The effect of phospholipases and proteases on the binding of γ-aminobutyric acid to junctional complexes of rat cerebellum

A preparation enriched in junctional complexes, as judged by marker enzymes and electron microscopy, was prepared from rat cerebellum. The junctional complexes were incubated with γ-amino[¹⁴C]butyric acid at 25°C for 10 min, using [³H]sucrose as a marker for entrapped space. Total binding was determined in the absence of, and non-specific binding in the presence of, an excess of unlabelled γ-aminobutyric acid. The difference between the two binding values, i.e. the specific binding, was saturable and reversible, and showed positive cooperativity with a Hill number of about 2. The specific binding was inhibited by N-methylbicuculline, picrotoxinine and imidazole-4-acetic acid, but not by curare, strychnine or l-2,4-diaminobutyric acid. The above compounds had little effect on the non-specific binding, but addition of ethylenediaminetetraacetic acid decreased non-specific binding by 80%. Trypsin, pronase, phospholipase A₂ (EC 3.1.1.4), lysolecithin and sodium dodecyl sulfate decreased binding. Phospholipase C (EC 3.1.4.3) increased the specific binding by 260%. Phospholipids competed with γ-aminobutyric acid for binding, with phosphatidylethanolamine being more potent than phosphatidylcholine. These results lend support for Watkins' hypothesis that phosphatidylethanolamine competes with γ-aminobutyric acid for binding to the receptor protein.     

The incorporation of radioactive fatty acids and of radioactive derivatives of glucose into the phospholipids of subsynaptosomal fractions of cerebral cortex.

1. Crude synaptosomal fractions (P2) from guinea-pig cerebral cortex were incubated in a Krebs-glucose medium containing labelled fatty acids and [3H]glucose. After the shortest incubation period (7.5 min) a high percentage (50-80%) of the total radioactive fatty acids was found in the P2 fractions. 2. After the incubation, the synaptosomal fractions were submitted to hypo-osmotic disruption and subsynaptosomal fractionation was carried out by using discontinuous-sucrose-gradient centrifugation. The specific radioactivities of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol were determined in fractions D (synaptic vesicles), E (microsomal preparation) and H (disrupted synaptosomes), as were the specific activities of a number of marker enzymes and the distribution of acetylcholine. 3. By using [14C]oleate, [14C]arachidonate, [3H]palmitate and [3H]glucose, the order to specific radioactivities in fraction D was found to be: phosphatidylinositol greater than phosphatidylcholine greater than phosphatidylserine greater than phosphatidylethanolamine. 4. The specific radioactivities of phosphatidylcholine and phosphatidylethanolamine were always higher in fraction D than in fraction E. As fraction E had higher specific activities of several membrane marker enzymes, the enhanced labelling found in fraction D was considered to be localized in the synaptic vesicles. In this fraction, phosphatidylinositol made particularly large contributions to the total phospholipid labelling derived from [14C]arachidonate and [3H]glucose. 5. The similar labelling ratios of fatty acid/glucose in the phospholipids of fractions D and E, and the high specific radioactivities in the total phospholipid of the soluble fraction O, suggested intrasynaptosomal phospholipid transport.     

A gamma-aminobutyric acid/benzodiazepine receptor complex of bovine cerebral cortex

The gamma-aminobutyric acid/benzodiazepine receptor from bovine cerebral cortex was solubilized with sodium deoxycholate and purified by affinity chromatography on benzodiazepine-agarose and ion exchange chromatography. The benzodiazepine binding protein was enriched 1800-fold. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and dithiothreitol showed the presence of two major bands of Mr = 57,000 and 53,000. [3H]Flunitrazepam, after UV irradiation, was incorporated irreversibly into both bands of the isolated protein. A high affinity binding site for gamma-aminobutyric acid was co-purified with the benzodiazepine binding site and the two sites were shown to reside on the same physical structure. The dissociation constants were 10 +/- 4 nM for [3H] flunitrazepam and 12 +/- 3 nM for the gamma-aminobutyric acid agonist [3H]muscimol. The maximum specific activity for [3H] muscimol binding was 4.3 nmol/mg of protein. The ratio of [3H]muscimol to [3H]flunitrazepam binding sites was between 3 and 4. Gel filtration and sucrose density gradient sedimentation studies gave a Stokes radius of 7.3 +/- 0.5 nm and a sedimentation coefficient of 11.1 +/- 0.3 S, respectively. The purified complex had a pharmacological profile that corresponds to the receptor specificity found in membranes and crude soluble extracts.     

