Irreversible labelling of delta-opioid receptors in rat brain and neuroblastoma cells by [3H]azido-DTLET: characterization of subunits and autoradiographic visualization of the covalent binding

[3H]Az-DTLET (Tyr-D-Thr-Gly-Phe(pN3)-Leu-Thr), a photoaffinity probe for delta opioid receptors binds to a single class of sites in rat brain membranes with a high affinity (KD = 1.66 nM). The selectivity index of Az-DTLET (KI delta/KI mu = 0.036) is better than that of its precursor DTLET (0.053). Rat brain or neuroblastoma glioma cells membranes were incubated with 10 nM [3H]Az-DTLET, washed and irradiated with U.V. After irradiation a fraction (20-30%) of specific binding was found to remain indissociable after 10 min at 60 degrees C and was considered as irreversible. This fraction increased as a function of the irradiation time. The radioactivity irreversibly bound to rat brain membranes, solubilized by sodium cholate, was associated with high molecular weight species (200,000 daltons). In denaturing conditions (SDS 2%), the [3H]Az-DTLET specific binding was associated with molecular components of 45-50 K and 90-100 K daltons. In contrast, when opioid receptors were prelabelled by [3H]Az-DTLET, solubilized by Na-cholate and irradiated, the radioactivity was only recovered with subunits of 45-50 K daltons. The autoradiographic localization of the irreversibly bound [3H]Az-DTLET in rat brain was identical to that of reversibly bound [3H]DTLET or [3H]Az-DTLET. These results suggest that [3H]Az-DTLET represents an adequate specific probe for studies on the structure, function and anatomical distribution at light and even electron microscopic level of delta-opioid receptors.     

Binding characteristics of [H]opioid ligands to active opioid binding sites solubilized from rat brain membranes by glycodeoxycholate and NaCl: the recovery of binding activity by dilution

This paper describes the binding properties of [³H]peptidergic opioi ligands to binding sites solubilized from rat brain membranes by the treatment with 0.125% sodium glycodeoxycholate and 1 M NaCl. The highest amount of the specific binding of [³H]-[d-Ala²-, Met⁵]enkephalinamide was obtainable when 10-fold diluted solubilized preparations were incubated in the presence of 0.1 mM MnCl₂ and 100 mM NaCl at 0 °C (on ice) for 3 h. With this assay condition, the significant binding of followint [³H]opioid ligands, which have been thought to be selective for receptor types, was also observed: [³H]-[d-A;a², MePhe⁴, Gly-ol⁵]enkephalin (μ-type), [³H]-[d-Ala²,d-Leu⁵]enkephalin (δ-type) and [³H]dynorphin_1–9 (K-type). The number of binding sites in solubilized preparations for each [³H]ligand corresponded to 40–50% recovery of original membrane-bound binding sites. The Scatchard plot of the concentration-saturation binding curve showed only one class of binding sites, with a high affinity for each [³H]ligand. Apparent dissociation constants between solubilized receptors and [³H]ligands were the same as membrane-bound ones, but the ligand specificity for each receptor-type, which was examined by binding inhibition tests with unlabeled ligands, decreased. Present results indicate that heterogeneous opioid receptors in rat brain membranes seem to be transformed into less heterogeneous forms through the treatment with glycogdeoxycholate and NaCl and the dilution process.     

Characterization of a polyclonal antibody to the μ opioid receptor

Active opioid receptors have been solubilized from bovine striatal synaptosomal membranes and purified approximately 4000-fold using a combination of affinity and hydroxyapatite chromatography. The affinity column was constructed by attaching hybromet, a newly synthesized opioid ligand with high affinity for the μ receptor, to a solid support matrix. A polyclonal antibody was generated to opioid receptors by injection of the purified receptor preparation into female New Zealand rabbits. The specificity of the antiserum was demonstrated by receptor competition and immunoprecipitation studies. Immunological titration of opioid binding activity from rat brain showed that the antibody was able to displace specific binding of [³H]etorphine (universal opioid) and [³H]dihydromorphine (μ opioid) from rat membranes, but was ineffective against the binding of [³H]ethylketocyclazocine (κ opioid), [³H]d-Ala²,d-Leu⁵-enkephalin (δ opioid) or [³H]phencyclidine (phencyclidine/σ receptor ligand). The antibody was able to precipitate the M_r 94 000 component of the ¹²⁵I-labeled affinity-purified receptor, a finding which suggests that this subunit may be an opioid recognition component. By indirect immunofluorescence, the antibody was shown to bind specifically to the plasma membranes of the neurotumor cell line NCB-20 (neuroblastoma × Chinese hamster brain hybrid cells), which has high affinity opioid receptors. The observed fluorescence in the neuroblastoma cells was prevented by pre-adsorption of the antibody with purified receptor from rat brain. These results indicate that the antibody is specific for opioid receptors and may prove useful in the precise localization of opioid receptors in the central and peripheral nervous systems by immunohistochemical procedures.     

