Yingxian Xiao

Measuring nicotinic receptors with characteristics of α4β2, α3β2 and α3β4 subtypes in rat tissues by autoradiography

Comparison of [125I]epibatidine and 5‐[125I]iodo‐3‐(2‐azetidinylmethoxy)pyridine ([125I]A‐85380) autoradiography showed evidence for nicotinic receptor heterogeneity. To identify the receptor subtypes, we performed [125I]epibatidine autoradiography in the presence of cytisine or A‐85380. By comparing these results with binding data from human embryonic kidney (HEK) 293 cells stably transfected with different combinations of rat nicotinic receptor subunits, we were able to quantify three distinct populations of [125I]epibatidine binding sites with characteristics of α4β2, α3β2 and α3β4 receptors. Although the predominant subtype in rat brain was α4β2, non‐α4β2 binding sites were prominent in many regions. In the habenulo‐peduncular system, cerebellum, substantia gelatinosa, and many medullary nuclei, α3β4‐like binding accounted for more than 40% of [125I]epibatidine binding, and nearly all binding in superior cervical ganglion and pineal gland. Other regions enriched in α3β4‐like binding included locus ceruleus, dorsal tegmentum, subiculum and anteroventral thalamic nucleus. Regions enriched in α3β2‐like binding included the habenulo‐peduncular system, many visual system structures, certain geniculate nuclei, and dopaminergic regions. The combination of autoradiography using a broad spectrum radioligand in the presence of selectivecompetitors, and data from binding to defined receptor subtypes in expression systems, allowed us to quantify the relative populations of these three subtypes.

Sazetidine-A, A Novel Ligand That Desensitizes α4β2 Nicotinic Acetylcholine Receptors without Activating Them

Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels found throughout the central and peripheral nervous systems. They are crucial to normal physiology and have been clearly implicated in nicotine addiction. In addition, they are possible therapeutic targets in a wide range of pathological conditions, including cognitive disorders, Parkinsons disease, and neuropathic pain. Nicotinic ligands are usually classified as agonists (or partial agonists), competitive antagonists, or noncompetitive antagonists. Sazetidine-A is a new nicotinic ligand that shows a different pharmacological profile from any of these known classes of ligands. Sazetidine-A competes with very high binding affinity (Ki ≈ 0.5 nM) and selectivity for the α4β2 nAChR subtype (Ki ratio α3β4/α4β2 ∼ 24,000). Despite its high affinity, sazetidine-A neither activates nAChR channel function nor prevents channel activation when it is applied simultaneously with nicotine. However, when it is pre-incubated for 10 min with the receptors, it potently blocks nicotine-stimulated α4β2 nAChR function (IC50 ≈ 30 nM). The action of sazetidine-A may be explained by its very low affinity for the resting conformation of the α4β2 nAChRs, and its very high affinity for the desensitized state of the receptor. We propose that sazetidine-A is a “silent desensitizer” of nAChRs, meaning that it desensitizes the receptor without first activating it. Furthermore, comparison of the effects of sazetidine-A and nicotine at α4β2 nAChRs suggests that the predominant effects of nicotine and other nicotinic agonists are related to desensitization of the receptors and that sazetidine-A potently mimics these effects.

Membrane potential fluorescence: A rapid and highly sensitive assay for nicotinic receptor channel function

Seven cell lines expressing native and transfected nicotinic receptor subtypes were evaluated functionally by using fluorescent assays based on membrane potential and calcium dynamics with “no-wash” dye systems. Both assays provided the same rank orders of potency for (±)-epibatidine, 2S-(−)-nicotine, 7R,9S-(−)-cytisine, and 1,1-dimethyl-4-phenylpiperazinium in a cell line expressing rat α3β4 receptors. Nicotinic antagonists mecamylamine and dihydro-β-erythroidine inhibited responses in both assays. Both agonist and antagonist activity were assessed within the same experiment. Agonists seemed more potent in the membrane potential assay than in the calcium assay, whereas the converse was true for antagonists. The membrane potential assay afforded robust responses in K-177 cells expressing human α4β2 receptors, in IMR-32 and SH-SY5Y cells expressing human ganglionic receptors, and in TE-671 cells expressing human neuromuscular receptors. These lines gave weak to modest calcium responses. Moreover, membrane potential responses were obtained in cell lines expressing rat α4β2 and α4β4 receptors, which were devoid of calcium responses. Thus, membrane potential serves as a sensitive measure of nicotinic activity, and the resulting depolarization may be as important as calcium in cell signaling.

