A Globin Fragment, LVV-Hemorphin-7, Induces [3H]Thymidine Incorporation in a Neuronal Cell Line via the AT4 Receptor

The AT4 receptor was characterized initially as a specific binding site for angiotensin IV, a C-terminal fragment of the vasoactive peptide angiotensin II. Recently, we found that LVV-hemorphin-7, a fragment of beta globin, is an abundant peptide in the brain and binds to the AT4 receptor with high affinity and specificity. In the neuroblastoma/glioma hybrid cell line, NG108-15, LVV-hemorphin-7 and angiotensin IV competed for 125I-angiotensin IV binding in a biphasic fashion with IC50 values of 1.2 x 10(-10) and 1.1 x 10(-9) M for the high-affinity site, respectively, and 6.7 x 10(-8) and 1.5 x 10(-8) M for the low-affinity site, respectively. Both peptides were internalized rapidly by the cells. However, LVV-hemorphin-7, but not angiotensin IV, elicited a 1.8-fold increase in DNA synthesis in a dose-dependent manner. Furthermore, co-incubation of the cells with an excess of angiotensin IV (10(-6) M) inhibited LVV-hemorphin-7-stimulated DNA synthesis. Therefore, whereas LVV-hemorphin-7 and angiotensin IV were capable of binding to the AT4 receptor, only LVV-hemorphin-7 elicited [3H]thymidine incorporation in NG108-15 cells. In contrast, angiotensin IV behaved as an antagonist. The current finding suggests that LVV-hemorphin-7 is a functional peptide in the central nervous system and in view of its abundance in neural tissue, compared with angiotensin IV, may be of significant physiological importance.     

Immunolabeling of Central Serotonin 5-HT1Dβ Receptors in the Rat, Mouse, and Guinea Pig with a Specific Anti-Peptide Antiserum

Abstract: A synthetic peptide (25 amino acids) corresponding to a specific portion of the third intracytoplasmic loop of the rat serotonin 5-HT_1B/1Dβ receptor was coupled to keyhole limpet hemocyanin and injected monthly into rabbits. Anti-peptide antibodies were detected by enzyme-linked immunosorbent assay and characterized by immunoprecipitation of the 5-HT_1B/1Dβ receptor in CHAPS-solubilized extracts from rat striatal membranes. Up to 60% of solubilized striatal serotonin- O -carboxymethylglycyl[¹²⁵I]iodotyrosinamide ([¹²⁵I]GTI; a selective 5-HT_1B/1D radioligand) binding sites were immunoprecipitated and subsequently pharmacologically identified as 5-HT_1B receptors. The remaining 40% of [¹²⁵I]GTI binding sites were shown to be 5-HT_1D receptors. In addition, these antibodies were successfully used in immunofluorescence experiments to detect the 5-HT_1B/1Dβ, but not the 5-HT_1D/1Dα, receptor in transiently transfected LLC-PK1 cells. Immunoautoradiographic experiments performed with brain sections from the rat, mouse, and guinea pig showed that the substantia nigra and globus pallidus contained the highest densities of 5-HT_1Dβ receptor-like immunoreactivity. Comparison of the regional distribution of immunolabeling with that of the specific binding of [¹²⁵I]GTI in the brain of these species further confirmed that the anti-peptide antibodies selectively recognized only the 5-HT_1Dβ component of [¹²⁵I]GTI specific receptor binding sites.     

Methyl 3β-(4-[125I]Iodophenyl)Tropane-2β-Carboxylate In Vitro Binding to Dopamine and Serotonin Transporters Under "Physiological" Conditions

