Characterisation and Molecular Identification of Adrenomedullin Binding Sites in the Rat Spinal Cord: A Comparison with Calcitonin Gene-Related Peptide Receptors

Abstract: Calcitonin gene-related peptide (CGRP) and its receptors are found in mammalian spinal cord. We show, for the first time, binding sites for the novel related peptide adrenomedullin in rat spinal cord microsomes. ¹²⁵I-Adrenomedullin binding showed high affinity ( K _D = 0.45 ± 0.06 n M ) and sites were abundant ( B _max = 723 ± 71 fmol/mg of protein). CGRP, amylin, and calcitonin did not compete at these sites ( K _i > 10 µ M ). High-affinity CGRP binding sites ( K _D = 0.18 ± 0.01 n M ) were much less numerous ( B _max = 17.7 ± 2.4 fmol/mg of protein) and showed competition by unlabeled adrenomedullin ( K _i = 34.6 ± 2.4 n M ). Chemical cross-linking revealed a major band for ¹²⁵I-adrenomedullin of M_r = 84,400 ± 1,200 and a minor band of M_r = 122,000 ± 8,700. ¹²⁵I-CGRP cross-linking showed bands of lower molecular weight (M_r = 74,500 ± 5,000 and 61,000 ± 2,200). Enzymic deglycosylation of the adrenomedullin binding site showed a considerable carbohydrate content. Neither adrenomedullin nor CGRP was able to increase cyclic AMP in spinal cord. Adrenomedullin mRNA was present in spinal cord, at one-third of its level in lung, and adrenomedullin immunoreactivity was present, at a low concentration (40 fmol/g of tissue). Thus, the presence of abundant binding sites and adrenomedullin mRNA and immunoreactivity anticipate an as yet undefined function for this peptide in spinal cord.     

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.     

Identification and Characterization of Endothelin Receptor Subtype B in Rat Retina

Abstract: The presence of immunoreactive (IR) endothelin (ET)-1 and ET-1 receptors in rat retina has been studied by radioimmunoassay and receptor assay, respectively. The specific binding of ¹²⁵I-ET-1 to rat retinal particulate preparations was saturable. Apparent equilibrium conditions were established within 120–140 min. Scatchard analysis of binding data indicated a single class of high-affinity binding sites with a K _D of 35 ± 11 p M and a B_max of 168 ± 60 fmol/mg of protein. ¹²⁵I-ET-1 binding to retinal particulate preparations was not inhibited by 1 μ M concentrations of somatostatin, atrial natriuretic factor, brain natriuretic peptide, thyroid-stimulating hormone, growth hormone, or insulin. The three endothelin isoforms, ET-1,-2, and-3, had similar affinity for the receptor. Cross-linking of ¹²⁵I-ET-1 to retinal particulate preparations with disuccinimidyl suberate resulted in the labeling of two bands with apparent molecular masses of 52 and 34 kDa. We have established a highly sensitive and specific radioimmunoassay for ET-1. The concentration of IR-ET-1 in rat retina was 35 ± 10 fmol/g wet weight. The demonstration of specific high-affinity ETB receptors and the presence of IR-ET-1 suggest that the peptide may act as a neurotransmitter or neuro-modulator in the retina.     

Pharmacological and Regional Characterization of [3H]LY278584 Binding Sites in Human Brain

Abstract: Binding of [³H]LY278584, which has been previously shown to label 5-hydroxytryptamine₃ (5-HT₃) receptors in rat cortex, was studied in human brain. Saturation experiments revealed a homogeneous population of saturable binding sites in amygdala ( K _D= 3.08 ± 0.67 n M, B _max= 11.86 ± 1.87 fmol/mg of protein) as well as in hippocampus, caudate, and putamen. Specific binding was also high in nucleus accumbens and entorhinal cortex. Specific binding was negligible in neocortical areas. Kinetic studies conducted in human hippocampus revealed a K _on of 0.025 ± 0.009 n M ⁻¹ min⁻¹ and a K _off of 0.010 ± 0.002 min⁻¹. The kinetics of [³H]LY278584 binding were similar in the caudate. Pharmacological characterization of [³H]LY278584 specific binding in caudate and amygdala indicated the compound was binding to 5-HT₃ receptors. We conclude that 5-HT₃ receptors labeled by [³H]LY278584 are present in both limbic and striatal areas in human brain, suggesting that 5-HT₃ receptor antagonists may be able to influence the dopamine system in humans, similarly to their effects in rodent studies.     

