Quantitative assessment of heterogeneous 3H-spiperone binding to rat neostriatum and frontal cortex

Anomalies of the binding of ³Hspiperone to rat cerebral membranes have been examined. By employing a very low ligand concentration ($\text{～ 25}\text{pM}{}^3\text{Hspiperone}$) we have demonstrated that even within the corpus striatum, ³Hspiperone appears to bind to multiple sites and that dopaminergic and serotonergic agents can selectively inhibit from these sites. In the corpus striatum, 75–80% of the ³Hspiperone specific binding can be inhibited with high affinity by dopaminergic drugs while some 20–30% is inhibited with high affinity by serotonergic compounds. The two ³Hspiperone sites, which we have shown to have affinities of 31 and 325 pM, may therefore represent dopaminergic and serotonergic sites. At higher concentrations of ³Hspiperone, however, the picture may be complicated by a further low affinity site. The great selectivity shown by dopaminergic agonists for the two ³Hspiperone sites explains the ‘flattened’ displacement curves reported for ³Hspiperone/agonist interactions. As dopaminergic agents show the greater affinity for the high affinity ³Hspiperone site, it is tempting to speculate that this site has the greatest association with the dopamine receptor.     

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.     

Effects of a series of substituted benzamides on rat prolactin secretion and 3H-spiperone binding to bovine anterior pituitary membranes

The potency of seven substituted benzamine drugs (AHR-5531B, AHR-5645B, AHR-6092, AHR-8764, bromopride, sultopride and tiapride) to stimulate rat prolactin (PRL) secretion $\text{in vivo}$ was found to be three orders of magnitude greater than that of non-benzamide neuroleptic drugs relative to their respective abilities to inhibit ³H-spiperone binding to bovine anterior pituitary membranes. Nevertheless, the IC₅₀ values for the inhibition of ³H-spiperone binding by the seven substituted benzamide drugs was significantly correlated with their high potency to stimulate rat PRL secretion $\text{in vivo}$. Further, the slope of the regression line for these substituted benzamides paralleled that of a series of butyrophenone, phenothiazine, morphanthridine and dibenzodiazepine neuroleptic drugs. Two benzamide (sulpiride and metoclopramide) and three non-benzamide neuroleptic drugs gave intermediate results. This data suggests that blockade of different subgroups of dopamine receptors in the anterior pituitary gland labeled by ³H-spiperone may be responsible for the $\text{in vivo}$ stimulation of PRL secretion by the benzamide and non-benzamide neuroleptioc drugs.     

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.     

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.     

Inhibition of 3H-clozapine binding in rat brain after oral administration of neuroleptics

Clozapine, thioridazine, perlapine, clothiapine, chlorpromazine, NT 104–252, loxapine or haloperidol were administered orally, and atropine subcutaneously, to rats. The animals were decapitated, brain tissue was removed and homogenized in tris buffer and incubated with ³H-clozapine. Total and nonspecifically bound ³H-clozapine were measured in each preparation. A dose-dependent inhibition of specific ³H-clozapine binding of more than 50% was observed only after the administration of clozapine or thioridazine. There was no correlation between inhibition of ³H-clozapine binding and that of ³H-haloperidol, a specific ligand for DA-receptors. ³H-Clozapine receptor density was much greater than ³H-haloperidol receptor density, suggesting that the majority of clozapine binding sites are not DA-receptors.     

