Cytotoxic furanosesquiterpenoids and steroids from Ircinia mutans sponges

ContextIrcinia mutans Wilson (Irciniidae) is a sponge with antimicrobial and cytotoxic constituents.ObjectiveOur objective was to characterise the cytotoxic constituents of two seasonal collections of I. mutans.Materials and methodsThe sponges were extracted in methanol-dichloromethane and their constituents were purified and characterised using column chromatography, GC-MS, 1 D and 2 D NMR. Anti-proliferative activities of the compounds, were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay (0.25–100 μg/mL, 72 h) against leukaemia (MOLT-4), breast (MCF-7) and colon cancer (HT-29) human cells.ResultsThree furanosesquiterpoids; furodysin (1), ent-furodysinin (2) and furoircin (3) and ten sterols were characterised in I. mutans, for the first time. Cholesterol (4), cholesta-5, 7-dien-3β-ol (5) and ergosterol (6) were determined in the sponge from the winter collections, while cholesta-5, 22-dien-3β-ol (7), 24-methyldesmosterol (8), campesterol (9), stigmasterol (10), γ-ergostenol (11), chondrillasterol (12) and γ-sitosterol (13) were detected in the summer samples. The steroids from the winter collection exhibited cytotoxic activity with IC₅₀ values of 13.0 ± 0.9, 11.1 ± 1.7 and 1.1 ± 0.4 µg/mL, against the mentioned cancer cell lines, respectively, while those from the summer sample, showed greater activity, IC₅₀ = 1.1 ± 0.2 μg/mL against MOLT-4. The purified steroids showed potent MOLT-4 cytotoxic activity, IC₅₀ values = 2.3–7.8 µg/mL.Discussion and conclusionThe present study suggests that I. mutans is a rich source of cytotoxic steroids, and introduces 3 as new natural product. Considering the high cytotoxic activity of the steroids, these structures could be candidates for anticancer drug development in future research.     

Structure Activity Relationships of αv Integrin Antagonists for Pulmonary Fibrosis by Variation in Aryl Substituents

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The effect of a transmembrane amino acid on etomidate sensitivity of an invertebrate GABA receptor

1. The γ-aminobutyric acid (GABA)-modulatory and GABA-mimetic actions of etomidate at mammalian GABA_A receptors are favoured by β₂- or β₃- versus β₁-subunit containing receptors, a selectivity which resides with a single transmembrane amino acid (β_{2 N290}, β_{3 N289}, β_{1 S290}). Here, we have utilized the Xenopus laevis oocyte expression system in conjunction with the two-point voltage clamp technique to determine the influence of the equivalent amino acid (M314) on the actions of this anaesthetic at an etomidate-insensitive invertebrate GABA receptor (Rdl) of Drosophila melanogaster.
2. Complementary RNA-injected oocytes expressing the wild type Rdl GABA receptor and voltage-clamped at −60 mV responded to bath applied GABA with a concentration-dependent inward current response and a calculated EC₅₀ for GABA of 20±0.4 μM. Receptors in which the transmembrane methionine residue (M314) had been exchanged for an asparagine (Rdl_M314N) or a serine (Rdl_M314S) also exhibited a concentration-dependent inward current response to GABA, but in both cases with a reduced EC₅₀ of 4.8±0.2 μM.
3. Utilizing the appropriate GABA EC₁₀, etomidate (300 μM) had little effect on the agonist-evoked current of the wild type Rdl receptor. By contrast, at Rdl_M314N receptors, etomidate produced a clear concentration-dependent enhancement of GABA-evoked currents with a calculated EC₅₀ of 64±3 μM and an E_max of 68±2% (of the maximum response to GABA).
4. The actions of etomidate at Rdl_M314N receptors exhibited an enantioselectivity common to that found for mammalian receptors, with 100 μM R-(+)-etomidate and S-(−)-etomidate enhancing the current induced by GABA (EC₁₀) to 52±6% and 12±1% of the GABA maximum respectively.
5. The effects of this mutation were selective for etomidate as the GABA-modulatory actions of 1 mM pentobarbitone at wild type Rdl (49±4% of the GABA maximum) and Rdl_M314N receptors (53±2% of the GABA maximum) were similar. Additionally, the modest potentiation of GABA produced by the anaesthetic neurosteroid 5α-pregnan-3α-ol-20-one (Rdl=25±4% of the GABA maximum) was not altered by this mutation (Rdl_M314N=18±3% of the GABA maximum).
6. Etomidate acting at β₁ (S290)-containing mammalian GABA_A receptors is known to produce only a modest GABA-modulatory effect. Similarly, etomidate acting at Rdl_M314S receptors produced an enhancement of GABA but the magnitude of the effect was reduced compared to Rdl_M314N receptors.
7. Etomidate acting at human α₆β₃γ_2L receptors is known to produce a large enhancement of GABA-evoked currents and at higher concentrations this anaesthetic directly activates the GABA_A receptor complex. Mutation of the human β₃ subunit asparagine to methionine (β_{3 N289M} found in the equivalent position in Rdl completely inhibited both the GABA-modulatory and GABA-mimetic action of etomidate (10–300 μM) acting at α₆β_{3 N289M}γ_2L receptors.
8. It was concluded that, although invertebrate and mammalian proteins exhibit limited sequence homology, allosteric modification of their function by etomidate can be influenced in a complementary manner by a single amino acid substitution. The results are discussed in relation to whether this amino acid contributes to the anaesthetic binding site, or is essential for transduction. Furthermore, this study provides a clear example of the specificity of anaesthetic action.

