Specific [3H]-spiroperidol binding sites in human prefrontal cortex: potential site multiplicity and overall serotonin-like selectivity

[3H]-Spiroperidol specifically binds at sites in human prefrontal cortex. The binding of this ligand is apparently anomalous at 37 degrees C, with a substantial loss of specific binding occurring between 5 and 40 min incubation. However, at 21 degrees C, this loss of binding is not observed even at 60 min. At 21 degrees C, [3H]-spiroperidol binding in human prefrontal cortex is apparently occurring at multiple sites or multiple affinity states of single classes of sites, or at a combination of both. The overall selectivity is predominantly serotonergic, rather than dopaminergic.     

Ontogenetic changes in [3H]-spiroperidol binding sites in posthatch chick brain

The ontogenetic development of [3H]-spiroperidol binding sites was measured in the optic tectum, cerebellum, forebrain base, and forebrain roof of 1-, 4-, and 16-day-old chicks. In the chick optic tectum and cerebellum both the density and the total number of [3H]-spiroperidol binding sites increased from 4- to 16-days-posthatch, but no significant differences were found in either brain area across the initial four posthatch days. In the forebrain base, [3H]-spiroperidol receptor density and total binding increased significantly between 1- and 4-days-posthatch, but at 16-days-posthatch there was a slight decrease in receptor density. Binding sites in the forebrain roof were minimal at all ages. As expected, saturation experiments yielded curvilinear plots indicating the presence of high- and low-affinity binding sites. The high-affinity sites probably reflect dopamine D-2 receptors; whereas, the low-affinity sites may reflect other receptor types, possibly serotonin S-2. These results suggest that large doses of haloperidol, which are normally used in chick behavioral research, may produce behavioral effects by antagonizing multiple receptors.     

Modulation of specific [3H]phenytoin binding by benzodiazepines

We have shown that diazepam (ED₅₀ 2.4 M), flunitrazepam (ED₅₀ 10.2 M) and Ro5-4864 (ED₅₀ 5 M) are able to enhance both total and specific [³H]phenytoin binding. Picrotoxin (IC₅₀ 1.43 M) and chloride, either NaCl or KCl (IC₅₀ 42.4 M) inhibit both the increase in total and specific binding of [³H]phenytoin, Ro 15-1788 does not. The optimum time for this enhancement was 3–4 hours. While the ED₅₀'s for the benzodiazepines are high their order of potency suggests that an involvement of both the peripheral type benzodiazepine receptor and the GABA-chloride ionophore complex is likely. Clonazepam (IC₅₀ 23 M), oxazepam (IC₅₀ 12 M) chlordiazepoxide (IC₅₀ 35 M) and Ro8682-10, a convulsant benzodiazepine (IC₅₀ 16 M) all inhibit both total and specific [³H]phenytoin binding. These effects were not blocked by chloride ions, picrotoxin or Ro 15-1788, and reached equilibrium within 45 minutes. This order of potency also parallels that for the peripheral benzodiazepine receptor in rat brain. These data suggest the presence of a micromolar benzodiazepine receptor site which may play a role in the control of CNS excitability. Nitrazepam, medazepam, bromazepam and the tetralobenzodiazepines U38335, U42794, U43434, and U37834 had no effect on total or specific [³H]phenytoin binding nor on the actions of the other benzodiazepines described in concentrations up to 50 M.     

