Characterization of the binding of [3H]-CGS 19755: a novel N-methyl-D-aspartate antagonist with nanomolar affinity in rat brain.

1. CGS 19755 (cis-4-phosphonomethyl-2-piperidine carboxylic acid), a rigid analogue of 2-amino-5-phosphonopentanoic acid (AP5), is one of the most potent competitive N-methyl-D-aspartate (NMDA) antagonists described. Using Triton-treated crude synaptic membranes from rat brain, binding studies indicated that [3H]-CGS 19755 bound with high affinity and selectivity to the NMDA-type excitatory amino acid receptor. 2. [3H]-CGS 19755 binding was saturable, reversible, heat-labile, pH-dependent and linear with protein concentration. Specific binding represented 80-85% of the total amount bound. 3. Using a centrifugation assay, saturation experiments revealed two distinct binding components with Kd values of 9 and 200 nM, and corresponding Bmax values of 0.55 and 1.00 pmol mg-1 protein. In contrast, a single binding component with a Kd value of 24 nM and an apparent Bmax value of 0.74 pmol mg-1 protein was observed with a filtration assay. 4. Competition experiments in which both assay techniques were used, showed that [3H]-CGS 19755 selectively labels the NMDA receptor. The most active inhibitors of [3H]-CGS 19755 binding were L-glutamate and CGS 19755 (IC50 values = 100 nM). 5. In the centrifugation assay, a number of excitatory amino acids were found to generate shallow inhibition curves, and computer analysis indicated the presence of two binding components. The quisqualate receptor ligand AMPA (D,L-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate), kainic acid and the non-competitive NMDA antagonists, such as phencyclidine, tiletamine and MK-801, were without activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

High affinity binding of [3H]ethylketocyclazocine to rat brain homogenate

Ethylketocyclazocine (Ekc), a potent κ-receptor agonist, binds to opiate receptor sites in rat brain tissue. The binding was saturable with respect to the concentration of [³H]Ekc. The dissociation constant of Ekc-receptor complex was 1.8 nM and the maximum number of binding sites in the whole brain was 9.6 pmol/g of tissue. Opiate agonists and antagonists have a high affinity for [³H]Ekc binding sites, but results of present investigations failed to differentiate opiates thought to be specific for the μ- and κ-receptors.     

High affinity and stereospecific binding of [3H]d-amphetamine to rat brain

Treatment for 21 days but not 3 days with clorgyline, a selective monoamine oxidase type A inhibitor with antidepressant effects, causes significant escape from clonidine's normal suppressant effects on locomotion in the rat. The effect is accompanied by a 57% decrease in [³H]yohimbine binding in cerebral cortical membranes. The data support and extend prior reports of the development of subsensitivity in the inhibitory presynaptic noradrenergic system as an important component of antidepressant efficacy.     

[3H]Tryptamine: High affinity binding sites in rat brain

[³H]Tryptamine binds to rat brain cortex. The binding is saturable and of high affinity. Drug displacement studies indicate that the binding site has a high degree of specificity for tryptamine and that the site is distinct from serotonin receptor binding sites.     

A quantitative study of [3H]D-Ala2-D-Leu5-enkephalin binding to rat brain membranes. Evidence that oxymorphone is a noncompetitive inhibitor of the lower affinity delta-binding site

