[3H]Thienylcyclohexylpiperidine Binding Activity in Brain Synaptic Membranes Treated with Triton X-100

Binding activity of [3H]thienylcyclohexylpiperidine was examined using rat brain synaptic membranes treated with Triton X-100. This compound is proposed to be a noncompetitive antagonist for the N-methyl-D-aspartate (NMDA)-sensitive subclass of brain excitatory amino acid receptors. The activity decreased in proportion to increasing concentrations of the detergent up to 0.08%. In vitro addition of L-glutamate (Glu) partially restored the decreased activity caused by this Triton treatment, whereas further addition of glycine (Gly) entirely reversed the loss of activity to the level found in membranes extensively washed but not treated with a detergent. These stimulatory effects were found to be due to the acceleration of the association of ligand. The rank order of potentiation of the activity coincided well with that of the affinity for the NMDA-sensitive subclass among numerous Glu analogs. The potentiation by Gly as well as Glu was invariably prevented by competitive NMDA antagonists, such as DL-2-amino-5-phosphonovalerate and (+/-)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonate, but not by strychnine. No significant difference was observed between pharmacological profiles of the activities in synaptic membranes treated and not treated with Triton X-100, except haloperidol. The potency of this sigma-ligand to inhibit the activity was greatly reduced by the Triton treatment in the presence of both Glu and Gly. These results suggest that the regulatory properties of Triton-treated synaptic membranes remain unchanged in terms of the interaction within the NMDA receptor complex.     

Inhibitory Modulation by Sodium Ions of the N-Methyl-D-Aspartate Recognition Site in Brain Synaptic Membranes

Specific binding of radiolabeled L-glutamic acid (Glu) was examined using rat brain synaptic membranes treated with a low concentration of Triton X-100. The binding drastically increased in proportion to increasing concentrations of the detergent used up to 0.1%. Addition of 100 mM sodium acetate significantly potentiated the binding in membranes not treated with Triton X-100, whereas it markedly inhibited the binding in Triton-treated membranes. The binding in Triton-treated membranes was inversely dependent on incubation temperature and reached a plateau within 10 min after the initiation of incubation at 2 degrees C, whereas the time required to attain equilibrium at 30 degrees C was less than 1 min. Sodium acetate invariably inhibited the binding detected at both temperatures independently of the incubation time via decreasing the affinity for the ligand. The binding was significantly displaced by agonists and antagonists for an N-methyl-D-aspartate (NMDA)-sensitive subclass of brain excitatory amino acid receptors, but not by those for the other subclasses. Inclusion of sodium acetate reduced the potencies of NMDA agonists to displace the binding without virtually affecting those of NMDA antagonists. Moreover, sodium ions inhibited the ability of Glu to potentiate the binding of N-[3H] [1-(2-thienyl)cyclohexyl]piperidine to open NMDA channels in Triton-treated membranes. These results suggest that sodium ions may play an additional modulatory role in the termination process of neurotransmission mediated by excitatory amino acids via facilitating a transformation of the NMDA recognition site from a state with high affinity for agonists to a state with low affinity.     

Abolition of the NMDA-mediated responses by a specific glycine antagonist, 6,7-dichloroquinoxaline-2,3-dione (DCQX)

Among various quinoxaline derivatives examined, only 6,7-dichloroquinoxaline-2,3-dione (DCQX) competitively displaced the strychnine-insensitive binding of [3H]glycine, without affecting the other binding sites on the N-methyl-D-aspartate (NMDA) receptor complex. This novel specific antagonist abolished the ability of L-glutamate to potentiate [3H]MK-801 binding activity in brain synaptic membranes treated with Triton X-100. Inclusion of glycine reversed this preventive action of DCQX on the potentiation induced by glutamate.     

