nAChR Agonist Research Articles

Acetylcholine Receptor Agonists Exerted Neuroprotection Against Doxorubicin‐Induced Chemobrain

AbstractBackgroundAlthough doxorubicin has been broadly used in treating a wide range of cancers, it frequently causes a serious neurological consequences referred to as chemobrain in cancer patients. Chemobrain shares several key pathological features with Alzheimer’s disease (AD) including neuroinflammation and Tau pathology. In addition to the inhibition of acetylcholinesterase, the activation of a7nicotinic and muscarinic acetylcholine receptors (a7nAChR and mAChR) using their agonists has been considered an effective therapeutic target against dementia. However, the benefit of intervention with nAChR and mAChR agonists in DOX‐induced chemobrain has never been investigated.MethodTwenty‐five male Wistar rats were assigned to received either (0.9% NSS) or doxorubicin (3 mg/kg; 6 doses) via intraperitoneal injection. Then, doxorubicin‐treated rats were divided into 4 intervention groups to treat with either vehicle, an a7nAChR agonist (PNU‐282987; 3 mg/kg), a mAChR agonist (bethanechol; 12 mg/kg), or a combined treatment of PNU‐282987 and bethanechol via intraperitoneal injection as a concomitant treatment with doxorubicin for a period of 30 days. After completion of the treatment paradigm, cognitive function was evaluated using the novel object location task (NOLT). The hippocampal tissues were dissected to enable further investigation.ResultSystemic administration of doxorubicin impaired long‐term cognitive function in rats as shown by a decreased preference index in the NOLT (Figure A). Furthermore, doxorubicin‐treated rats exhibited neuroinflammation in the hippocampus as demonstrated by an increase in TNF‐a protein expression (Figure B) and an alteration in microglial morphology to a pro‐inflammatory amoeboid‐liked phenotype (Figure C). Notably, injection of doxorubicin markedly reduced the phosphorylation of GSK3b at Ser9 (Figure D) and upregulated the phosphorylation of Tau at Thr181 (Figure E). Also, a decline in the dendritic spine density in the CA1 region was observed in doxorubicin‐treated rats compared with the controls (Figure F). Interestingly, all AChR agonists restored cognitive function in rats in a similar manner by attenuating neuroinflammation and Tau phosphorylation while preserving dendritic spine density (Figure A‐F).ConclusionTogether, activation of AChRs by their agonists protected against the neuropathology of doxorubicin‐induced chemobrain, indicating their potential role as a novel strategy to prevent chemobrain in patients receiving chemotherapy.

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 β2 nAChR subunits in the left ventricle of rat heart. In a rat model of myocardial ischemia, we studied the effect of α4β2 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-β-erythroidine, antagonist of α4β2 nAChR, as well as methyllycaconitine, antagonist of α7 and α6β2-containing nAChR, prevented protective effect of varenicline. This together with the presence of α4, α6 and β2 subunit mRNA in the left ventricule of rat heart raises the possibility that the varenicline effect is mediated by α4β2 as well as by α7 and/or α6β2-containing receptors. Our results point to a new way for the use of cytisine and varenicline as cardioprotective agents.

Exploring Cholinergic Compounds for Peripheral Neuropathic Pain Management: A Comprehensive Scoping Review of Rodent Model Studies.

Neuropathic pain affects about 7-8% of the population, and its management still poses challenges with unmet needs. Over the past decades, researchers have explored the cholinergic system (muscarinic and nicotinic acetylcholine receptors: mAChR and nAChR) and compounds targeting these receptors as potential analgesics for neuropathic pain management. This scoping review aims to provide an overview of studies on peripheral neuropathic pain (PNP) in rodent models, exploring compounds targeting cholinergic neurotransmission. The inclusion criteria were original articles on PNP in rodent models that explored the use of compounds directly targeting cholinergic neurotransmission and reported results of nociceptive behavioral assays. The literature search was performed in the PubMed and Web of Science databases (1 January 2000-22 April 2023). The selection process yielded 82 publications, encompassing 62 compounds. The most studied compounds were agonists of α4β2 nAChR and α7 nAChR, and antagonists of α9/α10 nAChR, along with those increasing acetylcholine and targeting mAChRs. Studies mainly reported antinociceptive effects in traumatic PNP models, and to a lesser extent, chemotherapy-induced neuropathy or diabetic models. These preclinical studies underscore the considerable potential of cholinergic compounds in the management of PNP, warranting the initiation of clinical trials.

