Characterization of [3H]Vesamicol binding in rat brain preparations

The binding of (1)-[³H]vesamicol was characterized in several subcellular fractions and brain regions of the rat. Binding to a lysed P₂ fraction from the rat cerebral cortex reached equilibrium within 4 min at 37°C and was reversible (dissociation half-time 4.9 min). At least two binding affinities were found in P₂ fractions from the cerebral cortex (K_d:21 nM and 980 nM), striatum (K_d:28 nM and 690 nM), and cerebellum (K_d:22 nM and 833 nM). High affinity B_max values were highest in striatum (1.17 pmol/mg protein), followed by cerebellum (0.67 pmol/mg protein), and cerebral cortex (0.38 pmol/mg protein). Low affinity B_max values were highest in cerebellum (5.2 pmol/mg protein), with similar values for cerebral cortex (3.7 pmol/mg protein) and striatum (3.8 pmol/mg protein). High affinity but not low affinity binding in each brain region was stereospecific. Another inhibitor of vesicular ACh-transport also displaced 1-vesamicol binding potently (IC₅₀:17 nM) and efficaciously (over 90%). Both high affinity and low affinity B_max values for [³H]vesamicol-binding were highest in a partially purified synaptic vesicle fraction, followed by puriffied synaptosomes, crude membranes and P₂ fractions. Specific binding was not observed in a mitochondria-enriched fraction. Crude membrane preparations of primary, neuron-enriched whole brain cultures also exhibited high (64 nM) and low affinity (1062 nM) [³H]vesamicol binding. Isoosmotic replaement of 0.18 M KCl in the binding-buffer with NaCl had no effect on binding. These results suggest that at least some high affinity [³H]vesamicol binding in rat brain preparations may be associated with synaptic vesicles, some of which may not be cholinergic in origin.     

Characterization and Subcellular Localization of l-[3H]Carnitine Binding Sites in Rat Brain

Abstract: In the present study, we investigated the existence of a binding site for l -carnitine in the rat brain. In crude synaptic membranes, l -[³H]carnitine bound with relatively high affinity (K_D = 281 nM) and in a saturable manner to a finite number (apparent B_max value = 7.3 pmol/mg of protein) of binding sites. Binding was reversible and dependent on protein concentration, pH, ionic strength, and temperature. Kinetic studies revealed a K_off of 0.018 min^?1 and a K_on of 0.187 × 10^?3 min^?1 nM^?1. Binding was highest in spinal cord, followed by medulla oblongata-pons ≥ corpus striatum ≥ cerebellum = cerebral cortex = hippocampus = hypothalamus = olfactory bulb. l -[³H]Carnitine binding was stereoselective for the l -isomers of carnitine, propionylcarnitine, and acetylcarnitine. The most potent inhibitor of l -[³H]carnitine binding was l -carnitine followed by propionyl-l -carnitine. Acetyl-l -carnitine and isobutyryl-l -carnitine showed an affinity ～500-fold lower than that obtained for l -carnitine. The precursor γ-butyrobetaine had negligible activity at 0.1 mM. l -Carnitine binding to rat crude synaptic membrane preparation was not inhibited by neurotransmitters (GABA, glycine, glutamate, aspartate, acetylcholine, dopamine, norepinephrine, epinephrine, 5-hydroxytryptamine, histamine) at a final concentration of 0.1 mM. In addition, the binding of these neuroactive compounds to their receptors was not influenced by the presence of 0.1 mMl -carnitine. Finally, a subcellular fractionation study showed that synaptic vesicles contained the highest density of l -carnitine membrane binding sites whereas l -carnitine palmitoyltransferase activity was undetectable, thus excluding the possibility of the presence of an active site for carnitine palmitoyltransferase. This finding indicated that the localization of the l -[³H]carnitine binding site should be essentially presynaptic.     

