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Prorocentrolides are members of the cyclic imine phycotoxins family. Their chemical structure includes a 26-membered carbo-macrocycle and a 28-membered macrocyclic lactone arranged around a hexahydroisoquinoline that incorporates the characteristic cyclic imine group. Six prorocentrolides are already known. However, their mode of action remains undetermined. The aim of the present work was to explore whether prorocentrolide A acts on nicotinic acetylcholine receptors (nAChRs), using competition-binding assays and electrophysiological techniques. Prorocentrolide-A displaced [125I]α-bungarotoxin binding to Torpedo membranes, expressing the muscle-type (α1₂β1γδ) nAChR, and in HEK-293 cells, expressing the chimeric chick neuronal α7-5HT₃ nAChR. Functional studies revealed that prorocentrolide-A had no agonist action on nAChRs, but inhibited ACh-induced currents in Xenopus oocytes that had incorporated the muscle-type α1₂β1γδ nAChR to their membranes, or that expressed the human α7 nAChR, as revealed by voltage-clamp recordings. Molecular docking calculations showed the absence of the characteristic hydrogen bond between the iminium group of prorocentrolide-A and the backbone carbonyl group of Trp147 in the receptor, explaining its weaker affinity as compared to all other cyclic imine toxins. In conclusion, this is the first study to show that prorocentrolide-A acts on both muscle and neuronal nAChRs, but with higher affinity on the muscle-type nAChR.

The cyclic imine toxin 20-methyl spirolide G (20-meSPX-G), produced by the toxigenic dinoflagellate Alexandrium ostenfeldii/Alexandrium peruvianum, has been previously reported to contaminate shellfish in various European coastal locations, as revealed by mouse toxicity bioassay. The aim of the present study was to determine its toxicological profile and its molecular target selectivity. 20-meSPX-G blocked nerve-evoked isometric contractions in isolated mouse neuromuscular preparations, while it had no action on contractions elicited by direct electrical stimulation, and reduced reversibly nerve-evoked compound muscle action potential amplitudes in anesthetized mice. Voltage-clamp recordings in Xenopus oocytes revealed that 20-meSPX-G potently inhibited currents evoked by ACh on Torpedo muscle-type and human α7 nicotinic acetylcholine receptors (nAChR), whereas lower potency was observed in human α4β2 nAChR. Competition-binding assays showed that 20-meSPX-G fully displaced [3H]epibatidine binding to HEK-293 cells expressing the human α3β2 (Ki = 0.040 nM), whereas a 90-fold lower affinity was detected in human α4β2 nAChR. The spirolide displaced [125I]α-bungarotoxin binding to Torpedo membranes (Ki = 0.028 nM) and in HEK-293 cells expressing chick chimeric α7-5HT3 nAChR (Ki = 0.11 nM). In conclusion, this is the first study to demonstrate that 20-meSPX-G is a potent antagonist of nAChRs, and its subtype selectivity is discussed on the basis of molecular docking models.

Prorocentrolides are members of the cyclic imine phycotoxins family. Their chemical structure includes a 26-membered carbo-macrocycle and a 28-membered macrocyclic lactone arranged around a hexahydroisoquinoline that incorporates the characteristic cyclic imine group. Six prorocentrolides are already known. However, their mode of action remains undetermined. The aim of the present work was to explore whether prorocentrolide-A acts on nicotinic acetylcholine receptors (nAChRs), using competition-binding assays and electrophysiological techniques. Prorocentrolide-A displaced [125I]α-bungarotoxin binding to Torpedo membranes, expressing the muscle-type (α12β1γδ) nAChR, and in HEK-293 cells, expressing the chimeric chick neuronal α7-5HT3 nAChR. Functional studies revealed that prorocentrolide-A had no agonist action on nAChRs, but inhibited ACh-induced currents in Xenopus oocytes that had incorporated the muscle-type α12β1γδ nAChR to their membranes, or that expressed the human α7 nAChR, as revealed by voltage-clamp recordings. Molecular docking calculations showed the absence of the characteristic hydrogen bond between the iminium group of prorocentrolide-A and the backbone carbonyl group of Trp147 in the receptor, explaining its weaker affinity as compared to all other cyclic imine toxins. In conclusion, this is the first study to show that prorocentrolide-A acts on both muscle and neuronal nAChRs, but with higher affinity on the muscle-type nAChR.

