Chronic exposure to cigarette smoke extract upregulates nicotinic receptor binding in adult and adolescent rats.

Heavy smokers display increased radioligand binding of nicotinic acetylcholine receptors (nAChRs). This "upregulation" is thought to be a contributing factor to tobacco dependence. Although cigarette smoke contains thousands of constituents that can contribute to nicotine dependence, it is not well understood whether non-nicotine constituents contribute to nAChR upregulation. In this study, we used an aqueous cigarette smoke extract (CSE), which contains nicotine and soluble constituents of cigarette smoke, to induce nAChR upregulation in adult and adolescent rats. To do this, male rats were exposed to nicotine or CSE (1.5 mg/kg/day nicotine equivalent, intravenously) daily for ten days. This experimental procedure produces equivalent levels of brain and plasma nicotine in nicotine- and CSE-treated animals. We then assessed nAChR upregulation using quantitative autoradiography to measure changes in three nAChR types. Adolescents were found to have consistently greater α4ß2 nAChR binding than adults in many brain regions. Chronic nicotine exposure did not significantly increase nAChR binding in any brain region at either age. Chronic CSE exposure selectively increased α4ß2 nAChR binding in adolescent medial amygdala and α7 binding in adolescent central amygdala and lateral hypothalamus. CSE also increased α3ß4 nAChR binding in the medial habenula and interpeduncular nucleus, and α7 binding in the medial amygdala, independent of age. Overall, this work provides evidence that cigarette smoke constituents influence nAChR upregulation in an age-, nAChR type- and region-dependent manner.

New insights in the mode of action of (+)-erythravine and (+)-11α-hydroxy-erythravine alkaloids.

Erythrinian alkaloids ((+)-erythravine and (+)-11-α-hydroxy-erythravine) have been pointed as the main responsible agents for the anticonvulsant and anxiolytic properties of Erythrina mulungu Mart ex Benth. The present work provides a new set of information about the mode of action of these alkaloids by the use of a complementary approach of neurochemical and electrophysiological assays. We propose here that the antiepileptic and anxiolytic properties exhibited by both alkaloids appear not to be related to the inhibition of glutamate binding or GABA uptake, or even to the increase of glutamate uptake or GABA binding, as investigated here by the use of rat cortical synaptosomes. Similarly, and even in a high concentration, (+)-erythravine and (+)-11-α-hydroxy-erythravine did not modulate the main sodium and potassium channel isoforms checked by the use of voltage-clamp studies on Xenopus laevis oocytes. However, unlike (+)-11-α-hydroxy-erythravine, which presented a little effect, it was possible to observe that the (+)-erythravine alkaloid produced a significant inhibitory modulation on α4ß2, α4ß4 and α7 isoforms of nicotinic acetylcholine receptors also checked by the use of voltage-clamp studies, which could explain at least partially its anxiolytic and anticonvulsant properties. Since (+)-11-α-hydroxy-erythravine and (+)-erythravine modulated nicotinic acetylcholine receptors to different extents, it is possible to reinforce that small differences between the chemical structure of these alkaloids can affect the selectivity and affinity of target-ligand interactions, conferring distinct potency and/or pharmacological properties to them, as previously suggested by differential experimental comparison between different erythrinian alkaloids.

Muscarinic and Nicotinic Modulation of Neocortical Layer 6A Synaptic Microcircuits Is Cooperative and Cell-Specific.

Acetylcholine (ACh) is known to regulate cortical activity during different behavioral states, for example, wakefulness and attention. Here we show a differential expression of muscarinic ACh receptors (mAChRs) and nicotinic ACh receptors (nAChRs) in different layer 6A (L6A) pyramidal cell (PC) types of somatosensory cortex. At low concentrations, ACh induced a persistent hyperpolarization in corticocortical (CC) but a depolarization in corticothalamic (CT) L6A PCs via M 4 and M1 mAChRs, respectively. At ~ 1 mM, ACh depolarized exclusively CT PCs via α4ß2 subunit-containing nAChRs without affecting CC PCs. Miniature EPSC frequency in CC PCs was decreased by ACh but increased in CT PCs. In synaptic connections with a presynaptic CC PC, glutamate release was suppressed via M4 mAChR activation but enhanced by nAChRs via α4ß2 nAChRs when the presynaptic neuron was a CT PC. Thus, in L6A, the interaction of mAChRs and nAChRs results in an altered excitability and synaptic release, effectively strengthening CT output while weakening CC synaptic signaling.

