Anti-smoking drugs cytisine and varenicline reduce cardiac reperfusion injury in rat model of myocardial ischemia.

Evidence to date indicates that activation of nicotinic acetylcholine receptors (nAChRs) can reduce cardiac injury from ischemia and subsequent reperfusion. The use of nAChR agonists in various animal models leads to a reduction in reperfusion injury. Earlier this effect was shown for the agonists of α7 nAChR subtype. In this work, we demonstrated the expression of mRNA encoding α4, α6 and ß2 nAChR subunits in the left ventricle of rat heart. In a rat model of myocardial ischemia, we studied the effect of α4ß2 nAChR agonists cytisine and varenicline, medicines used for the treatment of nicotine addiction, and found them to significantly reduce myocardium ischemia-reperfusion injury, varenicline manifesting a higher protection. Dihydro-ß-erythroidine, antagonist of α4ß2 nAChR, as well as methyllycaconitine, antagonist of α7 and α6ß2-containing nAChR, prevented protective effect of varenicline. This together with the presence of α4, α6 and ß2 subunit mRNA in the left ventricule of rat heart raises the possibility that the varenicline effect is mediated by α4ß2 as well as by α7 and/or α6ß2-containing receptors. Our results point to a new way for the use of cytisine and varenicline as cardioprotective agents.

Analogs of 6-Bromohypaphorine with Increased Agonist Potency for α7 Nicotinic Receptor as Anti-Inflammatory Analgesic Agents.

Hypaphorines, tryptophan derivatives, have anti-inflammatory activity, but their mechanism of action was largely unknown. Marine alkaloid L-6-bromohypaphorine with EC50 of 80 µM acts as an agonist of α7 nicotinic acetylcholine receptor (nAChR) involved in anti-inflammatory regulation. We designed the 6-substituted hypaphorine analogs with increased potency using virtual screening of their binding to the α7 nAChR molecular model. Fourteen designed analogs were synthesized and tested in vitro by calcium fluorescence assay on the α7 nAChR expressed in neuro 2a cells, methoxy ester of D-6-iodohypaphorine (6ID) showing the highest potency (EC50 610 nM), being almost inactive toward α9α10 nAChR. The macrophages cytometry revealed an anti-inflammatory activity, decreasing the expression of TLR4 and increasing CD86, similarly to the action of PNU282987, a selective α7 nAChR agonist. 6ID administration in doses 0.1 and 0.5 mg/kg decreased carrageenan-induced allodynia and hyperalgesia in rodents, in accord with its anti-inflammatory action. Methoxy ester of D-6-nitrohypaphorine demonstrated anti-oedemic and analgesic effects in arthritis rat model at i.p. doses 0.05-0.26 mg/kg. Tested compounds showed excellent tolerability with no acute in vivo toxicity in dosages up to 100 mg/kg i.p. Thus, combining molecular modelling and natural product-inspired drug design improved the desired activity of the chosen nAChR ligand.

Inhibition of nicotinic acetylcholine receptors by oligoarginine peptides and polyamine-related compounds.

Oligoarginine peptides, known mostly for their cell-penetrating properties, are also inhibitors of the nicotinic acetylcholine receptors (nAChRs). Since octa-arginine (R8) inhibits α9α10 nAChR and suppresses neuropathic pain, we checked if other polycationic compounds containing amino and/or guanidino groups could be effective and tested the activity of the disulfide-fixed "cyclo"R8, a series of biogenic polyamines (putrescine, spermidine, and spermine), C-methylated spermine analogs, agmatine and its analogs, as well as acylpolyamine argiotoxin-636 from spider venom. Their inhibitory potency on muscle-type, α7 and α9α10 nAChRs was determined using radioligand analysis, electrophysiology, and calcium imaging. "Cyclo"R8 showed similar activity to that of R8 against α9α10 nAChR (IC50 ≈ 60 nM). Biogenic polyamines as well as agmatine and its analogs displayed low activity on muscle-type Torpedo californica, as well as α7 and α9α10 nAChRs, which increased with chain length, the most active being spermine and its C-methylated derivatives having IC50 of about 30 µM against muscle-type T. californica nAChR. Argiotoxin-636, which contains a polyamine backbone and terminal guanidino group, also weakly inhibited T. californica nAChR (IC50 ≈ 15 µM), but it revealed high potency against rat α9α10 nAChR (IC50 ≈ 200 nM). We conclude that oligoarginines and similar polycationic compounds effectively inhibiting α9α10 nAChR may serve as a basis for the development of analgesics to reduce neuropathic pain.