The mechanism by which mu ligands inhibit the binding of prototypic delta agonists to preparations of brain membranes is controversial. Most investigators assume competitive inhibition. In this study, we examine the interaction of the mu agonist oxymorphone and delta agonist DSTLE (D-Ser2-Thr6-Leu-enkephalin) with [3H]D-Ala2-D-Leu5-enkephalin (DADL) binding to membranes of rat brain. According to the two-site competitive model, mu ligands are competitive inhibitors at both sites. The two-site allosteric model supposes that mu ligands are competitive inhibitors at one binding site, and noncompetitive inhibitors at the other binding site. Quantitative analysis of DSTLE and oxymorphone binding demonstrated that the two-site allosteric model fit the data significantly better than did the two-site competitive model, and that oxymorphone is a noncompetitive inhibitor of the lower affinity [3H]DADL-binding site. Autoradiographic studies demonstrated that the lower affinity [3H]DADL-binding site (mu-noncompetitive binding site) had an anatomical distribution apparently indistinguishable from that obtained with [3H]oxymorphone (type I pattern), supporting the hypothesis that the lower affinity delta-binding site is the delta-binding site of an opiate receptor complex consisting of interacting mu- and delta-binding sites.     

3H]CGP 39653: a new N-methyl-D-aspartate antagonist radioligand with low nanomolar affinity in rat brain.

CGP 39653 (D,L-(E)-2-amino-4-propyl-5-phosphono-3-pentenoic acid) was initially discovered to inhibit the binding of [3H]L-glutamate and [3H]3-[+/-)2-carboxypiperazin-4-yl)-propyl-1- phosphonic acid [( 3H]CPP) with Ki values of 230 and 5 nM, respectively. The radiolabeled compound [3H]CGP 39653 binds to rat frontal cortical membranes in a saturable and reversible manner. Analysis of saturation experiments revealed that the ligand labels one binding site with a Kd value of 6 nM. Competition experiments indicated that the order of potency of a number of competitive excitatory amino acid agonist and antagonist compounds was similar to that found previously for other N-methyl-D-aspartate (NMDA) receptor ligands. In contrast to these competitive inhibitors, which produced steep inhibition curves, glycine inhibited binding in a complex manner. When the functional activity of the unlabeled compound was explored, CGP 39653 blocked NMDA-evoked depolarizations in the rat cortical wedge in vitro and inhibited L-glutamate stimulated [3H]N(1-[2-thienyl]cyclohexyl)3,4-piperidine [( 3H]TCP) binding in cortical membranes. These results suggest that [3H]CGP 39653 selectively binds to the NMDA receptor as an antagonist with high affinity and is currently the ligand of choice for labeling the NMDA receptor.     

Identification and initial characterization of high-affinity [3H]dextrorphan binding sites in rat brain

We have identified specific high-affinity [3H]dextrorphan binding sites in rat forebrain. [3H]Dextrorphan binds saturably and reversibly to an apparently homogenous class of sites characterized by a Bmax of 2.62 +/- 0.06 pmol/mg protein and KD of 60 +/- 4 nM. Glycine and glutamate independently increase [3H]dextrorphan binding in a concentration-dependent manner. The pharmacological profiles of [3H]dextrorphan binding characterized by equilibrium competition experiments together with these data suggest that [3H]dextrorphan labels a site at or near the N-methyl-D-aspartate receptor.     

Characterization of the binding of [3H]L-689,560, an antagonist for the glycine site on the N-methyl-D-aspartate receptor, to rat brain membranes.

The binding characteristics of [3H]L-689,560 [(+/-)-4-(trans)-2-carboxy-5,7-dichloro-4-phenylaminocarbonylamino -1,2,3,4- tetrahydroquinoline], a selective antagonist for the glycine site on the N-methyl-D-aspartate receptor, have been evaluated using rat cortex/hippocampus P2 membranes. Specific [3H]L-689,560 binding was saturable, having a Kd of 2.97 nM and a Bmax of 4.15 pmol/mg of protein. The Bmax value was not significantly different from that obtained for [3H]glycine in the same membrane preparation, and L-689,560 and glycine were found to be mutually competitive. The specific binding of [3H]L-689,560 (1 nM) represented 96 +/- 0.02% (four experiments) of total binding. Association experiments at 4 degrees revealed that [3H]L-689,560 reached equilibrium in 120 min, with a t1/2 of 40 min. The dissociation of [3H]L-689,560 was slow at 4 degrees (t1/2 = 118 min), allowing the use of filtration to separate free from bound radioactivity. Both association and dissociation curves were best fitted by a double-exponential function, suggesting the presence of two components. Comparison of IC50 values obtained using [3H]glycine and [3H]L-689,560 binding for 21 glycine site ligands (including agonists, partial agonists, and antagonists, with affinities spanning 5 orders of magnitude) showed a 1:1 correlation, with a correlation coefficient of 0.97. This suggests that efficacy does not have a large influence on the affinity of glycine site ligands when an agonist or antagonist radioligand is used. Ligands for other amino acid recognition sites did not directly inhibit [3H]L-689,560 binding. [3H]L-689,560 is an improved radioligand for the glycine site that will facilitate further investigations of its properties.     