Sub-anesthetic concentrations of (R,S)-ketamine metabolites inhibit acetylcholine-evoked currents in α7 nicotinic acetylcholine receptors

The effect of the (R,S)-ketamine metabolites (R,S)-norketamine, (R,S)-dehydronorketamine, (2S,6S)-hydroxynorketamine and (2R,6R)-hydroxynorketamine on the activity of α7 and α3β4 neuronal nicotinic acetylcholine receptors was investigated using patch-clamp techniques. The data indicated that (R,S)-dehydronorketamine inhibited acetylcholine-evoked currents in α7-nicotinic acetylcholine receptor, IC(50) = 55 ± 6 nM, and that (2S,6S)-hydroxynorketamine, (2R,6R)-hydroxynorketamine and (R,S)-norketamine also inhibited α7-nicotinic acetylcholine receptor function at concentrations ≤ 1 μM, while (R,S)-ketamine was inactive at these concentrations. The inhibitory effect of (R,S)-dehydronorketamine was voltage-independent and the compound did not competitively displace selective α7-nicotinic acetylcholine receptor ligands [(125)I]-α-bungarotoxin and [(3)H]-epibatidine indicating that (R,S)-dehydronorketamine is a negative allosteric modulator of the α7-nicotinic acetylcholine receptor. (R,S)-Ketamine and (R,S)-norketamine inhibited (S)-nicotine-induced whole-cell currents in cells expressing α3β4-nicotinic acetylcholine receptor, IC(50) 3.1 and 9.1 μM, respectively, while (R,S)-dehydronorketamine, (2S,6S)-hydroxynorketamine and (2R,6R)-hydroxynorketamine were weak inhibitors, IC(50) >100 μM. The binding affinities of (R,S)-dehydronorketamine, (2S,6S)-hydroxynorketamine and (2R,6R)-hydroxynorketamine at the NMDA receptor were also determined using rat brain membranes and the selective NMDA receptor antagonist [(3)H]-MK-801. The calculated K(i) values were 38.95 μM for (S)-dehydronorketamine, 21.19 μM for (2S,6S)-hydroxynorketamine and>100 μM for (2R,6R)-hydroxynorketamine. The results suggest that the inhibitory activity of ketamine metabolites at the α7-nicotinic acetylcholine receptor may contribute to the clinical effect of the drug.

Sazetidine-A, a Selective α4β2 Nicotinic Receptor Desensitizing Agent and Partial Agonist, Reduces Nicotine Self-Administration in Rats

Adequate treatment of tobacco addiction remains problematic. Part of the problem with treatment is a poor understanding of the pharmacologic aspects of nicotine contributing to addiction. In addition to activating nicotinic acetylcholine receptors, nicotine also desensitizes them. It is currently not known how much of each of nicotines actions contribute to its particular behavioral effects. Sazetidine-A (saz-A) is a novel nicotinic receptor-desensitizing agent and partial agonist with high selectivity for α4β2 receptors. The current experiments were conducted to determine whether saz-A would reduce nicotine self-administration in rats and to characterize its ancillary effects. Adult male Sprague-Dawley rats were allowed to self-administer nicotine. After initial food pellet training followed by 10 sessions of nicotine self-administration training, the rats were administered saz-A (0.1–3 mg/kg s.c.) or the saline vehicle in a repeated-measures counterbalanced design. Saz-A at the 3 mg/kg dose significantly decreased nicotine self-administration relative to performance of the same rats after saline injections. In a second study, long-term administration of this dose of sazetidine-A over the course of 10 sessions significantly reduced nicotine self-administration with no apparent diminution of effect. Saz-A in this dose range had only modest effects on locomotor activity, without any overall decrease in activity over a 1-h-long session. Saz-A significantly reduced food self-administration, but this effect was smaller than its effect on nicotine self-administration. Saz-A, which is a selective α4β2-desensitizing agent and partial agonist, effectively reduces nicotine self-administration. This type of treatment holds promise for a new therapy to aid smoking cessation.