Snake Toxins Labeled by Green Fluorescent Protein or Its Synthetic Chromophore are New Probes for Nicotinic acetylcholine Receptors.

Fluorescence can be exploited to monitor intermolecular interactions in real time and at a resolution up to a single molecule. It is a method of choice to study ligand-receptor interactions. However, at least one of the interacting molecules should possess good fluorescence characteristics, which can be achieved by the introduction of a fluorescent label. Gene constructs with green fluorescent protein (GFP) are widely used to follow the expression of the respective fusion proteins and monitor their function. Recently, a small synthetic analogue of GFP chromophore (p-HOBDI-BF2) was successfully used for tagging DNA molecules, so we decided to test its applicability as a potential fluorescent label for proteins and peptides. This was done on α-cobratoxin (α-CbTx), a three-finger protein used as a molecular marker of muscle-type, neuronal α7 and α9/α10 nicotinic acetylcholine receptors (nAChRs), as well as on azemiopsin, a linear peptide neurotoxin selectively inhibiting muscle-type nAChRs. An activated N-hydroxysuccinimide ester of p-HOBDI-BF2 was prepared and utilized for toxin labeling. For comparison we used a recombinant α-CbTx fused with a full-length GFP prepared by expression of a chimeric gene. The structure of modified toxins was confirmed by mass spectrometry and their activity was characterized by competition with iodinated α-bungarotoxin in radioligand assay with respective receptor preparations, as well as by thermophoresis. With the tested protein and peptide neurotoxins, introduction of the synthetic GFP chromophore induced considerably lower decrease in their affinity for the receptors as compared with full-length GFP attachment. The obtained fluorescent derivatives were used for nAChR visualization in tissue slices and cell cultures.

Tetrapeptide Ac-HAEE-NH2 Protects α4ß2 nAChR from Inhibition by Aß.

The cholinergic deficit in Alzheimer's disease (AD) may arise from selective loss of cholinergic neurons caused by the binding of Aß peptide to nicotinic acetylcholine receptors (nAChRs). Thus, compounds preventing such an interaction are needed to address the cholinergic dysfunction. Recent findings suggest that the 11EVHH14 site in Aß peptide mediates its interaction with α4ß2 nAChR. This site contains several charged amino acid residues, hence we hypothesized that the formation of Aß-α4ß2 nAChR complex is based on the interaction of 11EVHH14 with its charge-complementary counterpart in α4ß2 nAChR. Indeed, we discovered a 35HAEE38 site in α4ß2 nAChR, which is charge-complementary to 11EVHH14, and molecular modeling showed that a stable Aß42-α4ß2 nAChR complex could be formed via the 11EVHH14:35HAEE38 interface. Using surface plasmon resonance and bioinformatics approaches, we further showed that a corresponding tetrapeptide Ac-HAEE-NH2 can bind to Aß via 11EVHH14 site. Finally, using two-electrode voltage clamp in Xenopus laevis oocytes, we showed that Ac-HAEE-NH2 tetrapeptide completely abolishes the Aß42-induced inhibition of α4ß2 nAChR. Thus, we suggest that 35HAEE38 is a potential binding site for Aß on α4ß2 nAChR and Ac-HAEE-NH2 tetrapeptide corresponding to this site is a potential therapeutic for the treatment of α4ß2 nAChR-dependent cholinergic dysfunction in AD.

Activation of α7 Nicotinic Acetylcholine Receptor Upregulates HLA-DR and Macrophage Receptors: Potential Role in Adaptive Immunity and in Preventing Immunosuppression.

