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Journal of Oral Biosciences Jun 2024Typical agonists of G protein-coupled receptors (GPCRs), including muscarinic acetylcholine receptors (mAChRs), activate both G-protein and β-arrestin signaling...
OBJECTIVES
Typical agonists of G protein-coupled receptors (GPCRs), including muscarinic acetylcholine receptors (mAChRs), activate both G-protein and β-arrestin signaling systems, and are termed balanced agonists. In contrast, biased agonists selectively activate a single pathway, thereby offering therapeutic potential for the specific activation of that pathway. The mAChR agonists carbachol and pilocarpine are known to induce phosphorylation of extracellular signal-regulated kinase-1/2 (ERK1/2) via G-protein-dependent and -independent pathways, respectively. We investigated the involvement of β-arrestin and its downstream mechanisms in the ERK1/2 phosphorylation induced by carbachol and pilocarpine in the human salivary ductal cell line, HSY cells.
METHODS
HSY cells were stimulated with pilocarpine or carbachol, with or without various inhibitors. The cell lysates were analyzed by western blotting using the antibodies p44/p42 and phosphor-p44/p42.
RESULTS
Western blot analysis revealed that carbachol elicited greater stimulation of ERK1/2 phosphorylation compared to pilocarpine. ERK1/2 phosphorylation was inhibited by atropine and gefitinib, suggesting that mAChR activation induces transactivation of epidermal growth factor receptors (EGFR). Moreover, inhibition of carbachol-mediated ERK1/2 phosphorylation was achieved by GF-109203X (a PKC inhibitor), a βARK1/GRK2 inhibitor, barbadin (a β-arrestin inhibitor), pitstop 2 (a clathrin inhibitor), and dynole 34-2 (a dynamin inhibitor). In contrast, pilocarpine-mediated ERK1/2 phosphorylation was only inhibited by barbadin (a β-arrestin inhibitor) and PP2 (a Src inhibitor).
CONCLUSION
Carbachol activates both G-protein and β-arrestin pathways, whereas pilocarpine exclusively activates the β-arrestin pathway. Additionally, downstream of β-arrestin, carbachol activates clathrin-dependent internalization, while pilocarpine activates Src.
Topics: Humans; Phosphorylation; Receptors, Muscarinic; Pilocarpine; Carbachol; Muscarinic Agonists; Signal Transduction; Salivary Ducts; beta-Arrestins; Cell Line; Extracellular Signal-Regulated MAP Kinases; Blotting, Western; Arrestins
PubMed: 38336259
DOI: 10.1016/j.job.2024.02.002 -
Iranian Journal of Medical Sciences Jan 2024Epidemic thunderstorm asthma is an observed increase in cases of acute bronchospasm following thunderstorms. This study aimed to compare the frequency of obstructive...
BACKGROUND
Epidemic thunderstorm asthma is an observed increase in cases of acute bronchospasm following thunderstorms. This study aimed to compare the frequency of obstructive airway disease or bronchial hyperresponsiveness in subjects with thunderstorm-associated respiratory symptoms with subjects with similar symptoms presented at other times.
METHODS
A cross-sectional study from June to November of 2013 was conducted on subjects with thunderstorm-associated respiratory symptoms living in Ahvaz City, Iran. Thunderstorm-associated subjects were presented with asthmatic symptoms in thunderstorms, and other patients presented with similar symptoms at other times. Baseline spirometry was performed on patients to examine the presence of obstructive airway disease. In all patients with normal spirometry, a provocation test was applied. A comparison of qualitative and quantitative variables was made using the Chi-square and independent test, respectively. All analyses were carried out using SPSS Statistics Version 22. A P value less than 0.05 was considered statistically significant.
RESULTS
Out of 584 subjects, 300 and 284 participants were in thunderstorm-associated and non-thunderstorm-associated groups, respectively. After the final analysis, 87 (30.6%) and 89 (33.3%) of the thunderstorm-associated subjects and non-thunderstorm-associated group, respectively, had pieces of evidence of airflow limitation (P=0.27). Among the patients with normal spirometry, 161 (81.72%) of the thunderstorm-associated patients and 100 (56.17%) patients of the non-thunderstorm-associated symptoms group had a positive methacholine challenge test result (P<0.001).