HIGH AFFINITY l-[3H]GLUTAMATE BINDING TO POSTSYNAPTIC RECEPTOR SITES ON RAT CEREBELLAR MEMBRANES

l [³H]glutamate binding was investigated in membrane preparations derived from rat cerebellum, an area of the brain where it is likely that a high density of postsynaptic glutamate receptors occurs. Glutamate was hound specifically and, in freshly prepared membranes, was optimal under physiological conditions of pH and temperature and was associated with the synaptic membrane fraction of the cell. Specific binding occurred through a single, high-affinity process with a K_D, of 744 nM and a capacity of 73 pmol/mg protein. Unlike the findings reported for GABA, the specific binding of glutamate to fresh membranes did not involve an uptake site. Comparison of the potencies of a wide range of compounds with known pharmacological activities, demonstrated that their ability to displace specific glutamate binding was consistent with specific interactions with glutamate receptors.     

Interaction of the synthetic peptide octarphin with rat adrenal cortex membranes

The synthetic peptide octarphin (TPLVTLFK, fragment 12?C19 of ??-endorphin), a selective agonist of the nonopioid ??-endorphin receptor, was labeled with tritium yielding specific activity of 28 Ci/mmol. The binding of [³H]octarphin to rat adrenal cortex membranes was studied under normal conditions as well as after cold and heat shocks. It was found that under normal conditions [³H]octarphin specifically binds to the membranes with high affinity: K _d1 = 36.3 ± 2.5 nM, B_max1 = 41.0 ± 3.8 pmol/mg protein. The specific binding of [³H]octarphin to the membranes was inhibited by unlabeled ??-endorphin (K _i = 33.9 ± 3.6 nM) and the agonist of the non-opioid receptor decapeptide immunorphin (K _i = 36.8 ± 3.3 nM). Unlabeled naloxone, [Leu⁵]- and [Met⁵]enkephalins, ??- and ??-endorphins, and corticotropin were inactive (K _i > 1 ??M). Both cold and heat shocks decreased the binding affinity: K _d2 = 55.6 ± 4.2 nM and K _d3 = 122.7 ± 5.6 nM, respectively. In both cases, the maximal binding capacity of the receptor did not change. Thus, even a short-term thermal shock significantly affects the sensitivity of the non-opioid ??-endorphin receptor of adrenal cortex membranes.     

Characterization of a dopamine-sensitive [3H]LSD binding site in honeybee (Apis mellifera) brain

[³H]Lysergic acid diethylamide ([³H]LSD) binds on membrane homogenate of honeybee brain to both a dopamine-sensitive site (D-site) and a serotonin-sensitive site (S-site). Under suitable conditions the properties of the two sites can be studied separately. Specific binding of [³H]LSD to both the D-site and the S-site has high affinity and is saturable. The mean equilibrium dissociation constants (K_D) were 3.8 nM for the D- and 0.89 nM for the S-site. The densities (B_max values) of both binding sites were 1.7 pmol/mg protein for the D-site and 0.79 pmol/mg protein for the S-site. [³H]LSD binding to the D-site was reversible and reached equilibrium in about 30 min. Pharmacological displacement studies display a high binding affinity of the putative natural agonist dopamine to the D-site (K_i = 22 nM). The most potent displacers of D-site binding were lisuride, (+)-bromocriptine, chlorpromazine, S(+)-butaclamol, and 6,7-ADTN. The [³H]LSD labelled D-site seems to be G-protein coupled, since addition of the stable GTP analogue GTPγS or NaCl to the incubation medium evoked a decrease of specific [³H]LSD binding to the D-site.     

Solubilization and partial purification of the GABA receptor from mouse brain and a binding assay for the solubilized receptor.

Abstract— The GABA receptor from mouse brain was solubilized with lysolecithin. A 56-fold overall purification and activation were achieved by discontinuous sucrose gradient centrifugation and solubilization. Activation of binding by both procedures was observed. The solubilized receptor has the following binding constants: K_D1= 3.5 _nM, K_D2= 52 _nM, B_{max 1}= 2.8 pmol/mg protein and B_{max 2}= 14 pmol/mg protein for muscimol; K_D1= 12 nM, K_D2= 470 nM, B_{max 1}= 1.4 pmol/mg protein and B_{max 2}= 17 pmol/mg protein for GABA. Specific GABA binding was inhibited by imidazoleacetic acid and bicuculline with IC₅₀ values of 250_nM and 1 _μM respectively. A rapid and sensitive filtration binding assay for the solubilized receptor has been developed. Lysolecithin was also found suitable for the solubilization of acetylcholine receptor from T. californica electroplaques.     

