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Susanna A Wood - One of the best experts on this subject based on the ideXlab platform.
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extracts from benthic Anatoxin producing phormidium are toxic to 3 macroinvertebrate taxa at environmentally relevant concentrations
Environmental Toxicology and Chemistry, 2018Co-Authors: Brian S Anderson, Jennifer P Voorhees, Bryn M Phillips, Rich Fadness, Rosalina Stancheva, Jeanette Nichols, Daniel Orr, Susanna A WoodAbstract:Toxin-producing cyanobacteria are increasing in rivers and streams globally, leading to growing concerns over their potential impacts on aquatic ecosystems. The present study was designed to culture field-collected Phormidium in the laboratory, identify individual species, conduct chemical analyses to identify cyanotoxins, and conduct toxicity tests to investigate the potential for this genera to impact stream health. Freshwater toxicity tests were conducted with standard US Environmental Protection Agency invertebrate test protocols with culture water used to grow 3 Phormidium strains isolated from the Russian River (CA, USA). Enzyme linked immunosorbent assays were used to measure total Anatoxin concentrations. Culture waters from the 3 Phormidium strains were highly toxic to Ceriodaphnia dubia, Hyalella azteca, and Chironomus dilutus. The C. dubia 7-d survival median lethal concentrations were 0.71, 0.49, and 0.56 μg/L Anatoxin for Phormidum strains 1, 2, and 3, respectively. The 7-d reproduction inhibitory concentrations, 25% were 0.55, 0.32, and 0.30 μg/L Anatoxin for strains 1, 2, and 3, respectively. Chironomus dilutus survival was reduced at concentrations <2 μg/L Anatoxin by all 3 strains, and the H. azteca 96-h lethal concentrations, 25% were 2.82, 1.26, and 5.30 μg/L for strains 1, 2, and 3, respectively. Additional liquid chromatography-mass spectrometry analyses demonstrated that the likely Anatoxin variant in these cultures was dihydro-Anatoxin-a. The results suggest that Anatoxins produced by Phormidium have the potential to impact stream macroinvertebrates. Environ Toxicol Chem 2018;37:2851-2859. © 2018 SETAC.
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development and application of a quantitative pcr assay to assess genotype dynamics and Anatoxin content in microcoleus autumnalis dominated mats
Toxins, 2018Co-Authors: Laura T Kelly, Susanna A Wood, Tara G Mcallister, Ken G. RyanAbstract:Microcoleus is a filamentous cyanobacteria genus with a global distribution. Some species form thick, cohesive mats over large areas of the benthos in rivers and lakes. In New Zealand Microcoleus autumnalis is an Anatoxin producer and benthic proliferations are occurring in an increasing number of rivers nationwide. Anatoxin content in M. autumnalis-dominated mats varies spatially and temporally, making understanding and managing proliferations difficult. In this study a M. autumnalis-specific TaqMan probe quantitative PCR (qPCR) assay targeting the anaC gene was developed. The assay was assessed against 26 non-M. autumnalis species. The assay had a detection range over seven orders of magnitude, with a limit of detection of 5.14 × 10-8 ng μL-1. The anaC assay and a cyanobacterial specific 16S rRNA qPCR were then used to determine toxic genotype proportions in 122 environmental samples collected from 19 sites on 10 rivers in New Zealand. Anatoxin contents of the samples were determined using LC-MS/MS and Anatoxin quota per toxic cell calculated. The percentage of toxic cells ranged from 0 to 30.3%, with significant (p < 0.05) differences among rivers. The Anatoxin content in mats had a significant relationship with the percentage of toxic cells (R² = 0.38, p < 0.001), indicating that changes in Anatoxin content in M. autumnalis-dominated mats are primarily related to the dominance of toxic strains. When applied to more extensive samples sets the assay will enable new insights into how biotic and abiotic parameters influence genotype composition, and if applied to RNA assist in understanding Anatoxin production.
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Anatoxins are consistently released into the water of streams with Microcoleus autumnalis-dominated (cyanobacteria) proliferations.
