Cytisine Binding Sites Research Articles

Genetic correlation between the free-choice oral consumption of nicotine and alcohol in C57BL/6J × C3H/HeJ F2 intercross mice

Previous studies in humans have demonstrated a high co-morbidity between alcoholism and smoking. This co-morbidity between alcohol and nicotine dependence can be attributed, in part, to common genetic factors. In rodents, behavioral and physiological responses to alcohol and nicotine also appear to share common genetic influences. In this report, the genetic correlation between free-choice oral nicotine and oral alcohol consumption was evaluated using an ascending two-bottle choice paradigm in C57BL/6 × C3H/HeJ F2 intercross mice. For all concentrations of nicotine (25, 50, and 100 μg/ml) and alcohol (3, 6, and 10%) tested, nicotine consumption was significantly correlated with alcohol consumption. Nicotine consumption at the highest nicotine concentration tested (100 μg/ml) showed low, but significant, correlations with the number of [ 3H]-cytisine binding sites in the hippocampus ( r = 0.307) and the number of [ 125I]-α-bungarotoxin binding sites in the cortex ( r = −0.328). No significant correlations between alcohol consumption and the number of either [ 3H]-cytisine or [ 125I]-α-bungarotoxin binding sites was observed. A polymorphism in the nicotinic receptor α4 subunit gene, Chrna4, showed a trend with nicotine consumption and a significant association with alcohol consumption in female but not male mice. These results indicate that common genetic factors influence nicotine and alcohol consumption in mice. However, neither individual differences in the expression of [ 3H]-cytisine or [ 125I]-α-bungarotoxin binding nicotinic receptors nor the polymorphism in Chrna4 likely contribute to the genetic overlap that influences the consumption of both of these drugs of abuse in C57BL/6 × C3H/HeJ F2 mice.

A Polymorphism in the Mouse Neuronal α4 Nicotinic Receptor Subunit Results in An Alteration in Receptor Function

Nicotine-stimulated (86)Rb(+) efflux and [(3)H]cytisine binding, both of which seem to measure the nicotinic acetylcholine receptor, composed of alpha4 and beta2 subunits, were assessed in eight brain regions obtained from 14 inbred mouse strains. The potential role of a single nucleotide polymorphism (SNP) in the nicotinic receptor alpha4 subunit gene (Chrna4) on nicotinic receptor binding and function in mice was also evaluated. This SNP leads to an alanine-to-threonine variation at amino acid position 529 of the nascent alpha4 subunit polypeptide. Both nicotine-stimulated (86)Rb(+) efflux and [(3)H]cytisine binding were found to vary across brain regions and among mouse strains. Variability in nicotine-stimulated (86)Rb(+) efflux was positively correlated (r > 0.9) within each strain with the number of [(3)H]cytisine binding sites. However, the number of [(3)H]cytisine binding sites was not correlated with nicotine-stimulated (86)Rb(+) efflux across mouse strains. In contrast, the Chrna4 polymorphism was associated with receptor function across mouse strains: (86)Rb(+) efflux was greater in seven of the eight brain regions studied in those mouse strains that carry the Ala-529 variant of Chrna4. The Chrna4 SNP did not seem to influence the number of [(3)H]cytisine binding sites across mouse strains. These data indicate that inbred mouse strains exhibit differences in receptor function that cannot be attributed to variation in receptor expression but may be explained, at least in part, by the missense polymorphism in the alpha4 subunit.

Effects of chronic treatment with (+)-nicotine on the stress-induced hypertension and downregulation of central nicotinic receptors in rats Comparative study with (−)-nicotine