CONCLUSION
Most of the patients with thunderstorm-associated respiratory symptoms had no obvious evidence of airflow limitation in spirometry.
Topics: Humans; Cross-Sectional Studies; Methacholine Chloride; Asthma; Bronchial Provocation Tests; Pulmonary Disease, Chronic Obstructive
PubMed: 38322159
DOI: 10.30476/ijms.2023.96337.2784 -
Epilepsia Open Apr 2024The goal of this research was to evaluate the effect of DM type 2 (DM2) on SE severity, neurodegeneration, and brain oxidative stress (OS) secondary to seizures.
OBJECTIVE
The goal of this research was to evaluate the effect of DM type 2 (DM2) on SE severity, neurodegeneration, and brain oxidative stress (OS) secondary to seizures.
METHODS
DM2 was induced in postnatal day (P) 3 male rat pups by injecting streptozocin (STZ) 100 mg/kg; control rats were injected with citrate buffer as vehicle. At P90, SE was induced by the lithium-pilocarpine administration and seizure latency, frequency, and severity were evaluated. Neurodegeneration was assessed 24 h after SE by Fluoro-Jade B (F-JB) staining, whereas OS was estimated by measuring lipid peroxidation and reactive oxygen species (ROS).
RESULTS
DM2 rats showed an increase in latency to the first generalized seizure and SE onset, had a higher number and a longer duration of seizures, and displayed a larger neurodegeneration in the hippocampus (CA3, CA1, dentate gyrus, and hilus), the piriform cortex, the dorsomedial nucleus of the thalamus and the cortical amygdala. Our results also show that only SE, neither DM2 nor the combination of DM2 with SE, caused the increase in ROS and brain lipid peroxidation.
SIGNIFICANCE
DM2 causes higher seizure severity and neurodegeneration but did not exacerbate SE-induced OS under these conditions.
PLAIN LANGUAGE SUMMARY
Our research performed in animal models suggests that type 2 diabetes mellitus (DM2) may be a risk factor for causing higher seizure severity and seizure-induced neuron cell death. However, even when long-term seizures promote an imbalance between brain pro-oxidants and antioxidants, DM2 does not exacerbate that disproportion.
Topics: Rats; Animals; Male; Diabetes Mellitus, Type 2; Reactive Oxygen Species; Pilocarpine; Seizures; Status Epilepticus; Oxidative Stress
PubMed: 38321819
DOI: 10.1002/epi4.12905 -
The Journal of Neuroscience : the... Mar 2024Gonadotropin-releasing hormone (GnRH)-synthesizing neurons orchestrate reproduction centrally. Early studies have proposed the contribution of acetylcholine (ACh) to...
Gonadotropin-releasing hormone (GnRH)-synthesizing neurons orchestrate reproduction centrally. Early studies have proposed the contribution of acetylcholine (ACh) to hypothalamic control of reproduction, although the causal mechanisms have not been clarified. Here, we report that in vivo pharmacogenetic activation of the cholinergic system increased the secretion of luteinizing hormone (LH) in orchidectomized mice. 3DISCO immunocytochemistry and electron microscopy revealed the innervation of GnRH neurons by cholinergic axons. Retrograde viral labeling initiated from GnRH-Cre neurons identified the medial septum and the diagonal band of Broca as exclusive sites of origin for cholinergic afferents of GnRH neurons. In acute brain slices, ACh and carbachol evoked a biphasic effect on the firing rate in GnRH neurons, first increasing and then diminishing it. In the presence of tetrodotoxin, carbachol induced an inward current, followed by a decline in the frequency of miniature postsynaptic currents (mPSCs), indicating a direct influence on GnRH cells. RT-PCR and whole-cell patch-clamp studies revealed that GnRH neurons expressed both nicotinic (α4β2, α3β4, and α7) and muscarinic (M1-M5) AChRs. The nicotinic AChRs contributed to the nicotine-elicited inward current and the rise in firing rate. Muscarine via M1 and M3 receptors increased, while via M2 and M4 reduced the frequency of both mPSCs and firing. Optogenetic activation of channelrhodopsin-2-tagged cholinergic axons modified GnRH neuronal activity and evoked cotransmission of ACh and GABA from a subpopulation of boutons. These findings confirm that the central cholinergic system regulates GnRH neurons and activates the pituitary-gonadal axis via ACh and ACh/GABA neurotransmissions in male mice.