Harmful algae, 2018Co-Authors: Susanna A Wood, Laura Biessy, Jonathan PuddickAbstract:Abstract Proliferations of potentially toxic, mat-forming Microcoleus are increasing in streams globally. A range of cyanotoxins are produced by Microcoleus , with the neurotoxic Anatoxins (Anatoxin-a, dihydro-Anatoxin-a, homoAnatoxin-a and dihydro-homoAnatoxin-a) the most commonly reported. The Anatoxins produced by Microcoleus are thought to be largely contained within the cells. More knowledge on whether Anatoxins are been released into the overlying stream water is required to better assess health risks to human, animals, and aquatic organisms. Field studies were conducted in three streams experiencing toxic Microcoleus autumnalis (basionym Phormidium autumnale )-dominated proliferations. Samples were collected every 1.5–3 h over a 24- or 26-h sampling period. Water samples were analyzed for total (intracellular and dissolved) and dissolved Anatoxins, and time-integrated Anatoxin samples were collected using solid phase adsorption tracking technology (SPATT). Anatoxins were detected in all stream water and SPATT samples (max. 0.91 ng mL −1 and 95 ng g -1 of strata-x hr −1 ). At two sites, Anatoxins were largely dissolved, whereas at the third site only total Anatoxins could be detected. Temporal variability in Anatoxin concentrations was observed, but there were no evident patterns between sampling sites. Linear regression showed a very weakstatistically significant relationship ( R 2 = 0.24, p = 0.002) between total Anatoxin concentrations in water and SPATT, however, when tested per site, only one of the three showed a significant relationship. These results highlight the potential for chronic exposure to Anatoxins for humans (i.e., through drinking water) and aquatic organisms in streams with M. autumnalis proliferations. The health implications of this are unknown.
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spatiotemporal dynamics of phormidium cover and Anatoxin concentrations in eight new zealand rivers with contrasting nutrient and flow regimes
Science of The Total Environment, 2018Co-Authors: Tara G Mcallister, Susanna A Wood, Javier Atalah, Ian HawesAbstract:Abstract Toxic benthic cyanobacterial proliferations, particularly of the genus Phormidium, are a major concern in many countries due to their increasing extent and severity. The aim of this study was to improve the current understanding of the dominant physicochemical variables associated with high Phormidium cover and toxin concentrations. Phormidium cover and Anatoxin concentrations were assessed weekly for 30 weeks in eight predominately cobble-bed rivers in the South Island of New Zealand. Phormidium cover was highly variable both spatially (among and within sites) and temporally. Generalized additive mixed models (GAMMs) identified site, month of the year, conductivity and nutrient concentrations over the accrual period as significant variables associated with Phormidium cover. Cover was greatest under low to intermediate accrual dissolved inorganic nitrogen (DIN) and dissolved reactive phosphorus (DRP) concentrations. Accrual nutrients had a strong, negative effect on cover at concentrations > 0.2 mg L− 1 DIN and 0.014 mg L− 1 DRP. The effect of flow was generally consistent across rivers, with cover accruing with time since the last flushing flow. Total Anatoxins were detected at all eight study sites, at concentrations ranging from 0.008 to 662.5 mg kg− 1 dried weight. GAMMs predicted higher total Anatoxin concentrations between November and February and during periods of accrual DRP
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the abundance of toxic genotypes is a key contributor to Anatoxin variability in phormidium dominated benthic mats
Marine Drugs, 2017Co-Authors: Susanna A Wood, Jonathan PuddickAbstract:The prevalence of benthic proliferations of the Anatoxin-producing cyanobacterium Phormidium are increasing in cobble-bed rivers worldwide. Studies to date have shown high spatial and temporal variability in Anatoxin concentrations among mats. In this study we determined Anatoxin quotas (toxins per cell) in field samples and compared these results to the conventionally-used concentrations (assessed per dry weight of mat). Three mats were selected at sites in two rivers and were sampled every 2–3 h for 24–26 h. The samples were lyophilized and ground to a fine homogenous powder. Two aliquots of known weights were analyzed for Anatoxin congeners using liquid chromatography-mass spectrometry, or digital droplet PCR with Phormidium-specific anaC primers to measure absolute quantities of gene copies. Anatoxin concentrations in the mats varied 59- and 303-fold in the two rivers over the study periods. A similar pattern was observed among gene copies (53- and 2828-fold). When converted to Anatoxin quotas there was markedly less variability (42- and 16-fold), but significantly higher Anatoxin quotas were observed in mats from the second river (p < 0.001, Student’s t-test). There were no obvious temporal patterns with high and low Anatoxin concentrations or quotas measured at each sampling time and across the study period. These results demonstrate that variability in Anatoxin concentrations among mats is primarily due to the abundance of toxic genotypes. No consistent modulation in Anatoxin production was observed during the study, although significant differences in Anatoxin quotas among rivers suggest that site-specific physiochemical or biological factors may influence Anatoxin production.