Autoradiographic localization of specific [3H]dexamethasone binding in fetal lung

The cellular and subcellular localization of specific [3H]dexamethasone binding was examined in fetal mouse lung at various stages of development and in human fetal lung at 8 weeks of gestation using a rapid in vitro steroid incubation technique followed by thaw-mount autoradiography. Competition studies with unlabeled steroids demonstrate the specificity of [3H]dexamethasone labeling, and indicate that fetal lung mesenchyme is a primary glucocorticoid target during lung development. Quantitative binding studies, involving incubation of intact tissue with competing ligand and subsequent subcellular fractionation, show this to be specific, nuclear binding characteristic of glucocorticoid receptors. Autoradiographs of [3H]dexamethasone binding in lung tissue at early stages of development demonstrate that the mesenchyme directly adjacent to the more proximal portions of the bronchiolar network is heavily labeled. In contrast, the epithelium which will later differentiate into bronchi and bronchioles, is relatively unlabeled. Distal portions of the growing epithelium, destined to become alveolar ducts and alveoli, do show nuclear localization of [3H]dexamethasone. Because of the known importance of the mesenchyme in controlling lung development and the ability of glucocorticoids to stimulate lung development, these results suggest that many of the growth-promoting effects of glucocorticoids may be mediated through the mesenchyme. In addition, by utilizing a technique which allows the simultaneous examination of extracellular matrix components and [3H]dexamethasone binding, a relationship is observed between extensive mesenchymal [3H]dexamethasone binding and extensive extracellular matrix accumulation. Since glucocorticoids stimulate the synthesis of many extracellular matrix components, these results suggest a role for these hormones in affecting mesenchymal-epithelial interactions during lung morphogenesis.     

[3H]Tyramine binding: A comparison with neuronal [3H]-dopamine uptake and [3H]mazindol binding processes

[³H]Spiperone ([³H]SPI) binding sites in rat or bovine striata have been solubilized using CHAPS or digitonin detergents. Solubilized sites retained the binding characteristics of those in native membrane preparations. The same solubilized material, however, did not bind [³H]tyramine ([³H]PTA), thus indicating that [³H]PTA binding sites and DA receptors are different chemico-physical entities. In membrane preparations or crude synaptosomes obtained from the c.striatum of neonatally-rendered hypothyroid rats, when central DA-pathways are impaired, both [³H]PTA binding and [³H]DA uptake processes were markedly decreased, with no effect on [³H]mazindol ([³H]MAZ) binding, compared to euthyroids. Reserpine, a well-known inhibitor of DA-uptake into a variety of secretory vesicles, and a potent in vivo andin vitro inhibitor of [³H]PTA binding, did not affect the [³H]MAZ binding process. This further supported the suggestion that while [³H]PTA binding sites are almost totally associated with the vesicular transporter for DA, [³H]MAZ does label a site involved in the DA-translocation across the neuronal membrane. The latter process seems to be rather insensitive to thyroid hypofunction, when however the intracellular storage of DA might be consistently impaired. In conclusion, PTA might be well exploited as a marker of the DA vesicular transporter through its molecular characterization, whenever possible.Special issue dedicated to Dr. Paola S. Timiras     

Different specific binding sites of [3H]glycine and [3H]strychnine in synaptosomal membranes isolated from frog retina

Synaptosomal fractions were isolated from frog retina: a fraction enriched in photoreceptor terminals (P1) and a second one (P2) containing interneurons terminals. We compared the binding of [³H]glycine and [³H]strychine to membranes of these synaptosomes. The binding of both radioactive ligands was saturable and Na⁺-independent. [³H]Glycine bound to a single site in P1 and P2 synaptosomal fractions, with K_D=12 and 82 nM and B_Max=3.1 and 3.06 pmol/mg protein respectively. [³H]Strychnine bound to two sites in each one of the synaptosomal fractions. For P1 K_D values were 3.9 and 18.7 nM, and B_Max values were 1.1 and 7.1 pmol/mg protein, respecitively. Membranes from the P2 synaptosomal fraction showed K_D's of 0.6 and 48 nM and B_Max's of 0.4 and 4.5 pmol/mg. Specific [³H]glycine binding was displaced by -alanine, l-serine, d-serine and HA966, but not by strychnine 7-chlorokynurenic or 5,7-dichloro-kynurenic acids. Specific [³H]strychnine, binding was partially displaced by glycine and related aminoacids and totally displaced only by 2-NH₂-strychnine. Our results indicate the presence of high affinity binding sites for glycine and strychnine in frog retinal synaptosomal membranes. The pharmacological binding pattern indicates the presence of the strychnine sensitive glycine receptor as well as other sites. These might not include the NMDA receptor-associated glycine site.     

