Contribution of Monoamine Oxidase Inhibition to Tobacco Dependence: A Review of the Evidence.

BACKGROUND: There is a hypothesis that substances present in, or derived from, tobacco smoke inhibit monoamine oxidase (MAO) in the brains of smokers, reducing the degradation of catecholamine neurotransmitters involved in central reward pathways and acting synergistically with nicotine to increase its addictive effects. OBJECTIVE: The objective of this review was to evaluate the evidence for a role of MAO inhibition by tobacco-derived substances in tobacco dependence. INVESTIGATIONAL PLAN: Relevant studies on the effects of tobacco use on MAO levels or activity in humans were identified by electronic searches. RESULTS: The identified data show a clear association between smoking and lower density of MAO-A and MAO-B binding sites in the brains of smokers and strong evidence that MAO is inhibited by a substance or substances in, or derived from, tobacco smoke. There was little evidence to support the hypothesis that low MAO levels/activity is a predictive factor for tobacco use. Substances that inhibit MAO in in vitro assays have been isolated from tobacco leaves and tobacco smoke; however, no single substance has been shown to be absorbed from tobacco smoke and to inhibit MAO in the brains of human smokers. Nevertheless, it is possible that MAO inhibition in smokers could result from additive or synergistic effects of several tobacco-derived substances. MAO inhibition potentiates the reinforcing effects of intravenous nicotine in rodents; however, no data were identified to support the hypothesis that MAO inhibitors in or derived from tobacco or tobacco additives affect tobacco dependence in human smokers. IMPLICATIONS: This comprehensive review describes the available evidence for the role of MAO inhibition in tobacco dependence and points the way for further research in this field. In view of the large number of MAO inhibitors identified in tobacco and tobacco smoke, identification of the putative inhibitors responsible for the lower level/activity of MAO in smokers may be impractical. Future studies must address whether the lower level/activity of MAO observed in smokers is also seen in users of other tobacco products and if this change is implicated in their dependence-inducing effects.

Hydrophobic residues at position 10 of α-conotoxin PnIA influence subtype selectivity between α7 and α3ß2 neuronal nicotinic acetylcholine receptors.

Neuronal nicotinic acetylcholine receptors (nAChRs) are a diverse class of ligand-gated ion channels involved in neurological conditions such as neuropathic pain and Alzheimer's disease. α-Conotoxin [A10L]PnIA is a potent and selective antagonist of the mammalian α7 nAChR with a key binding interaction at position 10. We now describe a molecular analysis of the receptor-ligand interactions that determine the role of position 10 in determining potency and selectivity for the α7 and α3ß2 nAChR subtypes. Using electrophysiological and radioligand binding methods on a suite of [A10L]PnIA analogs we observed that hydrophobic residues in position 10 maintained potency at both subtypes whereas charged or polar residues abolished α7 binding. Molecular docking revealed dominant hydrophobic interactions with several α7 and α3ß2 receptor residues via a hydrophobic funnel. Incorporation of norleucine (Nle) caused the largest (8-fold) increase in affinity for the α7 subtype (Ki=44nM) though selectivity reverted to α3ß2 (IC50=0.7nM). It appears that the placement of a single methyl group determines selectivity between α7 and α3ß2 nAChRs via different molecular determinants.

An automated system for intracellular and intranuclear injection.

The Xenopus oocyte expression system has played an important role in the study of cellular proteins, particularly in the field of membrane physiology; expression of transporters and ion channels has significantly advanced our knowledge of these membrane proteins and the rapid and easy expression of mutants has been crucial in many structure-function studies. Xenopus oocytes are an expression system in many ligand-binding assays and in functional screening for ion channel modulators. Several commercially available automated technologies use this system, generating a demand for large numbers of oocytes injected with ion channel genes. Injection of oocytes with genetic material is generally carried out manually. Here we describe an automated system capable of injecting up to 600 oocytes per hour. Oocytes are contained in microplates with conical wells, a simple calibration procedure by the operator is required and pipette filling and oocyte injection are carried out automatically. Following intracellular injection of mRNA coding for ligand-gated ion channels close to 100% of oocytes tested positive for expression, and intranuclear injection of cDNA gave a rate of expression >50%. Moreover, we demonstrate that this method can also be successfully applied to inject zebrafish embryos and could be extended to other cell types.

Bacterial expression, NMR, and electrophysiology analysis of chimeric short/long-chain alpha-neurotoxins acting on neuronal nicotinic receptors.

