To measure the sensitivity of the Ca2+ response of CalfluxCTN to carbachol stimulation in CHO-hM1R cells, we aliquoted serial dilutions of carbachol (Sigma-Aldrich) to a 384-well plate. The maximum final concentration of carbachol per well was 20 μM, and then it was diluted threefold serially until 12 concentrations were achieved, ranging between 0.17 nM and 20 μM carbachol. For Fluo-8 AM carbachol concentration response curves (CRCs), an identical protocol with the same concentrations was used, and the half-maximal effective concentration (EC50) was determined by curve fitting in R.
Carbachol
Manufactured by Merck Group
Sourced in United States, United Kingdom, Germany, Sao Tome and Principe, Italy, Australia, Japan, Hungary, Brazil, France
Carbachol is a chemical compound that acts as an acetylcholine receptor agonist. It is commonly used in laboratory settings as a research tool to study the effects of acetylcholine on various biological systems.
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Lab products found in correlation
Forskolin, by Merck Group (20 mentions)
Phenylephrine, by Merck Group (20 mentions)
Potassium chloride (kcl), by Merck Group (17 mentions)
Isoproterenol, by Merck Group (16 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (15 mentions)
Acetylcholine, by Merck Group (14 mentions)
Adenosine triphosphate (atp), by Merck Group (12 mentions)
L name, by Merck Group (11 mentions)
Indomethacin, by Merck Group (10 mentions)
Sodium bicarbonate, by Merck Group (10 mentions)
Bovine serum albumin (bsa), by Merck Group (10 mentions)
Isoprenaline, by Merck Group (9 mentions)
Calcium chloride, by Merck Group (9 mentions)
Magnesium chloride, by Merck Group (9 mentions)
Potassium dihydrogen phosphate, by Merck Group (8 mentions)
Acetylcholine chloride, by Merck Group (8 mentions)
Histamine, by Merck Group (8 mentions)
Sodium chloride (nacl), by Avantor (8 mentions)
Fura 2 am, by Thermo Fisher Scientific (8 mentions)
Thapsigargin, by Merck Group (7 mentions)
288 protocols using carbachol
1
Carbachol-Induced Ca2+ Response in CHO-hM1R
2
Pharmacological Characterization of Test Compounds
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The test compounds are highly hygroscopic and were stored in a dry place. The solutions were prepared fresh every day. The products used in this study were obtained from the following sources, and stock solutions were prepared as indicated below: acetylcholine (Sigma-Aldrich, Italy), carbachol (Sigma-Aldrich, Italy), histamine (Sigma-Aldrich, Italy), IbTX (Sigma-Aldrich, Italy), indomethacin (Sigma-Aldrich, Italy), glycopyrronium bromide (NVA237, Novartis), forskolin (Sigma-Aldrich, Italy), papaverine (Sigma-Aldrich, Italy), indacaterol fumarate (QAB149, Novartis), quinine (Sigma-Aldrich, Italy) and TeTX (Sigma-Aldrich, Italy).
acetylcholine, carbachol, histamine and papaverine were dissolved in distilled water; glycopyrronium, indacaterol and forskolin were dissolved in dimethylsulfoxide (DMSO); indomethacin was dissolved in ethanol and then diluted in KH buffer solution. The maximal amount of ethanol (0.02 %) did not influence isolated tissue response [7 (link), 13 (link)–15 (link), 17 (link), 19 (link), 33 (link)]. Compounds were stored in small aliquots at −80 °C until their use.
acetylcholine, carbachol, histamine and papaverine were dissolved in distilled water; glycopyrronium, indacaterol and forskolin were dissolved in dimethylsulfoxide (DMSO); indomethacin was dissolved in ethanol and then diluted in KH buffer solution. The maximal amount of ethanol (0.02 %) did not influence isolated tissue response [7 (link), 13 (link)–15 (link), 17 (link), 19 (link), 33 (link)]. Compounds were stored in small aliquots at −80 °C until their use.
3
Pharmacological Agents for Membrane Potential
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We used several pharmacological agents including Hm1a (Alomone Labs STH-601), linopirdine (Sigma L134), Retigabine (Alomone D-23129), the cholinomimetic carbamoylcholine chloride (carbachol; Sigma C4382), muscarine (Sigma M6532), and the nicotinic agonist 4-Acetyl-1,1-dimethylpiperazinium iodide (Tocris 0352). Hm1a, muscarine, and 4-Acetyl-1,1-dimethylpiperazinium iodide were dissolved in deionized water, aliquoted, and frozen at −20° C. carbachol was stored as a powder at RT, and both linopirdine and Retigabine were stored as 100 mM stock solutions in DMSO (Sigma) aliquoted in −20°C. For pharmacological experiments in which drug was dissolved in DMSO, the same concentration of DMSO was present in control external solution. After a baseline recording, drugs were perfused in at 3 mL/min while continuously recording membrane potential; repeat measurements were performed 10 min after wash-in. A subset of cells were recorded for up to 1 hr following washout of each drug, as needed in some cases to observe complete or near-complete washout. For experiments using carbachol, repeat measurements were made 5 min after wash-in.
