Modulation of GABA binding sites by CNS depressants and CNS convulsants

[³H]Muscimol binding at 23°C and muscimol stimulated [³H]flunitrazepam binding at 37°C to membranes of rat cerebral cortex have been investigated. In washed membrane preparations, 2 apparent populations of [³H]muscimol binding sites can be observed. At 23°C [³H]muscimol binding is more sensitive to inhibition by NaCl and by other salts than at 0°C. The CNS depressants etazolate and pentobarbital reversibly enhance [³H]muscimol binding and they increase the affinity of muscimol as a stimulator of [³H]flunitrazepam binding. Conversely the CNS convulsants picrotoxin, picrotoxinin and isopropylbicyclophosphate (IPTBO) reversibly interfere with [³H]muscimol binding when NaCl is present and these drugs antagonize the effects of etazolate. In the presence of NaCl, picrotoxin, picrotoxinin and IPTBO also decrease the apparent affinity of muscimol or GABA as stimulator of [³H]flunitrazepam binding. Binding of [³H]muscimol to GABA recognition sites of rat cerebral cortex is enhanced by Ag⁺, Hg⁺ and Cu²⁺ in μM concentrations, Ag⁺ being most potent. The effects of 100 μM AgNO₃ persist after repeated washing of the membranes. When membranes are pretreated with AgNO₃ only one apparent population of [³H]muscimol binding sites with high affinity (K_d: 6–8 nM) is found. In AgNO₃ pretreated membranes, the affinity of muscimol as stimulator of [³H]flunitrazepam binding is increased 18 times (EC₅₀ 14 nM) when compared to control membranes, (EC₅₀ 253 nM). In AgNO₃ pretreated membranes, etazolate, pentobarbital and IPTBO fail to perturb either [³H]muscimol binding or baseline and muscimol stimulated [³H]flunitrazepam binding. The results demonstrate that the apparent sensitivity of GABA binding sites of the GABA-benzodiazepine-picrotoxin receptor complex can be increased by etazolate and pentobarbital and decreased by picrotoxin and IPTBO. These drugs have in common that they interfere with [³H]dihydropicrotoxinin binding.     

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.     

Substrate Specificity of [3H] Muscimol Binding to a Particulate Fraction of a Neuron-enriched Culture of Embryonic Rat Brain

Abstract: The effects of some GABA analogues and some drugs on the binding of [³H]muscimol (3.08 nM) to thoroughly washed subcellular particles prepared from a neuron-enriched culture of embryonic rat brain were examined using Na+-free Tris-citrate medium and a centrifugation method. Competition for [³H]muscimol binding sites by excess(10^?5 M) unlabelled GABA provided estimates of “specific” binding. In accord with in vivo neuropharmacological studies on GABA receptors and with in vitro studies on cerebral membrane preparations, [³H]muscimol binding was potently inhibited by muscimol itself (IC₅₀, 2.5 nM), GABA (1C₅₀, 43 nM), isoguvacine (IC₅₀, 61 nM), and 3-aminopropanesulphonic acid (IC₅₀, 160 nM), and less potently inhibited by the GABA antagonist bicuculline methobromide (IC₅₀, 800 nM). δ- Aminovaleric acid (IC₅₀, 2.6 μM), the glycinelp-alanine antagonist strychnine (IC₅₀, 6.6 μM), and the predominantly glial GABA uptake inhibitors β-alanine (IC₅₀, 23 μM) and p-proline (IC₅₀, 66 μM) also inhibited [³H]muscimol binding. Other inhibitors of Na+-dependent GABA uptake, (±)-nipecotic acid, L- 2,4-diaminobutyric acid, and guvacine, as well as picrotoxinin, were relatively inactive as inhibitors of [³H]muscimol binding (IC₅₀≥ 1 mM). In addition to revealing that GABA receptors are present on neuronal membranes before the formation of most synapses, this binding of [³H]muscimol that occurs to neuronal, but not to glial, membranes might be useful as a “neuronal marker” and for the further characterization and isolation of GABA receptors.     