Elevation of [3H]cimetidine binding by CuCl2 in brain membranes of rats

Influences of dithiothreitol (DTT), p-chloromercuriphenyl sulfonate (PCMPS) and ascorbate on CuCl₂-induced elevation of [³H]cimetidine binding were investigated in brain membranes of rats. CuCl₂ (10–500 μM) elevated specific [³H]cimetidine binding in a concentration-dependent manner. There were two types of [³H]cimetidine binding in the presence of 50 μM CuCl₂: high affinity binding with K_d = 1.97 nM and low affinity with K_d = 21.6 nM. PCMPS (10 and 100 μM) reduced the binding in both media with and without CuCl₂. DTT (1–30 μM) or ascorbate (0.1 and 1.0 mM) markedly elevated the binding in the presence of CuCl₂ but showed no effect and ascorbate rather inhibited the binding in the absence of CuCl₂. DTT (0.1 mM) diminished the binding in the presence and absence of CuCl₂. CuCl₂ (50 μM) significantly (P < 0.01) increased the IC₅₀ of histamine for [³H]cimetidine binding and the effect was greater than that from 100 μM GTP. It is suggested that sulfhydryl groups sensitive to PCMPS could interact with Cu²⁺ and thus be involved in an elevation of cimetidine binding. Cu²⁺ seems to regulate affinity of agonist binding for cimetidine binding sites presumably by acting on cimetidine binding sites and/or GTP binding regulatory proteins.     

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.     

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.     

Change in the charactertistics of 3H-spiperone binding to rat striatal membranes after acute chlorpromazine administration: effects of buffer washing of membranes.

After intraperitoneal injections of ³H-spiperone into the rat, brain membrane preparations retain the majority of the radioactivity even after several buffer washes. With ³H-spiperone as ligand, dissociation constants were significantly elevated and maximum binding unchanged in rat striatal membranes after acute intraperitoneal injection of chlorpromazine (14 mg/kg). It is suggested that in studies of post-mortem brains of schizophrenics that contain neuroleptics specific ³H-spiperone binding will be lowered by competition from residual drug in membrane preparations and valid comparisons of ³H-spiperone binding to preparations from control and schizophrenic brains can only be made if maximum binding values are determined.     

Steroid hormone modulation of 3H-prostaglandin E1 binding to bovine corpus luteum cell membranes

The specific binding of ³H-prostaglandin E₁ (³H-PGE₁) to bovine corpus luteum cell membranes was not affected by cholesterol or various progestins at concentrations of up to 9.0 × 10⁻⁶M. At concentrations above 2.5 × 10⁻⁶M; estrone, 17β-estradiol (but not 17α-estradiol or 17β-estradiol glucuronide), estriol, equilin, D-equilenin, 17-ethynyl estradiol, diethylstilbestrol, cortisol, corticosterone, deoxycorticosterone and aldosterone inhibited specific binding of ³H-PGE₁. On the other hand, testosterone and dihydrotestosterone (DHT) (but not androstenedione) significantly enhanced ³H-PGE₁ binding. These findings permitted the following correlations between steroid structure and modulation of ³H-PGE₁ binding: steroids with a free phenolic ring and a 17β-hydroxyl or 17-keto group or C-21 steroids with a C-20 ketone and a C-21 hydroxy group decrease, whereas C-19 steroids with a C-17 hydroxy group enhance specific binding of ³H-PGE₁. PGE receptors are heterogeneous with respect to affinity for ³H-PGE₁. The steroids that decreased ³H-PGE₁ binding caused a lowering to a complete loss of low affinity PGE receptors. Steroids that increased ³H-PGE₁ binding caused appearance of new low affinity PGE receptors. Association rate constants for ³H-PGE₁ binding were decreased by 17β-estradiol (61%) and increased by DHT (59%).     

[3H] verapamil binding sites in skeletal muscle tranverse tubule membranes

[³H]verapamil binding to muscle tubule membrane has the following properties. K_D = 27 ± 5 nM and maximum binding capacity B_max = 50 ± 5 pmol/mg of protein. A 1 = 1 stoichiometry of binding was found for the ratio of [³H]verapamil versus [³H] nitrendipine binding sites. The dissociation constant found at equilibrium is near that determined from the ratio of the rate constants for association (k₁) and dissociation (k_?1). Antiarrhythmic drugs like D600, diltiazem and bepridil are competitive inhibitors of [³H]verapamil binding with K_D values between 40 and 200 nM. Dihydropyridine analogs are apparent non competitive inhibitors of [³H]verapamil binding with half-maximum inhibition values (K_0.5) between 1 and 5 nM.     