Immune response during sepsis is characterized by hyper-inflammation followed by immunosuppression. The crucial role of macrophages is well-known for both septic stages, since they are involved in immune homeostasis and inflammation, their dysfunction being implicated in immunosuppression. The cholinergic anti-inflammatory pathway mediated by macrophage α7 nicotinic acetylcholine receptor (nAChR) represents possible drug target. Although α7 nAChR activation on macrophages reduces the production of proinflammatory cytokines, the role of these receptors in immunological changes at the cellular level is not fully understood. Using α7 nAChR selective agonist PNU 282,987, we investigated the influence of α7 nAChR activation on the expression of cytokines and, for the first time, of the macrophage membrane markers: cluster of differentiation 14 (CD14), human leukocyte antigen-DR (HLA-DR), CD11b, and CD54. Application of PNU 282,987 to THP-1Mϕ (THP-1 derived macrophages) cells led to inward ion currents and Ca2+ increase in cytoplasm showing the presence of functionally active α7 nAChR. Production of cytokines tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-10 was estimated in classically activated macrophages (M1) and treatment with PNU 282,987 diminished IL-10 expression. α7 nAChR activation on THP-1Mϕ, THP-1M1, and monocyte-derived macrophages (MDMs) increased the expression of HLA-DR, CD54, and CD11b molecules, but decreased CD14 receptor expression, these effects being blocked by alpha (α)-bungarotoxin. Thus, PNU 282,987 enhances the macrophage-mediated immunity via α7 nAChR by regulating expression of their membrane receptors and of cytokines, both playing an important role in preventing immunosuppressive states.

Complex approach for analysis of snake venom α-neurotoxins binding to HAP, the high-affinity peptide.

Snake venom α-neurotoxins, invaluable pharmacological tools, bind with high affinity to distinct subtypes of nicotinic acetylcholine receptor. The combinatorial high-affinity peptide (HAP), homologous to the C-loop of α1 and α7 nAChR subunits, binds biotinylated α-bungarotoxin (αBgt) with nanomolar affinity and might be a protection against snake-bites. Since there are no data on HAP interaction with other toxins, we checked its binding of α-cobratoxin (αCtx), similar to αBgt in action on nAChRs. Using radioiodinated αBgt, we confirmed a high affinity of HAP for αBgt, the complex formation is supported by mass spectrometry and gel chromatography, but only weak binding was registered with αCtx. A combination of protein intrinsic fluorescence measurements with the principal component analysis of the spectra allowed us to measure the HAP-αBgt binding constant directly (29 nM). These methods also confirmed weak HAP interaction with αCtx (>10000 nM). We attempted to enhance it by modification of HAP structure relying on the known structures of α-neurotoxins with various targets and applying molecular dynamics. A series of HAP analogues have been synthesized, HAP[L9E] analogue being considerably more potent than HAP in αCtx binding (7000 nM). The proposed combination of experimental and computational approaches appears promising for analysis of various peptide-protein interactions.

Isomerization of Asp7 in Beta-Amyloid Enhances Inhibition of the α7 Nicotinic Receptor and Promotes Neurotoxicity.

Cholinergic dysfunction in Alzheimer's disease (AD) can be mediated by the neuronal α7 nicotinic acetylcholine receptor (α7nAChR). Beta-amyloid peptide (Aß) binds to the α7nAChR, disrupting the receptor's function and causing neurotoxicity. In vivo not only Aß but also its modified forms can drive AD pathogenesis. One of these forms, iso-Aß (containing an isomerized Asp7 residue), shows an increased neurotoxicity in vitro and stimulates amyloidogenesis in vivo. We suggested that such effects of iso-Aß are α7nAChR-dependent. Here, using calcium imaging and electrophysiology, we found that iso-Aß is a more potent inhibitor of the α7nAChR-mediated calcium current than unmodified Aß. However, Asp7 isomerization eliminated the ability of Aß to decrease the α7nAChR levels. These data indicate differences in the interaction of the peptides with the α7nAChR, which we demonstrated using computer modeling. Neither Aß nor iso-Aß competed with 125I-α-bungarotoxin for binding to the orthosteric site of the receptor, suggesting the allosteric binging mode of the peptides. Further we found that increased neurotoxicity of iso-Aß was mediated by the α7nAChR. Thus, the isomerization of Asp7 enhances the inhibitory effect of Aß on the functional activity of the α7nAChR, which may be an important factor in the disruption of the cholinergic system in AD.