The Binding of the GAB A Agonist [3H]THIP to Rat Brain Synaptic Membranes

Abstract: THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) is a specific GABA agonist with potent analgesic properties. The binding of radioactive THIP to thoroughly washed, frozen, and thawed membranes isolated from rat brains has been studied at 2°C under sodium ion-free conditions and compared with the binding of [³H]GABA and [³H]piperidine-4-sulphonic acid ([³H]P4S). The best computer fits to the experimental data were in all cases attained with a receptor model based on three independent binding sites, of which only the high- and medium-affinity sites could be characterised satisfactorily. While the K_D values were found to be comparable for all three ligands employed, the density of the high-affinity binding site (B_M1) was, with the exception of the membranes from the cerebellum, considerably lower for [³H]THIP than for [³H]GABA and [³ H]P4S. The regional distribution of the GABA receptors, which bind [³H]THIP, was different from those recognizing [³H]GABA and [³H]P4S. A number of analogues, including asymmetric compounds with known configuration, were tested as inhibitors of the binding of [³H]GABA, [³H]muscimol, [³H]THIP, [³H]isoguvacine, and [³H]P4S. The concentrations of the asymmetric compounds required for the inhibition of [³H]P4S binding were much higher than those required for the displacement of [³H]GABA, [³H]muscimol, [³H]THIP, and [³H]isoguvacine. The comparable relative potencies of inhibitors do, however, indicate that all of the ligands bind to the GABA receptors.     

Maximal GABA and muscimol binding to high-affinity sites differ in physiological and in non-physiological buffers

The sensitivity of [³H]GABA and [³H]muscimol high-affinity binding sites to physiological (Krebs-Ringer's bicarbonate) and non-physiological (Tris-citrate) buffers was examined using synaptosomal membranes from bovine retinas. The maximum number of sites (B_max) for [³H]GABA was present when the tissue was assayed in KRB. With only one exception, this effect was independent of the washing conditions used or a small change in pH. In contrast, [³H]muscimol binding sites were maximally present when the tissue was washed in Tris, regardless of the assaying conditions or the small change in pH. Neither [³H]GABA nor [³H]muscimol was displaced by ( - )baclofen. The apparent dissociation constants (K_d) of the ligands did not change under any of the conditions tested. These findings demonstrate a fundamental difference between GABA and muscimol binding sites.     

Binding sites for [3H]GABA, [3H]flunitrazepam and [35S]TBPS in insect CNS

The CNS of the cockroach Periplaneta americana contains saturable, specific binding sites for [³H]GABA, [³H]flunitrazepam and [³⁵S]TBPS. The [³H]GABA binding site exhibits a pharmacological profile distinct from that reported for mammalian GABA_A and GABA_B receptors. The most potent inhibitors of [³H]GABA binding were GABA and muscimol, whereas isoguvacine, thiomuscimol and 3-aminopropane sulphonic acid were less effective. Bicuculline methiodide and baclofen were ineffective. Binding of [³⁵S]TBPS was partially inhibited by 1.0 × 10⁻⁶ M GABA, whilst binding of [³H]flunitrazepam was enhanced by 1.0 × 10⁻⁷ M GABA. The pharmacological profile of the [³H]flunitrazepam binding site showed some similarities with the peripheral benzodiazepine binding sites of vertebrates, with Ro-5-4864 being a far more effective inhibitor of binding than clonazepam. Thus a class of GABA receptors with pharmacological properties distinct from mammalian GABA receptor subtypes is present in insect CNS.     

The specific binding of the platelet-activating factor (PAF) receptor antagonist WEB 2086 and the benzodiazepine flunitrazepam to rat hepatocytes

Thieno-triazolodiazepines WEB 2086 and BN 50739 have been described as the potent PAF receptor antagonists. Binding of radiolabeled [³H]WEB 2086 has been widely employed to characterize PAF receptors in different cells. In a search for a PAF receptor in isolated rat hepatocytes, we discovered that the binding of [³H]WEB to rat hepatocytes was highly specific but had a relatively low affinity with a K_d of 113 nM and B_max of 0.65 pmol/10⁶ cells in freshly isolated cell suspension and K_d of 1.65 μM and B_max of 2.0 pmol/plate in cultured hepatocytes. No consistent specific binding of [³H]PAF itself was found in the same cell preparations. The binding of [³H]flunitrazepam in the presence of the peripheral type of benzodiazepine receptor antagonist Ro 5-4864 was saturated and exhibited a K_i of 3.8 nM and B_max of 3.5 pmol/plate. The central type of benzodiazepine receptor antagonist clonazepam also competed for the [³H]flunitrazepam binding, however with a much lower affinity. Various antagonists inhibited the binding of [³H]WEB 2086 with a rank order BN 50739⪢Ro 5-4864≥clonazepam. Interestingly, bicuculline, a specific antagonist of GABA(A) recognition sites, also significantly reduced the binding of [³H]WEB 2086. The binding of [³H]flunitrazepam was inhibited with a rank potency BN 50739⪢WEB 2086. Taken together, these findings suggest that the specific binding of PAF receptor antagonists WEB 2086 and BN 50739 in rat hepatocytes does not involve PAF receptors and occurs via peripheral benzodiazepine and, possibly GABA(A) receptor sites.     

