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.     

Effect of Halide Ions on t-[35S]Butylbicyclophosphorothionate Binding

The binding of t-[35S]butylbicyclophosphorothionate [( 35S]TBPS) to a site on the GABAA receptor complex is ion dependent. This study was conducted to determine the effects of ion species and concentration on the time course, affinity, and number of sites of [35S]TBPS binding. At a concentration of 200 mM ion, the time to equilibrium for [35S]TBPS binding was shortest for I-, followed by Br- less than Cl- less than F-. A similar rank order was observed for the concentration of ion required to produce half-maximal [35S]TBPS binding. Saturation binding experiments were conducted to evaluate the effect of increasing ion concentration on the KD and Bmax of [35S]TBPS binding. The Bmax was independent of both ion species and concentration. The receptor affinity, however, increased with increasing concentration for each ion. Calculated maximal affinity values were not different between ions; however, the EC50 to produce those values was different among ions and ranked in the same order as that for time course and maximal binding data. Association and dissociation rates for [35S]TBPS binding were greater in I- than in Cl-. These data emphasize the importance of ion selection and incubation times on [35S]TBPS binding.     

t-[35S]Butylbicyclophosphorothionate Binding under Equilibrium and Nonequilibrium Conditions: Differential Effects of Barbiturates and γ-Aminobutyric Acid in the Long-Sleep and Short-Sleep Selected Mouse Lines

Significant differences were demonstrated between the long-sleep (LS) and short-sleep (SS) selected mouse lines in the abilities of barbiturates and gamma-aminobutyric acid (GABA) to inhibit t-[35S]butylbicyclophosphorothionate [( 35S]TBPS) binding to well-washed cerebral cortical membranes. Thus, using phenobarbital to initiate the dissociation of [35S]TBPS, the extent of inhibition was significantly greater in LS mice (but not SS mice) than would be predicted using equilibrium conditions. Pentobarbital had the opposite effect, causing [35S]TBPS to dissociate to a greater extent in SS than LS membranes. [35S]TBPS binding was dissociated from LS and SS membranes by GABA to a greater and lesser extent, respectively, than would be predicted from equilibrium studies. Because no line differences in the potencies of these drugs to inhibit [35S]TBPS binding were found using equilibrium conditions, these results indicate that the association rates of barbiturates and GABA may be different between these lines. These findings are consistent with neurochemical studies indicating differences in the benzodiazepine/GABA receptor-chloride channel complex in these selected lines and may explain their differential sensitivities to certain agents acting through this supramolecular complex.     

Age-Related Loss of t-[35S]Butylbicyclophosphorothionate Binding to the γ-Aminobutyric AcidA Receptor-Coupled Chloride Ionophore in Rat Cerebral Cortex

Muscimol and t-butylbicyclophosphorothionate (TBPS) are known to label two distinct sites within the gamma-aminobutyric acidA (GABAA) receptor complex, i.e., the GABA recognition site and the chloride ionophore, respectively. Age-dependent changes in the specific binding of [3H]muscimol and [35S]TBPS were compared in membranes prepared from the cerebral cortex of rats, 2-800 days old. Perinatal (day 2) binding of muscimol and TBPS represented 8 and 20% of the respective values for adults (day 180). After the first week, muscimol binding increased more rapidly than TBPS binding. Levels near those of adults were reached at day 20 and remained practically unchanged in adulthood (day 180). In aged (780-day-old) rats, the binding of TBPS was significantly reduced, whereas muscimol binding did not change compared with adult values. This decrease of TBPS binding derived from a reduced density of binding sites, rather than from affinity changes. The allosteric responsiveness of TBPS binding to exogenous GABA was also reduced in aged animals. These findings indicate an age-related change in the molecular (structural) organization of the GABAA receptor-chloride ionophore complex in rat cerebral cortex.     