Neurotensin receptors in canine intestinal smooth muscle: preparation of plasma membranes and characterization of (Tyr3−125I)-labelled neurotensin binding

To study the binding of (Tyr³−¹²⁵I)-labelled neurotensin to intestinal muscle, plasma membranes have been purified from dog intestinal circular smooth muscle. Purification was done by differential centrifugation followed by separation on a sucrose gradient. Electron microscopic study revealed that the dissected circular muscles used as the source of membranes were free of myenteric plexus and that the plasma membrane fraction obtained was free of any mitochondria or synaptosomes. The fraction used was obtained at the interface of 14%–33% sucrose density on the gradient and was 25-times enriched in the plasma membrane marker enzyme 5′-nucleotidase activity as compared to post-nuclear supernatant. This fraction contained negligible activity of mitochondrial membrane marker enzyme cytochrome c oxidase and low activity of a putative endoplasmic reticulum marker enzyme NADPH-cytochrome-c reductase. This membrane fraction contained a high density of neurotensin binding sites. This binding was studied by kinetic and by saturation approaches. Analysis of data from saturation binding studies by the computer programs (EBDA and LIGAND) suggested the presence of a two-site model (K_d1 = 0.118 nM, K_d2 = 3.18 nM, B_max1 = 9.73 fmol/mg and B_max2 = 129.8 fmol/mg). A part of specifically bound neurotensin was rapidly dissociated. No cooperativity between the two receptor types could be detected. A kinetic analysis of binding gave the K_d value equal to 0.107 nM. Carboxy terminal amino acid residues 8–13 were found to be essential for the binding activity and replacement of Tyr¹¹ by tryptophan reduced the affinity of the peptide by 10 times in displacement studies. Binding was modulated by sodium ions and a guanine nucleotide Gpp[NH]p. MgCl₂, CaCl₂ and KCl were also found to reduce the specific binding. Evidence was found of a high specific binding to another membrane fraction poor in plasma membranes and rich in synaptosomes. We concluded that plasma membrane of canine intestinal circular muscle contains neurotensin receptors with recognition properties distinct from those obtained in previous studies of neurotensin binding sites in murine tissues. Another neurotensin binding site may be present on neuronal membranes.     

Pharmacological characterization of the locust neuronal 3H-mianserin binding site,a putative histamine receptor

1. The ³H-mianserin binding protein in the nervous tissue of the locust Locusta migratoria reveals properties that are characteristic for ligand receptor interactions.2. ³H-mianserin binds with high affinity (K₁ = 7.05 nM) to its single binding site (B_max = 1.53 pmol/mg protein). This binding site shows pharmacological properties similar to those of vertebrate histamine H₁-receptors. All tested histamine H₁-antagonists, and sole H₁-antagonists, are able to displace ³H-rnianserin binding even at low concentrations.3. Quantitative comparison of the pharmacological properties of the locust ³H-mianserin binding site with human and rat H₁-receptors revealed striking similarities (r² of locust/rat = 0.793, of locust/human 0.852).4. The ³H-mianserin binding site shows affinity decrease if the incubation medium is depleted of Mg²⁺-ions. This decrease is characteristic for G-protein coupled receptors.     

Endogenous inhibitor of GABA binding in mammalian brain

Binding of radioactive gamma-aminobutyric acid to homogenates of mammalian brain was detected by a centrifugation assay. The binding capacity of the tissue was maximal and stable with time only if the tissue was thoroughly washed to remove an endogenous inhibitor of binding. With such washed tissue, binding to total rat or cow brain appeared to involve two populations of sites in the absence of sodium ions, the major site having a dissociation constant of 150 nM and saturating at 80 pmol/g brain, and a minor site with a K_D of 20 nM and saturating at 20 pmol/g wet tissue. This sodium-independent GABA binding as a whole was localized in the crude mitochondrial, microsomal, and synaptosomal membrane fractions.     