Effects of chronic ethanol exposure on the gaba-benzodiazepine receptor complex in rat brain

Chronic treatment of male Wistar rats with ethanol by inhalation did not affect the binding of [³H]flunitrazepam, [³H]GABA or [³H]muscimol to extensively washed synaptic membranes. Neither the affinity (K_d) nor the number of binding sites (Bmax) for these ligands was changed. However, GABA enhancement of [³H]flunitrazepam binding was significantly decreased by approx. 40% in ethanol-treated animals (172% compared to 215%). Acute treatment with ethanol did not produce changes in the binding of [³H]flunitrazepam or [³H]muscimol. These findings suggest that chronic ethanol treatment leads to uncoupling of the various receptor sites on the GABA—benzodiazepine receptor ionophore-complex in the brain.     

Stimulatory and protective effects of benzodiazepines on GABA receptors labeled with [3H]muscimol

We investigated the effects of benzodiazepines on [³H]muscimol binding to rat brain membranes and on heat inactivation of GABA receptors. Scatchard analysis of [³H]muscimol binding to frozen and 0.05% Triton X-100 treated membranes revealed two components; a higher affinity (Kd=2.2 nM, Bmax=1.2 pmol/mg protein) and a lower affinity component (Kd=15.9 nM, Bmax=4.4 pmol/mg protein). Diazepam and flurazepam (3 μM) increased significantly the specific binding of 40 nM but not of 2 nM [³H]muscimol. This stimulation was attributed to an increase in the affinity of the lower affinity component for GABA receptors. The time course of heat inactivation of GABA receptors revealed rapidly and then slowly denaturating Phases. These observations would suggest that there are multiple GABA receptors with different sensitivities to the heat treatment. Diazepam depressed remarkably the slowly denaturating phase(s). After heat treatment for 50 min, the single component of GABA receptors with Kd of 14.3 nM and Bmax of 0.6 pmol/mg protein survived, whereas in the membranes preincubated with 3 μM diazepam, the Kd and Bmax of the still viable GABA receptors were 14.8 nM and 1.14 pmol/mg protein, respectively. In light of these findings, the stimulation of the lower affinity component of GABA receptors may be related to the protective effect of these drugs against heat inactivation.     

Identification of stereospecific [3H]spiroperidol binding sites in mammalian lymphocytes

Direct binding to intact rat lymphocytes has been shown for the potent dopaminergic antagonist [³H]spiroperidol. The specific binding is saturable with two components (K_D1 = 1.9 nM, K_D2 = 36.2 nM). Determination of the K_D by kinetic studies measuring rate constants for association and dissociation provided K_D values similar to those obtained in equilibrium experiments. The specific binding is proportional to cell concentration and temperature dependent with a maximum at 37°C. [³H]spiroperidol binding is stereospecific since (+)butaclamol was more effective than (?)butaclamol. The relative potencies of different antidopaminergic agents in competing for [³H]spiroperidol binding sites parallel their activity in the striatum. Dopaminergic receptors have also been demonstrated in other mammalian lymphocytes (rabbit, dog, human). Lymphocyte dopaminergic receptors could be implicated in lymphocytes mediated immune response.     