Specific binding of [3H]phenytoin in the human brain

Competition between cold phenytoin and [3H]phenytoin binding was observed in normal human brain. Binding was observed in all areas examined. The highest number of sites was in the amygdala (a total of 717.71 fmol/mg protein) and the lowest in the Brodman area (BA) 4 of the motor cortex (153.91 fmol/mg protein) and cerebellar cortex (154.4 fmol/mg protein). In three areas, amygdala, cortex area BA 38 (inferior parietal lobe), and cortex area BA 8 (premotor cortex), two sets of binding sites were observed. In these areas the Kd for the higher affinity sites ranged from 35 to 116 nM, and for the lower affinity site, from 328 to 866 nM. In the four areas where only one binding site was observed the KdS ranged from 164 to 311 nM and the Scatchard plot was linear.     

Neurotensin decreases high affinity [3H]-ouabain binding to cerebral cortex membranes

Previous work from this laboratory showed the ability of neurotensin to inhibit synaptosomal membrane Na(+), K(+)-ATPase activity, the effect being blocked by SR 48692, a non-peptidic antagonist for high affinity neurotensin receptor (NTS1) [López Ordieres and Rodríguez de Lores Arnaiz 2000; 2001]. To further study neurotensin interaction with Na(+), K(+)-ATPase, peptide effect on high affinity [(3)H]-ouabain binding was studied in cerebral cortex membranes. It was observed that neurotensin modified binding in a dose-dependent manner, leading to 80% decrease with 1 × 10(-4)M concentration. On the other hand, the single addition of 1 × 10(-6)M, 1 × 10(-5)M and 1 × 10(-4)M SR 48692 (Sanofi-Aventis, U.S., Inc.) decreased [(3)H]-ouabain binding (in %) to 87 ± 16; 74 ± 16 and 34 ± 17, respectively. Simultaneous addition of neurotensin and SR 48692 led to additive or synergic effects. Partial NTS2 agonist levocabastine inhibited [(3)H]-ouabain binding likewise. Saturation assays followed by Scatchard analyses showed that neurotensin increased K(d) value whereas failed to modify B(max) value, indicating a competitive type interaction of the peptide at Na(+), K(+)-ATPase ouabain site. At variance, SR 48692 decreased B(max) value whereas it did not modify K(d) value. [(3)H]-ouabain binding was also studied in cerebral cortex membranes obtained from rats injected i. p. 30 min earlier with 100 μg and 250 μg/kg SR 48692. It was observed that the 250 μg/kg SR 48692 dose led to 19% decrease in basal [(3)H]-ouabain binding. After SR 48692 treatments, addition of 1 × 10(-6)M led to additive or synergic effect. Results suggested that [(3)H]-ouabain binding inhibition by neurotensin hardly involves NTS1 receptor.     

Evidence for the participation of disulfide and sulfhydril groups in the specific binding of [3H]prazosin in cerebral cortex

The tritiated ₁ antagonist prazosin [³H]PRZ binds specifically and with high affinity to postsynaptic adrenoceptors in membrane preparations from cerebral cortex. Since adrenoceptors are of protein nature, it was of interest of investigate the possible role of disulfide (—SS—) and sulfhydril (—SH) groups in the binding of [³H]PRZ. Pretreatment of the membranes with the disulfide and sulfhydryl reactivesdl0Dithiothreitol,l-Dithiothreitol, Dithioerythritol or 5,5-Dithiobis-(2-nitrobenzoic acid) (DTNB), alone or in combination with the alkylating agent N-Methylmaleimide (NMM), decreased specific [³HPRZ binding, with minor changes in the non-specific counts. Saturation experiments revealed that all these reagents reduced the affinity of the binding site for [³H]PRZ, as judged by theK _d 25°C, but only the alkylating agent NMM and the oxydizing reagent DTNB produced in addition to the increase inK _d, a decrease of the maximum binding capacity (B _max). The present results provide evidence for a participation of—SS—and/or—SH groups in the recognition site of the ₁-adrenoceptor of cerebral cortex.     