Enhancement of t-[35S]Butylbicyclophosphorothionate and [3H]Strychnine Binding by Monovalent Anions Reveals Similarities Between γ-Aminobutyric Acid- and Glycine-Gated Chloride Channels

The characteristics of [3H]strychnine and t-[35S]-butylbicyclophosphorothionate ([35S]TBPS) binding to sites associated with glycine- and gamma-aminobutyric acid (GABA)-gated chloride channels were compared in the presence of a series of anions with known permeabilities through these channels. Good correlations were found between (a) the potencies (EC50) of these anions to stimulate radioligand binding and their permeabilities relative to chloride; (b) the affinities (KD) of these radioligands in the presence of fixed concentrations of these anions and their relative permeabilities; (c) the potencies (EC50) of these anions to stimulate [35S]TBPS and [3H]strychnine binding; and (d) the affinities (KD) of [3H]strychnine and [35S]TBPS measured at a fixed concentration of these anions. These studies support electrophysiological and biochemical observations demonstrating similarities between glycine- and GABA-gated chloride channels, and suggest that anions enhance [3H]strychnine and [35S]TBPS binding through specific anion binding sites located at the channels.     

Separation of cardiac plasmalemma into cell surface and T-tubular components. Distribution of saxitoxin- and nitrendipine-binding sites

To compare surface sarcolemmal with T-tubular distributions of [3H]saxitoxin (STX)- and [3H]nitrendipine (NTD)-binding sites, we centrifuged membrane vesicles from sheep and bovine ventricles on a 10-40% linear sucrose gradient from which fractions were assayed for STX and NTD binding; for markers of surface sarcolemma (ouabain-sensitive Na,K-ATPase activity, [3H]quinuclidinyl benzilate binding); and for markers of junctional sarcoplasmic reticulum known to be preferentially associated with T-tubules (ryanodine-sensitive Ca2+ uptake, calsequestrin, an Mr 300,000 putative phosphorylatable "foot" protein, and electron microscopically visible junctional sarcoplasmic reticulum-plasmalemma complexes). We identified three distinct peaks in the sucrose gradient, each characterized by significant high and low affinity STX- and high affinity NTD-binding: Peak I (approximately 19% sucrose), highly enriched in surface sarcolemma; Peak III (approximately 36% sucrose), enriched in junctional sarcoplasmic reticulum markers and hence in junctional sarcoplasmic reticulum complexes with T-tubule; and Peak II (approximately 27% sucrose), showing greatest specific STX binding and only moderate NTD binding, enriched in T-tubular membrane, unassociated with junctional sarcoplasmic reticulum. For ventricular myocytes, the ratio NTD sites/STX sites was 2.5 for surface sarcolemma, but only approximately 1.0 for T-tubules. Unlike data published for mammalian skeletal muscle, sheep and beef cardiac NTD receptors were not significantly more concentrated in T-tubular than in surface plasmalemma.     

Interaction of enkephalins and des-tyrosyl-enkephalins with synaptosomal plasma membrane vesicles: enkephalin binding and inhibition of proline transport

Leucine- and methionine-enkephalins inhibit the Na+-dependent transport of proline into plasma membrane vesicles derived from synaptosomes. Glycine transport is weakly inhibited by enkephalins whereas there is no inhibition of transport of glutamic acid, aspartic acid, or gamma-aminobutyric acid. The inhibition of proline uptake is observed with des-tyrosyl-enkephalins but not with morphine, dynorphin(1-13), or beta-endorphins. Furthermore, enkephalin-induced inhibition of proline transport is not antagonized by naloxone. [Leu]enkephalinamide and modified [Leu]enkephalins with greater selectivity for the delta-subclass of enkephalin binding sites are less effective than [Leu]enkephalin in the inhibition of proline transport. Specific binding of [3H]Leu-enkephalin to the plasma membrane vesicles is demonstrated, and des-Tyr-[Leu]enkephalin competes with Leu-enkephalin for [Leu]enkephalin binding sites. The similarity in the concentrations of des-Tyr-[Leu]enkephalin required to compete for specific [Leu]enkephalin binding and to inhibit proline transport suggests that a specific subclass of enkephalin binding sites, distinguished by their recognition of both the enkephalins and their des-tyrosyl derivatives, may be associated with the synaptic proline transport system.     

Studies on peroxisomes. VI. Relationship between the peroxisomal core and urate oxidase.

