Nicotinic Binding Research Articles

Nicotine binding in human striatum: elevation in schizophrenia and reductions in dementia with Lewy bodies, Parkinson’s disease and Alzheimer’s disease and in relation to neuroleptic medication

Striatal nicotinic acetylcholine receptors with high affinity for nicotinic agonists are involved with the release of a number of neurotransmitters, including dopamine. Previous findings as to whether these receptors are changed in Parkinson’s disease and Alzheimer’s disease are inconsistent and no previous investigations have focused on these receptors in dementia with Lewy bodies and schizophrenia, which are also associated with disorders of movement. The present autoradiographic study of striatal [ 3H]nicotine binding in Alzheimer’s and Parkinson’s diseases, dementia with Lewy bodies and schizophrenia was conducted with particular reference to the potentially confounding variables of tobacco use and neuroleptic medication. [ 3H]Nicotine binding in both dorsal and ventral caudate and putamen was significantly reduced in Parkinson’s disease (43–67%, n=13), Alzheimer’s disease (29–37%, n=13) and dementia with Lewy bodies (50–61%, n=20) compared to age-matched controls ( n=42). Although tobacco use in the control group was associated with increased [ 3H]nicotine binding (21–38%), and neuroleptic treatment in dementia with Lewy bodies and Alzheimer’s disease was associated with reduced [ 3H]nicotine binding (up to 29%), differences between neurodegenerative disease groups and controls persisted in subgroups of Alzheimer’s disease cases (26–33%, n=6, in the ventral striatum) and dementia with Lewy body cases (30–49%, n=7, in both dorsal and ventral striatum) who had received no neuroleptic medication compared to controls who had not smoked ( n=10). In contrast, striatal [ 3H]nicotine binding in a group of elderly (56–85 years) chronically medicated individuals with schizophrenia ( n=6) was elevated compared with the entire control group (48–78%, n=42) and with a subgroup that had smoked (24–49%, n=8). The changes observed in [ 3H]nicotine binding are likely to reflect the presence of these receptors on multiple sites within the striatum, which may be differentially modulated in the different diseases. Further study is warranted to explore which nicotinic receptor subunits and which neuronal compartments are involved in the changes in [ 3H]nicotine binding reported, to aid development of potential nicotinic receptor therapy.

Neuroprotection in Alzheimer's disease - new strategies for treatment.

Alzheimer's disease is the most common dementia disorder characterized by multiple pathological changes in the brain leading to a progressive memory loss and other cognitive symptoms producing occupational and social disabilities. Although a great deal of progress has been made in recent years in further understanding the genetic aberrations and patho-physiological processes of Alzheimer's disease there is still no cure of the disease. The transmitter replacement therapy is so far the most explored therapy. Three cholinesterase inhibitors have so far been approved and presently in clinical use in many countries. Although the cholinesterase inhibitors generally appear to produce symptomatic effects with palliative effect on existing cognitive disturbances recent data suggest that they also may have effect on progression of the disease including possible neuroprotective effects. Possible interactions between Abeta and cholinergic neurotransmission may exist. Treatment of cells with Abeta causes decreased cholinergic activity. Pretreatment of PC12 cells with cholinesterase inhibitors such as tacrine and donepezil in clinical relevant concentrations can attenuate Abeta (25-35) toxicity through mechanisms which may be mediated via nicotinic receptors. Estrogen has been shown to protect against Abeta toxicity in different cell lines and also to reduce the formation of Abeta. Its mechanism for the neuroprotective effect is however not fully clarified. A potentiation of the clinical effect of cholinesterase inhibitors in Alzheimer patients has been given together with estrogen. Experimental data suggest that the neuroprotective effect of estrogen as studied in PC12 cells was mediated at least partly via the alpha(7) nicotinic receptor. Treatment with Abeta in nanomolar concentrations for 7 days in PC12 cells significantly decreased the number of nicotinic receptor binding sites and mRNA levels. The effects by Abeta on nicotinic receptors are prevented by nicotine pretreatment. The finding suggests a possible link between Abeta and nicotinic receptor deficits in Alzheimer patients in the early course of the disease.