Effects of Euterpe oleracea to Enhance Learning and Memory in a Conditioned Nicotinic and Muscarinic Receptor Response Paradigm by Modulation of Cholinergic Mechanisms in Rats.

Euterpe oleracea (EO) includes a large number of polyphenolic compounds such as phenolics, flavonoids, and anthocyanins that have antioxidant activities. E. oleracea was suggested to ease the oxidative stress and inflammation in brain cells. Our aim was to analyze the effects of E. oleracea on learning and memory. Seventy-two (250 ± 25 g) male Wistar albino rats were used for this study. The groups consisted of control, EO100 mg/kg, EO300 mg/kg, scopolamine 1.5 mg/kg, mecamylamine 7.5 mg/kg, combinations of scopolamine with EO100 mg/kg, EO300 mg/kg, and rivastigmine 1.5 mg/kg; and mecamylamine combined with EO100 mg/kg. Before the start of the study, E. oleracea doses were provided once a day for a period of 15 days and for a 6-day experimental period. Thirty minutes after intraperitoneal scopolamine and mecamylamine injections, gastrogavage was applied to each group. Ninety minutes after the drug treatments, locomotor activity and Morris water maze tests were performed. Rats were killed and each hippocampus was used for the quantification of acetylcholine (Ach). Statistical analyses were calculated using one-way and two-way analyses of variance (ANOVA), and a value of P < .05 was considered significant. In groups EO100 mg/kg and EO300 mg/kg the results did not show any significant changes on learning and memory compared with the control group. Mecamylamine and scopolamine enhanced the latency for the escape platform, and decreased the time spent in escape platform quadrant when the memory tests were applied in reference to the control value of P < .05. Scopolamine and mecamylamine combinations of EO100 mg/kg, EO300 mg/kg, and rivastigmine were proven to improve the memory. There was significant difference between the first and fifth days of the learning tests in all the groups, but no significant difference occurred between the groups. Ach levels in hippocampi supported all memory tests. We suggest that E. oleracea made no alterations on learning and memory, but still improved nicotinic and muscarinic receptor-mediated and impaired memory just as rivastigmine.

Qualitative Assay to Detect Dopamine Release by Ligand Action on Nicotinic Acetylcholine Receptors.

A pheochromocytoma of the rat adrenal medulla derived (a.k.a. PC12) cell-based assay for dopamine measurement by luminescence detection was customized for the qualitative evaluation of agonists and antagonists of nicotinic acetylcholine receptors (nAChRs). The assay mechanism begins with ligand binding to transmembrane nAChRs, altering ion flow into the cell and inducing dopamine release from the cell. Following release, dopamine is oxidized by monoamine oxidase generating hydrogen peroxide that catalyzes a chemiluminescence reaction involving luminol and horseradish peroxidase, thus producing a detectable response. Results are presented for the action of nAChR agonists (acetylcholine, nicotine, and cytisine), and antagonists (α-conotoxins (α-CTxs) MII, ImI, LvIA, and PeIA) that demonstrate a luminescence response correlating to the increase or decrease of dopamine release. A survey of cell growth and treatment conditions, including nerve growth factor, nicotine, ethanol, and temperature, led to optimal assay requirements to achieve maximal signal intensity and consistent response to ligand treatment. It was determined that PC12 cells treated with a combination of nerve growth factor and nicotine, and incubated at 37 °C, provided favorable results for a reduction in luminescence signal upon treatment of cells with α-CTxs. The PC12 assay is intended for use as a fast, efficient, and economic qualitative method to assess the bioactivity of molecules that act on nAChRs, in which testing of ligand-nAChR binding hypotheses and computational predictions can be validated. As a screening method for nAChR bioactivity, lead compounds can be assessed for their likelihood of exhibiting desired bioactivity prior to being subjected to more complex quantitative methods, such as electrophysiology or live animal studies.

Drug Discovery on Natural Products: From Ion Channels to nAChRs, from Nature to Libraries, from Analytics to Assays.

