Comparative Analysis of Nicotine-Like Receptor-Ligand Interactions in Rodent Brain Homogenate

The effects of different variables such as incubation time, temperature, tissue protein content, and pH on the interactions of various labelled nicotinic ligands with nicotine-like binding sites in vitro were studied in rodent brain preparations. The ligands tested were alpha-[3H]bungarotoxin (alpha-[3H]BTX), [3H]tubocurarine ([3H]TC), and [3H]nicotine ([3H]NIC). The regional distribution of the labelled nicotinic ligand binding was also studied and affinity constants and maximal binding (Bmax) values for the equilibrium [3H]NIC binding are given. Association kinetics for [3H]NIC and [3H]TC binding to brain homogenate were similar, with maximal binding within 5-10 min of incubation, followed by a continuous decrease. In contrast, the binding of alpha-[3H]BTX to brain homogenate was much slower, reaching equilibrium after 30-60 min of incubation. Scatchard analysis of equilibrium binding data for [3H]NIC in the hippocampus indicated two binding sites: a high-affinity site (Bmax, 60 pmol/g protein; KD, 6 nM) and a low-affinity site (Bmax, 230 pmol/g protein; KD, 125 nM). The data for the high-affinity [3H]NIC binding site are very similar to previously found data for the high-affinity binding site of [3H]TC and the binding site of alpha-[3H]BTX. Each ligand showed regional differences in binding, and the binding pattern also differed between the ligands.     

Pharmacological Characterization of Cholinergic Receptors in a Human Neuroblastoma Cell Line

A human neuroblastoma cell line, IMR32, has been characterized as far as morphology, membrane receptors for neurotransmitters, and uptake and release of [3H]3,4-dihydroxyphenylethylamine ([3H]dopamine). These cells expressed at their surface both nicotinic and muscarinic cholinergic receptors, revealed by [125I]alpha-bungarotoxin and [3H]quinuclidinylbenzilate ([3H]QNB) binding, respectively. [125I]alpha-Bungarotoxin binding was efficiently inhibited by alpha-bungarotoxin, nicotine, carbachol, and d-tubocurarine. [3H]QNB binding was competitively inhibited by atropine, pirenzepine, and carbachol. Hexamethonium did not affect the binding of either ligand. In competition experiments with [3H]QNB, pirenzepine recognized only one binding site with "low affinity," and carbachol recognized two sites with different affinities. beta-adrenergic receptors were present in a very low amount, whereas alpha-adrenergic and dopaminergic receptors were not detectable. IMR32 cells had an imipramine-sensitive [3H]dopamine uptake, but carbachol, high levels of K+, the calcium ionophore A23187, and alpha-latrotoxin were not able to induce release of [3H]dopamine that had been taken up. The ultrastructural analysis showed that IMR32 cells contained very few dense-core vesicles, suggesting a low storage capacity for neurotransmitter. These cells could be an useful in vitro model for studying neurotransmitter receptors of the human CNS.     

Postnatal Development of Cholinergic Enzymes and Receptors in Mouse Brain

The developmental profiles for the cholinergic enzymes acetylcholinesterase and choline acetyltransferase, and the muscarinic and nicotinic receptors were determined in whole mouse brain. The enzyme activities (per milligram of protein) increased steadily from birth, reaching adult levels at 20 days of age. These increases were primarily due to increases in Vmax. Muscarinic receptor numbers, measured by [3H]quinuclidinyl benzilate binding, also increased from birth to 25 days of age. Brain nicotinic receptors were measured with the ligands L-[3H]nicotine and alpha-[125I]-bungarotoxin. Neonatal mouse brain had approximately twice the number of alpha-bungarotoxin binding sites found in adult mouse brain. Binding site numbers rose slightly until 10 days of age, after which they decreased to adult values, which were reached at 25 days of age. The nicotine binding site was found in neonatal brain at concentrations comparable to those at the alpha-bungarotoxin site followed by a steady decline in nicotine binding until adult values were reached. Thus, brain nicotinic and muscarinic systems develop in totally different fashions; the quantity of muscarinic receptors increases with age, while the quantity of nicotinic receptors decreases. It is conceivable that nicotinic receptors play an important role in directing the development of the cholinergic system.     

