Etomidate stereospecifically stimulates forebrain,but not cerebellar, 3H-diazepam binding

³H-Diazepam binding to a total particulate fraction of rat forebrain is enhanced by (+)-etomidate and GABA, but not by (?)-etomidate. The enhancement of ³H-diazepam binding by (+)-etomidate was due to a two-fold increase in binding affinity, the maximal number of sites remained unchanged. The degree of stimulation with (+)-etomidate was higher than that obtained with GABA. THIP did not stimulate ³H-diazepam binding to forebrain, and did not reverse the enhanced binding seen with (+)-etomidate or GABA. In a synaptosomal membrane preparation of rat cerebellum, unlike (+)-etomidate, GABA and muscimol produced a marked stimulation of ³H-diazepam binding. (+)-Etomidate did not inhibit ³-muscimol binding to GABA receptors, nor did it activate or inhibit other $\text{in vitro}$ receptor binding assays. The effects of (+)-etomidate on the benzodiazepine binding are different from those of gabamimetic drugs. It is proposed that like barbiturates, (+)-etomidate may affect benzodiazepine binding by interaction with the chloride ionophore which is coupled to the GABA-receptor.     

Differential binding of 3H-imipramine and 3H-mianserin in rat cerebral cortex

Drug competition profiles, effect of raphé lesion, and sodium dependency of the binding of two antidepressant drugs ³H-imipramine and ³H-mianserin to rat cerebral cortex homogenate were compared to examine whether the drugs bound to a common “antidepressant receptor.” Of the neurotransmitters tested, only serotonin displaced binding of both ³H-imipramine and ³H-mianserin. ³H-mianserin binding was potently displaced by serotonin S₂ antagonists and exhibited a profile similar to that of ³H-spiperone binding. In the presence of the serotonin S₂ antagonist spiperone, antihistamines (H₁) potently displaced ³H-mianserin binding. ³H-Imipramine binding was displaced potently by serotonin uptake inhibitors. The order of potency of serotonergic drugs in displacing ³H-imipramine binding was not similar to their order in displacing ³H-spiperone or ³H-serotonin binding. Prior midbrain raphé lesions greatly decreased the binding of ³H-imipramine but did not alter binding of ³H-mianserin. Binding of ³H-imipramine but not ³H-mianserin was sodium dependent. These results show that ³H-imipramine and ³H-mianserin bind to different receptors. ³H-Imipramine binds to a presynaptic serotonin receptor which is probably related to a serotonin uptake recognition site, the binding of which is sodium dependent. ³H-Mianserin binds to postsynaptic receptors, possibly both serotonin S₂ and histamine H₁ receptors, the binding of which is sodium independent.     

Stereospecific binding of 3H-phencyclidine in brain membranes

Phencyclidine (PCP) displaceable binding of ³H-PCP to glass-fiber filters was eliminated and total binding markedly reduced by initial treatment of the discs with 0.05% polyethyleneimine. Assessed with treated filters, unlabeled PCP displaced ³H-PCP in both rat and pigeon brain membranes with an EC50 of 1 μM. Of similar high inhibitory potency were dextrorphan, levorphanol, SKF 10047 and ketamine, while morphine, naloxone and etorphine had EC50 values higher then 1 mM. Using the dissociative anesthetic dexoxadrol and its inactive isomer levoxadrol as displacing agents, stereospecific binding of ³H-PCP was obtained in rat and pigeon brain membranes. The markedly higher potency of dexoxadrol, relative to levoxadrol, in displacing bound ³H-PCP is compatible with behavioral data for these enantiomers. However, they were equipotent in displacing ³H-PCP bound to glass-fiber filters in the absence of tissue. Heat denaturation, but not freezing, abolished stereospecific binding of ³H-PCP, which was also absent in rat liver membranes. The stereospecific binding component in brain displayed biphasic saturability at 60–70 nM and 300–400 nM, respectively.     

High affinity 3H-cimetidine binding in guinea-pig tissues

Receptor binding studies have been carried out in guinea-pig cerebral cortex and gastric mucosa membrane preparations using ³H-cimetidine as the radioligand. The binding was found to be time dependent and saturable and confined to a single population of binding sites. However, the calculated K_D values were different for the two tissues, did not correlate with those reported from classical pharmacological experimentation and there was either no or limited displacement by known H₂ specific agonists. It was concluded that the observed high affinity binding site was probably related to an imidazole recognition site rather than the histamine H₂ receptor. The need for careful evaluation of the data is stressed.     

