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FASEB Journal : Official Publication of... Jul 2023Leucine-rich repeat containing 8A (LRRC8A) volume regulated anion channels (VRACs) are activated by inflammatory and pro-contractile stimuli including tumor necrosis...
Leucine-rich repeat containing 8A (LRRC8A) volume regulated anion channels (VRACs) are activated by inflammatory and pro-contractile stimuli including tumor necrosis factor alpha (TNFα), angiotensin II and stretch. LRRC8A associates with NADPH oxidase 1 (Nox1) and supports extracellular superoxide production. We tested the hypothesis that VRACs modulate TNFα signaling and vasomotor function in mice lacking LRRC8A exclusively in vascular smooth muscle cells (VSMCs, Sm22α-Cre, Knockout). Knockout (KO) mesenteric vessels contracted normally but relaxation to acetylcholine (ACh) and sodium nitroprusside (SNP) was enhanced compared to wild type (WT). Forty-eight hours of ex vivo exposure to TNFα (10 ng/mL) enhanced contraction to norepinephrine (NE) and markedly impaired dilation to ACh and SNP in WT but not KO vessels. VRAC blockade (carbenoxolone, CBX, 100 μM, 20 min) enhanced dilation of control rings and restored impaired dilation following TNFα exposure. Myogenic tone was absent in KO rings. LRRC8A immunoprecipitation followed by mass spectroscopy identified 33 proteins that interacted with LRRC8A. Among them, the myosin phosphatase rho-interacting protein (MPRIP) links RhoA, MYPT1 and actin. LRRC8A-MPRIP co-localization was confirmed by confocal imaging of tagged proteins, Proximity Ligation Assays, and IP/western blots. siLRRC8A or CBX treatment decreased RhoA activity in VSMCs, and MYPT1 phosphorylation was reduced in KO mesenteries suggesting that reduced ROCK activity contributes to enhanced relaxation. MPRIP was a target of redox modification, becoming oxidized (sulfenylated) after TNFα exposure. Interaction of LRRC8A with MPRIP may allow redox regulation of the cytoskeleton by linking Nox1 activation to impaired vasodilation. This identifies VRACs as potential targets for treatment or prevention of vascular disease.
Topics: Animals; Mice; Acetylcholine; Anions; Membrane Proteins; Mice, Knockout; Myosin-Light-Chain Phosphatase; Signal Transduction; Tumor Necrosis Factor-alpha; Muscle, Smooth, Vascular
PubMed: 37310356
DOI: 10.1096/fj.202300561R -
Trends in Neurosciences Feb 2022Cholinergic innervation of the hippocampus uses the neurotransmitter acetylcholine (ACh) to coordinate neuronal circuit activity while simultaneously influencing the... (Review)
Review
Cholinergic innervation of the hippocampus uses the neurotransmitter acetylcholine (ACh) to coordinate neuronal circuit activity while simultaneously influencing the function of non-neuronal cell types. The α7 nicotinic ACh receptor (nAChR) subtype is highly expressed throughout the hippocampus, has the highest calcium permeability compared with other subtypes of nAChRs, and is of high therapeutic interest due to its association with a variety of neurological disorders and neurodegenerative diseases. In this review, we synthesize research describing α7 nAChR properties, function, and relationship to cognitive dysfunction within the hippocampal circuit and highlight approaches to help improve therapeutic development.
Topics: Acetylcholine; Hippocampus; Humans; Neurons; Receptors, Nicotinic; alpha7 Nicotinic Acetylcholine Receptor
PubMed: 34916082
DOI: 10.1016/j.tins.2021.11.006 -
American Journal of Physiology. Cell... Feb 2021The innate and adaptive immune systems play an important role in the development of cardiac diseases. Therefore, it has become critical to identify molecules that can... (Review)
Review
The innate and adaptive immune systems play an important role in the development of cardiac diseases. Therefore, it has become critical to identify molecules that can modulate inflammation in the injured heart. In this regard, activation of the cholinergic system in animal models of heart disease has been shown to exert protective actions that include immunomodulation of cardiac inflammation. In this mini-review, we briefly present our current understanding on the cardiac cellular sources of acetylcholine (ACh) (neuronal vs. nonneuronal), followed by a discussion on its contribution to the regulation of inflammatory cells. Although the mechanism behind ACh-mediated protection still remains to be fully elucidated, the beneficial immunomodulatory role of the cholinergic signaling emerges as a potential key regulator of cardiac inflammation.
Topics: Acetylcholine; Animals; Anti-Inflammatory Agents; Cardiotonic Agents; Heart; Heart Diseases; Humans; Inflammation; Neurons
PubMed: 33264077
DOI: 10.1152/ajpcell.00315.2020 -
Anesthesiology Apr 2021Cholinergic drugs are known to modulate general anesthesia, but anesthesia responses in acetylcholine-deficient mice have not been studied. It was hypothesized that mice...