Abstract: Methyl 3β-(4-[¹²⁵I]iodophenyl)tropane-2β-carboxylate ([¹²³I]β-CIT) is a single photon emission computed tomographic radiotracer for in vivo labeling of dopamine (DA) and serotonin (5-HT) transporters. Single photon emission computed tomographic experiments in nonhuman primates showed that [¹²³I]β-CIT in vivo binding to DA transporters had a much slower washout than binding to 5-HT transporters. This observation was not predicted from previously published in vitro studies. These studies, performed at 22°C in nonphysiological buffer, reported similar affinity of [¹²⁵I]β-CIT for DA and 5-HT transporters. We now report [¹²⁵I]β-CIT binding parameters to fresh rat membranes at 22°C and 37°C, in a buffer mimicking the composition of cerebrospinal fluid. At both temperatures, binding to DA transporters was best fit by a twosite model, whereas binding to 5-HT transporters was compatible with one population of sites. At 22°C, [¹²⁵I]β-CIT showed similar affinity to high-affinity DA (0.39 n M ) and 5-HT transporter sites (0.47 n M ). Increasing the incubation temperature from 22°C to 37°C reduced binding to DA transporters by 60%, whereas binding to 5-HT transporters was only marginally affected. In vitro kinetic experiments failed to detect significant differences in on or off rates that could explain the observed in vivo kinetics. These experiments thus failed to explain [¹²³ I]β-CIT in vivo uptake kinetics, suggesting the existence of specific factors affecting the in vivo situation.     

Subnuclear Distribution of the S-100 Protein Specific Binding Sites in Rat Brain

Abstract: Fractionation of isolated brain nuclei previously reacted with ¹²⁵I-labelled S-100 showed that most of the specifically bound radioactivity associated with the nuclear membranes and the nucleoli. Labelling of nucleoli, which indicates the entrance of ¹²⁵I-labelled S-100 into the nucleus, was observed at 37°C, but not at 0–4°C. When tested separately for ¹²⁵I-labelled S-100 specific binding, both the nuclear membranes and the nucleoli were found to bind ¹²⁵I-labelled S-100 in a biphasic manner, the binding displaying a high affinity and a low affinity component, as observed with intact nuclei. However, the binding to nuclear membranes was largely irreversible, while that to nucleoli was fully reversible after any association time.     

Characterization of [3H]CP-96,501 as a Selective Radioligand for the Serotonin 5-HT1B Receptor: Binding Studies in Rat Brain Membranes

Abstract: 3-(1,2,5,6-Tetrahydro-4-pyridyl)-5- n -propoxyindole (CP-96,501) was found to be a more selective ligand at the serotonin 5-HT_1B receptor than the commonly used 5-HT_1B agonist, 3-(1,2,5,6-tetrahydro-4-pyridyl)-5-methoxyindole (RU 24969). In rat brain membranes, the tritiated derivative, [³H]CP-96,501, was found to bind with a high affinity ( K _D, 0.21 n M ) to a single binding site ( n _H, 1.0). The receptor density of this site ( B _max, 72 fmol/mg of protein) matched that of the 5-HT_1B receptor determined with [³H]5-HT. Competition curves of 16 serotonergic compounds in [³H]CP-96,501 binding also indicated a single binding site. The rank order of their binding affinities with this new radioligand showed a high degree of correlation with their affinities at the 5-HT_1B receptor determined with [³H]5-HT or [¹²⁵I]iodocyanopindolol. Serotonergic compounds displayed competitive inhibition of [³H]CP-96,501 binding. In the presence of 5'-guanylylimidodiphosphate [Gpp(NH)p], [³H]CP-96,501 binding was reduced, while the potency of CP-96,501 to displace [¹²⁵I]iodocyanopindolol binding was also decreased. These findings are consistent with the agonist nature of CP-96,501. The results of this study suggest that [³H]CP-96,501 is a useful agonist radioligand for the 5-HT_1B receptor.     

Compared Binding Properties of 125I-Labeled Analogues of Neurotensin and Neuromedin N in Rat and Mouse Brain

Abstract: Neurotensin and neuromedin N are two structurally related peptides that are synthesized by a common precursor. The purpose of the present work was to characterize neuromedin N receptors in rat and mouse brain and to compare these receptors with those of neurotensin. A radiolabeled analogue of neuromedin N has been prepared by acylation of the N-terminal amino group of the peptide with the ¹²⁵I-labeled Bolton-Hunter reagent. This ¹²⁵I-labeled derivative of neuromedin N bound to newborn mouse brain homogenate with high affinity (K _d = 0.5 n M ). Cross-competition experiments between radiolabeled and unlabeled neurotensin and neuromedin N indicated that each peptide was able to displace completely and specifically the other peptide from its interaction with its receptor. Independently of the radioligand used, the affinity of neurotensin was always better than that of neuromedin N. Quantitative radioautographic studies demonstrated that the ratio of labeling intensities obtained with ¹²⁵I-labeled analogues of neurotensin and neuromedin N remained constant in all the brain areas. Our results do not support the existence of a specific neuromedin N receptor in rat and mouse brain and can be explained by the presence of a common receptor for both peptides.     