The effects of chronic treatment with (+)-nicotine on the immobilization stress-induced changes in blood pressure, body weight, and [ 3H]cytisine binding to brain nicotinic receptors were examined in rats and were compared with those of (−)-nicotine. Immobilization stress (2 hr/day −1, for 2 weeks) induced a moderate elevation of blood pressure, loss of body weight gain, and downregulation of [ 3H]cytisine binding sites in cerebral cortex and midbrain. Chronic treatment with (+)- or (−)-nicotine inhibited the stress-induced hypertension but did not suppress the inhibition of body weight gain by stress. Body weight before stress load was also reduced by (−)-nicotine but not (+)-nicotine treatment. Treatment with each isomer increased the maximum number of [ 3H]cytisine binding sites (B max) of brain stem, cerebral cortex, and hippocampus. The B max in midbrain was also increased by (+)-nicotine but not by (−)-nicotine. Stress-induced downregulation in cerebral cortex was inhibited by both isomers. These results suggest that (+)- and (−)-nicotine cause various effects, including anti-stress effect, on the central nervous system in rats, which are slightly different between the isomers.

Nicotinic receptor binding sites in rat primary neuronal cells in culture: characterization and their regulation by chronic nicotine

We have characterized high affinity neuronal nicotinic acetylcholine receptors labeled by [ 3 H ]cytisine in primary neuronal cell cultures from fetal rat brains. After 15 days in culture, the highest density of [ 3 H ]cytisine binding sites ( B max≈57 fmol/mg protein) was found in cells from the brainstem, which includes the following subcortical brain areas: the septum, thalamus, hypothalamus, midbrain, pons and medulla. A lower density of sites was found in cells from the cerebral cortex, hippocampus, and caudate nucleus. [ 3 H ]Cytisine binds to receptors in primary cells from the brainstem and cerebral cortex with a K d of ≈0.5 nM, and the binding is inhibited by the agonists nicotine, acetylcholine, and epibatidine with IC 50 values of 1 to 20 nM, and by carbachol and the antagonist dihydro-β-erythroidine with IC 50 values of 0.5 to 1.5 μM. Chronic treatment of neuronal cultures with nicotine for 7 days differentially affected the number of nicotinic receptors in cells from different brain areas; it significantly increased the number of nicotinic binding sites in cells from the cerebral cortex, hippocampus, and caudate, but not in cells from the brainstem. The nicotine-induced increase of receptors in cerebral cortical cultures was not blocked by either mecamylamine or dihydro-β-erythroidine. These results indicate that primary cultures of rat neuronal cells provide a good model system in which to study and compare the properties and regulation of native neuronal nicotinic acetylcholine receptors.

Structure-activity studies related to ABT-594, a potent nonopioid analgesic agent: Effect of pyridine and azetidine ring substitutions on nicotinic acetylcholine receptor binding affinity and analgesic activity in mice

Analogs of A-98593 ( 1) and its enantiomer ABT-594 ( 2) with diverse substituents on the pyridine ring were prepared and tested for affinity to nicotinic acetylcholine receptor binding sites in rat brain and for analgesic activity in the mouse hot plate assay. Numerous types of modifications were consistent with high affinity for [ 3H]cytisine binding sites. By contrast, only selected modifications resulted in retention of analgesic potency in the same range as 1 and 2. Analogs of 2 with one or two methyl substituents at the 3-position of the azetidine ring also were prepared and found to be substantially less active in both assays.

Cognitive impairment in spontaneously hypertensive rats: role of central nicotinic receptors. I

Both human essential hypertension and genetically induced hypertension in rats have been associated with a range of impairments of cognitive ability. The spontaneous hypertensive rat (SHR) previously has been shown to exhibit a decrease in the expression of brain nicotinic acetylcholine receptors, a factor that could play a role in the impaired ability of this strain in the performance of learning and memory-related tasks. The purpose of this study was to help determine whether task impairment by SHR was related to the reduced expression of central nicotinic acetylcholine receptors. Twelve-week-old SHR were tested in two phases of a water maze (spatial memory) task, and their performance was compared with that of two age-matched normotensive strains, Wistar Kyoto (WKY) and Wistar rats. During Phase 1, SHR exhibited significantly increased latencies to locate a hidden platform as compared with either WKY or Wistar rats. During Phase 2 (subsequent series of trials after a 4-day inter-phase period), where rats were required to find a new platform location, SHR again exhibited significantly impaired performance compared to the normotensive strains. In a single trial passive avoidance paradigm, SHR again displayed significantly reduced avoidance behavior as compared with both WKY and Wistar rats. In consecutive coronal sections, the density of [ 3H]cytisine binding sites was decreased in SHR by up to 25% in about half of the brain regions examined, with the deficits particularly apparent in cephalic regions. The binding of [ 125I]α-bungarotoxin to brain sections also was decreased in SHR; however, only certain brain areas exhibited significant interstrain differences. These alterations in the expression of putative nicotinic receptor subtypes in SHR were not due to changes in the density of cholinergic neurons since there were no interstrain differences in the binding densities for [ 3H]vesamicol, which labels the vesicular acetylcholine transporter. Moreover, the magnitude of nicotine-stimulated rubidium efflux from cortical and striatal synaptosomes in vitro was significantly reduced in samples derived from SHR as compared with those from normotensive rats. These results are consistent with the possibility that a reduction in the expression of cortical nicotinic receptors in SHR plays a role in this strain's impaired performance of both spatial and non-spatial learning and memory-related tasks.