4
Preparation of Pharmacological Agents
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Cisplatin, phenylephrine, carbachol, and sodium nitroprusside were obtained from Sigma (Sigma Chemical Company, Poole, Dorset, UK).
phenylephrine, carbachol, and sodium nitroprusside were dissolved in distilled water. Cisplatin was dissolved in saline (0.9% NaCl).
phenylephrine, carbachol, and sodium nitroprusside were dissolved in distilled water. Cisplatin was dissolved in saline (0.9% NaCl).
5
Cholinergic and GABAergic Modulation of Hippocampal Theta
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Either carbachol (0.1 μl of 0.156 μg/μl; Sigma, United States; Jiang and Khanna, 2006 (link)) or nicotine tartrate (0.1 μl of 12 μg/μl; Sigma, United States; Ikemoto et al., 2006 (link)) was microinjected into the mSuM or the lSuM. carbachol is a broad spectrum cholinergic agonist (Ariffin et al., 2010 (link)) while nicotine is an agonist at the nicotinic cholinergic receptors. Muscimol hydrobromide (0.5 μl of 2 μg/μl; Sigma, United States; Lee et al., 2011 (link)), a GABA mimetic, was microinjected into the MS. All drugs were dissolved in vehicle made of 0.5% w/v Alcian blue dye (Sigma, United States) solution in saline (VWR, Germany). As microinjection control, vehicle was microinjected into SuM.
Functionally, intra-SuM microinjection of carbachol at the selected concentration elicits robust hippocampal theta activation (Jiang and Khanna, 2006 (link)). The concentration of intra-SuM nicotine used in this study is known to result in reinforcing behavioral effects (Ikemoto et al., 2006 (link)). While intraseptal muscimol at the aforementioned concentration results in suppression of hippocampal theta activation induced on RPO stimulation (Lee et al., 2011 (link)).
Functionally, intra-SuM microinjection of carbachol at the selected concentration elicits robust hippocampal theta activation (Jiang and Khanna, 2006 (link)). The concentration of intra-SuM nicotine used in this study is known to result in reinforcing behavioral effects (Ikemoto et al., 2006 (link)). While intraseptal muscimol at the aforementioned concentration results in suppression of hippocampal theta activation induced on RPO stimulation (Lee et al., 2011 (link)).
6
Murine Airway Reactivity Evaluation
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OVA-sensitized mice were sacrificed on day 21 by enflurane overdose, exsanguinated, and their lungs were removed. The main bronchi were rapidly dissected and cleaned from fat and connective tissue. Rings of 1–2 mm length were cut and mounted in 3 mL isolated organ baths containing Krebs solution, at 37 °C, oxygenated (95% O2 and 5% CO2), and connected to an isometric force transducer (type 7006, Ugo Basile, Comerio, Italy) associated to a Powerlab 800 (AD Instruments, Oxford, UK). Rings were initially stretched until a resting tension of 0.5 g was reached and allowed to equilibrate for at least 30 min during which tension was adjusted, when necessary, to 0.5 g and the bathing solution was periodically changed. In each experiment, bronchial rings were previously challenged with carbachol (1 × 10−6 M) (Sigma-Aldrich, Milan, Italy) and then a cumulative concentration-response curve of carbachol (1 × 10−9 M − 3 × 10−6 M) or salbutamol (1 × 10−8–3 × 10−5 M) was performed to evaluate bronchial reactivity. Results are expressed as dyne per mg tissue [37 (link),38 (link)].
7
Optimized Culture Medium for LGAC Isolation
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The culture medium was prepared based on the culture media used for LGAC cultures, as previously described (9, 15, 17) , and was applied for LGAC isolation, cell manipulation, and cell culture.
For the preparation of the medium, 250 mL of low glucose (1 g/L) Dulbecco's Modified Eagles Medium (DMEM) (Invitrogen, Camirillo, CA) was supplemented with gentamicin 25 µg/mL, putrescine 1.0 mM, reduced glutathione 10 µg/mL, ascorbic acid 50 µg/mL, dexamethasone 10 µg/mL, insulin 5.0 µg/mL, selenium 5.0 ng/mL, transferrin 5.0 µg/mL, and carbachol 100 μM (Sigma-Aldrich, St. Louis, MO, USA). The reagents were carefully weighed (Bioprecisa, FA 2104N, PR, Brazil) and mixed under aseptic conditions in a fume hood (Nuaire, NU-425, USA) (Table 1).