     

Subunit-dependent interaction of the general anaesthetic etomidate with the γ-aminobutyric acid type A receptor

1. The GABA modulating and GABA-mimetic actions of the general anaesthetic etomidate were examined in voltage-clamp recordings performed on Xenopus laevis oocytes induced, by cRNA injection, to express human recombinant γ-aminobutyric acid_A (GABA_A) receptor subunits.
2. Currents mediated by recombinant receptors with the ternary subunit composition α_xβ_yγ_2L (where x=1,2,3 or 6 and y=1 or 2), in response to GABA applied at the appropriate EC₁₀, were enhanced by etomidate in a manner that was dependent upon the identity of both the α and β subunit isoforms.
3. For the β₂-subunit containing receptors tested, the EC₅₀ for the potentiation of GABA-evoked currents by etomidate (range 0.6 to 1.2 μM) was little affected by the nature of the α subunit present within the hetero-oligomeric complex. However, replacement of the β₂ by the β₁ subunit produced a 9–12 fold increase in the etomidate EC₅₀ (6 to 11 μM) for all α-isoforms tested.
4. For α₁, α₂ and α₆, but not α₃-subunit containing receptors, the maximal potentiation of GABA-evoked currents by etomidate was greater for β₂- than for β₁-subunit containing receptors. This was most clearly exemplified by receptors composed of α₆β₁γ_2L compared to α₆β₂γ_2L subunits, where a maximally effective concentration of etomidate potentiated currents evoked by GABA at EC₁₀ to 28±2% and 169±4% of the maximal GABA response, respectively.
5. For α₁ subunit-containing receptors, the potency and maximal potentiating effect of either pentobarbitone or propofol was essentially unaffected by the β subunit isoform contained within the receptor complex. The potency of the anaesthetic neurosteroid 5α-pregnan-3α-ol-20-one was marginally higher for β₁ rather than the β₂ subunit-containing receptor, although its maximal effect was similar at the two receptor isoforms.
6. The GABA-mimetic action of etomidate was supported by β₂- but not β₁-subunit containing receptors, whereas that of pentobarbitone or propofol was evident with either β isoform. For β₂-subunit containing receptors, both the agonist EC₅₀ and the maximal current produced by etomidate were additionally influenced by the α isoform.
7. It is concluded that the subtype of β-subunit influences the potency with which etomidate potentiates GABA-evoked currents and that the β isoform is a crucial determinant of the GABA-mimetic activity of this compound. The nature of the α-subunit also impacts upon the maximal potentiation and activation that the compound may elicit. Such pronounced influences may aid the identification of the site that recognises etomidate. More generally, these results provide a clear example of structural specificity in anaesthetic action.