Cognitive impairment in spontaneously hypertensive rats: role of central nicotinic receptors. Part II

The adult spontaneously hypertensive rat (SHR) has been shown to exhibit a decrease in the expression and nicotine-stimulated function of brain nicotinic acetylcholine receptors, factors that could play a role in the impaired ability of this strain in the performance of learning and memory-related tasks. The purpose of this study was to determine whether either or both the impaired task performance and the loss of nicotinic receptors is directly related to the presence of the hypertensive state. To address this issue, two experimental approaches were taken. In the first series, 4-week-old pre-hypertensive SHR were tested in two phases of a water maze (spatial memory) task, and their performance was compared with that of two age-matched normotensive strains, Wistar Kyoto (WKY) and Wistar rats. During phase 1, SHR and WKY rats were not different in their ability to learn the task. In contrast, during phase 2 (subsequent series of trials after a 4 day inter-phase period), where rats were required to find a new platform location, SHR exhibited significantly impaired performance compared to both WKY and Wistar normotensive controls. In a single trial passive avoidance paradigm, SHR again displayed significantly reduced avoidance behavior as compared with both WKY and Wistar rats. In consecutive coronal sections the density of [ 3H]cytisine binding sites was decreased in pre-hypertensive SHR by up to 18% in about 40% of the brain regions examined, with the deficits particularly apparent in frontal cortex (layers 4–6), posterior subiculum, several thalamic regions, and the interpeduncular nucleus. In the second series, age-matched SHR and WKY were treated with the antihypertensive agent hydralazine administered in the drinking water beginning at 4 weeks of age. Hydralazine prevented the development of hypertension in adult SHR, but did not forestall the reduced expression of brain nicotinic receptors, nor the impairment in learning- and memory-related tasks normally observed in untreated adults with established hypertension. Moreover, the magnitude of nicotine-stimulated rubidium efflux from cortical and striatal synaptosomes in vitro was significantly reduced in samples derived from hydralazine-treated SHR as compared with those from hydralazine-treated, or untreated WKY. These results support the contention that the hypertensive state does not directly contribute to the reduced expression of nicotinic receptors in SHR. Therefore, the SHR may provide an important genetic model for the study of the role of central nicotinic receptors in cognitive and learning abnormalities.

Regulation of Human α4β2 Neuronal Nicotinic Acetylcholine Receptors by Cholinergic Channel Ligands and Second Messenger Pathways