Demonstration of specific "high affinity" binding sites for [3H] imipramine on human platelets

High affinity and saturable binding sites for [³H] imipramine have been demonstrated on human platelet membranes. These binding sites appear to be specific for tricyclic antidepressants and their pharmacologically-active metabolites. In contrast, inactive tricyclic compounds such as the parent iminodibenzyl and iminostilbenes do not inhibit [³H] imipramine binding. The binding of [³H] imipramine to human platelets is of high affinity $\text{(}\text{Kd}\text{? 1.4}\text{nM}\text{)}$, saturable $\text{(}\text{Bmax}\text{? 625}\text{fmols/mg prot}\text{)}$, and sensitive to proteolytic degradation. The effects of various drugs and neurotransmitter agonists and antagonists suggests that these binding sites are pharmacologically distinct from the previously reported binding of tricyclic antidepressants to alpha-adrenergic, muscarinic-cholinergic, and histaminergic receptors. The binding characteristics of [³H] imipramine to platelets is similar to that in rat and human brain and may thus serve as a useful model in elucidating the pharmacological and physiological significance of these binding sites.     

In situ imaging of specific binding of [3H]isatin in rat brain

Isatin is an endogenous indole that influences a range of processes both in vivo and in vitro. It has a distinct and discontinuous distribution in the brain, as well as in other mammalian tissues and body fluids. However, the distribution of isatin binding sites in the brain is not known. Using a real-time beta-imager we have investigated the distribution of [3H]isatin-specific binding in rat brain sections. The highest labeling was found in hypothalamic nuclei and in the cortex, hippocampus, and cerebellum. Administration of the mechanism based monoamine oxidase inhibitor, pargyline, reduced but did not abolish the specific binding of [3H]isatin in the rat brain. The distribution became cortex, cerebellum, hypothalamus > hippocampus > brain stem > thalamus approximately striatum.     

In vivo specific binding of [3H]1-nicotine in the mouse brain

[3H] 1-Nicotine was used as a receptor ligand in the intact mouse. It was injected i.v., and radioactivity in brain regions was assayed. Nonspecific binding was estimated by pretreatment with unlabelled 1-nicotine. Radioactivity entered the brain rapidly, was heterogeneously distributed, and declined after 5 min. Estimated specific binding was highest in the medial and posterior cortex, midbrain, thalamus/hypothalamus and medulla/pons; intermediate in the cerebellum, caudate/putamen, frontal and frontoparietal cortex; and lowest in the hippocampus and olfactory bulb. Autoradiography showed similar patterns. Coinjection of unlabelled 1-nicotine reduced specific binding so that it approached estimated nonspecific binding. Nicotinic agonists reduced radioactivity in the thalamus/hypothalamus, but nicotinic antagonists were less active. Non-nicotinic drugs did not reduce brain radioactivity. The results suggest that radiolabelled nicotine may be used for in vivo receptor studies despite problems in estimating nonspecific binding.     

