Calcium modulation and high calcium permeability of neuronal nicotinic acetylcholine receptors.

Two properties were found to distinguish neuronal from muscle nicotinic acetylcholine receptors (nAChRs). First, neuronal nAChRs have a greater Ca2+ permeability. The high Ca2+ flux through neuronal nAChRs activates a Ca(2+)-dependent Cl- conductance, and the Ca2+ to Cs+ permeability ratio (PCa/PCs) is 7 times greater for neuronal than for muscle nAChRs. A second difference between the receptor types is that neuronal nAChRs are potently modulated by physiological levels of external Ca2+. Neuronal nAChR currents are enhanced by external Ca2+ in a dose-dependent manner. The results indicate that changes in extracellular Ca2+ modulate neuronal nAChRs and may modulate cholinergic synapses in the CNS. Also, activation of neuronal nAChRs produces a significant influx of Ca2+ that could be an important intracellular signal.     

Activation of Nicotinic Acetylcholine Receptors Augments Calcium Channel-mediated Exocytosis in Rat Pheochromocytoma (PC12) Cells

The functional effect of activating Ca²⁺-permeable neuronal nicotinic acetylcholine receptors (nAChRs) on vesicle secretion was studied in PC12 cells. Single cells were patch-clamped in the whole-cell configuration and stimulated with either brief pulses of nicotine to activate the Ca²⁺-permeable nAChRs or with voltage steps to activate voltage-dependent Ca²⁺ channels. Membrane capacitance was used as a measure of vesicle secretion. Activation of nAChRs by nicotine application to cells voltage clamped at −80 mV evoked secretion. This secretion was completely abolished by nicotinic antagonists. When the cells were voltage clamped at +20 mV in the presence of Cd²⁺ to block voltage-activated Ca²⁺ channels, nicotine elicited a small amount of secretion. Most interestingly, when the nAChRs were activated coincidentally with voltage-dependent Ca²⁺ channels, secretion was augmented approximately twofold over the secretion elicited with voltage-dependent Ca²⁺ channels alone. Our data suggest that Ca²⁺ influx via nAChRs affects Ca²⁺-dependent cellular functions, including vesicle secretion. In addition to the secretion evoked by nAChR activation at hyperpolarized potentials, we demonstrate that even at depolarized potentials, nAChRs provide an important Ca²⁺ entry pathway underlying Ca²⁺-dependent cellular processes such as exocytosis.     

Calcium Dependency of Muscarinic and Nicotinic Agonist-Induced ATP and Catecholamine Secretion from Porcine Adrenal Chromaffin Cells

The secretion of catecholamines and ATP induced by cholinergic agonists and its dependence on extracellular Ca2+ were studied in cultured porcine adrenal chromaffin cells. Both nicotine and methacholine (a selective muscarinic agonist) induced secretion and increases in cytosolic free Ca2+ concentration ([Ca2+]in), although the activation of nicotinic receptors produced responses that were larger than those produced by activation of muscarinic receptors. The secretion and the increase in [Ca2+]in evoked by nicotine were completely dependent on extracellular Ca2+ and were blocked by prior depolarization of the cells with high extracellular K+ levels. In addition, nicotine induced significant 45Ca2+ influx. In contrast, the secretion and the increase in [Ca2+]in evoked by methacholine were partially dependent on extracellular Ca2+; methacholine also induced 45Ca2+ influx. Prior depolarization of the cells with high extracellular K+ levels did not block methacholine-induced secretion. In general, nicotinic responses were mediated by Ca2+ influx through voltage-dependent pathways. In contrast, muscarinic responses were dependent on both Ca2+ influx through an unknown mechanism that could not be inactivated by high K+ concentration-induced depolarization and presumably also intracellular Ca2+ mobilization.     