Topics: Mice; Animals; Male; Acetylcholine; Gonadotropin-Releasing Hormone; Carbachol; Neurons; Cholinergic Agents; Nicotine; Luteinizing Hormone; gamma-Aminobutyric Acid
PubMed: 38320853
DOI: 10.1523/JNEUROSCI.1780-23.2024 -
Zhonghua Jie He He Hu Xi Za Zhi =... Feb 2024The methacholine challenge test (MCT) is a standard evaluation method of assessing airway hyperresponsiveness (AHR) and its severity, and has significant clinical value...
The methacholine challenge test (MCT) is a standard evaluation method of assessing airway hyperresponsiveness (AHR) and its severity, and has significant clinical value in the diagnosis and treatment of bronchial asthma. A consensus working group consisting of experts from the Pulmonary Function and Clinical Respiratory Physiology Committee of the Chinese Association of Chest Physicians, the Task Force for Pulmonary Function of the Chinese Thoracic Society, and the Pulmonary Function Group of Respiratory Branch of the Chinese Geriatric Society jointly developed this consensus. Based on the "" published in 2014, the issues encountered in its use, and recent developments, the group has updated the Through an extensive collection of expert opinions, literature reviews, questionnaire surveys, and multiple rounds of online and offline discussions, the consensus addressed the eleven core issues in MCT's clinical practice, including indications, contraindications, preparation of provocative agents, test procedures and methods, quality control, safety management, interpretation of results, and reporting standards. The aim was to provide clinical pulmonary function practitioners in healthcare institutions with the tools to optimize the use of this technique to guide clinical diagnosis and treatment. Who is suitable for conducting MCT? What are contraindications for performing MCT?Patients with atypical symptoms and a clinical suspicion of asthma, patients diagnosed with asthma requiring assessment of the severity of airway hyperresponsiveness, individuals with allergic rhinitis who are at risk of developing asthma, patients in need of evaluating the effectiveness of asthma treatment, individuals in occupations with high safety risks due to airway hyperresponsiveness, patients with chronic diseases prone to airway hyperresponsiveness, others requiring assessment of airway reactivity.Absolute contraindications: (1) Patients who are allergic to methacholine (MCh) or other parasympathomimetic drugs, with allergic reactions including rash, itching/swelling (especially of the face, tongue, and throat), severe dizziness, and dyspnea; (2) Patients with a history of life-threatening asthma attacks or those who have required mechanical ventilation for asthma attacks in the past three months; (3) Patients with moderate to severe impairment of baseline pulmonary function [Forced Expiratory Volume in one second (FEV) less than 60% of the predicted value or FEV<1.0 L]; (4) Severe urticaria; (5) Other situations inappropriate for forced vital capacity (FVC) measurement, such as myocardial infarction or stroke in the past three months, poorly controlled hypertension, aortic aneurysm, recent eye surgery, or increased intracranial pressure.Relative contraindications: (1) Moderate or more severe impairment of baseline lung function (FEV%pred<70%), but individuals with FEV%pred>60% may still be considered for MCT with strict observation and adequate preparation; (2) Experiencing asthma acute exacerbation; (3) Poor cooperation with baseline lung function tests that do not meet quality control requirements; (4) Recent respiratory tract infection (<4 weeks); (5) Pregnant or lactating women; (6) Patients currently using cholinesterase inhibitors (for the treatment of myasthenia gravis); (7) Patients who have previously experienced airway spasm during pulmonary function tests, with a significant decrease in FEV even without the inhalation of provocative. How to prepare and store the challenge solution for MCT?Before use, the drug must be reconstituted and then diluted into various concentrations for provocation. The dilution concentration and steps for MCh vary depending on the inhalation method and provocation protocol used. It is important to follow specific steps. Typically, a specified amount of diluent is added to the methacholine reagent bottle for reconstitution, and the mixture is shaken until the solution becomes clear. The diluent is usually physiological saline, but saline with phenol (0.4%) can also be used. Phenol can reduce the possibility of bacterial contamination, and its presence does not interfere with the provocation test. After reconstitution, other concentrations of MCh solution are prepared using the same diluent, following the dilution steps, and then stored separately in sterile containers. Preparers should carefully verify and label the concentration and preparation time of the solution and complete a preparation record form. The reconstituted and diluted MCh solution is ready for immediate use without the need for freezing. It can be stored for two weeks if refrigerated (2-8 ℃). The reconstituted solution should not be stored directly in the nebulizer reservoir to prevent crystallization from blocking the capillary opening and affecting aerosol output. The temperature of the solution can affect the production of the nebulizer and cause airway spasms in the subject upon inhaling cold droplets. Thus, refrigerated solutions should be brought to room temperature before use. What preparation is required for subjects prior to MCT?