Increase in Striatal [3H]Muscimol Binding Following Intrastriatal Injection of Kainic Acid: A Denervation Supersensitivity Phenomenon

The effect of intrastriatal microinjection of kainic acid (KA) on specific binding of [³H]muscimol to the particulate fractions obtained from corpus striatum (CS), globus pallidus (GP), substantia nigra (SN), and cerebral cortex (CC) was examined. Seven days after the unilateral intrastriatal microinjection of KA, the amount of specifically bound [³H]muscimol was significantly increased at the injected site, whereas no significant alteration of [³H]muscimol binding was found in GP, SN, or CC. Scatchard analysis of striatal binding revealed that microinjection of KA significantly increased the affinity (K_D) of GABA receptors on the injected (lesioned) side of the CS without affecting the total number of binding sites (B_max) therein. This significant increase in [³H]muscimol binding, however, was eliminated by pretreating particulate fractions from the CS with Triton X-100, a non-ionic detergent. No statistically significant difference in amounts of [³H]muscimol binding was detected when the preparations from the KA-treated and non-treated CS were preincubated with 0.05% Triton X-100, respectively. Scatchard analysis using CS preparations treated with 0.05% Triton X-100 revealed that the affinity of the GABA receptor was increased by treatment with Triton X-100, while the total number of binding sites (B_max) was unchanged by this treatment. These results suggest that neuronal degeneration produced by KA in vivo and pretreatment of particulate preparations with Triton X-100 in vitro may increase the amount of specifically bound [³H]muscimol to CS preparations by a similar molecular mechanism.     

A comparison of GABA and beta-alanine transport and GABA membrane binding in the rat brain

It was found that rat brain nerve endings contain a high affinity and Na^- dependent transport system for [³H]β-alanine ([³H]β-ala). As determined from Michaelis-Menten plots, the [³H]β-ala K_m was 2.8 × 10^-5 M and the V_max was 0.29 nmol/mg protein/5 min. Under similar incubation conditions the [³H]GABA K_m was 3.8 x 10^-6M and the V_max was 6.3 nmol/mg protein/5 min. The [³H]β-ala and [³H]GABA transport systems were further characterized by determining the IC₅₀ values for a number of compounds. The compounds tested were GABA, β-ala, l -2,4-diaminobutyric acid. DL-3-hyd-roxy-GABA, β-guanidopropionic acid, strychnine, γ-guanidobutyric acid, imidazole-4-acetic acid, DL-proline, bicuculline, L-serine, glycine, l -α-ala and taurine. DABA, dl -3-hydroxy-GABA, β-guanidopro-pionic acid and γ-guanidobutyric acid were more potent inhibitors of [³H]GABA than [³H]β-ala transport. Strychnine, imidazole-4-acetic acid, proline and glycine were between 2 and 6 times more potent inhibitors of [³H]β-ala than [³H]GABA transport. β-Ala, bicuculline, serine, α-alanine and taurine were all markedly more potent (12–150 times) inhibitors of [³H]β-ala than [³H]GABA transport. IC₅₀ values were also determined for the above compounds for the sodium-dependent and the sodium-independent binding of [³H]GABA to both fresh and frozen brain membranes. In general, the potency of these compounds to inhibit either sodium-independent or sodium-dependent binding was greater in fresh tissue. It was also observed that the neurophysiologically‘glycine-like’amino acids were more potent inhibitors in the presence of NaCl. No significant correlations were found between [³H]GABA binding under any condition and [³H]GABA or [³H]β-ala transport into nerve endings.     

High Specific Binding of [3H]GABA and [3H]Muscimol to Membranes from Dendrodendritic Synaptosomes of the Rat Olfactory Bulb

Olfactory bulbs contain dendrodendritic synapses, which occur between granule cells and mitral cells, and gamma-aminobutyric acid (GABA) is thought to act as an inhibitory neurotransmitter at these synapses. Synaptosomes derived from the dendrodendritic synapses of the olfactory bulb were shown previously to contain considerable L-glutamate decarboxylase activity. The subcellular distribution and binding parameters of [3H]GABA and [3H]muscimol binding sites have now been determined in the rat olfactory bulb. Of all fractions examined, crude synaptic membranes (CSM) prepared from the dendrodendritic synaptosomes were shown to have the highest specific binding activity and accounted for nearly all of the total binding activity for both ligands. The specific binding activities for [3H]GABA and for [3H]muscimol were greatly increased after treating the CSM with 0.05% Triton X-100. Binding was shown to be Na+-independent, reversible, pharmacologically specific, and saturable. High- and low-affinity sites were detected for both ligands, and both classes of sites had appreciably lower KD values for muscimol (KD1 = 3.1 nM, KD2 = 25.1 nM) than for GABA (KD1 = 8.6 nM; KD2 = 63.7 nM). The amounts of the high-affinity binding sites for muscimol and GABA were similar (Bmax = 1.7 and 1.5 pmol/mg protein, respectively). The results of the present experiments indicate that the GABA and muscimol binding sites represent the GABA postsynaptic receptor, presumably on mitral cell dendrites, and provide further support for the hypothesis that GABA functions as a neurotransmitter at the dendrodendritic synapses in the olfactory bulb.     