Nicotine potentiates the neurogenic contractile response of rabbit bladder tissue via nicotinic acetylcholine receptors: nitric oxide and prostaglandins have no role in this process

Nicotine, a nicotinic acetylcholine receptors (nAChRs) agonist, has a role in modulation of the neurotransmitter release following nerve stimulation in both the central and peripheral nervous systems. The aim of this study was to determine whether electrical field stimulation (EFS)-evoked contractions are altered in rabbit bladder in the presence of nicotine and, if an alteration occurs, to investigate the effects of nitric oxide and prostaglandins on nicotine-induced alternation in isolated rabbit bladder. EFS-evoked contractile responses from rabbit bladder obtained were recorded with isometric force displacement transducers. Nicotine was added to preparations at various concentrations. The effects of hexamethonium, cadmium (Cd(2+)), indomethacin and N-nitro-L-arginine methyl ester (L-NAME) were tested on the EFS-evoked contractions in the presence of nicotine. Nicotine led to a dose-dependent increase in the amplitude of the EFS-evoked contractile responses. Cd(2+) and hexamethonium inhibited the nicotine-induced increase in EFS-evoked responses, whereas indomethacin and L-NAME had no effect. In conclusion, nicotine increased the EFS-evoked contractile responses possibly by facilitating release of neurotransmitters from nerve terminals by a mechanism dependent on the influx of Ca(2+) from voltage-gated Ca(2+) channels (VGCCs) via activation of nAChRs in isolated rabbit bladder. Nitric oxide and prostaglandins do not have a physiological role in the regulation of neurotransmitter release.     

nAChR-mediated calcium responses and plasticity in Drosophila Kenyon cells

In Drosophila, nicotinic acetylcholine receptors (nAChRs) mediate fast excitatory synaptic transmission in mushroom body Kenyon cells, a neuronal population involved in generation of complex behaviors, including responses to drugs of abuse. To determine whether activation of nAChRs can induce cellular changes that contribute to functional plasticity in these neurons, we examined nicotine-evoked responses in cells cultured from brains of late stage OK107-GAL4 pupae. Kenyon cells can be identified by expression of green fluorescent protein (GFP+). Nicotine activates alpha-bungarotoxin-sensitive nAChRs, causing a rapid increase in intracellular calcium levels in over 95% of the Kenyon cells. The nicotine-evoked calcium increase has a voltage-gated calcium channel (VGCC) dependent component and a VGCC-independent component that involves calcium influx directly through nAChRs. Thapsigargin treatment reduces the nicotine response consistent with amplification by calcium release from intracellular stores. The response to nicotine is experience-dependent: a short conditioning pulse of nicotine causes a transient 50% reduction in the magnitude of the response to a test pulse of nicotine when the interpulse interval is 4 h. This cellular plasticity is dependent on activation of the VGCC-component of the nicotine response and on cAMP-signaling, but not on protein synthesis. These data demonstrate that activation of nAChRs induces a calcium-dependent plasticity in Kenyon cells that could contribute to adult behaviors involving information processing in the mushroom bodies including responses to nicotine.     

Pretreatment of the cockroach cercal afferent/giant interneuron synapses with nicotinoids and neonicotinoids differently affects acetylcholine and nicotine-induced ganglionic depolarizations

We have recently demonstrated that neonicotinoid insecticides were able to act as agonists of postsynaptic nicotinic acetylcholine receptors (nAChRs) expressed at the synapse between the cercal nerve XI and the giant interneurons, in the sixth abdominal ganglion. In this work, we demonstrated that nicotinoids such as nornicotine acted as an agonist of nicotinic acetylcholine receptors expressed at cercal afferent/giant interneurons while cotinine was a poor agonist. Indeed, nornicotine induced a ganglionic depolarization which was blocked by the nicotinic antagonist mecamylamine. In addition, we found that pretreatment of the sixth abdominal ganglion with 1 and 10 μM nornicotine and cotinine had no significant effect on acetylcholine and nicotine-induced depolarization. But pretreatment with 1 and 10 μM acetamiprid and imidacloprid had a strong effect. 1 and 10 μM acetamiprid completely blocked acetylcholine-induced depolarization, whereas imidacloprid had a partial effect. The present work therefore suggests, in agreement with previous studies, that nornicotine and cotinine bind to distinct cockroach postsynaptic nAChRs, whereas acetamiprid and imidacloprid have competitive effects with acetylcholine and nicotine on ganglionic depolarization.     

