Retinal [125I]iodomelatonin binding sites in the tree shrew (Tupaiidae).

The characteristics of melatonin-binding sites labelled by [¹²⁵I]iodomelatonin in membrane preparations from the tree shrew retina were determined. Specific binding of [¹²⁵I]iodomelatonin to the membrane preparations of tree shrew retina was rapid, stable, saturable, and reversible. Among the indoles tested only 6-chloromelatonin, melatonin and N-acetylserotonin had significant affinities to the [¹²⁵I]iodomelatonin binding site. Scatchard analysis of the membrane preparations revealed a dissociation constant (K_d) of 51.0 ± 16 pM and total number of binding sites (B_max) of 1.97 ± 0.6 fmol/mg protein.     

125I]iodomelatonin binding sites in spleens of birds and mammals.

The specific binding of [125I]iodomelatonin to duck spleen membrane preparations was studied in detail. These binding sites were stable, saturable, reversible and of high affinity. Scatchard analysis of the binding revealed a equilibrium binding constant (Kd) of 73.1 +/- 5.4 pM and a total number of binding sites (Bmax) of 3.64 +/- 1.38 fmol/mg protein. Studies on the relative binding capacities of [125I]iodomelatonin to the spleen in different species showed the following order: duck greater than chicken greater than guinea pig greater than pigeon greater than mouse. No binding site was detected in the rat spleen. The presence of [125I]iodomelatonin binding sites in the spleen of birds and mammals suggested a direct action of pineal melatonin on the immune system.     

Identification ofβ 2-adrenoceptors on guinea pig alveolar macrophages using (−)-3-[125I]iodocyanopindolol

The -adrenoceptor antagonist (–)-3-[¹²⁵I]iodocyanopindolol ([¹²⁵I]ICYP) binds with high affinity and in a saturable way to membranes of guinea pig alveolar macrophages. The equilibrium dissociation constant for [¹²⁵I]ICYP is 24.3 ± 1.2 pM, and the number of binding sites is 166.3 ± 13.7 fmol/mg protein (N=4, ±SEM). Displacement studies with selective antagonists showed that [¹²⁵I]ICYP labels ₂-adrenoceptors on guinea pig alveolar macrophages.     

A radiolabeled peptide ligand of the hERG channel, [125I]-BeKm-1

The wild-type scorpion toxin BeKm-1, which selectively blocks human ether-a-go-go related (hERG) channels, was radiolabeled with iodine at tyrosine 11. Both the mono- and di-iodinated derivatives were found to be biologically active. In electrophysiological patch-clamp recordings mono-[¹²⁷I]-BeKm-1 had a concentration of half-maximal inhibition (IC₅₀ value) of 27 nM, while wild-type BeKm-1 inhibited hERG channels with an IC₅₀ value of 7 nM. Mono-[¹²⁵I]-BeKm-1 was found to bind in a concentration-dependent manner and with picomolar affinity to hERG channel protein in purified membrane vesicles from transfected human embryonic kidney cells (HEK-293). Under optimized conditions the equilibrium dissociation constant (K_d) values from saturation and kinetic binding analysis were 13 and 14 pM, respectively. Both the association and dissociation of [¹²⁵I]-BeKm-1 were fast (association rate constant, k_on=3.6×10⁷ M^–1s^–1; dissociation rate constant, k_off=0.005 s^–1). Wild-type BeKm-1 displaced binding of [¹²⁵I]-BeKm-1 with half-maximal inhibitory concentrations of 44 pM. In contrast, competition experiments with a BeKm-1 mutant BeKm-1-K18A, in which the toxin interaction site is disrupted, resulted in a drop in affinity by more than 300-fold as compared to the wild-type toxin. Iberiotoxin and apamin, peptide inhibitors of Ca²⁺-activated K⁺-channels, had no effect on [¹²⁵I]-BeKm-1 binding. Adding the classical rapid delayed rectifier current (I_Kr) blocker E-4031 reduced binding of [¹²⁵I]-BeKm-1 to the hERG channel to an IC₅₀ of 7 nM. In autoradiographic studies on rat hearts, binding of [¹²⁵I]-BeKm-1 was dose-dependent and could partially be displaced by the addition of excess amounts of non-radioactive BeKm-1. The density of the radioactive signal was equally distributed in the myocardium of both the ventricle and atria indicating a homogenous expression of hERG channels throughout the heart.     

