Nicotinic Receptors Underlying Nicotine Dependence: Evidence from Transgenic Mouse Models.

Nicotine underlies the reinforcing properties of tobacco cigarettes and e-cigarettes. After inhalation and absorption, nicotine binds to various nicotinic acetylcholine receptor (nAChR) subtypes localized on the pre- and postsynaptic membranes of cells, which subsequently leads to the modulation of cellular function and neurotransmitter signaling. In this chapter, we begin by briefly reviewing the current understanding of nicotine's actions on nAChRs and highlight considerations regarding nAChR subtype localization and pharmacodynamics. Thereafter, we discuss the seminal discoveries derived from genetically modified mouse models, which have greatly contributed to our understanding of nicotine's effects on the reward-related mesolimbic pathway and the aversion-related habenulo-interpeduncular pathway. Thereafter, emerging areas of research focusing on modulation of nAChR expression and/or function are considered. Taken together, these discoveries have provided a foundational understanding of various genetic, neurobiological, and behavioral factors underlying the motivation to use nicotine and related dependence processes, which are thereby advancing drug discovery efforts to promote long-term abstinence

Chemical chaperones exceed the chaperone effects of RIC-3 in promoting assembly of functional α7 AChRs.

Functional α7 nicotinic acetylcholine receptors (AChRs) do not assemble efficiently in cells transfected with α7 subunits unless the cells are also transfected with the chaperone protein RIC-3. Despite the presence of RIC-3, large amounts of these subunits remain improperly assembled. Thus, additional chaperone proteins are probably required for efficient assembly of α7 AChRs. Cholinergic ligands can act as pharmacological chaperones to promote assembly of mature AChRs and upregulate the amount of functional AChRs. In addition, we have found that the chemical chaperones 4-phenylbutyric acid (PBA) and valproic acid (VPA) greatly increase the amount of functional α7 AChRs produced in a cell line expressing both α7 and RIC-3. Increased α7 AChR expression allows assay of drug action using a membrane potential-sensitive fluorescent indicator. Both PBA and VPA also increase α7 expression in the SH-SY5Y neuroblastoma cell line that endogenously expresses α7 AChRs. VPA increases expression of endogenous α7 AChRs in hippocampal neurons but PBA does not. RIC-3 is insufficient for optimal assembly of α7 AChRs, but provides assay conditions for detecting additional chaperones. Chemical chaperones are a useful pragmatic approach to express high levels of human α7 AChRs for drug selection and characterization and possibly to increase α7 expression in vivo

A new synthetic medium for the optimization of docosahexaenoic acid production in Crypthecodinium cohnii.

The heterotrophic microalgae Crypthecodinium cohnii was usually cultivated in complex medium containing glucose, yeast extract and sea salt. For the preparation of DHA with highest purity, a new defined medium without the yeast extract was developed. Different inoculated densities, C/N ratios, temperatures, culture volumes and glucose additions were investigated to optimize the algal growth rate and DHA production. The growth period in C. cohnii was shortened from 12-14 days to 7-8 days, the OD600 was enhanced from 2.0 to 3.0, the glucose consumption was accelerated and used up on day 3-4, and the DHA content in culture were increased from 10 to 45 nmoles/300 μl batch. It was found that C. cohnii had optimal growth and DHA accumulation in 25 °C, 0.2 inoculated density, 5-10 C/N ratio, 5:1 air/culture volume ratio. This is the first time DHA production using C.cohnii has been optimized in synthetic medium. This allows preparation of uniformly radiolabeled 13C- and 14C-DHA

Acute activation, desensitization and smoldering activation of human acetylcholine receptors.

The behavioral effects of nicotine and other nicotinic agonists are mediated by AChRs in the brain. The relative contribution of acute activation versus chronic desensitization of AChRs is unknown. Sustained "smoldering activation" occurs over a range of agonist concentrations at which activated and desensitized AChRs are present in equilibrium. We used a fluorescent dye sensitive to changes in membrane potential to examine the effects of acute activation and chronic desensitization by nicotinic AChR agonists on cell lines expressing human α4β2, α3β4 and α7 AChRs. We examined the effects of acute and prolonged application of nicotine and the partial agonists varenicline, cytisine and sazetidine-A on these AChRs. The range of concentrations over which nicotine causes smoldering activation of α4β2 AChRs was centered at 0.13 µM, a level found in smokers. However, nicotine produced smoldering activation of α3β4 and α7 AChRs at concentrations well above levels found in smokers. The α4β2 expressing cell line contains a mixture of two stoichiometries, namely (α4β2)2β2 and (α4β2)2α4. The (α4β2)2β2 stoichiometry is more sensitive to activation by nicotine. Sazetidine-A activates and desensitizes only this stoichiometry. Varenicline, cytisine and sazetidine-A were partial agonists on this mixture of α4β2 AChRs, but full agonists on α3β4 and α7 AChRs. It has been reported that cytisine and varenicline are most efficacious on the (α4β2)2α4 stoichiometry. In this study, we distinguish the dual effects of activation and desensitization of AChRs by these nicotinic agonists and define the range of concentrations over which smoldering activation can be sustained