Binding of octopamine to membranes from the white-eyed and wild-type housefly heads

In attempting to develop an octopamine (OA) receptor preparation with ready access to large amounts of tissue, we examined the binding of OA to membranes from the heads of white and red houseflies (Musca domestica L.). Binding was dependent on the presence of L-ascorbic acid in the medium. However, equilibrium was reached only over 24–36 h at 4°C and reversal of binding was also slow and incomplete. Scatchard analysis revealed at least two binding sites in the white-eyed housefly. A high-affinity site (K_d = 13.9 nM and B_max = 3.9 pmol/mg protein) was present, but the majority of the binding had low affinity (K_d = 1130 nM and B_max = 165 pmol/mg protein). Scatchard analysis revealed a low affinity in the red-eyed housefly (K_d = 240 nM and B_max = 12 pmol/mg protein). Catecholamines were the best competitors for OA binding followed by phenolamines such as OA and synephrine. 5-Hydroxytryptamine was less effective. Phentolamine and mianserin, which are good antagonists of the ability of OA to stimulate adenylate cyclase in housefly head membranes, and formamidine and imidazolines, which are potent partial agonists of this adenylate cyclase, were poor competitors of OA binding. The slow kinetics, low affinity, large amount, and unconventional pharmacological profile of this binding is not congruent with it being a neuroreceptor. When the brain was dissected free from the head, less than 10% of the total specific binding of OA was found in the brain membrane fraction. This suggests that most of the binding of OA may be to cuticular sites that possibly are associated with the metabolism of catecholamines in cuticular synthesis. Thus, binding studies made with labeled catecholamines and phenolamines on insect tissues containing significant cuticular elements should be interpreted with caution.     

L-Glutamate Receptor Heterogeneity: Labeling of Distinct Receptor Sub-Types Using Radioligand Binding Techniques

Abstract

This study demonstrates (1) that L-[³H]glutamate labels 3 distinct binding sites (types A1, A2 and A4) in isolated rat brain membranes and (2) that only the N-methyl-aspartate (A1) and quisqualate (A2) receptor classes are associated with the postsynaptic density (PSD). L-[³H]glutamate bound to PSDs with K_d 339 nM and B_max 6.1 pmol/mg protein. These sites were resolved into 2 distinct sub-types on the basis of inhibition studies. N-Methyl-aspartate maximally inhibited 57% of PSD-located L-glutamate binding sites (the A1 site) and quisqualate 43% (the A2 site); the effects of both substances were additive. The ligand selectivities of these 2 sites indicated their identity with the N-methyl-D-aspartate and quisqualate receptor classes defined electrophysiologically. The Cl^?-dependent population of L-glutamate binding sites (the A4 site) which predominates in synaptic membranes was absent from PSDs.     

Binding of [3H]batrachotoxinin A-20-α-benzoate to a high affinity site associated with house fly head membranes

1. [³H]Batrachotoxinin A-20-α-benzoate (BTX-B), a radioligand that labels the alkaloid activator recognition site of the voltage-sensitive sodium channel, was bound specifically to high affinity, saturable sites in a subcellular preparation from house fly (Musca domestica L.) heads that was shown previously to contain binding sites for other sodium channel-directed ligands.2. Specific binding of [³H]BTX-B was observed in the presence of 140 mM sodium or potassium and was inhibited by choline ion.3. Saturating concentrations of scorpion (Leiurus quinquestriatus) venom stimulated the specific binding of [³H]BTX-B four-fold, increasing the proportion of specific binding of 10 nM [³H]BTX-B from less than 15% to 40%. Equilibrium dissociation studies in the presence of scorpion venom gave an equilibrium dissociation constant (K_D) for [³H]BTX-B of 80 nM and a maximal binding capacity (B_max) of 1.5 pmol/mg protein.4. Parallel experiments in the absence of venom gave a K_D value of 140 nM and a B_max of 1.3 pmol/mg protein, indicating that scorpion venom stimulated [³H]BTX-B binding by increasing the affinity of this site approximately two-fold.5. The specific binding of [³H]BTX-B was inhibited by the sodium channel activators aconitine and batrachotoxin and, to a lesser extent, by the anticonvulsant diphenylhydantoin. However, several other sodium channel-directed neurotoxins known to exert allosteric effects on the binding of [³H]BTX-B to mammalian brain preparations did not affect the binding of [³H]BTX-B to house fly head membranes.6. These studies provide evidence for a high affinity binding site in house fly head membrane preparations that exhibits properties expected of the activator recognition site of the voltage-sensitive sodium channel but does not respond to several compounds known to modify allosterically the binding of [³H]BTX-B to sodium channels in mammalian brain.     