Interaction of t-Butylbicyclophosphorothionate with γ-Aminobutyric Acid-Gated Chloride Channels in Cultured Cerebral Neurons

The role of t-butylbicyclophosphorothionate (TBPS) as an antagonist of gamma-aminobutyric acid (GABA) was studied with primary cultures of neurons from the chick embryo cerebrum. The addition of GABA stimulated the uptake of 36Cl- by neurons and the dose dependence of this effect followed hyperbolic kinetics with a K0.5 = 1.3 microM for GABA. TBPS proved to be a potent inhibitor of GABA-dependent Cl- uptake (IC50 = 0.30 microM). Analysis of the kinetics of this process revealed that TBPS is a noncompetitive inhibitor (Ki = 0.15 microM) with respect to GABA. Scatchard analysis of direct binding of [35S]TBPS to membranes isolated from neuronal cultures gave curvilinear plots. These could be resolved by nonlinear regression methods into two components with KD values of 3.1 nM and 270 nM. The TBPS binding constant for this lower affinity site agreed well with the IC50 and Ki values for inhibition of Cl- flux, suggesting that this site is physiologically relevant to GABA antagonism. GABA was a noncompetitive displacer of [35S]TBPS binding to the lower affinity site. The Ki value for this displacement by GABA (1.7 microM) was comparable to the value for GABA enhancement of Cl- flux. The binding of [35S]TBPS to its low-affinity site on neuronal membranes was ninefold higher in the presence of Cl- than with gluconate, an impermeant anion. The rank order for anion stimulation of [35S]TBPS binding was Br- greater than or equal to SCN- greater than Cl- greater than or equal to NO3- greater than I- greater than F- greater than gluconate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of [3H]Ro 15–1788 Binding to Membrane-Bound Rat Brain Benzodiazepine Receptors

Abstract: Ro 15–1788 is thought to interact specifically with the benzodiazepine receptor population in the mammalian CNS as an antagonist. We have compared the kinetics of interaction of this ligand with the benzodiazepine agonist flunitrazepam in the rat cerebellum. The association of [³H]Ro 15–1788 with the benzodiazepine receptor in this brain region is monoexponential, and the dissociation, initiated either by dilution or by displacement with a number of different ligands, also obeys simple monoexponential kinetics. There is no evidence of cooperative interactions with this ligand, and its dissociation is unaffected by the presence of 100 μ M γ-aminobutyric acid and/or 150 m M sodium chloride. In contrast, the dissociation of the agonist [³H]flunitrazepam is biphasic, and the possible interpretation of this data in terms of agonist-induced conformational change is discussed. Evidence is also presented that the mechanism of interaction of Ro 15–1788 with the benzodiazepine receptor population in hippocampal membranes is distinct from that found in the cerebellum.     

t-[35S]Butylbicyclophosphorothionate Binding Sites in Invertebrate Tissues

Specific high affinity binding of the cage convulsant t-[35S]butylbicyclophosphorothionate (TBPS) was observed in membrane homogenates of housefly heads and crayfish abdominal muscles. [35S]TBPS binding in these two invertebrate tissues was inhibited by biologically active cage convulsants, picrotoxin analogs, and barbiturates. The housefly binding sites were inhibited most potently by several insecticides. Approximately 50% of total binding was displaceable by excess (0.1 mM) nonradioactive TBPS, picrotoxinin, ethyl bicyclophosphate, or dieldrin. Optimal binding assay conditions for housefly homogenates included pH 7.5, 22 degrees C temperature, 0.3 M chloride concentration, and incubation for 60 min; for crayfish homogenates, 4 degrees C temperature and 150-min incubations were optimal. Scatchard plots of equilibrium binding indicated one site in both tissues (KD = 50 nM, Bmax = 250 fmol/mg protein in housefly; KD = 25 nM, Bmax = 100 fmol/mg protein in crayfish). Association kinetics in housefly were consistent with one rate constant (k+1 = 8 X 10(6) M-1 min-1), but dissociation was described better by two rate constants (k-1 = 0.28 min-1 and 0.042 min-1; calculated KD values of 80 nM and 12 nM). Displacement by cage convulsants showed Hill numbers near 0.5, also consistent with two populations of affinity, while displacement by other drugs showed Hill numbers near 1.0. [35S]TBPS binding in insects was most potently inhibited by the insecticides dieldrin (IC50 = 50 nM), aldrin, and lindane (200 nM), in a stereospecific manner, consistent with this binding site being the receptor for biological toxicity. [35S]TBPS binding was also inhibited by relatively high concentrations of some pyrethroid insecticides, such as deltamethrin and cypermethrin (1-2 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Foot-Shock Stress and Anxiogenic β-Carbolines Increase t-[35S]Butylbicyclophosphorothionate Binding in the Rat Cerebral Cortex, an Effect Opposite to Anxiolytics and γ-Aminobutyric Acid Mimetics