Curare alkaloids from Matis Dart Poison: Comparison with d-tubocurarine in interactions with nicotinic, 5-HT3 serotonin and GABAA receptors.

Several novel bisbenzylisoquinoline alkaloids (BBIQAs) have recently been isolated from a Matis tribe arrow poison and shown by two-electrode voltage-clamp to inhibit mouse muscle nicotinic acetylcholine receptors (nAChR). Here, using radioligand assay with Aplysia californica AChBP and radioiodinated α-bungarotoxin ([125I]-αBgt), we show that BBIQA1, BBIQA2, and d-tubocurarine (d-TC) have similar affinities to nAChR orthosteric site. However, a competition with [125I]-αBgt for binding to the Torpedo californica muscle-type nAChR revealed that BBIQAs1, 2, and 3 are less potent (IC50s = 26.3, 8.75, and 17.0 µM) than d-TC (IC50 = 0.39 µM), while with α7 nAChR in GH4C1 cells, BBIQA1 was less potent that d-TC (IC50s = 162 µM and 7.77 µM, respectively), but BBIQA2 was similar (IC50 = 5.52 µM). In inhibiting the Ca2+ responses induced by acetylcholine in Neuro2a cells expressing the mouse adult α1ß1εδ nAChR or human α7 nAChR, BBIQAs1 and 2 had similar potencies to d-TC (IC50s in the range 0.75-3.08 µM). Our data suggest that BBIQA1 and BBIQA2 can inhibit adult muscle α1ß1εδ nAChR by both competitive and noncompetitive mechanisms. Further experiments on neuronal α3ß2, α4ß2, and α9α10 nAChRs, expressed in Xenopus laevis oocytes, showed that similar potencies for BBIQAs1, 2, and d-TC. With α3ß2γ2 GABAAR currents were almost completely inhibited by d-TC at a high (100 µM) concentration, but BBIQAs1 and 2 were less potent (only 40-50% inhibition), whereas in competition with Alexa Fluor 546-α-cobratoxin for binding to α1ß3γ2 GABAAR in Neuro2a cells, d-TC and these analogs had comparable affinities. Especially interesting effects of BBIQAs1 and 2 in comparison with d-TC were observed for 5-HT3AR: BBIQA1 and BBIQA2 were 5- and 87-fold less potent than d-TC (IC50 = 22.63 nM). Thus, our results reveal that these BBIQAs differ from d-TC in their potencies towards certain Cys-loop receptors, and we suggest that understanding the reasons behind this might be useful for future drug design.

Makaluvamine G from the Marine Sponge Zyzzia fuliginosa Inhibits Muscle nAChR by Binding at the Orthosteric and Allosteric Sites.

Diverse ligands of the muscle nicotinic acetylcholine receptor (nAChR) are used as muscle relaxants during surgery. Although a plethora of such molecules exists in the market, there is still a need for new drugs with rapid on/off-set, increased selectivity, and so forth. We found that pyrroloiminoquinone alkaloid Makaluvamine G (MG) inhibits several subtypes of nicotinic receptors and ionotropic γ-aminobutiric acid receptors, showing a higher affinity and moderate selectivity toward muscle nAChR. The action of MG on the latter was studied by a combination of electrophysiology, radioligand assay, fluorescent microscopy, and computer modeling. MG reveals a combination of competitive and un-competitive inhibition and caused an increase in the apparent desensitization rate of the murine muscle nAChR. Modeling ion channel kinetics provided evidence for MG binding in both orthosteric and allosteric sites. We also demonstrated that theα1 (G153S) mutant of the receptor, associated with the myasthenic syndrome, is more prone to inhibition by MG. Thus, MG appears to be a perspective hit molecule for the design of allosteric drugs targeting muscle nAChR, especially for treating slow-channel congenital myasthenic syndromes.