Natural extracts are complex mixtures that may be rich in useful bioactive compounds and therefore are attractive sources for new leads in drug discovery. This review describes drug discovery from natural products and in explaining this process puts the focus on ion-channel drug discovery. In particular, the identification of bioactives from natural products targeting nicotinic acetylcholine receptors (nAChRs) and serotonin type 3 receptors (5-HT3Rs) is discussed. The review is divided into three parts: "Targets," "Sources," and "Approaches." The "Targets" part will discuss the importance of ion-channel drug targets in general, and the α7-nAChR and 5-HT3Rs in particular. The "Sources" part will discuss the relevance for drug discovery of finding bioactive compounds from various natural sources such as venoms and plant extracts. The "Approaches" part will give an overview of classical and new analytical approaches that are used for the identification of new bioactive compounds with the focus on targeting ion channels. In addition, a selected overview is given of traditional venom-based drug discovery approaches and of diverse hyphenated analytical systems used for screening complex bioactive mixtures including venoms.

Volatile Terpenes and Brain Function: Investigation of the Cognitive and Mood Effects of Mentha × Piperita L. Essential Oil with In Vitro Properties Relevant to Central Nervous System Function.

BACKGROUND: Extracts of several members of the monoterpene-rich Lamiaceae sub-family Nepetoideae, including those from the Salvia (sage), Melissa (Lemon balm) and Rosmarinus (rosemary) genera, evince cognitive and mood effects in humans that are potentially related to their effects on cholinergic and GABAergic neurotransmission. To date, despite promising in vitro properties, the cognitive and mood effects of the closely related Mentha spicata (spearmint) and Mentha piperita (peppermint) remain unexplored. This study therefore assessed the human cognitive/mood effects of the M. spicata/piperita essential oil with the most promising, brain-relevant in vitro properties according to pre-trial in vitro screening. Design: Organic spearmint and peppermint (Mentha spicata/piperita) essential oils were pre-screened for neurotransmitter receptor binding and acetylcholinesterase (AChE) inhibition. In a double-blind, placebo-controlled, balanced cross-over study, 24 participants (mean age 25.2 years) consumed single doses of encapsulated placebo and 50 µl and 100 µl of the most promising essential oil (peppermint with nicotinic/GABAA receptor binding and AChE inhibitory properties, that increased calcium influx in a CAD cell neuronal model). Psychological functioning was assessed with mood scales and a range of standardised, cognitively demanding tasks pre-dose and at 1, 3 and 6 h post-dose. Results: The highest (100 µL) dose of essential oil improved performance on the cognitively demanding Rapid Visual Information Processing task (RVIP) at 1 h and 3 h post-dose and both doses attenuated fatigue and improved performance of the Serial 3 s subtraction task at 3 h post-dose. Conclusion: Peppermint (Mentha piperita) essential oil with high levels of menthol/menthone and characteristic in vitro cholinergic inhibitory, calcium regulatory and GABAA/nicotinic receptor binding properties, beneficially modulated performance on demanding cognitive tasks and attenuated the increase in mental fatigue associated with extended cognitive task performance in healthy adults. Future investigations should consider investigating higher doses.

5-Desmethylnobiletin augments synaptic ACh levels and nicotinic ACh receptor activity: A potential candidate for alleviation of cholinergic dysfunction.

Cholinergic function is compromised in plethora of neurodegenerative disorders especially Alzheimer's disease. Increasing acetylcholine (ACh) levels has been the mainstay in majority of the therapeutic regimens, accepted for management of disease. The present study investigates the efficacy of 5-Desmethylnobiletin (DN), a polymethoxyflavone in augmenting cholinergic function using Caenorhabditis elegans as a model organism. The studies revealed significant elevation in cholinergic transmission mediated through increased levels of ACh and activity of nicotinic acetylcholine receptors (nAChR). Further investigation into the mechanistic aspects indicated that DN enhanced cholinergic function through down modulation of acetylcholinesterase activity at enzyme and transcript level along with upregulation of non alpha subunit, unc-29 which could be linked with enhanced nAChR activity as evident from levamisole assay. Additionally, studies on antioxidant properties, implicated significant potential of DN in curtailing ROS, both in vivo and in vitro. Our studies present DN as a phytomolecule with novel biological activities which could be exploited and researched upon for therapeutic avenues in terms of cholinergic function and antioxidant potential.

1-Methyl-4-propan-2-ylbenzene from Thymus vulgaris Attenuates Cholinergic Dysfunction.