Depressive Characteristics of FSL Rats: Involvement of Central Nicotinic Receptors

Antidepressant effects of acute or chronic nicotine treatments in swim test immobility of Flinders sensitive line (FSL) rats, an animal model of depression, were recently demonstrated (Tizabi et al. Psychopharmacology 142:193, 1999). In the present study we sought to determine whether the antidepressant effects of nicotine could be blocked by the nicotinic antagonist, mecamylamine (MEC). Moreover, the effects of chronic nicotine treatment on [ 3H]cytisine binding in discrete brain regions of FSL and their control Flinders resistant line (FRL) rats were also evaluated. Adult male FSL rats were treated with MEC (0.5 mg/kg) 20 min prior to an acute or chronic nicotine administration. MEC by itself did not affect the immobility in swim test. However, it completely blocked the acute or chronic nicotine effects. Daily nicotine injection (0.4 mg/kg/day for 14 days) resulted in an increase in [ 3H]cytisine binding primarily in the FRL rats. An increase in nicotinic receptor binding following chronic nicotine administration is believed to reflect desensitization of these receptors. These findings, coupled with previous observation of higher basal nicotinic receptors in FSL rats, further support the involvement of central nicotinic receptors in depressive characteristics of these rats. Moreover, the data suggest therapeutic potential for selective nicotinic receptor agonists in depressive disorders.

Does apolipoprotein E (APO E) genotype influence nicotinic receptor binding in Alzheimer's disease?

Does apolipoprotein E (APO E) genotype influence nicotinic receptor binding in Alzheimer's disease?

Investigation of the haem-nicotinate interaction in leghaemoglobin. Role of hydrogen bonding.

A strategic assessment of the contributions of two active-site hydrogen bonds in the binding of nicotinate to recombinant ferric soybean leghaemoglobin a (rLb) was carried out by mutagenic replacement of the hydrogen-bonding residues (H61A and Y30A variants) and by complementary chemical substitution of the carboxylate functionality on the nicotinate ligand. Dissociation constants, Kd (pH 5.5, mu = 0.10 M, 25.0 +/- 0.1 degrees C), for binding of nicotinate to ferric rLb, H61A and Y30A were 1.4 +/- 0.3 microM, 19 +/- 1 microM and 11 +/- 1 microM, respectively; dissociation constants for binding of nicotinamide were, respectively, 38 +/- 1 mM, 50 +/- 2 mM and 12 +/- 1 mM, and for binding of pyridine were 260 +/- 50 microM, 4.5 +/- 0.5 microM and 66 +/- 8 microM, respectively. Binding of cyanide and azide to the H61A and Y30A variants was unaffected by the mutations. The pH-dependence of nicotinate binding for rLb and Y30A was consistent with a single titration process (pKa values 6.9 +/- 0.1 and 6.7 +/- 0.2, respectively); binding of nicotinate to H61A was independent of pH. Reduction potentials for the rLb and rLb-nicotinate derivatives were 29 +/- 2 mV (pH 5.40, 25.0 degrees C, mu = 0.10 M) and - 65 +/- 2 mV (pH 5.42, 25.0 degrees C, mu = 0.10 M), respectively. The experiments provide a quantitative assessment of the role of individual hydrogen bonds in the binding process, together with a definitive determination of the pKa of His61 and unambiguous evidence that titration of His61 controls binding in the neutral to alkaline region.

Interaction of tobacco-specific toxicants with the neuronal α 7 nicotinic acetylcholine receptor and its associated mitogenic signal transduction pathway: potential role in lung carcinogenesis and pediatric lung disorders