Different specific binding sites of [3H]glycine and [3H]strychnine in synaptosomal membranes isolated from frog retina

Synaptosomal fractions were isolated from frog retina: a fraction enriched in photoreceptor terminals (P1) and a second one (P2) containing interneurons terminals. We compared the binding of [³H]glycine and [³H]strychine to membranes of these synaptosomes. The binding of both radioactive ligands was saturable and Na⁺-independent. [³H]Glycine bound to a single site in P1 and P2 synaptosomal fractions, with K_D=12 and 82 nM and B_Max=3.1 and 3.06 pmol/mg protein respectively. [³H]Strychnine bound to two sites in each one of the synaptosomal fractions. For P1 K_D values were 3.9 and 18.7 nM, and B_Max values were 1.1 and 7.1 pmol/mg protein, respecitively. Membranes from the P2 synaptosomal fraction showed K_D's of 0.6 and 48 nM and B_Max's of 0.4 and 4.5 pmol/mg. Specific [³H]glycine binding was displaced by -alanine, l-serine, d-serine and HA966, but not by strychnine 7-chlorokynurenic or 5,7-dichloro-kynurenic acids. Specific [³H]strychnine, binding was partially displaced by glycine and related aminoacids and totally displaced only by 2-NH₂-strychnine. Our results indicate the presence of high affinity binding sites for glycine and strychnine in frog retinal synaptosomal membranes. The pharmacological binding pattern indicates the presence of the strychnine sensitive glycine receptor as well as other sites. These might not include the NMDA receptor-associated glycine site.     

Localization and pharmacological characterization of nicotinic-cholinergic binding sites in cockroach brain using α- and neuronal bungarotoxin

[¹²⁵I]α-Bungarotoxinisusedasaprobetostudythenicotinic-cholinergicreceptorinmembrane preparations of the cockroach brain. Binding is restricted mainly to particulate fractions of brain homogenates, is time dependent and is saturable above 2 nM with very low non-specific binding. Scatchard analysis indicates that binding is associated with a single affinity site (K_d = 1.09 nM) having a B_max of 8926 fmol/mg protein which is the highest concentration of binding sites yet reported in insects. Association kinetics are best fit by a mono-exponential model with a k_obs = 4.37 × 10⁻³s⁻¹. Dissociation is best described by a bi-exponential model giving dissociation constants of 1.18 × 10⁻⁵ and 9.94 × 10⁻⁵s⁻¹. The K_ds calculated from kinetic data are 0.029 and 0.25 nM suggesting the possibility of heterogeneous binding sites not detected by saturation studies. Displacement studies indicate that binding follows a nicotinic pharmacology and demonstrate the high affinity of methyllycaconitine and the anthelmintics, morantel and pyrantel. Displacement by neuronal bungarotoxin shows the presence of two distinct binding sites not differentiated by α-bungarotoxin. Autoradiographic studies show α-bungarotoxin to be binding to neuropile regions of the brain, to be displaced from these regions by agents effective in binding studies and demonstrate that the neuronal bungarotoxin binding sites can be regionally localized.     

[3H] 5,7-Dichlorokynurenic Acid,a High Affinity Ligand for the NMDA Receptor Glycine Regulatory Site

Abstract

The NMDA subtype of glutamate receptors is allosterically linked to a strychnine-insensitive glycine regulatory site. Kynurenic acid and its halogenated derivatives are non-competitive NMDA antagonists acting at the glycine site. We have prepared [³H] 5,7-dichlorokyrurenic acid (DCKA) as an antagonist radioligand and have characterized its binding. 3-Bromo-5,7-DCKA was catalytically dehalogenated in the presence of tritium gas and HPLC purified to yield [³H] 5,7-DCKA with a specific activity of 17.6 Ci/mmol. [³H] 5,7-DCKA bound to rat brain synaptosomes with a K_d of 69 ± 23 nM and B_max = 14.5 ± 3.2 pmoles/mg protein. Binding was 65–70% specific at 10 nM [³H] 5,7-DCKA. This ligand is thus more selective and has higher affinity than [³H] glycine, in addition to being an antagonist.     