Changes in Nicotinic and Muscarinic Cholinergic Receptors in Alzheimer-Type Dementia

Nicotinic and muscarinic cholinergic receptors were studied in autopsied brains from four histologically normal controls and five histopathologically verified cases of Alzheimer-type dementia (ATD), using ligand binding techniques. Nicotinic and muscarinic cholinergic receptors were assessed by (-)-[3H]nicotine and [3H]quinuclidinyl benzilate [( 3H]QNB), respectively. Compared with the controls, (-)-[3H]nicotine binding sites in the ATD brain regions examined were significantly reduced in the putamen and the nucleus basalis of Meynert (NbM). [3H]QNB binding was significantly reduced in the hippocampus and NbM. These findings suggest that there are significant changes of nicotinic and muscarinic cholinergic receptors in selected regions of ATD brains.     

Muscarinic and Nicotinic Cholinergic Binding Sites in the Terminal Abdominal Ganglion of the Cricket (Acheta domesticus)

Cricket (Acheta domesticus) terminal abdominal ganglia (TG) contain high concentrations (approximately 2 pmol/mg protein) of muscarinic and nicotinic cholinergic binding sites, based on the capacity of TG to bind specifically the labelled ligands L-[3H]quinuclidinyl benzilate ([3H]QNB) and [125I]alpha-bungarotoxin ([125I]alpha-BGT) with high affinity. For both ligands, binding is saturable and reversible. Competitive displacement experiments indicate that the [3H]QNB and [125I]alpha-BGT binding sites probably represent pharmacologically distinct classes of putative TG acetylcholine receptors (AChRs). Results from physiological recording and autoradiographic localization experiments demonstrate that a portion of the putative nicotinic AChRs is localized in synaptic regions of the well-characterized cercal sensory-giant interneuron pathway in the TG, where they are likely to serve as functional synaptic AChRs. Unlike nicotinic ligands, muscarinic agents do not appear to be pharmacologically active in this pathway. Therefore, in the insect CNS, putative muscarinic and nicotinic AChRs coexist at high density, but can be pharmacologically distinguished from one another on the basis of criteria derived from both ligand binding and physiological methods.     

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.     

Evidence for Functional Activity of Up-Regulated Nicotine Binding Sites in Rat Striatal Synaptosomes

A number of studies have found that the chronic administration of nicotine causes an increase in the density of nicotinic binding sites in the brain, but it is not known whether these additional binding sites are functionally active receptors. In this study, the effects of 1-week administration of the potent nicotinic agonist, (+)-anatoxin-a (96 nmol/day via osmotic minipumps), was assessed on [3H]nicotine binding and [3H]dopamine uptake and release in rat striatal synaptosomes. Chronic (+)-anatoxin-a treatment resulted in a 32% increase in the Bmax of [3H]nicotine binding in anatoxin-treated animals compared to control. There was a 43% increase in the activity of 3 microM nicotine to release [3H]dopamine from synaptosomes of anatoxin-treated animals, but the release induced by 20 mM K+ depolarization was unaffected. There was no effect of chronic (+)-anatoxin-a treatment on the uptake of [3H]dopamine. A strong positive correlation (r = 0.64) was found between the density of [3H]nicotine binding sites and the nicotine-induced stimulation of [3H]dopamine release in individual animals. These results indicate that (+)-anatoxin-a, like nicotine, produces an up-regulation of nicotine binding sites following chronic administration, and that these additional sites are functional receptors capable of mediating the release of dopamine from striatal synaptosomes.     