Pulmonary neuroendocrine cells function as hypoxia-sensitive chemoreceptors, and they release peptides and biogenic amines that are important mediators of pulmonary neonatal adaptation. Some of these products additionally act as autocrine growth factors. Increased numbers of pulmonary neuroendocrine cells have been observed in several smoking-associated pediatric lung disorders such as bronchopulmonary dysplasia, cystic fibrosis, sudden infant death syndrome, and asthma. Disturbed pulmonary neuroendocrine function has been implicated in the etiology of this disease complex. One of the most common smoking-associated lung cancer types, small cell lung carcinoma, expresses phenotypic and functional features of pulmonary neuroendocrine cells. We, as well as others, have shown that the release of the autocrine growth factors 5-hydroxytryptamine (5-HT, serotonin) and mammalian bombesisn/gastrin releasing peptide (MB/GRP) by cell lines derived from human small cell lung carcinoma or fetal hamster pulmonary neuroendocrine cells are regulated by a neuronal nicotinic acetylcholine receptor comprised of α 7 subunits. In radio-receptor assays, nicotine and the nicotine-derived carcinogenic nitrosamines N′-nictrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) bound with high affinity to this receptor with affinitites NNK>nicotine>NNN. Binding of nicotine or NNK to the α 7 receptor resulted in calcium influx and overexpression and activation of the serine–threonine protein kinase Raf-1. In turn, this event lead to overexpression and activation of the mitogen activated (MAP) kinases extracellular signal regulated kinase 1 (ERK1) and extracellular signal regulated kinase 2 (ERK2) and stimulation of DNA synthesis accompanied by an increase in cell numbers in fetal pulmonary neuroendocrine cells and small cell carcinoma cells. Exposure of fetal pulmonary neuroendocrine cells for 6 days to NNK caused a prominant up-regulation of Raf-1. Our findings suggest that chronic exposure to nicotine and NNK in pregnant women who smoke may up-regulate the α 7 nicotinic receptor as well as components of its associated mitogenic signal transduction pathway, thus increasing the susceptibilities of the infants for the development of pediatric lung disorders. Similarly, up-regulation of one or several components of this nicotinic receptor pathway in smokers may be an important factor for the development of small cell lung carcinoma.

Pharmacological and null mutation approaches reveal nicotinic receptor diversity

We have developed an array of assays for nicotinic acetylcholine receptor binding and function. [ 125I]α-Bungarotoxin-, (−)-[ 3H]nicotine-, and [ 3H]epibatidine-binding nicotinic acetylcholine receptors were assayed in mouse brain membranes and sections. Nicotinic acetylcholine receptor function was quantified using synaptosomal [ 3H]dopamine, [ 3H]γ-aminobutyric acid ([ 3H]GABA), and 86Rb + efflux techniques. Additionally, the effects of β2 subunit deletion on each of the measures were assessed. Detailed pharmacological comparison revealed minimally six nicotinic binding subtypes: [ 125I]α-bungarotoxin-binding nicotinic acetylcholine receptors; β2-subunit-dependent and -independent high-affinity (−)-[ 3H]nicotine-binding sites; β2-dependent and -independent cytisine-resistant [ 3H]epibatidine-binding sites; and a β2-dependent low-affinity [ 3H]epibatidine binding site. Comparative pharmacology suggested that [ 3H]GABA and dihydro-β-erythroidine (DHβE)-sensitive 86Rb + efflux are mediated by the same (probably α4β2) nicotinic acetylcholine receptor subtype, while other nicotinic acetylcholine receptor subtypes evoke [ 3H]dopamine and DHβE-resistant 86Rb + efflux. In whole-brain preparations, each measure of nicotinic acetylcholine receptor function was β2 dependent. The majority of β2-independent [ 3H]epibatidine binding was located in small, scattered brain nuclei, suggesting that individual nuclei may prove suitable for identification of novel, native nicotinic acetylcholine receptors.

Nicotinic receptor subtypes in human brain ageing, Alzheimer and Lewy body diseases

Human brain ageing is associated with reductions in a variety of nicotinic receptors subtypes, whereas changes in age-related disorders including Alzheimer's disease or Parkinson's disease are more selective. In Alzheimer's disease, in the cortex there is a selective loss of the α4 (but not α3 or 7) subunit immunoreactivity and of nicotine or epibatidine binding but not α-bungarotoxin binding. Epibatidine binding is inversely correlated with clinical dementia ratings and with the level of Aβ1–42, but not related to plaque or tangle densities. In contrast, α-bungarotoxin binding is positively correlated with plaque densities in the entorhinal cortex. In human temporal cortex loss of acetylcholinesterase catalytic activity is positively correlated with decreased epibatidine binding and in a transgenic mouse model over expressing acetylcholinesterase, epibatidine binding is elevated. In Parkinson's disease, loss of striatal nicotine binding appears to occur early but is not associated with a loss of α4 subunit immunoreactivity. Tobacco use in normal elderly individuals is associated with increased α4 immunoreactivity in the cortex and lower densities of amyloid-β plaques, and with greater numbers of dopaminergic neurons in the substantia nigra pars compacta. These findings indicate an early involvement of the α4 subunit in β-amyloidosis but not in nigro-striatal dopaminergic degeneration.