Differential effects of the adenosine A1 receptor allosteric enhancer PD 81,723 on agonist binding to brain and adipocyte membranes

The benzoylthiophene analog, PD 81,723, has been shown to allosterically enhance agonist binding and functional activation of the mammalian adenosine (ADO) A₁ receptor subtype by putatively maintaining the receptor in a high affinity state. The present studies were conducted to evaluate the ability of PD 81,723 to enhance the binding of [³H]cyclohexyladenosine ([³H]CHA) to A₁ receptors of neural (cerebral cortex) and non-neural (adipocyte) origin in three different species; rat, guinea pig and dog. PD 81,723 (0.3–100 μM) produced a concentration-dependent enhancement of [³H]CHA binding to rat brain A₁ receptors. These effects were also species-dependent with larger enhancements (150–200% of control) observed in guinea pig and dog brain membranes as compared to the rat (120% of control). In contrast, PD 81,723 did not produce any enhancement of [³H]CHA binding to A₁ receptors in adipocyte membranes from any of the species examined. Additional binding studies were conducted using pharmacological manipulations that have previously been shown to enhance the allosteric effects of PD 81,723. In the presence of 1 mM GTP, the allosteric effects of PD 81,723 (15 μM) were increased in rat, guinea pig and dog brain membranes, however, in adipocyte membranes from each species, no significant alteration in agonist binding was observed. Similarly, the A₁ receptor selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (added to effectively reduce the intrinsic antagonist properties of PD 81,723) was found to enhance the allosteric effects of PD 81,723 (15 μM) in brain, but produce no alteration of agonist binding in adipocyte membranes from each species. Examination of the dissociation kinetics of [³H]CHA binding from rat brain and adipocyte membranes revealed that PD 81,723 (15 μM) differentially slowed agonist dissociation from brain, but not adipocyte, membranes. Taken together, the present data support the hypothesis that in tissues that are sensitive to PD 81,723, this benzyolthiophene functions to maintain the A₁ receptor in a high-affinity state and that the relative proportions of high-affinity A₁ receptors present in specific tissues may contribute, at least in part, to the apparent differential effects of PD 81,723 on agonist binding. The tissue specific modulation of A₁ receptor function by PD 81,723 also illustrates the possibility that the locus of allosteric modulation by PD 81,723 may be manifest via a specific, but indirect and tissue-dependent, interaction with the A₁ receptor.     

The presence of delta and mu-, but not kappa or ORL1 receptors in bovine pinealocytes

Physicians have noted since antiquity that their patients complained of less pain and required fewer analgesics at night-time. In humans, the circulating levels of melatonin, a pineal substance with analgesic and hypnotic properties, exhibit a pronounced circadian rhythm with serum levels being high at night and low during day-time. Moreover, pinealectomy abolishes the analgesic effects of melatonin, and naloxone disrupts the day–night rhythm of nociception. In this study, we have attempted to identify and characterize the nature and types of opioid receptor in bovine pinealocyte membranes, using a radioligand binding technique with the selective radioligands [³H]DAMGO, [³H]DPDPE, [³H]U69593 and [³H]orphanin-FQ (OFQ) for identifying mu (μ)-, delta (δ)-, kappa (κ)- and opioid receptor-like (ORL₁) receptors, respectively. The saturation experiments on bovine pinealocyte membranes for [³H]DPDPE binding provided B_max and K_d values of 553±24 fmol/mg protein and 1.3±0.6 nM; and for [³H]DAMGO binding provided B_max and K_d values of 6.3±1.3 fmol/mg protein and 1.2±0.4 nM, respectively. On the other hand, the specific radioligands ([³H]U69593 and [³H]OFQ) binding of κ and ORL₁ receptors were undetectable in bovine pinealocyte membranes. Furthermore, competitive experiments with opioid agonist and antagonist and related compounds confirmed the presence of μ- and δ-opioid binding sites in bovine pinealocyte membranes. These results indicate that neither κ nor ORL₁ receptors are present on the pinealocytes, and the majority of opioid receptors found in the bovine pineal gland are δ (possibly, both δ₁ and δ₂) types, with a minority being μ type, and that both are primarily located on the bovine pinealocyte membranes. These opioid receptors, by stimulating the activity of N-acetyltransferase, enhance the synthesis of melatonin.     

