Nicotine Modulates The Expression Of A Diverse Set Of Genes In The Neuronal Sh-Sy5Y Cell Line

Nicotine exposure can have long lasting effects on nervous system function, some of which must contribute to nicotine dependence. Up-regulation, an increase in numbers of radioligand-binding nicotinic acetylcholine receptors (nAChR), occurs on exposure to nicotine at high concentrations. To determine whether altered gene expression might account for long term changes and up-regulation following nicotine exposure, we assessed effects of 1 h of 1 mM nicotine exposure on alter. ation of gene expression in the neuron-like SH-SY5Y neuroblastoma clonal line. Repeat and cross-controlled microarray analyses yielded a list of 17 genes from the initially screened ∼5,000 whose expression was consistently altered following nicotine treatment. Subsequent quantitative, real time reverse transcriptase PCR analyses confirmed altered expression in 14 of 16 genes tested. Further, the general nAChR antagonist, d-tubocurarine, blocked all but two of the observed changes in gene expression, indicating that these changes are dependent on nAChR activation. Use of other antagonists revealed that nAChR subtypes can differentially affect gene expression. The genes affected code for proteins that may be broadly categorized into four groups: transcription factors, protein processing factors, RNA-binding proteins, and plasma membrane-associated proteins. Our results suggest that nicotinic activation of nAChR may have a broad role in affecting cellular physiology through modulating gene expression

Modulation Of Gain-Of-Function α6*-Nicotinic Acetylcholine Receptor By β3 Subunits

We previously have shown that β3 subunits either eliminate (e.g. for all-human (h) or all-mouse (m)α6β4β3-nAChR) or potentiate (e.g. for hybrid mα6hβ4hβ3- or mα6mβ4hβ3- nAChR containing subunits from different species) function of α6*-nAChR expressed in Xenopus oocytes, and that nAChR hα6 subunit residues Asn-143 and Met-145 in N-terminal domain loop E are important for dominant-negative effects of nAChR hβ3 subunits on hα6*-nAChR function. Here, we tested the hypothesis that these effects of β3 subunits would be preserved even if nAChR α6 subunits harbored gain-of-function, leucine- or valine-to-serine mutations at 9′ or 13′ positions (L9′S or V13′S) in their second transmembrane domains, yielding receptors with heightened functional activity and more amenable to assessment of effects of β3 subunit incorporation. However, coexpression with β3 subunits potentiates rather than suppresses function of all-human, all-mouse, or hybrid α6 (L9′S or V13′S)β4*- or α6(N143D+M145V) L9′Sβ2*-nAChR. This contrasts with the lack of consistent function when α6 (L9′S or V13′S) and β2 subunits are expressed alone or in the presence of wild-type β3 subunits. These results provide evidence that gain-of-function hα6hβ2*-nAChR (i.e. hα 6(N143D+M145V) L9′Shβ2hβ3 nAChR) could be produced in vitro. These studies also indicate that nAChR β3 subunits can be assembly partners in functional α6*-nAChR and that 9′ or 13′ mutations in the nAChR α6 subunit second transmembrane domain can act as gain-of-function and/or reporter mutations. Moreover, our findings suggest that β3 subunit coexpression promotes function of α6*-nAChR. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc

Reporter Mutation Studies Show That Nicotinic Acetylcholine Receptor (Nachr) α5 Subunits And/Or Variants Modulate Function Of α6*-Nachr

To further the understanding of functional α6α5*- nicotinic acetylcholine receptors (nAChR; the asterisk (*) indicates known or possible presence of other subunits), we have heterologously expressed in oocytes different, mouse or human, nAChR subunit combinations. Coexpression with wild-type α5 subunits or chimeric α5/β3 subunits (in which the human α5 subunit N-terminal, extracellular domain is linked to the remaining domains of the human β3 subunit) almost completely abolishes the very small amount of function seen for α6β4*-nAChR and does not induce function of α6β2*-nAChR. Coexpression with human α5 V9,S subunits bearing a valine 290 to serine mutation in the 9′ position of the second transmembrane domain does not rescue the function of α6β4*-nAChR or induce function of α6β2*-nAChR. However, coexpression with mutant chimeric α5/β3 V9,S subunits has a gain-of-function effect (higher functional expression and agonist sensitivity and spontaneous opening inhibited by mecamylamine) on α6β4*- nAChR. Moreover, N143D + M145V mutations in the α6 subunit N-terminal domain enable α5/β3 V9,S subunits to have a gain-of-function effect on α6β2*-nAChR. nAChR containing chimeric α6/α3 subunits plus either β2 or β4 subunits have some function that is modulated in the presence of α5 or α5/β3 subunits. Coexpression with α5/β3 V9,S subunits has a gain-of-function effect more pronounced than that in the presence of α5 V9,S subunits. Gain-of-function effects are dependent, sometimes subtly, on the nature and apparently the extracellular, cytoplasmic, and/or transmembrane domain topology of partner subunits. These studies yield insight into assembly of functional α6α5*-nAChR and provide tools for development of α6*- nAChR-selective ligands that could be important in the treatment of nicotine dependence, and perhaps other neurological diseases. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc

Roles For N-Terminal Extracellular Domains Of Nicotinic Acetylcholine Receptor (Nachr) β3 Subunits In Enhanced Functional Expression Of Mouse α6β2β3- And α6β4β3-Nachrs

Functional heterologous expression of naturally expressed mouse α6∗-nicotinic acetylcholine receptors (mα6∗-nAChRs; where \ ∗\ indicates the presence of additional subunits) has been difficult. Here we expressed and characterized wild-type (WT), gain-of-function, chimeric, or gain-of-function chimeric nAChR subunits, sometimes as hybrid nAChRs containing both human (h) and mouse (m) subunits, in Xenopus oocytes. Hybrid mα6mβ4hβ3- (∼5-8-fold) or WT mα6mβ4mβ3-nAChRs (∼2-fold) yielded higher function than mα6mβ4-nAChRs. Function was not detected when mα6 and mβ2 subunits were expressed together or in the additional presence of hβ3 or mβ3 subunits. However, function emerged upon expression of mα6mβ2mβ3V9′S-nAChRs containing β3 subunits having gain-of-function V9′S (valine to serine at the 9′-position) mutations in transmembrane domain II and was further elevated 9-fold when hβ3V9′Ssubunits were substituted for mβ3V9′Ssubunits. Studies involving WT or gain-of-function chimeric mouse/human β3 subunits narrowed the search for domains that influence functional expression of mα6∗-nAChRs. Using hβ3 subunits as templates for site-directed mutagenesis studies, substitution with mβ3 subunit residues in extracellular N-terminal domain loops \ C\ (Glu221and Phe223), \ E\ (Ser144and Ser148), and \ β2-β3\ (Gln94and Glu101) increased function of mα6mβ2∗- (′2-3-fold) or mα6mβ4∗(′2-4-fold)-nAChRs. EC50values for nicotine acting at mα6mβ4∗-nAChR were unaffected by β3 subunit residue substitutions in loop C or E. Thus, amino acid residues located in primary (loop C) or complementary (loops β2-β3 and E) interfaces of β3 subunits are some of the molecular impediments for functional expression of mα6mβ2β3- or mα6mβ4β3-nAChRs

Identification Of N-Terminal Extracellular Domain Determinants In Nicotinic Acetylcholine Receptor (Nachr) α6 Subunits That Influence Effects Of Wild-Type Or Mutant β3 Subunits On Function Of α6β2*- Or α6β4*-Nachr

Despite the apparent function of naturally expressed mammalian α6*-nicotinic acetylcholine receptors (α6*-nAChR; where*indicates the known or possible presence of additional subunits), their functional and heterologous expression has been difficult. Here, we report that coexpression with wild-type β3 subunits abolishes the small amount of function typically seen for all-human or all-mouse α6β4*-nAChR expressed in Xenopus oocytes. However, levels of function and agonist potencies are markedly increased, and there is atropine-sensitive blockade of spontaneous channel opening upon coexpression of α6 and β4 subunits with mutant β3 subunits harboring valine-to-serine mutations at 9′- or 13′-positions. There is no function when α6 and β2 subunits are expressed alone or in the presence of wild-type or mutant β3 subunits. Interestingly, hybrid nAChR containing mouse α6 and human (h) β4 subunits have function potentiated rather than suppressed by coexpression with wild-type hβ3 subunits and potentiated further upon coexpression with hβ3 V9,S subunits. Studies using nAChR chimeric mouse/human α6 subunits indicated that residues involved in effects seen with hybrid nAChR are located in the α6 subunit N-terminal domain. More specifically, nAChR hα6 subunit residues Asn-143 and Met-145 are important for dominant-negative effects of nAChR hβ3 subunits on hα6hβ4-nAChR function. Asn-143 and additional residues in the N-terminal domain of nAChR hα6 subunits are involved in the gain-of-function effects of nAChR hβ3 V9,S subunits on α6β2*-nAChR function. These studies illuminate the structural bases for effects of β3 subunits on α6*-nAChR function and suggest that unique subunit interfaces involving the complementary rather than the primary face of α6 subunits are involved. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc

Functional expression of nicotinic acetylcholine receptors containing rat α7 subunits in human SH-SY5Y neuroblastoma cells

AbstractNeuronal nicotinic acetylcholine receptors (nAChR) are made from different combinations of subunits encoded by a diverse family of genes. However, the recently cloned α7 gene codes for subunits that can form homooligomeric nAChR complexes when expressed in Xenopus oocytes. Electrophysiological studies reveal that these α7-nAChR function as α-bungarotoxin (Bgt)-sensitive, quickly activating/inactivating ion channels with a unique pharmacological profile and an unusually high permeability to calcium ions. Although similar observations have been made in studies of Bgt-sensitive, functional nAChR subtypes that are naturally expressed in neuronal cells, all attempts until now to reconstitute functional α7-nAChR in cell lines have failed. Here we report the successful use of SH-SY5Y human neuroblastoma cells, which naturally express low levels of endogenous α7 transcripts, to stably overexpress heterologous rat nAChR α7 transgenes. These transgenes are expressed as the appropriately-sized α7 messages and protein, and stably transfected SH-SY5Y cells have over 30-times higher levels of specific Bgt binding sites than do wild-type cells. Whole cell current recordings confirm that transfected cells express functional nAChR that are sensitive to blockade by Bgt and display the typical physiological and pharmacological profiles of α7-nAChR. We conclude that stable, functional expression of α7 transgenes in a mammalian cell line has been achieved for the first time

Distinctive Single-Channel Properties Of α4β2-Nicotinic Acetylcholine Receptor Isoforms

Central nervous system nicotinic acetylcholine receptors (nAChR) are predominantly of the α4β2 subtype. Two isoforms exist, with high or low agonist sensitivity (HS-(α4β2) 2 β2- and LS-(α4β2) 2 α4-nAChR). Both isoforms exhibit similar macroscopic potency and efficacy values at low acetylcholine (ACh) concentrations, mediated by a common pair of high-affinity α4(+)/(-)β2 subunit binding interfaces. However LS-(α4β2) 2 α4-nAChR also respond to higher concentrations of ACh, acting at a third α4(+)/(-)α4 subunit interface. To probe isoform functional differences further, HS- and LS-α4β2-nAChR were expressed in Xenopus laevis oocytes and single-channel responses were assessed using cell-attached patch-clamp. In the presence of a low ACh concentration, both isoforms produce low-bursting function. HS-(α4β2) 2 β2-nAChR exhibit a single conductance state, whereas LS-(α4β2) 2 α4-nAChR display two distinctive conductance states. A higher ACh concentration did not preferentially recruit either conductance state, but did result in increased LS-(α4β2) 2 α4-nAChR bursting and reduced closed times. Introduction of an α4(+)/(-)α4-interface loss-of-function α4W182A mutation abolished these changes, confirming this site’s role in mediating LS-(α4β2) 2 α4-nAChR responses. Small or large amplitude openings are highly-correlated within individual LS-(α4β2) 2 α4-nAChR bursts, suggesting that they arise from distinct intermediate states, each of which is stabilized by α4(+)/(-)α4 site ACh binding. These findings are consistent with α4(+)/(-)α4 subunit interface occupation resulting in allosteric potentiation of agonist actions at α4(+)/(-)β2 subunit interfaces, rather than independent induction of high conductance channel openings

Distinctive Roles For α7*- And α9*-Nicotinic Acetylcholine Receptors In Inflammatory And Autoimmune Responses In The Murine Experimental Autoimmune Encephalomyelitis Model Of Multiple Sclerosis

