α-Bungarotoxin Binds to Low-Affinity Nicotine Binding Sites in Rat Brain

Reported differences in the pharmacology and distribution of [3H]nicotine and [125I]alpha-bungarotoxin binding sites in mammalian brain suggest that these ligands label separate receptor sites. Affinity purification of an alpha-bungarotoxin binding protein from rat brain failed to copurify the high-affinity nicotine binding site, which remained in the nonbound soluble fraction after the affinity chromatography step. This confirms the independence of these putative receptor sites. Nevertheless, the binding of [125I]alpha-bungarotoxin to P2 membranes was inhibited by (-)-nicotine (Ki = 9 X 10(-6) M), and this sensitivity was preserved after affinity purification. It is proposed that alpha-bungarotoxin binds to a population of low-affinity nicotine binding sites. Comparison of the enantiomers of nicotine in competition studies at both radioligand binding sites revealed an 80-fold preference for the (-) form at the high-affinity [3H]nicotine binding site, whereas the site labelled by [125I]alpha-bungarotoxin displayed little stereoselectivity. In this respect, the brain alpha-bungarotoxin binding site resembles the nicotinic acetylcholine receptor from Torpedo electric organ.     

Interaction of monoclonal antibodies with alpha-bungarotoxin and (-)-nicotine binding sites in goldfish brain. Identification of putative nicotinic acetylcholine receptor subtypes

Monoclonal antibodies raised against the nicotinic acetylcholine receptor of Electrophorus electricus electroplaque have been used as probes to characterize putative nicotinic acetylcholine receptors in goldfish brain. One monoclonal antibody (mAb), mAb 47, recognized a protein which binds both (-)-[3H]nicotine and 125I-alpha-bungarotoxin with high affinity. Another monoclonal antibody (mAb 172) recognized a protein which binds (-)-[3H]nicotine but not 125I-alpha-bungarotoxin. Both antibodies precipitated a protein(s) (biosynthetically labeled with [35S]methionine) in the absence, but not in the presence, of excess purified nicotinic acetylcholine receptor from Torpedo nobiliana. The dilution of mAb 47 that precipitated half of the maximum amount of 125I-alpha-bungarotoxin binding protein was the same as that which precipitated half of the maximum amount of (-)-[3H]nicotine binding activity. When used in combination, the two antibodies precipitated more (-)-[3H]nicotine radioactivity than either antibody alone. The (-)-[3H]nicotine and 125I-alpha-bungarotoxin binding component-mAb complexes were characterized by sucrose density centrifugation. In the presence of either mAb 172 or 47, the (-)-[3H] nicotine binding component migrated further into the gradient, but only mAb 47 shifted the 125I-alpha-bungarotoxin peak. Incubation of solubilized brain extract with alpha-bungarotoxin-coupled Sepharose reduced the amount of (-)-[3H]nicotine radioactivity precipitated by mAb 47 but not by mAb 172. These data suggest that the antibodies may recognize distinct subtypes of (-)-nicotine binding sites in goldfish brain, one subtype which binds both 125I-alpha-bungarotoxin and (-)-[3H]nicotine and a second subtype which binds only (-)-[3H] nicotine.     

Effects of Chronic Nicotine Infusion on Kinetics of High-Affinity Nicotine Binding

Abstract: It is well established that chronic nicotine treatment produces a dose-dependent increase in high-affinity l -[³H]nicotine binding. This increase may be due to chronic desensitization of the receptor. Sophisticated kinetic analyses of high-affinity nicotine binding to rat brain have demonstrated that the association rate is biphasic; the fast phase may represent binding to a high-affinity predesensitized state and the slow phase may represent binding to a lower affinity ground state that then isomerizes to form the high-affinity binding site. This isomerization presumably leads to receptor desensitization. The studies reported here assessed whether binding to mouse brain nicotinic receptors shows these same properties and whether chronic intravenous infusion of nicotine results in changes in these kinetic properties. The results obtained indicate that mouse brain nicotine binding also shows biphasic association kinetics and uniphasic dissociation kinetics, which supports the assertion that the receptor exists in two interconvertible states. However, unlike other results obtained with rat brain, the rate of the slow association process did not change with ligand concentration. Chronic infusion resulted in a dose-dependent increase in l -[³H]-nicotine binding, but the ratio of fast/slow phases of binding was not changed by these treatments. These results suggest that chronic infusion does not alter measurably the kinetics of nicotinic receptor binding when measured in vitro.     

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.     