Influence of lipid peroxidation on [H]ketanserin binding to 5-HT2 prefrontal cortex receptors

The influence of lipid peroxidation on 5-HT₂ receptor binding was examined in prefrontal cortex membranes from sheep brain. Lipid peroxidation was induced with ascorbic acid and ferrous sulphate and measured by the thiobarbituric acid method. In lipid-peroxidized membranes, [³H]ketanserin specific binding was inhibited. The B_max values decreased by 80%, from 50.1±3.5 fmol/mg protein in control membranes to 10.1±2.0 fmol/mg protein in peroxidized membranes, indicating a decrease in the number of 5-HT₂ binding sites. However, the K_D values for the [³H]ketanserin specific binding did not significantly change. In order to further characterize [³H]ketanserin binding, the inhibition potency (IC₅₀ values) of antagonists or agonists of serotonin and dopamine receptors for [³H]ketanserin specific binding was determined. In control membranes, the order of the inhibition potency of the drugs tested was the following: ketanserin (−log [IC₅₀] = 8.56±0.70) ritanserin (−log [IC₅₀] = 8.13±0.30) methysergide (−log [IC₅₀] = 7.42±0.50) spiperone (−log [IC₅₀] = 7.23±0.18) serotonin (−log [IC₅₀] = 6.99±0.65) haloperidol (−log [IC₅₀] = 6.95±0.65) dopamine (−log [IC₅₀] = 5.82±0.76). After membrane lipid peroxidation, the IC₅₀ value for ritanserin was significantly increased, suggesting a decreased capacity for displacing [³H]ketanserin specific binding. Other antagonists of 5-HT₂ receptors showed apparent increases in IC₅₀ values upon peroxidation, whereas spiperone was shown to be the most potent drug (−log [IC₅₀] = 7.19±1.06) in inhibiting [³H]ketanserin specific binding. A decrease in polyunsaturated fatty acids, namely docosahexaenoic acid (22:6) was also observed in peroxidized membranes. These results indicate a modulating role of the surrounding lipids and of the physical properties of the membranes on the binding activity of 5-HT₂ receptors upon the lipid peroxidation process, which can be involved in the tissue impairment that occurs during the aging process and in post-ischemic situations.     

Alkylation of [3H]8-OH-DPAT binding sites in rat cerebral cortex and hippocampus

The binding of tritiated 8-hydroxy-2-(di-n-propyl-amino)tetralin, or [³H]8-OH-DPAT, to membranes from rat cerebral cortex and hippocampus could be inhibited by serotonin (5-HT) and buspirone, and by the 5-HT antagonists propranolol, NAN-190, pindolol, pindobind-5-HT_1A, WAY100135, spiperone and ritanserin. All competition curves, except for ritanserin, best fitted a two-site model. In vitro treatment of the membranes withN-ethylmaleimide (NEM), to alkylate sulfhydryl groups, caused dose-dependent decreases of binding; the inhibition curves were biphasic, and the effects irreversible. Reduction of disulfide bonds withl-dithiothreitol (L-DTT) also decreased binding, but in a monophasic way; these effects were fully reversible in cortex, but only partially reversible in hippocampus. In the latter region, but not in cerebral cortex, previous occupancy by [³H]8-OH-DPAT partially protected binding from the effects of bothL-DTT and NEM, suggesting that the thiol groups in the receptor recognition site(s) of this brain region are readily accessible. The binding characteristics were examined with the aid of saturation curves, carried out with increasing concentrations, up to 140 nM, of [³H]8-OH-DPAT. The saturation data were suggestive of a two-site receptor model incorporating a high-affinity site (Kh of 0.3–0.5 nM) corresponding to the 5-HT_1A receptor, and a low-affinity site (Kl ofca 25 nM). After in vivo alkylations, carried out by treating rats withN-ethoxycarbonyl-2-ethoxy-1,2-dihydro-quinoline (EEDQ), the saturation curves from both control and EEDQ-treated rats were again best fitted to a two-site model. For EEDQ-treated animals, a drastic decrease of 5-HT_1A receptor activity was noted; this loss was greater in hippocampus than in cerebral cortex. Since the decrease in 5-HT_1A receptors was not associated with changes in low-affinity binding, the results suggest independent regulations of the two [³H]8-OH-DPAT binding proteins. Altogether, the present data further supports the notion that [³H]8-OH-DPAT, besides labelling 5-HT_1A receptors, also binds to other structures in rat cerebral cortex and hippocampus. Special issue dedicated to Dr. Kinya Kuriyama     

3H]epinephrine and [3H]norepinephrine binding to alpha-noradrenergic.