Interaction of Strychnine-Insensitive Glycine Binding with MK-801 Binding in Brain Synaptic Membranes

Strychnine-insensitive [3H]glycine binding was detected in brain synaptic membranes treated with Triton X-100 using a filtration assay method. The binding was a time-dependent, inversely temperature-dependent, and reversible process with a relatively high affinity for the neuroactive amino acid. Scatchard analysis revealed that Triton treatment doubled both the affinity and density of the binding sites, which consisted of a single component. The binding was not only displaced by structurally-related amino acid such as D-serine and D-alanine, but also inhibited by some peptides containing glycine, including glycine methylester and N-methylglycine. These ligands invariably potentiated the binding of [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]- cyclohepten-5,10-imine ([3H]MK-801), a noncompetitive antagonist for the N-methyl-D-aspartate-sensitive subclass of the central excitatory amino acid receptors, in a concentration-dependent manner. Among various endogenous tryptophan metabolites, kynurenic acid significantly inhibited the strychnine-insensitive [3H]glycine binding. The Triton treatment did not affect the pharmacological profile of [3H]MK-801 binding sites. These results suggest that brain synaptic membranes treated with Triton X-100 are useful in evaluating the strychnine-insensitive and kynurenate-sensitive binding sites of glycine, which are functionally linked to N-methyl-D-aspartate- sensitive receptor channels.     

Solubilization, Characterization, and Partial Purification of α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid-, Quisqualate-, Kainate-Sensitive l-Glutamate Binding Sites from Porcine Brain Synaptic Junctions

L-[3H]Glutamate binding sites were solubilized from porcine brain synaptic junctions by Triton X-114 in the presence of KCl. The solubilized binding sites bound L-[3H]glutamate reversibly with KD and Bmax values of 1.48 +/- 0.18 microM and 178.2 +/- 15.9 pmol/mg of protein, respectively. These binding sites appeared to be integral membrane glycoproteins, with sugar moieties recognized by wheat germ agglutinin. A 49.3-fold purification of these binding sites was achieved by Triton X-114 solubilization, anion-exchange chromatography, and affinity chromatography using wheat germ agglutinin-Sepharose. The apparent molecular mass of the partially purified binding sites was 620 +/- 50 kDa. L-[3H]Glutamate bound to the solubilized preparation could be effectively displaced by agonists of non-N-methyl-D-aspartate (NMDA) L-glutamate receptors but not by NMDA or alpha-amino-4-phosphonobutyrate. The rank order for the competitive ligands in displacing L-[3H]glutamate was: quisqualate greater than alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid greater than L-glutamate greater than kainate.     

Further Evidence for Multiple Forms of an N-Methyl-d-Aspartate Recognition Domain in Rat Brain Using Membrane Binding Techniques

Abstract— Pretreatment with sulfhydryl-reactive agents, such as N-ethylmaleimide and p-chloromercuriphenylsul-fonic acid, invariably resulted in marked inhibition of the binding of dl -(E)-2-amino-4-[³H]propyl-5-phosphono-3-pentenoic acid ([³H]CGP 39653), a competitive antagonist at an N-methyl-d -aspartate (NMDA)-sensitive subclass of central excitatory amino acid receptors, in brain synaptic membranes extensively washed and treated with Triton X-100, but did not significantly affect the binding of L-[³H]-glutamic acid ([³H]Glu), an endogenous agonist. The pre-treatment was effective in reducing the binding of [³H]-CGP 39653 at equilibrium, without altering the initial association rate, and decreased the affinity for the ligand. Pretreatment with sulfhydryl-reactive agents also enhanced the potencies of NMDA agonists to displace [³H]-CGP 39653 binding and attenuated those of NMDA antagonists, but had little effect on the potencies of the agonists and antagonists to displace [³H]Glu binding. The binding of both [³H]CGP 39653 and [³H]Glu was similarly sensitive to pretreatment with four different proteases in Tritontreated membranes, whereas pretreatment with phospho-lipase A₂ or C markedly inhibited [³H]CGP 39653 binding without altering [³H]Glu binding. Moreover, both phospho-lipases not only induced enhancement of the abilities of NMDA agonists to displace the binding of [³H]CGP 39653 and [³H]Glu, but also caused diminution of those of NMDA antagonists. These results suggest that both sulfhydryl-reactive agents and phospholipases may predominantly interfere with radiolabeling of the NMDA recognition domain in a state favorable to an antagonist by [³H]CGP 39653, with concomitant facilitation of that in an agonist-preferring form by [³H]Glu. The possible presence of multiple forms of the NMDA recognition domain is further supported by these data.     