Chronic nicotine administration does not increase nicotinic receptors labeled by [125I]epibatidine in adrenal gland,superior cervical ganglia,pineal or retina

Neuronal nicotinic acetylcholine receptors (nAChRs) were measured in CNS and peripheral tissues following continuous exposure to saline or nicotine hydrogen tartrate (3.3 or 10 mg/kg/day) for 14 days via osmotic pumps. Initially, binding of [3H](-)nicotine, [3H]cytisine and [3H]epibatidine to nAChRs was compared to determine the suitability of each for these kinds of studies. The predominant nAChR labeled by agonists in the cerebral cortex is an alpha 4 beta 2 subtype, whereas the predominant nicotinic receptors in the adrenal gland, superior cervical ganglia and pineal gland contain an alpha 3 subunit, and they do not bind either [3H](-)nicotine or [3H]cytisine with high affinity. In retina some nAChRs bind all three ligands with high affinity, and others appear to bind only [3H]epibatidine. Thus, only [3H]epibatidine had high enough affinity to be useful for measuring the nAChRs in all of the tissues. The receptors from nicotine-treated rats were then measured using [125I]epibatidine, which has binding characteristics very similar to [3H]epibatidine. Treatment with the two doses of nicotine hydrogen tartrate increased binding sites in the cerebral cortex by 40% and 70%, respectively. In contrast, no significant changes in the density of receptor binding sites were found in the adrenal gland, superior cervical ganglia, pineal gland or retina. These data indicate that chronic administration of nicotine even at high doses does not increase all nicotinic receptor subtypes, and that receptors containing alpha 3 subunits may be particularly resistant to this nicotine-induced change.     

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.     

In Vivo Regulation of [3H]Acetylcholine Recognition Sites in Brain by Nicotinic Cholinergic Drugs

The in vivo regulation of [3H]acetylcholine [( 3H]ACh) recognition sites on nicotinic receptors in rat brain was examined by administering drugs that increase stimulation of nicotinic cholinergic receptors, either directly or indirectly. After 10 days of treatment with the cholinesterase inhibitor diisopropyl fluorophosphate, [3H]ACh binding in the cortex, thalamus, striatum, and hypothalamus was decreased. Scatchard analyses indicated that the decrease in binding in the cortex was due to a reduction in the apparent density of [3H]ACh recognition sites. In contrast, after repeated administration of nicotine (5-21 days), the number of [3H]ACh recognition sites was increased in the cortex, thalamus, striatum, and hypothalamus. Similar effects were observed in the cortex and thalamus following repeated administration of the nicotinic agonist cytisin. The nicotinic antagonists mecamylamine and dihydro-beta-erythroidine did not alter [3H]ACh binding following 10-14 days of administration. Further, concurrent treatment with these antagonists and nicotine did not prevent the nicotine-induced increase in these binding sites. The data indicate that [3H]ACh recognition sites on nicotinic receptors are subject to up- and down-regulation, and that repeated administration of nicotine results in a signal for up-regulation, probably through protracted desensitization at the recognition site.     

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.     

Neosurugatoxin blocks nicotinic acetylcholine receptors in the brain

Neosurugatoxin, a neurotoxin isolated from the Japanese ivory mollusc ($\text{Babylonia japonica}$) is a nicotinic antagonist with a specificity towards ganglionic nicotinic receptors. At low concentration (5 × 10^?8 M) neosurugatoxin inhibited the release of [³H]dopamine evoked by 1,1-dimethyl-4-phenylpiperazinium (DMPP) from rat striatal nerve terminals, without affecting the response to K⁺-depolarisation. In contrast, αbungarotoxin did not antagonise the action of DMPP. Neosurugatoxin also inhibited [³H] nicotine binding to rat brain membranes but had no effect on [¹²⁵I]αbungarotoxin binding to the same tissue preparation. These results support the view that functional nicotinic receptors in the CNS resemble ganglionic nicotinic receptors. Neosurugatoxin has considerable potential as a useful probe for such receptors in the brain.     