Monospecific antibodies against a synthetic peptide predicted from the alpha-3 nicotinic receptor cDNA inhibit binding of [3H]nicotine to rat brain nicotinic cholinergic receptor

Polyclonal antibodies were raised against a synthetic decapeptide (designated S3) predicted from a segment of the alpha-3 subunit cDNA (amino acid residues 130-139) encoding the rat brain nicotinic cholinergic receptor. This segment was selected because it may be proximate to the nicotine/acetylcholine-binding site of the receptor (1). By radioligand binding assays and sucrose density gradient centrifugation, these monospecific antibodies were shown to inhibit the binding of [3H]nicotine to both the large molecular weight rat brain receptor (240 kDa) and to an SDS-disaggregated nicotine-binding subunit species (80 kDa), in a dose-dependent manner. The neutralizing effect of the anti-S3 antibodies supports the view that this region of the protein is closely related to the agonist binding site.     

A solid-phase competitive radioimmunoassay for the insulin receptor

A radioimmunoassay for the insulin receptor has been developed. In this assay, unlabeled receptor competes with 125I-labeled receptor for binding to monoclonal anti-receptor antibodies immobilized on microtiter wells coated with affinity-purified anti-mouse immunoglobulin G. This assay was highly reproducible and could detect 7 ng (14 fmol) of insulin receptor. By utilizing monoclonal antibodies to various antigenic regions of the receptor, different parts of the receptor molecule could be examined. By utilizing antibodies to the cytoplasmic domain of the receptor, an assay was developed which was not influenced by the presence of insulin and could equally detect the insulin receptor from different species (rat and human) and different tissues (placenta and brain). By utilizing antibodies to an autophosphorylation site of the receptor, the assay was shown capable of detecting the extent of phosphorylation of the receptor. Finally, this assay was utilized to monitor the decrease in insulin receptors in lysates of insulin-treated human lymphocytes. This radioimmunoassay should be useful for monitoring both the number and status of the insulin receptor under a variety of physiological conditions.     

Evidence for a noncholinergic nicotine receptor on human phagocytic leukocytes

Summary A noncholinergic nicotine receptor on human phagocytic leukocytes has been characterized using the binding of³H-(d,l)-nicotine. The average affinity +/-standard deviation of (d,l)-nicotine for the receptor on neutrophils is 36 ± 18 nM (n=6). The binding is saturable with an average of 8.7 x 10⁴ sites per neutrophil. Monocytes and to a lesser extent lymphocytes but not erythrocytes also display specific binding. Bound nicotine is dissociable from the receptor and is not metabolized. Only close structural analogs of nicotine bind to the receptor, which is stereoselective for the (d)-isomer. The receptor can be occupied by (l)-nicotine at concentrations present in the blood of smokers. It is suggested that some of the adverse effects of smoking on leukocyte functions may be mediated by a specific nicotine receptor.     

Interaction of the nicotinic agonist (R,S)-3-pyridyl-1-methyl-2-(3-pyridyl)-azetidine (MPA) with nicotinic acetylcholine receptor subtypes expressed in cell lines and rat cortex

The interaction of the nicotinic agonist (R,S)-3-pyridyl-1-methyl-2-(3-pyridyl)-azetidine (MPA) with different nicotinic acetylcholine receptor (nAChR) subtypes was studied in cell lines and rat cortex. MPA showed an affinity (Ki = 1.21 nM) which was higher than anatoxin-a > (−)-nicotine > (+)-[R]nornicotine > (−)-[S]nornicotine > and (+)-nicotine, but lower than cytisine (Ki = 0.46 nM) in competing for (−)-[³H]nicotine binding in M10 cells, which stably express the recombinant ₄β₂ nAChR subtype. A one-binding site model was observed in all competing experiments between (−)-[³H]nicotine binding and each of the agonists studied in M10 cells. MPA showed a 13-fold higher affinity for (−)-[³H]nicotine binding sites compared to the [³H]epibatidine binding sites in rat cortical membranes. In human neuroblastoma SH-SY5Y cells, which predominantly express the ₃ nAChR subunit mRNA, MPA displaced [³H]epibatidine binding from a single population of the binding sites with an affinity in the same nM range as that observed MPA in displacing [³H]epibatidine binding in rat cortical membranes. Chronic treatment of M10 cells with MPA significantly up-regulated the number of (−)-[³H]nicotine binding sites in a concentration dependent manner. Thus MPA appears to have higher affinity to 4-subunit containing receptor subtype than ₃-subunit containing receptor subtype of nAChRs. Furthermore MPA binds to ₄β₂ receptor subtype with higher affinity than (−)-nicotine and behaves, opposite to cytisine, as a full agonist in up-regulating the number of nAChRs. © 1998 Elsevier Science Ltd. All rights reserved.     