Characterisation of [3H]gabapentin binding to a novel site in rat brain: homogenate binding studies

The binding characteristics of [³H]gabapentin, the radiolabelled analogue of the novel anticonvulsant gabapentin (1-(aminomethyl)cyclohexaneacetic acid) were studied using purified synaptic plasma membranes prepared from rat cerebral cortex. In 10 mM HEPES buffer [³H]gabapentin bound to a single population of sites with high affinity (K_D = 38 ± 2.8 mM) with a maximum binding capacity of 4.6 ± 0.4 pmol/mg protein, reaching equilibrium after 30 min at 20°C. This novel site was unique to the central nervous system with little or no specific [³H]gabapentin binding being measurable in a range of peripheral tissues. Binding was potently inhibited by a range of gabapentin analogues and 3-alkyl substituted γ-aminobutyric acid (GABA) derivatives although GABA itself and the selective GABA_B receptor ligand baclofen, were only weakly active. Gabapentin itself (IC₅₀ = 80 nM) and 3-isobutyl GABA (IC₅₀ = 80 nM) which also has anticonvulsant properties, showed the highest affinity for the binding site. Of a wide range of other pharmacologically active compounds only the polyamines spermine and spermidine influenced [³H]gabapentin binding, with both compounds producing a maximum of 50% inhibition of specific binding. Magnesium ions produced a similar pattern of inhibition but the effect of the polyamines and magnesium ions were not additive. The data provide evidence for the existence in brain of a novel binding site that may mediate the anticonvulsant effects of gabapentin and other potential anticonvulsant compounds.     

Evidence for a displaceable non-specific [3H]neurotensin binding site in rat brain

Summary Levocabastine is a potent antihistamine drug, structurally unrelated to neurotensin. In rat and mouse brain but not in other animal species, it inhibited 60% of the [³H]neurotensin binding displaced by unlabelled neurotensin or neurotensin (8–13).The levocabastine-sensitive site or site 1 displayed high affinity properties for levocabastine (IC₅₀=25 nM) and was highly stereospecific (IC₅₀-value higher than 10 M for one of the isomers). Binding to the site 1 in rat brain corresponded to the [³H]neurotensin binding displaceable by 1 M levocabastine, whereas binding to the site 2 corresponded to the binding displaced by 1 M neurotensin when the site 1 was occluded by 1 M levocabastine.Both site 1 and site 2 appeared to be saturable. Scatchard plots obtained in rat bulbus olfactorius allowed to calculate a K _D-values of 7.1 nM and a B _max-values of 37.2 fmol/mg original tissue for site 1, while site 2 displayed a K _D-value of 0.7 nM and a B _max-value of 16.3 fmol/mg original tissue. The regional distributions of both sites showed marked differences. The site 1 was homogeneously distributed throughout all rat brain areas, whereas the amount of site 2 binding was markedly different in separate brain areas: bulbus olfactorius and substantia nigra had the highest amounts (8.9 and 7.8 fmol/mg tissue) while cerebellum had the lowest (0.4 fmol/mg tissue).In spite of its high affinity and stereospecificity, site 1 has to be considered as an acceptor or recognition site for [³H]neurotensin because of its species-link, low saturability and homogeneous distribution in all rat brain areas.On the other hand, site 2 had the characteristics of a physiological receptor: high affinity, saturability in the low nanomolar range and marked regional distribution in rat brain. Site 2 corresponds therefore most probably to the physiological neurotensin receptor. The foregoing experiments provide evidence for the presence of a drug displaceable, non-specific (=unrelated to a physiological receptor) neurotensin binding site in rat brain; levocabastine should be an important tool to occlude this site in order to reveal, by means of in vitro binding assays, the specific neurotensin binding site in rat brain.     