The α9α10 nicotinic cholinergic receptor (nAChR) is a ligand-gated pentameric cation-permeable ion channel that mediates synaptic transmission between descending efferent neurons and mechanosensory inner ear hair cells. When expressed in heterologous systems, α9 and α10 subunits can assemble into functional homomeric α9 and heteromeric α9α10 receptors. One of the differential properties between these nAChRs is the modulation of their ACh-evoked responses by extracellular calcium (Ca 2+ ). While α9 nAChRs responses are blocked by Ca 2+ , ACh-evoked currents through α9α10 nAChRs are potentiated by Ca 2+ in the micromolar range and blocked at millimolar concentrations. Using chimeric and mutant subunits, together with electrophysiological recordings under two-electrode voltage-clamp, we show that the TM2-TM3 loop of the rat α10 subunit contains key structural determinants responsible for the potentiation of the α9α10 nAChR by extracellular Ca 2+ . Moreover, molecular dynamics simulations reveal that the TM2-TM3 loop of α10 does not contribute to the Ca 2+ potentiation phenotype through the formation of novel Ca 2+ binding sites not present in the α9 receptor. These results suggest that the TM2-TM3 loop of α10 might act as a control element that facilitates the intramolecular rearrangements that follow ACh-evoked α9α10 nAChRs gating in response to local and transient changes of extracellular Ca 2+ concentration. This finding might pave the way for the future rational design of drugs that target α9α10 nAChRs as otoprotectants.

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.

Blood levels of the acute phase reactant C-reactive protein (CRP) are frequently measured as a clinical marker for inflammation, but the biological functions of CRP are still controversial. CRP is a phosphocholine (PC)-binding pentraxin, mainly produced in the liver in response to elevated levels of interleukin-1β (IL-1β) and of the IL-1β-dependent cytokine IL-6. While both cytokines play important roles in host defense, excessive systemic IL-1β levels can cause life-threatening diseases such as trauma-associated systemic inflammation. We hypothesized that CRP acts as a negative feedback regulator of monocytic IL-1β maturation and secretion. Here, we demonstrate that CRP, in association with PC, efficiently reduces ATP-induced inflammasome activation and IL-1β release from human peripheral blood mononuclear leukocytes and monocytic U937 cells. Effective concentrations are in the range of marginally pathologic CRP levels (IC  = 4.9 µg/ml). CRP elicits metabotropic functions at nicotinic acetylcholine (ACh) receptors (nAChRs) containing subunits α7, α9, and α10 and suppresses the function of ATP-sensitive P2X7 receptors in monocytic cells. Of note, CRP does not induce ion currents at conventional nAChRs, suggesting that CRP is a potent nicotinic agonist controlling innate immunity without entailing the risk of adverse effects in the nervous system. In a prospective study on multiple trauma patients, IL-1β plasma concentrations negatively correlated with preceding CRP levels, whereas inflammasome-independent cytokines IL-6, IL-18, and TNF-α positively correlated. In conclusion, PC-laden CRP is an unconventional nicotinic agonist that potently inhibits ATP-induced inflammasome activation and might protect against trauma-associated sterile inflammation.

