84 research outputs found
Associations of Nicotinic Acetylcholine Receptor Subunits Expressed in the Rat Habenula
No full textPh.D.The goals of this study were to define the nicotinic acetylcholine receptor (nAChR) subtypes expressed in the rat habenula. This was done using three main techniques: single and sequential immunoprecipitation using subunit-specific antibodies, single-point binding with α-conotoxin mII, and saturation binding with [3H]-epibatidine and competition binding with nicotine, cytisine, AT-1001, and sazetidine-A.Single immunoprecipitation experiments revealed that every subunit probed was expressed in the habenula in significant quantities. The α5 subunit was expressed in approximately 26% of nAChRs, the α4 subunit was expressed in approximately 40% of receptors, α3 was expressed in approximately 32% of receptors, β2 was expressed in approximately 48% of receptors, and β4 was expressed in approximately 51% of nAChRs in the habenula. Sequential immunoprecipitations demonstrated that every subunit expressed was significantly associated with every other subunit with one notable exception: the α4 and α5 subunits were not discernably associated in the habenula. Additionally, we found that approximately 20% of nAChRs in the habenula contain both the β2 and β4 subunits. Finally, through binding assays with [3H]-epibatidine and α-conotoxin MII, we determined that there are no detectable α6-containing nAChRs in the habenula.Saturation binding assays determined that nAChRs in the habenula have a Kd for [3H]-epibatidine of 78 pM. Additionally, we found that approximately 20% of nAChRs have a high affinity for Sazetidine-A (a ligand with a high specificity for β2-containing nAChRs) and approximately 70% of nAChRs in the rat habenula had a high affinity for AT-1001 (a ligand with a high specificity for β4-containing nAChRs). From this data, and the data from the immunoprecipitation reactions, we hypothesized that the β2β4* nAChRs in the habenula indicate binding properties much like those of β4-containing nAChRs.Finally, nAChR subunits in the olfactory bulb and olfactory tubercle were probed by single and sequential immunoprecipitation. In the olfactory bulb, the α5 subunit was expressed in approximately 28% of nAChRs, the α4 subunit was expressed in approximately 48% of receptors, α3 was expressed in approximately 41% of receptors, β2 was expressed in approximately 71% of receptors, and β4 was expressed in approximately 33% of nAChRs
Pharmacological Properties of alpha4beta2 and alpha3beta4 Nicotine Acetylcholine Receptors: Ligand Binding, Activation, Desensitization, and Interspecies Differences
No full textPh.D.Smoking cessation medications using nicotinic ligands, including nicotine, cytisine, and varenicline, have traditionally revolved around selective activation of alpha4beta2 nicotinic acetylcholine receptors (nAChRs). However, nAChR desensitization may be the primary action of nicotine and may be a viable approach for smoking cessation medication. The alpha3beta4 nAChR subtype has been implicated in side effects of nicotinic drugs as well as nicotine addiction. The purpose of the work here is to investigate pharmacological properties of these two nAChR subtypes, with a focus on mechanisms of ligand binding, activation, desensitization, and interspecies differences between human and rat receptors.The first aim of this dissertation was to dissect the properties of six ligands (nicotine, cytisine, sazetidine-A, varenicline, epibatidine, and 5-I-A85380) at alpha4beta2 nAChRs. We developed a method for determining the dissociation rates of unlabeled ligands. These ligands are more potent at desensitizing than at activating alpha4beta2 nAChRs. A correlation was found between duration of desensitization and binding affinity.The second aim was to develop methods for using microfluidic laminar stream solution exchange (MLSSE) to study nAChRs expressed in HEK293 cell lines. The variables considered in this technique were drug exposure time and washout time. We demonstrated that MLSSE can be applied to a broad range of nAChRs.The final aim studied interspecies differences in alpha3beta4 drug affinity. All tested drugs besides acetylcholine have higher potency for human alpha3beta4 nAChRs than rat nAChRs. AT-1001, a new high affinity alpha3beta4 ligand, does not select for desensitization over activation in alpha3beta4 or alpha4beta2 nAChRs. Furthermore, the differences in affinity of AT-1001 between alpha3beta4 and alpha4beta2 nAChRs result in longer desensitization in alpha3beta4 nAChRs compared to alpha4beta2 nAChRs. This is consistent with the affinity of a ligand for a receptor subtype being the major determinant for desensitization length.The investigations in my dissertation contribute to a better understanding of the pharmacological properties of alpha4beta2 and alpha3beta4 nAChRs. The methods developed will help in future studies. Moreover, the results provide important insight in developing new nicotinic therapeutics based on nAChR desensitization and indicate that investigators should be aware of species differences in studying nicotinic ligands