Abstract 043: The Role Of Nicotine-induced Nitrosative And Oxidative Stress In The Development Of Podocyte Damage In The Dahl Ss Rat

Introduction: Patients with hypertension or renal disease are more likely to exhibit glomerular pathology associated with smoking. Smoking has been linked to lower nitric oxide (NO) bioavailability, oxidative and nitrosative stress. Nicotine triggers the formation of peroxynitrite (ONOO - ), a highly damaging redox molecule, and was also recently implicated as a contributor to hypertensive renal disease. Here we hypothesize that nicotine induces podocyte damage through peroxynitrite formation, thus causing glomerular function decline and aggravation of kidney damage in the Dahl SS rat. Methods: We performed live confocal imaging on human podocytes to detect the production of ONOO - (HPF), Ca 2+ (Fluo-8), and nitric oxide NO (DAF-FM) in response to nicotine. Seahorse assay was used to detect changes in mitochondrial health. Then, nicotine was chronically infused in the Dahl SS rats using Alzet pumps (0.2 mg/kg/day s.c., 0.4% NaCl, 21 days). Electron microscopy (EM) and Masson's trichrome staining were used to blindly evaluate glomerular and mitochondrial damage. ANOVA with post-hoc comparisons was used for statistical analysis. Results: IHC indicated that human and rat glomerular podocytes express nAChRs. Acute application of nicotine promoted intracellular Ca 2+ and ONOO - transients. The ONOO - response was blocked in the presence of SOD, indicating that ONOO - production requires superoxide. The application of specific nAChR agonists elicited Ca 2+ transients but did not produce ONOO - , suggesting that nitrosative processes may occur independently from Ca 2+ influx or nAChR activation. Incubation with nicotine (12-hrs) resulted in a significant decrease in podocytes’ NO bioavailability (p<0.05) and mitochondrial respiration (p<0.05). In vivo, in the Dahl SS rat nicotine infusion promoted mitochondrial damage in podocytes, which exhibited swelling, loss of cristae, and signs of mitophagy. Masson's trichrome staining and the assessment of glomerular damage showed deterioration of glomerular health. Conclusion: The pathological shift in redox balance in the podocyte is directly associated with nicotine. It may be the primary mechanism responsible for podocyte damage, renal function decline, and blood pressure development in smokers.

An in vitro assay to investigate venom neurotoxin activity on muscle-type nicotinic acetylcholine receptor activation and for the discovery of toxin-inhibitory molecules

Snakebite envenoming is a neglected tropical disease that causes over 100,000 deaths annually. Envenomings result in variable pathologies, but systemic neurotoxicity is among the most serious and is currently only treated with difficult to access and variably efficacious commercial antivenoms. Venom-induced neurotoxicity is often caused by α-neurotoxins antagonising the muscle-type nicotinic acetylcholine receptor (nAChR), a ligand-gated ion channel. Discovery of therapeutics targeting α-neurotoxins is hampered by relying on binding assays that do not reveal restoration of receptor activity or more costly and/or lower throughput electrophysiology-based approaches. Here, we report the validation of a screening assay for nAChR activation using immortalised TE671 cells expressing the γ-subunit containing muscle-type nAChR and a fluorescent dye that reports changes in cell membrane potential. Assay validation using traditional nAChR agonists and antagonists, which either activate or block ion fluxes, was consistent with previous studies. We then characterised antagonism of the nAChR by a variety of elapid snake venoms that cause muscle paralysis in snakebite victims, before defining the toxin-inhibiting activities of commercial antivenoms, and new types of snakebite therapeutic candidates, namely monoclonal antibodies, decoy receptors, and small molecules. Our findings show robust evidence of assay uniformity across 96-well plates and highlight the amenability of this approach for the future discovery of new snakebite therapeutics via screening campaigns. The described assay therefore represents a useful first-step approach for identifying α-neurotoxins and their inhibitors in the context of snakebite envenoming, and it should provide wider value for studying modulators of nAChR activity from other sources.

GTS-21 Enhances Regulatory T Cell Development from T Cell Receptor-Activated Human CD4+ T Cells Exhibiting Varied Levels of CHRNA7 and CHRFAM7A Expression.