Neuronal nicotinic receptor deficits in Alzheimer patients with the Swedish amyloid precursor protein 670/671 mutation.

The influence of beta-amyloid on cholinergic neurotransmission was studied by measuring alterations in nicotinic acetylcholine receptors (nAChRs) in autopsy brain tissue from subjects carrying the Swedish amyloid precursor protein (APP) 670/671 mutation. Significant reductions in numbers of nAChRs were observed in various cortical regions of the Swedish 670/671 APP mutation family subjects (-73 to -87%) as well as in sporadic Alzheimer's disease (AD) cases (-37 to -57%) using the nicotinic agonists [3H]epibatidine and [3H]nicotine, which bind with high affinity to both alpha3 and alpha4 and to alpha4 nAChR subtypes, respectively. Saturation binding studies with [3H]epibatidine revealed two binding sites in the parietal cortex of AD subjects and controls. A significant decrease in Bmax (-82%) for the high-affinity site was observed in APP 670/671 subjects with no change in K(D) compared with controls (0.018 nM APP 670/671; 0.036 nM control). The highest load of neuronal plaques (NPs) was observed in the parietal cortex of APP 670/671 brains, whereas the number of [3H]nicotine binding sites was less impaired compared with other cortical brain regions. Except for a positive significant correlation between the number of [3H]nicotine binding sites and number of NPs in the parietal cortex, no strict correlation was observed between nAChR deficits and the presence of NPs and neurofibrillary tangles, suggesting that these different processes may be closely related but not strictly dependent on each other.

Quantitative assessment of nicotinic acetylcholine receptor proteins in the cerebral cortex of Alzheimer patients

Cholinergic transmission has for long been known to be one of the most severely affected systems in Alzheimer's disease (AD), resulting clinically in massive cognitive deficits. The molecular basis of this dysfunction — on both the pre- and the postsynaptic sites — is still a matter of ongoing investigations. Here, we report on the quantitative assessment of nicotinic acetylcholine receptor isoform expression in AD vs. control cortices. For both subunit proteins assessed, the α4 and the α7 isoform, highly significant decreases in diseased vs. normal cortices were observed. Both α4 and α7 subunits are known to be important constituents in hetero- (α4β2) and homooligomeric (α7) receptor subtypes. Their decreased expression may contribute to the decreased nicotinic binding known to be accompanied by AD and severe cognitive deficits. The quantitative assessment of nicotinic acetylcholine receptor expression will help to determine those subunits suited as targets for pharmacological stimulation.

Nicotine Binding to Native and Substituted Peptides Comprising Residues 188–207 of Nicotinic Acetylcholine Receptor α1, α2, α3, α4, α5, and α7 Subunits

Structural determinants of L-[3H]nicotine binding to synthetic peptides comprising residues 188–207 of nicotinic acetylcholine receptor α subunits were invesitigated by equilibrium binding analysis. Two binding components were detected, one of low affinity (Kd ∼ 1.5 μM) that did not differ significantly among peptides and another of high affinity. The high affinity binding component was higher for the neuronal peptides (Kd = 14–23 nM) than the muscle α1 peptides (Kd = 52 nM). The following nonconservative substitutions in the α4 peptide resulted in a significant decrease in nicotine affinity for the peptide: Y190A, Y190D, C192G, E195A, E195–, P199A, P199–, and Y203A. Substitution of α4P199 with a leucine which is present in the α1 sequence decreased the affinity of the α4 peptide for nicotine and substitution of α1L199 with a proline (α4) or a glutamine (α3) increased the affinity of the α1 peptide. It is concluded that aromatic residues contribute to the binding site for nicotine on the α4 subunit and that the residue present at position 199 partly determines differences in nicotine affinity for different α subunits.

