma line, which has been reported to express M3 receptors, on the basis of pharmacology and molecular size, was found to express a mixture of subtypes (m1 = 31%, m2 = 21%, m3 = 43%). Interestingly, SH-SY-5Y and SH-IN cells, both derived from SK-N-SH cells, exhibit predominantly m3 receptors (74% for SH-SY-5Y; 58% for SH-IN), with lower levels of m1 and m2 receptors (5% and 8% for SH-SY-5Y; 4% and 23% for SH-IN, respectively.) Another commonly studied cell line, 132-1-N1 astrocytoma cells, reportedly expressing M3 receptors, based upon mRNA measurements and second messenger linkage, also expresses a predominance of m3 receptors (91% of total). This m3-selective antiserum should prove useful not only for localizing and quantifying m3 muscarinic receptors but also for examining mechanisms involved in the regulation of receptor expression in human tissues or animal models of disease, as well as in cell culture.
Mol Pharmacol 1991 Nov
PMID:Development of an antiserum against m3 muscarinic receptors: distribution of m3 receptors in rat tissues and clonal cell lines. 194 43

Thyroid hormone receptors (TRs) are nuclear proteins that regulate gene expression through interactions with specific DNA sequences. It is well known that thyroid hormones have critical functions in the control of normal brain development. In the rat brain, at least three mRNA species are generated by differential processing of the TR alpha transcript. Only one of the isoforms, TR alpha-1, is a transcriptional activator, while the regulatory roles of the carboxy-terminal variants TR alpha-2 and TR alpha-2v remain unclear. In this study we have used polymerase chain reaction amplification of total RNA to compare TR alpha-1, TR alpha-2, and TR alpha-2v mRNA levels in the brainstem, cerebellum, cerebrum, midbrain, and olfactory bulbs of developing neonatal brains in rats. RNA was collected 5, 10, 15, 20, and 25 days after birth from both normal and hypothyroid animals. Coordinate expression of all three isoforms was observed in most tissues during development, with TR alpha-2 generally maintaining the highest level of expression, and TR alpha-1 the lowest. In hypothyroid tissues, TR alpha-1 message was generally increased, while TR alpha-2 was not. To explore the possible roles of the TR alpha isoforms, we have compared their DNA-binding activities. We report that compared to TR alpha-1, the carboxy-terminal variants TR alpha-2 and TR alpha-2v show different binding patterns with a thyroid hormone response element, suggesting that they bind only poorly as monomers. The varying ratios of the TR alpha isoform expression together with their distinct binding patterns and reported repressor functions suggest that TR alpha isoforms have important roles during brain development and function, and may serve to fine-tune the biological responses to thyroid hormone.
Mol Endocrinol 1991 Aug
PMID:Coordinate expression of functionally distinct thyroid hormone receptor alpha isoforms during neonatal brain development. 194 7

After ovulation, female African catfish are strongly attracted by the odor of male conspecifics. This attraction depends on the presence of the seminal vesicle, a part of the male reproductive organs. Removal of the seminal vesicle illustrates this fact. A low dose of seminal vesicle fluid, added to the water, appears to be highly attractive for catfish which have ovulated. Fractionation of the fluid and testing of the different fractions shows that steroid glucuronides could be responsible for the attraction. These steroid glucuronides can be identified with gas chromatographic-mass spectrometric analysis. A mixture of glucuronides, prepared to resemble the composition of the seminal vesicle fluid, evokes a dose-dependent attraction. The most potent odorant, observed by measuring electrical responses from the olfactory epithelium and from the olfactory tract appears to be 3 alpha,17 alpha-dihydroxy-5 beta-pregnan-20-one-3 alpha-glucuronide.
J Steroid Biochem Mol Biol 1991
PMID:Steroid glucuronides as male pheromones in the reproduction of the African catfish Clarias gariepinus--a brief review. 195 57


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