Increases of kinin B1 and B2 receptors binding sites after brain infusion of amyloid-beta 1-40 peptide in rats

Although numerous inflammation pathways have been implicated in Alzheimer's disease, the involvement of the kallikrein–kinin system is still under investigation. We anatomically localized and quantified the density of kinin B₁ and B₂ receptors binding sites in the rat brain after the infusion of amyloid-β (Aβ) peptide in the right lateral brain ventricle for 5 weeks. The conditioned avoidance test showed a significant reduction of memory consolidation in rats infused with Aβ (68.6 ± 20.9%, P < 0.05) when compared to control group (90.8 ± 4.1%; infused with vehicle). Autoradiographic studies performed in brain samples of both groups using [¹²⁵I]HPP-[des-Arg¹⁰]-Hoe-140 (150 pM, 90 min, 25 °C) showed a significant increase in density of B₁ receptor binding sites in the ventral hippocampal commissure (1.23 ± 0.07 fmol/mg), fimbria (1.31 ± 0.05 fmol/mg), CA1 and CA3 hippocampal areas (1.05 ± 0.03 and 1.24 ± 0.02 fmol/mg, respectively), habenular nuclei (1.30 ± 0.04 fmol/mg), optical tract (1.30 ± 0.05 fmol/mg) and internal capsule (1.26 ± 0.05 fmol/mg) in Aβ group. For B₂ receptors ([¹²⁵I]HPP-Hoe-140, 200 pM, 90 min, 25 °C), a significant increase in density of binding sites was observed in optical tract (2.04 ± 0.08 fmol/mg), basal nucleus of Meynert (1.84 ± 0.18 fmol/mg), lateral septal nucleus – dorsal and intermediary portions (1.66 ± 0.29 fmol/mg), internal capsule (1.74 ± 0.19 fmol/mg) and habenular nuclei (1.68 ± 0.11 fmol/mg). In control group, none of these nuclei showed [¹²⁵I]HPP-Hoe-140 labeling. This significant increase in densities of kinin B₁ and B₂ receptors in animals submitted to Aβ infusion was observed mainly in brain regions related to cognitive behavior, suggesting the involvement of the kallikrein–kinin system in Alzheimer's disease in vivo.     

Distribution and characterization of melatonin receptors in the brain of the Japanese quail, Coturnix japonica

2-[¹²⁵I]iodomelatonin was used to study the distribution and properties of the melatonin receptor in the Japanese quail brain. High receptor density was detected in the major targets of direct retinal input (optic tectum, nucleus of the optic basal rout, ventrolateral geniculate nucleus), as well as areas representing terminals in the visual pathways (nucleus rotundus, ectostriatum, thalamo-hyperstriatal pathway). Binding was also found in the piriform cortex, the hypophyseal pars tuberalis, the oculomotorius nucleus and the associated Edinger-Westphal nucleus, and in the nuclei of the third, fourth and sixth cranial nerves. A comparison of the receptor pharmacological profile to that of the mammalian brain demonstrated pharmacological identity of the two binding sites. In the saturation experiments, GPT_γS decreased the binding affinity, numerical K_d values increasing from ≈ 35 pM to ≈ 150 pM.     

Oocytes from Xenopus laevis contain an intrinsic σ2-like binding site

In preparation for expression studies for rat brain σ-binding sites, Xenopus oocytes were tested for the presence of [³H]di-o-tolylguanidine (DTG)-binding sites. Native oocytes were found to contain two intrinsic [³H]DTG-binding sites, a high-affinity site (K_d = 32 ± 6 nM, B_max of 45.7 ± 19 pmol/mg protein) and a low-affinity binding site (K_d = 1.3 ± 0.7 μM, B_max of 3.2 ± 0.7 nmol/mg protein). In a series of radioligand-binding-displacement studies, the high-affinity binding sites were found to have a binding profile which has a similar K_d to that of the mammalian σ₂-binding site (32 vs. 38 nM). Comparison of the IC₅₀ values for inhibition of [³H]DTG binding in rat liver and oocytes for DTG, haloperidol (HAL), (−)-pentazocine, (+)-3-(3-hydroxyphenyl)-N-propylpiperidine hydrochloride ((+)-3-PPP), (+(-pentazocine and Zn²⁺, showed similarity in rank (r² = 0.913) but a 7-fold lower potency in oocytes. These results suggest that the high-affinity [³H]DTG-binding site in oocytes represents a σ₂-like binding site.     

