Nicotinic Cholinergic Signaling Research Articles

Co-activation of selective nicotinic acetylcholine receptor subtypes is required to reverse hippocampal network dysfunction and prevent fear memory loss in Alzheimer's disease.

Alzheimer's disease (AD) is the most common form of dementia with no known cause and cure. Research suggests that a reduction of GABAergic inhibitory interneurons' activity in the hippocampus by beta-amyloid peptide (Aβ) is a crucial trigger for cognitive impairment in AD via hyperexcitability. Therefore, enhancing hippocampal inhibition is thought to be protective against AD. However, hippocampal inhibitory cells are highly diverse, and these distinct interneuron subtypes differentially regulate hippocampal inhibitory circuits and cognitive processes. Moreover, Aβ unlikely affects all subtypes of inhibitory interneurons in the hippocampus equally. Hence, identifying the affected interneuron subtypes in AD to enhance hippocampal inhibition optimally is conceptually and practically challenging. We have previously found that Aβ selectively binds to two of the three major hippocampal nicotinic acetylcholine receptor (nAChR) subtypes, α7- and α4β2-nAChRs, but not α3β4-nAChRs, and inhibits these two receptors in cultured hippocampal inhibitory interneurons to decrease their activity, leading to hyperexcitation and synaptic dysfunction in excitatory neurons. We have also revealed that co-activation of α7- and α4β2-nAChRs is required to reverse the Aβ-induced adverse effects in hippocampal excitatory neurons. Here, we discover that α7- and α4β2-nAChRs predominantly control the nicotinic cholinergic signaling and neuronal activity in hippocampal parvalbumin-positive (PV+) and somatostatin-positive (SST+) inhibitory interneurons, respectively. Furthermore, we reveal that co-activation of these receptors is necessary to reverse hippocampal network dysfunction and fear memory loss in the amyloid pathology model mice. We thus suggest that co-activation of PV+ and SST+ cells is a novel strategy to reverse hippocampal dysfunction and cognitive decline in AD.

Spontaneous enteric nervous system activity generates contractile patterns prior to maturation of gastrointestinal motility.

Spontaneous neuronal network activity is essential to the functional maturation of central and peripheral circuits, yet whether this is a feature of enteric nervous system development has yet to be established. Although enteric neurons are known exhibit electrophysiological properties early in embryonic development, no connection has been drawn between this neuronal activity and the development of gastrointestinal (GI) motility patterns. We use exvivo GI motility assays with newly developed unbiased computational analyses to identify GI motility patterns across mouse embryonic development. We find a previously unknown pattern of neurogenic contractions termed "clustered ripples" that arises spontaneously at embryonic day 16.5, an age earlier than any identified mature GI motility patterns. We further show that these contractions are driven by nicotinic cholinergic signaling. Clustered ripples are neurogenic contractile activity that arise from spontaneous ENS activity and precede all known forms of neurogenic GI motility. This earliest motility pattern requires nicotinic cholinergic signaling, which may inform pharmacology for enhancing GI motility in preterm infants.

Cholinergic Mechanisms in Gastrointestinal Neoplasia.

Acetylcholine-activated receptors are divided broadly into two major structurally distinct classes: ligand-gated ion channel nicotinic and G-protein-coupled muscarinic receptors. Each class encompasses several structurally related receptor subtypes with distinct patterns of tissue expression and post-receptor signal transduction mechanisms. The activation of both nicotinic and muscarinic cholinergic receptors has been associated with the induction and progression of gastrointestinal neoplasia. Herein, after briefly reviewing the classification of acetylcholine-activated receptors and the role that nicotinic and muscarinic cholinergic signaling plays in normal digestive function, we consider the mechanics of acetylcholine synthesis and release by neuronal and non-neuronal cells in the gastrointestinal microenvironment, and current methodology and challenges in measuring serum and tissue acetylcholine levels accurately. Then, we critically evaluate the evidence that constitutive and ligand-induced activation of acetylcholine-activated receptors plays a role in promoting gastrointestinal neoplasia. We focus primarily on adenocarcinomas of the stomach, pancreas, and colon, because these cancers are particularly common worldwide and, when diagnosed at an advanced stage, are associated with very high rates of morbidity and mortality. Throughout this comprehensive review, we concentrate on identifying novel ways to leverage these observations for prognostic and therapeutic purposes.