The effect of foot-shock stress on t-[35S]butylbicyclophosphorothionate [( 35S]TBPS) binding to fresh unwashed membrane preparations from rat cerebral cortex was studied and was compared to those of GABAA receptor agonists and antagonists and to positive and negative modulators of the GABAergic transmission. [35S]TBPS binding was increased in the cerebral cortex of rats exposed to foot shock compared to that of nonstressed rats. Scatchard analysis revealed that the effect of foot shock was due to an increase in the total number of [35S]TBPS binding sites. In contrast, the in vitro addition of muscimol or GABA induced a dose-dependent inhibition of [35S]TBPS binding, an effect abolished by the concomitant addition of the GABA receptor antagonist, bicuculline, which, per se, enhanced [35S]TBPS binding by 73%. Thus, bicuculline, similar to stress, increased [35S]TBPS binding in the same membrane preparation. In contrast to stress, the anxiolytic and positive modulators of the GABAergic transmission (ZK 93423, ZK 91296, and diazepam) inhibited the specific binding of [35S]TBPS in a concentration-dependent manner. The greatest inhibitory effect was produced by ZK 93423 at 30 microM (31% of control), followed by diazepam (54% of control) and by the partial agonist ZK 91296 (61% of control). Scatchard plot analysis indicated that the inhibition induced by ZK 93423 and diazepam was due to a decrease in the density of [35S]TBPS recognition sites. On the other hand, the anxiogenic beta-carbolines DMCM and FG 7142 mimicked the effect of stress. Thus, at a 10 microM concentration, DMCM and FG 7142 increased [35S]TBPS binding by 22% and 26%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding Characteristics of t-[35S]Butylbicyclophosphorothionate in Discrete Brain Regions of Rats Made Tolerant to and Dependent on Pentobarbital

The effects of acute and continuous pentobarbital administration by pellet implantation on binding characteristics of t-[35S]butylbicyclophosphorothionate ([35S]TBPS) in discrete regions of rat brains were examined. Acute administration of pentobarbital (60 mg/kg, s.c.) affected neither the KD nor the Bmax values of [35S]TBPS binding in any of the regions studied. The cerebella of pentobarbital-tolerant rats had an increased density of [35S]TBPS binding sites with no change in their apparent affinity. There were no significant changes in the binding characteristics in the frontal cortex (FC), the striatum (ST), and the substantia nigra (SN) of these animals. Twenty-four hours after removal of the pentobarbital pellets, a significant decrease in the latency of onset of first twitch response induced by pentylenetetrazol (PTZ) (50 mg/kg, i.p.) was observed. In addition, the density of [35S]TBPS binding sites was significantly increased in the FC, the SN, and the cerebellum but not in the ST. In all brain regions studied, placebo pellet implantation and pentobarbital tolerance and dependence caused no changes in the apparent affinity of [35S]TBPS binding or the IC50 of pentobarbital for the inhibition of [35S]TBPS binding. These results suggest that [35S]TBPS binding was significantly increased following the withdrawal of the pentobarbital pellets without altering intrinsic coupling activity of barbiturate recognition sites and convulsant binding sites and that these increases in [35S]TBPS binding are related to the increased susceptibility to seizures induced by PTZ in rats made dependent on pentobarbital.     

t-[3H]Butylbicycloorthobenzoate: New Radioligand Probe for the γ-Aminobutyric Acid–Regulated Chloride Ionophore

t-[3H]Butylbicycloorthobenzoate [( 3H]TBOB; 22 Ci/mmol) was prepared by reductive dechlorination of its 4-chlorophenyl analog with tritium gas. This new radioligand binds reversibly to fresh washed rat brain P2 membranes in 500 mM NaCl plus 50 mM sodium-potassium phosphate buffer (pH 7.4) at 25 degrees C, with 80-90% specific relative to total binding, a KD of 61 +/- 15 nM, and a Bmax of 1.6 +/- 0.5 pmol/mg of protein. [3H]TBOB association with its binding site(s) is monophasic, but its dissociation is biphasic. The binding characteristics of [3H]TBOB are essentially identical to those of t-[35S]butylbicyclophosphorothionate [( 35S]TBPS) with respect to pH dependence, stimulation by anions, regional distribution in the brain, and pharmacological profile. Saturation analyses and dissociation studies further indicate that TBOB and TBPS have a common binding site. However, binding of the two radioligands differs in respect to temperature effects. In contrast to [35S]TBPS, which exhibits negligible binding at 0 degrees C, [3H]TBOB binds to rat brain membranes at 0, 25, and 37 degrees C with similar KD values. [3H]TBOB with its long radioactive half-life and temperature-independent KD is a valuable supplement to [35S]TBPS in further biochemical and pharmacological characterization of the gamma-aminobutyric acid receptor-ionophore complex.     