3H-haloperidol binding: Some theoretical aspects

Evidence is provided to indicate that non-specific binding may in some cases be overlooked due to the non-saturating nature of this type of binding. Some of the theoretical aspects of competition for receptor binding sites are examined.     

3H-clozapine binding to rat brain membranes

³H-Clozapine binds specifically and with high affinity (K_D = 1.3 nM) to rat brain membranes. About two thirds of reversibly bound ³H-clozapine are displaced by hyoscyamine in a stereospecific manner, suggesting interaction of clozapine with muscarinic cholinergic receptors. Most of the remaining ³H-clozapine binding is stereospecifically inhibited by butaclamol, but this binding component seems not to be related to dopamine receptors.     

Imipramine binding site Temperature dependence of the binding of 3H-labeled imipramine and 3H-labeled paroxetine to human platelet membrane

The characteristics of ³H-labeled imipramine and ³H-labeled paroxetine binding to human platelet membranes were determined at various temperatures between 0 and 37°C. Both paroxetine and imipramine probably bind to the same molecular complex in the platelet membrane, but the binding characteristics are different for the two molecules. The dissociation constant ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}$) for imipramine increases from 0.3 nM to 7.0 nM with increasing incubation temperature in a continuous way, whereas $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ for paroxetine is almost constant, about 0.05 nM, between 0 and 19°C, and first begins to increase from 0.06 nM to 0.16 nM between 20 and 37°C. This suggests that the binding of paroxetine to the binding site induces a conformational change in the molecular complex of the binding site, whereas the binding of imipramine takes place without conformational changes in the binding site.     

Properties of 3H-cimetidine binding in rat brain membrane fractions

In an attempt to characterize the brain histamine H₂ receptor, experiments were undertaken to study the binding properties of (N-methyl-³H) -cimetidine, an H₂ receptor antagonist, in rat brain membranes. Using a centrifugation assay, ³H-cimetidine binding having a K_d of 0.40μM and a Bmax of 3.9 pmoles/mg protein was detected. Of fourteen anions and cations tested, one, Cu⁺⁺, dramatically increased specific ³H-cimetidine binding, the increase being due mainly to a change in Bmax. Studies of substrate specificity for ³H-cimetidine binding revealed that Cu⁺⁺, while not significantly affecting the potency of H₂ receptor agonists and antagonists, dramatically decreases the potency of H₁ receptor substances on the ³H-cimetidine binding site. In addition, both the relative and absolute potencies of various H₂ receptor agonistsv and antagonists in displacing the ligand in the presence of Cu⁺⁺ parallels their potencies in biological systems. These findings suggest that, under these conditions, ³H-cimetidine may be labelling a biologically relevant H₂ binding site in brain and that Cu⁺⁺ may regulate the substrate specificity for this site. The brain regional distribution and kinetic analysis of the binding suggest that it is not localized solely to the synaptic receptor for histamine, but may also be associated with histamine receptors at other neuronal, glial or vascular sites.     

[3H]muscimol binding in the rat retina

Crude membrane fractions were prepared from rat retinae and used to study the specific binding of [³H]muscimol, a potent GABA agonist. Specific [³H]muscimol binding was enhanced 2–3 fold by pretreatment of the membranes with 0.025% Triton X-100. Two muscimol binding sites were demonstrated with K_D values of 4.4 and 12.3 nM. GABA, muscimol, and 3-aminopropanesulfonic acid were the most potent inhibitors of specific [³H]muscimol binding with K_I values of 15, 10, and 50 nM, respectively. These data are consistent with binding to the synaptic GABA receptor.     

Dopamine receptors in the goldfish retina: 3H-spiroperidol and 3H-domperidone binding; and dopamine-stimulated adenylate cyclase activity

Dopamine receptors in the goldfish retina have been examined by binding studies using ³H-spiroperidol and ³H-domperidone as specific ligands, and by measuring retinal adenylate cyclase activities in the presence and absence of dopamine. Our results indicate that washed membranes from goldfish retinal homogenate bind a variety of dopamine agonists and antagonists with high affinities and with characteristics similar to those reported for the brain, with the exception that in this retina there is virtually no binding of the specific D₂ receptor antagonist, ³H-domperidone. In addition, there is a very low basal activity of adenylate cyclase which can be greatly stimulated by dopamine, possibly reflecting a high degree of coupling between this enzyme and the dopamine receptor. Taken together, our findings indicate that the goldfish retina contains a high density of D₁ type dopamine receptors and few, if any, D₂ type receptors.     