Vitor Vasconcelos - One of the best experts on this subject based on the ideXlab platform.
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Production of Anatoxin-a by cyanobacterial strains isolated from Portuguese fresh water systems.
Ecotoxicology (London England), 2009Co-Authors: Joana Osswald, Sandra Rellán, Ana Gago-martínez, Vitor VasconcelosAbstract:The occurrence of Anatoxin-a in several freshwater systems in Portugal and its production by Portuguese cyanobacterial strains, after cultivation in laboratory, were studied. Surface water samples from 9 water bodies, for recreational and human consumption usage, were surveyed for Anatoxin-a presence and for obtaining cultures of pure cyanobacterial strains. Anatoxin-a analysis was performed by high performance liquid chromatography (HPLC) with fluorescence detection (FLD) followed by Mass Spectrometry (MS) confirmation. No Anatoxin-a was detected in all the natural water samples (limit of detection (LOD) = 25 ng l−1) but among the 22 isolated cyanobacterial strains, 13 could produce Anatoxin-a in laboratory conditions (LOD = 3 ng g−1 dw). This proportion of Anatoxin-a producing strains (59.1%) in laboratory is discussed considering the hypothesis that Anatoxin-a is a more frequent metabolite in cyanobacteria than it was thought before and making its occurrence in Portuguese freshwaters almost certain. Therefore, health and ecological risks caused by Anatoxin-a in Portugal, should be seriously considered.
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First detection of Anatoxin-a in human and animal dietary supplements containing cyanobacteria.
Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association, 2009Co-Authors: Sandra Rellán, Joana Osswald, Ana Gago-martínez, Martin L. Saker, Vitor VasconcelosAbstract:Anatoxin-a is a potent neurotoxin produced by several species of cyanobacteria. This alkaloid may cause fatal intoxication to exposed organisms and this has raised concerns over the increasing popularity of food supplements containing cyanobacteria. These are being marketed with alleged health properties for animal and human consumption. These supplements most commonly contain the genera Spirulina (Arthrospira) and Aphanizomenon and their consumption represent a potential route for Anatoxin-a exposure in cases where adequate quality control is not undertaken. In this work, several dietary supplements containing cyanobacteria from different commercial suppliers were evaluated for the presence of Anatoxin-a by high performance liquid chromatography with fluorescence detection. Additionally, the presence of the previously derivatized Anatoxin-a was confirmed by using Gas chromatography-mass spectrometry. A total of 39 samples were analysed in the study. Results showed that three of the samples (7.7%) contained Anatoxin-a, at concentrations ranging from 2.50 to 33 microg g(-1). Quality control of cyanobacterial food supplements is required to avoid potential health effects in humans and animals.
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Effects of cyanobacterial extracts containing Anatoxin-a and of pure Anatoxin-a on early developmental stages of carp.