The peroxisomal core from the liver of rats was purified 450-fold as a marker of urate oxidase [EC 1.7.3.3.] activity. This preparation has a high specific activity of urate oxidase but not of other peroxisomal enzymes: D-amino acid oxidase [EC 1.4.3.3.], L-alpha-hydroxy acid oxidase [EC 1.1.3.15], or catalase [EC 1.11.1.6]. No activity of marker enzymes for other subcellular particles; cytochrome c oxidase [EC1.9.3.1] (mitochondria), acid phosphatase [EC 3.1.3.2] (lysosomes), or glucose-6-phosphatase [EC 3.1.3.9] (microsomes), was detected in this preparation. The core obtained showed a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the position of the band was found to correspond to a molecular weight 35,000. When the peroxisomal core was subjected to treatment at various pH's with 0.1 M carbonate buffer, urate oxidase was almost completely solubulized at pH 11.0, although approximately 35% of the core protein still remained in the pellet After solubilization of the core at pH 11.0, the specific activity of urate oxidase in the supernatant increased about 1.6 times; the density of the insoluble protein remaining in the pellet was identical with the that of the original core on sucrose density gradient centrifugation.     

Modulation of [3H]Muscimol Binding in Rat Cerebellar and Cerebral Cortical Membranes by Picrotoxin, Pentobarbitone, and Etomidate

Modulation of [3H]muscimol binding by picrotoxin, pentobarbitone, and etomidate was investigated in rat cerebellar and cerebral cortical membranes. In cerebellum, at 37 degrees C in the presence of chloride ions (150 mM), picrotoxin and picrotoxinin decreased specific [3H]muscimol binding to 43 +/- 3% of control, with an EC50 of 1.2 +/- 0.1 microM. [3H]Muscimol saturation experiments in the presence and absence of picrotoxin indicated that the picrotoxin effect was primarily due to a loss of high-affinity muscimol sites with KD approximately equal to 10 nM. Pentobarbitone enhanced specific [3H]muscimol binding to 259 +/- 3% of control, with EC50 = 292 +/- 37 microM, and etomidate increased binding to 298 +/- 18%, with EC50 = 7.1 +/- 1.0 microM. The influence of temperature and chloride ion concentration on these effects was investigated by comparing experiments at 37 and 0 degrees C in the presence or absence of chloride at constant ionic strength. The results indicate that studies at 0 degrees C underestimate the coupling between GABA receptors and barbiturate sites and that they greatly overestimate the importance of chloride ions in this phenomenon. In cerebral cortical membranes (37 degrees C, 150 mM Cl-), the effect of picrotoxin was similar to that observed in cerebellum, whereas the effects of pentobarbitone and etomidate were greater, but occurred at higher concentrations.     

Actions of dietary orotic acid on liver synthesis of phosphatidylcholine and phosphatidylethanolamine in rats

The in vivo rates of the reactions of the cytidine pathways of liver phosphatidylcholine and phosphatidylethanolamine synthesis were measured in rats after 1 day of feeding on a semisynthetic diet containing 1% orotic acid. The calculations were made from the specific and total radioactivity versus time curves of the precursors and products following intraportal injection of [1,2-14C]choline, [2-14C]ethanolamine, and [2-3H]glycerol. The liver CTP level was increased twofold and the rates of CDP-choline and phosphatidylcholine synthesis were stimulated 4.5-fold in the rats fed orotic acid. The rate of CDP-ethanolamine synthesis was increased but could not be accurately quantified because of its extreme rapidity. No change occurred in the rate of the ethanolaminephosphotransferase reaction and the overall rate of phosphatidylethanolamine synthesis was unchanged by orotic acid feeding. The catalytic activities of the enzymes of the cytidine pathways of phosphatidylcholine and phosphatidylethanolamine synthesis were not affected by feeding orotic acid for 1 day. Similar findings were obtained 3 h following intragastric administration of 100 mg of orotic acid. The results suggest the possibility that changes in the levels of liver CTP may play a role in regulation of the cytidine pathway of liver phosphatidylcholine synthesis but not of phosphatidylethanolamine synthesis, because the latter pathway appears to be tightly controlled at the ethanolaminephosphotransferase step.     