Distribution of Specific High-Affinity Binding Sites for [3H]Imipramine in Human Brain

Abstract: [³H]Imipramine binds with high affinity to membranes from different regions of the human brain. The highest density of binding sites was observed in the hypothalamus and substantia nigra and the lowest density in the white matter and cerebellum. As found in rat brain, tricyclic antidepressant drugs are potent inhibitors of [³H]imipramine binding. Atypical antidepressants are, however, much weaker at inhibiting the specific binding. The [³H]imipramine binding site in human cortex is apparently identical to the site already described in the rat brain and in human platelets.     

Binding Characteristics of a Potent AMPA Receptor Antagonist [3H]Ro 48-8587 in Rat Brain

Abstract: A new AMPA receptor antagonist, Ro 48-8587, was characterized pharmacologically in vitro. It is highly potent and selective for AMPA receptors as shown by its effects on [³H]AMPA, [³H]kainate, and [³H]MK-801 binding to rat brain membranes and on AMPA- or NMDA-induced depolarization in rat cortical wedges. [³H]Ro 48-8587 bound with a high affinity ( K _D = 3 n M ) to a single population of binding sites with a B _max of 1 pmol/mg of protein in rat whole brain membranes. [³H]Ro 48-8587 binding to rat whole brain membranes was inhibited by several compounds with the following rank order of potency: Ro 48-8587 > 6-nitro-7-sulphamoylbenzo[ f ]quinoxaline-2,3-dione (NBQX) > YM 90K > 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) > quisqualate > AMPA > glutamate > kainate > NMDA. The distribution and abundance of specific binding sites (∼95% of total) in sections of rat CNS, revealed by quantitative receptor radioautography and image analysis, indicated a very discrete localization. Highest binding values were observed in cortical layers (binding in layers 1 and 2 > binding in layers 3–6), hippocampal formation, striatum, dorsal septum, reticular thalamic nucleus, cerebellar molecular layer, and spinal cord dorsal horn. At 1 n M , the values for specific binding were highest in the cortical layers 1 and 2 and lowest in the brainstem (∼2.6 and 0.4 pmol/mg of protein, respectively). Ro 48-8587 is a potent and selective AMPA receptor antagonist with improved binding characteristics (higher affinity, selectivity, and specific binding) compared with those previously reported.     

Binding of Cholecystokinin-8 (CCK-8) Peptide Derivatives to CCKA and CCKB Receptors

Abstract: The structural requirements for the selective binding of cholecystokinin-8 (CCK-8)-related peptides to peripheral (CCK_A) receptors are not sufficiently understood. In this study, the interaction of a series of newly shortened analogues of CCK-8 with both receptor subtypes was analyzed by displacement studies using [³H]-CCK-8 and ¹²⁵l-Bolton-Hunter (BH)-CCK-8 as radioligands. The pentapeptide derivative of CCK-8, succinyl-Tyr (SO₃H)-Met-Gly-Trp-Met-phenethylamide, was found to bind selectively with high affinity to the CCK_A receptor. The replacement of Met²⁸ and/or Met³¹ by norleucine and of L-Trp³⁰ by its D-analogue had no significant effect on the binding properties of the peptide. Further C-terminal shortening resulted in a drastic loss of affinity and selectivity of the CCK receptor binding.     