Polyamines allosterically modulate [3H]nitrendipine binding to the voltage-sensitive calcium channel in rat brain.

The effects of polyamines on radioligand binding to the slow voltage-dependent Ca2+ channel were studied using membranes from the rat cerebral cortex. [3H]Diltiazem binding was inhibited by arcaine (IC50 = 55 microM) and, in decreasing order of potency, by agmatine, spermidine, spermine and putrescine. Under control conditions, only spermidine and spermine allosterically inhibited [3H]nitrendipine binding while arcaine, agmatine and putrescine were inactive. Nevertheless, putrescine antagonized the effect of spermine as well as the allosteric effects of diltiazem and verapamil on the binding of [3H]nitrendipine, in a manner analogous to that shown previously for Ca2+. Thus, polyamines may function as endogenous modulators of the voltage-dependent Ca2+ channel.     

[3H] sertraline binding to rat brain membranes

Tritiated sertraline, a radiolabeled form of a potent and selective inhibitor of serotonin uptake, was found to bind with high affinity to rat whole brain membranes. Characterization studies showed that [³H] sertraline binding occurred at a single site with the following parameters:K _d 0.57 nM,B _max 821 fmol/mg protein,n _h 1.06. This binding was reversible; the dissociation constant calculated from kinetic measurements (K _d 0.81 nM) agreed with that determined by saturation binding experiments. [³H] Sertraline binding in the presence of serotonin, paroxetine, fluoxetine or imipramine suggested competitive inhibition of binding (large increase inK _d with little change inB _max). The rank order of potency of inhibition of [³H] sertraline binding was similar to that of inhibition of serotonin uptake for known uptake inhibitors and the 1-amino-4-phenyltetralin uptake blockers. A marked decrease in ex vivo [³H] sertraline binding in the brain of rats 7 days after treatment withp-chloroamphetamine was consistent with the loss of serotonin uptake sites induced by this agent. The results of our study indicated that [³H] sertraline labels serotonin uptake sites in rat brain.     

High affinity binding of [3H]paroxetine and [3H]imipramine to human platelet membranes

Paroxetine, one of the most potent and specific serotonin uptake inhibitors, was tritiated and used for binding studies with human platelet membranes. Specific, high affinity binding was demonstrated. The binding was compared with [3H]imipramine binding; it was found that the maximal binding (Bmax) was the same for [3H]paroxetine and [3H]imipramine, whereas the affinity was much higher for [3H]paroxetine (KD 0.08 nM and 0.56 nM for paroxetine and imipramine binding, respectively). IC50 was calculated for the inhibition of [3H]paroxetine and [3H]imipramine binding by a number of antidepressants; the corresponding Hill coefficients were also calculated.     

Possible involvement of membrane phospholipids in inhibition by ferrous ions of [3H]MK-801 binding to the native N-methyl-D-aspartate channel in rat brain.