Competitive Inhibition of γ-Aminobutyric Acid Receptor Binding by N-2-Hydroxyethylpiperazine-N''-2- Ethanesulfonic Acid and Related Buffers

Several Good buffers (MOPS, ACES, BES, HEPES, ADA, and PIPES) competitively inhibited both high-affinity and low-affinity [3H]gamma-aminobutyric acid receptor binding to rat brain synaptic membranes. The most potent inhibitor was MOPS, which had Ki values of 180 nM and 79 nM for the high- and low-affinity binding sites, respectively. HEPES had Ki values of 2.25 mM and 115 microM. The buffers had no appreciable effect on sodium-dependent GABA binding or on gamma-aminobutyrate aminotransferase activity. Surprisingly, the buffers were extremely ineffectual as inhibitors of either high- or low-affinity [3H]muscimol binding. Indeed, they were of the order of 10(5) times less effective in this case than against [3H]GABA binding. These results clearly show (a) that the use of such buffers as MOPS or HEPES should be avoided in studying the interaction of GABA with its receptor, and (b) the binding sites of [3H]GABA and [3H]muscimol are not identical.     

Characterization of the Binding of the GABA Agonist [3H]Piperidine-4-Sulphonic Acid to Bovine Brain Synaptic Membranes

Abstract: The binding of radioactive piperidine-4-sulphonic acid ([³H]P4S) to thoroughly washed, frozen, and thawed membranes isolated from cow and rat brains has been studied. Quantitative computer analysis of the binding curves for four regions of bovine brain revealed the general presence of two binding sites. In these brain regions less satisfactory computer fits were obtained for receptor models showing one or three binding sites or negative cooperativity. With the use of Tris-citrate buffer at 0°C the two affinity classes for P4S in bovine cortex membranes revealed the following binding parameters: K_D= 17 ± 7 nM (B_max= 0.15 ± 0.07 pmol/mg protein) and K_D= 237 ± 100 nM (B_max= 0.80 ± 0.20 pmol/mg protein). Heterogeneity was also observed for association and dissociation rates of [³H]P4S. The slow binding component (k_on= 5.6 × 10⁷ or 8.8 × 10⁷ M^-1 min^-1, k_Off= 0.83 min^-1, and K_D= 14.7 or 9.4 nM, determined by two different methods in phosphate buffer containing potassium chloride) corresponds to the high-affinity component of the equilibrium binding curve (K_D= 11 nM, B_max= 0.12 pmol/mg protein in the same buffer system). The association and dissociation rates for the subpopulation of rapidly dissociating sites, apparently corresponding to the low-affinity sites, were too rapid to be measured accurately. The binding of [³H]P4S appears to involve the same two populations of sites with B_max values similar to those for [³H]GABA binding to the same tissue, although the kinetic parameters for the two ligands are somewhat different. Furthermore, comparative studies on the inhibition of [³H]P4S and [³H]GABA binding by various GABA analogues, strongly suggest that P4S binds to the GABA receptors. The different effects of P4S and GABA on benzodiazepine binding are discussed.     

Properties of [3H] ß-carboline-3-carboxylate ethyl ester binding to the benzodiazepine receptor

β-Carbolines are inhibitors of [³H] diazepam binding with the most potent inhibitor being β-carboline-3-carboxylate ethyl ester (β-CCE). In this report the binding of [³H] β-CCE to extensively washed rat forebrain membranes is characterized. [³H] ß-CCE binds with high affinity (K_D = 1.4 nM) to an apparently homogenous population of benzodiazepine receptor. The rank order of potency for inhibition of [³H] ß-CCE binding by different benzodiazepines is clonazepam > diazepam > chlordiazepoxide, which is similar to that observed for inhibition of [³H] diazepam binding. In marked contrast to [³H] diazepam, the binding of [³H] ß-CCE is not modulated by GABA since concentrations of GABA as high as 10^?3 M had no effect. [³H] ß-CCE is also less potent than [³H] diazepam in its interaction with the peripheral type kidney benzodiazepine receptor indicating that this ligand has a higher degree of specificity for the central brain type benzodiazepine receptor.     