The alpha4beta2 nicotinic acetylcholine receptors (nAChRs), a major subtype in the brain, have been shown to be modulated by chronic treatment with nicotine. In this study, the regulation of recombinant human alpha4beta2 nAChR subtype by (-)-nicotine and other cholinergic channel modulators was studied using human embryonic kidney 293 cells stably expressing this subunit combination. The treatment of transfected cells with (-)-nicotine and other activator ligands, including (-)-cytisine, 1,1-dimethyl-4-phenylpiperazinium, (S)-3-methyl-5-(1-methyl-2-pyrrolidinyl)isoxazole, and (+/-)-epibatidine, resulted in concentration-dependent increases in the levels of alpha4beta2 nAChRs. The increase in [3H]cytisine binding sites was initiated by low concentrations of (-)-nicotine (<100 nM); was maximal at 10 microM (15-fold), rapid (t0.5 = 4.0 +/- 0.5 hr), and totally reversible (t0.5 = 11.7 +/- 0.1 hr); and occurred with no change in ligand binding affinity. Antagonists, including dihydro-beta-erythroidine, d-tubocurarine, and methyllycaconitine, also elicited significant increases in receptor levels. A good correlation was observed between the Ki values for binding inhibition and the EC50 values for receptor up-regulation. Treatment of cells with mecamylamine, a noncompetitive antagonist, did not change receptor levels or alter (-)-nicotine-evoked up-regulation. (-)-Nicotine-evoked up-regulation was blocked by cycloheximide, suggesting a role for protein synthesis. Treatment of cells with (-)-nicotine or dihydro-beta-erythroidine differentially modulated the efficacy of acetylcholine to activate cation efflux. Both 6-beta-[beta'(piperidino)propionyl]forskolin and phorbol-12-myristate-13-acetate increased [3H]cytisine binding sites and nAChR function and enhanced the effects of chronic (-)-nicotine treatment in a synergistic manner. These results collectively demonstrate that human alpha4beta2 nAChRs can be differentially up-regulated by chronic treatment with nAChR ligands and activation of protein kinase A- and protein kinase C-dependent mechanisms.

Spinal nicotinic receptor expression in spontaneously hypertensive rats.

Intrathecal administration of nicotinic agonists previously has been shown to result in exaggerated pressor and heart rate responses as well as greater nociceptive behavior in adult (12-week-old) spontaneously hypertensive rats (SHR) than in age-matched normotensive Wistar-Kyoto rats (WKY). Paradoxical to these augmented responses to nicotinic agonists in SHR, nicotinic receptor number in the spinal cord as measured by cytisine binding sites is lower in adult SHR than normotensive WKY and Sprague-Dawley rats. Using the high-affinity agonist epibatidine, we found similar differences in receptor number between strains in both in vitro ligand binding experiments with spinal cord membranes and in situ autoradiographic analyses. Spinal nicotinic receptor number did not differ in 5-week-old prehypertensive SHR and age-matched WKY; however, receptor numbers were higher in young rats of both strains than in their adult counterparts. Antihypertensive treatment (25 mg/kg per day hydralazine PO) in 6-week-old SHR from 6 to 12 weeks of age markedly reduced the progressive rise in blood pressure yet did not alter nicotinic receptor number compared with untreated rats. Similar treatment of WKY with hydralazine produced a slight fall in blood pressure but no change in receptor number. Thus, normalization of blood pressure by hydralazine in SHR does not result in a return of receptor expression to levels seen in normotensive rats. Higher centrally mediated pressor activity or augmented postcoupling events after neuronal nicotinic receptor stimulation may slowly downregulate expression of spinal nicotinic receptors in this genetically hypertensive rat strain.

Chronic nicotine treatment delays the developmental increase in brain muscarinic receptors in rat neonate

Developmental increase in the muscarinic receptors ([ 3H]quinuclidinyl benzylate binding sites) of rat neonate brain (cerebral cortex and cerebellum) was significantly inhibited by chronic nicotine treatment of the dams during pregnancy and lactation. However, development of the nicotinic receptors ([ 3H]cytisine binding sites) was not inhibited and rather was up-regulated in the cerebral cortex and brainstem by the nicotine treatment. Such inhibition and up-regulation were not seen in nicotine-withdrawn rats after birth. These results suggest that nicotine treatment during lactation may cause a remarkable delay in development of muscarinic neurotransmission in rat neonates.