Prior treatment with ferrous chloride led to the marked inhibition of [3H](+)-5-methyl-10, 11-dihydro-5H-dibenzo[a, d]cyclohepten-5, 10-imine (MK-801) binding to an open ion channel associated with the N-methyl-D-aspartate (NMDA) receptor in a concentration-dependent manner at concentrations of higher than 1 microM in rat brain synaptic membranes. Both phospholipases A2 and C significantly prevented the inhibition when treated before the treatment with ferrous chloride, while neither superoxide dismutase nor alpha-tocopherol affected the inhibition even when treated simultaneously with ferrous chloride. Of various saturated and unsaturated fatty acids, moreover, both oleic and arachidonic acids exclusively decreased the potency of ferrous chloride to inhibit binding when membranes were treated with fatty acids, followed by a second treatment with ferrous chloride. These results suggest that membrane phospholipids may be, at least in part, responsible for the interference by ferrous ions in the opening processes of the native NMDA channel through molecular mechanisms associated with the release of unsaturated fatty acids in rat brain.     

Polyamines allosterically modulate [3H]nitrendipine binding to the voltage-sensitive calcium channel in rat brain

The effects of polyamines on radioligand binding to the slow voltage-dependent Ca²⁺ channel were studied using membranes from the rat cerebral cortex. [³H]Diltiazem binding was inhibited by arcaine (IC₅₀ = 55 μM) and, in decreasing order of potency, by agmatine, spermidine, spermine and putrescine. Under control conditions, only spermidine and spermine allosterically inhibited [³H]nitrendipine binding while arcaine, agmatine and putrescine were inactive. Nevertheless, putrescine antagonized the effect of spermine as well as the allosteric effects of diltiazem and verapamil on the binding of [³H]nitrendipine, in a manner analogous to that shown previously for Ca²⁺. Thus, polyamines may function as endogenous modulators of the voltage-dependent Ca²⁺ channel.     

High and low affinity binding of [3H]cholate to rat liver plasma membranes

The transport of bile acids across sinusoidal and canalicular membranes of hepatocytes is characterized as carrier mediated. Such a carrier should specifically bind bile acids at physiological concentrations. We examined the binding of [3H]cholate to rat liver plasma membranes using a microcentrifugation technique and detected high (KD = 1.23 +/- 0.44 microM, Bmax = 21.8 +/- 3.3 pmol/mg protein) and low (KD = 1.97 +/- 1.33 mM, Bmax = 41.5 +/- 25.3 nmol/mg protein) affinity binding sites. Maximal binding was achieved within 15-45 sec and was stable for 2 min at 37 degrees. Binding to the high affinity site was reversible, was not Na+ dependent or attributable to vesicular uptake, and exhibited a broad pH optimum. Binding to this site was negligible or not detected in liver mitochondrial and microsomal fractions, was saturable, and was inhibited by other bile acids. The IC50 values for bile acids as inhibitors of [3H]cholate binding at the high affinity site were: taurocholate, 1.9 nM; glycodeoxycholate, 3.1 nM; chenodeoxycholate, 5.6 nM; taurochenodeoxycholate, 7.3 nM; glycochenodeoxycholate, 11 nM; lithocholate, 13 nM; taurodeoxycholate, 20 nM; glycocholate, 3.6 microM; and deoxycholate, 5.6 microM. [3H]Cholate specific binding was inhibited by 10(-5) M bromosulfophthalein, bilirubin and indocyanin green. These data support the hypothesis that the high affinity binding site represents a carrier which is shared by bile acids and nonbile acid organic anions.     

Characteristics of [3H]sultopride binding to rat brain

The binding of [3H]sultopride, a benzamide drug, to rat brain was investigated in vitro. Specific [3H]sultopride binding was observed in dopaminergic regions: striatum, nucleus accumbens, olfactory tubercle, substantia nigra, frontal cortex and anterior pituitary. Specific [3H]sultopride binding to striatum was saturable and had one high affinity binding site with a KD of 5.8 nM and a total density of receptors 25.7 pmol/g. [3H]Sultopride binding was stereoselectively displaced by (-)- and (+)-sultopride. Inhibition studies indicated that all neuroleptic drugs and dopamine were capable of displacing sultopride from its binding sites. A highly significant correlation was observed between IC50 values against [3H]sultopride and those against [3H]spiperone binding. Specific [3H]sultopride binding was highly dependent on the presence of sodium ions. The results suggest that the characteristics of sultopride binding sites seem to be similar to those of the D2-receptor labeled by spiperone and haloperidol. The sultopride binding site was highly dependent on the presence of sodium ions and may thus be characterized as a sodium-dependent D2-receptor.     