Effects of Cerebral Ischemia on Regional Dopamine Release and D1 and D2 Receptors

Abstract: To expand on the nature of regional cerebral vulnerability to ischemia, the release of dopamine (DA) and dopaminergic (D₁ and D₂) receptors were investigated in Mongolian gerbils subjected to bilateral carotid artery occlusion (15 min) alone or with reflow (1–2 h). Extracellular cortical and striatal content of DA and its metabolites was measured by microdialysis using HPLC with electrochemical detection. The kinetic properties of D₁ and/or D₂ receptor binding sites were determined in cortical and striatal membranes with the use of radiolabeled ligands (¹²⁵I-SCH23982 and [³H]YM-09151-2, respectively). The ischemic release of DA from the striatum was greater (400-fold over preischemic level) than that from the cortex (12-fold over preischemic content). The affinity for the D₁-receptor ligand was lower ( K _D= 1.248 ± 0.047 n M ) after ischemia than that for sham controls ( K _D= 0.928 ± 0.032 n M, p < 0.001). The number of binding sites for D₂ receptors decreased in striatum ( B _max= 428 ± 18.4 fmol/mg of protein) after ischemia compared with sham controls ( B _max= 510 ± 25.2 fmol/mg of protein, p < 0.05). D₁ or D₂ binding sites were not changed either in the ischemic cortex or postischemic striatum and cortex. The findings strongly suggest that the ischemic release of DA from striatum is associated with early transient changes in D₁- and D₂-mediated DA neurotransmission.     

Characterization, Distribution, and Protein Kinase C-Mediated Regulation of [35S]Glutathione Binding Sites in Mouse and Human Spinal Cord

Abstract: We have characterized a high-affinity [³⁵S]-glutathione ([³⁵S]GSH) binding site in mouse and human spinal cord. [³⁵S]GSH binding sites in mouse and human spinal cord were observed largely within the gray matter in both the dorsal and ventral horns of spinal cord at cervical, thoracic, and lumbosacral segments. High-affinity [³⁵S]GSH binding was saturable, showing a B _max of 72 fmol/mg of protein and a K _D of 3.0 n M for mouse spinal cord and a B _max of 52 fmol/mg of protein and a K _D of 1.6 n M for human spinal cord. [³⁵S]GSH binding was displaceable by GSH, l -cysteine, and S -hexyl-GSH, but not by glutamate, glycine, or NMDA. These [³⁵S]GSH binding sites exhibited kinetic and saturation characteristics similar to GSH binding sites in rat brain astrocytes. To determine whether [³⁵S]GSH binding sites could be regulated by protein kinase C, we exposed human spinal cord sections to phorbol 12,13-diacetate for 1 h before ligand binding. Phorbol ester treatment increased [³⁵S]GSH binding by ∼60%, an effect that could be blocked by exposure of spinal cord sections to 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, a general protein kinase inhibitor. [³⁵S]GSH binding sites in the spinal cord of both species exhibited many of the characteristics of a receptor including saturable binding, high affinity, ligand specificity, and modulation by kinase activity. These data suggest that GSH is a neurotransmitter in the CNS.     

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.     