(1) Detailed medical history inquiry and exclusion of contraindications.(2) Inquiring about factors and medications that may affect airway reactivity and assessing compliance with medication washout requirements: When the goal is to evaluate the effectiveness of asthma treatment, bronchodilators other than those used for asthma treatment do not need to be discontinued. Antihistamines and cromolyn have no effect on MCT responses, and the effects of a single dose of inhaled corticosteroids and leukotriene modifiers are minimal, thus not requiring cessation before the test. For patients routinely using corticosteroids, whether to discontinue the medication depends on the objective of the test: if assisting in the diagnosis of asthma, differential diagnosis, aiding in step-down therapy for asthma, or exploring the effect of discontinuing anti-inflammatory treatment, corticosteroids should be stopped before the provocation test; if the patient is already diagnosed with asthma and the objective is to observe the level of airway reactivity under controlled medication conditions, then discontinuation is not necessary. Medications such as IgE monoclonal antibodies, IL-4Rα monoclonal antibodies, traditional Chinese medicine, and ethnic medicines may interfere with test results, and clinicians should decide whether to discontinue these based on the specific circumstances.(3) Explaining the test procedure and potential adverse reactions, and obtaining informed consent if necessary. What are the methods of the MCT? And which ones are recommended in current clinical practice?Commonly used methods for MCT in clinical practice include the quantitative nebulization method (APS method), Forced Oscillalion method (Astograph method), 2-minute tidal breathing method (Cockcroft method), hand-held quantitative nebulization method (Yan method), and 5-breath method (Chai 5-breath method). The APS method allows for precise dosing of inhaled Methacholine, ensuring accurate and reliable results. The Astograph method, which uses respiratory resistance as an assessment indicator, is easy for subjects to perform and is the simplest operation. These two methods are currently the most commonly used clinical practice in China. What are the steps involved in MCT?The MCT consists of the following four steps:(1) Baseline lung function test: After a 15-minute rest period, the subjects assumes a seated position and wear a nose clip for the measurement of pulmonary function indicators [such as FEV or respiratory resistance (Rrs)]. FEV should be measured at least three times according to spirometer quality control standards, ensuring that the best two measurements differ by less than 150 ml and recording the highest value as the baseline. Usually, if FEV%pred is below 70%, proceeding with the challenge test is not suitable, and a bronchodilation test should be considered. However, if clinical assessment of airway reactivity is necessary and FEV%pred is between 60% and 70%, the provocation test may still be conducted under close observation, ensuring the subject's safety. If FEV%pred is below 60%, it is an absolute contraindication for MCT.(2) Inhalation of diluent and repeat lung function test for control values: the diluent, serving as a control for the inhaled MCh, usually does not significantly impact the subject's lung function. the higher one between baseline value and the post-dilution FEV is used as the reference for calculating the rate of FEV decline. If post-inhalation FEV decreases, there are usually three scenarios: ①If FEV decreases by less than 10% compared to the baseline, the test can proceed, continue the test and administer the first dose of MCh. ②If the FEV decreases by≥10% and<20%, indicating a heightened airway reactivity to the diluent, proceed with the lowest concentration (dose) of the provoking if FEV%pred has not yet reached the contraindication criteria for the MCT. if FEV%pred<60% and the risk of continuing the challenge test is considerable, it is advisable to switch to a bronchodilation test and indicate the change in the test results report. ③If FEV decreases by≥20%, it can be directly classified as a positive challenge test, and the test should be discontinued, with bronchodilators administered to alleviate airway obstruction.