N-methyl-D-aspartate-sensitive [3H]glutamate binding sites in brain synaptic membranes treated with Triton X-100

Specific binding activity of radiolabeled L-glutamic acid, a putative central excitatory neutrotransmitter, was drastically increased with increasing concentrations of Triton X-100 used for pretreatment of rat brain synaptic membranes. The binding in these Triton-treated membranes was a protein dependent, inversely temperature-dependent, stereospecific, structure-selective and saturable process with a high affinity for the amino acid. The binding activity was invariably inhibited by agonists and antagonists for the N-methyl-D-aspartic acid (NMDA)-sensitive subclass, but not by agonists for the other subclasses of excitatory amino acid neurotransmitter receptors in the brain. Scatchard analysis revealed that the binding sites consisted of a single component with a Kd of 24.4 +/- 2.5 nM and a Bmax of 0.94 +/- 0.09 pmol/mg protein. Some endogenous tryptophan metabolites such as kynurenic acid and quinolinic acid also inhibited the binding. These results suggest that synaptic membranes may indeed contain the NMDA-sensitive receptors which are disclosed by Triton X-100 treatment.     

Characterization of γ-Aminobutyric Acid Binding Sites on Crude Synaptic Membranes

[3H]GABA binding to crude synaptic membranes of rat brain was studied in an attempt to identify GABA binding to its synaptic receptor in the presence of Na+. Membrane vesicles prepared from crude synaptic membrane fractions were useful as a tool to differentiate synaptic GABA receptors from GABA uptake sites. The crude synaptic membranes treated with Triton X-100 [membranes (TX)] involved two classes of GABA binding sites (KD = 38.7 and 78.0 nM) in the absence of Na+, but the high-affinity sites disappeared in the presence of Na+ and a single class of GABA binding sites (KD = 75.0 nM) was detected. The failure to detect an active uptake of [3H]GABA into the vesicles prepared from membranes (TX) suggests that the [3H]GABA binding in the presence of Na+ was related to synaptic GABA receptors. It is probable that Na+ could mask the presence of the high-affinity class of GABA receptor.     

6,7-Dichloroquinoxaline-2,3-Dione is a Competitive Antagonist Specific to Strychnine-Insensitive [3H]Glycine Binding Sites on the N-Methyl-D-Aspartate Receptor Complex

Multiple binding sites on the N-methyl-D-aspartate (NMDA) receptor complex were examined using rat brain synaptic membranes treated with Triton X-100. Binding of [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imi ne ([3H]MK-801), a noncompetitive NMDA antagonist, in the presence of 10 microM L-glutamate not only was inhibited by different types of antagonists, such as 6,7-dichloro-3-hydroxy-2-quinoxaline-carboxylate, 7-chlorokynurenate, and 6,7-dichloroquinoxaline-2,3-dione (DCQX), but also was abolished by non-NMDA antagonists, including 6-cyano-7-nitroquinoxaline-2,3-dione and 6,7-dinitroquinoxaline-2,3-dione. The inhibition of [3H]MK-801 binding by these compounds was invariably reversed or attenuated by addition of 10 microM glycine. Among these novel antagonists with an inhibitory potency on [3H]MK-801 binding, only DCQX abolished [3H]glycine binding without inhibiting [3H]glutamate and [3H](+-)-3-(2-carboxypiperazine-4-yl)propyl-1-phosphonate bindings. Other antagonists examined were all effective as displacers of the latter two bindings. These results suggest that DCQX is an antagonist highly selective to the strychnine-insensitive glycine binding sites with a relatively high affinity.     

Inhibition by Calmodulin Antagonists of [3H]MK-801 Binding in Brain Synaptic Membranes

In brain synaptic membranes not extensively washed, (+)-5-[3H]methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5, 10-imine ([3H]MK-801) binding was markedly inhibited in a concentration-dependent manner (at concentrations above 1 microM) by several compounds having antagonistic activity at the Ca(2+)-binding protein calmodulin. Scatchard analysis revealed that N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibited the binding through a significant decrease in the density of binding sites without affecting the affinity at 10 microM. In membranes extensively washed and treated with a low concentration of Triton X-100, L-glutamic acid (Glu) drastically accelerated the initial association rate of [3H]MK-801 binding with glycine (Gly), almost doubling the initial association rate found in the presence of Glu alone. The addition of W-7 invariably reduced the initial association rate observed in the presence of either Glu alone or both Glu and Gly, without significantly altering the dissociation rate of bound [3H]-MK-801, irrespective of the presence of the two stimulatory amino acids. The maximal potencies of Glu, Gly, and spermidine in potentiating the binding were all attenuated by W-7. These results suggest that calmodulin antagonists may interfere with opening processes of an ion channel associated with an N-methyl-D-aspartate-sensitive subclass of excitatory amino acid receptors in rat brain.     