Modulation of Human Neuronal ␣3 Nicotinic Receptors 1

ABSTRACT Functional effects of human ␣5 nicotinic ACh receptor (AChR) subunits coassembled with ␣3 and ␤2 or with ␣3 and ␤4 subunits, were investigated in Xenopus oocytes. The presence of ␣5 subunits altered some properties of both ␣3 AChRs and differentially altered other properties of ␣3␤2 AChRs vs. ␣3␤4 AChRs. ␣5 subunits increased desensitization and Ca ϩϩ permeability of all ␣3 AChRs. The Ca ϩϩ permeabilities of both ␣3␤2␣5 and ␣3␤4␣5 AChRs were comparable to that of ␣7 AChRs. As we have shown previously, ␣5 subunits increased the ACh sensitivity of ␣3␤2 AChRs 50-fold but had little effect on ␣3␤4 AChRs. ␣5 caused only subtle changes in the activation potencies of ␣3 AChRs for nicotine, cytisine and 1,1-dimethyl-4-plenylpiperazinium (DMPP). However, ␣5 increased the efficacies of nicotine and DMPP on ␣3␤2 AChRs but decreased them on ␣3␤4 AChRs. Immunoisolation of cloned human AChRs expressed in oocytes showed that ␣5 efficiently coassembled with ␣3 plus ␤2 and/or ␤4 subunits. As expected, human AChRs immunoisolated from SH-SY5Y neuroblastoma cells showed that AChRs containing ␣3 and probably ␣5 subunits were present, but ␣4 AChRs were not. In brain, by contrast, ␣4␤2 AChRs were shown to predominate over ␣3 AChRs. Some of the brain ␣4␤2 AChRs were found to contain ␣5 subunits. Neuronal nicotinic AChRs are thought to be formed by pentameric assemblies of certain combinations of ␣2, ␣3, ␣4, ␣5, ␣6, ␣7, ␣8, ␣9, ␤2, ␤3 and ␤4 subunits Our initial studies of human ␣5 subunits expressed in Xenopus oocytes showed that they assembled efficiently with human ␣3 and ␤2 or human ␣3 and ␤4 subunits to form AChRs that desensitized more rapidly and that, especially in the case of ␣3␤2␣5 AChRs, exhibited altered pharmacological propertie

¹²⁵I αBgt binding to the α7/RIC-3 cell line suggests that some of the α7 synthesized is not assembled into mature AChRs.

<p>A) Binding of ¹²⁵I αBgt to the surface of live cells revealed 2.95±0.08 fmol/well of high affinity (K_D = 0.841±0.048 nM, n = 6) binding sites characteristic of mature α7 AChRs. B) Binding to parallel cultures after fixation and permeabilization with the detergent Triton X-100 revealed a similar amount of high affinity binding sites (2.91±0.48 fmol/well, n = 6) but also additional low affinity binding sites presumably corresponding to unassembled or partially assembled α7 subunits. The affinity for ¹²⁵I αBgt of these sites was too low to measure accurately, and their low affinity prevented measurement of their total amount. Cells were plated on poly-D-lysine coated microwell plates then fixed by adding 100 µl/well of 10% phosphate buffered formalin to the 100 µl of culture medium for 1 hour at room temperature. After washing with PBS, cells were permeabilized using 0.1% Triton X-100 in PBS. Both live and fixed culture plates were labeled with ¹²⁵I αBgt for two hours. After washing each well 3 times with 200 µl of PBS, bound αBgt was determined by γ counting of individual wells. Nonspecific binding determined by binding in the presence of 5 mM nicotine.</p

Sucrose gradient sedimentation revealed that nearly all of the α7 subunits synthesized in the α7/RIC-3 cell line were not assembled into mature AChRs.

<p>α7/RIC-3 cell line was transfected with additional FLAG tagged RIC-3. α7 AChRs were immuno-isolated from aliquots of each gradient fraction using microwells coated with mAb 319 to α7 subunit. These AChRs were labeled with 10 nM ¹²⁵I αBgt. Mature AChRs sedimented at the size of <i>Torpedo</i> AChR monomers. <i>Torpedo</i> AChR 9.5 S monomers and 13 S dimers were sedimented on the gradient as internal standards. <i>Torpedo</i> AChRs were isolated from aliquots of each fraction using microwells coated with mAb 210 to α1 subunit. These AChRs were labeled with 1 nM ¹²⁵I αBgt. The positions of the <i>Torpedo</i> AChR monomer and dimer peaks are shown by arrows. FLAG-tagged RIC-3 was visualized using western blots of pools of aliquots from four fractions using antibodies to the FLAG tag. Most of the RIC-3 sedimented near the top of the gradient and was not associated with mature AChRs or denatured α7 protein. α7 protein was visualized using western blots of pools of aliquots from four fractions using mAb 319 to α7. Most of the α7 protein was in large amorphous aggregates near the bottom of the gradient, and very little was in the fractions containing mature AChRs.</p

Chemical chaperones increase α7 AChR expression on the cell surface.

<p>A) Chemical chaperones PBA, VPA or NaB can increase surface expression in the α7/RIC-3 cell line. PBA or VPA greatly increase surface expression of α7 AChRs in the cell line transfected with both α7 and RIC-3 after 5 days treatment. In combination, PBA and VPA produce a larger effect, together increasing surface α7 AChR expression 10 fold. B) Growth at 29°C for the last 24 hours increases α7 AChR expression. PBA (6 mM) acting as a chemical chaperone and MLA (0.1 mM) acting as a pharmacological chaperone each substantially increased expression of AChRs on the cell surface. PBA was also tested in combination with 5% human serum. These effects were larger when the cells were grown at low temperature. Together, PBA+MLA produced a maximum effect, independent of temperature.</p