Ascorbic acid enables reversible dopamine receptor 3H-agonist binding

The effects of ascorbic acid on dopaminergic ³H-agonist receptor binding were studied in membrane homogenates of bovine anterior pituitary and caudate, and rat striatum. In all tissues virtually no stereospecific binding (defined using luM (+)butaclamol) of the ³H-agonists N-propylnorapomorphine (NPA), apomorphine, or dopamine could be demonstrated in the absence of ascorbic acid. Although levels of total ³H-agonist binding were three to five times greater in the absence than in the presence of 0.1% ascorbic acid, the increased binding was entirely non-stereospecific. Greater amounts of dopamine-inhibitable ³H-NPA binding could be demonstrated in the absence of 0.1% ascorbic acid, but this measure of “specific binding” was demonstrated not to represent dopamine receptor binding since several other catecholamines and catechol were equipotent with dopamine and more potent than the dopamine agonist (±)amino-6,7-dihydroxy-1,2,3,4-tetrahydronapthalene (ADTN) in inhibiting this binding. High levels of dopamine-displaceable ³H-agonist binding were detected in fresh and boiled homogenates of cerebellum, an area of brain which receives no dopaminergic innervation, further demonstrating the non-specific nature of ³H-agonist binding in the absence of ascorbic acid. These studies emphasize that under typical assay conditions ascorbic acid is required in order to demonstrate reversible and specific ³H-agonist binding to dopamine receptors.     

Direct binding of 3H-lisuride to adrenergic and serotonergic receptors

The characteristics of the specific binding of ³H-lisuride hydrogen maleate (³H-LHM) to homogenates of rat striatum and bovine frontal cortex tissue were investigated. In rat striatum 50% of ³H-LHM binding was inhibited potently by spiperone and haloperidol indicating a component of ³H-LHM binding to D₂ dopamine receptors. Specific ³H-LHM binding was detected in rat striatum after selective blockade of the D₂ dopamine component indicating specific ³H-LHM binding to other striatal sites. In bovine frontal cortex clonidine and serotonin competition curves for specific ³H-LHM binding included high affinity phases indicating alpha₂ adrenergic and high affinity serotonergic components of binding. Blockade of the adrenergic component by 10^?7M clonidine resulted in the specific ³H-LHM binding exhibiting distinctly serotonergic characteristics. Conversely, blockade of the serotonergic component by 2 × 10^?7M serotonin resulted in the specific ³H-LHM binding exhibiting distinct alpha₂ receptor characteristics. These data demonstrate the specific binding of ³H-LHM to alpha₂ adrenergic receptors, to a high affinity serotonin site, and to D₂ dopamine receptors.     

Effect of phencyclidine on [3H]QNB binding

Intraperitoneal injection of phencyclidine before intravenous injection of [³H] Quinuclidinyl benzilate (QNE, 1.6 μg/kg) significantly increased the amount of radioactivity found in the brains of female C57BL/6J mice one hour after the ³H-QNB administration. This effect was found in hypothalamus, cortex, hippocampus and striatum and was decreased by pretreatment of the animal with atropine. The magnitude of the enhancement varied as a function of dose but did not change across the time span studied. These data are in contrast to our findings and those of others of inhibition of the specific binding of ³H-QNB to muscarinic cholinergic receptors by PCP $\text{in vitro}$. When atropine or PCP was administered $\text{in vivo}$ and the tissue later analyzed $\text{in vitro}$, no effects of the drugs were observed on ³H-QNB binding. The reasons for the differences remain a matter of speculation.     

The binding of 3H-Isoguvacine to mouse brain synaptic membranes

³H-Isoguvacine, a gamma aminobutyric acid (GABA) agonist, has been shown to bind to a mouse forebrain synaptic membrane preparation. The specific binding is displaceable by GABA, muscimol and bicuculline but not by picrotoxin or diaminobutyric acid. Kinetic data suggest two binding affinities. Highest levels of binding are observed in the cerebellum, cortex and hippocampus. It is suggested that isoguvacine binds to GABA binding sites and therefore represents a new ligand for measuring GABA receptor binding.     