Immune cells such as T cells and macrophages express α7 nicotinic acetylcholine receptors (α7 nAChRs), which contribute to the regulation of immune and inflammatory responses. Earlier findings suggest α7 nAChR activation promotes the development of regulatory T cells (Tregs) in mice. Using human CD4+ T cells, we investigated the mRNA expression of the α7 subunit and the human-specific dupα7 nAChR subunit, which functions as a dominant-negative regulator of ion channel function, under resting conditions and T cell receptor (TCR)-activation. We then explored the effects of the selective α7 nAChR agonist GTS-21 on proliferation of TCR-activated T cells and Treg development. Varied levels of mRNA for both the α7 and dupα7 nAChR subunits were detected in resting human CD4+ T cells. mRNA expression of the α7 nAChR subunit was profoundly suppressed on days 4 and 7 of TCR-activation as compared to day 1, whereas mRNA expression of the dupα7 nAChR subunit remained nearly constant. GTS-21 did not alter CD4+ T cell proliferation but significantly promoted Treg development. These results suggest the potential ex vivo utility of GTS-21 for preparing Tregs for adoptive immunotherapy, even with high expression of the dupα7 subunit.

Varenicline enhances recognition memory via α7 nicotinic acetylcholine receptors in the medial prefrontal cortex in male mice

Previous studies postulated that chronic administration of varenicline, a partial and full agonist at α4β2 and α7 nicotinic acetylcholine receptors (nAChRs), respectively, enhances recognition memory. However, whether its acute administration is effective, on which brain region(s) it acts, and in what signaling it is involved, remain unknown. To address these issues, we conducted a novel object recognition test using male C57BL/6J mice, focusing on the medial prefrontal cortex (mPFC), a brain region associated with nicotine-induced enhancement of recognition memory. Systemic administration of varenicline before the training dose-dependently enhanced recognition memory. Intra-mPFC varenicline infusion also enhanced recognition memory, and this enhancement was blocked by intra-mPFC co-infusion of a selective α7, but not α4β2, nAChR antagonist. Consistent with this, intra-mPFC infusion of a selective α7 nAChR agonist augmented object recognition memory. Furthermore, intra-mPFC co-infusion of U-73122, a phospholipase C (PLC) inhibitor, or 2-aminoethoxydiphenylborane (2-APB), an inositol trisphosphate (IP3) receptor inhibitor, suppressed the varenicline-induced memory enhancement, suggesting that α7 nAChRs may also act as Gq-coupled metabotropic receptors. Additionally, whole-cell recordings from mPFC layer V pyramidal neurons in vitro revealed that varenicline significantly increased the summation of evoked excitatory postsynaptic potentials, and this effect was suppressed by U-73122 or 2-APB. These findings suggest that varenicline might acutely enhance recognition memory via mPFC α7 nAChR stimulation, followed by mPFC neuronal excitation, which is mediated by the activation of PLC and IP3 receptor signaling. Our study provides evidence supporting the potential repositioning of varenicline as a treatment for cognitive impairment.

Target identification and validation of the alpha7 nicotinic acetylcholine receptor as a potential therapeutic target in retinal disease.