Ecotoxicology and environmental safety, 2008Co-Authors: Joana Osswald, A. P. Carvalho, João Claro, Vitor VasconcelosAbstract:This study compares the effects of pure Anatoxin-a and cyanobacterial extracts of an Anatoxin-a producing strain on early stages of development of carp. Carp eggs were exposed from 2:30 h to 4 days post-fertilization to different ecologically relevant concentrations of Anatoxin-a, provided as pure toxin or contained in the cyanobacterial extracts. Data on time to mortality, mortality rate, time to hatching, hatching rate, skeletal malformations rate, and larval standard length were registered until 8 days post-fertilization. At any tested concentration of Anatoxin-a, the pure toxin was almost harmless to carp early stages of development, contrarily to cell extracts that were highly toxic. Only an adverse effect on the larval length was found at the highest concentration of pure toxin, while increasing concentrations of cell extracts caused increasing adverse effects in all the analyzed parameters. Anatoxin-a producing cyanobacteria should be regarded as putative modulators of aquatic ecosystems communities.
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Uptake and depuration of Anatoxin-a by the mussel Mytilus galloprovincialis (Lamarck, 1819) under laboratory conditions.
Chemosphere, 2008Co-Authors: Joana Osswald, Sandra Rellán, Ana Gago, Vitor VasconcelosAbstract:Abstract Cyanobacterial blooms tend to be more common in warm and nutrient-enriched waters and are increasing in many aquatic water bodies due to eutrophication. The aim of this work is to study the accumulation and depuration of Anatoxin-a by Mytilus galloprovincialis a widespread distributed mussel living in estuarine and coastal waters and recognized worldwide as a bioindicator (e.g. Mussel Watch programs). Research on the distribution and biological effects of Anatoxin-a in M. galloprovincialis is important. Nevertheless, the risk of human intoxication due to the consumption of contaminated bivalves should also be considered. A toxic bloom was simulated in an aquarium with 5 × 105 cell ml−1 of Anabaena sp. (ANA 37), an Anatoxin-a producing strain. Mussels were exposed to Anabaena for 15 days and then 15 days of depuration followed. Three or more animals were sampled every 24 h for total toxin quantification and distribution in soft tissues (edible parts). Water samples were also taken every 24 h in order to calculate total dissolved and particulate Anatoxin-a concentrations. Anatoxin-a was quantified by HPLC with fluorescence detection. No deaths occurred during accumulation and depuration periods. One day after the beginning of depuration, the toxin could not be detected in the animals. Anatoxin-a is distributed in the digestive tract, muscles and foot and is probably actively detoxified.
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Toxicology and detection methods of the alkaloid neurotoxin produced by cyanobacteria, Anatoxin-a
Environment international, 2007Co-Authors: Joana Osswald, Sandra Rellán, Ana Gago, Vitor VasconcelosAbstract:Freshwater resources are under stress due to naturally occurring conditions and human impacts. One of the consequences is the proliferation of cyanobacteria, microphytoplankton organisms that are capable to produce toxins called cyanotoxins. Anatoxin-a is one of the main cyanotoxins. It is a very potent neurotoxin that was already responsible for some animal fatalities. In this review we endeavor to divulgate much of the internationally published information about toxicology, occurrence and detection methods of Anatoxin-a. Cyanobacteria generalities, Anatoxin-a occurrence and production as well as Anatoxin-a toxicology and its methods of detection are the aspects focused in this review. Remediation of Anatoxin-a occurrence will be addressed with a public health perspective. Final remarks call the attention for some important gaps in the knowledge about this neurotoxin and its implication to public health. Alterations of aquatic ecosystems caused by Anatoxin-a is also addressed. Although Anatoxin-a is not the more frequent cyanotoxin worldwide, it has to be regarded as a health risk that can be fatal to terrestrial and aquatic organisms because of its high toxicity.
M. Alfonso - One of the best experts on this subject based on the ideXlab platform.
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Evaluation of in vivo and in vitro recovery rate of Anatoxin-a through the microdialysis probe.