Effect of Chronic Nicotine Treatment on Nicotinic Autoreceptor Function and N-[3H]Methylcarbamylcholine Binding Sites in the Rat Brain

It has been reported that N-methylcarbamylcholine (MCC), a nicotinic agonist, binds to central nicotinic receptors and causes an increase of acetylcholine (ACh) release from certain central cholinergic nerve terminals. The present experiments determine whether these two phenomena change in response to the chronic administration of nicotine, a procedure known to result in an increase in nicotinic binding sites. Chronic nicotine caused a brain region-specific up-regulation of [3H]MCC sites; binding increased in the frontal cortex, parietal cortex, striatum, and hippocampus, but not in the occipital cortex or cerebellum. The effect of nicotine was selective to nicotinic binding sites, because muscarinic sites, both M1 ([ 3H]pirenzepine) and M2 ([3H]ACh), were unaffected by chronic nicotine treatment. MCC increased the release of ACh from the frontal cortex and hippocampus by a calcium-dependent mechanism; MCC did not alter ACh release from striatum or occipital cortex of control animals. The MCC-induced increase in ACh release was not apparent in those animals which had been treated with nicotine. There was a partial recovery of nicotinic autoreceptor function when animals were allowed to recover (4 days) following chronic nicotine treatment, but the density of binding sites remained increased compared to control. Chronic nicotine did not change the potassium-evoked release of ACh from the frontal cortex or hippocampus, but decreased this measure from striatum. It also decreased the ACh content of the striatum, but not that of the cortex or the hippocampus; the activity of choline acetyltransferase was not altered in any of the regions tested.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of two radiolabeled quinuclidinyl benzilate ligands for the characterization of the human peripheral lung muscarinic receptor

Quinuclidinyl benzilate, a muscarinic antagonist, has previously been used in its tritiated form ([3H]-QNB) to study the lung muscarinic receptor. We investigated whether a newer iodinated form of QNB ([125I]-QNB) of higher specific activity would be an appropriate ligand to study the human peripheral lung muscarinic receptor. Both the tritiated and iodinated ligands bound specifically to human lung at 23 degrees C. At 37 degrees C the specific binding of [3H]-QNB increased slightly, but no specific binding of [125I]-QNB was found. The data from multiple equilibrium binding experiments covering a wide range of radiolabeled QNB concentrations were combined and analyzed using the computer modeling program, LIGAND. The tritiated QNB identified a single affinity human lung binding site with a Kd of 46 +/- 9 pM and a receptor concentration of 34 +/- 3 fmol/mg protein. The iodinated QNB identified a single higher affinity human lung binding site (Kd = 0.27 +/- 0.32 pM) of much smaller quantity (0.62 +/- 0.06 fmol/mg protein). Competition studies comparing the binding of unlabeled QNB relative to labeled QNB indicated that unlabeled QNB had the same Kd as that measured for [3H]-QNB, but a 5 log greater Kd than that measured for [125I]-QNB. Other muscarinic receptor agonists and antagonists competed with [3H]-QNB, but not [125I]-QNB for binding to muscarinic receptors with the expected magnitude and rank order of potency. We conclude that of the 2 radiolabeled forms of QNB available, only the tritiated form should be used to study the human peripheral lung muscarinic receptor.     

Nicotinic Modulation of [3H]Dopamine Release from Striatal Synaptosomes: Pharmacological Characterisation