The role of acetylcholine (ACh) in visual processing in the mammalian retina has been the focus of research for many decades. Pioneering work on the localization of ACh discovered that the neurotransmitter is synthesized and stored in a distinct subpopulation of amacrine (starburst) cells. It has been shown that ACh release is regulated to a low resting "tonic" level, much like what is observed at the neuromuscular junction (NMJ). If there were a dysfunction in the tonic release of ACh, might post-synaptic changes render the targets of ACh [i.e., retinal ganglion cells (RGCs)] vulnerable to disease? During my time at Pharmacia & Upjohn (PNU), selective nicotinic ACh receptor (nAChR) agonists (e.g., PNU-282987) were developed as a possible therapy for central nervous system (CNS) diseases. As RGCs are the main targets of neurodegeneration in glaucoma, could the activation of this target provide neuroprotection? In response to this question, experiments to identify alpha7 nAChRs in the retina (i.e., target ID studies) followed by "proof-of-concept" experiments were conducted. Target ID studies included binding studies with retinal homogenates, [125I]-alpha-bungarotoxin (α-BTX) autoradiography, and fluorescently tagged α-BTX binding in retinal slices. Imaging studies of intracellular calcium dynamics in the retinal slice were conducted. Reverse transcription-polymerase chain reaction (RT-PCR) analysis with alpha7 nAChR knockout mice using the "laser-capture microdissection" technique, in situ hybridization studies, and RT-PCR analysis of the human retina were conducted. Collectively, these experiments confirmed the presence of alpha7 nAChRs on specific cells in the retina. "Proof-of-concept" neuroprotection studies demonstrated that PNU-282987 provided significant protection for RGCs. This protection was dose dependent and was blocked with selective antagonists. More recently, evidence for the generation of new RGCs has been reported with PNU-282987 in rodents. Interestingly, the appearance of new RGCs is more pronounced with eye-drop application than with intravitreal injection. One could postulate that this reflects the neurogenic activation of alpha7 receptors on the retinal pigment epithelium (RPE) (eye drops) vs. a neuroprotective effect on RGCs (injections). In conclusion, there does appear to be a cholinergic retinal "tone" associated with RGCs that could be utilized as a neuroprotective therapy. However, a distinct cholinergic neurogenic mechanism also appears to exist in the outer retina that could possibly be exploited to generate new RGCs lost through various disease processes.

Optical Control of Invertebrate nAChR and Behaviors with Dithienylethene-Imidacloprid.

Photopharmacology involving azobenzene or dithienylethene has changed established methods of studying receptor functions, allowing for increasing the spatiotemporal resolution. There are no photopharmacological tools available for the invertebrate nicotinic acetylcholine receptor (nAChR). Here, we report a photochromic ligand, dithienylethene-imidacloprid (DitIMI), using a dithienylethene photoswitch embedded in the nAChR agonist imidacloprid. It was found that DitIMI displayed good photochromism and fluorescence switching behaviors upon irradiation with UV/vis light in aqueous solution. We demonstrated that open-DitIMI has low spontaneous in vitro and in vivo activity but can be photoisomerized to a highly active closed-form. Surprisingly, the photoswitchable DitIMI showed a large difference in insecticidal activity between the open and closed forms, in which the insecticidal activity against Aphis craccivora of the ring-closed isomers for DitIMI was 355 times that of the corresponding ring-open isomers. This photoisomerization can further be translated to photomodulation of neuron membrane potential and behavioral responses of living mosquito larvae and American cockroaches. The photomanipulation of nACh neurotransmission opens new avenues to understanding inhibitory circuits in intact animals.

Expression and role of nicotinic acetylcholine receptors during midbrain dopaminergic neuron differentiation from human induced pluripotent stem cells.

Nicotinic acetylcholine receptors (nAChRs) expressed in midbrain dopaminergic (mDA) neurons modulate mDA neuronal activity. However, their expression patterns and functional roles during mDA neuronal development remain unknown. Here, we profiled the expression and function of nAChR subtypes during mDA neuron differentiation from human induced pluripotent stem cells (hiPSCs). Midbrain dopaminergic neurons were differentiated from hiPSCs using a recently developed proprietary method that replicates midbrain development. The expression patterns of developmental marker proteins were monitored during mDA neuronal differentiation using immunohistochemical analysis. Gene expression of nAChR subtypes was analyzed by reverse transcription polymerase chain reaction. Pharmacological nAChR agonists and antagonists were used to reveal the role of the α6 nAChR subunit in the differentiation of mDA neurons from hiPSCs. CHRNA4 expression was detected at the mDA neural progenitor stage, whereas CHRNA6 expression began during the mDA neuronal stage. CHRNA7 was expressed throughout the differentiation process, including in the undifferentiated hiPSCs. We also found that LMO3, a gene expressed in a subset of substantia nigra pars compacta (SNC) DA neurons in the midbrain, showed increased expression following nicotine treatment in a concentration-dependent manner. Additionally, 5-iodo A85380, a selective α6 nAChR agonist, also increased LMO3 expression in hiPSC-derived mDA neurons, and this increase was suppressed by simultaneous treatment with bPiDi, a selective α6 nAChR antagonist. Our findings suggest that stimulating the α6 nAChR subunit on hiPSC-derived mDA neurons may induce neuronal maturation that is biased toward SNC DA neurons.