     

The effect of atorvastatin treatment on serum oxysterol concentrations and cytochrome P450 3A4 activity

Aims

Atorvastatin is known to both inhibit and induce the cytochrome P450 3A4 (CYP3A4) enzyme in vitro. Some clinical studies indicate that atorvastatin inhibits CYP3A4 but there are no well-controlled longer term studies that could evaluate the inducing effect of atorvastatin. We aimed to determine if atorvastatin induces or inhibits CYP3A4 activity as measured by the 4β-hydroxycholesterol to cholesterol ratio (4βHC : C).

Methods

In this randomized, double-blind, placebo-controlled 6 month study we evaluated the effects of atorvastatin 20 mg day⁻¹ (n = 15) and placebo (n = 14) on oxysterol concentrations and determined if atorvastatin induces or inhibits CYP3A4 activity as assessed by the 4βHC : C index. The respective 25-hydroxycholesterol and 5α,6α-epoxycholesterol ratios were used as negative controls.

Results

Treatment with atorvastatin decreased 4βHC and 5α,6α-epoxycholesterol concentrations by 40% and 23%, respectively. The mean 4βHC : C ratio decreased by 13% (0.214 ± 0.04 to 0.182 ± 0.04, P = 0.024, 95% confidence interval (CI) of the difference –0.0595, –0.00483) in the atorvastatin group while no significant change occurred in the placebo group. The difference in change of 4βHC : C between study arms was statistically significant (atorvastatin –0.032, placebo 0.0055, P = 0.020, 95% CI of the difference –0.069, –0.0067). The ratios of 25-hydroxycholesterol and 5α,6α-epoxycholesterol to cholesterol did not change.

Conclusions

The results establish atorvastatin as an inhibitor of CYP3A4 activity. Furthermore, 4βHC : C is a useful index of CYP3A4 activity, including the conditions with altered cholesterol concentrations.     

The Rhizomes of Acorus gramineus and the Constituents Inhibit Allergic Response In vitro and In vivo

The rhizomes of Acorus gramineus have frequently been used in traditional medicine mainly for sedation as well as enhancing brain function. In this study, the anti-allergic activity of A. gramineus was investigated. The 70% ethanol extract of the rhizomes of A. gramineus was found to inhibit the allergic response against 5-lipoxygenase (5-LOX)-catalyzed leukotriene (LT) production from rat basophilic leukemia (RBL)-1 cells and β-hexosaminidase release from RBL-2H3 cells with IC₅₀’s of 48.9 and >200 μg/ml, respectively. Among the 9 major constituents isolated, β-asarone, (2R,3R,4S,5S)-2,4-dimethyl-1,3-bis (2'',4'',5''-trimethoxyphenyl)tetrahydrofuran (AF) and 2,3-dihydro-4,5,7-trimethoxy-1-ethyl-2-methyl-3-(2,4,5-trimethoxyphenyl)indene (AI) strongly inhibited 5-LOX-catalyzed LT production in {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187-treated RBL-1 cells, AI being the most potent (IC₅₀=6.7 μM). Against β-hexosaminidase release by antigen-stimulated RBL-2H3 cells, only AI exhibited strong inhibition (IC₅₀=7.3 μM) while β-asarone and AF showed 26.0% and 39.9% inhibition at 50 μM, respectively. In addition, the ethanol extract of A. gramineus showed significant inhibitory action against the hapten-induced delayed hypersensitivity reaction in mice by oral administration at 200 mg/kg. Therefore, it is suggested that A. gramineus possesses anti-allergic activity and the constituents including β-asarone and AI certainly contribute to the anti-allergic activity of the rhizomes of A. gramineus.     