(3) Inhalation of MCh and repeat lung function test to assess decline: prepare a series of MCh concentrations, starting from the lowest and gradually increasing the inhaled concentration (dose) using different methods. Perform pulmonary function tests at 30 seconds and 90 seconds after completing nebulization, with the number of measurements limited to 3-4 times. A complete Forced Vital Capacity (FVC) measurement is unnecessary during testing; only an acceptable FEV measurement is required. The interval between two consecutive concentrations (doses) generally should not exceed 3 minutes. If FEV declines by≥10% compared to the control value, reduce the increment of methacholine concentration (dose) and adjust the inhalation protocol accordingly. If FEV declines by≥20% or more compared to the control value or if the maximum concentration (amount) has been inhaled, the test should be stopped. After inhaling the MCh, close observation of the subject's response is necessary. If necessary, monitor blood oxygen saturation and auscultate lung breath sounds. The test should be promptly discontinued in case of noticeable clinical symptoms or signs.(4) Inhalation of bronchodilator and repeat lung function test to assess recovery: when the bronchial challenge test shows a positive response (FEV decline≥20%) or suspiciously positive, the subject should receive inhaled rapid-acting bronchodilators, such as short-acting beta-agonists (SABA) or short-acting muscarinic antagonists (SAMA). Suppose the subject exhibits obvious symptoms of breathlessness, wheezing, or typical asthma manifestations, and wheezing is audible in the lungs, even if the positive criteria are not met. In that case, the challenge test should be immediately stopped, and rapid-acting bronchodilators should be administered. Taking salbutamol as an example, inhale 200-400 μg (100 μg per puff, 2-4 puffs, as determined by the physician based on the subject's condition). Reassess pulmonary function after 5-10 minutes. If FEV recovers to within 10% of the baseline value, the test can be concluded. However, if there is no noticeable improvement (FEV decline still≥10%), record the symptoms and signs and repeat the bronchodilation procedure as mentioned earlier. Alternatively, add Ipratropium bromide (SAMA) or further administer nebulized bronchodilators and corticosteroids for intensified treatment while keeping the subject under observation until FEV recovers to within 90% of the baseline value before allowing the subject to leave. What are the quality control requirements for the APS and Astograph MCT equipment?(1) APS Method Equipment Quality Control: The APS method for MCT uses a nebulizing inhalation device that requires standardized flowmeters, compressed air power source pressure and flow, and nebulizer aerosol output. Specific quality control methods are as follows:a. Flow and volume calibration of the quantitative nebulization device: Connect the flowmeter, an empty nebulization chamber, and a nebulization filter in sequence, attaching the compressed air source to the bottom of the chamber to ensure airtight connections. Then, attach a 3 L calibration syringe to the subject's breathing interface and simulate the flow during nebulization (typically low flow:<2 L/s) to calibrate the flow and volume. If calibration results exceed the acceptable range of the device's technical standards, investigate and address potential issues such as air leaks or increased resistance due to a damp filter, then recalibrate. Cleaning the flowmeter or replacing the filter can change the resistance in the breathing circuit, requiring re-calibration of the flow.b. Testing the compressed air power source: Regularly test the device, connecting the components as mentioned above. Then, block the opening of the nebulization device with a stopper or hand, start the compressed air power source, and test its pressure and flow. If the test results do not meet the technical standards, professional maintenance of the equipment may be required.c. Verification of aerosol output of the nebulization chamber: Regularly verify all nebulization chambers used in provocation tests. Steps include adding a certain amount of saline to the chamber, weighing and recording the chamber's weight (including saline), connecting the nebulizer to the quantitative nebulization device, setting the nebulization time, starting nebulization, then weighing and recording the post-nebulization weight. Calculate the unit time aerosol output using the formula [(weight before nebulization-weight after nebulization)/nebulization time]. Finally, set the nebulization plan for the provocation test based on the aerosol output, considering the MCh concentration, single inhalation nebulization duration, number of nebulization, and cumulative dose to ensure precise dosing of the inhaled MCh.