Behavioral Study of 3- and 5-Halocytisine Derivatives in Zebrafish Using the Novel Tank Diving Test (NTT)

Anxiety is a serious mental disorder, and recent statistics have determined that 35.12% of the global population had an anxiety disorder during the COVID-19 pandemic. A mechanism associated with anxiolytic effects is related to nicotinic acetylcholine receptor (nAChR) agonists, principally acting on the α4β2 nAChR subtype. nAChRs are present in different animal models, including murine and teleosteos ones. Zebrafish has become an ideal animal model due to its high human genetic similarities (70%), giving it high versatility in different areas of study, among them in behavioral studies related to anxiety. The novel tank diving test (NTT) is one of the many paradigms used for studies on new drugs related to their anxiolytic effect. In this work, an adult zebrafish was used to determine the behavioral effects of 3- and 5-halocytisine derivatives, using the NTT at different doses. Our results show that substitution at position 3 by chlorine or bromine decreases the time spent by the fish at the bottom compared to the control. However, the 3-chloro derivative at higher doses increases the bottom dwelling time. In contrast, substitution at the 5 position increases bottom dwelling at all concentrations showing no anxiolytic effects in this model. Unexpected results were observed with the 5-chlorocytisine derivative, which at a concentration of 10 mg/L produced a significant decrease in bottom dwelling and showed high times of freezing. In conclusion, the 3-chloro and 3-bromo derivatives show an anxiolytic effect, the 3-chlorocytisine derivative being more potent than the 3-bromo derivative, with the lowest time at the bottom of the tank at 1mg/L. On the other hand, chlorine, and bromine at position 5 produce an opposite effect.

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.

Elimination Resistance: Characterizing Multi-compartment Toxicokinetics of the Neonicotinoid Thiacloprid in the Amphipod Gammarus pulex Using Bioconcentration and Receptor-Binding Assays.

Delayed toxicity is a phenomenon observed for aquatic invertebrates exposed to nicotinic acetylcholine receptor (nAChR) agonists, such as neonicotinoids. Furthermore, recent studies have described an incomplete elimination of neonicotinoids by exposed amphipods. However, a mechanistic link between receptor binding and toxicokinetic modeling has not been demonstrated yet. The elimination of the neonicotinoid thiacloprid in the freshwater amphipod Gammarus pulex was studied in several toxicokinetic exposure experiments, complemented with in vitro and in vivo receptor-binding assays. Based on the results, a two-compartment model was developed to predict the uptake and elimination kinetics of thiacloprid in G. pulex. An incomplete elimination of thiacloprid, independent of elimination phase duration, exposure concentrations, and pulses, was observed. Additionally, the receptor-binding assays indicated irreversible binding of thiacloprid to the nAChRs. Accordingly, a toxicokinetic-receptor model consisting of a structural and a membrane protein (including nAChRs) compartment was developed. The model successfully predicted internal thiacloprid concentrations across various experiments. Our results help in understanding the delayed toxic and receptor-mediated effects toward arthropods caused by neonicotinoids. Furthermore, the results suggest that more awareness toward long-term toxic effects of irreversible receptor binding is needed in a regulatory context. The developed model supports the future toxicokinetic assessment of receptor-binding contaminants.

Desensitizing nicotinic agents normalize tinnitus-related inhibitory dysfunction in the auditory cortex and ameliorate behavioral evidence of tinnitus.

Tinnitus impacts between 10-20% of the population. Individuals most troubled by their tinnitus have their attention bound to and are distracted by, their tinnitus percept. While numerous treatments to ameliorate tinnitus have been tried, no therapeutic approach has been clinically accepted. The present study used an established condition-suppression noise-exposure rat model of tinnitus to: (1) examine tinnitus-related changes in nAChR function of layer 5 pyramidal (PNs) and of vasoactive intestinal peptide (VIP) neurons in primary auditory cortex (A1) and (2) examine how the partial desensitizing nAChR agonists, sazetidine-A and varenicline, can act as potential therapeutic agents in the treatment of tinnitus. We posited that tinnitus-related changes in layer 5 nAChR responses may underpin the decline in attentional resources previously observed in this animal model (Brozoski et al., 2019). In vitro whole-cell patch-clamp studies previously revealed a significant tinnitus-related loss in nAChR-evoked excitatory postsynaptic currents from A1 layer 5 PNs. In contrast, VIP neurons from animals with behavioral evidence of tinnitus showed significantly increased nAChR-evoked excitability. Here we hypothesize that sazetidine-A and varenicline have therapeutic benefits for subjects who cannot divert their attention away from the phantom sound in their heads. We found that sazetidine-A or varenicline normalized tinnitus-related reductions in GABAergic input currents onto A1 layer 5 PNs. We then tested sazetidine-A and varenicline for the management of tinnitus using our tinnitus animal model. Subcutaneous injection of sazetidine-A or varenicline, 1 h prior to tinnitus testing, significantly decreased the rat's behavioral evidence of tinnitus in a dose-dependent manner. Collectively, these results support the need for additional clinical investigations of partial desensitizing nAChR agonists sazetidine-A and varenicline for the treatment of tinnitus.