Extraction temperature affects the activities of antioxidation,carbohydrate-digestion enzymes,and angiotensin-converting enzyme of Pleurotus citrinopileatus extract

Extraction temperature can potentially affect the chemical compositions and bioactivities of the extracts obtained. The objective of this study was to investigate the effect of extraction temperature on the distribution of bioactive compounds and the bioactivities of Pleurotus citrinopileatus. The antioxidant activities (2,2-diphenyl-1-picrylhydrazyl and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)⁺ scavenging capabilities) and the inhibitory capabilities on pancreatic α-amylase, intestinal α-glucosidase, and hypertension-linked angiotensin-converting enzyme of hot water P. citrinopileatus extract and cold water P. citrinopileatus extract were determined. The results showed that the antioxidant capabilities and inhibitory effects on α-amylase, α-glucosidase, and angiotensin-converting enzyme of cold water P. citrinopileatus extract were significantly higher than those of hot water P. citrinopileatus extract. The cold water P. citrinopileatus extracted was further precipitated with 100% ammonium sulfate to obtain a polysaccharide fraction or with 75% ethanol to obtain a protein fraction. The inhibitory activities of the protein fraction of the cold water P. citrinopileatus extract on α-amylase, α-glucosidase, and angiotensin-converting enzyme were significantly higher than those of the polysaccharide fraction. In conclusion, the protein fraction of the cold water P. citrinopileatus extract could be responsible for its bioactivities.     

Biomarkers of Exposure to Zearalenone in In Vivo and In Vitro Studies

The measurement of human exposure to mycotoxins is necessary for its association with adverse health effects. This exposure is usually estimated from contamination levels of foodstuffs, which are the primary source of toxin exposure, and data on food consumption patterns. However, variations in contamination level, intestinal absorption, toxin distribution, and excretion lead to individual variations in toxin exposure that can be more readily measured with a biomarker. This review deals with the latest literature information about ZEN biomarkers in humans, animals, and cell line cultures. Their presence in urine, biomarkers that have effects in the kidney, liver, reproductive system and blood and biomarkers of cell response have been reported. It has highlighted the importance of determining α-zearalenol and β-zearalenol biomarkers to estimate the probable dietary intake (PDI) of a specific population or to characterize the severity of exposure to ZEN in animals or cell lines. α-ZEL and β-ZEL are cytotoxic by inhibiting cell proliferation, total protein and DNA syntheses, in this sense, an induction of expression proteins Hsp27 and Hsp70 was observed, and an increase in gene expression (TLR4, NF-kBp65, TNF-α, IL-1β, IL-6, IL-8, MGMT, α-GST, Hsp70, Nrf2, L-Fabp, HO-1, MAPK8), the determination of which indicates an oxidative stress effect. The integrity of the cell or tissue membrane is assessed by lactate dehydrogenase (LDH), which increase at exposure of ZEN (84.2 µM), and the proportions of some fatty acids of the renal tissue membrane were increased at treatments with ZEN. This review allows starting future studies of animal and population exposure in parallel with those of health effects works.     

Action potential shortening through the putative β4-adrenoceptor in ferret ventricle: comparison with β1- and β2-adrenoceptor-mediated effects

The electrophysiological responses to (−)-CGP 12177 ((−)-4-(3-tertiarybutylamino-2-hydroxypropoxy) benzimidazol-2-one), an agonist for the putative β₄-adrenoceptor, were investigated on isolated perfused ferret hearts paced at 100 min⁻¹ and compared to those of (−)-noradrenaline and (−)-adrenaline, mediated through β₁- and β₂-adrenoceptors respectively. The three agonists decreased ventricular monophasic action potential duration but prolonged the action potential plateau; β₃-adrenoceptor-selective agonists had no effect. (−)-CGP 12177 was the most potent, but (−)-noradrenaline the most efficacious; both agonists caused ventricular extra-systoles. Because only (−)-noradrenaline but not (−)-CGP 12177 elicited shortening of the refractory period, the mechanism of arrhythmias mediated through β₁- and putative β₄-adrenoceptors may be different.     