Heme symmetry, vibronic structure, and dynamics in heme proteins: ferrous nicotinate horse myoglobin and soybean leghemoglobin.

We report the visible and Soret absorption bands, down to cryogenic temperatures, of the ferrous nicotinate adducts of native and deuteroheme reconstituted horse heart myoglobin in comparison with soybean leghemoglobin-a. The band profile in the visible region is analyzed in terms of vibronic coupling of the heme normal modes to the electronic transition in the framework of the Herzberg-Teller approximation. This theoretical approach makes use of the crude Born-Oppenheimer states and therefore neglects the mixing between electronic and vibrational coordinates; however, it takes into account the vibronic nature of the visible absorption bands and allows an estimate of the vibronic side bands for both Condon and non-Condon vibrational modes. In this framework, an x-y splitting of the Q transition for native and deuteroheme reconstituted horse myoglobin is clearly assessed and attributed to electronic perturbations that, in turn, are caused by a reduction of the typical D(4h) symmetry of the system due to heme distortions of B(1g)-type symmetry and/or to an x-y asymmetric position of the nicotinate ring; in deuteroheme reconstituted horse myoglobin the asymmetric heme peripheral substituents add to the above effect(s). On the contrary, in leghemoglobin-a no spectral splitting upon nicotinate binding is observed, pointing to a planar heme configuration in which only distortions of A(1g)-type symmetry are effective and to which the nicotinate ring is bound in an x - y symmetric position. The local dynamic properties of the heme pocket of the three proteins are investigated through the temperature dependence of spectral line broadening. Leghemoglobin-a behaves as a softer matrix with respect to horse myoglobin, thus validating the hypothesis of a looser heme pocket conformation in the former protein, which allows a nondistorted heme configuration and a symmetric binding of the bulky nicotinate ligand.

Changes in nicotinic acetylcholine receptor subunit mRNAs and nicotinic binding in spontaneously hypertensive stroke prone rats

We have studied nicotinic acetylcholine receptors in spontaneously hypertensive ‘stroke prone’ (SHsp) rats. We found a significant decrease in 125I- α-bungarotoxin binding and α7 subunit mRNA levels in cortical areas of the SHsp rats with respect to Wistar Kyoto (WKy) normotensive rats. Antihypertensive drug treatment counteracted these changes in cerebral cortex but not in hippocampus. No significant change was instead found in [ 3H]-epibatidine binding and α4 and β2 subunit mRNA levels. SHsp rats showed decreased latency at the active avoidance test and transiently increased threshold at both hot-plate and tail-flick tests in comparison with WKy rats. None of these behavioral parameters was correlated with 125I-α-bungarotoxin binding in cortical areas. In conclusion, present data show a preferential impairment of α-bungarotoxin sensitive nAChRs in SHsp rats.

Adolescent nicotine exposure causes persistent upregulation of nicotinic cholinergic receptors in rat brain regions

Whereas numerous studies have explored the consequences of fetal or adult nicotine exposure, little or no basic research has been conducted for nicotine exposure during adolescence, the developmental period in which regular cigarette use typically begins. We administered nicotine to adolescent rats on postnatal days 30–47 via continuous infusion with implanted osmotic minipumps, using a dose rate (3–6 mg kg −1 day −1) set to achieve plasma nicotine levels found in smokers; results were compared to exposure of adult rats. During and after exposure, we assessed nicotinic cholinergic receptor binding in the midbrain, cerebral cortex, and hippocampus, using [ 3 H ]cytisine. Robust receptor upregulation was observed with both adolescent and adult nicotine exposure but there were major differences in the regional specificity and persistence of effect. In adolescents, upregulation was uniform across all regions during the infusion period, whereas in adults, there was a distinct regional hierarchy: midbrain<cerebral cortex<hippocampus; accordingly, receptors in the adolescent midbrain were upregulated far more than with adult exposure. In addition, adolescent nicotine treatment produced long-lasting effects on the receptors, with significant increases still apparent in male rats 1 month after the termination of drug exposure. We also obtained evidence for hippocampal cell damage in adolescent female rats exposed to nicotine, characterized by increases in total membrane protein concentration indicative of a decrease in overall cell size. Adolescent nicotine exposure thus elicits region- and gender-selective effects that differ substantially from those in adults, effects that may contribute to increased addictive properties and lasting deficits in behavioral performance.

