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Notes: Abstract: We report the isolation and characterization of a rat cDNA clone encoding a μ-opioid receptor. This receptor, a 398 amino acid protein, shares 59% overall identity with the mouse Δ-and K-opioid receptors. Transient expression of the receptor in COS cells revealed high-affinity binding of μ-selective opioid antagonists and agonists, with a KD for naloxone ∼1.5 nM, and for [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO) and morphine at the high-affinity site of 2–4 nM, confirming a μ-opioid pharmacological profile. Northern blotting and in situ hybridization histoohemistry revealed that the μ-opioid receptor mRNA was expressed in many brain regions, including cerebral cortex, caudate putamen, nucleus accumbens, olfactory tubercle, septal nuclei, thalamus, hippocampus, and medial habenular nucleus, in keeping with the known distribution of the μ-opioid receptor.

Notes: Abstract: At D2 3,4-dihydroxyphenylethylamine (dopamine) receptors in anterior pituitary tissue, magnesium ions shifted receptors to agonist high-affinity states, but decreased the affinity of the antagonist [3H]spiperone. Conversely, sodium ions shifted the receptors to agonist low-affinity states, but increased the affinity of [3H]spiperone. Magnesium is proposed to stabilize the hormone–receptor–guanine nucleotide regulatory protein complex, whereas sodium appears to destabilize this ternary complex. Thus, magnesium and sodium appear to mediate their regulatory effects via a common component at the D2 dopamine-receptor ternary complex.

Notes: 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.

Notes: Abstract: The apelin peptide was recently discovered and demonstrated to be the endogenous ligand for the G protein-coupled receptor, APJ. A search of the GenBank databases retrieved a rat expressed sequence tag partially encoding the preproapelin sequence. The GenBank search also revealed a human sequence on chromosome Xq25-26.1, containing the gene encoding preproapelin. We have used the rat sequence to screen a rat brain cDNA library to obtain a cDNA encoding the full-length open reading frame of rat preproapelin. This cDNA encoded a protein of 77 amino acids, sharing an identity of 82% with human preproapelin. Northern and in situ hybridization analyses revealed both human and rat apelin and APJ to be expressed in the brain and periphery. Both sequence and mRNA expression distribution analyses revealed similarities between apelin and angiotensin II, suggesting they that share related physiological roles. A synthetic apelin peptide was injected intravenously into male Wistar rats, resulting in immediate lowering of both systolic and diastolic blood pressure, which persisted for several minutes. Intraperitoneal apelin injections induced an increase in drinking behavior within the first 30 min after injection, with a return to baseline within 1 h.

Notes: 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 Ammon's 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.

Notes: Co-localization of dopamine D1 and D3 receptors in striatal neurons suggests that these two receptors interact at a cellular level in mediating dopaminergic function including psychostimulant-induced behaviour. To study D1 and D3 receptor interactions in cocaine-mediated effects, cocaine-induced locomotion and reward in mice lacking either D1, D3 or both receptors were analysed. Spontaneous locomotor activity was increased in D1–/– and D1–/–D3–/– mice and D1–/–D3–/– mice did not exhibit habituation of spontaneous rearing activity. Cocaine (20 mg/kg) increased locomotor activity in wild-type and D3–/– mice, failed to stimulate activity in D1–/– mice and reduced activity in D1–/–D3–/– mice. In the conditioned place preference, all groups exhibited reward at 5, 10 and 20 mg/kg of cocaine. D1–/–D3–/– mice did not demonstrate preference at 2.5 mg/kg of cocaine although preference was observed in wild-type, D1–/– and D3–/– mice. The transcription factor cAMP-responsive element binding protein (CREB) is activated by phosphorylation in striatal regions following dopamine receptor activation. Striatal pCREB levels following acute cocaine were increased in wild-type and D3–/– mice and decreased in D1–/– and D1–/–D3–/– mice. After repeated administration of 2.5 mg/kg of cocaine, D1–/– mice had lower pCREB levels in caudate–putamen and nucleus accumbens. Our findings suggest that, although spontaneous and cocaine-induced horizontal activity depended mainly on the presence of the D1 receptor, there may be crosstalk between D1 and D3 receptors in rearing habituation and the perception of cocaine reward at low doses of the drug. Furthermore, alterations in pCREB levels were associated with changes in cocaine-induced locomotor activity but not reward.