4-Oxystilbene compounds are selective ligands for neuronal nicotinic αBungarotoxin receptors

1. Starting from the structure of an old 4-oxystilbene derivate with ganglioplegic activity (MG624), we synthesized two further derivates (F2 and F3) and two stereoisomers of F3 (F3A and F3B), and studied their selective effect on neuronal nicotinic acetylcholine receptor (AChR) subtypes.
2. MG 624, F3, F3A and F3B inhibited of ¹²⁵I-αBungarotoxin (αBgtx) binding to neuronal chick optic lobe (COL) membranes, with nM affinity, but inhibited ¹²⁵I-αBgtx binding to TE671 cell-expressed muscle-type AChR only at much higher concentrations.
3. We immobilized the α7, β2 and β4 containing chick neuronal nicotinic AChR subtypes using anti-subunit specific antibodies. MG 624, F3, F3A and F3B inhibited ¹²⁵I-αBgtx binding to the α7-containing receptors with nM affinity, but inhibited ³H-Epi binding to β2-containing receptors only at very high concentrations (more than 35 μM); their affinity for the β4-containing receptors was ten times more than for the β2-containing subtype.
4. Both MG624 and F3 compounds inhibited the ACh evoked currents in homomeric oocyte-expressed chick α7 receptors with an IC₅₀ of respectively 94 and 119 nM.
5. High doses of both MG 624 and F3 depressed the contractile response to vagus nerve stimulation in guinea pig nerve-stomach preparations although at different IC₅₀s (49.4 vs 166.2 μM) The effect of MG624 on rat nerve-hemidiaphragm preparations was 33 times less potent than that of F3 (IC₅₀ 486 vs 14.5 μM).
6. In conclusion, MG624 and F3 have a high degree of antagonist selectivity for neuronal nicotinic αBgtx receptors containing the α7 subunit.

     

The involvement of tumour necrosis factor-α in the protective effects of 17β oestradiol in splanchnic ischaemia-reperfusion injury

1. Tumour necrosis factor-α (TNF-α) is a cytokine that is implicated in the pathogenesis of ischaemic states and atherosclerosis. We tested the hypothesis that the vasoprotective effects of the oestrogens may be mediated in vivo by inhibition of the formation of TNF-α.
2. Anaesthetized rats, subjected to total occlusion of the superior mesenteric artery and the coeliac trunk for 45 min developed a severe shock state resulting in a fatal outcome within 75–90 min after the release of occlusion. Sham-operated animals were used as controls.
3. Splanchnic artery occlusion (SAO) shocked rats had a marked hypotension, enhanced levels of TNF-α in serum and macrophages, leukopenia and increased ileal leukocyte accumulation, studied by means of myeloperoxidase activity (MPO). Furthermore, aortae from SAO rats showed a marked hyporeactivity to phenylephrine (PE, 1 nM–10 μM), reduced responsiveness to acetylcholine (ACh, 10 nM–10 μM) and an increased staining for intercellular adhesion molecule-1 (ICAM-1).
4. In vivo administration of 17β oestradiol (500 μg kg⁻¹, i.m., three hours before the induction of SAO) increased survival rate (100%, 4 h after SAO), enhanced mean arterial blood pressure; reduced serum TNF-α (25±5 u ml⁻¹ vs 379±16 u ml⁻¹), ameliorated leukopaenia and reduced ileal MPO (0.7±0.02 u 10⁻³ g⁻¹ tissue vs 4.2±0.4 u 10⁻³ g⁻¹ tissue). Furthermore aortae from SAO rats treated with 17β oestradiol exhibited a greater contractile response to phenylephrine, improved responsiveness to ACh and a blunted staining of ICAM-1. Finally 17β oestradiol, added in vitro to peritoneal macrophages collected from untreated SAO rats, significantly reduced TNF-α production.
5. Our results suggest that inhibition of TNF-α in vivo may explain, at least in part, the vasoprotective effects of oestrogens.