Modification of opioid agonist binding by pertussis toxin

Membrane fractions prepared from rat striate, cortex and midbrain were treated with pertussis toxin, which has been shown to adenosine diphosphate (ADP)-ribosylate the GTP-binding protein G_i, reducing its coupling with receptors. In striatal membranes, treatment with 40 μg toxin per mg membrane protein labeled 60% of the G_i present and 70% of another G protein, G_o; this treatment reduced binding of the opioid agonist [³H]d-Ala²-d-Leu⁵-enkephalin ([³H]DADLE) 20–50%, with the decrease largerly reflecting a decreased affinity. In cortex, toxin treatment reduced [³H]DADLE binding by 35–70%, corresponding to ADP-ribosylation of 50% of G_i and 40% of G_o. In midbrain, [³H]DADLE binding was unaffected by toxin treatment that ADP-ribosylated 86% of the G_i and 72% of the G_o. These results provide further evidence that opioid receptors are associated with GTP-binding proteins in striatum and cortex, where they have also been shown to inhibit adenylate cyclase. Despite the presence of G_i and G_o in midbrain, however, there appears to be no coupling between them and opioid receptors.     

Pharmacological and anatomical evidence of selective μ, δ, and χ opioid receptor binding in rat brain

While the distribution of opioid receptors can be differentiated in the rat central nervous system, their precise localization has remained controversial, due, in part, to the previous lack of selective ligands and insensitive assaying conditions. The present study analyzed this issue further by examining the receptor selectivity of [³H]DAGO (Tyr-d-Ala-Gly-MePhe-Gly-ol), [³H]DPDPE (2-d-penicillamine-5-d-penicillamine-enkephalin), [³H]DSLET (Tyr-d-Ser-Gly-Phe-Leu-Thr) and [³H](−)bremazocine, and their suitability in autoradiographically labelling selective subpopulations of opoiod receprtors in rat brain. The results from saturation, competitions, and autoradiographic experiments indicated that the three opioid receptor subtypes can be differentiated in the rat brain and that [³H]-DAGO and [³H]DPDPE selectively labelled μ and δ binding sites, respectively. In contrast, [³H]DSLET was found to be relatively non-selective, and labelled both μ and δ sites. [³H]Bremazocine was similarly non-selective in the absence of μ and δ ligands and labelled all three opioid receptor subtypes. However, in the presence of 100 nM DAGO and DPDPE, concentrations sufficient to saturate the μ and δ sites, [³H]bremazocine did label χ sites selectively. The affinity [³H]bremazocine binding sites showed a unique distribution with relatively dense χ labelling in the hypothalamus and median eminence, areas with extremely low μ and δ binding. These results point to the selectivity, under appropriate conditions, of [³H]DAGO, [³H]DPDPE and [³H]bremazocine and provide evidence for the differential distribution of μ, δ, and χ opioid receptors in rat brain.     

Mu-opioid ([H]DAGO) binding in slices of rat brain: Inhibition by sodium ions, guanyl-5′-yl imidodiphosphate and by the adrenergic neurotoxins DSP4 and xylamine

We have characterized and quantified the specific binding of the μ-agonist [³H] DAGO to 300 μM slices of hypothalamus and cerebral cortex. The receptors have many of the opioid characteristics previously demonstrated in homogenate assays. Binding is reversible, saturable, stereospecific and of high affinity. The δ-opioids DTLET and DSLET are 36- and 30-fold respectively, less effective than DAGO in competing for the binding site. Assays can be routinely performed in the presence of physiological concentrations of sodium, though in TRIS buffer the affinity and the number of receptors is increased. Protection of the ligand against proteolytic degradation is unnecessary. Unexpectedly, we observed that GppNHp inhibits [³H] DAGO binding to brain slices. This suggests an allosteric modification of the μ-receptor in the membranes of intact cells. The μ-receptors are also blocked by the adrenergic neurotoxins DSP4 and xylamine. This re-emphasizes our contention that care should be exercised in the use of these drugs. The technique is simple, rapid and involves minimal disruption of tissue. It should provide new opportunities for the study of cell surface opioid receptor subtypes in intact tissue.     