Neuronal nicotinic acetylcholine receptor alpha3 subunit protein in rat brain and sympathetic ganglion measured using a subunit-specific antibody: regional and ontogenic expression.

A synthetic peptide corresponding to the C‐terminus of the α3 subunit of the rat neuronal nicotinic acetylcholine receptor (nAChR) was used to generate a rabbit polyclonal α3 antibody. The specificity of this antibody was characterized by immunoblotting, immunohistochemical and immunoprecipitation techniques. Using this antibody, the relative densities of the α3 subunit were quantitatively determined in different brain regions and in superior cervical ganglion (SCG). Among these regions, SCG, interpeduncular nucleus (IPN) and pineal gland showed the highest levels of α3 protein expression. Habenula and superior colliculi had intermediate levels of expression. Low levels were found in cerebral cortex, hippocampus and cerebellum. The ontogenic profile of the α3 subunit in the SCG was also determined. The α3 protein level is low at postnatal day (P 1), but increases rapidly during the first seven postnatal days. This level then plateaus and remains stable through postnatal day 35. These findings suggest that neuronal nAChRs containing the α3 subunit participate in important roles in specific regions of the rat brain and the SCG.

Analgesic Effects of Sazetidine-A, a New Nicotinic Cholinergic Drug

Background:The use of nicotinic agonists for analgesia is limited by their unacceptable side effects. Sazetidine-A is a new partial agonist nicotinic ligand that has very high selectivity for &bgr;2-containing nicotinic acetylcholine receptors. It potently and selectively desensitizes &agr;4&bgr;2 nicotinic acetylcholine receptors without measurable effects on &agr;3&bgr;4 receptors. The authors investigated the analgesic effects of Sazetidine-A using the formalin model of chronic inflammatory pain. Methods:The formalin test was conducted after rats received intraperitoneal saline, Sazetidine-A (0.125, 0.25, 0.5, 1, 2 mg/kg), or subcutaneous epibatidine (2.5–5–10 &mgr;g/kg). In other experiments, Sazetidine-A was preceded by naloxone (0.5 mg/kg) or mecamylamine (10 mg). Effects of Sazetidine-A and epibatidine on locomotor were tested in an open field, and seizure activity was measured using the Racine scale. Locus coeruleus neuron extracellular single-unit spontaneous discharge was recorded in anesthetized animals after Sazetidine-A and epibatidine. Results:Higher doses of Sazetidine-A (0.5, 1, or 2 mg/kg) induced analgesia, with pain scores significantly lower than those seen after saline, lower doses of Sazetidine-A, and epibatidine (P < 0.001). Naloxone did not antagonize the effects of Sazetidine-A, and mecamylamine had partial, dose-dependent antagonistic effects. Epibatidine excited locus coeruleus neurons, whereas Sazetidine-A had no effect on these neurons. Epibatidine and Sazetidine-A affected animals’ locomotor activity for the initial 20 min. While analgesic doses of epibatidine caused seizures, no seizure activity or other neurologic complications were seen in animals that received as much as four times the minimum analgesic dose of Sazetidine-A. Conclusions:Sazetidine-A seems to be a potent analgesic without causing neurologic side effects.