Presynaptic nicotinic acetylcholine receptors on striatal nerve terminals modulate the release of dopamine. We have compared the effects of a number of nicotinic agonists and antagonists on a perfused synaptosome preparation preloaded with [3H]dopamine. (-)-Nicotine, acetylcholine, and the nicotinic agonists cytisine and 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), at micromolar concentrations, stimulated the release of [3H]dopamine from striatal nerve terminals. Carbamylcholine was a much weaker agonist. The actions of (-)-nicotine, cytisine, and DMPP were inhibited by low concentrations of the nicotinic antagonists dihydro-beta-erythroidine, mecamylamine, pempidine, and neosurugatoxin; alpha-bungarotoxin was without effect, and extending the time of exposure to this toxin resulted in only very modest inhibition. This pharmacology points to a specific nicotinic receptor mechanism that is clearly distinct from that at the neuromuscular junction. Atropine failed to antagonise the effects of acetylcholine and carbamylcholine, suggesting that no muscarinic component is involved. The nicotinic receptor ligands (-)-[3H]nicotine and 125I-alpha-bungarotoxin bound to specific sites enriched in the synaptosome preparation. Drugs tested on the perfused synaptosomes were examined for their ability to interact with these two ligand binding sites in brain membranes. The differential sensitivity to the neurotoxins alpha-bungarotoxin and neosurugatoxin of the 125I-alpha-bungarotoxin and (-)-[3H]nicotine binding sites, respectively, leads to a tentative correlation of the (-)-[3H]nicotine site with the presynaptic nicotinic receptor on striatal nerve terminals.     

Characterization of Muscarinic Cholinergic Receptors in the Crab Nervous System

The selective muscarinic antagonist L-[3H]-quinuclidinyl benzilate (L-[3H]QNB) binds reversibly and with high affinity (KD = 0.3 nM) to a single population (Bmax = 105 fmol/mg protein) of specific sites in nervous tissue of the crab Cancer magister. The binding site is stereoselective; (-)QNB is over 200 times more potent than (+)QNB as an inhibitor of specific L-[3H]QNB binding. The muscarinic antagonists scopolamine and atropine are over 10,000 times more potent inhibitors of L-[3H]QNB binding than the nicotinic antagonists decamethonium and d-tubocurarine. The muscarinic agonists oxotremorine, pilocarpine, arecoline, and carbachol also compete effectively for the L-[3H]QNB binding site. This pharmacological profile strongly suggests the presence of classical muscarinic receptors in the crab nervous system. These receptors are localized to nervous tissue containing cell bodies and neuropil, whereas specific L-[3H]QNB binding is low or absent in peripheral nerve, skeletal muscle, and artery.     

Thymopoietin, a Thymic Polypeptide, Specifically Interacts at Neuronal Nicotinic α-Bungarotoxin Receptors

alpha-Bungarotoxin (alpha-BGT), a snake venom polypeptide, interacts potently and specifically with a nicotinic receptor population in neuronal tissue. However, the identity of this site is unclear, because, unlike at the neuromuscular junction and in electroplax, in nervous tissue the toxin does not block nicotinic cholinergic responses. Therefore, we sought endogenous compounds other than acetylcholine that could interact with the neuronal alpha-BGT site. In the present experiments, thymopoietin, a polypeptide isolated from the thymus, is shown to inhibit potently alpha-BGT binding to brain membranes in a dose-dependent manner (IC50 = 3.1 nM). This effect was not shared by a wide variety of other peptides, including thysplenin, a closely related polypeptide. Thymopoietin did not inhibit the binding of other radioligands known to interact with different populations of cholinergic receptors, such as [3H]nicotine and [3H]methylcarbachol, which bind to nicotinic receptors, or [3H]quinuclidinylbenzilate, which binds to muscarinic receptors. These results show that thymopoietin potently and specifically affects 125I-alpha-BGT binding to brain membranes and suggest that thymopoietin might be an endogenous ligand for alpha-BGT receptors in neuronal tissue.     