Glial Regulation of α7-Type Nicotinic Acetylcholine Receptor Expression in Cultured Rat Cortical Neurons

Abstract: Primary embryonic cortical cultures were used as an in vitro model to evaluate the influence of glia on developmental expression of α7-type nicotinic acetylcholine receptors in rat brain. In cells cultured in serum-containing medium without mitotic inhibitors, specific ¹²⁵I-α-bungarotoxin binding to α7-type nicotinic receptors was maximal 4–8 days after plating. Treatment with 5'-fluorodeoxyuridine (80 µ M ) from 1 to 3 days in vitro significantly reduced glial proliferation and concomitantly increased ¹²⁵I-α-bungarotoxin binding, whereas plating onto a glial bed layer decreased binding. There was no significant binding to pure glial cultures. Treatment-induced changes in neuronal binding resulted from alterations in receptor density, with no change in affinity. 5'-Fluorodeoxyuridine treatment also increased cellular expression of α7 receptor mRNA but had no effect on N -[³H]methylscopolamine binding to muscarinic receptors. Glial conditioned medium decreased ¹²⁵I-α-bungarotoxin binding in both control and 5'-fluorodeoxyuridine-treated cultures, suggesting the release of a soluble factor that inhibits α7-type nicotinic receptor expression. An additional mechanism of glial regulation may involve removal of glutamate from the surrounding medium, as added glutamate (200 µ M ) increased ¹²⁵I-α-bungarotoxin binding in astrocyte-poor cultures but not in those that were astrocyte enriched. These results suggest that glia may serve a physiological role in regulating α7-type nicotinic receptors in developing brain.     

Evidence for κ- and μ-Opioid Receptor Expression in C6 Glioma Cells

Abstract: The astrocytoma cell line rat C6 glioma has been used as a model system to study the mechanism of various opioid actions. Nevertheless, the type of opioid receptor(s) involved has not been established. Here we demonstrate the presence of high-affinity U69,593, endomorphin-1, morphine, and β-endorphin binding in desipramine (DMI)-treated C6 cell membranes by performing homologous and heterologous binding assays with [³H]U69,593, [³H]morphine, or ¹²⁵I-β-endorphin. Naive C6 cell membranes displayed U69,593 but neither endomorphin-1, morphine, nor β-endorphin binding. Cross-linking of ¹²⁵I-β-endorphin to C6 membranes gave labeled bands characteristic of opioid receptors. Moreover, RT-PCR analysis of opioid receptor expression in control and DMI-treated C6 cells indicate that both κ- and μ-opioid receptors are expressed. There does not appear to be a significant difference in the level of μ nor κ receptor expression in naive versus C6 cells treated with DMI over a 20-h period. Collectively, the data indicate that κ- and μ-opioid receptors are present in C6 glioma cells.     

Calcium Regulation of Agonist Binding to α7-Type Nicotinic Acetylcholine Receptors in Adult and Fetal Rat Hippocampus

Abstract: Quantitative autoradiography was used to compare the binding properties of α7-type nicotinic acetylcholine receptors in fetal and adult rat hippocampus. Whereas there were high levels of ¹²⁵I-α-bungarotoxin (¹²⁵I-α-BTX) binding throughout fetal hippocampal field CA1, there was a significant decrease in binding site density in the adult. The affinity of ¹²⁵I-α-BTX binding, as well as α-cobratoxin and nicotine potency to displace ¹²⁵I-α-BTX, did not change with age. Addition of Ca²⁺ to the assay buffer did not alter ¹²⁵I-α-BTX binding, or α-cobratoxin inhibition of ¹²⁵I-α-BTX binding, although it significantly increased nicotine affinity at both ages. The effect of Ca²⁺ on agonist affinity was dose-dependent, with an EC₅₀ value of 0.25–0.5 m M . Ca²⁺ also significantly increased the cooperativity of nicotine displacement curves in stratum oriens of the adult, but not in the fetus. These findings indicate that the properties of hippocampal ¹²⁵I-α-BTX binding sites are largely similar across age. Ca²⁺ selectively enhances the affinity of agonist binding, with no change in antagonist binding. This ionic effect may result from potentiation of agonist binding to a desensitized state of the α7 nicotinic acetylcholine receptor and may represent an important neuroprotective mechanism.     