Previous studies have demonstrated immunosuppressive and anti-inflammatory effects of nicotine, including in the experimental autoimmune encephalomyelitis (EAE) model in mice of some forms of multiple sclerosis (MS). Other studies using knock-out (KO) mice have implicated nicotinic acetylcholine (ACh) receptors containing α7, α9, or β2 subunits (α7*-, α9*- or β2*-nAChR) in different, disease exacerbating or disease-ameliorating processes. These outcomes are in harmony with gene expression analyses showing nAChR subunit mRNA in many classes of immune system cell types. Consistent with influences on disease status, predictable effects of nAChR subunit (and subtype) KO, or of nicotine exposure, are seen on immune cell numbers and distribution and on cytokine levels or other markers of immunity, inflammation, demyelination, and axonal degradation. Providing support for our hypotheses about distinctive roles for nAChR subtypes in EAE, here we have used direct and adoptive EAE induction and a nAChR subunit gene double knock-out (DKO) strategy. Immune cell expression of nAChR α9 subunits as protein is demonstrated by immunostaining of isolated CD4+, CD8+, CD11b+ and CD11c+ cells from wild-type (WT) mice, but not in cells from nAChR α9 subunit KO animals. Nicotine exposure is protective against directly-induced EAE in WT or α7/α9 DKO animals relative to effects seen in WT/vehicle-treated mice, but, remarkably, EAE is exacerbated in vehicle-treated α7/α9 DKO mice. Brain lesion volume and intracranial inflammatory activity similarly are higher in DKO/vehicle than in WT/vehicletreated animals, although nicotine’s protective effects are seen in each instance. By contrast, in adoptive transfer studies, disease severity is attenuated and disease onset is delayed in recipients of splenocytes from WT animals treated with nicotine rather than with vehicle. Moreover, protection as seen in nicotine-treated WT animals is the same in recipients of splenocytes from nAChR α7/α9 DKO mice irrespective of their exposure to nicotine or vehicle. When combined with previous observations, these findings are consistent with disease exacerbation (or even induction) being mediated at least in part via α9*-nAChR in peripheral immune cells. They also suggest protective roles of central nervous system (CNS) α7*-nAChR. The results suggest that both α7*- and α9*-nAChR are potential targets of therapeutic ligands to modulate inflammation and autoimmunity

Chemistry And Pharmacological Characterization Of Novel Nitrogen Analogues Of Amop-H-Oh (Sazetidine-A 6-[5-(Azetidin-2-Ylmethoxy)Pyridin-3-Yl]Hex-5-Yn-1- Ol) As α4β2-Nicotinic Acetylcholine Receptor-Selective Partial Agonists

In order to advance therapeutic applications of nicotinic ligands, continuing research efforts are being directed toward the identification and characterization of novel nicotinic acetylcholine receptor (nAChR) ligands that are both potent and subtype selective. Herein we report the synthesis and pharmacological evaluation of members of a new series of 3-alkoxy-5- aminopyridine derivatives that display good selectivity for the α4β2-nAChR subtype based on ligand binding and functional evaluations. The most potent ligand in this series, compound 64, showed high radioligand binding affinity and selectivity for rat α4β2-nAChR with a Ki value of 1.2 nM and 4700-fold selectivity for α4β2- over α3β4-nAChR, and ∼100-fold selectivity for functional, high-sensitivity, human α4β2-nAChR over α3β4*-nAChR. In the mouse forced swim test, compound 64 exhibited antidepressant-like effects. Structure-activity relationship (SAR) analyses suggest that the introduction of additional substituents to the amino group present on the pyridine ring of the N-demethylated analogue of compound 17 can provide potent α4β2-nAChR-selective ligands for possible use in treatment of neurological and psychiatric disorders including depression. © 2010 American Chemical Society

Identification Of Fresh Cardiorespiration Of 2018 Nusa Cendana University Of Penjaskesrek Students During Covid-19

During the pandemic, online lectures are mandatory however, how can students' physical activity be maintained or not. The goal is that students who have good cardiorespiratory freshness are expected to have good endurance. This research is a qualitative descriptive study. The method used is a survey with data collection techniques using measurement tests. The instrument used to determine the endurance level of a student's physical fitness was a multi-stage running test or the bleep test. The sample in this study using simple random sampling, the research sample was selected as many as 30 students. The results of the data from 30 Penjaskesrek class 2018 Nusa Cendana University students who took the multistage fitness test to find out the level of cardiorespiratory freshness was in the very poor classification of 10 people with a percentage of 33.33%, the lack of classification was 3 people (10%), the classification is quite good (33.33%), good classification 5 people (16.66%), very good classification 2 people (6.66%), and no student is in the special classification (0%). The results show that the level of cardiorespiratory freshness The 2018 Nusa Cendana University Penjaskesrek students are in a sufficient classification