Azemiopsin, a Selective Peptide Antagonist of Muscle Nicotinic Acetylcholine Receptor: Preclinical Evaluation as a Local Muscle Relaxant.

Azemiopsin (Az), a linear peptide from the Azemiops feae viper venom, contains no disulfide bonds, is a high-affinity and selective inhibitor of nicotinic acetylcholine receptor (nAChR) of muscle type and may be considered as potentially applicable nondepolarizing muscle relaxant. In this study, we investigated its preclinical profile in regard to in vitro and in vivo efficacy, acute and chronic toxicity, pharmacokinetics, allergenic capacity, immunotoxicity and mutagenic potency. The peptide effectively inhibited (IC50 ~ 19 nM) calcium response of muscle nAChR evoked by 30 µM (EC100) acetylcholine but was less potent (IC50 ~ 3 µM) at α7 nAChR activated by 10 µM (EC50) acetylcholine and had a low affinity to α4ß2 and α3-containing nAChR, as well as to GABAA or 5HT3 receptors. Its muscle relaxant effect was demonstrated at intramuscular injection to mice at doses of 30-300 µg/kg, 30 µg/kg being the initial effective dose and 90 µg/kg-the average effective dose. The maximal muscle relaxant effect of Az was achieved in 10 min after the administration and elimination half-life of Az in mice was calculated as 20-40 min. The longest period of Az action observed at a dose of 300 µg/kg was 55 min. The highest acute toxicity (LD50 510 µg/kg) was observed at intravenous injection of Az, at intramuscular or intraperitoneal administration it was less toxic. The peptide showed practically no immunotoxic, allergenic or mutagenic capacity. Overall, the results demonstrate that Az has good drug-like properties for the application as local muscle relaxant and in its parameters, is not inferior to the relaxants currently used. However, some Az modification might be effective to extend its narrow therapeutic window, a typical characteristic and a weak point of all nondepolarizing myorelaxants.

Interaction of Synthetic Human SLURP-1 with the Nicotinic Acetylcholine Receptors.

Human SLURP-1 is a secreted protein of the Ly6/uPAR/three-finger neurotoxin family that co-localizes with nicotinic acetylcholine receptors (nAChRs) and modulates their functions. Conflicting biological activities of SLURP-1 at various nAChR subtypes have been based on heterologously produced SLURP-1 containing N- and/or C-terminal extensions. Here, we report the chemical synthesis of the 81 amino acid residue human SLURP-1 protein, characterization of its 3D structure by NMR, and its biological activity at nAChR subtypes. Radioligand assays indicated that synthetic SLURP-1 did not compete with [125I]-α-bungarotoxin (α-Bgt) binding to human neuronal α7 and Torpedo californica muscle-type nAChRs, nor to mollusk acetylcholine binding proteins (AChBP). Inhibition of human α7-mediated currents only occurred in the presence of the allosteric modulator PNU120596. In contrast, we observed robust SLURP-1 mediated inhibition of human α3ß4, α4ß4, α3ß2 nAChRs, as well as human and rat α9α10 nAChRs. SLURP-1 inhibition of α9α10 nAChRs was accentuated at higher ACh concentrations, indicating an allosteric binding mechanism. Our results are discussed in the context of recent studies on heterologously produced SLURP-1 and indicate that N-terminal extensions of SLURP-1 may affect its activity and selectivity on its targets. In this respect, synthetic SLURP-1 appears to be a better probe for structure-function studies.

Pancreatic and snake venom presynaptically active phospholipases A2 inhibit nicotinic acetylcholine receptors.