Plasma Metabolomics Analysis of Polyvinyl Chloride Workers Identifies Altered Processes and Candidate Biomarkers for Hepatic Hemangiosarcoma and Its Development.

Background: High-level occupational vinyl chloride (VC) exposures have been associated with hepatic hemangiosarcoma, which typically develops following a long latency period. Although VC is genotoxic, a more comprehensive mode of action has not been determined and diagnostic biomarkers have not been established. The purpose of this study is to address these knowledge gaps through plasma metabolomics. Methods: Plasma samples from polyvinyl chloride polymerization workers who developed hemangiosarcoma (cases, n = 15) and VC exposure-matched controls (n = 17) underwent metabolomic analysis. Random forest and bioinformatic analyses were performed. Results: Cases and controls had similar demographics and routine liver biochemistries. Mass spectroscopy identified 606 known metabolites. Random forest analysis had an 82% predictive accuracy for group classification. 60 metabolites were significantly increased and 44 were decreased vs. controls. Taurocholate, bradykinin and fibrin degradation product 2 were up-regulated by greater than 80-fold. The naturally occurring anti-angiogenic phenol, 4-hydroxybenzyl alcohol, was down-regulated 5-fold. Top affected ontologies involved: (i) metabolism of bile acids, taurine, cholesterol, fatty acids and amino acids; (ii) inflammation and oxidative stress; and (iii) nicotinic cholinergic signaling. Conclusions: The plasma metabolome was differentially regulated in polyvinyl chloride workers who developed hepatic hemangiosarcoma. Ontologies potentially involved in hemangiosarcoma pathogenesis and candidate biomarkers were identified.

Chronic Nicotine Exposure Alters the Neurophysiology of Habenulo-Interpeduncular Circuitry.

Antagonism of nicotinic acetylcholine receptors (nAChRs) in the medial habenula (MHb) or interpeduncular nucleus (IPN) triggers withdrawal-like behaviors in mice chronically exposed to nicotine, implying that nicotine dependence involves the sensitization of nicotinic signaling. Identification of receptor and/or neurophysiological mechanisms underlying this sensitization is important, as it could promote novel therapeutic strategies to reduce tobacco use. Using an approach involving photoactivatable nicotine, we previously demonstrated that chronic nicotine (cNIC) potently enhances nAChR function in dendrites of MHb neurons. However, whether cNIC modulates downstream components of the habenulo-interpeduncular (Hb-IP) circuit is unknown. In this study, cNIC-mediated changes to Hb-IP nAChR function were examined in mouse (male and female) brain slices using molecular, electrophysiological, and optical techniques. cNIC enhanced action potential firing and modified spike waveform characteristics in MHb neurons. Nicotine uncaging revealed nAChR functional enhancement by cNIC on proximal axonal membranes. Similarly, nAChR-driven glutamate release from MHb axons was enhanced by cNIC. In IPN, the target structure of MHb axons, neuronal morphology, and nAChR expression is complex, with stronger nAChR function in the rostral subnucleus [rostral IPN (IPR)]. As in MHb, cNIC induced strong upregulation of nAChR function in IPN neurons. This, coupled with cNIC-enhanced nicotine-stimulated glutamate release, was associated with stronger depolarization responses to brief (1 ms) nicotine uncaging adjacent to IPR neurons. Together, these results indicate that chronic exposure to nicotine dramatically alters nicotinic cholinergic signaling and cell excitability in Hb-IP circuits, a key pathway involved in nicotine dependence.SIGNIFICANCE STATEMENT This study uncovers several neuropharmacological alterations following chronic exposure to nicotine in a key brain circuit involved in nicotine dependence. These results suggest that smokers or regular users of electronic nicotine delivery systems (i.e., "e-cigarettes") likely undergo sensitization of cholinergic circuitry in the Hb-IP system. Reducing the activity of Hb-IP nAChRs, either volitionally during smoking cessation or inadvertently via receptor desensitization during nicotine intake, may be a key trigger of withdrawal in nicotine dependence. Escalation of nicotine intake in smokers, or tolerance, may involve stimulation of these sensitized cholinergic pathways. Smoking cessation therapeutics are only marginally effective, and by identifying cellular/receptor mechanisms of nicotine dependence, our results take a step toward improved therapeutic approaches for this disorder.

Electroacupuncture Pretreatment Attenuates Acute Lung Injury Through α7 Nicotinic Acetylcholine Receptor-Mediated Inhibition of HMGB1 Release in Rats After Cardiopulmonary Bypass.