     

Natural Compounds Interacting with Nicotinic Acetylcholine Receptors: From Low-Molecular Weight Ones to Peptides and Proteins

Nicotinic acetylcholine receptors (nAChRs) fulfill a variety of functions making identification and analysis of nAChR subtypes a challenging task. Traditional instruments for nAChR research are d-tubocurarine, snake venom protein α-bungarotoxin (α-Bgt), and α-conotoxins, neurotoxic peptides from Conus snails. Various new compounds of different structural classes also interacting with nAChRs have been recently identified. Among the low-molecular weight compounds are alkaloids pibocin, varacin and makaluvamines C and G. 6-Bromohypaphorine from the mollusk Hermissenda crassicornis does not bind to Torpedo nAChR but behaves as an agonist on human α7 nAChR. To get more selective α-conotoxins, computer modeling of their complexes with acetylcholine-binding proteins and distinct nAChRs was used. Several novel three-finger neurotoxins targeting nAChRs were described and α-Bgt inhibition of GABA-A receptors was discovered. Information on the mechanisms of nAChR interactions with the three-finger proteins of the Ly6 family was found. Snake venom phospholipases A₂ were recently found to inhibit different nAChR subtypes. Blocking of nAChRs in Lymnaea stagnalis neurons was shown for venom C-type lectin-like proteins, appearing to be the largest molecules capable to interact with the receptor. A huge nAChR molecule sensible to conformational rearrangements accommodates diverse binding sites recognizable by structurally very different compounds.     

Synthesis and Evaluation of the Metabolites of AMG 221, a Clinical Candidate for the Treatment of Type 2 Diabetes

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Nimodipine inhibits IL-1β release stimulated by amyloid β from microglia

BACKGROUND AND PURPOSE

There is growing evidence that inflammation plays a major role in the pathogenesis of neural damage caused by deposition of amyloid β (Aβ) in the brain. Nimodipine has received attention as a drug that might improve learning and reduce cognitive deficits in Alzheimer''s disease, but the mechanism of action is poorly known. In this study, we tested the hypothesis that nimodipine inhibited Aβ-stimulated IL-1β release from microglia.

EXPERIMENTAL APPROACH

Cultures of N13 microglia cells or primary mouse microglia were treated with nimodipine, and intracellular accumulation and release of IL-1β in response to Aβ or to the P2 receptor agonists ATP and benzoyl ATP (BzATP) were measured. Accumulation of IL-1β was measured in vivo after intrahippocampal inoculation of Aβ in the absence or presence of nimodipine. The effect of nimodipine on Aβ-triggered cytotoxicity was also investigated.

KEY RESULTS

We show here that nimodipine dose-dependently inhibited Aβ-stimulated IL-1β synthesis and release from primary microglia and microglia cell lines. Furthermore, nimodipine also inhibited Aβ-induced IL-1βin vivo accumulation at concentrations known to be reached in the CNS. Finally, nimodipine protected microglia from Aβ-dependent cytotoxicity.

CONCLUSION AND IMPLICATIONS

These data suggest that alleviation of symptoms of Alzheimer''s disease following nimodipine administration might be due to an anti-inflammatory effect and point to a novel role for nimodipine as a centrally acting anti-inflammatory drug.     

Production of β-Cyclocitral and Its Precursor β-Carotene in Microcystis aeruginosa: Variation at Population and Single-Cell Levels

Bloom-forming cyanobacteria produce and release odorous compounds and pose threats to the biodiversity of aquatic ecosystem and to the drinking water supply. In this study, the concentrations of β-cyclocitral in different bacterial growth phases were investigated using GC–MS to determine the growth stage of Microcystis aeruginosa at high risk for β-cyclocitral production. Moreover, the synchronicity of the production of β-cyclocitral and its precursor β-carotene at both population and single-cell levels was assessed. The results indicated that β-cyclocitral was the main odorous compound produced by M. aeruginosa cells. The intracellular concentration of β-cyclocitral (C_β-cc) as well as its cellular quota (Q_β-cc) increased synchronously in the log phase, along with the increase of cell density. However, they reached the maximum values of 415 μg/L and 10.7 fg/cell in the late stationary phase and early stationary phase, respectively. The early stage of the stationary phase is more important for β-cyclocitral monitoring, and the sharp increase in Q_β-cc is valuable for anticipating the subsequent increase in C_β-cc. The molar concentrations of β-cyclocitral and β-carotene showed a linear relationship, with an R² value of 0.92, suggesting that the production of β-cyclocitral was linearly dependent on that of β-carotene, especially during the log phase. However, the increase in Q_β-cc was slower than that in β-carotene during the stationary phase, suggesting that β-cyclocitral production turned to be carotene oxygenase-limited when the growth rate decreased. These results demonstrate that variations of β-cyclocitral production on a single-cell level during different bacterial growth phases should be given serious consideration when monitoring and controlling the production of odorous compounds by M. aeruginosa blooms.     