Toxicon : official journal of the International Society on Toxinology, 2008Co-Authors: F. Campos, R. Durán, Sandra Rellán, Lilian Rosana Ferreira Faro, Ana Gago, M. AlfonsoAbstract:In vivo microdialysis is a versatile sampling technique commonly employed to observe changes in neurotransmitters levels that occur in response to different treatments, being these treatments administered through a microdialysis probe implanted into a specific brain region in living animals. In previous works we have used this technique to study the effects of the drug Anatoxin-a, a nicotinic acetylcholine receptor agonist, on dopamine release in striatum. The aim of the present study was to assess the recovery of Anatoxin-a through the microdialysis probe. This information allows knowing the exact amount of the drug crossing the microdialysis membrane, acting on extracellular tissue. High Performance Liquid Chromatography (HPLC) with Fluorescence Detection (FLD) has been used for the analysis of Anatoxin-a. We observed that the recovery of Anatoxin-a was about 0.5%. Under our experimental conditions, the results suggest that Anatoxin-a can be used as an important tool in the study of neuronal nicotinic receptors by in vivo microdialysis technique and also show a reliable estimation of the Anatoxin-a recovery through the microdialysis probe under both in vivo and in vitro conditions.
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Mediation of glutamatergic receptors and nitric oxide on striatal dopamine release evoked by Anatoxin-a. An in vivo microdialysis study.
European Journal of Pharmacology, 2006Co-Authors: F. Campos, L. Vidal, M. Alfonso, Lilian Rosana Ferreira Faro, R. DuránAbstract:In this work, the involvement of ionotropic glutamatergic receptors and nitric oxide on striatal dopamine release induced by Anatoxin-a was investigated in conscious and freely-moving rats. To study the participation of glutamatergic receptors, the effects of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate receptors antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and N-methyl-D-aspartate (NMDA) receptor antagonists, dizocilpine (MK-801) andd(�)-2-amino-5-phosphonopentanoicacid(APV),wereexamined.Theperfusionof3.5mMAnatoxin-aincreasedtheextracellulardopaminelevelsto 701% relative to the basal.When CNQX was administered with 3.5mM Anatoxin-a, the increase of dopamine levels was 29% smaller than that observed with Anatoxin-a alone. When MK-801 and APV were administered, the effect of Anatoxin-a was attenuated 26% and 25% respectively in terms of that observed with Anatoxin-a alone. And with CNQX plus MK-801, the effect of Anatoxin-a was 53% inhibited in terms of the effect of Anatoxin-a alone. These results suggest that the striatal dopamine release induced by Anatoxin-a is partly mediated by activation of both ionotropic glutamatergic receptors. Since the neuronal form of nitric oxide synthase (nNOS) produces nitric oxide (NO) primarily in response to activation of NMDA receptors, it was tested if NO could play any role in the effect of Anatoxin-a. Treatment with NOS inhibitors, L-nitro-arginine methyl ester (L-NAME) and d(�)2-amino-5-phosphonopentanoic acid (7-NI), induced decreased Anatoxin-a effects of 22% and 26% respectively. In conclusion, the present in vivo results demonstrate that Anatoxin-a induced an indirect activation of ionotropic glutamatergic receptors (NMDA and AMPA/kainite receptors), which stimulate striatal dopamine release. On the other hand, activation of NMDA receptors may elicit NO increased levels enhancing dopamine release.
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In vivo Effects of the Anatoxin-a on Striatal Dopamine Release
Neurochemical research, 2006Co-Authors: F. Campos, R. Durán, L. Vidal, L. R. F. Faro, M. AlfonsoAbstract:Anatoxin-a is an important neurotoxin that acts a potent nicotinic acetylcholine receptor agonist. This characteristic makes Anatoxin-a an important tool for the study of nicotinic receptors. Anatoxin-a has been used extensively in vitro experiments, however Anatoxin-a has never been studied by in vivo microdialysis studies. This study test the effect of Anatoxin-a on striatal in vivo dopamine release by microdialysis.