Magnocellular neurons of the rat supraoptic nucleus are endowed with functional nicotinic acetylcholine receptors

Acetylcholine can stimulate the release of vasopressin. 25 In organ-cultured hypothalamo-neurohypophyseal systems, acetylcholine enhanced vasopressin release by acting in or near the supraoptic nucleus. 28 Extracellular recordings 3,9,13 suggested that acetylcholine can increase supraoptic neuron excitability. These effects could be mimicked, in part, by nicotine or blocked by nicotinic antagonists, suggesting that they might be mediated by nicotinic acetylcholine receptors. Autoradiography indicated that α-bungarotoxin binding sites are present in the supraoptic nucleus; 8,14,19,27 however, neither acetylcholine nor nicotine binding sites could be detected. 8,14,27 Thus, the existence, let alone the nature, of nicotinic receptors in the supraoptic nucleus has so far remained elusive. The present work attempts to determine: (i) whether functional nicotinic receptors are present in this nucleus; (ii) whether they are located on neurosecretory magnocellular cells or at presynaptic sites; (iii) what their pharmacological and biophysical properties are; (iv) whether they influence the activity of all or only part of supraoptic neurons. Whole-cell recordings were performed in hypothalamic slices or in acutely dissociated supraoptic neurons and the effect of nicotinic agonists was tested under voltage-clamp conditions. Autoradiography was done in coronal hypothalamic sections, using [ 3H]epibatidine and [ 125I]α-bungarotoxin as ligands. Our results indicate that supraoptic neurons possess functional nicotinic receptors containing the α7 subunit.

Characterisation of multiple α-bungarotoxin binding sites in the aphid Myzus persicae (Hemiptera: Aphididae)

The nicotinic antagonist α-bungarotoxin (α-BgTx) has been extensively used in studies of the vertebrate nicotinic acetylcholine receptor (nAChR) and is used here to investigate putative nicotinic acetylcholine receptors of the peach potato aphid Myzus persicae. Saturable binding is consistent with the presence of both high and low affinity binding sites for [ 125I]-α-BgTx, with dissociation constants of 1.2 and 34 nM, and maximal binding capacities of 167 and 640 fmol mg −1, respectively, with a Hill value of 0.71. Displacement studies with various nicotinic ligands, including neonicotinoid insecticides, indicate that the pharmacology of the high affinity [ 125I]-α-BgTx binding site of M. persicae is similar to that of α-BgTx sites in Apis mellifera and Manduca sexta. However, low Hill values in the displacement studies suggest the presence of either multiple receptor sub-types or cooperativity between nicotinic binding sites on the same nAChR. Isotopic dissociation of [ 3H]-α-BgTx initiated by 1μM methyllycaconitine was accelerated and biphasic in character in contrast to monophasic dissociation initiated by imidacloprid, epibatidine or α-BgTx itself. This is consistent with a model in which there is allosteric interaction between at least two nicotinic binding sites per aphid nAChR.

Neuronal nicotinic receptors in dementia with Lewy bodies and schizophrenia: alpha-bungarotoxin and nicotine binding in the thalamus.

Neuronal nicotinic receptors have been implicated in schizophrenia on the basis of the high incidence of tobacco smoking in patients, abnormalities in cytisine and alpha-bungarotoxin (alphaBGT) binding in the hippocampus, and linkage between auditory P50 deficits and the region of chromosome 15 coding the alpha7 subunit. In another disease associated with psychosis, dementia with Lewy bodies (DLB), in which visual hallucinations predominate, reductions in nicotine binding have been identified in various cortical and subcortical regions. We investigated both alphaBGT and nicotine binding autoradiographically in different thalamic nuclei in autopsy brain tissue from patients with schizophrenia and DLB. AlphaBGT binding in the reticular nucleus was moderately reduced (25%) in schizophrenia and more extensively reduced (50%) in DLB. There were no significant alterations in nicotine binding in schizophrenia, and in DLB, a trend towards moderate reductions in most nuclei reached significance in the lateral dorsal nucleus. It is concluded that widespread abnormalities of thalamic nicotine are not implicated in schizophrenia or DLB, but that reticular alphaBGT binding may be involved to a lesser and greater extent in the pathophysiology or psychopathology of both disorders.