Nicotine-mediated signaling in glomerular podocytes

Introduction: Although nicotine’s harmful effects on renal function are established, the precise cellular mechanisms of smoking-related damage are understudied. Smoking-Related Glomerulopathy (SRG) is a renal disease phenotype associated with smoking. This condition histologically mimics diabetic nephropathy, however, SRG patients present with proteinuria and renal insufficiency without diabetes. Here we investigated the acute and chronic nicotine-related oxidative and nitrosative stress in glomerular podocytes. We hypothesized that nicotine may promote nitrosative stress in the kidney cells through the rapid production of peroxynitrite (ONOO - ) and a decline in nitric oxide (NO) bioavailability. Methods: To test the hypothesis, we used a conditionally immortalized human podocyte line and confocal imaging to detect the production of ONOO - (Hydroxyl Radical and Peroxynitrite Sensor; HPF), intracellular Ca 2+ (Fluo-8), and NO (DAF-FM). The presence of nicotinic acetylcholine receptors (nAChR) receptor subunits in podocytes was confirmed with immunocytochemistry and live imaging using commercially available pharmacology. NOS activity was analyzed in response to Ang II with the specific blockers for NOS1 (NΩ-Propyl-L-arginine hydrochloride) and NOS2 (L-NIL) subunits. One-way ANOVA (OriginPro) was used for statistical analysis. Results: Immunostaining indicated that human and rat kidneys express nicotinic acetylcholine receptors (nAChR). Notably, we detected specific expression of α7 nAChR in glomerular podocytes. In podocytes, acute nAChR activation promoted the mobilization of intracellular Ca 2+ controlled by intracellular store activation, and fast ONOO - transients. Multiple consecutive applications of nicotine resulted in repeated intracellular Ca 2+ and ONOO - transients. Nicotine-mediated ONOO - response was efficiently blocked in the presence of superoxide dismutase (SOD). The application of specific α7 or α4β2, α2β4, α4β4 and α3β4 nAChR agonists elicited Ca 2+ transients but did not reproduce the ONOO - response to nicotine, suggesting that nitrosative processes may occur independently from Ca 2+ influx or nAChR function. Chronic exposure to nicotine (12-hrs) resulted in a significant decline in podocytes’ NO bioavailability (p<0.05). While under normal conditions, NO is produced primarily by NOS1 (70%), and the rest is attributed to NOS2, chronic nicotine exposure led to an elevation of NOS2 (75%) and a decline in NOS1 activity (p<0.001). We observed similar NOS remodeling under hyperglycemic conditions, suggesting similar nitrosative processes in response to nicotine and high glucose. Conclusions: Nicotine promotes superoxide-stimulated nitrosative stress and peroxynitrite formation in podocytes leading to decline in NO bioavailability and pathological activation of NOS2. R01 NIDDK DK126720 (OP), DK129227 (OP), HL148114 (DVI), NIH/NCATS/SCTR UL1TR001450/SCTR 2214 (OP). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Acetylcholine potentiates glutamate transmission from the habenula to the interpeduncular nucleus in losers of social conflict