Initial detection of [H]clonidine binding to solubilized rat brainα2-adrenergic receptors: regulation by guanine nucleotides

α₂-adrenergic receptors labeled by [³H]clonidine (α₂-antagonist) can be solubilized from the rat brain with a zwitterionic detergent, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS). CHAPS-solubilized receptors have the same characteristics of membrane-boundα₂-receptors in the brain, and the regulation of receptor binding by guanine nucleotides is retained in the soluble state.     

Identification and characterization of adrenergic receptors and catecholamine-stimulated adenylate cyclase in hog pial membranes

In order to define the adrenergic receptors in small pial blood vessels, we studied [³H]dihydroalprenolol (DHA) binding, [³H]dihydroergocryptine (DHE) binding, and catecholamine-stimulated adenylate cyclase in membranes prepared from the hog pia. [³H]DHA bound to a saturable number of sites (0.09 pmol/mg protein) with a high affinity (10 nM). The ability of a small series of adrenergic agents to compete for [³H]DHA binding was as expected for binding to β-adrenergic membrane receptors. The receptor binding appeared to be composed largely ofβ₂-adrenergic sites but some sites withβ₁ properties were detected. The β-adrenergic agonist, isoproterenol, stimulated adenylate cyclase activity by 20% in pial membranes in the presence of GTP. The potency of catecholamines in stimulating adenylate cyclase correlated well with their ability to compete for [³H]DHA binding. [³H]DHE also bound to a saturable number of sites (0.39 pmol/mg) on these membranes. α-Adrenergic agents were potent competitors for [³H]DHE binding but dopamine, serotonin and histamine had effects only at high concentrations. These data demonstrate the presence of α- and β-adrenergic membrane receptors and β-adrenergically stimulated adenylate cyclase activity in small pial blood vessels from the hog.     

Autoradiographic localization of α5 subunit-containing GABAA receptors in rat brain

Multiple subtypes of GABA_A receptors are expressed in the rat central nervous system (CNS). To determine the distribution and proportion of α5 subunit containing receptors, quantitative autoradiographic analyses were performed with both [³H]L-655,708 and [³H]Ro15-1788, an α5 selective and a non selective benzodiazepine binding site ligand, respectively. High densities of [³H]L-655,708 binding sites were observed in hippocampus and olfactory bulb, where α5 receptors accounted for 20–35% of total [³H]Ro15-1788 binding sites. Low levels of [³H]L-655,708 sites were associated with the cortex as well as amygdala, thalamic, hypothalamic and midbrain nuclei. These observations indicate that although [³H]L-655,708 binding sites have an overall low expression in rat CNS, they may contribute significantly to GABAergic inhibition in specific brain regions.     

σ Binding parameters in developing rats predict behavioral efficacy of a σ ligand

The relationship between σ binding and the behavioral efficacy of a selective σ ligand was examined in rats of varying ages (30,45,60,75,90, and 150 days old). Scatchard analyses of the binding of the σ radioligand [³H]1,3-di-o-tolyguanidine ([³H]DTG) to brain membranes revealed significant age-related differences in binding to both crude synaptosomal and microsomal fractions. The functional significance of these developmental changes in σ ligand binding was studied by determining the postural effects of rubral microinjections of DTG in age-matched littermates of rats used in the binding studies. The degree of dystonia produced by a single dose of DTG was significantly correlated with the amount of [³H]DTG bound to rat brain synaptosomal membranes at low but not at high concentrations. No significant correlation between binding to the microsomal fraction and drug efficacy was observed. These experimental results were in good agreement with predicted amounts bound as estimated from a Scatchard analysis of the data. The results suggest that σ binding sites found in brain synaptosomal membranes are functional receptors involved in the control of movement and posture.     