Cholinergic dysfunction is manifested in a plethora of neurodegenerative and psychiatric disorders such as Alzheimer's, Parkinson's, and Huntington's diseases. The extent of cholinergic affliction is maximum in Alzheimer's disease which is a progressive neurodegenerative disorder involving death of cholinergic neurons. To this date, the therapeutic management of cholinergic dysfunction is limited to provide symptomatic relief through the use of acetylcholinesterase (Ache) inhibitors only. The present study elaborates the potential of thyme oil and its individual components in curtailing cholinergic deficits. We found that thyme oil augments neurotransmission by modulating synaptic acetylcholine (Ach) levels and nicotinic acetylcholine receptor activity, being orchestrated through upregulation of genes cho-1, unc-17 and unc-50. Studies on individual components revealed para-cymene (1-methyl-4-propan-2-ylbenzene) as the active component of thyme oil, contributing its effects through upregulation of cho-1, cha-1, unc-17 and unc-50, while downregulating ace-1 and ace-2. Interestingly, thymol and gamma-terpinene which although were devoid of any activity individually, exhibited significantly enhanced synaptic Ach levels and nicotinic acetylcholine receptor (nAchR) responsiveness, when administered in combination. Our findings advocate thyme oil and its constituents as potential candidates for amelioration of cholinergic dysfunction. The study is speculated to make a way for a new line of "phytomolecules-based drugs" from the diverse pool of natural compounds.

Pancuronium enhances isoflurane anesthesia in rats via inhibition of cerebral nicotinic acetylcholine receptors.

PURPOSE: This study was conducted to elucidate the mechanism of enhancement of volatile anesthetics by neuromuscular blocking agents in rats and to consider the relevance of this enhancement to clinical anesthesia. METHODS: Male Sprague-Dawley rats were used. After confirming a movement in response to tail clamping under 1.1 % isoflurane anesthesia, response was determined when the tail clamp was applied at several points after microinjection of pancuronium into the lateral ventricle. Arousal responses to microinjection of nicotine into the lateral ventricle were assessed with or without pretreatment with intraventricular pancuronium. The intravenous 50 % effective dose (ED50) and 95 % effective dose (ED95) for neuromuscular blockade with pancuronium administered in a cumulative fashion at 1.1 % isoflurane were calculated. RESULTS: Intraventricular pancuronium dose-dependently reduced the response to tail clamping, and the dose required to show immobilization of 50 % of rats (intraventricular ED50) was 1.62 µg/kg. Pretreatment with pancuronium at 6 µg/kg significantly reduced the effect of awakening by nicotine under isoflurane anesthesia (P = 0.044). The intravenous ED50 and ED95 for neuromuscular blockade were 63 µg/kg (90 % confidence interval [CI] 52-75 µg/kg) and 133 µg/kg (90 % CI 109-158 µg/kg), respectively. The ratio of intraventricular ED50 to intravenous ED50 was 0.026. CONCLUSION: Pancuronium microinjection into the lateral ventricle dose-dependently enhances the depth of isoflurane anesthesia, which might be caused by inhibition of neuronal nicotinic acetylcholine receptor transmission in the cerebrum. Intravenous injection of pancuronium at high doses might increase the cerebrospinal concentration to a level at which an effect can be observed.

Maternal exposure to hexachlorophene targets intermediate-stage progenitor cells of the hippocampal neurogenesis in rat offspring via dysfunction of cholinergic inputs by myelin vacuolation.

Hexachlorophene (HCP) is known to induce myelin vacuolation corresponding to intramyelinic edema of nerve fibers in the central and peripheral nervous system in animals. This study investigated the effect of maternal exposure to HCP on hippocampal neurogenesis in rat offspring using pregnant rats supplemented with 0 (controls), 100, or 300 ppm HCP in the diet from gestational day 6 to day 21 after delivery. On postnatal day (PND) 21, the numbers of T box brain 2(+) progenitor cells and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling(+) apoptotic cells in the hippocampal subgranular zone (SGZ) decreased in female offspring at 300 ppm, which was accompanied by myelin vacuolation and punctate tubulin beta-3 chain staining of nerve fibers in the hippocampal fimbria. In addition, transcript levels of the cholinergic receptor, nicotinic beta 2 (Chrnb2) and B-cell CLL/lymphoma 2 (Bcl2) decreased in the dentate gyrus. HCP-exposure did not alter the numbers of SGZ proliferating cells and reelin- or calcium-binding protein-expressing γ-aminobutyric acid (GABA)-ergic interneuron subpopulations in the dentate hilus on PND 21 and PND 77. Although some myelin vacuolation remained, all other changes observed in HCP-exposed offspring on PND 21 disappeared on PND 77. These results suggest that maternal HCP exposure reversibly decreases type-2b intermediate-stage progenitor cells via the mitochondrial apoptotic pathway in offspring hippocampal neurogenesis at 300 ppm HCP. Neurogenesis may be affected by dysfunction of cholinergic inputs into granule cell lineages and/or GABAergic interneurons as indicated by decreased transcript levels of Chrnb2 and numbers of Chrnb2(+) interneurons caused by myelin vacuolation in the septal-hippocampal pathway.