Kinetic and Equilibrium Studies of (-)125Iodocyanopindolol Binding to β-Adrenoceptors on Human Lymphocytes: Evidence for the Existence of two Classes of Binding Sites

Abstract

Saturation experiments were performed on intact human peripheral mononuclear leucocytes (MNL) and MNL membranes with (-)¹²⁵Iodocyanopindolol (¹²⁵ICYP) over a large concentration range (1.5-600pmol/l). The corresponding Scatchard plots were curvilinear suggesting two saturable classes of binding sites: A high affinity binding site (B_max1=1000±400 sites/cell, K_d1= 2.1±0.9 pmol/l for intact MNL and B_max1=550±190 sites/cell, K_d1=4.1±0.9 pmol/l for MNL membranes)and a low affinity binding site (B_max2=9150±3590 binding sites/cell, K_d2=440±50 pmol/l for intact MNL and B_max2=11560±4690 sites/cell, K_d2=410±70 pmol/l for MNL membranes). Dissociation of (-)¹²⁵ICYP from MNL was biphasic consisting of a slow dissociating component (dissociation rate constant k_-1=(0.5±0.2)x10^?3 min^?1 for intact MNL and k_-1=(1.0±0.1)x10^?3min^?1 for MNL membranes) and a fast dissociating component (k_-2=(80±20)x10^?3min^?1 for intact MNL and k_-2=(60±10)x10^?3min^?1 for MNL membranes). In dissociation experiments started after equilibration with various (-)¹²⁵ICYP concentrations k_-1 and k_-2 were independent of the equilibrium concentration, whereas the percentual occupancy of the slow and the fast dissociating component varied and was similar to the estimated fractional occupancy of either binding site at the same (-)¹²⁵ICYP concentrations in saturation experiments. The association rate constant was in the same order of magnitude for both binding sites. These results suggest two independent classes of binding sites for (-)¹²⁵ICYP on MNL.     

Two classes of arginine vasopressin binding sites on rat brain membranes

Specific binding sites for arginine vasopressin (AVP) were demonstrated on rat brain membranes using [³H]AVP of high specific activity. At pH 7.4 in the presence of 5 mM MgCl₂, one class of sites was measured with aK _D of 0.56 nM and aB _max of 4.3 fmol/mg protein. At pH 8.0 in the presence of MgCl₂, two distinct sites were observed, havingK _D values of 0.42 and 13 nM andB _max values of 5.6 and 68 fmol/mg protein, respectively, and similar results were obtained at pH 7.4 after repeatedly freezing and thawing the membranes. Binding increased with pH, apparently representing increased occupancy of the high capacity, lower affinity site. Binding to the lower affinity site was also enhanced by freezing and thawing membranes, or by adding 5 mM NiCl₂ or 10 M ZnCl₂ to the incubation medium, whereas binding to the high affinity site was dependent on the addition of Mg. AVP was over 35 times more active in displacing 0.4 nM AVP than oxytocin or arginine-vasotocin, and 10,000 times more active than somatostatin. A number of other peptides had no effect on [³H]AVP binding at concentrations up to 10^–5 M. Autoradiography and regional dissection studies revealed a marked concentration of high affinity AVP-binding sites in the supraoptic and paraventricular nuclei of the hypothalamus, and Mg significantly enhanced the binding in these regions.     

Binding of [3H] γ-Aminobutyric Acid and [3H]Muscimol in Purified Rat Brain Synaptic Plasma Membranes and the Effects of Bicuculline