Switching behaviors from aggression to submission in losers at the end of conspecific social fighting is essential to avoid serious injury or death. We have previously shown that the experience of defeat induces a loser-specific potentiation in the habenula (Hb)-interpeduncular nucleus (IPN) and show here that this is induced by acetylcholine. Calcium imaging and electrophysiological recording using acute brain slices from winners and losers of fighting behavior in zebrafish revealed that the ventral IPN (vIPN) dominates over the dorsal IPN in the neural response to Hb stimulation in losers. We also show that GluA1 α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits on the postsynaptic membrane increased in the vIPN of losers. Furthermore, these loser-specific neural properties disappeared in the presence of an α7 nicotinic acetylcholine receptor (nAChR) antagonist and, conversely, were induced in brain slices of winners treated with α7 nAChR agonists. These data suggest that acetylcholine released from Hb terminals in the vIPN induces activation of α7 nAChR followed by an increase in postsynaptic membrane GluA1. This results in an increase in active synapses on postsynaptic neurons, resulting in the potentiation of neurotransmissions to the vIPN. This acetylcholine-induced neuromodulation could be the neural foundation for behavioral switching in losers. Our results could increase our understanding of the mechanisms of various mood disorders such as social anxiety disorder and social withdrawal.

Unravelling the allosteric binding mode of αD-VxXXB at nicotinic acetylcholine receptors.

αD-conotoxins are 11kDa homodimers that potently inhibit nicotinic acetylcholine receptors (nAChRs) through a non-competitive (allosteric) mechanism. In this study, we describe the allosteric binding mode of the granulin-like C-terminal (CTD) of VxXXB bound to Lymnea stagnalis acetylcholine binding protein (Ls-AChBP), a soluble homologue of the extracellular ligand-binding domain of nAChRs. This co-crystal complex revealed a novel allosteric binding site for nAChR antagonists outside the C-loop that caps the orthosteric site defined by the nAChR agonist nicotine and the antagonist epibatidine. Mutational and docking studies on Ls-AChBP supported a two-site binding mode for full-length VxXXB, with the first CTD binding site located outside the C-loop as seen in the co-crystal complex, with a second CTD binding site located near the N-terminal end of the adjacent subunit of AChBP. These results provide new structural insight into a novel allosteric mechanism of nAChR inhibition and define the cooperative binding mode of the N-terminal domain linked granulin core domains of αD-conotoxins.

MP03-10 BRAIN α7 NICOTINIC ACETYLCHOLINE RECEPTORS SUPPRESS THE RAT MICTURITION REFLEX VIA BRAIN HYDROGEN SULFIDE

You have accessJournal of UrologyCME1 Apr 2023MP03-10 BRAIN α7 NICOTINIC ACETYLCHOLINE RECEPTORS SUPPRESS THE RAT MICTURITION REFLEX VIA BRAIN HYDROGEN SULFIDE Nobutaka Shimizu, Takahiro Shimizu, Shogo Shimizu, Youichirou Higashi, Suo Zou, Hideo Fukuhara, Takashi Karashima, Keiji Inoue, and Motoaki Saito Nobutaka ShimizuNobutaka Shimizu More articles by this author , Takahiro ShimizuTakahiro Shimizu More articles by this author , Shogo ShimizuShogo Shimizu More articles by this author , Youichirou HigashiYouichirou Higashi More articles by this author , Suo ZouSuo Zou More articles by this author , Hideo FukuharaHideo Fukuhara More articles by this author , Takashi KarashimaTakashi Karashima More articles by this author , Keiji InoueKeiji Inoue More articles by this author , and Motoaki SaitoMotoaki Saito More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003214.10AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: We previously reported that brain α7 nicotinic acetylcholine receptors (α7 nAChRs) and brain hydrogen sulfide (H2S) suppressed the rat micturition reflex via brain GABA receptors. However, it is unclear how the brain α7 nAChRs and brain H2S are related to each other in regulation of the micturition reflex. In this study, we examined whether brain H2S is involved in the suppression of micturition induced by stimulation of brain α7 nAChR. METHODS: Urethane anesthetized (0.8 g/kg, ip) male Wistar rats (350-420 g) were used. A catheter was inserted into the bladder to perform continuous cystometry (12 ml/h saline infusion). We examined effects of intracerebroventricularly (icv) pretreated GYY4137 (GYY, H2S donor, 1 or 3 nmol/rat) or AOAA (non-selective inhibitor of H2S synthesis, 3 or 10 μg/rat) on PHA568487 (PHA, α7 nAChR agonist, 0.3 or 1 nmol/rat, icv)-induced intercontraction intervals (ICI) prolongation. Continuous cystometry and evaluation of ICI and maximal voiding pressure (MVP) was started 120 min after the surgery and the first icv administration was performed 60 min after starting cystometry. RESULTS: PHA at a lower dose (0.3 nmol/rat) showed no significant effect on ICI, while under pretreatment with GYY, PHA (0.3 nmol/rat) significantly prolonged ICI even at a lower dose (Figure 1). PHA at a higher dose (1 nmol/rat) induced ICI prolongation and the PHA-induced prolongation was significantly suppressed by AOAA (Figure 2). The AOAA-induced suppression of the PHA-induced ICI prolongation was cancelled by supplementation of brain H2S via GYY (Figure 3). PHA at each dose showed no effect on MVP (data not shown). CONCLUSIONS: Brain α7 nAChRs suppressed the rat micturition reflex via brain H2S, therefore, brain α7 nAChRs and H2S might be new therapeutic targets for neurogenic bladder overactivity. Source of Funding: Smoking Research Foundation in Japan, KAKENHI (#20K07827), The Kochi Medical School Hospital President’s Discretionary Grant © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e25 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Nobutaka Shimizu More articles by this author Takahiro Shimizu More articles by this author Shogo Shimizu More articles by this author Youichirou Higashi More articles by this author Suo Zou More articles by this author Hideo Fukuhara More articles by this author Takashi Karashima More articles by this author Keiji Inoue More articles by this author Motoaki Saito More articles by this author Expand All Advertisement PDF downloadLoading ...