Phospholipases A2 (PLA2s) are enzymes found throughout the animal kingdom. They hydrolyze phospholipids in the sn-2 position producing lysophospholipids and unsaturated fatty acids, agents that can damage membranes. PLA2s from snake venoms have numerous toxic effects, not all of which can be explained by phospholipid hydrolysis, and each enzyme has a specific effect. We have earlier demonstrated the capability of several snake venom PLA2s with different enzymatic, cytotoxic, anticoagulant and antiproliferative properties, to decrease acetylcholine-induced currents in Lymnaea stagnalis neurons, and to compete with α-bungarotoxin for binding to nicotinic acetylcholine receptors (nAChRs) and acetylcholine binding protein. Since nAChRs are implicated in postsynaptic and presynaptic activities, in this work we probe those PLA2s known to have strong presynaptic effects, namely ß-bungarotoxin from Bungarus multicinctus and crotoxin from Crotalus durissus terrificus. We also wished to explore whether mammalian PLA2s interact with nAChRs, and have examined non-toxic PLA2 from porcine pancreas. It was found that porcine pancreatic PLA2 and presynaptic ß-bungarotoxin blocked currents mediated by nAChRs in Lymnaea neurons with IC50s of 2.5 and 4.8 µM, respectively. Crotoxin competed with radioactive α-bungarotoxin for binding to Torpedo and human α7 nAChRs and to the acetylcholine binding protein. Pancreatic PLA2 interacted similarly with these targets; moreover, it inhibited radioactive α-bungarotoxin binding to the water-soluble extracellular domain of human α9 nAChR, and blocked acetylcholine induced currents in human α9α10 nAChRs heterologously expressed in Xenopus oocytes. These and our earlier results show that all snake PLA2s, including presynaptically active crotoxin and ß-bungarotoxin, as well as mammalian pancreatic PLA2, interact with nAChRs. The data obtained suggest that this interaction may be a general property of all PLA2s, which should be proved by further experiments.

Peptide from Sea Anemone Metridium senile Affects Transient Receptor Potential Ankyrin-repeat 1 (TRPA1) Function and Produces Analgesic Effect.

The transient receptor potential ankyrin-repeat 1 (TRPA1) is an important player in pain and inflammatory pathways. It is a promising target for novel drug development for the treatment of a number of pathological states. A novel peptide producing a significant potentiating effect on allyl isothiocyanate- and diclofenac-induced currents of TRPA1 was isolated from the venom of sea anemone Metridium senile. It is a 35-amino acid peptide cross-linked by two disulfide bridges named τ-AnmTX Ms 9a-1 (short name Ms 9a-1) according to a structure similar to other sea anemone peptides belonging to structural group 9a. The structures of the two genes encoding the different precursor proteins of Ms 9a-1 were determined. Peptide Ms 9a-1 acted as a positive modulator of TRPA1 in vitro but did not cause pain or thermal hyperalgesia when injected into the hind paw of mice. Intravenous injection of Ms 9a-1 (0.3 mg/kg) produced a significant decrease in the nociceptive and inflammatory response to allyl isothiocyanate (the agonist of TRPA1) and reversed CFA (Complete Freund's Adjuvant)-induced inflammation and thermal hyperalgesia. Taken together these data support the hypothesis that Ms 9a-1 potentiates the response of TRPA1 to endogenous agonists followed by persistent functional loss of TRPA1-expressing neurons. We can conclude that TRPA1 potentiating may be useful as a therapeutic approach as Ms 9a-1 produces significant analgesic and anti-inflammatory effects in mice models of pain.

High-Affinity α-Conotoxin PnIA Analogs Designed on the Basis of the Protein Surface Topography Method.

Despite some success for small molecules, elucidating structure-function relationships for biologically active peptides - the ligands for various targets in the organism - remains a great challenge and calls for the development of novel approaches. Some of us recently proposed the Protein Surface Topography (PST) approach, which benefits from a simplified representation of biomolecules' surface as projection maps, which enables the exposure of the structure-function dependencies. Here, we use PST to uncover the "activity pattern" in α-conotoxins - neuroactive peptides that effectively target nicotinic acetylcholine receptors (nAChRs). PST was applied in order to design several variants of the α-conotoxin PnIA, which were synthesized and thoroughly studied. Among the best was PnIA[R9, L10], which exhibits nanomolar affinity for the α7 nAChR, selectivity and a slow wash-out from this target. Importantly, these mutations could hardly be delineated by "standard" structure-based drug design. The proposed combination of PST with a set of experiments proved very efficient for the rational construction of new bioactive molecules.