Temperature Dependence of [3H]Ro 15–1788 Binding to Benzodiazepine Receptors in Human Postmortem Brain Homogenates

The temperature dependence of in vitro binding of [3H]Ro 15-1788 to benzodiazepine receptors in human postmortem neocortex and neocerebellum homogenates was studied. An increase of the equilibrium dissociation constants (KD) from 1.40 nmol/L and 1.04 nmol/L at 4 degrees C to 6.10 nmol/L and 8.91 nmol/L at 37 degrees C was found for neocortex and neocerebellum, respectively. In contrast, maximal binding (Bmax) remained in the range of 30-35 fmol/mg for neocortex and 24-27 fmol/mg of tissue (wet weight) for neocerebellum at all the temperatures. The KD of 6.10 nmol/L for neocortex at 37 degrees C in vitro is of the same order as the KD of 10 nmol/L obtained by positron emission tomography for [11C]Ro 15-1788 binding to benzodiazepine receptors in the human neocortex in vivo. The differences in KD between in vitro and in vivo benzodiazepine receptor binding to human neocortex and cerebellum seem to be due at least partially to temperature differences of in vitro and in vivo studies.     

Comparison of Tryptic Peptides of Benzodiazepine Binding Proteins Photolabeled with [3H]Flunitrazepam or [3H]Ro 15–4513

When rat brain membranes were incubated with the benzodiazepine agonist [3H]flunitrazepam or the partial inverse benzodiazepine agonist [3H]Ro 15-4513 in the presence of ultraviolet light one protein (P51) was specifically and irreversibly labeled in cerebellum and at least two proteins (P51 and P55) were labeled in hippocampus. After digestion of the membranes with trypsin, protein P51 was degraded into several peptides. When P51 was photolabeled with [3H]Ro 15-4513, four peptides with apparent molecular weights of 39,000, 29,000, 21,000, and 17,000 were observed. When P51 was labeled with [3H]flunitrazepam, only two peptides with apparent molecular weights of 39,000 and 25,000 were obtained. Protein P55 was only partially degraded by trypsin, and whether it was labeled with [3H]flunitrazepam or [3H]Ro 15-4513 it yielded the same two proteolytic peptides with apparent molecular weights of 42,000 and 45,000. These results support the existence of at least two different benzodiazepine receptor subtypes associated with proteins P51 and P55. The different receptors seem to be differentially protected against treatment with trypsin. In addition, these results indicate that in the benzodiazepine receptor subtype associated with P51 benzodiazepine agonists and partial inverse benzodiazepine agonists irreversibly bind to different parts of the molecule.     

CL 218,872 Binding to Benzodiazepine Receptors in Rat Spinal Cord: Modulation by γ-Aminobutyric Acid and Evidence for Receptor Heterogeneity

The binding of the triazolopyridazine CL 218,872 to central benzodiazepine receptors identified with [3H]Ro 15-1788 was studied in extensively washed homogenates of rat spinal cord and cerebral cortex. CL 218,872 displacement curves were shallow in both spinal cord (nH = 0.67) and cortex (nH = 0.54), suggesting the presence of type 1 and type 2 benzodiazepine receptors in both tissues. CL 218,872 had lower affinity in spinal cord (IC50 = 825 nM) than cortex (IC50 = 152 nM), possibly reflecting the presence of fewer type 1 sites in the cord. Activating gamma-aminobutyric acid (GABA) receptors with 10 microM muscimol resulted in a two- to threefold increase in CL 218,872 affinity in both tissues without changes in the displacement curve slope. This indicates that GABA enhances CL 218,872 affinity for both type 1 and type 2 sites in both spinal cord and cerebral cortex.     