Comparison of quantitative calcium flux through NMDA, ATP, and ACh receptor channels.

NMDA receptors, ATP receptors, and nicotinic ACh receptors respond to agonist by undergoing conformational changes that open weakly selective cationic channels that are permeable to calcium. We determined the fraction of the current carried by calcium by simultaneously measuring membrane current using whole-cell patch-clamp techniques and intracellular Ca2+ using the fluorescent indicator Fura-2. The Fura-2 response to free Ca2+ was calibrated individually for each cell. Two different calibration methods are compared: one uses voltage-activated Ca2+ channels, and the other uses the same ligand-gated channels that are being tested but in a pure Ca2+ solution. The two methods give quantitatively different results. The method using pure Ca2+ currents through ligand-gated channels calibrates the Fura-2 signal through the same influx pathway that generates the test response, thus controlling for the distribution of channels and ensuring a similar interaction between the incoming Ca2+ and Fura-2. In a physiologic solution containing 2.5 mM Ca2+ at a holding potential of -50 mV, the percentage of inward current carried by Ca2+ through NMDA receptors in hippocampal neurons is 12.4%. By comparison, in sympathetic neurons the percentage of current carried by Ca2+ through neuronal nAChRs is 4.7%, and through ATP-activated purinergic receptors it is 6.5%. These percentages can be used to estimate the amount of Ca2+ entry through these receptors during synaptic activation, but care must be exercised in considering the many subtypes of each receptor.     

Calcium influx through nicotinic receptor in rat central neurons: its relevance to cellular regulation.

The Ca2+ permeability of a nicotinic acetylcholine receptor (nAChR) in the rat CNS was determined using both current and fluorescence measurements on medial habenula neurons. The elementary slope conductance of the nAChR channel was 11 pS in pure external Ca2+ (100 mM) and 42 pS in standard solution. Ca2+ influx through nAChRs resulted in the rise of cytosolic Ca2+ concentration ([Ca2+]i) to the micromolar range. This increase was maximal under voltage conditions (below -50 mV) in which Ca2+ influx through voltage-activated channels was minimal. Ca2+ influx through nAChRs directly activated a Ca(2+)-dependent Cl- conductance. In addition, it caused a decrease in the GABAA response that outlasted the rise in [Ca2+]i. These results underscore the physiological significance of Ca2+ influx through nAChR channel in the CNS.     

Endogenous Cholinergic Inputs and Local Circuit Mechanisms Govern the Phasic Mesolimbic Dopamine Response to Nicotine

Nicotine exerts its reinforcing action by stimulating nicotinic acetylcholine receptors (nAChRs) and boosting dopamine (DA) output from the ventral tegmental area (VTA). Recent data have led to a debate about the principal pathway of nicotine action: direct stimulation of the DAergic cells through nAChR activation, or disinhibition mediated through desensitization of nAChRs on GABAergic interneurons. We use a computational model of the VTA circuitry and nAChR function to shed light on this issue. Our model illustrates that the α4β2-containing nAChRs either on DA or GABA cells can mediate the acute effects of nicotine. We account for in vitro as well as in vivo data, and predict the conditions necessary for either direct stimulation or disinhibition to be at the origin of DA activity increases. We propose key experiments to disentangle the contribution of both mechanisms. We show that the rate of endogenous acetylcholine input crucially determines the evoked DA response for both mechanisms. Together our results delineate the mechanisms by which the VTA mediates the acute rewarding properties of nicotine and suggest an acetylcholine dependence hypothesis for nicotine reinforcement.     