Peptides from puff adder Bitis arietans venom, novel inhibitors of nicotinic acetylcholine receptors.

Phospholipase A2 (named bitanarin) possessing capability to block nicotinic acetylcholine receptors (nAChRs) was isolated earlier (Vulfius et al., 2011) from puff adder Bitis arietans venom. Further studies indicated that low molecular weight fractions of puff adder venom inhibit nAChRs as well. In this paper, we report on isolation from this venom and characterization of three novel peptides called baptides 1, 2 and 3 that reversibly block nAChRs. To isolate the peptides, the venom of B. arietans was fractionated by gel-filtration and reversed phase chromatography. The amino acid sequences of peptides were established by de novo sequencing using MALDI mass spectrometry. Baptide 1 comprised 7, baptides 2 and 3-10 amino acid residues, the latter being acetylated at the N-terminus. This is the first indication for the presence of such post-translational modification in snake venom proteins. None of the peptides contain cysteine residues. For biological activity studies the peptides were prepared by solid phase peptide synthesis. Baptide 3 and 2 blocked acetylcholine-elicited currents in isolated Lymnaea stagnalis neurons with IC50 of about 50 µM and 250 µM, respectively. In addition baptide 2 blocked acetylcholine-induced currents in muscle nAChR heterologously expressed in Xenopus oocytes with IC50 of about 3 µM. The peptides did not compete with radioactive α-bungarotoxin for binding to Torpedo and α7 nAChRs at concentration up to 200 µM that suggests non-competitive mode of inhibition. Calcium imaging studies on α7 and muscle nAChRs heterologously expressed in mouse neuroblastoma Neuro2a cells showed that on α7 receptor baptide 2 inhibited acetylcholine-induced increasing intracellular calcium concentration with IC50 of 20.6 ± 3.93 µM. On both α7 and muscle nAChRs the suppression of maximal response to acetylcholine by about 50% was observed at baptide 2 concentration of 25 µM, the value being close to IC50 on α7 nAChR. These data are in accord with non-competitive inhibition as follows from α-bungarotoxin binding experiments. The described peptides are the shortest peptides without disulfide bridges isolated from animal venom and capable to inhibit nAChR by non-competitive way.

Nicotinic receptor involvement in regulation of functions of mouse neutrophils from inflammatory site.

Participation of nicotinic acetylcholine receptors (nAChRs) in functioning of polymorphonuclear neutrophils (PMNs) isolated from inflammatory site of mice and expression of different nAChR subunits were studied. Nicotine and acetylcholine (ACh) modified respiratory burst induced by a chemotactic peptide N-formyl-MLF in neutrophils of male (but not female) mice. Antagonists of nAChRs α-cobratoxin (αCTX), α-conotoxins MII and [A10L]PnIA at concentrations of 0.01-5µM, 0.2µM and 1µM, respectively, eliminated nAChR agonist effects. ACh also affected adhesion of PMNs, this effect was also prevented by αCTX (100nM) and MII (1nM). Neutrophils of female mice after chronic nicotine consumption acquired sensitivity to nAChR agonists. Changes of free intracellular Ca(2+) concentration in neutrophils under the action of nAChR ligands were analyzed. In cells with no Ca(2+) oscillations and relatively low resting level of intracellular Ca(2+), nicotine triggered Ca(2+)-spikes, the lag of the response shortened with increasing nicotine concentration. A nicotinic antagonist caramiphen strongly decreased the effect of nicotine. RT-PCR analysis revealed mRNAs of α2, α3, α4, α5, α6, α7, α9, ß2, ß3, and ß4 nAChR subunits. Specific binding of [(125)I]-α-bungarotoxin was demonstrated. Thus in view of the effects and binding characteristics the results obtained suggest a regulatory role of α7, α3ß2 or α6* nAChR types in specific functions of PMNs.