Molecular organization of glutamate-sensitive chemo-stimulated nerve cell membranes. Interaction of monoclonal antibodies with glutamate-binding membrane proteins in the rat brain, human neuroblastoma and molluscan neurons

Hybrid cells obtained by fusion of myeloma PX63-Ag8-653 with immune splenocytes of BALB/c mice were found to produce monoclonal antibodies with a high degree of specificity to rat and human brain. The kinetics of specific IgG binding to purified fractions of glutamate-binding membrane proteins from rat and human brain were analyzed in Scatchard plots. The presence of a single type of binding sites with Kd = 100 nM was demonstrated. The monoclonal antibodies were shown to inhibit the specific binding of tritium-labeled L-glutamate to different brain synaptic membranes. Addition of monoclonal antibodies to the incubation medium induced a modulating effect of physiological responses to L-glutamate in Planorbarius corneus neurons. The possible use of specific antibodies to glutamate-binding proteins as immunochemical markers for the study of glutamate receptor topography on membrane surface was demonstrated with the aid of neuroblastoma cells N18 Tg2a and rat brain tissue slices. An analysis of glutamate receptor binding sites with the use of monoclonal antibodies revealed that these antibodies specifically recognize the active center in the receptor molecules which have identical antigen determinant sites in different biological systems.     

Anti-angiotensin II anti-idiotypic antibodies bind to angiotensin II receptor

An anti-idiotypic serum from a rabbit immunized with one anti-angiotensin II (AII) monoclonal antibody (A25) was shown to identify a cross-reactive idiotope (CRI) shared by six anti-AII monoclonal antibodies, in addition to a binding site-associated private idiotope. This anti-idiotypic reagent bound to rat liver membranes bearing AII receptors; binding was abolished after pretreatment of the membranes with AII. In immunoblotting experiments with rat liver membranes, as well as with rat pituitary homogenates, a 63,000 +/- 2,000 dalton protein was revealed that co-migrated with the AII receptor. After purification by affinity chromatography on an immobilized CRI+-antibody (A41), anti-CRI antibodies could immunoprecipitate the hormone binding activity from detergent-treated rat liver membranes and still recognize the 63,000 dalton protein. In contrast, anti-idiotypic antibodies specific for the private idiotope failed to interact with the AII receptor. Similar results were obtained with a second anti-idiotypic serum produced by immunization with another CRI+ anti-AII monoclonal antibody (A22). The sharing of the CRI determinant between the AII receptor and anti-AII antibodies might account for the reactivity of anti-idiotypic antibodies towards the AII receptor.     

Evidence that the acetylcholine binding site is not formed by the sequence alpha 127-143 of the acetylcholine receptor

The sequence alpha 127-143 of the alpha subunit of the acetylcholine receptor has been proposed to contain several important features: (1) the acetylcholine binding site, (2) the only N-glycosylation site of the alpha subunit, at asparagine-alpha 141, and (3) two cysteine residues, at alpha 128 and alpha 142, that may participate in a disulfide bond known to be near the binding site. We tested these hypotheses by using antisera to receptor and its subunits and monoclonal antibodies to the synthetic peptide alpha 127-143 cyclized by a disulfide bond between alpha 128 and alpha 142. Antisera to receptor and its alpha subunit were able to immunoprecipitate the iodinated peptide, and this reaction was inhibited by soluble receptor, but not by membrane-bound receptor. alpha-Bungarotoxin did not inhibit antiserum binding to solubilized receptor. Similarly, cholinergic ligands had little or no effect on binding to immobilized receptors of anti-peptide monoclonal antibodies. In addition, these monoclonal antibodies, when bound to the receptor, did not affect toxin binding kinetics. By contrast, preincubation with concanavalin A did inhibit monoclonal antibody binding. Reduction of the receptor significantly decreased the binding of three of the monoclonal antibodies, but subsequent alkylation with N-ethylmaleimide or the affinity labeling reagent bromoacetylcholine had no additional effect on binding. A dithiothreitol concentration about 100-fold higher that the one needed to reduce the disulfide near the acetylcholine binding site was necessary to inhibit monoclonal antibody binding. We conclude that the sequence alpha 127-143 is not fully exposed on the surface when the receptor is in the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of monoclonal anti-enzyme antibodies for analytical purposes.