BACKGROUND
Cholinergic drugs are known to modulate general anesthesia, but anesthesia responses in acetylcholine-deficient mice have not been studied. It was hypothesized that mice with genetic deficiency of forebrain acetylcholine show increased anesthetic sensitivity to isoflurane and ketamine and decreased gamma-frequency brain activity.
METHODS
Male adult mice with heterozygous knockdown of vesicular acetylcholine transporter in the brain or homozygous knockout of the transporter in the basal forebrain were compared with wild-type mice. Hippocampal and frontal cortical electrographic activity and righting reflex were studied in response to isoflurane and ketamine doses.
RESULTS
The loss-of-righting-reflex dose for isoflurane was lower in knockout (mean ± SD, 0.76 ± 0.08%, n = 18, P = 0.005) but not knockdown (0.78 ± 0.07%, n = 24, P = 0.021), as compared to wild-type mice (0.83 ± 0.07%, n = 23), using a significance criterion of P = 0.017 for three planned comparisons. Loss-of-righting-reflex dose for ketamine was lower in knockout (144 ± 39 mg/kg, n = 14, P = 0.006) but not knockdown (162 ± 32 mg/kg, n = 20, P = 0.602) as compared to wild-type mice (168 ± 24 mg/kg, n = 21). Hippocampal high-gamma (63 to 100 Hz) power after isoflurane was significantly lower in knockout and knockdown mice compared to wild-type mice (isoflurane-dose and mouse-group interaction effect, F[8,56] = 2.87, P = 0.010; n = 5 to 6 mice per group). Hippocampal high-gamma power after ketamine was significantly lower in both knockout and knockdown mice when compared to wild-type mice (interaction effect F[2,13] = 6.06, P = 0.014). The change in frontal cortical gamma power with isoflurane or ketamine was not statistically different among knockout, knockdown, and wild-type mice.
CONCLUSIONS
These findings suggest that forebrain cholinergic neurons modulate behavioral sensitivity and hippocampal gamma activity during isoflurane and ketamine anesthesia.
Topics: Acetylcholine; Anesthetics, Inhalation; Animals; Isoflurane; Ketamine; Male; Mice; Mice, Knockout; Models, Animal; Prosencephalon
PubMed: 33635947
DOI: 10.1097/ALN.0000000000003713 -
Shock (Augusta, Ga.) Oct 2022Introduction: Perioperative alterations in perfusion lead to ischemia and reperfusion injury, and supplemental oxygen is administered during surgery to limit hypoxic...
Introduction: Perioperative alterations in perfusion lead to ischemia and reperfusion injury, and supplemental oxygen is administered during surgery to limit hypoxic injury but can lead to hyperoxia. We hypothesized that hyperoxia impairs endothelium-dependent and endothelium-independent vasodilation but not the vasodilatory response to heme-independent soluble guanylyl cyclase activation. Methods: We measured the effect of oxygen on vascular reactivity in mouse aortas. Mice were ventilated with 21% (normoxia), 60% (moderate hyperoxia), or 100% (severe hyperoxia) oxygen during 30 minutes of renal ischemia and 30 minutes of reperfusion. After sacrifice, the thoracic aorta was isolated, and segments mounted on a wire myograph. We measured endothelium-dependent and endothelium-independent vasodilation with escalating concentrations of acetylcholine (ACh) and sodium nitroprusside (SNP), respectively, and we measured the response to heme-independent soluble guanylyl cyclase activation with cinaciguat. Vasodilator responses to each agonist were quantified as the maximal theoretical response ( Emax ) and the effective concentration to elicit 50% relaxation (EC 50 ) using a sigmoid model and nonlinear mixed-effects regression. Aortic superoxide was measured with dihydroethidium probe and high-performance liquid chromatography quantification of the specific superoxide product 2-hydroxyethidium. Results: Hyperoxia impaired endothelium-dependent (ACh) and endothelium-independent (SNP) vasodilation compared with normoxia and had no effect on cinaciguat-induced vasodilation. The median ACh Emax was 76.4% (95% confidence interval = 69.6 to 83.3) in the normoxia group, 53.5% (46.7 to 60.3) in the moderate hyperoxia group, and 53.1% (46.3 to 60.0) in the severe hyperoxia group ( P < 0.001, effect across groups), while the ACh EC 50 was not different among groups. The SNP Emax was 133.1% (122.9 to 143.3) in normoxia, 128.3% (118.1 to 138.6) in moderate hyperoxia, and 114.8% (104.6 to 125.0) in severe hyperoxia ( P < 0.001, effect across groups), and the SNP EC 50 was 0.38 log M greater in moderate hyperoxia than in normoxia (95% confidence interval = 0.18 to 0.58, P < 0.001). Cinaciguat Emax and EC 50 were not different among oxygen treatment groups (median range Emax = 78.0% to 79.4% and EC 50 = -18.0 to -18.2 log M across oxygen groups). Aorta 2-hydroxyethidium was 1419 pmol/mg of protein (25th-75th percentile = 1178-1513) in normoxia, 1993 (1831-2473) in moderate hyperoxia, and 2078 (1936-2922) in severe hyperoxia ( P = 0.008, effect across groups). Conclusions: Hyperoxia, compared with normoxia, impaired endothelium-dependent and endothelium-independent vasodilation but not the response to heme-independent soluble guanylyl cyclase activation, and hyperoxia increased vascular superoxide production. Results from this study could have important implications for patients receiving high concentrations of oxygen and at risk for ischemia reperfusion-mediated organ injury.