Role of acetylcholine receptors in proliferation and differentiation of P19 embryonal carcinoma cells

Coordinated proliferation and differentiation of progenitor cells is the base for production of appropriate numbers of neurons and glia during neuronal development in order to establish normal brain functions. We have used murine embryonal carcinoma P19 cells as an in vitro model for early differentiation to study participation of nicotinic (nAChR) and muscarinic acetylcholine (mAChR) receptors in the proliferation of neural progenitor cells and their differentiation to neurons. We have previously shown that functional nicotinic acetylcholine receptors (nAChRs) already expressed in embryonic cells mediate elevations in cytosolic free calcium concentration ([Ca2+]i) via calcium influx through nAChR channels whereas intracellular stores contribute to nAChR- and mAChR-mediated calcium fluxes in differentiated cells [Resende et al., Cell Calcium 43 (2008) 107-121]. In the present study, we have demonstrated that nicotine provoked inhibition of proliferation in embryonic cells as determined by BrdU labeling. However, in neural progenitor cells nicotine stimulated proliferation which was reversed in the presence of inhibitors of calcium mobilization from intracellular stores, indicating that liberation of intracellular calcium contributed to this proliferation induction. Muscarine induced proliferation stimulation in progenitor cells by activation of Galphaq/11-coupled M1, M3 and M5 receptors and intracellular calcium stores, whereas Galphai/o-protein coupled M2 receptor activity mediated neuronal differentiation.     

The lymphocytic cholinergic system and its contribution to the regulation of immune activity

Lymphocytes express most of the cholinergic components found in the nervous system, including acetylcholine (ACh), choline acetyltransferase (ChAT), high affinity choline transporter, muscarinic and nicotinic ACh receptors (mAChRs and nAChRs, respectively), and acetylcholinesterase. Stimulation of T and B cells with ACh or another mAChR agonist elicits intracellular Ca2+ signaling, up-regulation of c-fos expression, increased nitric oxide synthesis and IL-2-induced signal transduction, probably via M3 and M5 mAChR-mediated pathways. Acute stimulation of nAChRs with ACh or nicotine causes rapid and transient Ca2+ signaling in T and B cells, probably via alpha7 nAChR subunit-mediated pathways. Chronic nicotine stimulation, by contrast, down-regulates nAChR expression and suppresses T cell activity. Activation of T cells with phytohemagglutinin or antibodies against cell surface molecules enhances lymphocytic cholinergic transmission by activating expression of ChAT and M5 mAChR, which is suggestive of local cholinergic regulation of immune system activity. This idea is supported by the facts that lymphocytic cholinergic activity reflects well the changes in immune system function seen in animal models of immune deficiency and immune acceleration. Collectively, these data provide a compelling picture in which lymphocytes constitute a cholinergic system that is independent of cholinergic nerves, and which is involved in the regulation of immune function.     

Nicotine-induced Ca<Superscript>2+</Superscript>-myristoyl Switch of Neuronal Ca<Superscript>2+</Superscript> Sensor VILIP-1 in Hippocampal Neurons: A Possible Crosstalk Mechanism for Nicotinic Receptors