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.     

Stereospecific 3H-nicotine binding to intact and solubilized rat brain membranes and evidence for its noncholinergic nature

³H-nicotine binding was performed on intact and solubilized rat brain membranes as well as membranes from the electric organ of the $\text{Torpedo}$ fish. The K_d for binding to intact and solubilized rat brain membranes was 5.6 × 10^?9 M and 1.1 × 10^?8M respectively, and the binding capacity 2.0 × 10^?14 and 3.0 × 10^?13 moles /mg protein respectively. The K_d for Torpedo membranes was 3.1 × 10^?7M and the binding capacity 6.8 × 10^?13 moles/mg protein. The binding was stereospecific with the affinity of the (?)-nicotine being about 8 times greater than the (+)-nicotine with all three preparations. The relative affinity for the nicotine binding site of nicotinic cholinergic drugs was considerably less in rat brain than in $\text{Torpedo}$ membranes, where the sites are mainly cholinergic. A comparison was made of the ability of a variety of cholinergic drugs and nicotine derivatives to compete with ³H-nicotine binding and their relative pharmacologic potency to produce or inhibit a characteristic prostration syndrome caused by (?)-nicotine administered intraventricularly to rats. From such studies it was concluded that nicotine, in part, may be interacting at noncholinergic sites in rat brain.     

Mechanism of Cu2+-induced elevation of [3H]cimetidine binding to membranes in rat brain

The mechanism of increasing effect of CuCl₂ on specific [³H]cimetidine binding was examined in brain membranes of rats. CuCl₂-Induced elevation of [³H]cimetidine binding was high in Krebs-Ringer solution (pH 7.4) compared to those in 50 mM Na, K-phosphate buffer (pH 7.4) and in 50 mM Tris-HCl buffer (pH 7.4). CaCl₂ (5–50 mM) inhibited effect of CuCl₂, but NaCl (25–200 mM), KCl (5–100 mM) or MgCl₂ (5–50 mM) did not. CuCl₂ (50 μM) elevated 9.3- and 2.5-fold the binding in phosphate- and Tris—HCl buffer, respectively. EDTA-2Na decreased the binding elevated by 50 μM CuCl₂ in phosphate buffer to the similar level in Tris-HCl buffer, whereas it did not affect those in Tris-HCl buffer. The absorption spectra of cimetidine and CuCl₂ mixture showed a peak at 317 nm in phosphate buffer that was not observed in Tris-HCl buffer. It is suggested that cimetidine-Cu²⁺ chelate complex could be formed in phosphate buffer, resulting in higher amount of binding in phosphate buffer than in Tris-HCl buffer. PdCl₂ also caused a marked elevation in [³H]cimetidine binding, seeming to be due to formation of cimetidine-Pd²⁺ chelate complex. There were two types of [³H]cimetidine binding in the presence of 20 nM PdCl₂: high affinity binding with K_d = 0.7 ± 0.1 nM and low affinity binding with K_d = 44.3 ± 3.0 nM. It is suggested that cimetidine-Cu²⁺ complex binds to cimetidine binding sites in brain with higher affinity than cimetidine alone.     

