α9α10 nicotinic acetylcholine receptors regulate murine bone marrow granulocyte functions.

Polymorphonuclear neutrophilic granulocytes (PMNs) are extremely important in defense of the organism against infections and in inflammatory processes including neuroinflammation and pain sensation. Different subtypes of nicotinic acetylcholine receptors (nAChRs) are involved in modulation of PMN activities. Earlier we determined expression of α2-7, α9, ß3, ß4 subunits and regulatory role of α7 and α3ß2 nAChR subtypes in functions of inflammatory PMNs. Other authors detected mRNA of α9 subunit in bone marrow neutrophils (BM-PMNs). Murine BM-PMNs coming out from the bone marrow, where they develop, to blood were characterized as mature. There was no data for α10 and for the presence of functionally active α9α10 nAChRs in BM-PMNs. Here we detected for the first time mRNA expression of the α10 nAChR subunit in BM-PMNs and confirmed the expression of mRNA for α9 nAChR. With the help of α-conotoxins RgIA and Vc1.1, highly selective antagonists of α9α10 nAChRs, we have revealed participation of α9 and/or α9α10 nAChRs in regulation of cytosolic Ca2+ concentration, cell adhesion, and in generation of reactive oxygen species (ROS). Nicotine, choline, RgIA, and Vc1.1 induced Ca2+ transients in BM-PMNs, enhanced cell adhesiveness and decreased production of ROS indicating involvement of α9, possibly co-assembled with α10, nAChRs in the BM-PMN activity for recruitment and cytotoxicity.

PNU-120596, a positive allosteric modulator of mammalian α7 nicotinic acetylcholine receptor, is a negative modulator of ligand-gated chloride-selective channels of the gastropod Lymnaea stagnalis.

Excitatory α7 neuronal nicotinic receptors (nAChR) are widely expressed in the central and peripheral nervous and immune systems and are important for learning, memory, and immune response regulation. Specific α7 nAChR ligands, including positive allosteric modulators are promising to treat cognitive disorders, inflammatory processes, and pain. One of them, PNU-120596, highly increased the neuron response to α7 agonists and retarded desensitization, showing selectivity for α7 as compared to heteromeric nAChRs, but was not examined at the inhibitory ligand-gated channels. We studied PNU-120596 action on anion-conducting channels using voltage-clamp techniques: it slightly potentiated the response of human glycine receptors expressed in PC12 cells, of rat GABAA receptors in cerebellar Purkinje cells and mouse GABAA Rs heterologously expressed in Xenopus oocytes. On the contrary, PNU-120596 exerted an inhibitory effect on the receptors mediating anion currents in Lymnaea stagnalis neurons: two nAChR subtypes, GABA and glutamate receptors. Acceleration of the current decay, contrary to slowing down desensitization in mammalian α7 nAChR, was observed in L. stagnalis neurons predominantly expressing one of the two nAChR subtypes. Thus, PNU-120596 effect on these anion-selective nAChRs was just opposite to the action on the mammalian cation-selective α7 nAChRs. A comparison of PNU-120596 molecule docked to the models of transmembrane domains of the human α7 AChR and two subunits of L. stagnalis nAChR demonstrated some differences in contacts with the amino acid residues important for PNU-120596 action on the α7 nAChR. Thus, our results show that PNU-120596 action depends on a particular subtype of these Cys-loop receptors.

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.

An unusual phospholipase A2 from puff adder Bitis arietans venom--a novel blocker of nicotinic acetylcholine receptors.

The venoms of snakes from Viperidae family mainly influence the function of various blood components. However, the published data indicate that these venoms contain also neuroactive components, the most studied being neurotoxic phospholipases A2 (PLA2s). Earlier we have shown (Gorbacheva et al., 2008) that several Viperidae venoms blocked nicotinic acetylcholine receptors (nAChRs) and voltage-gated Ca²+ channels in isolated identified neurons of the fresh-water snail Lymnaea stagnalis. In this paper, we report on isolation from puff adder Bitis arietans venom and characterization of a novel protein bitanarin that reversibly blocks nAChRs. To isolate the protein, the venom of B. arietans was fractionated by gel-filtration, ion-exchange and reversed phase chromatography and fractions obtained were screened for capability to block nAChRs. The isolated protein competed with [¹²5I]iodinated α-bungarotoxin for binding to human α7 and Torpedo californica nAChRs, as well as to acetylcholine-binding protein from L. stagnalis, the IC50 being 20 ± 1.5, 4.3 ± 0.2, and 10.6 ± 0.6 µM, respectively. It also blocked reversibly acetylcholine-elicited current in isolated L. stagnalis neurons with IC50 of 11.4 µM. Mass-spectrometry analysis determined the molecular mass of 27.4 kDa and the presence of 28 cysteine residues forming 14 disulphide bonds. Edman degradation of the protein and tryptic fragments showed its similarity to PLA2s from snake venoms. Indeed, the protein possessed high PLA2 activity, which was 1.95 mmol/min/µmol. Bitanarin is the first described PLA2 that contains 14 disulphide bonds and the first nAChR blocker possessing PLA2 activity.