γ-Aminobutyric Acid: Temperature Dependence in Benzodiazepine Receptor Binding

The effects of muscimol and/or incubation temperature on the inhibition of [3H]flunitrazepam receptor binding by benzodiazepine receptor ligands were investigated. At 0 degree C muscimol decreased the Ki values for some ligands as displacers of [3H]flunitrazepam binding to brain-specific sites while increasing or having no effect on the Ki values for other ligands. The Ki values for some ligands are higher at 37 degrees C than at 0 degree C but are reduced by muscimol at both 0 degrees and 37 degrees C. In contrast, the ligands whose Ki values are increased by muscimol either decreased or did not alter the Ki values at 37 degrees C as compared to those at 0 degree C. Incubation of membranes at 37 degrees C for 30 min accelerated gamma-aminobutyric acid (GABA) release by 221% over that at 0 degree C. These results indicate that changes in incubation temperature alter benzodiazepine receptor affinity for ligands via GABA.     

An improved procedure for the synthesis of 14C-labeled phosphatidylserine from cerebral phosphatidic acid

A complete procedure to prepare a highly labeled phosphatidyl-L-[U-14C]serine possessing the same fatty acid composition of brain phospholipids is reported. CDP-diglyceride was synthesized by reaction between phosphatidic acid and CMP-morpholidate as the dicyclohexylcarboxamidium salt. The reaction between CDP-diglyceride and L-[U-14C]serine to produce the labeled phosphatidylserine was catalyzed by the CDP-diglyceride: L-serine phosphatidyl transferase (EC 2.7.8.8) from E. coli. A selective inhibition of phosphatidylserine decarboxylase activity, present as contaminant in the enzyme extract, was introduced in order to avoid a low yield of product. Traces of phosphatidylethanolamine (about 1%) were easily removed by preparative thin-layer chromatography. The yield of the labeled product was as high as 87% and it specific radioactivity was 170 mCi/mmol.     

Angiotensinase activity and angiotensin receptor binding in guinea pig aorta.

The angiotensinase (EC 3.4.99.3) activity of the subcellular fractions of guinea pig aorta has been studied in relation to their [14C]angiotensin binding capacity. The enzyme activity occurs in the following decreasing order: supernatant greater than plasma membrane fraction greater than 105 000 X g pellet greater than mitochondrial fraction. The specific binding of [14C]angiotensin to these fractions follows the same pattern. Pretreatment of the subcellular fractions at 47 degrees C for 20 min was performed in an attempt to differentiate binding of angiotensin to the pharmacological receptor from binding to the destroying enzymes. This procedure decreased the angiotensinase activity in the plasma membrane fraction only whereas the specific binding of [14C]angiotensin to this fraction was not significantly decreased, suggesting that the plasma membrane angiotensinase is a thermolabile enzyme.     

In Vivo Release from Cerebral Cortex of [14C]Glutamate Synthesized from [U-14C]Glutamine

Awake, unrestrained, and behaviourally normal animals with superfusion cannulae implanted over the sensorimotor cortex were used in a study of the capacity of infused [U-14C]glutamine for labelling glutamate and other amino acids released by depolarising stimuli. A spontaneous background release of [14C]glutamate was detected. This was increased by tityustoxin (1 microM). The specific radioactivity of glutamate increased eightfold during the evoked-release period. [14C]Aspartate was also detected and showed increased release, but not increased specific labelling, in response to depolarisation. Evoked gamma-aminobutyric acid (GABA) release occurred but only small amounts of [14C]GABA were detected. Glutamine showed increased rates of uptake to the sensorimotor cortex during stimulation periods, suggesting an accelerated breakdown via glutaminase.     

Synthesis and biodistribution of a new radiotracer for in vivo labeling of serotonin uptake sites by PET,cis-N,N-[11C]dimethyl-3-(2′,4′-dichlorophenyl)-indanamine (cis-[11C]DDPI)

A new PET radiotracer for in vivo labeling of serotonin (5-HT) uptake sites, cis-N, N-[¹¹C]dimethyl-3-(2′,4′-dichlorophenyl)-indanamine, cis-[¹¹C]DDPI, was synthesized and its biological behavior was studied. The radiosynthesis of cis-[¹¹C]DDPI was performed by N-methylation of cis-N-methyl-3-(2′,4′-dichlorophenyl)-indanamine with [¹¹C]iodomethane. The average radiochemical yield was approx. 8%, with an average specific activity of 600mCi/μmol. Following intravenous administration, cis-[¹¹C]DDPI accumulated in mouse brain regions rich in 5-HT uptake sites, such as olfactory tubercles, hypothalamus and frontal cortex. Following pre-injection of 1 mg/kg of paroxetine, a high affinity 5-HT uptake blocker, the binding of cis-[¹¹C]DDPI in the olfactory tubercles, hypothalamus and frontal cortex was decreased by 23, 25 and 16%; this corresponds to 73, 82 and 59% of the specific binding in these regions. These results suggest that the accumulation of cis-[¹¹C]DDPI in the tissues rich in 5-HT sites is a result of specific binding of cis-[¹¹C]DDPI to 5-HT uptake sites. Due to the relatively high non-specific uptake and slow clearance of this compound from non-specific binding sites, the ratio between specific and non-specific binding increased slowly with time, reaching 1.5:1 at 60 min after injection.     