Binding characteristics and regional distribution of [I]iodomelatonin binding sites in the brain of the human fetus

The binding and pharmacological characteristics of [¹²⁵I]iodomelatonin binding sites in human fetal brain membrane preparations were determined. Membrane preparations of the whole brain revealed a equilibrium binding constant (K_d) of 17.5 pmol/l with a total number of binding sites (B_max) at 0.8 fmol/mg protein. Among the various brain regions studied, [¹²⁵I]iodomelatonin binding was highest in the hypothalamus, and lowest in the mid-brain, pons-medulla, and cerebral cortex. The K_d of the hypothalamus was calculated to be 26.1 pmol/l and the B_max 5.4 fmol/mg protein. Only 6-chloromelatonin, melatonin and N-acetylserotonin had significant inhibition in the binding. Our results suggest that melatonin receptors are present in the human brain and that melatonin may exert a central action.     

Interaction of ivermectin with γ-aminobutyric acid receptors in Trichinella spiralis muscle larvae

The value of the γ-aminobutyric acid (GABA) receptor of nematodes as a target for ivermectin's mode of action remains unclear. Using binding assays, we examined extracts from Trichinella spiralis muscle larvae for the presence of [³H]-ivermectin and [³H]-GABA binding sites. Tissue preparations displayed affinity binding sites for [³H]-ivermectin with a dissociation constant (K _d) of 83 nM and a receptor density (B_max) of 145 fmol/mg protein. We also identified a specific [³H]-GABA binding activity with a K _d of 1.2 μM and a B_max of 4.78 pmol/mg protein. In competition studies, ivermectin was found to be a competitive inhibitor of specific [³H]-GABA binding activity with an inhibition constant (K _i) of 3.39 nM, suggesting that GABA receptors could be implicated in the mechanism of action of ivermectin in nematodes. Received: 7 August 1998 / Accepted: 22 September 1998     

Localisation of dopamine D3 receptor in the rat cerebellar cortex: a light microscope autoradiographic study

The pharmacological properties and the anatomical localisation of dopamine D₃ receptor were assessed in the rat cerebellar cortex using radioligand binding techniques associated with light microscope autoradiography and 7-[³H]hydroxy-N,N-di-n-propyl-2-aminotetralin (7-[³H]OH-DPAT) as a ligand. 7-[³H]OH-DPAT was specifically bound to sections of rat cerebellar cortex with a dissociation constant (K_d) of 0.5 nM and a maximum density of binding sites (B_max) of 97 ± 4 fmol/mg tissue. The rank order of potency of competitors of 7-[³H]OH-DPAT binding and the observation that guanosine triphosphate did not affect radioligand binding suggest the labelling of a dopamine D₃ receptor. 7-[³H]OH-DPAT binding sites are located mainly in the molecular layer and in lesser amounts in the Purkinje neuron layer, primarily within the cell body of Purkinje neurons. No specific accumulation of silver grains was observed in the granule neuron layer or in the white matter of the cerebellar cortex. The localisation of a putative dopamine D₃ receptor within Purkinje neurons suggests that this site may have functional relevance in the cerebellar cortex.     