Visinin-like protein (VILIP-1) belongs to the neuronal Ca²⁺ sensor family of EF-hand Ca²⁺-binding proteins that regulate a variety of Ca²⁺-dependent signal transduction processes in neurons. It is an interaction partner of α4β2 nicotinic acetylcholine receptor (nAChR) and increases surface expression level and agonist sensitivity of the receptor in oocytes. Nicotine stimulation of nicotinic receptors has been reported to lead to an increase in intracellular Ca²⁺ concentration by Ca²⁺-permeable nAChRs, which in turn might lead to activation of VILIP-1, by a mechanism described as the Ca²⁺-myristoyl switch. It has been postulated that this will lead to co-localization of the proteins at cell membranes, where VILIP-1 can influence functional activity of α4-containing nAChRs. In order to test this hypothesis we have investigated whether a nicotine-induced and reversible Ca²⁺-myristoyl switch of VILIP-1 exists in primary hippocampal neurons and whether pharmacological agents, such as antagonist specific for distinct nAChRs, can interfere with the Ca²⁺-dependent membrane localization of VILIP-1. Here we report, that only α7- but not α4-containing nAChRs are able to elicit a Ca²⁺-dependent and reversible membrane-translocation of VILIP-1 in interneurons as revealed by employing the specific receptor antagonists dihydro-beta-erythroidine and methylallylaconitine. The nAChRs are associated with processes of synaptic plasticity in hippocampal neurons and they have been implicated in the pathology of CNS disorders, including Alzheimer’s disease and schizophrenia. VILIP-1 might provide a novel functional crosstalk between α4- and α7-containing nAChRs.     

Cellular and synaptic mechanisms of nicotine addiction

The tragic health effects of nicotine addiction highlight the importance of investigating the cellular mechanisms of this complex behavioral phenomenon. The chain of cause and effect of nicotine addiction starts with the interaction of this tobacco alkaloid with nicotinic acetylcholine receptors (nAChRs). This interaction leads to activation of reward centers in the CNS, including the mesoaccumbens DA system, which ultimately leads to behavioral reinforcement and addiction. Recent findings from a number of laboratories have provided new insights into the biologic processes that contribute to nicotine self-administration. Examination of the nAChR subtypes expressed within the reward centers has identified potential roles for these receptors in normal physiology, as well as the effects of nicotine exposure. The high nicotine sensitivity of some nAChR subtypes leads to rapid activation followed in many cases by rapid desensitization. Assessing the relative importance of these molecular phenomena in the behavioral effects of nicotine presents an exciting challenge for future research efforts.     

Methadone increases intracellular calcium in SH-SY5Y and SH-EP1-halpha7 cells by activating neuronal nicotinic acetylcholine receptors

(-)-Methadone acts as an agonist at opioid receptors. Both (+)- and (-)-enantiomers of methadone have been suggested to be potent non-competitive antagonists of alpha3beta4 neuronal nicotinic acetylcholine receptors (nAChRs). In the present study, we have examined interactions of methadone with nAChRs by using receptor binding assays, patch-clamp recording and calcium fluorometry imaging with SH-SY5Y cells naturally expressing alpha7 and alpha3* nAChR subtypes and SH-EP1-halpha7 cells heterologously expressing human alpha7 nAChRs. Methadone potently inhibited binding of [3H]methyllycaconitine to alpha7 nAChRs and that of [3H]epibatidine to alpha3* nAChRs. Methadone pretreatment induced up-regulation of epibatidine binding sites in SH-SY5Y cells. Using whole-cell patch-clamp recording, both isomers of methadone activated cation currents via mecamylamine-sensitive nAChRs in SH-SY5Y cells. Nicotine and both (+)- and (-)-methadone evoked increases in [Ca2+]i in both fluo-3AM loaded cell lines, and these effects were blocked by mecamylamine and by the alpha7 selective antagonist methyllycaconitine, suggesting effects of methadone as alpha7-nAChR agonist. Sensitivity of sustained nicotine and methadone effects to blockade by CdCl2, ryanodine and xestospongin-c implicates voltage-operated Ca2+ channels and intracellular Ca2+ stores as downstream modulators of elevated [Ca2+]i. Collectively, our results suggest that methadone engages in complex and potentially pharmacologically significant interactions with nAChRs.     