Role of carnitine and carnitine palmitoyltransferase as integral components of the pathway for membrane phospholipid fatty acid turnover in intact human erythrocytes.

The deacylation and reacylation process of phospholipids is the major pathway of turnover and repair in erythrocyte membranes. In this paper, we have investigated the role of carnitine palmitoyltransferase in erythrocyte membrane phospholipid fatty acid turnover. The role of acyl-L-carnitine as a reservoir of activated acyl groups, the buffer function of carnitine, and the importance of the acyl-CoA/free CoA ratio in the reacylation process of erythrocyte membrane phospholipids have also been addressed. In intact erythrocytes, the incorporation of [1-14C]palmitic acid into acyl-L-carnitine, phosphatidylcholine, and phosphatidylethanolamine was linear with time for at least 3 h. The greatest proportion of the radioactivity was found in acyl-L-carnitine. Competition experiments using [1-14C]palmitic and [9,10-3H]oleic acid demonstrated that [9,10-3H]oleic acid was incorporated preferentially into the phospholipids and less into acyl-L-carnitine. When an erythrocyte suspension was incubated with [1-14C]palmitoyl-L-carnitine, radiolabeled palmitate was recovered in the phospholipid fraction, and the carnitine palmitoyltransferase inhibitor, 2-tetradecylglycidic acid, completely abolished the incorporation. ATP depletion decreased incorporation of [1-14C]palmitic and/or [9,10-3H]oleic acid into acyl-L-carnitine, but the incorporation into phosphatidylcholine and phosphatidylethanolamine was unaffected. In contrast, ATP depletion enhanced the incorporation into phosphatidylcholine and phosphatidylethanolamine of the radiolabeled fatty acid from [1-14C]palmitoyl-L-carnitine. These data are suggestive of the existence of an acyl-L-carnitine pool, in equilibrium with the acyl-CoA pool, which serves as a reservoir of activated acyl groups. The carnitine palmitoyltransferase inhibition by 2-tetradecylglycidic acid or palmitoyl-D-carnitine caused a significant reduction of radiolabeled fatty acid incorporation into membrane phospholipids, only when intact erythrocytes were incubated with [9,10-3H]oleic acid. These latter data may be explained by the differences in rates and substrates specificities between acyl-CoA synthetase and the reacylating enzymes for palmitate and oleate, which support the importance of carnitine palmitoyltransferase in modulating the optimal acyl-CoA/free CoA ratio for the physiological expression of the membrane phospholipids fatty acid turnover.     

Solubilization of the central benzodiazepine receptor and study of its interaction with nicotinamide

It was shown that nicotinamide and NAD inhibit the specific binding of [3H]flunitrazepam to benzdiazepine receptors without causing a direct influence of gamma-aminobutyric acid (GABA) receptors. The GABA-benzdiazepine complex was separated by solubilization with 0.5% lubrol PX. The solubilized preparations contain the binding sites for [3H]flunitrazepam alone (Kd = 5.9 nm). The Kd value for the membrane-bound benzdiazepine receptor is 2.9 nM. NAD inhibited the specific binding of [3H]flunitrazepam to the solubilized membrane preparation when used at concentrations by several orders of magnitude lower than that of nicotinamide. Using gel filtration on Sepharose 6B-CL, the molecular mass of the soluble benzdiazepine receptor protein was determined.     

Substantia Nigra γ-Aminobutyric Acid Receptors in Huntington''s Disease

The specific binding of [3H]gamma-aminobutyric acid (GABA) to nigral GABA receptors has been studied in postmortem brains from controls and patients with Huntington's disease (HD). A specific increase in the number of high-affinity binding sites for [3H]GABA was observed in HD patients, analogous to changes observed in rat substantia nigra [3H]GABA binding after striatal kainic acid (KA) lesion. The results provide further support for the striatal KA lesion in the rat as an animal model of HD. The implications of the results for the proposed therapeutic potential of GABA agonists in HD are discussed.     