This review discusses the analytical applications of monoclonal antibodies specific for enzymes. One important, but not well-studied, application of these monoclonal antibodies is their use in immobilizing enzymes on solid supports. This method is based on binding the enzymes to an immobilized antibody through the antigen binding site of the antibody. Enzymes immobilized this way retain much of their activity. The utility of immobilized enzyme reactors prepared by immobilizing the enzymes through antibodies is demonstrated by using them in the determination of acetylcholine and choline in brain tissue extracts. Currently available methods for immobilizing antibodies and enzymes are reviewed. Other issues discussed in this review include the problems and advantages of immobilized enzyme reactors, especially when used in conjunction with HPLC. In addition, the applications of monoclonal antibodies for the detection and measurement of enzymes and their isoforms are summarized.     

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.     

Monoclonal antibodies to the rat liver glucocorticoid receptor.

Monoclonal antibodies against the 90 000 mol. wt. form of the activated rat liver glucocorticoid receptor were generated from mice immunized with a partially purified receptor preparation. The screening assay was based on the precipitation of liver cytosol, labelled with [3H]triamcinolone acetonide, with monoclonal antibodies bound to immobilized rabbit anti-mouse IgG. Out of 102 hybridomas obtained, 76 produced immunoglobulin and eight of them were found to react with the receptor molecule. Only one of the positive clones secreted IgG whereas the other seven produced IgM. The complexes of receptor and antibodies were identified by sucrose density gradient centrifugation. All seven monoclonal antibodies tested reacted with the 90 000 mol. wt. form of the receptor but not with the 40 000 mol. wt. form that contains the steroid and DNA binding domains. None of the monoclonal antibodies interfered with the binding of the receptor to DNA cellulose, thus suggesting that the antigenic determinants are located in a region of the receptor that is not directly implicated in either steroid binding or DNA binding. These antigenic determinants were common to glucocorticoid receptors from several tissues of the rat, whereas glucocorticoid receptors from other species react only with some of the antibodies.     

Stereoselective Nicotine-Induced Release of Dopamine from Striatal Synaptosomes: Concentration Dependence and Repetitive Stimulation

Using a sensitive perfusion system we have studied the nicotine-induced release of [3H]dopamine ([( 3H]DA) from striatal synaptosomes. Nicotine-evoked release was concentration dependent with an EC50 of 3.8 microM. The response to 1 microM nicotine was comparable to that to 16 mM K+; 10 microM veratridine evoked a larger response. All three stimuli were Ca2+ dependent but only the response to veratridine was blocked by tetrodotoxin. Repetitive stimulations by 1 microM (-)-nicotine (100 microliters) at 30-min intervals resulted in similar levels of [3H]DA release; higher concentrations of (-)-nicotine resulted in an attenuation of the response particularly following the third stimulation. This may reflect desensitisation or tachyphylaxis of the presynaptic nicotinic receptor. The action of nicotine was markedly stereoselective: a 100-fold higher concentration of (+)-nicotine was necessary to evoke the same level of response as 1 microM (-)-nicotine. It is proposed that these presynaptic nicotinic receptors on striatal terminals are equivalent to high-affinity nicotine binding sites described in mammalian brain.     

Nicotine Indirectly Inhibits [3H]Dopamine Uptake at Concentrations That Do Not Directly Promote [3H]Dopamine Release in Rat Striatum

The effects of both (-)- and (+)-nicotine isomers were examined on in vitro uptake and release of [3H]dopamine in rat striatum. Both isomers inhibited uptake of [3H]dopamine in chopped tissue at concentrations well below those necessary for promoting release of preloaded [3H]dopamine. (-)-Nicotine was more potent than (+)-nicotine both at inhibiting uptake and at promoting release. Unlike other dopamine uptake inhibitors, however, nicotine inhibited only 50% of the total uptake. In the presence of 1 nM nicotine, the residual [3H]dopamine uptake was less sensitive to inhibition by cocaine than uptake in the absence of nicotine. Nicotine did not compete against the binding of [3H]GBR 12935, a selective dopamine uptake inhibitor. The nicotinic receptor agonists carbachol and 1,1-dimethyl-4-phenylpiperazinium iodide also inhibited uptake, whereas the nicotinic antagonists chlorisondamine and mecamylamine blocked nicotine's effect. Thus, the effect of nicotine on dopamine uptake appears to be mediated by a receptor similar to the nicotinic acetylcholine receptor. These receptors do not seem to be on the terminals that are accumulating dopamine, however, since tetrodotoxin prevented the effect of nicotine on [3H]dopamine uptake and nicotine had no effect on uptake in a synaptosomal preparation.     