Characteristics of albumin binding to opossum kidney cells and identification of potential receptors

 Albumin re-absorption in the kidney proximal tubule may be pathophysiological in disease. Opossum kidney (OK) cell monolayers were used to investigate the characteristics of [¹²⁵I]-labelled albumin binding at 4°C. Two binding sites were identified, one with high affinity (K _D 154.8 ±7 mg/l) and low capacity, the other with low affinity (K _D 8300 ± 1000 mg/l) and high capacity. Binding was sensitive to lectins Glycine max and Ulex europaeus I, but not other lectins, indicating involvement of a glycoprotein(s) in the binding process. Binding was also sensitive to a number of agents known to inhibit binding to scavenger receptors. [¹²⁵I]-Labelled albumin ligand blotting of OK cell membrane proteins identified several albumin-binding proteins with identical lectin affinities to those proteins mediating albumin binding to OK cell monolayers. These results provide initial evidence of the identity of albumin receptors in kidney tubules, and suggest that they may be members of the family of scavenger receptors. Received: 24 July 1996 / Received after revision: 16 October 1996 / Accepted: 18 October 1996     

Distribution of angiotensin IV binding sites (AT4 receptor) in the human forebrain, midbrain and pons as visualised by in vitro receptor autoradiography

Angiotensin IV and other AT₄ receptor agonists, improve memory retention and retrieval in the passive avoidance and swim maze learning paradigms. Angiotensin IV binding sites (also known as the AT₄ receptors) are widely distributed in guinea pig and monkey (Macaca fascicularis) brains where high densities of the binding sites have been detected in the hippocampus, neocortex and motor nuclei. However, the distribution of the binding sites in the human brain is not known. We have recently localised the angiotensin IV binding sites (AT₄ receptors) in post-mortem human brain using iodinated Nle-angiotensin IV, a higher affinity and more stable analogue of angiotensin IV. This radioligand bound with relatively high affinity and specificity to angiotensin IV binding sites. In competition studies on consecutive sections through the prefrontal cortex and claustrum, angiotensin IV, Nle-angiotensin IV and LVV-hemorphin 7 competed for the binding of ¹²⁵I[Nle]-angiotensin IV with nanomolar affinities. Angiotensin II and the AT₁ and AT₂ receptor antagonists were ineffective in competing for the binding at concentrations of up to 10 μM. We found high densities of ¹²⁵I[Nle]-angiotensin IV binding sites throughout the cerebral cortex including the insular, entorhinal, prefrontal and cingulate cortices. Very high densities of the binding sites were observed in the claustrum, choroid plexus, hippocampus and pontine nucleus. Some thalamic nuclei displayed high densities of binding including the anteroprincipal, ventroanterior, anteromedial, medial dorsal and ventrolateral nuclei. The caudate nucleus, putamen, many amygdaloid nuclei and the red nucleus all displayed moderate densities of binding with a higher level detected in the substantia nigra pars compacta. In the hypothalamus, high densities binding sites were found in the ventromedial nucleus with lower levels in the dorsomedial and paraventricular nuclei. The distribution of ¹²⁵I[Nle]-angiotensin IV binding sites in the human brain is similar to that found in other species and supports multiple roles for the binding sites in the central nervous system, including facilitation of memory retention and retrieval.     

The ontogeny of 2-[125I]iodomelatonin binding sites in chicken brain.

The characteristics of the binding sites for 2-[125I]iodomelatonin were studied in chicken brain membranes during development. Specific binding, defined using cold melatonin (1 microM), was detected as early as 8-day-old embryos. Scatchard analysis of saturation experiments showed that 2-[125I]iodomelatonin binds to a single class of site at all ages tested (8-day-old embryos to 3-month-old chicks). Binding affinity (Kd) did not change during development (18-31 pM), but the maximal number of binding sites (Bmax) increased until embryonic day 18, and then remained relatively constant until 30 days of age. A further increase in Bmax was seen at 3 months of age. Guanosine 5'-triphosphate (GTP, 1 mM) inhibited 2-[125I]iodomelatonin binding at all ages suggesting that the melatonin binding site is coupled to a guanine nucleotide binding protein at a very early stage of development. Competition experiments with a number of melatonin analogues indicated that the binding site detected in the brain at embryonic day 8 was pharmacologically identical to that observed 15 days after hatching.     