Abstract: The binding of [³H] γ-aminobutyric acid ([³H]GABA) and [³H]muscimol has been studied in purified synaptic plasma membrane (SPM) preparations from rat brain. Scatchard analysis of specific binding (defined as that displaced by 100 μMγ-aminobutyrate) indicated that the binding of both radiolabelled ligands was best described by a two component Langmuir adsorption isotherm. The apparent K_D and B_max values for [³H]GABA at 4°C were K_D1, 20 nM; K_D2,165 nM; B_max1, 0.48 pmol;B_max2, 6.0 pmol. mg^?1; for [³H]muscimol at 4°C they were: K_D1, 1.75 nM; K_D2, 17.5 nM; B_maxl, 0.84 pmol. mg^?1; B_max2, 4.8 pmol.mg^?1; and for [³H]muscimol at 37°C they were: K_D1, 7.0 nM; K_m, 60 nM; B_max], 0.5 pmol-mg^?1; B_max2, 7.2 pmol-mg¹. Under the experimental conditions used, the similar B_milx values for [³H]GABA and [³H]muscimol binding to the SPM preparations suggests that the high- and low-affinity components for the two radiolabeled ligands are identical. The effects of the GAB A antagonist bicuculline on the binding of [³H]muscimol at 4^CC and 37°C were studied. At 4°C, antagonism of muscimol binding appeared to be competitive at the high-affinity site but noncompetitive at the low-affinity site. At 37°C, antagonism was again competitive at the high-affinity site but was of a mixed competitive/noncompetitive nature at the low-affinity site. Assuming that binding to the high-affinity site is associated with the pharmacological actions of bicuculline, the apparent K_D values obtained suggest a pA₂ value of 5.3 against [³H]muscimol at 4°C and 37°C. This figure is in good agreement with several estimates of the potency of bicuculline based on pharmacological measurements. Results from displacement studies using [³H]GABA and [³H]muscimol suggest that [³H]GABA might be a more satisfactory ligand than [³H]muscimol in GABA radioreceptor assays.     

Glomerular thromboxane A2/prostaglandin H2 receptors: characterization and effect of adriamycin-induced nephrotic syndrome

We have characterized the thromboxane (TX) A₂/prostaglandin (PG) H₂ receptor in glomeruli isolated from the rat using the agonist radioligand [¹²⁵I]-BPO. Binding of [¹²⁵]-BOP was highly specific, stereoselective, and to a single class of high affinity binding sites (K_d = 1/16 ± 0.22 nM and B_max = 348 ± 32 fol/mg protein; n = 6). Binding of [¹²⁵I]-BOP was competed for by the agonist ONO11113 (K_d = 50.8 ± 8.0 nM; n = 4) and the antagonists SQ29548 (K_d = 15.8 ± 1.0 nM; n = 3), L657925 (K_d = 12.1 ± 2.2 nM; n = 3) and L65796 (K_d = 1642 ± 135 nM; n = 3). I-BOP also produced a TXA₂/PGH₂ receptor-mediated rise in [CA²⁺]_i in isolated glomeruli In adriamycin-induced nephrotic syndrome in the rat, the development of proteinuria is reported to be dependent on increased renal TXA₂ production. We therefore examined whether or not changes in glomerular TXA₂/PGH₂ receptors occur between control and nephrotic rats. No changes in expression of affinity of either glomerular or platelet TXA₂/PGH₂ receptors were observed. K_d and B_max values for isolated isolated glomeruli were 1.45 ± 0.24 nM and 406 ± 72 fmol/gm for controls and 1.22 ± 0.25 nM and 321 ± 62 fmol/gm for nephrotic rats (n = 6).     

Leukotriene C4 ([3H] - LTC4) binding to membranes isolated from a hamster smooth muscle cell line (DDTIMF2)

Leukotriene C₄ (LTC₄) has been demonstrated to induce contraction of the smooth muscle cell line DDTIMF2. A partially purified membrane fraction obtained from these cells exhibited a high affinity binding site for LTC₄. Binding of [³H]-LTC₄ was saturable, specific and reversible with a dissociation constant (K_d) of 21 ± 4 nM. The maximum number of binding sites (B_max) was 55 ± 5 pmol/mg of protein. Specificity was demonstrated in competition studies in which the Ki of LTC₄ against specifically bound [³H] - LTC₄ was 12 nM whereas Leukotriene D₄ (LTD₄) and Leukotriene E₄ (LTE₄) had a Ki of 38 ± 4 and 4.7 ± 0.5 nM respectively. A previously described antagonist of leukotriene-induced smooth muscle contraction PFL 55712 had a K_i of 23 ± 2 nM as determined by competition binding experiments.