Concanavalin A Receptors Associated with Rat Brain Synaptic Junctions Are High Mannose-Type Oligosaccharides

Abstract: Glycoproteins were isolated from a rat brain synaptic junction fraction by affinity chromatography on Concanavalin A-agarose. The isolated glycoproteins were digested with pronase and radiolabeled with ¹²⁵I-Bolton Hunter reagent, and ¹²⁵I-Concanavalin A-binding glycopeptides were isolated by chromatography on Concanavalin A-agarose. Treatment of the ¹²⁵I-Concanavalin A-binding glycopeptides with either α-mannosidase or endo-β- N -acetylglucosaminidase-C₁₁ abolished their interaction with Concanavalin A. The pronase digest was reacted with endo-β-N-acetylglucosaminidase-C₁₁ and released oligosaccharides were reduced with NaB³H₄. Following affinity chromatography on Concanavalin A-agarose, Concanavalin A-binding [³H]oligosaccharides were chromatographed on Biogel P₄. Two major oligosaccharides corresponding to standard carbohydrates containing eight and five mannose residues were identified. Treatment of these oligosaccharides with α-mannosidase converted them to smaller saccharides having a mobility on Biogel P₄ columns equal to the standard disaccharide mannose-β-1-4- N '-acetylglucosamine. These results demonstrate that the Concanavalin A receptor activity associated with CNS synaptic junctions resides in asparaginelinked oligosaccharides of the high-mannose type.     

Interaction of Guanine Nucleotides with [3H]Kainate and 6-[3H]Cyano-7-Nitroquinoxaline-2,3-dione Binding in Goldfish Brain

Abstract— Recent reports have suggested that a major proportion of [³H]kainate binding in goldfish brain is to a novel form of G-protein-linked glutamate receptor. Here we confirm that guanine nucleotides decrease [³H]kainate binding in goldfish brain membranes, but that binding is also reduced to a similar extent under conditions where G-protein modulation should be minimised. Inclusion of GTPγS resulted in an approximately twofold decrease in the affinity of [³H]kainate binding and a 50% reduction in the apparent B _max values in both Mg²⁺/Na⁺ and Mg²⁺/Na⁺-free buffer when assayed at 0°c. The pharmacology of [³H]kainate binding is similar to that of well-characterised ionotropic kainate receptors but unlike that of known me-tabotropic glutamate receptors, with neither 1 S ,3 R -amino-1,3-cyclopentanedicarboxylic acid (1 S ,3 R -ACPD) nor ibo-tenic acid being effective competitors. The molecular mass of the [³H]kainate binding protein, as determined by radiation inactivation, was 40 kDa, similar to the subunit sizes of other lower vertebrate kainate binding proteins that are believed to comprise ligand-gated ion channels. Furthermore, GTP-γS also inhibited the binding of the non-NMDA receptor-selective antagonist 6-[³H]cyano-7-ni-troquinoxaline-2,3-dione. These data strongly suggest that the regulatory interaction between guanine nucleotides and [³H]kainate and 6-[³H]cyano-7-nitroquinoxaline-2,3-dione binding is complex and involves competition at the agonist/antagonist binding site in addition to any G-protein-mediated modulation.     

Taurine Interactions with Chick Retinal Membranes

Abstract: Binding of [³H]taurine to whole retinal membranes and to membranes obtained from retinal subcellular fractions was studied. [³H]Taurine bound to chick retinal membranes with high affinity and specificity. Two types of [³H]taurine binding associated to retinal membranes were observed, one with a K_D= 0.68 μM and the other one with a K_D,= 9.32 μM. Both types of binding were highly Na⁻-dependent. The Na⁺-dependent taurine binding was antagonized by strychnine. Bound [³H]taurine was effectively displaced by β-alanine but not by GABA or glycine. Taurine binding was preferentially localized in membranes obtained from the crude synaptosomal fraction, although it is also present in substantial amounts in all retinal membranes. A Na⁺-independent [³H]taurine binding exhibiting properties which might represent interaction with postsynaptic receptor sites could not be demonstrated in the chick retina.     