Topics: Mice; Animals; Soluble Guanylyl Cyclase; Nitroprusside; Acetylcholine; Superoxides; Hyperoxia; Endothelium, Vascular; Vasodilation; Vasodilator Agents; Heme; Oxygen; Nitric Oxide
PubMed: 36018251
DOI: 10.1097/SHK.0000000000001982 -
Current Biology : CB Jun 2023Switching 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...
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.
Topics: Animals; Interpeduncular Nucleus; Receptors, Nicotinic; Glutamic Acid; Acetylcholine; Habenula; Zebrafish
PubMed: 37105168
DOI: 10.1016/j.cub.2023.03.087 -
FEBS Letters Jul 2020Acetylcholine (ACh) signaling orchestrates mammalian movement, mental capacities, and inflammation. Dysregulated ACh signaling associates with many human mental... (Review)
Review
Acetylcholine (ACh) signaling orchestrates mammalian movement, mental capacities, and inflammation. Dysregulated ACh signaling associates with many human mental disorders and neurodegeneration in an individual-, sex-, and tissue-related manner. Moreover, aged patients under anticholinergic therapy show increased risk of dementia, but the underlying molecular mechanisms are incompletely understood. Here, we report that certain cholinergic-targeting noncoding RNAs, named Cholino-noncoding RNAs (ncRNAs), can modulate ACh signaling, agonistically or antagonistically, via distinct direct and indirect mechanisms and at different timescales. Cholino-ncRNAs include both small microRNAs (miRNAs) and long noncoding RNAs (lncRNAs). The former may attenuate translation and/or induce destruction of target mRNAs that code for either ACh-signaling proteins or transcription factors controlling the expression of cholinergic genes. lncRNAs may block miRNAs via 'sponging' events or by competitive binding to the cholinergic target mRNAs. Also, single nucleotide polymorphisms in either Cholino-ncRNAs or in their recognition sites in the ACh-signaling associated genes may modify ACh signaling-regulated processes. Taken together, both inherited and acquired changes in the function of Cholino-ncRNAs impact ACh-related deficiencies, opening new venues for individual, sex-related, and age-specific oriented research, diagnosis, and therapeutics.
Topics: Acetylcholine; Aging; Animals; Female; Humans; Male; RNA, Untranslated; Signal Transduction; Spatio-Temporal Analysis
PubMed: 32330292
DOI: 10.1002/1873-3468.13789 -
Circulation Research Sep 2023The phrase complete vagal withdrawal is often used when discussing autonomic control of the heart during exercise. However, more recent studies have challenged this...
BACKGROUND
The phrase complete vagal withdrawal is often used when discussing autonomic control of the heart during exercise. However, more recent studies have challenged this assumption. We hypothesized that cardiac vagal activity increases during exercise and maintains cardiac function via transmitters other than acetylcholine.
METHODS
Chronic direct recordings of cardiac vagal nerve activity, cardiac output, coronary artery blood flow, and heart rate were recorded in conscious adult sheep during whole-body treadmill exercise. Cardiac innervation of the left cardiac vagal branch was confirmed with lipophilic tracer dyes (DiO). Sheep were exercised with pharmacological blockers of acetylcholine (atropine, 250 mg), VIP (vasoactive intestinal peptide; [4Cl-D-Phe6,Leu17]VIP 25 µg), or saline control, randomized on different days. In a subset of sheep, the left cardiac vagal branch was denervated.
RESULTS
Neural innervation from the cardiac vagal branch is seen at major cardiac ganglionic plexi, and within the fat pads associated with the coronary arteries. Directly recorded cardiac vagal nerve activity increased during exercise. Left cardiac vagal branch denervation attenuated the maximum changes in coronary artery blood flow (maximum exercise, control: 63.5±5.9 mL/min, n=8; cardiac vagal denervated: 32.7±5.6 mL/min, n=6, 2.5×10), cardiac output, and heart rate during exercise. Atropine did not affect any cardiac parameters during exercise, but VIP antagonism significantly reduced coronary artery blood flow during exercise to a similar level to vagal denervation.