Curare Alkaloids: Constituents of a Matis Dart Poison.

A phytochemical study of dart and arrow poison from the Matis tribe led to the identification of D-(-)-quinic acid, L-malic acid, ethyldimethylamine, magnoflorine, and five new bisbenzyltetrahydroisoquinoline alkaloids (BBIQAs), 1-5. D-Tubocurarine could not be identified among these products. BBIQA (3) contains a unique linkage at C-8 and C-11'. All structures were characterized by a combination of NMR and HRESIMS data. The effects of Matis poison and individual BBIQAs (1-3) on rat muscle nAChR expressed in Xenopus oocytes have been investigated using the two-electrode voltage clamp technique.

Neurotoxins from snake venoms and α-conotoxin ImI inhibit functionally active ionotropic γ-aminobutyric acid (GABA) receptors.

Ionotropic receptors of γ-aminobutyric acid (GABAAR) regulate neuronal inhibition and are targeted by benzodiazepines and general anesthetics. We show that a fluorescent derivative of α-cobratoxin (α-Ctx), belonging to the family of three-finger toxins from snake venoms, specifically stained the α1ß3γ2 receptor; and at 10 µm α-Ctx completely blocked GABA-induced currents in this receptor expressed in Xenopus oocytes (IC50 = 236 nm) and less potently inhibited α1ß2γ2 ≈ α2ß2γ2 > α5ß2γ2 > α2ß3γ2 and α1ß3δ GABAARs. The α1ß3γ2 receptor was also inhibited by some other three-finger toxins, long α-neurotoxin Ls III and nonconventional toxin WTX. α-Conotoxin ImI displayed inhibitory activity as well. Electrophysiology experiments showed mixed competitive and noncompetitive α-Ctx action. Fluorescent α-Ctx, however, could be displaced by muscimol indicating that most of the α-Ctx-binding sites overlap with the orthosteric sites at the ß/α subunit interface. Modeling and molecular dynamic studies indicated that α-Ctx or α-bungarotoxin seem to interact with GABAAR in a way similar to their interaction with the acetylcholine-binding protein or the ligand-binding domain of nicotinic receptors. This was supported by mutagenesis studies and experiments with α-conotoxin ImI and a chimeric Naja oxiana α-neurotoxin indicating that the major role in α-Ctx binding to GABAAR is played by the tip of its central loop II accommodating under loop C of the receptors.

6-bromohypaphorine from marine nudibranch mollusk Hermissenda crassicornis is an agonist of human α7 nicotinic acetylcholine receptor.

6-Bromohypaphorine (6-BHP) has been isolated from the marine sponges Pachymatisma johnstoni, Aplysina sp., and the tunicate Aplidium conicum, but data on its biological activity were not available. For the nudibranch mollusk Hermissenda crassicornis no endogenous compounds were known, and here we describe the isolation of 6-BHP from this mollusk and its effects on different nicotinic acetylcholine receptors (nAChR). Two-electrode voltage-clamp experiments on the chimeric α7 nAChR (built of chicken α7 ligand-binding and glycine receptor transmembrane domains) or on rat α4ß2 nAChR expressed in Xenopus oocytes revealed no action of 6-BHP. However, in radioligand analysis, 6-BHP competed with radioiodinated α-bungarotoxin for binding to human α7 nAChR expressed in GH4C1 cells (IC50 23 ± 1 µM), but showed no competition on muscle-type nAChR from Torpedo californica. In Ca2+-imaging experiments on the human α7 nAChR expressed in the Neuro2a cells, 6-BHP in the presence of PNU120596 behaved as an agonist (EC50 ~80 µM). To the best of our knowledge, 6-BHP is the first low-molecular weight compound from marine source which is an agonist of the nAChR subtype. This may have physiological importance because H. crassicornis, with its simple and tractable nervous system, is a convenient model system for studying the learning and memory processes.