(±)-[³H]Epinephrine and (?)-[³H]norepinephrine bind saturably to calf cerebral cortex membranes under appropriate incubation conditions in a fashion indicating that they label α-noradrenergic receptors. Binding of the two [³H]catecholamines is saturable with dissociation constants of 20–30 nM. Binding is stereoselective with (?)-norepinephrine displaying about twenty times greater affinity than (+)-norepinephrine. The relative potencies of catecholamines in competing for these binding sites parallels their relative pharmacologic effects at α-noradrenergic receptors in numerous tissues. Thus, (?)-epinephrine is 2–3 times more potent than (?)-norepinephrine and 500 times more potent than (?)-isoproterenol. Binding is inhibited by low concentrations of the α-antagonists phentolamine and phenoxybenzamine but not by the β-antagonist propranolol.     

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.     

Characterization of [3H]-forskolin binding sites in young and adult rat brain cortex: identification of suramin as a competitive inhibitor of [3H]-forskolin binding

Little is know about forskolin binding in the rat brain during ontogenetic development. For this paper, we have characterized specific binding sites for [3H]-forskolin in cerebrocortical membranes from young (12-day-old) and adult (90-day-old) rats. High-affinity, as well as super-high-affinity, [3H]-forskolin binding sites were detected in samples from both age groups tested, and the binding parameters of these sites differed significantly. Whereas the number of high-affinity [3H]-forskolin binding sites was higher by about 50% in adult than in young rats, their affinity was markedly (about 4 times) lower. In the presence of AlF4-, the number high-affinity [3H]-forskolin binding sites in samples from young rats rose to the level determined in samples from adult animals, and the number of super-high-affinity sites considerably increased in both age groups. The different characteristics of [3H]-forskolin binding found in cerebrocortical membranes from young and adult rats may be closely related to markedly diminished adenyl cyclase activity in preparations from adult animals. Results of our experiments with suramin indicated that this drug may act as a competitive inhibitor of [3H]-forskolin binding.     

Characterization of specific binding sites for [3H]-staurosporine on various protein kinases

Binding of [3H]-staurosporine to different protein kinases was time-dependent, reversible and saturable. Scatchard analysis of saturation isotherms indicated one class of binding sites for [3H]-staurosporine with dissociation constants (KD) of 9.6, 2.0, 3.0 and 7.4 nM for protein kinase C, cAMP-dependent protein kinase, tyrosine protein kinase and calcium/calmodulin-dependent protein kinase respectively. [3H]-staurosporine binding was fully displaced by unlabelled staurosporine or the related compound K-252a whereas other protein kinase inhibitors (H-7, H-8 and W-7) did not compete with [3H]-staurosporine. These data confirm that sataurosporine shows no selectivity for different protein kinases and suggest the putative existence of distinct, specific binding sites for [3H]-staurosporine on these enzymes.     

Benzodiazepine binding in human brain: characterization using [3H]flunitrazepam.

[³H]Flunitrazepam was used to characterize benzodiazepine binding sites in human brain. The benzodiazepine binding sites exhibited high affinity, pharmacological specificity and saturability in their binding of [³H]flunitrazepam. The dissociation constant (K_D) of [³H]flunitrazepam binding was determined by three different methods and found to be in the range of 2–3 nM. The potency of several benzodiazepine analogs to inhibit specific [³H]-flunitrazepam binding $\text{in}$$\text{vitro}$ correlated well with their potency in several $\text{in}$$\text{vivo}$ human and animal tests. The density of [³H]-flunitrazepam binding sites was highest in the cerebrocortical and rhinencephalic areas, intermediate in the cerebellum, and lowest in the brain stem and commissural tracts.