Ascorbic Acid Inhibits 125I-SCH 23982 Binding but Increases the Affinity of Dopamine for D1 Dopamine Receptors

Abstract: Effects of ascorbic acid (AA) on ¹²⁵I-SCH 23982 binding to D₁ dopaminergic receptors in membrane preparations from rat striatum were investigated. AA in the range of 0.03 µ M –0.33 m M inhibited 75% of specific binding of ¹²⁵I-SCH 23982 in a dose-dependent manner. At higher concentrations, this inhibition of binding activity by AA was less potent, and 3.3 m M AA inhibited only 30% of specific binding. Reduced glutathione did not alter the inhibition of binding by 0.33 m M AA, but reduced the inhibition by 3.3 m M AA to 8% of specific binding. The loss of specific binding by AA was rescued by 1 m M EDTA, an inhibitor of lipid peroxidation. In the absence of AA, competition experiments with the agonist, dopamine, revealed the presence of high-affinity ( K _h = 224.9 ± 48.9 n M ) and low-affinity ( K _l = 21,100 ± 2,400 n M ) binding sites. Although the maximum binding of ¹²⁵I-SCH 23982 decreased to 40% without affecting the K _D value in the presence of 1.67 m M AA, the value of the high-affinity site for dopamine was increased ( K _h = 23.3 ± 9.4 n M ) and that of the low-affinity site was decreased ( K _l = 136,800 ± 40,900 n M ). These results suggest that AA may affect D₁ dopamine receptor function by lipid peroxidation, competition with dopamine for low-affinity sites, and reduced oxidation of dopamine.     

Human Y-79 Retinoblastoma Cells Exhibit Specific Corticotropin-Releasing Hormone Binding Sites

Abstract: In this study we have identified specific binding sites for corticotropin-releasing hormone (CRH) in human Y-79 retinoblastoma cell membranes by using ¹²⁵I-Tyrovine CRH (¹²⁵I-oCRH) as radioligand. Binding at 19°C was rapid with steady state being reached within 20 min, reversible and linear with membrane protein concentration. The ¹²⁵I-oCRH binding was enhanced by Mg²⁺ and inhibited by the GTP analogue guanosine 5'- O -(3'-thiotriphosphate). Y-79 cell membranes exhibited two populations of binding sites, a high-affinity site with an apparent dissociation constant ( K _D) of 1 n M and a low-affinity site with an apparent K _D of 500 n M . ¹²⁵I-oCRH binding was completely antagonized by human/rat CRH, [Met(O)²¹]oCRH, α-helical CRH_9–41, urotensin I, and sauvagine with a rank order of potency similar to that displayed by CRH receptors of other tissues. These data describe for the first time the presence of specific CRH-binding sites in retinal cells. The Y-79 cell line may therefore constitute a valuable model in which to study CRH action on retinal cells.     

Sensitization of G Protein-Coupled Benzodiazepine Receptors in the Striatum of 6-Hydroxydopamine-Lesioned Rats

Abstract: The nonselective benzodiazepine (BZ) agonist diazepam is a potent inhibitor of adenylyl cyclase (AC) activity in the rat striatum. To examine this inhibitory action of diazepam further, its effects were examined in 6-hydroxydopamine-lesioned animals, which reportedly exhibit sensitization of the striatal AC pathway. As previously observed, inhibition of AC activity by diazepam was biphasic, with the first phase being receptor-mediated, whereas the second phase involves a direct action on the enzyme itself. In the presence of NaCl (120 m M ), a marked sensitization to the receptor-mediated inhibitory effect of diazepam on AC activity was observed in striatal membranes of lesioned animals. EC₅₀ values were 10.4 ± 1.1 and 4.8 ± 0.9 n M ( p < 0.05) for intact and lesioned striata, respectively. An examination of [³H]diazepam binding revealed a significant increase in the density of binding sites in denervated striata, with no change in affinity. A time-dependent increase in [α-³²P]GTP labeling of two distinct striatal proteins with apparent molecular masses of 40 and 45 kDa, suggestive of the α subunits of G_i and G_s, respectively, was observed. There was a significant increase in basal [α-³²P]GTP binding to both proteins in lesioned striata. In addition, diazepam stimulated [α-³²P]GTP binding to the 40-kDa protein, especially in lesioned striata. These data indicate that the sensitization of the receptor-mediated inhibitory effect of diazepam on AC activity in denervated striata may involve up-regulation of BZ receptors as well as enhanced functional coupling of these receptors to inhibitory G proteins.     