Characterization of [3H]-imidazenil binding to rat brain membranes.

1. The binding of [3H]-imidazenil, an imidazobenzodiazepine carboxamide, to rat cerebellar membranes was characterized at different temperatures. 2. Specific binding was linear with tissue concentrations and reached maximum after 90, 30 and 5 min incubation at 0, 21 and 37 degrees C, respectively. The binding was of high affinity, specific and saturable; non linear regression and Scatchard analysis of the data was compatible with the presence of a single population of receptor sites with Bmax of 0.74 +/- 0.020, 0.90 +/- 0.011 and 1.0 +/- 0.036 pmol mg-1 protein at 0, 21 and 27 degrees C, respectively. Binding affinity decreased with increasing temperature: Kd were 0.29 +/- 0.051 nM (0 degrees C), 1.0 +/- 0.080 nM (21 degrees C) and 2.4 +/- 0.38 nM (37 degrees C). 3. At all tested temperatures, [3H]-imidazenil binding was reversible and the Kd calculated from the dissociation and association rate constants approximated the equilibrium Kd. 4. In the presence of gamma-aminobutyric acid (GABA), Kd increased 4 fold at 0 degrees C, whereas Bmax increased, albeit slightly, at all temperatures. 5. Benzodiazepines (BZDs), imidazopyridines and methyl-beta-carboline-3-carboxylate (beta CCM) were effective inhibitors of [3H]-imidazenil binding. Conversely, GABAA antagonists, barbiturates, picrotoxin and peripheral BZD receptor ligands were devoid of any activity. 6. Comparing [3H]-imidazenil to [3H]-flumazenil binding in various brain areas, similar densities of recognition sites as well as like regional differences in the distribution of binding sites for both radioligands were observed (cortex = striatum > cerebellum > spinal cord).(ABSTRACT TRUNCATED AT 250 WORDS)     

High affinity binding of [3H]-tyramine in the central nervous system.

Optimum assay conditions for the association of [3H]-para-tyramine [( 3H]-pTA) with rat brain membranes were characterized, and a saturable, reversible, drug-specific, and high affinity binding mechanism for this trace amine was revealed. The binding capacity (Bmax) for [3H]-pTA in the corpus striatum was approximately 30 times higher than that in the cerebellum, with similar dissociation constants (KD). The binding process of [3H]-pTA involved the dopamine system, inasmuch as (a) highest binding capacity was associated with dopamine-rich regions; (b) dopamine and pTA equally displaced specifically bound [3H]-pTA; (c) there was a severe loss in striatal binding capacity for [3H]-pTA and, reportedly, for [3H]-dopamine, following unilateral nigrostriatal lesion; (d) acute in vivo reserpine treatment markedly decreased the density of [3H]-pTA and, reportedly, of [3H]-dopamine binding sites. In competition experiments [3H]-pTA binding sites, though displaying nanomolar affinity for dopamine, revealed micromolar affinities for the dopamine agonists apomorphine and pergolide, and for several dopamine antagonists, while having very high affinity for reserpine, a marker for the catecholamine transporter in synaptic vesicles. The binding process of [3H]-pTA was both energy-dependent (ouabain-sensitive), and ATP-Mg2+-insensitive; furthermore, the potencies of various drugs in competing for [3H]-pTA binding to rat striatal membranes correlated well (r = 0.96) with their reported potencies in inhibiting [3H]-dopamine uptake into striatal synaptosomes. It is concluded that [3H]-pTA binds at a site located on/within synaptic vesicles where it is involved in the transport mechanism of dopamine.