Transferrin receptors on the plasma membrane of cultured rat astrocytes

It is generally accepted that transferrin receptor is located in the endothelial cells of the brain, but its existence in other brain cell types is less established. In this study, a [¹²⁵I]transferrin binding assay was used to determine whether there is transferrin receptor on the membrane of cultured rat cortical astrocytes (type 1) in vitro. The results demonstrated that cortical astrocytes (type 1) in suspension attracted [¹²⁵I]transferrin with a saturable and specific binding. Scatchard and Hill plot analysis showed that the dissociation constant (K_D) of the binding was about 3.5×10^–8 M and the number of receptors was about 7.1×10⁴/cell. The Hill coefficient was 0.99, approaching 1, indicating the absence of cooperativity. The receptor was specific both for rat and human transferrin. The binding of rat [¹²⁵I]transferrin could be competitively and specifically inhibited by unlabeled iron-saturated rat and human transferrin, and no difference was found between interaction of rat or human transferrin with this receptor. The interaction of duck or camel transferrin with this receptor was found to be very weak. This study provides evidence for the presence of transferrin receptor on the plasma membrane of cultured rat brain astrocytes. Received: 25 May 1999 / Accepted: 18 August 1999     

Kinetics of Receptor-Mediated Uptake and Processing of Interferon-α2a and Tumor Necrosis Factor-α by Human Tumor Cells

Abstract

The kinetics of uptake and processing of recombinant human interferon-a₂a (IFN) and recombinant human tumor necrosis factor-a (TNF) were studied in human epithelial rumor cell lines differing in sensitivity to growth inhibition by IFN and TNF. Concentrations of [¹²⁵I]IFN or [¹²⁵I]TNF at the cell surface and internalized by confluent cell monolayers incubated at 37°C were measured as a function of time. Cells incubated with [¹²⁵I]IFN exhibited transient maxima of surface-associated and internalized [^I25I]IFN after 1-2 hr of incubation followed by a steady-state attained after approximately 6 hr of exposure to [¹²⁵I]IFN. Concentration of [¹²⁵I]TNF associated with the plasma membrane displayed a maximum within 20 min of incubation. Internalized radioactivity increased with time and did not achieve a steady state. We analyzed the time-dependent concentrations of membrane-associated and internalized cytokines by use of a compartmental model. This model describes changes in concentrations of free surface receptors, of ligand-receptor complex at the membrane and of internalized ligand and contains a term for receptor recycling. The rate constants for concentration changes were evaluated by fitting model functions to data. The best fits for IFN were obtained without receptor recycling. The best-fit values for the endo-cytotic rate constant (k_tlFN) varied among cell lines from 2.4x10^?4 to 7.8 x10^?4 sec^?1. To obtain fits to time-dependent concentrations of surface-associated and internalized [¹²⁵I]TNF, introduction of a term for receptor recycling was required. Best-fit values for k, _TNF ranged among cell lines from 8.4x10^?4 to 2.5x10^?3 sec^?1. For every cell line, the value of k_lTNF was larger than the value of k_clFN. We tested the significance of these differences by substituting K.IFN ^tor K.TNT ^as fixed parameters in fits to data for TNF and v.v., respectively. Under these conditions, fits were significantly worse. Our data indicate that recycling is insignificant in kinetics of Type-I IFN receptor; recycling is necessary to explain the kinetics of TNF receptor. Upon binding, TNF is taken up by cells faster than IFN and is eliminated from cells more slowly than IFN.     

Insulin receptors along the rat nephron: [125I] Insulin binding in microdissected glomeruli and tubules