Endogenous inhibitors of Na+-independent [3H]GABA binding to crude synaptic membranes

The characteristics of the Na⁺-independent high-affinity binding of [³H]GABA to various types of crude synaptic membranes (CSM) prepared from rat brain cortex were studied. In freshly prepared CSM the content of GABA was so high that the high-affinity [³H]GABA binding could not be determined. In contrast when the frozen-thawed CSM were incubated at 37° for 30 min with or without Triton X-100 or phospholipase C and then washed repeatedly, there was a virtual disappearance of GABA from the supernatant extracts and the binding constants of [³H]GABA to CSM could be determined. Two apparent populations of [³H]GABA binding sites, one with a low- and the other with a high-affinity constant, were detected. The ratio of the number of high- to low-affinity binding sites varies with the method used to prepare the membranes. The lowest value of this ratio was observed with membranes incubated at 37° for 30 min. However, when frozen-thawed CSM were treated with 0.05% Triton X-100 repeatedly, the ratio of the number of high- to low-affinity binding sites increased progressively. This increase in ratio is due to a selective increase in the number of the high-affinity sites without significant changes in the number of the low-affinity sites. The extent of the increase in the number of sites that bind [³H]GABA with high affinity after repeated Triton X-100 treatments was paralleled by a decrease of an endogenous protein which inhibits GABA binding. The reapplication of this endogenous material to membranes repeatedly treated with Triton X-100 reduces the number of high-affinity binding sites for [³H]GABA to values similar to those measured in membranes that were not treated with Triton X-100. The inhibitory preparation extracted from CSM incubated with Triton X-100 was shown to be free of GABA or phospholipids. The gel filtration chromatography reveals the presence of two molecular forms of the inhibitor; of these, the high-molecular-weight material fails to bind GABA, whereas the low-molecular-weight material appears to bind GABA. The high-molecular-weight endogenous inhibitor has been termed GABA modulin.     

Inhibition of [3H]MK-801 Binding by Ferrous (II) but Not Ferric (III) Ions in a Manner Different from That by Sodium Nitroprusside (II) in Rat Brain Synaptic Membranes

Abstract: The addition of sodium nitroprusside (SNP) significantly inhibited binding of (+)-5-[³H]methyl-10,11-dihydro-5 H -dibenzo[ a,d ]cyclohepten-5,10-imine ([³H]MK-801) to an ion channel associated with the N -methyl- d -aspartate (NMDA) receptor in a concentration-dependent manner at concentrations of >1 µ M in rat brain synaptic membranes not extensively washed. However, neither S -nitroso- N -acetylpenicillamine nor S -nitroso- l -glutathione inhibited binding even at 100 µ M . Of the two compounds structurally related to SNP (II), similarly potent inhibition was induced by potassium ferrocyanide (II) but not by potassium ferricyanide (III). In addition, ferrous chloride (II) induced much more potent inhibition of binding than ferric chloride (III), at a similar concentration range. In contrast, iron chelators prevented the inhibition by ferrous chloride (II) without markedly affecting that by SNP (II) and potassium ferrocyanide (II). Pretreatment with ferrous chloride (II) also led to potent inhibition of [³H]MK-801 binding in a manner insensitive to subsequent addition of the iron chelators. Pretreatment with Triton X-100 resulted in significant potentiation of the ability of ferrous chloride (II) to inhibit [³H]MK-801 binding irrespective of the addition of agonists, moreover, although binding of other radioligands to the non-NMDA receptors was unaltered after pretreatment first with Triton X-100 and then with ferrous chloride (II). These results suggest that ferrous ions (II) may interfere selectively with opening processes of the NMDA channel through mechanisms entirely different from those underlying the inhibition by both SNP (II) and potassium ferrocyanide (II) in rat brain.     