Acute lung injury is a common complication after cardiopulmonary bypass (CPB). α7 Nicotinic acetylcholine receptors (α7nAChR) and α7nAChR-dependent cholinergic signaling are implicated in suppressing the release of high-mobility group box 1 (HMGB1) and reducing the inflammatory response. A previous study has shown the electroacupuncture (EA) pretreatment induces tolerance against lung injury. However, the role of EA in CPB is poorly understood. This study used EA and a rat model of CPB to determine whether EA was associated with CPB-induced lung injury. Rats were treated with EA at "Zusanli (ST36)" and "Feishu (BL13)" acupoints for 5 days before being subjected to CPB. Two hours post-CPB, samples of blood, bronchoalveolar lavage fluid (BALF), and lung tissues were processed for investigations. Our results showed that the expression of α7nAChR in lung tissue was significantly decreased after CPB. EA pretreatment prevented the reduction in the expression of α7nAChR, EA pretreatment reduced lung edema, inhibited inflammatory cytokines release in serum and lung as well as protein concentrations in BALF and HMGB1 release after CPB, and the beneficial effects were attenuated by α-BGT. Our study demonstrates that EA pretreatment plays a protective role in CPB-induced ALI, and inhibits HMGB1 release through α7nAChR activation in rats.

The prototoxin LYPD6B modulates heteromeric α3β4-containing nicotinic acetylcholine receptors, but not α7 homomers.

Prototoxins are a diverse family of membrane-tethered molecules expressed in the nervous system that modulate nicotinic cholinergic signaling, but their functions and specificity have yet to be completely explored. We tested the selectivity and efficacy of leukocyte antigen, PLAUR (plasminogen activator, urokinase receptor) domain-containing (LYPD)-6B on α3β4-, α3α5β4-, and α7-containing nicotinic acetylcholine receptors (nAChRs). To constrain stoichiometry, fusion proteins encoding concatemers of human α3, β4, and α5 (D and N variants) subunits were expressed in Xenopus laevis oocytes and tested with or without LYPD6B. We used the 2-electrode voltage-clamp method to quantify responses to acetylcholine (ACh): agonist sensitivity (EC50), maximal agonist-induced current (Imax), and time constant (τ) of desensitization. For β4-α3-α3-β4-α3 and β4-α3-β4-α3-α3, LYPD6B decreased EC50 from 631 to 79 μM, reduced Imax by at least 59%, and decreased τ. For β4-α3-α5D-β4-α3 and β4-α3-β4-α-α5D, LYPD6B decreased Imax by 63 and 32%, respectively. Thus, LYPD6B acted only on (α3)3(β4)2 and (α3)2(α5D)(β4)2 and did not affect the properties of (α3)2(β4)3, α7, or (α3)2(α5N)(β4)2 nAChRs. Therefore, LYPD6B acts as a mixed modulator that enhances the sensitivity of (α3)3(β4)2 nAChRs to ACh while reducing ACh-induced whole-cell currents. LYPD6B also negatively modulates α3β4 nAChRs that include the α5D common human variant, but not the N variant associated with nicotine dependence.

Muscarinic and nicotinic cholinergic receptor antagonists differentially mediate acquisition of fructose-conditioned flavor preference and quinine-conditioned flavor avoidance in rats