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In vivo Effects of the Anatoxin-a on Striatal Dopamine Release
Neurochemical Research, 2006Co-Authors: F. Campos, R. Durán, L. Vidal, L. R. F. Faro, M. AlfonsoAbstract:Anatoxin-a is an important neurotoxin that acts a potent nicotinic acetylcholine receptor agonist. This characteristic makes Anatoxin-a an important tool for the study of nicotinic receptors. Anatoxin-a has been used extensively in vitro experiments, however Anatoxin-a has never been studied by in vivo microdialysis studies. This study test the effect of Anatoxin-a on striatal in vivo dopamine release by microdialysis. The results of this work show that Anatoxin-a evoked dopamine release in a concentration-dependent way. Atropine had not any effect on dopamine release evoked by 3.5 mM Anatoxin-a. However, perfusion of nicotinic antagonists mecamylamine and α-bungarotoxin induced a total inhibition of the striatal dopamine release. Perfusion of α7*-receptors antagonists, metillycaconitine or α-bungarotoxin, partially inhibits the release of dopamine stimulated by Anatoxin-a. These results show that Anatoxin-a can be used as an important nicotinic agonist in the study of nicotinic receptor by in vivo microdialysis technique and also support further in vivo evidences that α7*nicotinic AChRs are implicated in the regulation of striatal dopamine release.
Susan Wonnacott - One of the best experts on this subject based on the ideXlab platform.
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The Chemistry and Pharmacology of Anatoxin-a and Related Homotropanes with respect to Nicotinic Acetylcholine Receptors
Marine Drugs, 2006Co-Authors: Susan Wonnacott, Timothy GallagherAbstract:This chapter covers the chemistry and nicotinic pharmacology of naturally occurring homotropane alkaloids, with an emphasis of Anatoxin-a. In addition to Anatoxin-a, homoAnatoxin and pinnamine, as well as the major classes of synthetic derivatives of Anatoxin-a including UB-165, are discussed.
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Direct C-11 functionalisation of Anatoxin-a. Application to the synthesis of new ligand-based structural probes
Journal of the Chemical Society Perkin Transactions 1, 1997Co-Authors: Nicholas A. Magnus, Susan Wonnacott, Laurent Ducry, Valérie Rolland, Timothy GallagherAbstract:A variety of methods have been evaluated for the functionalisation of the C-11 methyl group of Anatoxin-a. Reaction of N-Boc Anatoxin-a 9 with PhI(OH)OTs (Koser’s reagent) represents the method of choice and gives the synthetically versatile α-tosyloxy ketone 10. This intermediate provides a convenient vehicle for the attachment of spacer units to C-11 via a thioether linkage which has been applied to the synthesis of the dansylated [N-(5-dimethylamino-1-naphthylsulfonyl)] Anatoxin-a derivatives. Preliminary biological data relating to the α-thiomethyl Anatoxin-a derivative 16 and the dansylated ligands, 25 and 26, are also reported.
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Synthesis of UB-165: A novel nicotinic ligand and Anatoxin-a/epibatidine hybrid
Bioorganic & Medicinal Chemistry Letters, 1997Co-Authors: Emma Wright, Timothy Gallagher, Christopher G. V. Sharples, Susan WonnacottAbstract:Abstract UB-165 (5), a hybrid corresponding to natural Anatoxin-a and epibatidine, has been synthesised and shows significant potency at the high affinity nicotine binding site in rat brain. Ent-(5) shows a much lower level of activity which parallels the sense of enantiospecificity associated with Anatoxin-a.
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Anatoxin-a is a potent agonist of the nicotinic acetylcholine receptor of bovine adrenal chromaffin cells
European journal of pharmacology, 1995Co-Authors: Leah Molloy, Susan Wonnacott, Timothy Gallagher, Paul A. Brough, Bruce G. LivettAbstract:(+)-Anatoxin-a is a neurotoxic alkaloid produced by the cyanobacterium Anabaena flos-aquae. In this study synthetic (+/-)-Anatoxin-a was tested on isolated bovine adrenal chromaffin cells to determine its ability to evoke secretion of endogenous catecholamines through neuronal-type nicotinic receptor activation. Anatoxin-a was found to act as a potent agonist of the secretory response of chromaffin cells with an EC50 of 1-2 microM, compared with an EC50 of 4-5 microM for nicotine. The cells responded to Anatoxin-a and nicotine with bell-shaped concentration-response curves consistent with desensitisation at concentrations of Anatoxin-a greater than 5 microM and of nicotine greater than 20 microM. The secretion of catecholamines stimulated by Anatoxin-a was completely inhibited in a non-competitive manner by the nicotinic antagonist mecamylamine with an IC50 of 0.4-0.5 microM. In the presence of depolarising concentrations of K+ (15 or 50 mM), Anatoxin-a increased the secretion of catecholamines in a concentration-dependent manner up to the same maximum as that achieved by Anatoxin-a alone. It is concluded that Anatoxin-a acts as a potent and selective nicotinic agonist, capable of evoking secretion of endogenous catecholamines from chromaffin cells via their neuronal-type nicotinic receptor.
