“…mRNA for the α9 nAChR subunit was previously detected in BM-PMNs ( St-Pierre et al, 2016 ) and recently confirmed by us ( Safronova et al, 2021 ). In addition, we identified for the first time mRNA of the α10 subunit in these cells ( Safronova et al, 2021 ).…”
Section: Resultssupporting
confidence: 69%
“…In collaboration with crystallographers from Hellenic Pasteur Institute (Athens, Greece), we contributed to the determination of the X-ray structure of α-conotoxin RgIA in complex with the LBD of the α9 subunit ( Zouridakis et al, 2019 ). We further found that α-conotoxins RgIA and Vc1.1 influence cytosolic Ca 2+ concentration, cell adhesion, and generation of reactive oxygen species in murine bone marrow granulocytes ( Safronova et al, 2021 ). In this special issue on the α9α10 nAChR subtype, we will briefly discuss these findings and (1) report the selectivity and potency of novel α-neurotoxins from Naja melanoleuca snake venom at human α9α10 nAChRs and (2) present new data showing that α9α10-selective α-conotoxins potentiate release of the anti-inflammatory cytokine interleukin-10 (IL-10) from murine granulocytes.…”
Section: Introductionmentioning
confidence: 74%
“…The synthesis of α-conotoxins MII, RgIA, and Vc1.1 was described in Safronova et al (2021) , GeXIVA and PeIA in Kryukova et al (2018) . α-Neurotoxins were isolated from snake venoms: long-chain Tx-NM2 and Tx-NM3-1 from N. melanoleuca venom ( Son et al, 2021 ); long-chain neurotoxin I (NT I) and short-chain neurotoxin II (NT II) from N. oxiana and α-bungarotoxin (α-Btx) from Bungarus multicinctus ( Kudryavtsev et al, 2015 ); non-conventional WTX and long-chain α-cobratoxin (α-Ctx) from N. kaouthia ( Utkin et al, 2001 ; Osipov et al, 2008 , respectively).…”
Section: Methodsmentioning
confidence: 99%
“…Polymorphonuclear neutrophilic granulocytes (PMNs) were isolated from murine bone marrow using the previously described method ( Safronova et al, 2021 ). Shortly, a cell suspension was obtained after washing out murine femur, tibia, and humerus with cold RPMI-1640 medium and layered on a Percoll gradient (78, 62.5, and 55% in PBS).…”
Unlike most neuronal nicotinic acetylcholine receptor (nAChR) subunits, α7, α9, and α10 subunits are able to form functional homo- or heteromeric receptors without any β subunits. While the α7 subtype is widely distributed in the mammalian brain and several peripheral tissues, α9 and α9α10 nAChRs are mainly found in the cochlea and immune cells. α-Conotoxins that specifically block the α9α10 receptor showed anti-nociceptive and anti-hyperalgesic effects in animal models. Hence, this subtype is considered a drug target for analgesics. In contrast to the α9α10-selective α-conotoxins, the three-finger toxin α-bungarotoxin inhibits muscle-type and α7 nAChRs in addition to α9α10 nAChRs. However, the selectivity of α-neurotoxins at the α9α10 subtype was less intensively investigated. Here, we compared the potencies of α-conotoxins and α-neurotoxins at the human α9α10 nAChR by two-electrode voltage clamp analysis upon expression in Xenopus oocytes. In addition, we analyzed effects of several α9α10-selective α-conotoxins on mouse granulocytes from bone marrow to identify possible physiological functions of the α9α10 nAChR subtype in these cells. The α-conotoxin-induced IL-10 release was measured upon LPS-stimulation. We found that α-conotoxins RgIA, PeIA, and Vc1.1 enhance the IL-10 expression in granulocytes which might explain the known anti-inflammatory and associated analgesic activities of α9α10-selective α-conotoxins. Furthermore, we show that two long-chain α-neurotoxins from the cobra Naja melanoleuca venom that were earlier shown to bind to muscle-type and α7 nAChRs, also inhibit the α9α10 subtype at nanomolar concentrations with one of them showing a significantly slower dissociation from this receptor than α-bungarotoxin.