Binding of [¹²⁵I] Tyr A¹⁴ human insulin ([¹²⁵I] insulin) was measured at 4°C in glomeruli and pieces of tubule microdissected from collagenase-treated rat kidneys. For glomeruli and all segments tested, total and non specific binding increased linearly with glomeruli number or tubular length. When determined with 4.0 nM labelled hormone, the distribution of specific binding sites (expressed as 10^–18 mol [¹²⁵I] insulin bound per glomerulus or mm tubule length) was as follows: glomerulus, 2.5±0.3; proximal convoluted tubule (PCT), 12.6±0.6; pars recta (PR), 4.0±2.3; thin descending limb (TDL), 0.6±0.2; thin ascending limb (TAL), 0.6±0.2; medullary thick ascending limb (MAL), 0.8±0.1; cortical ascending limb (CAL), 2.1±0.1; distal convoluted tubule (DCT), 5.6±1.1; cortical collecting tubule (CCT), 3.2±0.3 and outer medullary collecting tubule (MCT), 2.3±0.1. Specific [¹²⁵I] insulin binding to glomeruli and tubule segments was time- and dose-dependent, saturable, reversible after elimination of free labelled ligand, and inhibited by unlabelled human insulin. When analysed in Scatchard and Hill coordinates, the binding data revealed a negative cooperation in the interaction processes between [¹²⁵I] insulin and glomerular and tubular binding sites, with apparent dissociation constants and Hill coefficients of the following values: glomerulus, 0.6 nM and 0.60; PCT, 10.0 nM and 0.55; MAL, 4.3 nM and 0.80; CAL, 2.0 nM and 0.74; CCT, 7.6 nM and 0.80 and MCT, 1.0 nM and 0.57 respectively. The stereospecificity of nephron binding sites was assessed in competitive experiments showing that unlabelled bovine and procine insulins were as efficient as human insulin for displacing [¹²⁵I] insulin, whereas A and B chains of insulin and unrelated peptide hormones were almost inactive. These results indicate that the detected [¹²⁵I] insulin binding sites may correspond to physiological insulin receptors.Abbreviations used [¹²⁵I] Insulin [¹²⁵I] Tyr A¹⁴ human insulin - PCT proximal convoluted tubule - PR pars recta - TDL thin descending limb - TAL thin ascending limb - MAL medullary thick ascending limb - CAL cortical ascending limb - DCT distal convoluted tubule - CCT cortical collecting tubule - MCT outer medullary collecting tubule     

鸟类及鼠类的胸腺、腔上囊及脾脏褪黑素受体的鉴定及意义

应用放射配体结合法检测大鼠、小鼠、仓鼠、鸡、鸭、鸽子及鹌鹑的胸腺、腔上囊及脾脏的[~(125)I]褪黑素(M)特异结合位点。结果显示鸟类及鼠类免疫器官均存在[~(125)I]M特异结合位点,并具低结合容量、高亲和力特点;动力学研究表明具可饱和性及可逆性,符合受体的结合及解离过程;特异性研究显示对M及激动剂有高度特异性,符合特异结合位点的全部条件,亚细胞分布的研究表明以细胞核含量最高,线粒体次之,且该结合位点具年龄依赖性降低。结果证实免疫器官存在根黑素受体(MR),免疫组织是M作用的靶器官,M对免疫系统的调节作用是通过免疫组织上MR直接作用的结果。     

[3H]Adenosine binding to rat mast cells — pharmacologic and functional characterization

Rat serosal mast cell adenosine receptors were characterized by [³H]adenosine binding to cell membrane particulates, and functional changes in mast cell mediator release and cyclic AMP levels were assessed, utilizing various adenosine analogs. [³H]adenosine binding to sonicated mast cell membrane preparations at 0°C in the presence of deoxycoformycin is linear with initial cell number, rapid and reversible. The cells display 16,400±1600 high affinity [³H]adenosine binding sites/cell, equivalent to 118 fmol bound/mg protein, with an equilibrium dissociation constant of 27.97±3.0 nM. Competition studies reveal that adenosine>2-chloroadenosine>NECA>l-PIA>d-PIA in competing for [³H]adenosine binding sites and that aminophylline and cromolyn sodium also bind to the putative receptor. Adenosine and its analogs, NECA, andl-PIA, appear to activate adenylate cyclase in resting mast cells by raising cyclic AMP, suggesting anR _a cell surface adenosine receptor subtype; these same analogs potentiate mast cellb-hexosaminidase release stimulated by specific antigen. The identification of rat mast cell [³H]adenosine binding sites whose stimulation augments resting cell cyclic AMP levels and antigen-induced mediator release suggests that these receptors may be important in the biochemical mechanisms of allergic diseases. The ability to assess the number and affinity of mast cell adenosine receptors will enable one to monitor receptor alterations during pharmacologic manipulation and in disease states.     