Mutation screening of the CHRNA4 and CHRNB2 nicotinic cholinergic receptor genes in Alzheimer's disease.

Potential genomic changes leading to decreased nicotine binding, crucial for cognitive dysfunction in Alzheimer's disease (AD), have not yet been studied. A search for mutations of the genes coding for the most widely distributed nicotinic receptor subtype alpha4beta2 (CHRNA4/CHRNB2) has been performed in AD patients by screening the coding regions of both genes by single strand conformation analysis and heteroduplex analysis of fibroblast-derived genomic DNA. Polymorphisms in CHRNA4, none of which led to amino acid changes in the predicted sequence, were found in three patients. Although the other receptor subunits have yet to be screened, it appears likely that the reduction of nicotine binding sites in AD is not due to genomic changes.

Up-regulation of Cell-surface α4β2 Neuronal Nicotinic Receptors by Lower Temperature and Expression of Chimeric Subunits

The predominant nicotinic acetylcholine receptor (nAChR) expressed in vertebrate brain is a pentamer containing alpha4 and beta2 subunits. In this study we have examined how temperature and the expression of subunit chimeras can influence the efficiency of cell-surface expression of the rat alpha4beta2 nAChR. Functional recombinant alpha4beta2 nAChRs, showing high affinity binding of nicotinic radioligands (K(d) = 41 +/- 22 pM for [(3)H]epibatidine), are expressed in both stably and transiently transfected mammalian cell lines. Despite this, only very low levels of alpha4beta2 nAChRs can be detected on the cell surface of transfected mammalian cells maintained at 37 degrees C. At 30 degrees C, however, cells expressing alpha4beta2 nAChRs show a 12-fold increase in radioligand binding (with no change in affinity), and a 5-fold up-regulation in cell-surface receptors with no increase in total subunit protein. In contrast to "wild-type" alpha4 and beta2 subunits, chimeric nicotinic/serotonergic subunits ("alpha4chi" and "beta2chi") are expressed very efficiently on the cell surface (at 30 degrees C or 37 degrees C), either as hetero-oligomeric complexes (e.g. alpha4chi+beta2 or alpha4chi+beta2chi) or when expressed alone. Compared with alpha4beta2 nAChRs, expression of complexes containing chimeric subunits typically results in up to 20-fold increase in nicotinic radioligand binding sites (with no change in affinity) and a similar increase in cell-surface receptor, despite a similar level of total chimeric and wild-type protein.

Altered disposition of repeated nicotine doses in rats immunized against nicotine.

Active immunization of rats against nicotine reduces the distribution of a single intravenous dose of nicotine to brain. The current study examined the effects of immunization on the distribution of repeated doses of nicotine, and on estimates of nicotine pharmacokinetic parameters. Groups of rats received five repeated doses of nicotine over 80 min (total dose equivalent to the nicotine intake from one, three or 10 cigarettes in a human). The serum nicotine concentration in immunized rats was 160-430% higher than controls after the fifth dose, demonstrating binding of nicotine to antibody. Brain nicotine concentration in immunized rats was reduced by 30-46%. The reduction in distribution of nicotine to brain correlated with the serum hapten-specific antibody concentration, the percentage of nicotine bound in serum, and with the unbound nicotine concentration in serum. In immunized rats, nicotine had a smaller steady state volume of distribution, lower systemic clearance, and longer terminal half-life than in controls. These data demonstrate that immunization against nicotine reduces nicotine distribution to brain, even after multiple nicotine doses at rates approximating heavy cigarette smoking. Whether this reduction in nicotine distribution is large enough to alter nicotine's physiological or behavioral effects remains to be studied.