Phospholipase A2 in macrophage plasma membrane releases arachidonic acid from phosphatidylinositol

A high level of arachidonic acid release from [2-14C]arachidonylphosphatidylinositol (PI) was observed at neutral pH (6.0-7.0) in the presence of purified plasma membranes of guinea pig peritoneal macrophages. This activity was at least 10-fold higher than that with arachidonylphosphatidylcholine (PC) or phosphatidylethanolamine (PE) as substrate. The accumulation of [14C]diacylglycerol and [14C]phosphatidic acid was not detected at any time, and arachidonic acid release from [14C]arachidonyldiacylglycerol was not detectable either. The data suggest that arachidonic acid release from PI may not occur via the phospholipase C pathway. In this paper, we demonstrate the possibility that arachidonic acid release from PI at neutral pH in the macrophage plasma membrane is dependent on the action of phospholipase A2 (EC 3.1.1.4) -like activity. The maximum arachidonic acid release was dependent upon both pH and substrate. Particularly, the activity of arachidonic acid release from PI at neutral pH was very high compared with that from PC or PE. We suggest that phosphatidylinositol phospholipase A2 (EC 3.1.1.52) may play an important role in providing arachidonic acid for subsequent metabolic activity in the macrophages.     

Two Saturable Recognition Sites for (-)[125I]Iodo-N6-(4-Hydroxyphenyl-Isopropyl)-Adenosine Binding on Purified Cardiac Sarcolemma

Abstract

Analysis of (-)[¹²⁵]iodo-N⁶-(4-hydroxyphenylisopropyl)-adenosine ([¹²⁵I]HPIA) binding to purified sarcolemmal preparations of guinea pig and bovine hearts revealed two classes of binding sites when unlabeled iodo-HPIA (100 μmol/1) was used as non-specific binding marker. In the presence of 1 mmol/1 theophylline, however, only the high affinity component was detected. Adenosine receptor agonists caused biphasic displacement of [¹²⁵I]HPIA binding, with a high affinity potency rank order typical of interaction with A₁-adenosine receptors. Biphasic competition curves were also observed with 8-phenyltheophylline and isobutylmethylxanthine, whereas the theophylline curve was monophasic up to 1 mmol/1. In brain membranes, specific binding of [¹²⁵I]HPIA as well as of [³H]PIA was further reduced when unlabeled iodo-HPIA replaces theophylline as the non-specific binding marker. These results suggest the presence of two [¹²⁵I]HPIA binding sites on cardiac sarcolemma and brain membranes, but receptor function can only be ascribed to the high affinity sites. The low affinity site probably represents an artefact, which is often observed when non-specific binding is defined with the unlabeled counterpart or a structurally related ligand of the radioligand used.     

Modulation of [3H]Diazepam Binding in Rat Cortical Membranes by GABAA Agonists

GABAA receptor agonists modulate [3H]diazepam binding in rat cortical membranes with different efficacies. At 23 degrees C, the relative potencies for enhancement of [3H]diazepam binding by agonists parallel their potencies in inhibiting [3H]gamma-aminobutyric acid [( 3H]GABA) binding. The agonist concentrations needed for enhancement of [3H]diazepam binding are up to 35 times higher than for [3H]GABA binding and correspond closely to the concentrations required for displacement of [3H]bicuculline methochloride (BMC) binding. The maximum enhancement of [3H]diazepam varied among agonists: muscimol = GABA greater than isoguvacine greater than 3-aminopropane sulphonic acid (3APS) = imidazoleacetic acid (IAA) greater than 4,5,6,7-tetrahydroisoxazolo (4,5,6)-pyridin-3-ol (THIP) = taurine greater than piperidine 4-sulphonic acid (P4S). At 37 degrees C, the potencies of agonists remained unchanged, but isoguvacine, 3 APS, and THIP acquired efficacies similar to GABA, whereas IAA, taurine, and P4S maintained their partial agonist profiles. At both temperatures the agonist-induced enhancement of [3H]diazepam binding was reversible by bicuculline methobromide and by the steroid GABA antagonist RU 5135. These results stress the importance of studying receptor-receptor interaction under near-physiological conditions and offer an in vitro assay that may predict the agonist status of putative GABA receptor ligands.