(2) Astograph method equipment quality control: Astograph method equipment for MCT consists of a respiratory resistance monitoring device and a nebulization medication device. Perform zero-point calibration, volume calibration, impedance verification, and nebulization chamber checks daily before tests to ensure the resistance measurement system and nebulization system function properly. Calibration is needed every time the equipment is turned on, and more frequently if there are significant changes in environmental conditions.a. Zero-point calibration: Perform zero-point calibration before testing each subject. Ensure the nebulization chamber is properly installed and plugged with no air leaks.b. Volume calibration: Use a 3 L calibration syringe to calibrate the flow sensor at a low flow rate (approximately 1 L/s).c. Resistance verification: Connect low impedance tubes (1.9-2.2 cmHO·L·s) and high impedance tubes (10.2-10.7 cmHO·L·s) to the device interface for verification.d. Bypass check: Start the bypass check and record the bypass value; a value>150 ml/s is normal.e. Nebulization chamber check: Check each of the 12 nebulization chambers daily, especially those containing bronchodilators, to ensure normal spraying. The software can control each nebulization chamber to produce spray automatically for a preset duration (e.g., 2 seconds). Observe the formation of water droplets on the chamber walls, indicating normal spraying. If no nebulization occurs, check for incorrect connections or blockages. How to set up and select the APS method in MCT?The software program of the aerosol provocation system in the quantitative nebulization method can independently set the nebulizer output, concentration of the methacholine agent, administration time, and number of administrations and combine these parameters to create the challenge test process. In principle, the concentration of the methacholine agent should increase from low to high, and the dose should increase from small to large. According to the standard, a 2-fold or 4-fold incremental challenge process is generally used. In clinical practice, the dose can be simplified for subjects with good baseline lung function and no history of wheezing, such as using a recommended 2-concentration, 5-step method (25 and 50 g/L) and (6.25 and 25 g/L). Suppose FEV decreases by more than 10% compared to the baseline during the test to ensure subject safety. In that case, the incremental dose of the methacholine agent can be reduced, and the inhalation program can be adjusted appropriately. If the subject's baseline lung function declines or has recent daytime or nighttime symptoms such as wheezing or chest tightness, a low concentration, low dose incremental process should be selected. What are the precautions for the operation process of the Astograph method in MCT?(1) Test equipment: The Astograph method utilizes the forced oscillation technique, applying a sinusoidal oscillating pressure at the mouthpiece during calm breathing. Subjects inhale nebulized MCh of increasing concentrations while continuous monitoring of respiratory resistance (Rrs) plots the changes, assessing airway reactivity and sensitivity. The nebulization system employs jet nebulization technology, comprising a compressed air pump and 12 nebulization cups. The first cup contains saline, cups 2 to 11 contain increasing concentrations of MCh, and the 12th cup contains a bronchodilator solution.(2) Provocation process: Prepare 10 solutions of MCh provocant with gradually increasing concentrations.(3) Operational procedure: The oscillation frequency is usually set to 3 Hz (7 Hz for children) during the test. The subject breathes calmly, inhales saline solution nebulized first, and records the baseline resistance value (if the subject's baseline resistance value is higher than 10 cmHO·L·s, the challenge test should not be performed). Then, the subject gradually inhales increasing concentrations of methacholine solution. Each concentration solution is inhaled for 1 minute, and the nebulization system automatically switches to the next concentration for inhalation according to the set time. Each nebulizer cup contains 2-3 ml of solution, the output is 0.15 ml/min, and each concentration is inhaled for 1 minute. The dose-response curve is recorded automatically. Subjects should breathe tidally during the test, avoiding deep breaths and swallowing. Continue until Rrs significantly rises to more than double the baseline value, or if the subject experiences notable respiratory symptoms or other discomfort, such as wheezing in both lungs upon auscultation. At this point, the inhalation of the provocant should be stopped and the subject switchs to inhaling a bronchodilator until Rrs returns to pre-provocation levels. If there is no significant increase in Rrs, stop the test after inhaling the highest concentration of MCh. How to interpret the results of the MCT?The method chosen for the MCT determines the specific indicators used for interpretation. The most commonly used indicator is FEV, although other parameters such as Peak Expiratory Flow (PEF) and Rrs can also be used to assess airway hyperresponsiveness. The test results can be classified as positive, suspiciously positive, or negative, based on a combination of the judgment indicators and changes in the subject's symptoms. If FEV decreases by≥20% compared to the baseline value after not completely