Multiple [35S]t-Butylbicyclophosphorothionate Binding Sites in Rat and Chicken Cerebral Hemispheres

Specific binding of [35S]t-butylbicyclophosphorothionate (TBPS) to membranes from cerebral hemispheres of adult rat and chicken was determined over a range of radioligand concentrations from 0.25 to 500 nM. Scatchard plots of these data were curvilinear and nonlinear regression analysis indicated binding to two sites that differ in affinity. For rat cerebrum, KD(1) = 1.15 nM, Bmax(1) = 0.085 pmol/mg; KD(2) = 232 nM, Bmax(2) = 16.9 pmol/mg. For chicken cerebrum, KD(1) = 1.39 nM, Bmax(1) = 0.111 pmol/mg; KD(2) = 166 nM, Bmax(2) = 17.6 pmol/mg. This multiplicity of [35S]TBPS binding was further confirmed when unlabeled TBPS or picrotoxinin displaced radioligand. The displacement curves were biphasic and yielded Hill coefficients from 0.65 to 0.70. These displacement curves were also resolved into two components with distinct IC50 values for unlabeled TBPS (rat, 1.55 and 271 nM; chicken, 2.40 and 224 nM). The IC50 values were similar to the dissociation constants obtained from equilibrium binding measurements.     

Lindane Administration to the Rat Induces Modifications in the Regional Cerebral Binding of [3H]Muscimol, [3H]-Flunitrazepam, and t-[35S]Butylbicyclophosphorothionate: An Autoradiographic Study

Abstract: The effect of lindane administration on the specific binding of ligands to different sites on the GABA_A receptor-ionophore complex was studied in the rat brain by receptor mapping autoradiography. [³H]Muscimol (Mus), [³H]flunitrazepam (Flu), and t -[³⁵S]butylbicyclophosphorothionate (TBPS) were used as specific ligands of GABA, benzodiazepine, and picrotoxinin binding sites, respectively. Rats received a single oral dose of 30 mg/kg lindane and they were classified into two groups according to the absence or presence of convulsions. Vehicle-treated groups acted as controls. The effect of the xenobiotic on ligand binding was measured in different brain areas and nuclei 12 min or 5 h after its administration. Lindane induced a generalized decrease in [³⁵S]TBPS binding, which was present shortly after dosing. In addition, [³H]Flu binding was increased in lindane-treated animals, this modification also appearing shortly after administration but diminishing during the studied time. Finally, lindane induced a decrease in [³H]Mus binding, which became more evident over time. These modifications were observed both in the presence and in the absence of convulsions. However, an increase in [³H]-Mus binding was detected shortly after lindane-induced convulsions. The observed decrease in [³⁵S]TBPS binding is in agreement with the postulated action of lindane at the picrotoxinin binding site of the GABA_A receptor chloride channel. The effects observed on the binding of [³H]Flu and [³H]Mus may be secondary to the action of lindane as an allosteric antagonist of the GABA_A receptor.     

γ-Aminobutyric Acid and Pentobarbital Enhance 2-[3H]Oxoquazepam Binding to Type I Benzodiazepine Recognition Sites in Rat and Human Brain

2-Oxoquazepam (2oxoquaz) is a novel benzodiazepine which shows preferential affinity for type I benzodiazepine recognition sites. In the present study, we analyzed the effect of gamma-aminobutyric acid (GABA), pentobarbital, and chloride ions on [3H]2oxoquaz and [3H]flunitrazepam ( [3H]FNT) binding to membrane preparations from rat and human brain. GABA stimulated [3H]-2oxoquaz and [3H]FNT binding in a concentration-dependent manner. The maximal enhancement produced by GABA on [3H]2oxoquaz binding was higher than that produced on [3H]FNT binding in both rat and human tissues. In the rat brain, the effect of GABA on [3H]2oxoquaz was similar throughout different brain areas, whereas the effect on [3H]FNT binding was lower in the cerebral cortex and hippocampus than in the cerebellum. Moreover, both [3H]2oxoquaz and [3H]FNT binding were stimulated by chloride ions and pentobarbital. The results are consistent with the hypothesis that type I benzodiazepine recognition sites are linked functionally to the GABA recognition site and the chloride ionophore.     

γ-Aminobutyric Acid-Activated 36Cl- Influx: A Functional In Vitro Assay for CNS γ-Aminobutyric Acid Receptors of Insects

The effects of gamma-aminobutyric acid (GABA) on the uptake of 36Cl- into a membrane microsac preparation from isolated nerve cords of the cockroach Periplaneta americana was studied. On addition of 1 microM GABA (after 4-s incubation, then rapid quenching) the influx of 36Cl- was stimulated to a level 75% above that of the control value. This stimulation was reduced by picrotoxin (100 microM), but was not significantly affected by bicuculline (100 microM). Results of 36Cl- influx experiments are in agreement with data obtained from radiolabelled ligand binding assays and electrophysiological investigations on the same tissue. The method described represents a functional in vitro assay for CNS GABA receptors of insects.     