Selection of a Novel Anti-Nicotine Vaccine: Influence of Antigen Design on Antibody Function in Mice

Anti-nicotine vaccines may aid smoking cessation via the induction of anti-nicotine antibodies (Ab) which reduce nicotine entering the brain, and hence the associated reward. Ab function depends on both the quantity (titer) and the quality (affinity) of the Ab. Anti-nicotine vaccines tested previously in clinical studies had poor efficacy despite high Ab titer, and this may be due to inadequate function if Ab of low affinity were induced. In this study, we designed and synthesized a series of novel nicotine-like haptens which were all linked to diphtheria toxoid (DT) as carrier, but which differed in the site of attachment of linker to nicotine, the nature of linker used, and the handle used to attach the hapten to DT. The resulting hapten conjugates were evaluated in a mouse model, using CpG (a TLR9 agonist) and aluminum hydroxide (Al(OH)₃) as adjuvants, whereby Ab titers, affinity and function were evaluated using a radiolabeled nicotine challenge model. A series of additional linkers varying in length, rigidity and polarity were used with a single hapten to generate additional DT-conjugates, which were also tested in mice. Conjugates made with different haptens resulted in various titers of anti-nicotine Ab. Several haptens gave similarly high Ab titers, but among these, Ab affinity and hence function varied considerably. Linker also influenced Ab titer, affinity and function. These results demonstrate that immune responses induced in mice by nicotine-conjugate antigens are greatly influenced by hapten design including site of attachment of linker to nicotine, the nature of linker used, and the handle used to attach the hapten to DT. While both Ab titer and affinity contributed to function, affinity was more sensitive to antigen differences.     

Purification and characterization of the human brain insulin receptor

The insulin receptor from human brain cortex was purified by a combination monoclonal antibody affinity column and a wheat germ agglutinin column. This purified receptor preparation exhibited major protein bands of apparent Mr = 135,000 and 95,000, molecular weights comparable to those for the alpha and beta subunits of the purified human placental and rat liver receptors. A minor protein band of apparent Mr = 120,000 was also observed in the brain receptor preparation. Crosslinking of 125I-insulin to all three receptor preparations was found to preferentially label a protein of apparent Mr = 135,000. In contrast, cross-linking of 125I-labeled insulin-like growth factor I to the brain preparation preferentially labeled the protein of apparent Mr = 120,000. The purified brain insulin receptor was found to be identical with the placental insulin receptor in the amount of neuraminidase-sensitive sialic acid and reaction with three monoclonal antibodies to the beta subunit of the placental receptor. In contrast, a monoclonal antibody to the insulin binding site recognized the placental receptor approximately 300 times better than the brain receptor. These results indicate that the brain insulin receptor differs from the receptor in other tissues and suggests that this difference is not simply due to the amount of sialic acid on the receptor.     

Demonstration and purification of an endogenous benzodiazepine from the mammalian brain with a monoclonal antibody to benzodiazepines

Four hybridoma lines secreting monoclonal antibodies to benzodiazepines were produced after BALB/c mice were immunized with a benzodiazepine-bovine serum albumin conjugate. The monoclonal antibodies were purified from ascites fluids, and their binding affinities for benzodiazepines and other benzodiazepine receptor ligands were determined. These antibodies have very high binding affinities for diazepam, flunitrazepam, Ro5-4864, Ro5-3453, Ro11-6896, and Ro5-3438 (the Kd values are in the 10(-9) M range). However, these antibodies have very low affinities for the benzodiazepine receptor inverse agonists (beta-carbolines) and antagonists (Ro15-1788 and CGS-8216). One of the monoclonal antibodies (21-7F9) has been used to demonstrate the existence of benzodiazepine-like molecules in the brain and for the purification of these molecules. Immunocytochemical experiments show that these molecules are neuronal and not glial and that they are ubiquitously distributed throughout the brain. Immunoblots indicate the presence of benzodiazepine-like epitopes in several brain peptides. An endogenous substance that binds to the central-type benzodiazepine receptor with agonist properties has been purified to homogeneity from the bovine brain. The purification consisted on immunoaffinity chromatography on immobilized monoclonal anti-benzodiazepine antibody followed by gel filtration on Sephadex G-25 and two reverse phase HPLCs. The purified substance has a small molecular weight and its activity is protease resistant. The endogenous substance blocks the binding of agonists, inverse agonists and antagonists to the central-type benzodiazepine receptor but it does not inhibit the binding of Ro5-4864 to the peripheral-type benzodiazepine receptor. The neurotransmitter gamma-aminobutyric acid increases the affinity of the benzodiazepine receptor for the purified substance. Preliminary evidence indicates that the purified substance is a benzodiazepine with a molecular structure that is identical or very close to N-desmethyldiazepam.