Stereoselectivity of the Inhibition of [3H]Hemicholinium-3 Binding to the Sodium-Dependent High-Affinity Choline Transporter by the Enantiomers of a- and β-Methylcholine

Abstract: In a previous report, we showed that the enantiomers of α- and β-methylcholine inhibited choline uptake with Stereoselectivity, but that their transport by the choline carrier of nerve terminals showed stereospecificity. The present experiments used the same choline analogues to determine if either of the above characteristics pertains to their ability to interact with the [³H]-hemicholinium-3 binding site present on striatal membranes and synaptosomes. [³H]Hemicholinium-3 binding to striatal membranes could be inhibited stereoselectively by the enantiomers of β-methylcholine, but R (+)-α-methyl-choline was little better than its enantiomer in this test. However, [³H]hemicholinium-3 binding to striatal synaptosomes was inhibited stereoselectively by the enantiomers of both α- and β-methylcholine. This difference between the properties of [³H]hemicholinium-3 binding to membranes or to synaptosomes appears related to the presence of two ligand binding states. The [³H]hemicholinium-3 binding site could be shifted to a low-affinity state by ATP treatment and to a high-affinity state by EDTA washing. When the [³H]hemicholinium-3 binding site existed in its low-affinity state, binding was inhibited stereoselectively by the enantiomers of both a- and β-methylcholine, but when shifted to its high-affinity state, it was inhibited stereoselectively only by the enantiomers of β–methylcholine. We conclude that hemicholinium-3 interacts with the substrate recognition site of the high-affinity choline transporter, but that the Stereoselectivity of this site changes depending on its affinity state.     

Characterization of the Binding of [3H]NS 257, a Novel Competitive AMPA Receptor Antagonist, to Rat Brain Membranes and Brain Sections

Abstract: The binding of [³H]NS 257 {1,2,3,6,7,8-hexahydro-3-(hydroxyimino)- N,N -[³H]dimethyl-7-methyl-2-oxobenzo[2,1- b :3,4- c '] dipyrrole-5-sulfonamide} to rat cortical membranes was characterized in the absence and presence of thiocyanate. Specific [³H]NS 257 binding was saturable and reversible, and the stimulating effect of thiocyanate on binding was optimal at 100 m M . In the presence of thiocyanate [³H]NS 257 bound to a single population of binding sites with an affinity of 225 ± 8 n M and a binding site density of 0.61 ± 0.04 pmol/mg of original tissue. Thiocyanate increased the affinity of the binding site labeled by [³H]NS 257 for both α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and l -glutamate by a factor of 20 and 5, respectively. However, the affinity of the agonist domoate and the antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo( f )-quinoxaline (NBQX) was decreased in the presence of thiocyanate. Apparently, the affinities of antagonists as well as agonists for the AMPA receptor can be either increased or decreased by thiocyanate. The rank order of potency of the putative agonists quisqualate > AMPA > l -glutamate > domoate > kainate and of the antagonists NBQX > CNQX is consistent with the labeling of AMPA receptors. Autoradiographic studies showed that the distribution of [³H]NS 257 binding sites in rat brain was similar to that of [³H]AMPA binding sites. NS 257 is the first AMPA antagonist to be described showing an increased affinity for the AMPA receptor in the presence of thiocyanate.     

Identification of Endothelin Receptor Subtype (ETB) in Human Cerebral Cortex Using Subtype-Selective Ligands

Abstract: Specific endothelin (ET) binding sites were characterized in membranes prepared from human cerebral cortices using binding assay and cross-linking analysis. The presence of immunoreactive (IR) ET-1 was studied by radioimmunoassay. Saturation binding experiments revealed that the K _D and B _max for ¹²⁵I-ET-1 and ¹²⁵l-ET-3 to membranes from gray matter were 25 ± 6 pM and 115 ± 15 fmol/mg of protein and 24 ± 5 p M and 108 ± 13 fmol/mg of protein, respectively. Similar results were obtained for white matter. In the presence of 10 n M sarafotoxin-6c, which is selective for ET_B receptors, ¹²⁵I-ET-1 and ¹²⁵l-ET-3 binding was totally abolished. However, in the presence of 1 μ M BQ123, which is selective for ET_Areceptors, both bindings were not affected. These results suggest that the human cerebral cortex contains only ET_Breceptors. Cross-linking of ¹²⁵I-ET-1 and ¹²⁵l-ET-3 to membranes with disuccinimidyl suberate resulted in the labeling of two bands of 48 and 31 kDa. Concentrations of IR-ET-1 in the gray and white matter were 7.0 ± 3.2 and 2.5 ± 1.7 fmol/g wet weight, respectively. The demonstration of high-affinity ET_B receptors and the presence of IRET-1 suggest that the peptide may act as a neurotransmitter or neuromodulator in the human cerebral cortex.     