Support for Radiolabeling of a Glycine Recognition Domain on the N-Methyl-d-Aspartate Receptor Ionophore Complex by 5,7-[3H]Dichlorokynurenate in Rat Brain

Abstract: Pretreatment with Triton X-100 more than doubled the binding of radiolabeled 5,7-dichlorokynurenic acid (DCKA), a proposed antagonist at a glycine (Gly) recognition domain on the N-methyl-d -aspartate (NMDA) receptor ionophore complex, in rat brain synaptic membranes. The binding exhibited an inverse temperature dependency, reversibility, and saturability, the binding sites consisting of a single component with a high affinity (27.5 nM) and a relatively low density (2.87 pmol/mg of protein). The binding of both [³H]DCKA and [³H]Gly was similarly displaced by numerous putative agonists and antagonists at the Gly domain in a concentration-dependent manner at a concentration range of 100 nM to 0.1 mM. Among the 24 putative ligands tested, DCKA was the second most potent displacer of the binding of both radioligands with no intrinsic affinity for the binding of [³H]kainic acid and α-amino-3-hydroxy-5-[³H]methylisoxazole-4-propionic acid (AMPA) to the non-NMDA receptors. In contrast, the other proposed potent Gly antagonist, 5,7-dinitroquinoxaline-2,3-dione, was active in displacing the binding of [³H]glutamic ([³H]Glu) and D,L-(E)-2-amino-4-[³H]propyl-5-phosphono-3-pentenoic acids to the NMDA recognition domain with a relatively high affinity for the non-NMDA receptors. In addition, the proposed antagonist at the AMPA-sensitive receptor, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline, not only displaced weakly the binding of both [³H]- Gly and [³H]DCKA, but also inhibited the binding of (+)-5-[³H]methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine ([³H]MK-801) to an ion channel associated with the NMDA-sensitive receptor in the presence of added Glu alone in a manner sensitive to antagonism by further added Gly. Clear correlations were seen between potencies of the displacers to displace [³H]DCKA binding and [³H]Gly binding, in addition to between the potencies to displace [³H]-DCKA or [³H]Gly binding and to potentiate or inhibit [³H]MK-801 binding. All quinoxalines tested were invariably more potent displacers of [³H]DCKA binding than [³H]Gly binding, whereas kynurenines were similarly effective in displacing the binding of both [³H]Gly and [³H]-DCKA. These results undoubtedly give support to the proposal that [³H]DCKA is one useful radioligand available in terms of its high selectivity and affinity for the Gly domain in the brain. Possible multiplicity of the Gly domain is suggested by the differential pharmacological profiles between the binding of [³H]Gly and [³H]DCKA.     

Effects of ifenprodil on the N-methyl-D-aspartate receptor ionophore complex in rat brain.

The effects of a cerebral anti-ischemic drug ifenprodil on the receptor ionophore complex of an N-methyl-D-aspartate (NMDA)-sensitive subclass of central excitatory amino acid receptors were examined using [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10- imine (MK-801) binding in rat brain synaptic membrane preparations as a biochemical measure. The binding in membrane preparations not extensively washed was markedly inhibited not only by competitive NMDA antagonists such as (+/-)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic, D-2-amino-5-phosphonovaleric and D-2-amino-7-phosphonoheptanoic acids, but also by competitive antagonists at the strychnine-insensitive glycine (Gly) site including 7-chlorokynurenic acid and 6,7-dichloroquinoxaline-2,3-dione. Among several proposed ligands for alpha-adrenergic receptors tested, ifenprodil most potently inhibited the binding in these membrane preparations due to a decrease in the density of the binding sites without significantly affecting the affinity. Ifenprodil also inhibited the binding of [3H]N-[1-(2-thienyl)cyclohexyl]piperidine as well as of [3H]MK-801 to open NMDA channels in a concentration-dependent manner at concentrations above 10 nM in membrane preparations extensively washed but not treated by a detergent, with a Hill coefficient of less than unity. Further treatment of extensively washed membrane preparations with a low concentration of Triton X-100 resulted in an almost complete abolition of [3H]MK-801 binding, and the binding was restored to the level found in membrane preparations not extensively washed following the addition of both L-glutamic acid (Glu) and Gly. Ifenprodil was effective in inhibiting [3H]MK-801 binding via reducing both initial association and dissociation rates in Triton-treated membrane preparations, irrespective of the presence of Glu and Gly added. The binding in Triton-treated membrane preparations was additionally potentiated by the polyamine spermidine in a concentration-dependent manner at concentrations above 10 microM in the presence of both Glu and Gly at maximally effective concentrations. Ifenprodil invariably diminished the abilities of these three stimulants to potentiate [3H]MK-801 binding at concentrations over 1 microM in a manner that the maximal responses each were reduced. These results suggest that ifenprodil does not interfere with the NMDA receptor complex as a specific isosteric antagonist at the polyamine domain in contrast to the prevailing view.     

gamma-Aminobutyric acid receptors in cerebellar membranes of rat brain after a treatment with Triton X-100.