Rats display both conditioned flavor preference (CFP) for fructose, and conditioned flavor avoidance (CFA) following sweet adulteration with quinine. Previous pharmacological analyses revealed that fructose-CFP expression was significantly reduced by dopamine (DA) D1 or D2 antagonists, but not NMDA or opioid antagonists. Fructose-CFP acquisition was significantly reduced by DA D1, DA D2 or NMDA antagonists, but not opioid antagonists. Quinine-CFA acquisition was significantly enhanced and prolonged by DA D1, NMDA or opioid, but not DA D2 antagonists. Cholinergic interneurons and projections interact with DA systems in the nucleus accumbens and ventral tegmental area. Further, both muscarinic and nicotinic cholinergic receptor signaling have been implicated in sweet intake and development of food-related preferences. Therefore, the present study examined whether systemic administration of muscarinic (scopolamine: SCOP) or nicotinic (mecamylamine: MEC) cholinergic receptor antagonists mediated fructose-CFP expression, fructose-CFP acquisition and quinine-CFA acquisition. For fructose-CFP expression, rats were trained over 10 sessions with a CS+ flavor in 8% fructose and 0.2% saccharin and a CS− flavor in 0.2% saccharin. Two-bottle choice tests with CS+ and CS− flavors mixed in 0.2% saccharin occurred following vehicle, SCOP (0.1–10mg/kg) and MEC (1–8mg/kg). For fructose-CFP acquisition, six groups of rats received vehicle, SCOP (1 or 2.5mg/kg), MEC (4 or 6mg/kg) or a limited intake vehicle control 0.5h prior to 10 CS+ and CS− training sessions followed by six 2-bottle CS+ and CS− choice tests in 0.2% saccharin. For quinine-CFA acquisition, five groups of rats received vehicle, SCOP (1 or 2.5mg/kg) or MEC (4 or 6mg/kg) 0.5h prior to 8 one-bottle CS− (8% fructose+0.2% saccharin: FS) and CS+ (fructose+saccharin+quinine (0.030%: FSQ) training sessions followed by six 2-bottle CS− and CS+ choice tests in fructose–saccharin solutions. Fructose-CFP expression was significantly reduced by SCOP (2.5–10mg/kg: 65–68%) and MEC (4–8mg/kg: 67–73%) relative to vehicle (89–90%), that occurred only when antagonist doses reduced total saccharin intake but in which CS+ intake was still significantly higher than CS− intake. Fructose-CFP acquisition was eliminated by SCOP at doses of 1 (40–54%) and 2.5 (45–58%)mg/kg, and was accompanied by a failure to observe CS+ and CS− intake differences during testing relative to vehicle (85–92%) and limited control (74–88%) conditions. In contrast, MEC failed to alter fructose-CFP acquisition. Quinine-CFA acquisition was significantly enhanced and prolonged by MEC at 4 (18–24%) and 6 (11–13%) mg/kg relative to vehicle (34–48%). In contrast, SCOP failed to alter quinine-CFA acquisition. These data implicate the cholinergic receptor system in mediating acquisition (learning) of sugar-induced preferences and quinine-induced aversions with muscarinic receptor signaling controlling the former and nicotinic receptor signaling controlling the latter.

Cholinergic signaling in the gut: a novel mechanism of barrier protection through activation of enteric glia cells.

Enteric glia cells (EGCs) play an important role in maintaining proper intestinal barrier function. We have shown that vagal nerve stimulation (VNS) increases EGC activation, which is associated with better gut barrier integrity. Enteric neurons communicate with EGCs through nicotinic cholinergic signaling, which may represent a pathway by which VNS activates EGCs. This study sought to define further the mechanism by which VNS prevents intestinal barrier failure using an in vitro model. We hypothesized that a nicotinic cholinergic agonist would increase EGC activation, prevent intestinal nuclear factor kappa-B (NF-κB) activation, and result in better intestinal barrier function. Cultured EGCs were exposed to the nicotinic cholinergic agonist nicotine. Expression of glial fibrillary acidic protein (GFAP) was measured by immunoblot to determine changes in EGC activation. Caco-2 cells were grown to confluence and incubated alone or in co-culture with EGCs. Cells were then stimulated with Cytomix for 24 h in the presence or absence of nicotine, and barrier integrity was assessed by permeability to 4-kDa FITC-dextran. Changes in phosphorylated inhibitor of NF-κb (P-IκBα) and phosphorylated NF-κB (P-NF-κB) were assessed by immunoblot. Stimulation with nicotine resulted in EGC activation, as demonstrated by an increase in GFAP expression. Cytomix stimulation increased permeability in Caco-2 cells cultured alone or with EGCs. Treatment of stimulated Caco-2/EGC co-cultures with nicotine reduced permeability similar to control. Nicotine failed to prevent barrier permeability in Caco-2 cells alone. Co-culture of stimulated Caco-2 cells with nicotine-activated EGCs prevented Cytomix-induced increases in P-IκBα and P-NF-κB expression. A pharmacologic nicotinic cholinergic agonist increased EGC activation and improved intestinal epithelial barrier function in an in vitro model of intestinal injury. Nicotine-activated EGCs appear to modulate barrier function by preventing the activation of the NF-κB pathway. Therapies aimed at activating EGCs may have important clinical applications for improving intestinal barrier function after injury.