Purification of γ-aminobutyric acid (GABA) receptor from rat brain by affinity column chromatography using a new benzodiazepine, 1012-S, as an immobilized ligand

The purification of γ-aminobutyric acid (GABA) and benzodiazepine receptors from the rat brain was employed by the affinity column using a new benzodiazepine, 1012-S, as immobilized ligand. The 1012-S has a aliphatic primary amino group and exhibited an extremely high potency for displacing [³H]flunitrazepam binding to solubilized benzodiazepine receptor preparation (IC₅₀ = 6.0 × 10⁻¹¹M). This benzodiazepine affinity gel retained almost all of the solubilized GABA receptors from synaptic membranes applied to the column, and 25.6% of the receptor was eluted bio-specifically following the application of 1 mM 1012-S. The highest purification fold thus obtained was 4576 (specific activity: 0.99 nmol/mg protein). Furthermore, the successive application of 1–2 M NaSCN also resulted the elution of a highly enriched GABA receptor (specific activity: 0.41 nmol/mg protein; purification fold: 1889). SDS-polyacrylamide gel electrophoretic profiles of the bio-specifically eluted fraction with 1012-S showed the existence of two major bands having the molecular weights of approximately 48,500 and 54,500 in which the former band was selectively photoaffinity-labelled with [³H]flunitrazepam. On the other hand, it was found that the non-specifically eluted fraction with NaSCN contained 4 additional minor bands having molecular weights of 41,000 to 51,000. These results indicate that GABA receptor of the rat brain is coupled, at least in part, with benzodiazepine receptor and is readily purified by the use of highly specific benzodiazepine affinity gel, 1012-S-acetamide adipic hydrazide Sepharose 4B. The present results also suggest that the purified GABA/benzodiazepine receptor complex may contain two different kinds of subunits having the molecular weights of 48,000 and 54,500, in which the former subunit may possess benzodiazepine binding sites.     

Decrease in δ-opioid receptor density in rat brain after chronic [d-Ala,d-Leu]enkephalin treatment

Chronic treatment of Sprague-Dawley rats with [d-Ala²,d-Leu⁵]enkephalin (DADLE) resulted in the development of tolerance to the antinociceptive effect of this opioid peptide. When opioid receptor binding was measured, time-dependent decreases in [³H]diprenorphine binding to the P₂ membranes prepared from the cortex, midbrain and striatum were observed. Scatchard analysis of the saturation binding data revealed a decrease in B_max values and no change in the K_d values of [³H]diprenorphine binding to these brain regions, indicative of down-regulation of the receptor. This reduction in the opioid receptor binding activities could be demonstrated to be due to the DADLE effect on the δ-opioid receptors in these brain regions. When [³H]DADLE binding was carried out in the presence of morphiceptin, a significant reduction in the δ-opioid receptor binding was observed in all brain areas tested. μ-Opioid receptor binding decrease was observed only in the striatum after 5 days of DADLE treatment. Additionally, the onset of δ-opioid receptor decrease in the midbrain area was rapid, within 6 h of the initiation of the chronic DADLE treatment. Thus, analogous to previous observations in which chronic etorphine treatment preferentially reduced μ-opioid receptor binding, chronic DADLE treatment preferentially reduced δ-opioid receptor binding activity.     

Characterization of a polyclonal antibody to the mu opioid receptor

Active opioid receptors have been solubilized from bovine striatal synaptosomal membranes and purified approximately 4000-fold using a combination of affinity and hydroxyapatite chromatography. The affinity column was constructed by attaching hybromet, a newly synthesized opioid ligand with high affinity for the mu receptor, to a solid support matrix. A polyclonal antibody was generated to opioid receptors by injection of the purified receptor preparation into female New Zealand rabbits. The specificity of the antiserum was demonstrated by receptor competition and immunoprecipitation studies. Immunological titration of opioid binding activity from rat brain showed that the antibody was able to displace specific binding of [3H]etorphine (universal opioid) and [3H]dihydromorphine (mu opioid) from rat membranes, but was ineffective against the binding of [3H]ethylketocyclazocine (kappa [3H]D-Ala2,D-Leu5-enkephalin (delta opioid) or [3H]phencyclidine (phencyclidine/sigma receptor ligand). The antibody was able to precipitate the Mr 94,000 component of the 125I-labeled affinity-purified receptor, a finding which suggests that this subunit may be an opioid recognition component. By indirect immunofluorescence, the antibody was shown to bind specifically to the plasma membranes of the neurotumor cell line NCB-20 (neuroblastoma X Chinese hamster brain hybrid cells), which has high affinity opioid receptors. The observed fluorescence in the neuroblastoma cells was prevented by pre-adsorption of the antibody with purified receptor from rat brain. These results indicate that the antibody is specific for opioid receptors and may prove useful in the precise localization of opioid receptors in the central and peripheral nervous systems by immunohistochemical procedures.     