“…mRNA for the α9 nAChR subunit was previously detected in BM-PMNs ( St-Pierre et al, 2016 ) and recently confirmed by us ( Safronova et al, 2021 ). In addition, we identified for the first time mRNA of the α10 subunit in these cells ( Safronova et al, 2021 ).…”
Section: Resultssupporting
confidence: 69%
“…In collaboration with crystallographers from Hellenic Pasteur Institute (Athens, Greece), we contributed to the determination of the X-ray structure of α-conotoxin RgIA in complex with the LBD of the α9 subunit ( Zouridakis et al, 2019 ). We further found that α-conotoxins RgIA and Vc1.1 influence cytosolic Ca 2+ concentration, cell adhesion, and generation of reactive oxygen species in murine bone marrow granulocytes ( Safronova et al, 2021 ). In this special issue on the α9α10 nAChR subtype, we will briefly discuss these findings and (1) report the selectivity and potency of novel α-neurotoxins from Naja melanoleuca snake venom at human α9α10 nAChRs and (2) present new data showing that α9α10-selective α-conotoxins potentiate release of the anti-inflammatory cytokine interleukin-10 (IL-10) from murine granulocytes.…”
Section: Introductionmentioning
confidence: 74%
“…The synthesis of α-conotoxins MII, RgIA, and Vc1.1 was described in Safronova et al (2021) , GeXIVA and PeIA in Kryukova et al (2018) . α-Neurotoxins were isolated from snake venoms: long-chain Tx-NM2 and Tx-NM3-1 from N. melanoleuca venom ( Son et al, 2021 ); long-chain neurotoxin I (NT I) and short-chain neurotoxin II (NT II) from N. oxiana and α-bungarotoxin (α-Btx) from Bungarus multicinctus ( Kudryavtsev et al, 2015 ); non-conventional WTX and long-chain α-cobratoxin (α-Ctx) from N. kaouthia ( Utkin et al, 2001 ; Osipov et al, 2008 , respectively).…”
Section: Methodsmentioning
confidence: 99%
“…Polymorphonuclear neutrophilic granulocytes (PMNs) were isolated from murine bone marrow using the previously described method ( Safronova et al, 2021 ). Shortly, a cell suspension was obtained after washing out murine femur, tibia, and humerus with cold RPMI-1640 medium and layered on a Percoll gradient (78, 62.5, and 55% in PBS).…”
Unlike most neuronal nicotinic acetylcholine receptor (nAChR) subunits, α7, α9, and α10 subunits are able to form functional homo- or heteromeric receptors without any β subunits. While the α7 subtype is widely distributed in the mammalian brain and several peripheral tissues, α9 and α9α10 nAChRs are mainly found in the cochlea and immune cells. α-Conotoxins that specifically block the α9α10 receptor showed anti-nociceptive and anti-hyperalgesic effects in animal models. Hence, this subtype is considered a drug target for analgesics. In contrast to the α9α10-selective α-conotoxins, the three-finger toxin α-bungarotoxin inhibits muscle-type and α7 nAChRs in addition to α9α10 nAChRs. However, the selectivity of α-neurotoxins at the α9α10 subtype was less intensively investigated. Here, we compared the potencies of α-conotoxins and α-neurotoxins at the human α9α10 nAChR by two-electrode voltage clamp analysis upon expression in Xenopus oocytes. In addition, we analyzed effects of several α9α10-selective α-conotoxins on mouse granulocytes from bone marrow to identify possible physiological functions of the α9α10 nAChR subtype in these cells. The α-conotoxin-induced IL-10 release was measured upon LPS-stimulation. We found that α-conotoxins RgIA, PeIA, and Vc1.1 enhance the IL-10 expression in granulocytes which might explain the known anti-inflammatory and associated analgesic activities of α9α10-selective α-conotoxins. Furthermore, we show that two long-chain α-neurotoxins from the cobra Naja melanoleuca venom that were earlier shown to bind to muscle-type and α7 nAChRs, also inhibit the α9α10 subtype at nanomolar concentrations with one of them showing a significantly slower dissociation from this receptor than α-bungarotoxin.
“…We previously found in the whole BM‐PMNs population that nAChR ligands (choline, nicotine, RgIA, and Vc1.1.) increased the proportions of BM‐PMN with Ca 2+ ‐spikes and number of adhered cells and decreased ROS production (Safronova et al, 2020). This is in agreement with data on decreasing of ROS production by isolated human peripheral blood neutrophils in response to nicotine (Matthews et al, 2011).…”
Polymorphonuclear neutrophilic granulocytes (PMNs) are the largest proportion of leukocytes in adult human blood that perform numerous functions, including phagocytosis, degranulation, generation of reactive oxygen species, and NETosis. Excessive neutrophil activity associates with hyperinflammation and tissue damage during pathologies such as inflammatory bowel disease, diabetes mellitus, tuberculosis, and coronavirus disease 2019. Nicotinic acetylcholine receptors (nAChRs) can modulate immune cells, including neutrophils, functions, therefore, nAChR ligands are considered as the potent agents for therapy of inflammation. Earlier it was shown, that about 30% of PMNs from the acute inflammatory site responded to nicotine by calcium spikes. In this study, we studied the generation of calcium spikes in murine granulocytes with different maturity level (evaluated by Gr‐1 expression) isolated from bone marrow in response to ligands of nAChRs in control and under chronic nicotine consumption. It was found that nearly 20%–25% cells in the granulocyte population responded to nicotine or selective antagonists of different type of nAChRs (α‐cobratoxin, GIC, and Vc1.1). We demonstrated that in the control group Ca2+‐mobilizing activity was regulated through α7 and α9α10 nAChRs in immature granulocytes (Gr‐1int), whereas in mature granulocytes (Gr‐1hi) it was regulated through α7, α3β2, and α9‐contained nAChRs. Sensitivity of PMNs to nicotine depended on their maturity level after chronic nicotine consumption. Gr‐1int cells responded to nicotine through α7 and α9‐contained nAChRs, while Gr‐1hi did not respond to nicotine. Thus, calcium response to nAChR ligands in bone marrow PMNs depends on their maturity level.
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