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Anatoxin-a-evoked [3H]dopamine release from rat striatal synaptosomes
Neuropharmacology, 1995Co-Authors: L. Soliakov, Timothy Gallagher, Susan WonnacottAbstract:Abstract Presynaptic nicotinic acetylcholine receptors on striatal nerve terminals modulate the release of dopamine. Using rat striatal synaptosomes loaded with [3H]dopamine, we have characterized the action of the selective nicotinic agonist, (±)Anatoxin-a, with respect to [3H]dopamine release, in order to explore the mechanisms coupling nicotinic receptor activation to exocytosis. Anatoxin-a evoked [3H]dopamine release in a concentration-dependent and mecamylamine-sensitive manner, EC50 = 0.11 μM. The maximum [3H]dopamine release elicited by Anatoxin-a was only 20% of the maximum elicited by KC1 depolarization; there was no additivity between Anatoxin-a and sub-maximal concentrations of KC1. Both agents stimulated Ca2+-dependent release that was equally sensitive to inhibition by 200 μM Cd2+. This result suggests that Anatoxin-a-stimulated exocytosis is mediated by Ca2+ influx via voltage-sensitive Ca2+ channels, with little contribution from Ca2+ entering directly through the nicotinic receptor channel. This view is supported by the abolition of Anatoxin-a-evoked [3H]dopamine release in Na+-depleted medium. A partial (40%) inhibition by tetrodotoxin was observed. These data suggest that activation of presynaptic nicotinic acetylcholine receptors by Anatoxin-a results in an influx of Na+, producing sufficient local depolarization to open voltage-sensitive Ca2+ and Na+ channels. The latter may then amplify the response, activating further Ca2+ channels. The particular voltage-sensitive Ca2+ channels involved remain to be determined.
F. Campos - One of the best experts on this subject based on the ideXlab platform.
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Evaluation of in vivo and in vitro recovery rate of Anatoxin-a through the microdialysis probe.
Toxicon : official journal of the International Society on Toxinology, 2008Co-Authors: F. Campos, R. Durán, Sandra Rellán, Lilian Rosana Ferreira Faro, Ana Gago, M. AlfonsoAbstract:In vivo microdialysis is a versatile sampling technique commonly employed to observe changes in neurotransmitters levels that occur in response to different treatments, being these treatments administered through a microdialysis probe implanted into a specific brain region in living animals. In previous works we have used this technique to study the effects of the drug Anatoxin-a, a nicotinic acetylcholine receptor agonist, on dopamine release in striatum. The aim of the present study was to assess the recovery of Anatoxin-a through the microdialysis probe. This information allows knowing the exact amount of the drug crossing the microdialysis membrane, acting on extracellular tissue. High Performance Liquid Chromatography (HPLC) with Fluorescence Detection (FLD) has been used for the analysis of Anatoxin-a. We observed that the recovery of Anatoxin-a was about 0.5%. Under our experimental conditions, the results suggest that Anatoxin-a can be used as an important tool in the study of neuronal nicotinic receptors by in vivo microdialysis technique and also show a reliable estimation of the Anatoxin-a recovery through the microdialysis probe under both in vivo and in vitro conditions.
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Mediation of glutamatergic receptors and nitric oxide on striatal dopamine release evoked by Anatoxin-a. An in vivo microdialysis study.