Long-term potentiation of excitatory inputs to brain reward areas by nicotine

Nicotine reinforces smoking behavior by activating nicotinic acetylcholine receptors (nAChRs) in the midbrain dopaminergic (DA) reward centers, including the ventral tegmental area (VTA). Although nicotine induces prolonged excitation of the VTA in vivo, the nAChRs on the DA neurons desensitize in seconds. Here, we show that activation of nAChRs on presynaptic terminals in the VTA enhances glutamatergic inputs to DA neurons. Under conditions where the released glutamate can activate NMDA receptors, long-term potentiation (LTP) of the excitatory inputs is induced. Both the short- and the long-term effects of nicotine required activation of presynaptic alpha7 subunit-containing nAChRs. These results can explain the long-term excitation of brain reward areas induced by a brief nicotine exposure. They also show that nicotine alters synaptic function through mechanisms that are linked to learning and memory.     

Involvement of alpha7 nicotinic acetylcholine receptors in activation of tyrosine hydroxylase and dopamine beta-hydroxylase gene expression in PC12 cells

Nicotine treatment increases intracellular free Ca(2+) concentration [Ca(2+)](i), stimulates catecholamine release, and elevates gene expression for the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH). However, the type of nicotinic acetylcholine receptors (nAChRs) mediating these events is unclear. The nAChR receptor antagonists alpha-bungarotoxin (alphaBTX) and methyllycaconitine greatly reduced the nicotine-triggered initial transient rise in [Ca(2+)](i) and prevented the second prolonged elevation of [Ca(2+)](i), suggesting the involvement of alpha7 nAChRs. Two specific alpha7 nicotinic agonists, 3-(2,4-dimethoxybenzilidene)anabaseine (DMXB) and E, E-3-(cinnamylidene)anabaseine (3-CA), were found to elicit a small, delayed increase in [Ca(2+)](i) with kinetics and magnitude similar to the second elevation observed with nicotine. This increase was inhibited by the inositol trisphosphate receptor antagonist xestospongin C. Exposure to 3-CA or DMXB for 6 or 24 h elevated TH and DBH mRNA levels two- to fourfold over control levels. These agonists were more effective than nicotine alone in increasing TH and DBH gene expression and significantly elevated [Ca(2+)](i) for up to 6 h. The increase in [Ca(2+)](i) or the elevation in TH mRNA by 3-CA was completely inhibited by alphaBTX. This study, for the first time, implicates stimulation of alpha7 nAChRs in the activation of TH and DBH gene expression.     

Muscarinic receptors couple to modulation of nicotinic ACh receptor desensitization in myenteric neurons

Signaling mechanisms coupled to activation of different neurotransmitter receptors interact in the enteric nervous system. ACh excites myenteric neurons by activating nicotinic ACh receptors (nAChRs) and muscarinic receptors expressed by the same neurons. These studies tested the hypothesis that muscarinic receptor activation alters the functional properties of nAChRs in guinea pig small intestinal myenteric neurons maintained in primary culture. Whole cell patch-clamp techniques were used to measure inward currents caused by ACh (1 mM) or nicotine (1 mM). Currents caused by ACh and nicotine were blocked by hexamethonium (100 microM) and showed complete cross desensitization. The rate and extent of nAChR desensitization was greater when recordings were obtained with ATP/GTP-containing compared with ATP/GTP-free pipette solutions. These data suggest that ATP/GTP-dependent mechanisms increase nAChR desensitization. The muscarinic receptor antagonist scopolamine (1 microM) decreased desensitization caused by ACh but not by nicotine, which does not activate muscarinic receptors. Phorbol 12,13-dibutyrate (10-100 nM), an activator of protein kinase C (PKC), but not 4-alpha-phorbol 12-myristate 13-acetate (a PKC inactive phorbol ester), increased nAChR desensitization caused by ACh and nicotine. Forskolin (1 microM), an activator of adenylate cyclase, increased nAChR desensitization, but this effect was mimicked by dideoxyforskolin, an adenylate cyclase inactive forskolin analog. These data indicate that simultaneous activation of nAChRs and muscarinic receptors increases nAChR desensitization. This effect may involve activation of a PKC-dependent pathway. These data also suggest that nAChRs and muscarinic receptors are coupled functionally through an intracellular signaling pathway in myenteric neurons.