Different snake venom neurotoxins block distinct subtypes of nicotinic acetylcholine receptors (nAChR). Short-chain alpha-neurotoxins preferentially inhibit muscle-type nAChRs, whereas long-chain alpha-neurotoxins block both muscle-type and alpha7 homooligomeric neuronal nAChRs. An additional disulfide in the central loop of alpha- and kappa-neurotoxins is essential for their action on the alpha7 and alpha3beta2 nAChRs, respectively. Design of novel toxins may help to better understand their subtype specificity. To address this problem, two chimeric toxins were produced by bacterial expression, a short-chain neurotoxin II Naja oxiana with the grafted disulfide-containing loop from long-chain neurotoxin I from N. oxiana, while a second chimera contained an additional A29K mutation, the most pronounced difference in the central loop tip between long-chain alpha-neurotoxins and kappa-neurotoxins. The correct folding and structural stability for both chimeras were shown by (1)H and (1)H-(15)N NMR spectroscopy. Electrophysiology experiments on the nAChRs expressed in Xenopus oocytes revealed that the first chimera and neurotoxin I blockalpha7 nAChRs with similar potency (IC(50) 6.1 and 34 nM, respectively). Therefore, the disulfide-confined loop endows neurotoxin II with full activity of long-chain alpha-neurotoxin and the C-terminal tail in neurotoxin I is not essential for binding. The A29K mutation of the chimera considerably diminished the affinity for alpha7 nAChR (IC(50) 126 nM) but did not convey activity at alpha3beta2 nAChRs. Docking of both chimeras toalpha7 andalpha3beta2 nAChRs was possible, but complexes with the latter were not stable at molecular dynamics simulations. Apparently, some other residues and dimeric organization of kappa-neurotoxins underlie their selectivity for alpha3beta2 nAChRs.

Partial agonists as therapeutic agents at neuronal nicotinic acetylcholine receptors.

Improved understanding of how brain function is altered in neurodegenerative disease states, pain and conditions, such as schizophrenia and attention deficit disorder, has highlighted the role of nicotinic acetylcholine receptors (nAChRs) in these conditions and identified them as promising therapeutic targets. nAChRs are widely expressed throughout the peripheral and central nervous system, and this widespread nature underlines the need for new ligands with different selectivities and pharmacological profiles if we are to avoid the adverse side effects associated with many of the nAChR modulators currently identified. Partial agonists have the unique property of being able to act both as agonists or antagonists depending on the concentration of endogenous neurotransmitter. Moreover, the agonist action of partial agonists has a 'ceiling' effect, giving them a large safety margin and making them an attractive proposition for therapeutic molecules. Partial agonists of nAChRs are currently being developed as a nicotine replacement therapy for smoking cessation and for the treatment of a number of neurological diseases associated with a loss of cholinergic function. This commentary will discuss the pharmacological properties of partial agonists and review recent research developments in the field of partial agonists acting at nicotinic receptors.

Novel approaches to pain relief using venom-derived peptides.

More than one and a half billion people worldwide suffer from moderate to severe chronic pain, the National Institute of Health estimates that pain costs health services approximately 100 billion US Dollar annually. Existing drugs for the treatment of pain are often associated with serious side effects and rapid development of tolerance, thus, there is a need for new, more selective, molecules. Ion channels play an important role throughout the pain response, from nociception via transient receptor potential (TRP) channels or ATP-sensitive receptors, propagation of action potentials by voltage-sensitive sodium and potassium channels to control of the release of neurotransmitters from presynaptic terminals of dorsal root ganglion (DRG) neurones in the dorsal horn by voltage-gated calcium channels. Venoms are complex mixtures of bioactive molecules that have evolved for prey capture and defence, many of these molecules have a high selectivity for physiological processes, including modulation of ion channel function, which has not been matched by man made molecules. Thus, venoms represent an extensive source of molecules for the development of therapeutic agents. This report will review the key ion channel targets for pain relief, and venom-derived molecules and their analogues acting at these targets. We will concentrate particularly on peptides isolated from Conus venom as these represent one of the best-characterised toxin families. Our current knowledge of the molecular pharmacology of these toxin molecules will be reviewed and problems associated with using peptides as therapeutics will be discussed, along with strategies to overcome these.

Allosteric modulation of ligand-gated ion channels.

Ligand-gated ion channels (LGICs) are cell surface proteins that play an important role in fast synaptic transmission and in the modulation of cellular activity. Due to their intrinsic properties, LGICs respond to neurotransmitters and other effectors (e.g. pH) and transduce the binding of a ligand into an electrical current on a microsecond timescale. Following activation, LGICs open allowing an ion flux across the cell membrane. Depending upon the charge and concentration of ions, the flux can cause a depolarization or hyperpolarization, thus modulating excitability of the cell. While our understanding of LGICs has significantly progressed during the past decade, many properties of these proteins are still poorly understood, in particular their modulation by allosteric effectors. LGICs are often thought as a simple on-off switches. However, a closer look at these receptors reveals a complex behavior and a wide repertoire of subtle modulation by intrinsic and extrinsic factors. From a physiological point of view, this modulation can be seen as an additional level of complexity in the cell signaling process. Here we review the allosteric modulation of LGICs in light of the latest findings and discuss the suitability of this approach to the design of new therapeutic molecules.