Chronic Sazetidine-A at Behaviorally Active Doses Does Not Increase Nicotinic Cholinergic Receptors in Rodent Brain

Chronic nicotine administration increases α4β2 neuronal nicotinic acetylcholine receptor (nAChR) density in brain. This up-regulation probably contributes to the development and/or maintenance of nicotine dependence. nAChR up-regulation is believed to be triggered at the ligand binding site, so it is not surprising that other nicotinic ligands also up-regulate nAChRs in the brain. These other ligands include varenicline, which is currently used for smoking cessation therapy. Sazetidine-A (saz-A) is a newer nicotinic ligand that binds with high affinity and selectivity at α4β2* nAChRs. In behavioral studies, saz-A decreases nicotine self-administration and increases performance on tasks of attention. We report here that, unlike nicotine and varenicline, chronic administration of saz-A at behaviorally active and even higher doses does not up-regulate nAChRs in rodent brains. We used a newly developed method involving radioligand binding to measure the concentrations and nAChR occupancy of saz-A, nicotine, and varenicline in brains from chronically treated rats. Our results indicate that saz-A reached concentrations in the brain that were ∼150 times its affinity for α4β2* nAChRs and occupied at least 75% of nAChRs. Thus, chronic administration of saz-A did not up-regulate nAChRs despite it reaching brain concentrations that are known to bind and desensitize virtually all α4β2* nAChRs in brain. These findings reinforce a model of nicotine addiction based on desensitization of up-regulated nAChRs and introduce a potential new strategy for smoking cessation therapy in which drugs such as saz-A can promote smoking cessation without maintaining nAChR up-regulation, thereby potentially increasing the rate of long-term abstinence from nicotine.

Toxicological analysis of low-nicotine and nicotine-free cigarettes.

Low-nicotine and nicotine-free cigarettes are commercially available under the brand-name Quest. Some consumers may believe that these are safer cigarettes, and they may smoke more cigarettes or inhale more smoke to compensate for low nicotine yields. Thus, we have studied the toxicological effects of these two cigarettes and compared them with the Kentucky reference cigarette 2R4F. Also, the availability of nicotine-free cigarettes allows for the assessing the role of nicotine in cigarette smoke. In addition to nicotine, some tobacco-specific nitrosamines, aldehydes, and volatile organic compounds were also reduced in the Quest cigarettes compared to the 2R4F. However, aromatic amines were higher in the nicotine-free compared with low nicotine cigarettes. The Ames test revealed that cigarette smoke condensates from the nicotine-free (CSC-F), low nicotine (CSC-L) and 2R4F (CSC-R) cigarettes had a similar mutagenic potency. Exposure to any CSC caused a similar dose-dependent LDH leakage from normal human bronchial epithelial cells. However, CSC-F had more inhibitory effects on the cell growth than CSC-L and CSC-R. Adding nicotine to the CSC-F attenuated this inhibition. Both Quest CSCs decreased gap junction intercellular communication and caused cell cycle arrest. CSC exposure increased cytoplasmic nucleosomes, sub-G1/G0 population and apoptotic comet tails. Proapoptotic protein Bax increased independent of p53 induction after exposure to CSC-F. In conclusion, these studies are not consistent with a perception that low-nicotine or nicotine-free cigarettes may have less toxicity in human cells. Nicotine, as it exists in CSC, attenuates cytotoxicity possibly in part through inhibition of apoptotic pathways.

Liquid chromatographic studies with immobilized neuronal nicotinic acetylcholine receptor stationary phases : effects of receptor subtypes, pH and ionic strength on drug-receptor interactions

Nicotinic acetylcholine receptor (nAChR) alpha3-subunits, beta4-subunits, alpha3/beta4-subunit combination and alpha4/beta2-subunit combination were immobilized on chromatographic stationary phases and the binding affinities of the different nAChR subtypes were chromatographically evaluated. The observed relative binding affinities of epibatidine were alpha4/beta2>alpha3/beta4 and epibatidine did not bind at alpha3-subunits and beta4-subunits. No significant difference in binding affinities was observed on the alpha4/beta2 nAChRs immobilized in immobilized artificial membrane (IAM) particles and those sterically immobilized on Superdex 200 beads. The effects of mobile phase pH and ionic strength on the binding affinities of the alpha3/beta4 nAChRs support were also investigated. The results are consistent with the proposed ligand-nAChR binding model in which a cationic center exists at the binding site.