Binding Characteristics and Interactions of Depressant Drugs with [35S]t-Butylbicyclophosphorothionate, a Ligand that Binds to the Picrotoxinin Site

Abstract: [³⁵S]r-Butylbicyclophosphorothionate (TBPT), a cage convulsant with picrotoxinin-like activity, binds to rat brain membranes to a single site with an apparent K_D of 25.1 ± 5.6 n M and a B_max of 1.40 ± 0.22 pmol/mg protein. TBPT binding to rat brain membranes was inhibited by a variety of convulsant, depressant, anxiolytic, and anticonvulsant drugs that had previously been shown to inhibit [³H]a-dihydropicrotoxinin binding. Depressant drugs such as pentobarbital and the nonbarbiturate (+)etomidate inhibited TBPT binding in an uncompetitive manner. Thus, pentobarbital and (+)etomidate decreased both the affinity and the number of binding sites of TBPT to whole brain membranes. The IC₅₀ values of (+)etomidate (9 μ M ) and pentobarbital (90 μ M ) are similar to the EC₅₀ values at which they enhance both [³H]-γ-aminobutyric acid and [³H]diazepam binding in cerebral cortex membranes. RO5–4864, which has recently been shown to be a convulsant, also inhibited TBPT binding (IC₅₀= 10 μ M ). These results suggest that TBPT binds to the picrotoxinin site and further supports the notion that the picrotoxinin site is an important modulatory site at the benzodiazepine-GABA receptor-ionophore complex.     

Differences in the Biophysical Properties of the Benzodiazepine/γ-Aminobutyric Acid Receptor Chloride Channel Complex in the Long-Sleep and Short-Sleep Mouse Lines

Significant differences in the thermal stability of benzodiazepine receptors were found in cerebral cortical membranes prepared from the long-sleep (LS) and short-sleep (SS) selected mouse lines. Thus, benzodiazepine receptors from LS mice were heat inactivated (55 degrees C) at a significantly faster rate than those from SS mice. Although gamma-aminobutyric acid (GABA) reduced the rate of heat inactivation in both lines, the more rapid rate of inactivation in the LS line was maintained. Furthermore, the potency of GABA to enhance [3H]flunitrazepam binding decreased threefold in membranes from LS mice as the incubation temperature was increased from 0 degrees to 37 degrees C, but was unaltered in membranes from SS mice. These differences in the biophysical properties of the benzodiazepine/GABA receptor chloride channel complex ("supramolecular complex"), together with a higher KD for t-[35S]butylbicyclophosphorothionate in membranes from LS compared to SS mice, suggest that the supramolecular complex may modulate the differential sensitivity to some depressants and convulsants in these lines.     

Endomorphin-Stimulated [35S]GTPγS Binding in Rat Brain: Evidence for Partial Agonist Activity at μ-Opioid Receptors

Abstract: Endomorphin-1 is a peptide whose binding selectivity suggests a role as an endogenous ligand at μ-opioid receptors. In the present study, the effect of endomorphin-1 on μ receptor-coupled G proteins was compared with that of the μ agonist DAMGO by using agonist-stimulated [³⁵S]GTPγS binding in rat brain. [³⁵S]GTPγS autoradiography revealed a similar localization of endomorphin-1 and DAMGO-stimulated [³⁵S]GTPγS binding in areas including thalamus, caudate-putamen, amygdala, periaqueductal gray, parabrachial nucleus, and nucleus tractus solitarius. Naloxone blocked endomorphin-1-stimulated labeling in all regions examined. Although the distribution of endomorphin-1-stimulated [³⁵S]GTPγS binding resembled that of DAMGO, the magnitude of endomorphin-1-stimulated binding was significantly lower than that produced by DAMGO. Concentration-effect curves of endomorphin-1 and DAMGO in thalamic membranes confirmed that endomorphin-1 produced only 70% of DAMGO-stimulated [³⁵S]GTPγS binding. Differences in maximal stimulation of [³⁵S]GTPγS binding between DAMGO and endomorphin-1 were magnified by increasing GDP concentrations, and saturation analysis of net endomorphin-1-stimulated [³⁵S]GTPγS binding revealed a lower apparent B _max value than that obtained with DAMGO. Endomorphin-1 also partially antagonized DAMGO stimulation of [³⁵S]GTPγS binding. These results demonstrate that endomorphin-1 is a partial agonist for G protein activation at the μ-opioid receptor in brain.