European Journal of Pharmacology, 2006Co-Authors: F. Campos, L. Vidal, M. Alfonso, Lilian Rosana Ferreira Faro, R. DuránAbstract:In this work, the involvement of ionotropic glutamatergic receptors and nitric oxide on striatal dopamine release induced by Anatoxin-a was investigated in conscious and freely-moving rats. To study the participation of glutamatergic receptors, the effects of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate receptors antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and N-methyl-D-aspartate (NMDA) receptor antagonists, dizocilpine (MK-801) andd(�)-2-amino-5-phosphonopentanoicacid(APV),wereexamined.Theperfusionof3.5mMAnatoxin-aincreasedtheextracellulardopaminelevelsto 701% relative to the basal.When CNQX was administered with 3.5mM Anatoxin-a, the increase of dopamine levels was 29% smaller than that observed with Anatoxin-a alone. When MK-801 and APV were administered, the effect of Anatoxin-a was attenuated 26% and 25% respectively in terms of that observed with Anatoxin-a alone. And with CNQX plus MK-801, the effect of Anatoxin-a was 53% inhibited in terms of the effect of Anatoxin-a alone. These results suggest that the striatal dopamine release induced by Anatoxin-a is partly mediated by activation of both ionotropic glutamatergic receptors. Since the neuronal form of nitric oxide synthase (nNOS) produces nitric oxide (NO) primarily in response to activation of NMDA receptors, it was tested if NO could play any role in the effect of Anatoxin-a. Treatment with NOS inhibitors, L-nitro-arginine methyl ester (L-NAME) and d(�)2-amino-5-phosphonopentanoic acid (7-NI), induced decreased Anatoxin-a effects of 22% and 26% respectively. In conclusion, the present in vivo results demonstrate that Anatoxin-a induced an indirect activation of ionotropic glutamatergic receptors (NMDA and AMPA/kainite receptors), which stimulate striatal dopamine release. On the other hand, activation of NMDA receptors may elicit NO increased levels enhancing dopamine release.
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In vivo Effects of the Anatoxin-a on Striatal Dopamine Release
Neurochemical research, 2006Co-Authors: F. Campos, R. Durán, L. Vidal, L. R. F. Faro, M. AlfonsoAbstract:Anatoxin-a is an important neurotoxin that acts a potent nicotinic acetylcholine receptor agonist. This characteristic makes Anatoxin-a an important tool for the study of nicotinic receptors. Anatoxin-a has been used extensively in vitro experiments, however Anatoxin-a has never been studied by in vivo microdialysis studies. This study test the effect of Anatoxin-a on striatal in vivo dopamine release by microdialysis.
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In vivo Effects of the Anatoxin-a on Striatal Dopamine Release
Neurochemical Research, 2006Co-Authors: F. Campos, R. Durán, L. Vidal, L. R. F. Faro, M. AlfonsoAbstract:Anatoxin-a is an important neurotoxin that acts a potent nicotinic acetylcholine receptor agonist. This characteristic makes Anatoxin-a an important tool for the study of nicotinic receptors. Anatoxin-a has been used extensively in vitro experiments, however Anatoxin-a has never been studied by in vivo microdialysis studies. This study test the effect of Anatoxin-a on striatal in vivo dopamine release by microdialysis. The results of this work show that Anatoxin-a evoked dopamine release in a concentration-dependent way. Atropine had not any effect on dopamine release evoked by 3.5 mM Anatoxin-a. However, perfusion of nicotinic antagonists mecamylamine and α-bungarotoxin induced a total inhibition of the striatal dopamine release. Perfusion of α7*-receptors antagonists, metillycaconitine or α-bungarotoxin, partially inhibits the release of dopamine stimulated by Anatoxin-a. These results show that Anatoxin-a can be used as an important nicotinic agonist in the study of nicotinic receptor by in vivo microdialysis technique and also support further in vivo evidences that α7*nicotinic AChRs are implicated in the regulation of striatal dopamine release.