P-393 - Chronic nicotine treatment delays the developmental increase in brain muscarinic receptors in rat neonate

Developmental increase in the muscarinic receptors ([3H]quinuclidinyl benzylate binding sites) of rat neonate brain (cerebral cortex and cerebellum) was significantly inhibited by chronic nicotine treatment of the dams during pregnancy and lactation. However, development of the nicotinic receptors ([3H]cytisine binding sites) was not inhibited and rather was up-regulated in the cerebral cortex and brainstem by the nicotine treatment. Such inhibition and up-regulation were not seen in nicotine-withdrawn rats after birth. These results suggest that nicotine treatment during lactation may cause a remarkable delay in development of muscarinic neurotransmission in rat neonates.

Alteration of brain nicotinic receptors induced by immobilization stress and nicotine in rats

Immobilization stress (2 h· day −1, for 2 weeks) caused downregulation of the nicotinic receptors ([ 3H]cytisine binding sites) in the rat brain (cerebral cortex and midbrain). However, chronic nicotine treatment abrogated stress induced downregulation of the [ 3H]cytisine binding sites, suggesting that chronic nicotine treatment may block stress activated nicotinic receptor-mediated neurotransmission.

Interaction of p-aminophenyldichloroarsine, an arsenical with specificity for vicinal cysteines, with [ 3H]cytisine binding sites in rat brain membranes

The arsenical compound p-aminophenyldichloroarsine (APA) is selective for spatially close thiols with which it forms a stable complex. The α subunits of nicotinic acetylcholine receptors are defined by the presence of a pair of adjacent cysteines close to the agonist binding site. Here the interaction of APA with [ 3H]cytisine binding sites, which correspond to the major subtype of nicotinic receptors in rat brain has been examined. Incubation of brain membranes with 10 μM APA abolished [ 3H]cytisine binding. The action of APA was dependent on prior reduction of sulphydryls with dithiothreitol. APA effects could not be reversed by oxidizing agents but could be reversed by the antiarsenical reagent 2,3-dimercapto-1-propane sulphonic acid. Under the conditions used, the concentration of APA producing a half-maximal decrease in binding was 130 nM. The loss of [ 3H]cytisine binding was due to a decrease in the number of binding sites ( B max) with no effect on affinity for the radioligand ( K d). Nicotinic ligands failed to protect against the reduction and arsenylation of neuronal receptor sites. These observations are consistent with the potent interaction of APA with this neuronal nicotinic recept.

Characterization of [ 3H]cytisine binding to human brain membrane preparations

The binding characteristics of [ 3H]cytisine, a putative CNS nicotinic receptor ligand, were examined in 4 regions of the human brain. [ 3H]Cytisine was found to bind non-cooperatively with high affinity to a single site in tissue homogenates and to exhibit low non-specific association. The binding characteristics of this ligand were evaluated in thalamus at 4°C and 24°C. The association constants were found to be 0.234 and 0.308 min −1nM −1, while the dissociation constants were 0.007 and 0.098 min −1, respectively. Saturation analysis of thalamus revealed the equilibrium K d to be 147 pM (4°C) and 245 pM (24°C), values in good agreement with those determined kinetically. The Hill coefficient varied slightly between brain regions; however, the mean values in all regions were examined were close to 1.0 at 0.95 ± 0.03 (4°C) and 0.91 ± 0.04 (24°C). [ 3H]Cytisine binding could be displaced using both nicotinic agonists and antagonists. Cytisine was the most potent displacer of [ 3H]cytisine binding with an K i of 250 pM. Nicotine and acetylcholine were also potent displacers with K i values of 1.8 and 8.1 nM, respectively. The nicotinic antagonists α-bungarotoxin and mecamylamine were ineffective competitors for the [ 3H]cytisine binding site while dihydro-β-erythroidine had an K i value of 109 nM. Thalamus showed the highest density of cytisine binding sites of all the regions examined (48 fmol/mg protein) while the hippocampus, cingulate gyrus and the cortex showed B max values of 18.9, 19.3 and 8.8 fmol/mg protein, respectively. The results of this characterization of [ 3H]cytisine binding are in excellent agreement with previously published reports on the nicotinic cholinergic receptor population in the human brain. We conclude that [ 3H]cytisine exhibits both high affinity for the receptor and a very low non-specific binding component which makes this a useful ligand for the quantitation of nicotinic receptors in tissues with low receptor density.