Alzheimer’s disease (AD), marked by cognitive impairment, predominantly affects the brain regions regulated by cholinergic innervation, such as the cerebral cortex and hippocampus. Cholinergic dysfunction, a key contributor to age-related cognitive decline, has spurred investigations into potential therapeutic interventions. We have previously shown that choline alphoscerate (α-GPC), a cholinergic neurotransmission-enhancing agent, protects from Aβ-mediated neurotoxicity. Herein, we investigated the effects of α-GPC on the microglial phenotype in response to Aβ via modulation of the nicotinic alpha-7 acetylcholine receptor (α7 nAChR). BV2 microglial cells were pre-treated for 1 h with α-GPC and were treated for 24, 48, and 72 h with Aβ1–42 and/or α-BTX, a selective α7nAchR antagonist. Fluorescent immunocytochemistry and Western blot analysis showed that α-GPC was able to antagonize Aβ-induced inflammatory effects. Of note, α-GPC exerted its anti-inflammatory effect by directly activating the α7nAChR receptor, as suggested by the induction of an increase in [Ca2+]i and Ach-like currents. Considering that cholinergic transmission appears crucial in regulating the inflammatory profiles of glial cells, its modulation emerges as a potential pharmaco-therapeutic target to improve outcomes in inflammatory neurodegenerative disorders, such as AD.

Amyloid β peptide (Aβ) is a key player in the development of Alzheimer's disease (AD). It is the primary component of senile plaques in AD patients and is also found in soluble forms. Cholinergic activity mediated by α7 nicotinic receptors has been shown to be affected by Aβ soluble forms. To shed light into the molecular mechanism of this effect, we explored the direct actions of oligomeric Aβ and Aβ on human α7 by fluorescence spectroscopy and single-channel recordings. Fluorescence measurements using the conformational sensitive probe crystal violet (CrV) revealed that in the presence of Aβ α7 undergoes concentration-dependent conformational changes. Exposure of α7 to 100 pM Aβ changes CrV K towards that of the desensitized state. However, α7 is still reactive to high carbamylcholine (Carb) concentrations. These observations are compatible with the induction of active/desensitized states as well as of a novel conformational state in the presence of both Aβ and Carb. At 100 nM Aβ, α7 adopts a resting-state-like structure which does not respond to Carb, suggesting stabilization of α7 in a blocked state. In real time, we found that Aβ is capable of eliciting α7 channel activity either in the absence or presence of the positive allosteric modulator (PAM) PNU-120596. Activation by Aβ is favored at picomolar or low nanomolar concentrations and is not detected at micromolar concentrations. At high Aβ concentrations, the mean duration of activation episodes elicited by ACh in the presence of PNU-120596 is significantly reduced, an effect compatible with slow open-channel block. We conclude that Aβ directly affects α7 function by acting as an agonist and a negative modulator. Whereas the capability of low concentrations of Aβ to activate α7 could be beneficial, the reduced α7 activity in the presence of higher Aβ concentrations or its long exposure may contribute to the cholinergic signaling deficit and may be involved in the initiation and development of AD.

Objective This study aimed to observe the impact of Tthoracic paravertebral nerve blockade(TPVB) at left T7 level on the α7nAChR-dependent cholinergic anti-inflammatory pathway in patients undergoing thoracoscopic lobectomy. Methods Scheduled thoracoscopic lung surgery patients at the First Affiliated Hospital of Nanchang University from August to September 2023 were divided into two groups according to the surgical site. The experimental group underwent left T7 paravertebral nerve blockade (LTPVB group), while the control group underwent right T7 paravertebral nerve blockade (RTPVB group). Relevant clinical data were collected, and Doppler ultrasound was used to measure the resistive index (RI) of the splenic artery before and after blockade. Additionally, perioperative α7nAChR levels and the expression levels of the inflammatory factors IL-1β, IL-6, and TNF-α were determined. Results There were no significant differences in general conditions, perioperative blood pressure, heart rate, or pain VAS scores between the two groups ( p  > 0.05). Splenic Doppler ultrasound showed that compared to before blockade, the RI of the splenic artery in the LTPVB group significantly decreased ( p  < 0.05). The α7nAChR levels at 12 h and 24 h postoperatively were significantly increased ( p  < 0.05) in both groups, and the levels of IL-1β, IL-6, and TNF-α gradually increased over time in both groups. However, the levels were significantly lower in the LTPVB group compared to the RTPVB group at 12 h and 24 h postoperatively ( p  < 0.05). Conclusion TPVB at left T7 can activate the α7nAChR-dependent cholinergic anti-inflammatory pathway, thereby alleviating the postoperative inflammatory response in patients undergoing thoracoscopic lobectomy.