Characterization of subtypes of gamma-aminobutyric acid receptors in an Ascaris muscle preparation by binding assay and binding of PF1022A,a new anthelmintic,on the receptors

 We examined the effect of PF1022A, one of the gabergic anthelmintics newly developed in Japan, on gamma-aminobutyric acid (GABA) receptors using a radioligand binding technique in isolated membrane preparations of the nematode Ascaris suum. Membrane protein was prepared from the homogenate of somatic muscle cells after ultracentrifugation. In addition to the basic binding of [2,3-³H-(N)]-GABA, the radioligand [methyl-³H]-bicuculline is used to identify the GABA_A receptor, whereas [butyl-4-³H]-baclofen is employed for GABA_B receptor sites. The dissociation constants (K _d values) and the maximal numbers of binding sites (B_max values) from Scatchard plotting for GABA receptors are close to those obtained in mammalian brain. PF1022A displaced in a concentration-dependent way the binding of [2,3-³H(N)]-GABA and [methyl-³H]-bicuculline as did other specific gabergic agents. In addition, PF1022A decreased the binding of [butyl-4-³H]-baclofen at a higher concentration, although this binding did not represent GABA_B sites. In a comparison of the inhibition constants (K_i values) of PF1022A with those of other agents, it is conclusive that PF1022A bound with GABA receptors. A direct effect of PF1022A on GABA receptors can thus be postulated. Received: 10 March 1995 / Accepted: 27 June 1995     

Evidence for heterogenous glycine domains but conserved multiple states of the excitatory amino acid recognition site of the NMDA receptor: regional binding studies with [3H]glycine and [3H]L-glutamate

Summary The possible heterogeneity of the agonist and glycine sites of the N-methyl-D-aspartate (NMDA) receptor-complex was examined using receptor binding techniques. Binding of [³H]L-glutamate ([³H]GLU) and [³H]glycine to synaptic membranes of cerebral and cerebellar cortices, and membranes of a granule cell preparation of rat cerebellum, was characterized. [³H]Glycine always labelled a single population of sites; densities of binding sites (B_max) in cortical, cerebellar and granule membranes were 3.1, 0.87 and 3.6 pmol/mg protein, respectively. Dissociation constants (K_d) in the same three preparations were 0.13, 0.31 and 1.9 M, respectively. In competition studies, D-cycloserine, but not D-serine and 7-chlorokynurenate, showed varying potency between the membrane preparations, and analysis of variance (ANOVA) revealed a significant interaction between ligands and membrane fractions. Binding of [³H]GLU was saturable and to a single population of sites: K_d 0.5–0.9 M and B_max 3.2–3.6 pmol/mg protein. In all three membrane preparations the rank order of potency of NMDA agonists as inhibitors of the binding of [³H]GLU was always L-aspartate>L-cysteate>L-cysteinesulphinate>L-serine-O-sulphate>ibotenate>L-homocysteate. NMDA, quinolinate and competitive NMDA antagonists were only weak inhibitors of the binding of [³H]GLU and never fully inhibited specific binding. Other subtype-selective excitatory amino acids were very weak or ineffective inhibitors of binding. Binding of NMDA agonists was better described by a two site model whereby the proportion of high affinity sites did not vary significantly across the three membrane preparations. Although the binding of [³H]GLU was relatively insensitive to NMDA itself and competitive NMDA antagonists, binding may be to a recognition site for NMDA-like agonists, since they fully inhibited specific binding. This excitatory amino acid recognition for NMDA agonists was conserved in the three membrane preparations. In cortical and granule membranes the B_max values for the binding of [³H]GLU and [³H]glycine had a stoichiometry of 1:1, whilst in cerebellar synaptic membranes this ratio was 4:1. Receptor autoradiography of NMDA-related [³H]GLU and [³H]glycine binding in tissue sections failed to reveal any differential labelling patterns in cerebral cortex and cerebellum. In the cerebellum, densities of silver grains found with both [³H]ligands were concentrated in the granule cell layer relative to the molecular layer, but the differences detected in membrane binding studies were not observed in cerebellum. Our findings suggest the existence of three types of heterogeneity for the glycine domain of the NMDA receptor: (1) differing affinities for glycine, (2) differing pharmacological profiles, and (3) differing stoichiometry in relation to the putative NMDA-like agonist site. Our evidence supports an hypothesis for the existence of multiple glycine domains which might differentially modulate NMDA-mediated neurotransmission.