Modulation of nicotinic receptor channels by adrenergic stimulation in rat pinealocytes

Melatonin secretion from the pineal gland is triggered by norepinephrine released from sympathetic terminals at night. In contrast, cholinergic and parasympathetic inputs, by activating nicotinic cholinergic receptors (nAChR), have been suggested to counterbalance the noradrenergic input. Here we investigated whether adrenergic signaling regulates nAChR channels in rat pinealocytes. Acetylcholine or the selective nicotinic receptor agonist 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) activated large nAChR currents in whole cell patch-clamp experiments. Norepinephrine (NE) reduced the nAChR currents, an effect partially mimicked by a β-adrenergic receptor agonist, isoproterenol, and blocked by a β-adrenergic receptor antagonist, propranolol. Increasing intracellular cAMP levels using membrane-permeable 8-bromoadenosine (8-Br)-cAMP or 5,6-dichlorobenzimidazole riboside-3',5'-cyclic monophosphorothioate (cBIMPS) also reduced nAChR activity, mimicking the effects of NE and isoproterenol. Further, removal of ATP from the intracellular pipette solution blocked the reduction of nAChR currents, suggesting involvement of protein kinases. Indeed protein kinase A inhibitors, H-89 and Rp-cAMPS, blocked the modulation of nAChR by adrenergic stimulation. After the downmodulation by NE, nAChR channels mediated a smaller Ca(2+) influx and less membrane depolarization from the resting potential. Together these results suggest that NE released from sympathetic terminals at night attenuates nicotinic cholinergic signaling.

Nicotinic Cholinergic Signaling in Adipose Tissue and Pancreatic Islets Biology: Revisited Function and Therapeutic Perspectives

Nicotinic acetylcholine receptors (nAChRs) are membrane ligand-gated cation channels whose activation is triggered by the binding of the endogenous neurotransmitter acetylcholine or other biologic compounds including nicotine. Their roles in synaptic transmission in the central and peripheral nervous system as well as in the neuromuscular junction have been extensively studied. Recent implications of nAChRs in intracellular signaling and their detection in peripheral nonneural cells (including epithelial cells and immune cells) have renewed the interest for this class of ionotropic receptors. In the present review, we focus our attention on the potential use of nicotinic cholinergic signaling in the treatment of metabolic diseases (such as obesity and diabetes) in browsing functions of nAChRs in adipose tissue and pancreatic islet biology. In fact, different nAChR subunits can be detected in these metabolic tissues, as well as in immune cells interacting with them. Various rodent models of obesity and diabetes benefit from stimulation of the nicotinic cholinergic pathway, whereas mice deficient for some nAChRs, in particular the α7 nAChR subunit, harbor a worsened metabolic phenotype. In contrast to potential therapeutic applications in metabolic diseases, an overstimulation of this signaling pathway during the early stage of development (typically through nicotine exposure during fetal life) presents deleterious consequences on ontogeny and functionality of adipose tissue and the endocrine pancreas which persist throughout life.

Activation of α7‐containing nicotinic receptors on astrocytes triggers AMPA receptor recruitment to glutamatergic synapses

Astrocytes, an abundant form of glia, are known to promote and modulate synaptic signaling between neurons. They also express α7-containing nicotinic acetylcholine receptors (α7-nAChRs), but the functional relevance of these receptors is unknown. We show here that stimulation of α7-nAChRs on astrocytes releases components that induce hippocampal neurons to acquire more α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors post-synaptically at glutamatergic synapses. The increase is specific in that no change is seen in synaptic NMDA receptor clusters or other markers for glutamatergic synapses, or in markers for GABAergic synapses. Moreover, the increases in AMPA receptors on the neuron surface are accompanied by increases in the frequency of spontaneous miniature synaptic currents mediated by the receptors and increases in the ratio of evoked synaptic currents mediated by AMPA versus NMDA receptors. This suggests that stimulating α7-nAChRs on astrocytes can convert 'silent' glutamatergic synapses to functional status. Astrocyte-derived thrombospondin is necessary but not sufficient for the effect, while tumor necrosis factor-α is sufficient but not necessary. The results identify astrocyte α7-nAChRs as a novel pathway through which nicotinic cholinergic signaling can promote the development of glutamatergic networks, recruiting AMPA receptors to post-synaptic sites and rendering the synapses more functional. We find that activation of nicotinic receptors on astrocytes releases a component that specifically recruits AMPA receptors to glutamatergic synapses. The recruitment appears to occur preferentially at what may be 'silent synapses', that is, synapses that have all the components required for glutamatergic transmission (including NMDA receptors) but lack sufficient AMPA receptors to generate a response. The results are unexpected and open up new possibilities for mechanisms underlying network formation and synaptic plasticity.