AT-1001: A High Affinity and Selective α3β4 Nicotinic Acetylcholine Receptor Antagonist Blocks Nicotine Self-Administration in Rats

Genomic and pharmacologic data have suggested the involvement of the α3β4 subtype of nicotinic acetylcholine receptors (nAChRs) in drug seeking to nicotine and other drugs of abuse. In order to better examine this receptor subtype, we have identified and characterized the first high affinity and selective α3β4 nAChR antagonist, AT-1001, both in vitro and in vivo. This is the first reported compound with a Ki below 10 nM at α3β4 nAChR and >90-fold selectivity over the other major subtypes, the α4β2 and α7 nAChR. AT-1001 competes with epibatidine, allowing for [³H]epibatidine binding to be used for structure-activity studies, however, both receptor binding and ligand-induced Ca²⁺ flux are not strictly competitive because increasing ligand concentration produces an apparent decrease in receptor number and maximal Ca²⁺ fluorescence. AT-1001 also potently and reversibly blocks epibatidine-induced inward currents in HEK cells transfected with α3β4 nAChR. Importantly, AT-1001 potently and dose-dependently blocks nicotine self-administration in rats, without affecting food responding. When tested in a nucleus accumbens (NAcs) synaptosomal preparation, AT-1001 inhibits nicotine-induced [³H]dopamine release poorly and at significantly higher concentrations compared with mecamylamine and conotoxin MII. These results suggest that its inhibition of nicotine self-administration in rats is not directly due to a decrease in dopamine release from the NAc, and most likely involves an indirect pathway requiring α3β4 nAChR. In conclusion, our studies provide further evidence for the involvement of α3β4 nAChR in nicotine self-administration. These findings suggest the utility of this receptor as a target for smoking cessation medications, and highlight the potential of AT-1001 and congeners as clinically useful compounds.     

Effects of the α subunit on imidacloprid sensitivity of recombinant nicotinic acetylcholine receptors

1. Imidacloprid is a new insecticide with selective toxicity for insects over vertebrates. Recombinant (α4β2) chicken neuronal nicotinic acetylcholine receptors (AChRs) and a hybrid nicotinic AChR formed by co-expression of a Drosophila melanogaster neuronal α subunit (SAD) with the chicken β2 subunit were heterologously expressed in Xenopus oocytes by nuclear injection of cDNAs. The agonist actions of imidacloprid and other nicotinic AChR ligands ((+)-epibatidine, (−)-nicotine and acetylcholine) were compared on both recombinant nicotinic AChRs by use of two-electrode, voltage-clamp electrophysiology.
2. Imidacloprid alone of the 4 agonists behaved as a partial agonist on the α4β2 receptor; (+)-epibatidine, (−)-nicotine and acetylcholine were all full, or near full, agonists. Imidacloprid was also a partial agonist of the hybrid Drosophila SAD chicken β2 receptor, as was (−)-nicotine, whereas (+)-epibatidine and acetylcholine were full agonists.
3. The EC₅₀ of imidacloprid was decreased by replacing the chicken α4 subunit with the Drosophila SAD α subunit. This α subunit substitution also resulted in an increase in the EC₅₀ for (+)-epibatidine, (−)-nicotine and acetylcholine. Thus, the Drosophila (SAD) α subunit contributes to the greater apparent affinity of imidacloprid for recombinant insect/vertebrate nicotinic AChRs.
4. Imidacloprid acted as a weak antagonist of ACh-mediated responses mediated by SADβ2 hybrid receptors and as a weak potentiator of ACh responses mediated by α4β2 receptors. This suggests that imidacloprid has complex effects upon these recombinant receptors, determined at least in part by the α subunit.