Functional maturation of isolated neural progenitor cells from the adult rat hippocampus.

Although neural progenitor cells (NPCs) may provide a source of new neurons to alleviate neural trauma, little is known about their electrical properties as they differentiate. We have previously shown that single NPCs from the adult rat hippocampus can be cloned in the presence of heparan sulphate chains purified from the hippocampus, and that these cells can be pushed into a proliferative phenotype with the mitogen FGF2 [Chipperfield, H., Bedi, K.S., Cool, S.M. & Nurcombe, V. (2002) Int. J. Dev. Biol., 46, 661-670]. In this study, the active and passive electrical properties of both undifferentiated and differentiated adult hippocampal NPCs, from 0 to 12 days in vitro as single-cell preparations, were investigated. Sparsely plated, undifferentiated NPCs had a resting membrane potential of approximately -90 mV and were electrically inexcitable. In > 70%, ATP and benzoylbenzoyl-ATP evoked an inward current and membrane depolarization, whereas acetylcholine, noradrenaline, glutamate and GABA had no detectable effect. In Fura-2-loaded undifferentiated NPCs, ATP and benzoylbenzoyl-ATP evoked a transient increase in the intracellular free Ca(2+) concentration, which was dependent on extracellular Ca(2+) and was inhibited reversibly by pyridoxalphosphate-6-azophenyl-2'-4'-disulphonic acid (PPADS), a P2 receptor antagonist. After differentiation, NPC-derived neurons became electrically excitable, expressing voltage-dependent TTX-sensitive Na(+) channels, low- and high-voltage-activated Ca(2+) channels and delayed-rectifier K(+) channels. Differentiated cells also possessed functional glutamate, GABA, glycine and purinergic (P2X) receptors. Appearance of voltage-dependent and ligand-gated ion channels appears to be an important early step in the differentiation of NPCs.

Alpha-conotoxins PnIA and [A10L]PnIA stabilize different states of the alpha7-L247T nicotinic acetylcholine receptor.

The effects of the native alpha-conotoxin PnIA, its synthetic derivative [A10L]PnIA and alanine scan derivatives of [A10L]PnIA were investigated on chick wild type alpha7 and alpha7-L247T mutant nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes. PnIA and [A10L]PnIA inhibited acetylcholine (ACh)-activated currents at wtalpha7 receptors with IC50 values of 349 and 168 nm, respectively. Rates of onset of inhibition were similar for PnIA and [A10L]PnIA; however, the rate of recovery was slower for [A10L]PnIA, indicating that the increased potency of [A10L]PnIA at alpha7 receptors is conveyed by its slower rate of dissociation from the receptors. All the alanine mutants of [A10L]PnIA inhibited ACh-activated currents at wtalpha7 receptors. Insertion of an alanine residue between position 5 and 13 and at position 15 significantly reduced the ability of [A10L]PnIA to inhibit ACh-evoked currents. PnIA inhibited the non-desensitizing ACh-activated currents at alpha7-L247T receptors with an IC50 194 nm. In contrast, [A10L]PnIA and the alanine mutants potentiated the ACh-activated current alpha7-L247T receptors and in addition [A10L]PnIA acted as an agonist. PnIA stabilized the receptor in a state that is non-conducting in both the wild type and mutant receptors, whereas [A10L]PnIA stabilized a state that is non-conducting in the wild type receptor and conducting in the alpha7-L247T mutant. These data indicate that the change of a single amino acid side-chain, at position 10, is sufficient to change the toxin specificity for receptor states in the alpha7-L247T mutant.

A new level of conotoxin diversity, a non-native disulfide bond connectivity in alpha-conotoxin AuIB reduces structural definition but increases biological activity.

alpha-Conotoxin AuIB and a disulfide bond variant of AuIB have been synthesized to determine the role of disulfide bond connectivity on structure and activity. Both of these peptides contain the 15 amino acid sequence GCCSYPPCFATNPDC, with the globular (native) isomer having the disulfide connectivity Cys(2-8 and 3-15) and the ribbon isomer having the disulfide connectivity Cys(2-15 and 3-8). The solution structures of the peptides were determined by NMR spectroscopy, and their ability to block the nicotinic acetylcholine receptors on dissociated neurons of the rat parasympathetic ganglia was examined. The ribbon disulfide isomer, although having a less well defined structure, is surprisingly found to have approximately 10 times greater potency than the native peptide. To our knowledge this is the first demonstration of a non-native disulfide bond isomer of a conotoxin exhibiting greater biological activity than the native isomer.