Stimulation of nicotine reward and central cholinergic activity in Sprague-Dawley rats exposed perinatally to a fat-rich diet.

While clinical studies show maternal consumption of palatable fat-rich diets during pregnancy to negatively impact the children's behaviors and increase their vulnerability to drug abuse, the precise behavioral and neurochemical mechanisms mediating these phenomena have yet to be examined. The study examined in rats whether gestational exposure to a high-fat diet (HFD) can increase the offspring's propensity to use nicotine and whether disturbances in central nicotinic cholinergic signaling accompany this behavioral effect. Rat offspring exposed perinatally to a HFD or chow diet were characterized in terms of their nicotine self-administration behavior in a series of operant response experiments and the activity of acetylcholinesterase (AChE) and density of nicotinic ACh receptors (nAChRs) in different brain areas. Perinatal HFD compared to chow exposure increased nicotine-self administration behavior during fixed ratio and dose-response testing and caused an increase in breakpoint using progressive ratio testing, while nicotine seeking in response to nicotine prime-induced reinstatement was reduced. This behavioral change induced by the HFD was associated with a significant reduction in activity of AChE in the midbrain, hypothalamus, and striatum and increased density of β2-nAChRs in the ventral tegmental area and substantia nigra and of α7-nAChRs in the lateral and ventromedial hypothalamus. Perinatal exposure to a HFD increases the vulnerability of the offspring to excessive nicotine use by enhancing its reward potential, and these behavioral changes are accompanied by a stimulation of nicotinic cholinergic signaling in mesostriatal and hypothalamic brain areas important for reinforcement and consummatory behavior.

Glutamatergic Synapse Formation is Promoted by α7-Containing Nicotinic Acetylcholine Receptors

Glutamate is the primary excitatory transmitter in adult brain, acting through synapses on dendritic spines and shafts. Early in development, however, when glutamatergic synapses are only beginning to form, nicotinic cholinergic excitation is already widespread; it is mediated by acetylcholine activating nicotinic acetylcholine receptors (nAChRs) that generate waves of activity across brain regions. A major class of nAChRs contributing at this time is a species containing α7 subunits (α7-nAChRs). These receptors are highly permeable to calcium, influence a variety of calcium-dependent events, and are diversely distributed throughout the developing CNS. Here we show that α7-nAChRs unexpectedly promote formation of glutamatergic synapses during development. The dependence on α7-nAChRs becomes clear when comparing wild-type (WT) mice with mice constitutively lacking the α7-nAChR gene. Ultrastructural analysis, immunostaining, and patch-clamp recording all reveal synaptic deficits when α7-nAChR input is absent. Similarly, nicotinic activation of α7-nAChRs in WT organotypic culture, as well as cell culture, increases the number of glutamatergic synapses. RNA interference demonstrates that the α7-nAChRs must be expressed in the neuron being innervated for normal innervation to occur. Moreover, the deficits persist throughout the developmental period of major de novo synapse formation and are still fully apparent in the adult. GABAergic synapses, in contrast, are undiminished in number under such conditions. As a result, mice lacking α7-nAChRs have an altered balance in the excitatory/inhibitory input they receive. This ratio represents a fundamental feature of neural networks and shows for the first time that endogenous nicotinic cholinergic signaling plays a key role in network construction.

Molecular mechanism of interactions of the physiological anti-hypertensive peptide catestatin with the neuronal nicotinic acetylcholine receptor