REGULATION AND STOICHIOMETRY OF α4β2* NEURONAL NICOTINIC RECEPTORS
No full textPh.D.Nicotinic acetylcholine receptors (nAChRs) are present throughout the nervous system where they mediate and modulate neurotransmission. The α4β2* subtype is the most prevalent high affinity nAChR expressed in the brain. Models of nicotine addiction and dependence implicate this receptor subtype as central to smoking behavior. This work examines the regulation, expression, and pharmacology of this receptor subtype.In Part I, the density of nAChRs in the rat cortex is examined following a two phase treatment paradigm which emulates a chronic smoker who quits and begins a smoking cessation therapy. Rats were administered saline or nicotine then maintained on these treatments or switched to treatment with saline, varenicline, a currently approved FDA smoking cessation therapy, or sazetidine-A, a new ligand efficacious in reducing nicotine self-administration in animals. Assessments of nAChR radioligand binding density and α4 and β2 subunit protein densities reveal that varenicline maintains nicotine-induced up-regulation while sazetidine-A allows receptors to return to basal levels.Part II examines high and low sensitivity α4β2 nAChRs, corresponding to subunit stoichiometries of (α4)2(β2)3 and (α4)3(β2)2, respectively. In this section, transfection ratios of rat α4 and β2 subunit cDNA in HEK293 cells are used to create a model of high and low sensitivity α4β2 nAChRs. Through characterization of this model system, a method to detect nAChR stoichiometry using two concentrations of acetylcholine was validated. Applying this method to the rat motor cortex indicates that expression of the low sensitivity α4β2 nAChR predominates in this area. The effect of nicotine treatment on α4β2 nAChR stoichiometry was also assessed.In Part III, the pharmacology of six concatameric α4β2β4* nAChRs expressed in HEK293 cells is characterized alongside non-linked α4β2 and α4β4 nAChRs. Stable cell lines for each unique nAChR permutation were created and demonstrated to produce nAChRs that assemble as full length products which bind [3H]-Epibatidine with high affinity. Sazetidine-A, a β2 selective ligand, and AT-1001, a β4 selective ligand, were used to determine the presence of α4β2 and α4β4 binding sites on each concatamer permutation. These data suggest that the 3rd subunit in the pentameric sequence occupies the non-ligand binding site of the receptor
Title Page Title: A New Radioligand Binding Assay to Measure the Concentration of Drugs in Rodent Brain Ex Vivo Running Title Page Running Title: A New Competitive-Saturation Binding Assay
No full textAbstract We have developed a new radioligand binding assay method to measure the concentration of non-radiolabeled drugs in the brain ex vivo. This new method fuses the concepts of standard competition and saturation binding assays, and utilizes a transformed version of the Cheng-Prusoff equation to calculate the drug concentration. After testing the validity of this method, we demonstrated its utility by measuring the brain concentration of sazetidine-A, a newly developed nicotinic receptor ligand, and its elimination rate after a single subcutaneous administration. Our results indicate that sazetidine-A reaches brain concentrations that are known to occupy and desensitize the majority of nAChR binding sites. Furthermore, using this method, we estimated the half-life of sazetidine-A in the rat brain to be ~65 min. It is important to note that the method described here to measure sazetidine-A in brain should be generalizable to other drugs acting at any receptor that can be reliably measured with a radiolabeled ligand
A New Radioligand Binding Assay to Measure the Concentration of Drugs in Rodent Brain Ex Vivo
No full textABSTRACT We have developed a new radioligand binding assay method to measure the concentration of nonradiolabeled drugs in the brain ex vivo. This new method fuses the concepts of standard competition and saturation binding assays and uses a transformed version of the Cheng-Prusoff equation (Biochem Pharmacol 22: 3099 -3108, 1973) to calculate the drug concentration. After testing the validity of this method, we demonstrated its utility by measuring the brain concentration of sazetidine-A, a newly developed nicotinic receptor ligand, and its elimination rate after a single subcutaneous administration. Our results indicate that sazetidine-A reaches brain concentrations that are known to occupy and desensitize the majority of neuronal nicotinic acetylcholine receptor binding sites. Furthermore, using this method, we estimated the half-life of sazetidine-A in the rat brain to be ϳ65 min. It is important to note that the method described here to measure sazetidine-A in brain should be generalizable to other drugs acting at any receptor that can be reliably measured with a radiolabeled ligand
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