GABA binding and bicuculline in spinal cord and cortical membranes from adult rat and from mouse neurons in cell culture

Sodium-independent [³H]GABA and [³H]muscimol binding was determined in adult rat cerebral cortical and spinal cord membranes and in membranes from fetal mouse cortical and spinal cord neurons in primary dissociated cell culture. In adult rat cerebral cortical membranes, [³H]GABA bound to two sites (K_d=8nM,B_max=0.62pmol/mg protein; K_d=390nM,B_max=3.9pmol/mg protein) whereas the GABA agonist, [³H]muscimol, bound only to a high affinity site (K_d=5.6nM,B_max=1.9pmol/mg protein). In adult rat spinal cord, only a low affinity site was seen with [³H]GABA (K_d=340nM,B_max=9.8pmol/mg protein) and only a high affinity site was seen with [³H]muscimol (K_d=5.6nM,B_max=0.25pmol/mg protein). The inability to measure a high affinity [³H]GABA binding site in spinal cord probably reflects the high ratio of low to high affinity sites in spinal cord (39:1). In membranes from mouse neurons in cel; culture, [³H]GABA bound to two sites on cortical neurons (K_d=9nM,B_max=0.24pmol/mg protein; K_d=510nM,B_max=1.3pmol/mg protein) and spinal cord neurons (K_d=13nM,B_max=0.12pmol/mg protein; K_d=640nM,B_max=3.2pmol/mg protein). Again, the ratio of low to high affinity sites in cultured mouse spinal cord neurons was high (27:1).The effects of the potent GABA antagonist, (+)bicuculline, on both low and high affinity [³H]GABA binding was determined. Bicuculline appeared to inhibit binding to both sites competitively but theK_i for inhibiting the high affinity site was 5 μM and for inhibiting the low affinity site was 115 μM. Bicuculline inhibited [³H]muscimol binding in both brain and spinal cord competitively withK_is of 4μM and 10 μM respectively. Bicuculline inhibition of [³H]muscimol binding in cultured neuronal membranes was similar to that in adult rat membranes.The binding of the potent GABA agonist, muscimol, only to the high affinity site in both adult rat and cultured mouse neuronal membranes suggests that the high affinity site is the physiologically relevant postsynaptic GABA receptor. The fact that bicuculline inhibits the high affinity site (but not the low affinity site) in concentrations similar to those needed to block GABA-responses in physiological experiments²⁸ supports this hypothesis.     

Muscarinic M1 and M3 receptors are present and increase intracellular calcium in adult rat anterior pituitary gland

Physiological and biochemical evidence indicates the existence of functional muscarinic cholinergic receptors in the anterior pituitary. The selectivity of these receptors has been characterised by studying the binding of [³H]quinuclidinyl benzilate ([³H]QNB) and [³H]diphenyl-acetoxy-N-methyl-piperidine ([³H]4-DAMP) in membrane preparation of male rat anterior pituitary at 25°C. Competition experiments with receptor selective muscarinic antagonists were used to characterise specific selective muscarinic receptor binding. Both [³H]QNB and [³H]4-DAMP bound to anterior pituitary membranes at low concentrations, binding was saturable and was potently displaced by 4-DAMP (M₁, M₃ subtypes selective antagonist) > atropine (general) > pirenzepine (M₁). Methoctramine (M₂) didn’t antagonise the [³H]QNB binding efficiently. Acetylcholine and carbachol increased the intracellular Ca²⁺ level in 62% and 65% of cultured rat anterior pituitary cells in a dose-dependent manner, and this effect was prevented by pirenzepine. Based on these results we suggest that both M₁ and M₃ muscarinic receptors are present and active in the majority of cells in the rat anterior pituitary gland, but their physiological role in the adult rat remains to be examined.     