Catestatin (CST), a chromogranin-A-derived peptide, is a potent endogenous inhibitor of the neuronal nicotinic acetylcholine receptor (nAChR). It exerts an anti-hypertensive effect by acting as a 'physiological brake' on transmitter release into the circulation. However, the mechanism of interaction of CST with nAChR is only partially understood. To unravel molecular interactions of the wild-type human CST (CST-WT) as well as its naturally occurring variants (CST-364S and CST-370L, which have Gly→Ser and Pro→Leu substitutions, respectively) with the human α3β4 nAChR, we generated a homology-modeled human α3β4 nAChR structure and solution structures of CST peptides. Docking and molecular dynamics simulations showed that ~90% of interacting residues were within 15 N-terminal residues of CST peptides. The rank order of binding affinity of these peptides with nAChR was: CST-370L>CST-WT>CST-364S; the extent of occlusion of the receptor pore by these peptides was also in the same order. In corroboration with computational predictions, circular dichroism analysis revealed significant differences in global structures of CST peptides (e.g. the order of α-helical content was: CST-370L>CST-WT>CST-364S). Consistently, CST peptides blocked various stages of nAChR signal transduction, such as nicotine- or acetylcholine-evoked inward current, rise in intracellular Ca(2+) and catecholamine secretion in or from neuron-differentiated PC12 cells, in the same rank order. Taken together, this study shows molecular interactions between human CST peptides and human α3β4 nAChR, and demonstrates that alterations in the CST secondary structure lead to the gain of potency for CST-370L and loss of potency for CST-364S. These findings have implications for understanding the nicotinic cholinergic signaling in humans.

Plenary: Plenary 1: Nicotinic synapse formation and the regulation of neural development

Nicotinic cholinergic signaling uses the transmitter acetylcholine (ACh) to activate ligand-gated ion channels termed nicotinic ACh receptors (nAChRs). It provides the main form of excitation to autonomic neurons in the periphery and exerts modulatory control over many non-nicotinic signaling pathways in the CNS. In both cases nicotinic signaling drives the early developmental conversion of GABAergic signaling from excitation to inhibition with important consequences for network function. Recent results indicate a more fundamental role for nicotinic signaling during development: it can accelerate glutamate synapse formation in the CNS and determine the location of synapses formed. Signaling through the two major classes of nAChRs exerts distinct but complementary and convergent effects to fashion functional glutamatergic contacts. Both in the autonomic nervous system and in the CNS, members of the PSD-95 family of PDZ-containing scaffold proteins shape the nature of nicotinic input a neuron receives. On autonomic neurons this includes transsynaptic indirect regulation by PDZ-proteins from the postsynaptic side to control separately synchronous and asynchronous ACh release from terminals contacting the neuron. Moreover, recent single-particle-tracking studies with Quantum Dots reveal that the PDZ-scaffold, along with cytoskeletal elements and the lipid environment, regulate the lateral mobility of nAChRs in ways that are both domain-specific and receptor subtype-dependent. These mechanisms may enable neurons to recruit appropriate receptors for location-specific tasks and thereby determine the kinds of nicotinic signaling that will occur.

Nicotinic Attenuation of Central Nervous System Inflammation and Autoimmunity

The expression of nicotinic acetylcholine receptors by neurons, microglia, and astrocytes suggests possibly diverse mechanisms by which natural nicotinic cholinergic signaling and exposure to nicotine could modulate immune responses within the CNS. In this study, we show that nicotine exposure significantly delays and attenuates inflammatory and autoimmune responses to myelin Ags in the mouse experimental autoimmune encephalomyelitis model. In the periphery, nicotine exposure inhibits the proliferation of autoreactive T cells and alters the cytokine profile of helper T cells. In the CNS, nicotine exposure selectively reduces numbers of CD11c(+) dendritic and CD11b(+) infiltrating monocytes and resident microglial cells and down-regulates the expression of MHC class II, CD80, and CD86 molecules on these cells. The results underscore roles of nicotinic acetylcholine receptors and nicotinic cholinergic signaling in inflammatory and immune responses and suggest novel therapeutic options for the treatment of inflammatory and autoimmune disorders, including those that affect the CNS.

Natural agents targeting the α7‐nicotinic‐receptor in NSCLC: A promising prospective in anti‐cancer drug development

Nicotinic acetylcholine receptors (nAChR) are expressed on normal bronchial epithelial and nonsmall cell lung cancer (NSCLC) cells and are involved in cell growth regulation. Nicotine induced cell proliferation. The purpose of this study was to determine if interruption of autocrine nicotinic cholinergic signaling might inhibit A549 NSCLC cell growth. For this purpose alpha-Cobratoxin (alpha-CbT), a high affinity alpha7-nAChR antagonist was studied. Cell growth decrease was evaluated by Clonogenic and MTT assays. Evidence of apoptosis was identified staining cell with Annexin-V/PI. Characterization of the basal NF-kappaB activity was done using the Trans-AM NF-kappaB assay colorimetric kit. "In vivo" antitumour activity was evaluated in orthotopically transplanted nude mice monitored by In vivo Imaging System technology. alpha-CbT caused concentration-dependent cell growth decrease, mitochondrial apoptosis caspases-9 and 3-dependent, but caspase-2 and p53-independent and down-regulation of basal high levels of activated NF-kappaB. alpha-CbT treatment determines a significant reduction of tumor growth in nude mice orthotopically engrafted with A549-luciferase cells (4.6% of living cells vs. 31% in untreated mice). No sign of toxicity was reported related to treatment. These findings suggest that alpha7-nAChR antagonists namely alpha-CbT may be useful adjuvant for treatment of NSCLC and potentially other cancers.