Abstract— The treatment of cerebellar membranes of rat brain with a low concentration of Triton X-100 followed by sufficient washing results in an increase of the Na⁺-independent binding of [³H]GABA and a total loss of the Na ⁺-dependent binding of [³H]GABA. The Na⁺-independent binding of [³H]GABA was more abundant in membranes of cerebellum than in membranes of other rat brain regions and mainly localized in the synaptic membrane fraction of a cerebellar homogenate. In the Triton-treated membranes, the Na⁺-independent binding of [³H]GABA was a saturable process, which could be resolved into two components, a high and a low affinity component with dissociation constants of 4.5 and 30 nm , respectively. The neurophysiological agonists, muscimol, GABA, and imidazole acetic acid, and the antagonist, bicuculline, inhibited the high affinity Na⁺-independent binding of [³H]GABA by 50% at 0.003, 0.012, 0.3 and 10 μm respectively. These data suggest that the Na⁺-independent binding of [³H]GABA in the Triton-treated cerebellar membranes represents the synaptic receptors of GABA. It is emphasized that extensive washing of the membranes after a Triton treatment is necessary in order to detect the high affinity Na⁺-independent binding of [³H]GABA.     

Differential effect of detergents on [3H]Ro 5-4864 and [3H]PK 11195 binding to peripheral-type benzodiazepine-binding sites

The present study demonstrates a differential effect of various detergent treatments on [3H]Ro 5-4864 and [3H]PK 11195 binding to peripheral benzodiazepine binding sites (PBS). Triton X-100 (0.0125%) caused a decrease of about 70% in [3H]Ro 5-4864 binding to membranes from various peripheral tissues of rat, but had only a negligible effect on [3H]PK 11195 binding. A similar effect of Triton X-100 was observed on guinea pig and rabbit kidney membranes. The decrease in [3H]Ro 5-4864 binding after treatment with Triton X-100 was apparently due to a decrease in the density of PBS, since the affinity remained unaltered. The detergents 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS), Tween 20, deoxycholic acid, or digitonin (0.0125%) caused only a minor change in [3H]Ro 5-4864 and [3H]PK 11195 binding to rat kidney membranes; but when concentrations were substantially increased (0.1%), all detergents caused a decrease of at least 50% in [3H]Ro 5-4864 binding, while [3H]PK 11195 binding to rat kidney membranes remained unaffected by the first three detergents, with only a minor decrease (15%) after treatment with digitonin. These results may further support the assumption that Ro 5-4864 and PK 11195 are agonist and antagonist, respectively, of PBS and interact with two different conformations or domains in the peripheral-type benzodiazepine binding site molecule.     

Heterogeneity Between Rat and Calf Peripheral-Type Benzodiazepine Binding Sites: Differential Sensitivity to Triton X-100

Abstract

The effect of various detergents treatment on the specific binding of [³H]PK 11195 (2nM) to peripheral-type benzodiazepine binding sites (PBS) in calf and rat kidney, adrenal gland, and cerebral cortex membranes was studied. At a concentration of 0.025%, Triton X-100 increased [³H]PK 11195 specific binding to calf kidney, adrenal gland, and cerebral cortex membranes by 20–40%. At the same concentration, Triton X-100 scarcely affected specific binding of [³H]PK 11195 to rat cerebral cortex but decreased binding to rat kidney and adranal gland membranes by 20–30%. At a concentration of 0.05% of Triton X-100, [³H]PK 11195 specific binding to calf kidney, adrenal gland, and cerebral cortex membranes was increased by 10–20%; whereas [³H]PK 11195 specific binding to rat kidney, adrenal gland, and cerebral cortex membranes was decreased by more than 40%. The increase in [³H]PK 11195 specific binding to calf kidney membranes following Triton X-100 (0.05%) treatment was apparently due to an increase in the binding affinity of PBS, since the density remained unaltered; whereas, the decrease in [³H]PK 11195 specific binding to rat kidney membranes was due to a decrease in both binding affinity and density of PBS. On the other hand, the detergents 3- [(3- cholamidopropyl)- dimethylammonio] - 1 - propane sulfonate (CHAPS), Tween 20, deoxycholic acid, and digitonin have a similar effect on [³H]PK 11195 specific binding to PBS in both calf and rat kidney membranes.     