A novel treatment target for Parkinson's disease.

We hypothesize that GPR109A message and expression are up-regulated in individuals with Parkinson's disease (PD). GPR109A is a high-affinity niacin receptor. Niacin is a precursor for NAD-NADH which is needed for dopamine production. Thus, niacin supplementation may serve three purposes: reduce inflammation through GPR109A-related mechanisms, increase dopamine synthesis in the striatum through NADPH supply and increase NAD/NADH ratio to boost mitochondrial functions. GPR109A and its agonists are known to exert anti-inflammatory actions in the skin, gut and retina. However these roles are neither anticipated nor established in the CNS. For the first time here we propose the roles of GPR109A and its agonists including niacin in CNS pathology. Moreover we predict that the neuroprotective roles of either niacin or butyrates in CNS occur via GPR109A.

Discovery of a potent and selective α3ß4 nicotinic acetylcholine receptor antagonist from an α-conotoxin synthetic combinatorial library.

α-Conotoxins are disulfide-rich peptide neurotoxins that selectively inhibit neuronal nicotinic acetylcholine receptors (nAChRs). The α3ß4 nAChR subtype has been identified as a novel target for managing nicotine addiction. Using a mixture-based positional-scanning synthetic combinatorial library (PS-SCL) with the α4/4-conotoxin BuIA framework, we discovered a highly potent and selective α3ß4 nAChR antagonist. The initial PS-SCL consisted of a total of 113 379 904 sequences that were screened for α3ß4 nAChR inhibition, which facilitated the design and synthesis of a second generation library of 64 individual α-conotoxin derivatives. Eleven analogues were identified as α3ß4 nAChR antagonists, with TP-2212-59 exhibiting the most potent antagonistic activity and selectivity over the α3ß2 and α4ß2 nAChR subtypes. Final electrophysiological characterization demonstrated that TP-2212-59 inhibited acetylcholine evoked currents in α3ß4 nAChRs heterogeneously expressed in Xenopus laevis oocytes with a calculated IC50 of 2.3 nM and exhibited more than 1000-fold selectivity over the α3ß2 and α7 nAChR subtypes. As such, TP-2212-59 is among the most potent α3ß4 nAChRs antagonists identified to date and further demonstrates the utility of mixture-based combinatorial libraries in the discovery of novel α-conotoxin derivatives with refined pharmacological activity.

Chronic sazetidine-A maintains anxiolytic effects and slower weight gain following chronic nicotine without maintaining increased density of nicotinic receptors in rodent brain.

Chronic nicotine administration increases the density of brain α4ß2* nicotinic acetylcholine receptors (nAChRs), which may contribute to nicotine addiction by exacerbating withdrawal symptoms associated with smoking cessation. Varenicline, a smoking cessation drug, also increases these receptors in rodent brain. The maintenance of this increase by varenicline as well as nicotine replacement may contribute to the high rate of relapse during the first year after smoking cessation. Recently, we found that sazetidine-A (saz-A), a potent partial agonist that desensitizes α4ß2* nAChRs, does not increase the density of these receptors in brain at doses that decrease nicotine self-administration, increase attention in rats, and produce anxiolytic effects in mice. Here, we investigated whether chronic saz-A and varenicline maintain the density of nAChRs after their up-regulation by nicotine. In addition, we examined the effects of these drugs on a measure of anxiety in mice and weight gain in rats. After increasing nAChRs in the rodent brain with chronic nicotine, replacing nicotine with chronic varenicline maintained the increased nAChR binding, as well as the α4ß2 subunit proteins measured by western blots. In contrast, replacing nicotine treatments with chronic saz-A resulted in the return of the density of nAChRs to the levels seen in saline controls. Nicotine, saz-A and varenicline each demonstrated anxiolytic effects in mice, but only saz-A and nicotine attenuated the gain of weight over a 6-week period in rats. These findings suggest that apart from its modest anxiolytic and weight control effects, saz-A, or drugs like it, may be useful in achieving long-term abstinence from smoking.