CONCLUSIONS
Our study demonstrates that cardiac vagal nerve activity actually increases and is crucial for maintaining cardiac function during exercise. Furthermore, our findings show the dynamic modulation of coronary artery blood flow during exercise is mediated by VIP.
Topics: Animals; Sheep; Acetylcholine; Heart; Coronary Vessels; Cardiac Output; Atropine
PubMed: 37641938
DOI: 10.1161/CIRCRESAHA.123.323017 -
ELife Jul 2020The mouse cerebral cortex contains neurons that express choline acetyltransferase (ChAT) and are a potential local source of acetylcholine. However, the...
The mouse cerebral cortex contains neurons that express choline acetyltransferase (ChAT) and are a potential local source of acetylcholine. However, the neurotransmitters released by cortical ChAT neurons and their synaptic connectivity are unknown. We show that the nearly all cortical ChAT neurons in mice are specialized VIP interneurons that release GABA strongly onto other inhibitory interneurons and acetylcholine sparsely onto layer 1 interneurons and other VIP/ChAT interneurons. This differential transmission of ACh and GABA based on the postsynaptic target neuron is reflected in VIP/ChAT interneuron pre-synaptic terminals, as quantitative molecular analysis shows that only a subset of these are specialized to release acetylcholine. In addition, we identify a separate, sparse population of non-VIP ChAT neurons in the medial prefrontal cortex with a distinct developmental origin that robustly release acetylcholine in layer 1. These results demonstrate both cortex-region heterogeneity in cortical ChAT interneurons and target-specific co-release of acetylcholine and GABA.
Topics: Acetylcholine; Animals; Brain; Cerebral Cortex; Choline O-Acetyltransferase; Heterozygote; Interneurons; Mice; Neurons; Prefrontal Cortex; Presynaptic Terminals; gamma-Aminobutyric Acid
PubMed: 32613945
DOI: 10.7554/eLife.57749 -
Journal of Experimental & Clinical... Sep 2023Chronic stress promotes most hallmarks of cancer through impacting the malignant tissues, their microenvironment, immunity, lymphatic flow, etc. Existing studies mainly...
BACKGROUND
Chronic stress promotes most hallmarks of cancer through impacting the malignant tissues, their microenvironment, immunity, lymphatic flow, etc. Existing studies mainly focused on the roles of stress-induced activation of systemic sympathetic nervous system and other stress-induced hormones, the organ specificity of chronic stress in shaping the pre-metastatic niche remains largely unknown. This study investigated the role of chronic stress in remodeling lung pre-metastatic niche of breast cancer.
METHODS
Breast cancer mouse models with chronic stress were constructed by restraint or unpredictable stress. Expressions of tyrosine hydroxylase, vesicular acetylcholine transporter (VAChT), EpCAM and NETosis were examined by immunofluorescence and confocal microscopy. mRNA and protein levels of choline acetyltransferase (ChAT), VAChT, and peptidylarginine deiminase 4 were detected by qRT-PCR and Western blotting, respectively. Immune cell subsets were analyzed by flow cytometry. Acetylcholine (ACh) and chemokines were detected by ELISA and multi chemokine array, respectively. ChAT in lung tissues from patients was examined by immunohistochemistry.
RESULTS
Breast cancer-bearing mice suffered chronic stress metastasized earlier and showed more severe lung metastasis than did mice in control group. VAChT, ChAT and ChAT epithelial cells were increased significantly in lung of model mice undergone chronic stress. ACh and chemokines especially CXCL2 in lung culture supernatants from model mice with chronic stress were profoundly increased. Chronic stress remodeled lung immune cell subsets with striking increase of neutrophils, enhanced NETosis in lung and promoted NETotic neutrophils to capture cancer cells. ACh treatment resulted in enhanced NETosis of neutrophils. The expression of ChAT in lung tissues from breast cancer patients with lung metastasis was significantly higher than that in patients with non-tumor pulmonary diseases.
CONCLUSIONS
Chronic stress promotes production of CXCL2 that recruits neutrophils into lung, and induces pulmonary epithelial cells to produce ACh that enhances NETosis of neutrophils. Our findings demonstrate for the first time that chronic stress induced epithelial cell derived ACh plays a key role in remodeling lung pre-metastatic niche of breast cancer.
Topics: Humans; Mice; Animals; Female; Acetylcholine; Membrane Transport Proteins; Breast Neoplasms; Lung; Epithelial Cells; Lung Neoplasms; Chemokines; Tumor Microenvironment
PubMed: 37773152
DOI: 10.1186/s13046-023-02836-5