Cation specificity of 3H-sulpiride binding involves alteration in the number of striatal binding sites

Specific ³H-sulpiride binding to rat striatal membranes shows an absolute requirement for the presence of sodium ions in the incubation buffer. Potassium, rubidium and caesium ions were unable to initiate specific ³H-sulpiride binding in a sodium free buffer, and lithium ionscould only partially replace sodium ions. Specific ³H-spiperone binding was unaffected by variation of the cation content of the incubation buffer. The alteration in ³H-sulpiride binding caused by sodium and lithium ions was due predominantly to an increase in the number of available binding sites, rather than to altered receptor affinity. Sodium ions may be essential for the accessability of ³H-sulpiride to a single site labelled also by ³H-spiperone. However, the Ki value for sulpiride displacement of ³H-spiperone in the presence of sodium ions was 20 times greater than the K_D value for ³H-sulpiride binding. So, ³H-sulpiride may interact with a highly sodium dependent binding site distinct from that labelled by ³H-spiperone.     

Decreased [3H]serotonin and [3H]spiperone binding consequent to lipid peroxidation in rat cortical membranes

The effect of lipid peroxidation on two types of serotonin binding sites was investigated. Incubation of rat cortical membranes with an ascobate-dependent peroxidizing system resulted in the formation of malonyldialdehyde and significant decreases in the specific binding of [³H]serotonin and [³H]spiperone to the treated membranes. When ascorbate concentrations were varied from 0.025 to 6.0 mM, malonyldialdehyde production increased to a maximum at 0.5 mM ascorbate and then declined. Conversely, the specific binding of [³H]serotonin and [³H]spiperone decreased to a minimum at 0.5 mM ascorbate and then increased. Regression analysis of the data revealed that the decrease in the two binding sites was linearly correlated to lipid peroxidation.     

Decrease in lymphocyte [3H]spiroperidol binding sites in parkinsonism

[³H]spiroperidol binding has been measured in lymphocytes from patients with Parkinson's disease and age matched healthy volunteers. A dramatic decrease (73%) in the number of binding sites (^Bmax) without any variation of the affinity (^KD) has been observed in Parkinsonian patients. This decrease in ^Bmax is linearly correlated with the degree of disability of the Parkinsonian patients (r = 0.891, p <0.001). This decrease appeared to be relatively selective since no variation was observed with patients suffering of other neurological disorders (vascular hemiplegia, Alzeihmer's disease, olivopontocerebellar degeneration, Huntington's chorea).     

The effect of varying 3H-shiperone concentration on its binding parameters

Higher concentrations of spiperone were required to compete against the binding of ³H-spiperone to calf caudate when higher concentrations of ³H-spiperone were used. The Cheng-Prusoff equation did not adequately apply to this hydrophobic ligand, since the K_i values varied directly with the ³H-spiperone concentration. The equation was only adequate and gave a constant K_i when the total concentrations of ³H-spiperone were replaced by the measured free concentrations. It is suggested that IC₅₀ values are best reported as such, unless the free concentrations are actually determined directly.     

Intracellular disposition of [3H]-cocaine, [3H]-norcocaine, [3H]-benzoylecgonine and [3H]-benzoylnorecgonine in the brain of rats

[³H]-cocaine, [³H]-norcocaine, [³H]-benzoylecgonine and [³H]-benzoylnorecgonine were administered i.c. in equi-potent pharmacologic doses and the intracellular disposition and metabolism of each drug determined. Norcocaine and cocaine rapidly entered and egressed from the brain so that 4.8–6.1% of the radioactivity present in brain at one minute was observed at 30 minutes. The highest levels of subcellular radioactivity were generally found in the microsomal plus supernatant, followed by the nuclear and shocked mitochondrial fractions. No apparent localization of the radioactivity occured in synaptic membranes. The brain/plasma (B/P) ratio curves for cocaine and norcocaine were similar; however, the norcocaine values were considerably higher at each time interval. Benzoylecgonine and benzoylnorecgonine had higher comparative B/P ratios than cocaine or norcocaine and persisted in brain for a longer period of time so that 0.6–2.1% of the radioactivity present in brain at 1 hour was detected at 24 hours. Cocaine and norcocaine were extensively metabolized to the benzoylmetabolites. Benzoylecgonine was metabolized to benzoylnorecgonine and benzoylnorecgonine was unmetabolized. The brain disposition data and B/P ratios agreed quite well with the overall pharmacologic action of cocaine and its metabolites.