Photoaffinity Labeling with 2-[2-(4-Azido-3-[125I]-Iodophenyl)ethylamino]adenosine and Autoradiography with 2-[2-(4-Amino-3-[125I]Iodophenyl)ethylamino]adenosine of A2a Adenosine Receptors in Rat Brain

Abstract: The A_2a adenosine receptor agonist 2-[2-(4-amino-3-iodophenyl)ethylamino]adenosine is a potent coronary vasodilator. The corresponding radioiodinated ligand, [¹²⁵I]APE, discriminates between high- and low-affinity conformations of A_2a adenosine receptors. In this study, [¹²⁵I]APE was used for rapid (24-h) autoradiography in rat brain sections. The pattern of [¹²⁵I]APE binding is consistent with that expected of an A_2a-selective radioligand. It is highest in striatum, nucleus accumbens, and olfactory tubercle, with little binding to cortex and septal nuclei. Specific [¹²⁵I]APE binding to these brain regions is abolished by 1 µ M 2- p -(2-carboxyethyl)phenethylamino-5'- N -ethylcarboxamidoadenosine (CGS-21680) but is little affected by 100 n M 8-cyclopentyl-1,3-dipropylxanthine. Conversion of [¹²⁵I]APE to the corresponding arylazide results in [¹²⁵I]AzPE. The rank-order potency of compounds to compete for [¹²⁵I]AzPE binding in the dark is CGS-21680 > d -( R )- N ⁶-phenylisopropyladenosine > N ⁶-cyclopentyladenosine, indicating that it also is an A_2a-selective ligand. Specific photoaffinity labeling by [¹²⁵I]AzPE of a single polypeptide (42 kDa) corresponding to A_2a adenosine receptors is reduced 55 ± 4% by 100 µ M guanosine 5'- O -(3-thiotriphosphate) and 91 ± 1.3% by 100 n M CGS-21680. [¹²⁵I]APE and [¹²⁵I]AzPE are valuable new tools for characterizing A_2a adenosine receptors and their coupling to GTP-binding proteins by autoradiography and photoaffinity labeling.     

Cerebral clearance of human amyloid-beta peptide (1-40) across the blood-brain barrier is reduced by self-aggregation and formation of low-density lipoprotein receptor-related protein-1 ligand complexes

Soluble amyloid-β peptide (Aβ) exists in the form of monomers and oligomers, and as complexes with Aβ-binding molecules, such as low-density lipoprotein receptor-related protein-1 (LRP-1) ligands. The present study investigated the effect of self-aggregation and LRP-1 ligands on the elimination of human Aβ(1–40) [hAβ(1–40)] from the rat brain across the blood–brain barrier. Incubation of [¹²⁵I]hAβ(1–40) monomer resulted in time-dependent and temperature-dependent dimer formation, and the apparent elimination rate of [¹²⁵I]hAβ(1–40) dimer was significantly decreased by 92.7% compared with that of [¹²⁵I]hAβ(1–40) monomer. Pre-incubation with LRP-1 ligands, such as activated α2-macroglobulin (α2M), apolipoprotein E2 (apoE2), apoE3, apoE4, and lactoferrin, reduced the elimination of [¹²⁵I]hAβ(1–40). By contrast, pre-administration of the same concentration of these molecules in the rat brain did not significantly inhibit [¹²⁵I]hAβ(1–40) monomer elimination. Purified [¹²⁵I]hAβ(1–40)/activated α2M complex and [¹²⁵I]activated α2M were not significantly eliminated from the rat brain up to 60 min. MEF-1 cells, which have LRP-1-mediated endocytosis, exhibited uptake of [¹²⁵I]activated α2M, and enhancement of [¹²⁵I]hAβ(1–40) uptake upon pre-incubation with apoE, suggesting that [¹²⁵I]activated α2M and [¹²⁵I]hAβ(1–40)/apoE complex function as LRP-1 ligands. These findings indicate that dimerization and LRP-1-ligand complex formation prevent the elimination of hAβ(1–40) from the brain across the blood–brain barrier.