Solubilisation of a Glutamate Binding Protein from Rat Brain

Rat brain synaptic plasma membranes were solubilised in either 1% Triton X-100 or potassium cholate and subjected to batch affinity adsorption on L-glutamate/bovine serum albumin reticulated glass fibre. The fibre was extensively washed, and bound proteins eluted with 0.1 mM L-glutamate in 0.1% detergent, followed by repeated dialysis to remove the glutamate from the eluted proteins. Aliquots of the dialysed extracts were assayed for L-[3H]glutamate binding activity in the presence or absence of 0.1 mM unlabelled L-glutamate (to define displaceable binding). Incubations were conducted at room temperature and terminated by rapid filtration through nitrocellulose membranes. Binding to solubilised fractions could be detected only following affinity chromatography. Binding was saturable and of relatively low affinity: KD = 1.0 and 1.8 microM for Triton X-100 and cholate extracts, respectively. The density of binding sites was remarkably high: approximately 18 nmol/mg protein for Triton X-100-solubilised preparations, and usually double this when cholate was employed. Analysis of structural requirements for inhibition of binding revealed that only a very restricted number of compounds were effective, i.e., L-glutamate, L-aspartate, and sulphur-containing amino acids. Binding was not inhibited significantly by any of the selective excitatory amino acid receptor agonists--quisqualate, N-methyl-D-aspartate, or kainate. The implication from this study is that the glutamate binding protein is similar if not identical to one previously isolated and probably is not related to the pharmacologically defined postsynaptic receptor subtypes, unless solubilisation of synaptic membranes resulted in major alterations to binding site characteristics. Since solubilisation with Triton X-100 is known to preserve synaptic junctional complexes, it seems likely that the origin of the glutamate binding protein may be extrajunctional, although its functional role is unknown.     

α-[3H]Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid Binding to Rat Striatal Membranes: Effects of Selective Brain Lesions

The binding of alpha-[3H]amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ([3H]AMPA), a structural Glu analog, to rat striatal membranes was studied. In the absence of potassium thiocyanate and Cl-/Ca2+, saturation-curve analysis of [3H]AMPA binding suggested that a single class of noninteracting binding sites with a KD value of 340 +/- 27 nM was involved, although AMPA inhibition of [3H]AMPA binding set at a concentration of 100 nM suggested, in contrast, the presence of multiple populations of striatal binding sites. Several other excitatory amino acid receptor agonists and antagonists were tested, and the most potent and selective quisqualic acid (QA) receptor agonists (QA, L-Glu, and AMPA) were found to represent the most potent inhibitors of [3H]AMPA binding. N-Methyl-D-aspartate receptor agonists and antagonists were ineffective as displacers of the [3H]AMPA binding. Lesions of intrastriatal neurons (using kainic acid local injections) and of corticostriatal afferent fibers led 2-3 weeks later to large decreases (63 and 30%, respectively) in striatal [3H]AMPA binding, whereas selective lesion of the nigrostriatal dopaminergic pathway (using nigral injection of 6-hydroxy-dopamine) was without any influence. Taken together, these results suggest that [3H]AMPA binding is primarily associated with postsynaptic intrastriatal neurons. Some [3H]AMPA binding sites may also be located presynaptically on corticostriatal nerve endings. So, in addition to the possibility that [3H]AMPA binding sites may be involved in corticostriatal synaptic transmission, it is interesting that these putative QA-preferring excitatory amino acid receptor sites may also play some role in autoregulatory processes underlying this excitatory synaptic transmission.     

The effect of triton X-100 on bovine brain synaptic membranes

Bovine brain synaptic membranes which were frozen and then extensively washed showed low affinity [³H]muscimol binding. These membranes contained GABA and calmodulin, apparently tightly bound within the membrane fraction. Membranes which were additionally treated with the detergent Triton X-100 showed high affinity [³H]muscimol binding. These membranes did not appear to contain GABA or calmodulin. Transmission electron microscopy studies demonstrated that the washed membrane fraction contained many synaptosomal and vesicular structures. Triton treatment led to the extensive rupture of these structures. These studies explain the well-reported findings of tightly bound GABA and calmodulin in brain membrane fractions, as being due to the entrapment of these compounds inside sealed membrane-bound structures which are still present after a freezethaw and extensive wash treatment, their complete removal requiring Triton-treatment to rupture the vesicles.