Characterisation of central adenosine A1 receptors and adenosine transporters in mice lacking the adenosine A2a receptor

The present study was designed to assess whether adenosine A_2a receptor knockout mice exhibit altered purine utilisation in brain nuclei. Specifically, the properties of adenosine transporters and adenosine A₁ receptors were characterised in brain membranes and on slide-mounted sections. The B_MAX for [³H]nitrobenzylthioinosine ([³H]NBTI) binding (adenosine transporter density) was significantly reduced in brainstem membranes of homozygotes (560±52 fmol/mg protein, n=5, P<0.05, Kruskal–Wallis ANOVA) compared to wildtype (1239±213 fmol/mg protein) and heterozygous mice (1300±558 fmol/mg protein). Quantitative autoradiography data indicated that [³H]NBTI binding in the medulla oblongata of heterozygous mice was seen to decrease significantly (P<0.05) in the subpostremal nucleus tractus solitarius (NTS), medial NTS, inferior olive and area postrema (AP). On the other hand, in the homozygous mice a decrease was seen in the medial NTS and AP. In the pons, [³H]1,3-dipropyl-8-cyclopentylxanthine ([³H]DPCPX) (adenosine A₁ receptor density) binding increased significantly (P<0.05, Kruskal–Wallis ANOVA) in the lateral parabrachial nucleus, caudal pontine reticular nucleus and locus coeruleus of homozygotes compared to wildtype. In higher brain centres, [³H]NBTI binding was reduced in the paraventricular thalamic nucleus of both heterozygous and homozygous mice, whereas [³H]DPCPX binding was reduced in the hippocampus and lateral hypothalamus of heterozygotes. In homozygotes, [³H]DPCPX binding in the hippocampus increased compared to wildtype mice. The present study indicates that deletion of the A_2a receptor may have contributed to region-specific compensatory changes in purine utilisation in brain nuclei associated with autonomic, neuroendocrine and behavioural regulation.     

Identification, partial characterization, and hypothalamic distribution of κ, μ, and δ opioid receptors in a passerine songbird (Junco hyemalis)

Brain tissues obtained from a passerine songbird (dark-eyed junco, Junco hyemalis) were used to identify and partially characterize central opioid receptors. We found that [³H]EKC (putative κ ligand), [³H]DAMGO (putative μ ligand), and [³H]DPDPE and [³H]pCl-DPDPE (two putative δ ligands) bind to brain tissue preparations specifically, in a time-dependent fashion, and with a high affinity (K_d's 5 nM). Binding sites are present at low concentrations (B_max < 120 fmol/mg protein), and they are pharmacologically selective. In vitro autoradiography studies revealed a high density of δ receptors in hypothalamic regions (ventromedial and lateral hypothalamus) that regulate feeding behavior. Together with previous studies, these observations suggest that the central influence of opioids on avian food consumption depends on mechanisms located in hypothalamic regions.     

Binding of []insulin to specific receptors and stimulation of nucleotide incorporation in cells cultured from rat brain

The occurrence of insuling receptors and biological responses to insulin has been investigated in trypsin-dissociated fetal rat brain cells maintained in culture for 8 days. Binding of [¹²⁵]insulin to brain cells in culture was time- and pH-dependent and 85–90% specific. Porcine insulin competed for [¹²⁵]insulin binding in a dose-dependent manner. Unrelated polypeptides, including angiotensin II, glucagon, bovine growth hormone, and bovine prolactin did not compete for [¹²⁵]insulin binding. The half-life of [¹²⁵]insulin dissociation from receptors at 24°C was 15 min and a plot of ln[B/Bo] vs time suggested two dissociation rate constants of2.7 × 10⁻⁴ sec⁻¹ and5.0 × 10⁻⁵ sec⁻¹. Scatchard analysis of the binding data gave a curvelinear plot which may indicate negative cooperativity or the occurrence of both high affinity(K_a = 2 × 10¹¹M⁻¹) and low affinity(K_a = 4 × 10¹⁰M⁻¹) sites. Of the estimated total of 4.9 × 10⁴ binding sites per cell, 28–30% appear to be high affinity sites.Incubation of cultures with insuling caused a time- and dose-dependent stimulation of [³H]thymidine and [³H]uridine incorporation into TCA-precipitable material. Maximum stimulation of thymidine incorporation (2–5-fold) occured 11 h after incubation with 167 nM insulin. The same concentration of insulin caused a 2.2-fold increase in [³H]uridine incorporation in 2 h. These results indicate that cells cultured from rat brain contain specific insulin receptors capable of mediating effects of insulin on macromolecular synthesis in the central nervous system.