inhaling at the highest concentration, the result can be judged as positive for Methacholine bronchial challenge test. If the patient has obvious wheezing symptoms or wheezing is heard in both lungs, but the challenge test does not meet the positive criteria (the highest dose/concentration has been inhaled), and FEV decreases between 10% and 20% compared to the baseline level, the result can also be judged as positive. If FEV decreases between 15% and 20% compared to the baseline value without dyspnea or wheezing attacks, the result can be judged as suspiciously positive. Astograph method: If Rrs rises to 2 times or more of the baseline resistance before reaching the highest inhalation concentration, or if the subject's lungs have wheezing and severe coughing, the challenge test can be judged as positive. Regardless of the result of the Methacholine bronchial challenge test, factors that affect airway reactivity, such as drugs, seasons, climate, diurnal variations, and respiratory tract infections, should be excluded. When using the APS method, the severity of airway hyperresponsiveness can be graded based on PD-FEV or PC-FEV. Existing evidence suggests that PD shows good consistency when different nebulizers, inhalation times, and starting concentrations of MCh are used for bronchial provocation tests, whereas there is more variability with PC. Therefore, PD is often recommended as the quantitative assessment indicator. The threshold value for PD with the APS method is 2.5 mg.The Astograph method often uses the minimum cumulative dose (Dmin value, in Units) to reflect airway sensitivity. Dmin is the minimum cumulative dose of MCh required to produce a linear increase in Rrs. A dose of 1 g/L of the drug concentration inhaled for 1-minute equals 1 unit. It's important to note that with the continuous increase in inhaled provocant concentration, the concept of cumulative dose in the Astograph method should not be directly compared to other methods. Most asthma patients have a Dmin<10 Units, according to Japanese guidelines. The Astograph method, having been used in China for over twenty years, suggests a high likelihood of asthma when Dmin≤6 Units, with a smaller Dmin value indicating a higher probability. When Dmin is between 6 and 10 Units, further differential diagnosis is advised to ascertain whether the condition is asthma.A negative methacholine challenge test (MCT) does not entirely rule out asthma. The test may yield negative results due to the following reasons:(1) Prior use of medications that reduce airway responsiveness, such as β2 agonists, anticholinergic drugs, antihistamines, leukotriene receptor antagonists, theophylline, corticosteroids, etc., and insufficient washout time.(2) Failure to meet quality control standards in terms of pressure, flow rate, particle size, and nebulization volume of the aerosol delivery device.(3) Poor subject cooperation leads to inadequate inhalation of the methacholine agent.(4) Some exercise-induced asthma patients may not be sensitive to direct bronchial challenge tests like the Methacholine challenge and require indirect bronchial challenge tests such as hyperventilation, cold air, or exercise challenge to induce a positive response.(5) A few cases of occupational asthma may only react to specific antigens or sensitizing agents, requiring specific allergen exposure to elicit a positive response.A positive MCT does not necessarily indicate asthma. Other conditions can also present with airway hyperresponsiveness and yield positive results in the challenge test, such as allergic rhinitis, chronic bronchitis, viral upper respiratory infections, allergic alveolitis, tropical eosinophilia, cystic fibrosis, sarcoidosis, bronchiectasis, acute respiratory distress syndrome, post-cardiopulmonary transplant, congestive heart failure, and more. Furthermore, factors like smoking, air pollution, or exercise before the test may also result in a positive bronchial challenge test. What are the standardized requirements for the MCT report?The report should include: (1) basic information about the subject; (2) examination data and graphics: present baseline data, measurement data after the last two challenge doses or concentrations in tabular form, and the percentage of actual measured values compared to the baseline; flow-volume curve and volume-time curve before and after challenge test; dose-response curve: showing the threshold for positive challenge; (3) opinions and conclusions of the report: including the operator's opinions, quality rating of the examination, and review opinions of the reviewing physician. What are the adverse reactions and safety measures of MCT?During the MCT, the subject needs to repeatedly breathe forcefully and inhale bronchial challenge agents, which may induce or exacerbate bronchospasm and contraction and may even cause life-threatening situations. Medical staff should be fully aware of the indications, contraindications, medication use procedures, and emergency response plans for the MCT.