Changes in [3H]-ouabain and [3H]-neurotensin binding to rat cerebral cortex membranes after administration of antipsychotic drugs haloperidol and clozapine

Evidences indicate the relationship between neurotensinergic and dopaminergic systems. Neurotensin inhibits synaptosomal membrane Na⁺, K⁺-ATPase activity, an effect blocked by SR 48692, antagonist for high affinity neurotensin receptor (NTS1) type. Assays of high affinity [³H]-ouabain binding (to analyze K⁺ site of Na⁺, K⁺-ATPase) show that in vitro addition of neurotensin decreases binding. Herein potential interaction between NTS1 receptor, dopaminergic D2 receptor and Na⁺, K⁺-ATPase was studied. To test the involvement of dopaminergic D2 receptors in [³H]-ouabain binding inhibition by neurotensin, Wistar rats were administered i.p.with antipsychotic drugs haloperidol (2 mg/kg) and clozapine (3, 10 and 30 mg/kg). Animals were sacrificed 18 h later, cerebral cortices harvested, membrane fractions prepared and high affinity [³H]-ouabain binding assayed in the absence or presence of neurotensin at a 10 micromolar concentration. No differences versus controls for basal binding or for binding inhibition by neurotensin were recorded, except after 10 mg/kg clozapine. Rats were administered with neurotensin (3, 10 y 30 μg, i.c.v.) and 60 min later, animals were sacrificed, cerebral cortices harvested and processed to obtain membrane fractions for high affinity [³H]-ouabain binding assays. Results showed a slight but statistically significant decrease in binding with the 30 μg neurotensin dose. To analyze the interaction between dopaminergic D2 and NTS1 receptors, [³H]-neurotensin binding to cortical membranes from rats injected with haloperidol (2 mg/kg, i.p.) or clozapine (10 mg/kg) was assayed. Saturation curves and Scatchard transformation showed that the only statistically significant change occurred in Bmax after haloperidol administration. Hill number was close to the unit in all cases. Results indicated that typical and atypical antipsychotic drugs differentially modulate the interaction between neurotensin and Na⁺, K⁺-ATPase. At the same time, support the notion of an interaction among dopaminergic and neurotensinergic systems and Na⁺, K⁺-ATPase at central synapses.     

Preferential release of newly synthesized [3H]GABA from striatal slices to preloaded [3H]GABA

The release of [³H]GABA which is newly synthesized from [³H]l-glutamic acid (GLU) has been examined using striatal slices obtained from the rat brain. It was found that 8–10% of [³H]GLU transported was converted to [³H]GABA during the incubation of striatal slices in the presence of nipecotic acid (5 × 10^?5 M). Nipecotic acid was added to the medium in order to prevent possible reuptake of [³H]GABA released during its synthesis, and it was found to have no significant effect on the formation of [³H]GABA from [³H]GLU as well as on the uptake of [³H]GLU. The application of high potassium (60 mM) stimulation exhibited a significant enhancement of the release of this newly synthesized [³H]GABA in a Ca²⁺ dependent manner. Kinetic analysis revealed that the evoked release of newly synthesized [³H]GABA was approximately two times greater than that of previously-loaded [³H]GABA, whereas no significant difference was observed in the spontaneous release. An immobilization stress in water failed to affect the release of newly synthesized [³H]GABA from striatal slices despite the occurrence of a significant enhancement of GABA formation in this structure.These results suggest that newly synthesized GABA may be preferentially released from its nerve terminals in response to the excitation of neurons at least in the striatum as compared with previously accumulated GABA.     

Characteristics of high-affinity [3H]adenosine binding to rat brain synaptosomes and turkey erythrocyte membranes

High affinity binding sites for [³H]adenosine in rat brain and in turkey erythrocytes can be identified by binding experiments. Displacement experiments using a number of adenosine analogs indicate that these high affinity sites do not represent the R-type adenosine receptors which mediate activation of adenylate cyclase, although the binding is theophylline sensitive. Similarly, the binding of [³H]adenosine is not to the P-site, which mediates inhibition of adenylate cyclase, since the high affinity binding persists in the presence of 2′,5′-dideoxyadenosine. Furthermore, these results remain qualitatively similar also in the presence of dipyridamole which blocks adenosine transport sites. We conclude that theophylline sensitivity does not indicate that [³H]adenosine binding sites correspond to adenosine receptors coupled to adenylate cyclase.     

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.