Acetylcholine is an autocrine or paracrine hormone synthesized and secreted by airway bronchial epithelial cells.

The role of acetylcholine (ACh) as a key neurotransmitter in the central and peripheral nervous system is well established. However, the role of ACh may be broader because ACh may also function as an autocrine or paracrine signaling molecule in a variety of nonneuronal tissues. To begin to establish ACh of nonneuronal origin as a paracrine hormone in lung, we have examined neonatal and adult monkey bronchial epithelium for the components involved in nicotinic cholinergic signaling. Using immunohistochemistry and RT-PCR, we have demonstrated in lung bronchial epithelial cells (BECs) expression of choline acetyltransferase, the vesicular ACh transporter, the choline high-affinity transporter, alpha7, alpha4, and beta2 nicotinic ACh receptor (nAChR) subunits, and the nAChR accessory protein lynx1. Confocal microscopy demonstrates that these factors are expressed in epithelial cells and are clearly distinct from neighboring nerve fibers. Confirmation of RNA identity has been confirmed by partial sequence analysis of PCR products and by cDNA cloning. Primary culture of BECs confirms the synthesis and secretion of ACh and the activity of cholinesterases. Thus, ACh meets all the criteria for an autocrine/paracrine hormone in lung bronchial epithelium. The nonneuronal cholinergic signaling pathway in lung provides a potentially important target for cholinergic drugs. This pathway may also explain some of the effects of nicotine on fetal development and also provides additional mechanisms by which smoking affects lung cancer growth and development.

Catecholamine Secretory Vesicle Stimulus-Transcription Coupling in Vivo: DEMONSTRATION BY A NOVEL TRANSGENIC PROMOTER/PHOTOPROTEIN REPORTER AND INHIBITION OF SECRETION AND TRANSCRIPTION BY THE CHROMOGRANIN A FRAGMENT CATESTATIN

Stimulation of chromaffin cell secretion in vitro triggers not only secretion but also resynthesis of just released catecholamines and chromogranin A, the precursor of the catecholamine release-inhibitory, nicotinic cholinergic antagonist peptide catestatin. Does stimulus-transcription coupling occur in vivo? And does catestatin antagonize secretion and transcription in vivo? To answer these questions, we employed a novel mouse strain harboring a chromogranin A promoter/firefly luciferase reporter transgene. Tissue-specific expression of the reporter was established by both luminescence and reverse transcription-PCR. Secretion and transcription in vivo were triggered by either direct nicotinic stimulation or vesicular transmitter depletion. Nicotinic blockade in vivo was attempted with either the classical antagonist chlorisondamine or the novel antagonist catestatin. Luciferase reporter expression was exquisitely sensitive over a large dynamic range, was specific for the transgenic animals, and paralleled typical neuroendocrine distribution of endogenous chromogranin A. Adrenal ontogeny revealed a rise of embryonic transgene expression until embryonal day 18, with an abrupt postnatal decline. Direct nicotinic stimulation of chromaffin cells caused catecholamine release and transgene transcription, each of which was nearly completely blocked by chlorisondamine. Similar adrenal results were obtained during vesicular catecholamine depletion. Both secretion and transcription were substantially blocked in the adrenal gland by catestatin. In brain and sympathetic nerve, stimulation of transcription was more modest, and reserpine responses were only incompletely blocked by chlorisondamine or catestatin, perhaps because of limited blood-brain barrier penetration by these cationic antagonists. Thus, nicotinic cholinergic stimulus-transcription coupling occurs in vivo and can be provoked either directly or indirectly (by vesicular transmitter depletion). Such coupling triggers the biosynthesis of chromogranin A, the precursor of catestatin. Catestatin itself blocks stimulation of both secretion and transcription in vivo. Thus, chromogranin A and its catestatin fragment may lie at the nexus of nicotinic cholinergic signaling in vivo.