Notes: Dopaminergic systems are thought to mediate the rewarding and reinforcing effects of palatable food. However, the relative contribution of different dopamine receptor subtypes is not clear. We used dopamine D1 receptor deficient mice (D^{\,-/-}_{1}) and their wild-type and heterozygous littermates to study the role of the D1 receptor in palatable food reinforced behaviour using operant responding and free access paradigms. Non-deprived mice were trained to press a lever for sucrose pellets under three schedules of reinforcement including fixed ratios (FR-1 and FR-4) and a progressive ratio (PR). Responding on one lever was reinforced by the delivery of a sucrose pellet or solution while responding on a second lever had no programmed consequences. Initially, D1 mutant mice took longer to learn to discriminate between the two levers and had significantly lower operant responding for sucrose pellets and solution than wild-type and heterozygous mice under all schedules of reinforcement. Food deprivation enhanced responding on the active lever in all mice although it remained significantly lower in D^{\,-/-}_{1} mice than in control mice. Following extinction of sucrose reinforcement and reversal of the levers, D^{\,-/-}_{1} mice showed deficits in extinguishing and reversing previously learned responses. Home cage intake and preference of sucrose pellets and solutions when given under free-choice access paradigms were similar among the groups.These results suggest that the dopamine D1 receptor plays a role in the motivation to work for reward (palatable food) but not in reward perception and is critical in learning new but relevant information and discontinuing previously learned responses.

Notes: Abstract: Although dopamine agonists can recognize two states of the D2 dopamine receptor in the anterior pituitary (Dhigh2 and Dlow2), we examined whether the dopamine antagonists such as [3H]spiperone could recognize these two sites with different affinities. Using up to 30 concentrations of [3H]spiperone, however, we could only detect a single population of binding sites (porcine anterior pituitary homogenates) with a dissociation constant (KD) of 130 pM. When specific [3H]spiperone binding was defined by a low concentration of (+)-butaclamol (100 nM), the apparent density was low. When defined by a high concentration of (+)-butaclamol (10 μM), nonspecific sites became detectable, thus revealing two apparent populations of sites for [3H]spiperone, only one of which was specific for dopamine. Sodium chloride reduced the KD of the single population of specific D2 sites to 64 pM. Guanine nucleotide by itself had no effect on the KD, but enhanced the density by 25%. Since the density-enhancement could be eliminated by extensive washing of membranes, and could be restored by preincubation with dopamine, the nucleotide-induced elevation of D2 density appeared to be a result of the release of tightly bound endogenous dopamine. Thus, monovalent cations and guanine nucleotides appear to have separate regulatory effects on the anterior pituitary D2 receptor that modulate antagonist-receptor interactions. Several maneuvers were used to test whether [3H]spiperone could differentiate between the two agonist-detected subpopulations of sites. Twentyfold different concentrations of [3H]spiperone (47 pM and 1000 pM) were found to label identical proportions of receptors in the Dhigh2 and Dlow2 states as detected by the agonist 6,7-dihydroxyaminotetralin (ADTN), suggesting that spiperone labelled equal proportions of Dhigh2 and Dlow2 sites without differential affinity for them. In addition, competition of spiperone for Dhigh2 sites selectively labelled by the agonist [3H]n-propylnora pomorphine (NPA) had a virtually identical KD for spiperone as did the total D2 receptor population as determined by direct binding studies (75 pM versus 64 pM). [3H]Spiperone also bound to a uniform population of Dlow2 sites induced by preincubation with guanine nucleotide with identical affinity as to the total D2 population. Thus, these data do not support a “reciprocal model’ for the D2 receptor (i.e., antagonist having low affinity for Dhigh2 and high affinity for Dlow2 in a manner reciprocal to agonists). It is therefore concluded from these studies that spiperone recognizes the Dhigh2 and Dlow2 states of the receptor with equal affinity and no evidence is provided in support for reciprocal modulation of antagonist/agonist affinities.

Notes: Summary (−)-3-PPP, is a unique dopamine analogue, reported to have selective agonist actions at dopamine autoreceptors and antagonist actions at postsynaptic receptors. The interactions of D2 dopamine receptors with (−)-3-PPPin vitro were examined, using [3H]spiperone to label D2 receptors in brain regions containing both pre- and postsynaptic D2 receptors (caudate nucleus, corpus striatum) and a region containing nonsynaptic D2 receptors (anterior pituitary). In the absence of sodium ions, (−)-3-PPP detected D2 receptors in high- and low-affinity states in all regions examined, as is typical of dopamine agonists. That these two subpopulations of (−)-3-PPP-detected sites were dopaminergic in nature was assured by precluding [3H]spiperone binding to serotonergic receptors. In the presence of sodium ions, there was a significant increase in the affinity of some D2 receptors detected by (−)-3-PPP, and (−)-3-PPP in the presence of sodium was unable to discriminate between the two D2 affinity states in pituitary and striatum. The addition of guanine nucleotide led to (−)-3-PPP recognition of a single D2 binding site; the enhanced affinity of D2 receptors for (−)-3-PPP in the presence of sodium was retained in the presence of guanine nucleotide. Thesein vitro characteristics of (−)-3-PPP recognition of dopamine D2 receptor binding sites, when compared with dopamine and spiperone are seen to have clear features of both typical agonist and antagonist interactions with D2 receptors in both brain and pituitary.