The ß2 nicotinic acetylcholine receptor subunit differentially influences ethanol behavioral effects in the mouse.

The high co-morbidity between alcohol (ethanol) and nicotine abuse suggests that nicotinic acetylcholine receptors (nAChRs), thought to underlie nicotine dependence, may also be involved in alcohol dependence. The ß2* nAChR subtype serves as a potential interface for these interactions since they are the principle mediators of nicotine dependence and have recently been shown to modulate some acute responses to ethanol. Therefore, the aim of this study was to more fully characterize the role of ß2* nAChRs in ethanol-responsive behaviors in mice after acute exposure to the drug. We conducted a battery of tests in mice lacking the ß2* coding gene (Chrnb2) or pretreated with a selective ß2* nAChR antagonist for a range of ethanol-induced behaviors including locomotor depression, hypothermia, hypnosis, and anxiolysis. We also tested the effect of deletion on voluntary escalated ethanol consumption in an intermittent access two-bottle choice paradigm to determine the extent of these effects on drinking behavior. Our results showed that antagonism of ß2* nAChRs modulated some acute behaviors, namely by reducing recovery time from hypnosis and enhancing the anxiolytic-like response produced by acute ethanol in mice. Chrnb2 deletion had no effect on ethanol drinking behavior, however. We provide further evidence that ß2* nAChRs have a measurable role in mediating specific behavioral effects induced by acute ethanol exposure without affecting drinking behavior directly. We conclude that these receptors, along with being key components in nicotine dependence, may also present viable candidates in the discovery of the molecular underpinnings of alcohol dependence.

[Tobacco--once a medicinal plant. Does it contain substances with medicinal properties?]. / Tyton--niegdys roslina lecznicza. Czy zawiera substancje o wlasciwosciach leczniczych?

Tobacco and its use was discovered by Christopher Columbus in parallel with the discovery of America. Soon after, tobacco became a known medicinal plant in Europe. Its harmful effects were gradually discovered, especially those of tobacco smoke, and now it is considered a toxic plant. Tobacco leaf has a monograph in German "Hagers Enzyklopädie derArzneistoffe und Drogen", which describes its old, already not valid, medicinal use and clearly shows the toxic effects. Epidemiological studies indicate about 50% lower incidence of Parkinson's disease in smokers than in non-smokers. In turn, studies of the brains of smokers using positron emission tomography showed significantly decreased level of monoamine oxidase B--an enzyme which degrades dopamine--the neurotransmitter which the significant insufficiency of about 80-85%, is responsible for the symptoms of Parkinson's disease. From the tobacco leaves there were isolated MAO-B inhibitors--naphthoquinone--2,3,6-trimethyl-1,4-naphthoquinone and diterpenoid -trans,trans-farnesol, which occur also in tobacco smoke. In the last decade many papers have appeared on the neuroprotective activity of nicotine, the best known component of tobacco. through the effect of this compound on specific nicotinic cholinergic receptors (nAChRs), which interacts with nigrostriatal dopaminergic system as well as the possibility of using nicotine for the treatment of Parkinson's disease and other neurodegenerative diseases. Moreover, tobacco was also found to contain inhibitors of neuronal nitric oxide synthase (nNOS). Tobacco cannot be considered a medicinal plant, but some compounds occurring in that plant may find therapeutic use.

An efficient analytical platform for on-line microfluidic profiling of neuroactive snake venoms towards nicotinic receptor affinity.