[3H]SCH 58261, a Selective Adenosine A2A Receptor Antagonist, Is a Useful Ligand in Autoradiographic Studies

Abstract: We have characterized the new potent and selective nonxanthine adenosine A_2A receptor antagonist SCH 58261 as a new radioligand for receptor autoradiography. In autoradiographic studies using agonist radioligands for A_2A receptors ([³H]CGS 21680) or A₁ receptors ( N ⁶-[³H]cyclohexyladenosine), it was found that SCH 58261 is close to 800-fold selective for rat brain A_2A versus A₁ receptors ( K _i values of 1.2 n M versus 0.8 µ M ). Moreover, receptor autoradiography showed that [³H]SCH 58261, in concentrations below 2 n M , binds only to the dopamine-rich regions of the rat brain, with a K _D value of 1.4 (0.8–1.8) n M . The maximal number of binding sites was 310 fmol/mg of protein in the striatum. Below concentrations of 3 n M , the nonspecific binding was <15%. Three adenosine analogues displaced all specific binding of [³H]SCH 58261 with the following estimated K _i values (n M ): 2-hex-1-ynyl-5'- N -ethylcarboxamidoadenosine, 3.9 (1.8–8.4); CGS 21680, 130 (42–405); N ⁶-cyclohexyladenosine, 9,985 (3,169–31,462). The binding of low concentrations of SCH 58261 was not influenced by either GTP (100 µ M ) or Mg²⁺ (10 m M ). The present results show that in its tritium-labeled form, SCH 58261 appears to be a good radioligand for autoradiographic studies, because it does not suffer from some of the problems encountered with the currently used agonist radioligand [³H]CGS 21680.     

Binding Characteristics and Interactions of Depressant Drugs with [35S]t-Butylbicyclophosphorothionate, a Ligand that Binds to the Picrotoxinin Site

Abstract: [³⁵S]r-Butylbicyclophosphorothionate (TBPT), a cage convulsant with picrotoxinin-like activity, binds to rat brain membranes to a single site with an apparent K_D of 25.1 ± 5.6 n M and a B_max of 1.40 ± 0.22 pmol/mg protein. TBPT binding to rat brain membranes was inhibited by a variety of convulsant, depressant, anxiolytic, and anticonvulsant drugs that had previously been shown to inhibit [³H]a-dihydropicrotoxinin binding. Depressant drugs such as pentobarbital and the nonbarbiturate (+)etomidate inhibited TBPT binding in an uncompetitive manner. Thus, pentobarbital and (+)etomidate decreased both the affinity and the number of binding sites of TBPT to whole brain membranes. The IC₅₀ values of (+)etomidate (9 μ M ) and pentobarbital (90 μ M ) are similar to the EC₅₀ values at which they enhance both [³H]-γ-aminobutyric acid and [³H]diazepam binding in cerebral cortex membranes. RO5–4864, which has recently been shown to be a convulsant, also inhibited TBPT binding (IC₅₀= 10 μ M ). These results suggest that TBPT binds to the picrotoxinin site and further supports the notion that the picrotoxinin site is an important modulatory site at the benzodiazepine-GABA receptor-ionophore complex.     

Desensitization of Nicotine-Stimulated [3H]Dopamine Release from Mouse Striatal Synaptosomes

Potential desensitization of brain nicotinic receptors was studied using a [³H]dopamine release assay. Nicotine-stimulated [³H]dopamine release from mouse striatal synaptosomes was concentration-dependent with an EC₅₀ of 0.33 ± 0.13 μ M and a Hill coefficient of 1.44 ± 0.18. Desensitization by activating concentrations of nicotine had a similar EC₅₀ and a half-time of 35 s. Concentrations of nicotine that evoked little release also induced a concentration-dependent desensitization (EC₅₀=6.9 plusmn; 3.6 n M , t_1/2= 1.6-2.0 min, n _H=1.02 ± 0.01). Both types of desensitization produced a maximum 75% decrease in [³H]dopamine release. Recovery from desensitization after exposure to low or activating concentrations of nicotine was time-dependent with half-times of 6.1 min and 12.4 min, respectively. Constants determined for binding of [³H]nicotine to striatal membrane at 22°C included a K _Dof 3.7 ± 0.5 n M , B_max of 67.5 ± 2.2 fmol/mg, and Hill coefficient of 1.07 ± 0.06. Association of nicotine with membrane binding sites was biphasic with half-times of 9 s and 1.8 min. The fast rate process contributed 37% of the total reaction. Dissociation was a uniphasic process with a half-time of 1.6 min. Comparison of constants determined by the release and binding assays indicated that the [³H]-nicotine binding site could be the presynaptic receptor involved in [³H]dopamine release in mouse striatal synaptosomes.     