Nicotine Reduces Reactive Oxygen Species and Enhances Cell Proliferation via the α4 Nicotinic Acetylcholine Receptor Subunit in Human Induced Pluripotent Stem Cells.

The effects of smoking on fetal development and stem cell differentiation are not fully understood. Although nicotinic acetylcholine receptors (nAChRs) are expressed in many organs of the human body, their significance in human induced pluripotent stem cells (hiPSCs) remains unclear. After expression levels of nAChR subunits in hiPSCs were determined, the effects of the nAChR agonist, nicotine, on undifferentiated hiPSCs were evaluated using a Clariom S Array. We also determined the effect of nicotine alone and with a nAChR subunit antagonist on hiPSCs. nAChR α4, α7, and β4 subunits were strongly expressed in hiPSCs. cDNA microarray, gene ontology, and enrichment analyses showed that exposing hiPSCs to nicotine altered expression of genes associated with immune responses, neurological system, carcinogenesis, cell differentiation, and cell proliferation. Particularly affected was metallothionein, which acts to decrease reactive oxygen species (ROS). The nicotine-induced reduction of ROS in hiPSCs was canceled by an α4 subunit or nonselective nAChR antagonist. HiPSC proliferation was increased by nicotine, and this effect, too, was canceled by an α4 antagonist. In conclusion, nicotine reduces ROS and enhances cell proliferation through the α4 nAChR subunit in hiPSCs. These findings provide new insight into the significance of nAChRs on human stem cells and fertilized human ova.

The SARS-CoV-2 Virus and the Cholinergic System: Spike Protein Interaction with Human Nicotinic Acetylcholine Receptors and the Nicotinic Agonist Varenicline

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is responsible for the worldwide coronavirus disease 2019 (COVID-19) pandemic. Although the pathophysiology of SARS-CoV-2 infection is still being elucidated, the nicotinic cholinergic system may play a role. To evaluate the interaction of the SARS-CoV-2 virus with human nicotinic acetylcholine receptors (nAChRs), we assessed the in vitro interaction of the spike protein of the SARS-CoV-2 virus with various subunits of nAChRs. Electrophysiology recordings were conducted at α4β2, α3β4, α3α5β4, α4α6β2, and α7 neuronal nAChRs expressed in Xenopus oocytes. In cells expressing the α4β2 or α4α6β2 nAChRs, exposure to the 1 µg/mL Spike-RBD protein caused a marked reduction of the current amplitude; effects at the α3α5β4 receptor were equivocal and effects at the α3β4 and α7 receptors were absent. Overall, the spike protein of the SARS-CoV-2 virus can interact with select nAChRs, namely the α4β2 and/or α4α6β2 subtypes, likely at an allosteric binding site. The nAChR agonist varenicline has the potential to interact with Spike-RBD and form a complex that may interfere with spike function, although this effect appears to have been lost with the omicron mutation. These results help understand nAChR's involvement with acute and long-term sequelae associated with COVID-19, especially within the central nervous system.