Dual effects of nicotine on oxidative stress and neuroprotection in PC12 cells

In order to identify the properties of nicotine in relation to oxidative stress or neuroprotection, differentiated PC12 cells were treated with nicotine, beta-amyloid peptide (Abeta(25-35)), free radical inducer and antioxidant by a separate addition or a combination way. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, lipid peroxidation, [3H]epibatidine binding sites for nicotinic receptor and [3H]quinuclidinyl benzilate (QNB) for muscarinic receptor have been detected. The significant decrease of MTT reduction and increase of lipid peroxidation in PC12 cells were only observed at treatments with high concentrations of nicotine (1 and 10 mM), while Vitamin E (VitE), an antioxidant, can prevent the neurotoxic effects. In addition, nicotine in low dosage (10 microM) rescued the decreased rates of cell viability and inhibited the production of lipid peroxidation resulted from H(2)O(2) and Abeta in the cultured cells. Significant increases in [3H]epibatidine binding sites were observed in PC12 cells exposed to nicotine, while no change was detected in [3H]QNB. The decreased number of nicotinic receptor binding sites due to the toxicity of Abeta was prevented by the addition of nicotine with low concentration. It is plausible that nicotine treatment may play dual effects on oxidative stress and neuroprotection, in which the effects are dependent on the differences in dosage of the drug used and their mechanisms of action. Generally, high dose of nicotine may induce neurotoxicity and stimulate oxidative stress, while reasonably low concentration may act as an antioxidant and play an important role for neuroprotective effect.     

Effects of chronic nicotine on heteromeric neuronal nicotinic receptors in rat primary cultured neurons

Nicotine increases the number of neuronal nicotinic acetylcholine receptors (nAChRs) in brain. This study investigated the effects of chronic nicotine treatment on nAChRs expressed in primary cultured neurons. In particular, we studied the chronic effects of nicotine exposure on the total density, surface expression and turnover rate of heteromeric nAChRs. The receptor density was measured by [12?I]epibatidine ([12?I]EB) binding. Untreated and nicotine-treated neurons were compared from several regions of embryonic (E19) rat brain. Twelve days of treatment with 10 μM nicotine produced a twofold up-regulation of nAChRs. Biotinylation and whole-cell binding studies indicated that up-regulation resulted from an increase in the number of cell surface receptors as well as intracellular receptors. nAChR subunit composition in cortical and hippocampal neurons was assessed by immunoprecipitation with subunit-selective antibodies. These neurons contain predominantly α4, β2 and α5 subunits, but α2, α3, α6 and β4 subunits were also detected. Chronic nicotine exposure yielded a twofold increase in the β2-containing receptors and a smaller up-regulation in the α4-containing nAChRs. To explore the mechanisms of up-regulation we investigated the effects of nicotine on the receptor turnover rate. We found that the turnover rate of surface receptors was > 2 weeks and chronic nicotine exposure had no effect on this rate.     

[1251]2α-BUNGAROTOXIN AND [3H]QUINUCLIDINYLBENZILATE BINDING IN CENTRAL NERVOUS SYSTEMS OF DIFFERENT SPECIES

Abstract— [¹²⁵I]Diiodo α-bungarotoxin ([¹²⁵I]₂BuTx) and [³H]quinuclidinylbenzilate ([³H]QNB) binding sites were measured in post-nuclear membrane fractions prepared from whole brains or brain regions of several species. Species studied included Drosophila melanogaster (fruit fly), Torpedo californiea (electric ray), Carassius auratus (goldfish), Ram pipiens (grass frog), Kana cutesheiana (bullfrog), Rattus norvegicus (rat, Sprague-Dawley), Mus muscalus (mouse, Swiss random, C58/J, LG/J), Oryctolagus cuniculus (rabbit, New Zealand Whitc), and Bos (cow). Acetyl-CoA: choline O -acetyltransferase (EC 2.3.1.6) levels were also determined in the post nuclear supernatants and correlated with the number of binding sites.
All species and regions except Drosophila had 16–150 fold more [³H]QNB binding sites than [¹²⁵I]₂BuTx binding sites. Brain regions with the highest levels of [¹²⁵I]₂BuTx binding were Drosophila heads (300 fmol/mg), goldfish optic tectum (80fmol/mg), and rat and mouse hippocampus (3040 fmol/mg). The highest levels of [³H]QNB binding were seen in rat and mouse caudate (1.3–1.6 pmol/mg). Lowest levels of [³H]QNB and [¹²⁵I]₂BuTx binding were seen in cerebellum. The utility of [¹²⁵I]₂BuTx and [³H]QNB binding as quantitative measures of nicotinic and muscarinic acetylcholine receptors in CNS is discussed.