Participation of Tubulin in the Stimulatory Regulation of Adenylyl Cyclase in Rat Cerebral Cortex Membranes

Abstract: This study examined effects of tubulin on the activation of adenylyl cyclase in rat cerebral cortex membranes. Tubulin, prepared from rat brain by polymerization with the hydrolysis-resistant GTP analogue 5'-guanylylimidodiphosphate (GppNHp) caused significant activation of the enzyme by ∼156% under conditions in which stimulation rather than inhibition of the enzyme was favored. Tubulin-GppNHp activated isoproterenol-sensitive adenylyl cyclase, potentiated forskolin-stimulated activity of the enzyme, and reduced agonist binding affinity for β-adrenergic receptors. When tubulin, polymerized with the hydrolysis-resistant photoaffinity GTP analogue [³²P] P ³(4-azidoanilido)- P ¹-5'-GTP ([³²P]AAGTP), was incubated with cerebral cortex membranes, AAGTP was transferred from tubulin to G_sα as well as G_iα. These results suggest that, in rat cerebral cortex membranes, the tubulin dimer participates in the stimulatory regulation of adenylyl cyclase by transferring guanine nucleotide to G_sα, as well as affecting the G_i-mediated inhibitory pathway.     

[3H]LY303870, a Novel Nonpeptide Radioligand for the NK-1 Receptor

Abstract: We synthesized a potent and selective antagonist radioligand for the neurokinin (NK)-1 receptor and characterized its binding to guinea pig striatal membranes. ( R ) - N - [2 - [Acetyl[³H₃][(2 - methoxyphenyl) - methyl]amino] - 1 - (1 H - indol - 3 - ylmethyl)ethyl][1,4' - bipiperidine]-1'-acetamide ([³H]LY303870) binds to a single class of sites with an equilibrium K _D of 0.22 n M and a B _max of 723 fmol/mg of protein. Unlabeled LY303870 potently inhibited the binding with an IC₅₀ of 0.56 n M , whereas the less active ( S )-enantiomer (LY306155) was substantially less potent. The nonpeptide NK-1 antagonists (±)-CP96,345 and (±)-RP 67580 had IC₅₀ values of 0.74 and 49 n M , respectively. Substance P (SP) was also a potent inhibitor with with an IC₅₀ of 3.1 n M . The inhibition by SP could be separated into two components: a high-affinity component with a K _i of 0.53 n M and a lower-affinity component with a K _i of 155 n M . Addition of 100 µ M guanylyl 5'-imidodiphosphate [Gpp(NH)p] in the incubation increased the relative amount of the low-affinity agonist state of the receptor. Consistent with the antagonist properties of LY303870, the dissociation rate of [³H]LY303870 was not changed by the presence of 100 µ M Gpp(NH)p. The distribution of [³H]LY303870 binding sites in the guinea pig brain closely matched the distribution of NK-1 receptors labeled by [³H]SP. Therefore, [³H]LY303870 is a potent and selective antagonist radioligand for NK-1 receptors in guinea pig brain. In addition, regulation of NK-1 agonist affinity by guanine nucleotides is similar to that seen for monoaminergic receptors.     