After buffering the pH to 7.6 (Model 215, Denver Instrument Company, USA), the culture medium was filtered with disposable 22-µm sterile filters (Corning Inc, NY, USA) connected to a vacuum pump.
Epithelial growth factor (EGF) was added (Sigma-Aldrich, St. Louis, MO) at a concentration of 10 ng/mL. The medium was stored at 4°C and removed 30 min prior to use to warm it up slowly to room temperature.
As specifically mentioned in the assays described below, the medium was supplemented with fetal bovine serum (FBS) (Invitrogen, Camirillo, CA, USA) or variable concentrations of insulin and carbachol (Sigma-Aldrich, St. Louis, MO, USA).
For the preparation of the medium, 250 mL of low glucose (1 g/L) Dulbecco's Modified Eagles Medium (DMEM) (Invitrogen, Camirillo, CA) was supplemented with gentamicin 25 µg/mL, putrescine 1.0 mM, reduced glutathione 10 µg/mL, ascorbic acid 50 µg/mL, dexamethasone 10 µg/mL, insulin 5.0 µg/mL, selenium 5.0 ng/mL, transferrin 5.0 µg/mL, and carbachol 100 μM (Sigma-Aldrich, St. Louis, MO, USA). The reagents were carefully weighed (Bioprecisa, FA 2104N, PR, Brazil) and mixed under aseptic conditions in a fume hood (Nuaire, NU-425, USA) (Table 1).
After buffering the pH to 7.6 (Model 215, Denver Instrument Company, USA), the culture medium was filtered with disposable 22-µm sterile filters (Corning Inc, NY, USA) connected to a vacuum pump.
Epithelial growth factor (EGF) was added (Sigma-Aldrich, St. Louis, MO) at a concentration of 10 ng/mL. The medium was stored at 4°C and removed 30 min prior to use to warm it up slowly to room temperature.
As specifically mentioned in the assays described below, the medium was supplemented with fetal bovine serum (FBS) (Invitrogen, Camirillo, CA, USA) or variable concentrations of insulin and carbachol (Sigma-Aldrich, St. Louis, MO, USA).
8
Vascular Smooth Muscle Cell Stimulation
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Human adult aortic VSMCs (passage 3–10) were purchased from Cell Applications Inc. (354-05a). VSMCs were grown in growth media (Cell Applications Inc.), prior to being washed with Earle’s Balanced Salt Solution (Thermo) and seeded in basal media (Cell Applications Inc.) onto 12 kPa hydrogels, 18 h prior to drug treatment. Standard VSMCs culture was performed as described previously (Ragnauth et al., 2010 (link); Warren et al., 2015 (link)).
Quiescent VSMCs were stimulated with either angiotensin II (0.01–100 µM) (Merck) or carbachol (0.01–100 µM) (Merck) for 30 min. For all other drug treatments, quiescent VSMCs were pretreated with the stated dose for 30 min, prior to co-treatment with angiotensin II (10 µM) for an additional 30 min. Please seeSupplementary Table S1 for a list of compounds used in this study.
Quiescent VSMCs were stimulated with either angiotensin II (0.01–100 µM) (Merck) or carbachol (0.01–100 µM) (Merck) for 30 min. For all other drug treatments, quiescent VSMCs were pretreated with the stated dose for 30 min, prior to co-treatment with angiotensin II (10 µM) for an additional 30 min. Please see
9
Quantifying Lacrimal Gland Organoid Secretion
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Secretory function of lacrimal gland organoids was quantified by measuring N-acetyl-β-glucosaminidase (NAG), a lysosomal enzyme in the tear fluid. In brief, organoids were treated either with 10 µM forskolin (Tocris) or 100 µM carbachol (Merck) for 30, 60, and 90 min. Bright field images of the treated cultures were acquired at the initiation of treatment and after 30 min, 60 min and 24 h. Acquired images were quantified using the measure function of the ImageJ software to assess the organoid swelling. Treated culture medium and control medium for background was collected and centrifuged at 10,000 x g for 3 min. NAG concentration in supernatants was quantified using a NAG assay kit (Abcam, ab204705) following the manufacturer’s protocol. Reaction product was detected colorimetrically at 400 nm using a microplate reader (Multiskan GO, Thermo Fisher Scientific).
10
Pharmacological Modulation of Smooth Muscle
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BIM (I), carbachol and ET‐1 was supplied by Merck Life Science UK Ltd. (Gillingham, UK) and BOP, phenylephrine and U46619 by Bio‐Techne Ltd. (Abingdon, UK). Nifedipine was supplied by Cayman Chemicals (Ann Arbor, USA) and Y27632 by STEMCELL Technologies (Cambridge, UK).
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