A novel pyrazoloquinoline that interacts with brain benzodiazepine receptors: Characterization of some and properties of CGS 9896.

The novel pyrazoloquinoline, CGS, 9896, was a potent inhibitor of specific [³H]-flunitrazepam binding in several brain regions with subnanomolar K_I values. The inhibition of [³H] propyl beta-carboline-3-carboxylate ([³H]-PCC) binding by CGS 9896 was enhanced by gamma-aminobutyric acid (GABA) but not by chloride ion. GABA enhancement of CGS 9896 inhibition of [³H]-PCC binding predicts this compound has benzodiazepine (BZD) agonist-type activity. Behavioral studies support this prediction. CGS 9896 was found to protect mice against bicuculline and metrazol induced seizures at doses that did not induce ataxia or sedation. CGS 9896 may represent a class of compounds with potential therapeutic value. The high affinity of this non-BZD compound suggests that CGS 9896 may also be of value as a high affinity ligand for the continued study of BZD receptors.     

Specific Binding of L-[3h]-Glutamic Acid to Rat Substantia Nigra Synaptic Membranes

Abstract

The specific binding of L-[³H] -glutamic acid (GLU) was investigated in synaptic membranes from rat substantia nigra. L-[³H]-GLU binding to the membrane preparations occurred in a reversible and saturable way. The specific binding was stimulated by the presence of CaCl₂ and was reduced by freezing and thawing the membranes. Scatchard analysis of the saturation isotherms yielded a non-linear plot suggesting that the binding reaction does not occur through a simpla bimolecular association. Assuming non-interacting binding sites, a high (K_D1, 139 nM; Bmax1, 3.5 pmoles/mg protein) and a low (K_D2, 667 nM; Bmax2, 15.1 pmoles/mg protein) affinity L-[³H]-GLU binding site were obtained. The kinetics of dissociation of bound L-[³H]-GLU was biphasic; the respective dissociation rate constant (k-1) being 0.20 min^?1 and 0.013 min^?1. A series of amino acid receptor agonists and antagonists were tested as inhibitors of L-[³H]-GLU specific binding. Quisqualic acid, L-GLU and D-α-aminoadipate (D-α-AA) were the most potent inhibitors. DL-2-amino-4-phosphonobutyrate (APB), N-Methy1-D-aspartate (NMDA) and D-GLU were moderate inhibitors, whereas diamino-pimelic acid (DAPA) and glutamate diethyl ester (GDEE) exhibited the lowest relative potency. Kainic acid (KA), γ-aminobutyric acid (GABA) and bicuculline were not able to modify at any concentration used the specific binding of L-[³H]-GLU. These data demonstrate the presence of specific GLU binding sites in synaptic structures at substantia nigra level and support the idea that excitatory amino acids may play a role in synaptic transmission in this brain region.     

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.     

Behavior of rat hypothalamic and extrahypothalamic immuno-reactive TRF in thin-layer chromatography (TLC) and high pressure liquid chromatography (HPLC)

[³H] quinuclidinyl benzilate (QNB), a specific muscarinic antagonist, was utilized to identify muscarinic cholinergic receptors on dispersed anterior pituitary cells. Scatchard analysis of [³H] QNB binding to receptors departs from linearity with upward concavity. A high affinity binding site having a dissociation constant (K_d) of 1.5 nM was observed when the [³H] QNB concentration was varied from 0.15 to 20 nM. A low affinity binding site (K_d 20 nM) was observed when [³H] QNB concentration was above 20 nM. Using 10 nM [³H] QNB for binding, the second order association rate constant (k₁) of 0.064 nM^?1 min^?1 and first order dissociation rate constant (k₂) of 0.078 min^?1$\text{(}\text{T}\text{1}\text{2}\text{8}\text{min}\text{)}$ were observed. k₂/k₁ = K_d of 1.22 nM is in good agreement with K_d = 1.5 nM from equilibrium data. Muscarinic cholinergic receptor antagonists, atropine and scopolamine, and agonist oxtoremorine potently competed with [³H] QNB binding. A nicotinic cholinergic receptor agonist was 50 times less potent as a competitor of [³H] QNB binding than the muscarinic agonist.