Characterization of Receptors for Cholecystokinin and Related Peptides in Mouse Cerebral Cortex

Abstract: The characteristics of cholecystokinin (CCK) binding to its receptors in a particulate membrane fraction of mouse cerebral cortex were studied by employing biologically active radioiodinated CCK prepared by conjugation with ¹²⁵I-Bolton-Hunter (¹²⁵I-BH) reagent. At 24°C binding was rapid, reversible, and linearly related to protein content. Binding was maximal at acidic pH (6.5) and reduced by the presence of monovalent cations. Under physiological conditions (pH 7.4, 118 mM-NaC1, 4.7 mM-KCl) Scatchard plots of CCK binding were linear with a K _D value of 1.27 nM and binding capacity of 115 fmol/mg protein. Optimal binding required the presence of both Mg²⁺ and EGTA, and was inhibited by the addition of micromolar concentrations of Cu²⁺ (ID₅₀= 30 μM). The cortical receptor recognized all major forms of CCK, with an order of potency of cholecystokinin octapeptide (CCK₈) > CCK > cholecystokinin tetrapeptide (CCK₄). Desulfated cholecystokinin octapeptide (dCCK₈) had a 10-fold lower affhity than CCK₈. Dibutyryl cyclic GMP, a potent competitive inhibitor of CCK binding to receptors in pancreas, was not a specific inhibitor of CCK binding to brain receptors. These present results support the concept that CCK may function as a regulatory peptide in brain, and that the cortical CCK receptor is different from the receptors mediating the peripheral action of CCK.     

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.     

Identification of GABAA/Benzodiazepine Receptors on Clathrin-Coated Vesicles from Rat Brain

Abstract: To investigate the subcellular compartments that are involved in the endocytosis and intracellular trafficking of GABA_A/benzodiazepine receptors, we have studied the distribution and properties of clonazepam-displaceable binding of [³H]flunitrazepam to membrane fractions from rat brain. The microsomal fraction was subjected to density centrifugation and gel filtration to isolate clathrin-coated vesicles. Homogeneity of the coated-vesicle fraction was demonstrated by using electron microscopy and by analysis of clathrin subunits and clathrin light-chain kinase. Vesicles exhibiting specific binding of [³H]flunitrazepam eluted from the sieving gel as a separate peak, which was coincident with that for coated vesicles. Scatchard analysis of equilibrium binding of [³H]flunitrazepam to coated vesicles yielded a K_D value of 21 ± 4.7 nM and a B_max value of 184 ± 28 fmol/mg. The K_D value for coated vesicles was 12-19-fold that found with microsomal or crude synaptic membranes. This low-affinity benzodiazepine receptor was not identified on any other subcellular fraction and thus appears to be a novel characteristic of coated vesicles. The B_maxvalue for coated vesicles, expressed per milligram of protein, corresponded to 16 and 115% of that found for crude synaptic and microsomal membrane fractions, respectively. Because the trafficking of neurotransmitter receptors via clathrin-coated vesicles is most likely to occur through endocytosis, the data suggest that an endocytotic pathway may be involved in the removal of GABA_A/benzodiazepine receptors from the neuronal surfaces of the rat brain. This mechanism could play a role in receptor sequestration and down-regulation that is produced by exposure to GABA and benzodiazepine agonists.     

Specific High-Affinity Binding of L-[3H]Aspartate to Rat Brain Membranes

Abstract: The binding of L-[³H]aspartate was investigated in washed membranes prepared from whole rat brain. We were able to differentiate two separate binding sites differing in their Na dependence. The Na-independent binding was saturable, reversible, and optimal at 20°C and at pHs in the neutral range. The dissociation constant (K_d) at 20°C was about 200 n M . This binding site seemed to be modulated by magnesium and calcium at physiological concentrations. None of the amino acids tested was a potent competitor for Na-independent L-[³H]aspartate binding. This binding site was unevenly distributed in the rat central nervous system: cerebellum = cerebral cortex > ponsmedulla > spinal cord. Destruction of the intrinsic neurons of the cerebellum by injecting kainic acid 30 days before sacrifice resulted in a 53% reduction in Na-independent binding in this region. The Na-dependent binding of L-[³H]-aspartate (K_d= 484 n M ) was strongly inhibited by D-aspartate, L-glutamate, D,L-aspartate β-hydroxamate; was unaffected by calcium and magnesium; and showed a different pattern of distribution: cerebral cortex > cerebellum = pons-medulla = spinal cord. This binding in cerebellum was unaffected by injections of kainic acid.