Venomous snakes have evolved their efficient venomous arsenals mainly to immobilize prey. The highly variable toxic peptides in these venoms target a myriad of neurotoxic and haemotoxic receptors and enzymes and comprise highly interesting candidates for drug discovery. Discovery of bioactive compounds from snake venoms, however, is a challenge to achieve. We have developed and applied a methodology to rapidly assess bioactives in a snake venom proteome. Our microfluidic platform opens up efficient and rapid profiling of venomous anti-cholinergic receptor compounds. The key advantages of our methodology are: (i) nano amounts of venom needed; and (ii) a direct correlation of selected bioaffinities with accurate mass. To achieve this, we have for the first time successfully constructed a functional post nano-LC split to MS and bioaffinity profiling. In our method, comprehensive venom profiles with accurate masses and corresponding bioaffinities are obtained in one analytical run and will subsequently allow immediate purification of bioactive peptides with LC-MS, guided by accurate masses of the bioactives only. We profiled several neurotoxic Elapidae snake venoms using our methodology in combination with the acetylcholine binding protein (AChBP) as biological target protein. The latter is a homologue of nicotinic acetylcholine receptors (nAChRs), a drug target in neurodegenerative diseases and cognitive decline such as Parkinson's and Alzheimer's, and in pain related diseases. Our methodology was evaluated and validated with high-affinity α-bungarotoxin and haemotoxic/proteolytic Vipera ammodytes venom spiked with α-bungarotoxin. Thereafter, the methodology was applied to profile the venom proteomes of Dendroaspis jamesoni kaimosae, Naja annulifera and Naja nivea. Gathering comprehensive profiling data took less than 2 h per snake venom measured. The data yielded 20 AChBP ligands of which the corresponding accurate masses were used to retrieve information from literature regarding their function and targeting specificity. We found that from these 20 ligands, 11 were previously reported on, while information on the others could not be found. From these 11 peptides, five have been reported to have nAChR affinity, while the others are reported as cytotoxic, cardiotoxic or as orphan toxin. Our methodology has the potential to aid the field of profiling complex animal venoms for drug discovery.

Carboetomidate inhibits alpha4/beta2 neuronal nicotinic acetylcholine receptors at concentrations affecting animals.

BACKGROUND: Carboetomidate is an etomidate derivative that produces hypnosis without inhibiting adrenal corticosteroid synthesis. Similar to etomidate, carboetomidate modulates γ-aminobutyric acid type A receptors, but its effects on other ion channel targets of general anesthetics are unknown. METHODS: We compared etomidate and carboetomidate effects on human N-methyl-d-aspartate receptors or neuronal nicotinic acetylcholine receptors (nnAChRs) expressed in Xenopus oocytes, using 2-microelectrode voltage clamp electrophysiology. RESULTS: Etomidate did not affect either type of receptor at clinically relevant concentrations, whereas carboetomidate concentrations near 50% effective concentration for anesthesia significantly inhibited nnAChRs. CONCLUSIONS: Compared with etomidate, carboetomidate's higher hydrophobicity is associated with greater inhibition of nnAChRs.

The Yin and Yang of non-neuronal α7-nicotinic receptors in inflammation and autoimmunity.

The alkaloid nicotine, a major addictive component of tobacco, exerts anti-inflammatory and immunemodulating activities on multiple cell types, such as T cells, B cells, dendritic cells, mononuclear phagocytes and polymorphonuclear leukocytes, in lung, spleen, liver, kidney and gastrointestinal tract. In addition, nicotine may blunt pro-inflammatory cytokine release, with prominent effects on T helper type 1 (Th1) and Th17 cytokines. The nonneuronal α7-nicotinic cholinergic receptors are a primary target for nicotine through the JAK2 and STAT3/NF-κB pathways, ultimately mediating the inhibition of pro-inflammatory gene transcription. The present paper reviews the growing evidence in favor of detrimental as well as beneficial effects of nicotine and other α7-nicotininc receptor agonists in pre-clinical models of organ-specific and systemic inflammatory and autoimmune diseases. These data may portend favorable implications for the targeted treatment of chronic and debilitating human disorders, such as diabetes, arthritis, asthma and inflammatory bowel disease, with α7-selective ligands.

Evidence-based pharmacological treatment of substance use disorders and pathological gambling.

This review summarizes our current knowledge of the pharmacological treatment of substance use disorders and pathological gambling using data mainly from randomized controlled trials and meta-analyses regarding these randomized controlled trials. The review is restricted to the selection of first and second line pharmacological treatments for smoking, alcohol dependence, opioid dependence, cocaine dependence, cannabis dependence and pathological gambling. It is concluded that great progress has been made in the last three decades and that currently evidence-based pharmacological treatments are available for smoking cessation, alcohol and opioid dependence and pathological gambling. At the same time a series of existing and new pharmacological compounds are being tested in cocaine and cannabis dependence. The review concludes with a summary of additional strategies to increase the effect size of already available pharmacological interventions, including polypharmacy, combining pharmacotherapy with psychotherapy and psychosocial support, and improved patient-treatment matching.