Topics: Child; Humans; Female; Aged; Methacholine Chloride; Bronchial Provocation Tests; Bronchodilator Agents; Respiratory Sounds; Lactation; Respiratory Aerosols and Droplets; Asthma; Respiratory Hypersensitivity; Dyspnea; Adrenal Cortex Hormones; Antibodies, Monoclonal; Histamine Antagonists; Phenols; Rhinitis, Allergic
PubMed: 38309959
DOI: 10.3760/cma.j.cn112147-20231019-00247 -
Journal of Comparative Physiology. B,... Feb 2024In salivary acinar cells, cholinergic stimulation induces elevations of cytosolic [Ca] to activate the apical exit of Cl through TMEM16A Cl channels, which acts as a...
In salivary acinar cells, cholinergic stimulation induces elevations of cytosolic [Ca] to activate the apical exit of Cl through TMEM16A Cl channels, which acts as a driving force for fluid secretion. To sustain the Cl secretion, [Cl] must be maintained to levels that are greater than the electrochemical equilibrium mainly by Na-K-2Cl cotransporter-mediated Cl entry in basolateral membrane. Glucose transporters carry glucose into the cytoplasm, enabling the cells to produce ATP to maintain Cl and fluid secretion. Sodium-glucose cotransporter-1 is a glucose transporter highly expressed in acinar cells. The salivary flow is suppressed by the sodium-glucose cotransporter-1 inhibitor phlorizin. However, it remains elusive how sodium-glucose cotransporter-1 contributes to maintaining salivary fluid secretion. To examine if sodium-glucose cotransporter-1 activity is required for sustaining Cl secretion to drive fluid secretion, we analyzed the Cl currents activated by the cholinergic agonist, carbachol, in submandibular acinar cells while comparing the effect of phlorizin on the currents between the whole-cell patch and the gramicidin-perforated patch configurations. Phlorizin suppressed carbachol-induced oscillatory Cl currents by reducing the Cl efflux dependent on the Na-K-2Cl cotransporter-mediated Cl entry in addition to affecting TMEM16A activity. Our results suggest that the sodium-glucose cotransporter-1 activity is necessary for maintaining the oscillatory Cl secretion supported by the Na-K-2Cl cotransporter activity in real time to drive fluid secretion. The concerted effort of sodium-glucose cotransporter-1, Na-K-2Cl cotransporter, and apically located Cl channels might underlie the efficient driving of Cl secretion in different secretory epithelia from a variety of animal species.
Topics: Animals; Mice; Acinar Cells; Carbachol; Chlorides; Glucose; Phlorhizin; Sodium; Sodium-Potassium-Chloride Symporters
PubMed: 38308715
DOI: 10.1007/s00360-024-01532-w -
European Journal of Pharmacology Mar 2024The role of the Wnt/β-catenin signaling pathway in epilepsy and the effects of its modulators as efficacious treatment options, though postulated, has not been...
The role of the Wnt/β-catenin signaling pathway in epilepsy and the effects of its modulators as efficacious treatment options, though postulated, has not been sufficiently investigated. We evaluated the involvement of β-catenin and GSK-3β, the significant proteins in this pathway, in the lithium chloride-pilocarpine-induced status epilepticus model in rodents to study acute phase of temporal lobe epilepsy (TLE). The modulators studied were 6-BIO, a GSK-3β inhibitor and Sulindac, a Dvl protein inhibitor. The disease group exhibited increased seizure score and seizure frequency, and the assessment of neurobehavioral parameters indicated notable alterations. Furthermore, histopathological examination of hippocampal brain tissues revealed significant neurodegeneration. Immunohistochemical study of hippocampus revealed neurogenesis in 6-BIO and sulindac groups. The gene and protein expression by RT-qPCR and western blotting studies indicated Wnt/β-catenin pathway downregulation and increased apoptosis in the acute phase of TLE. 6-BIO was very efficient in upregulating the Wnt pathway, decreasing neuronal damage, increasing neurogenesis in hippocampus and decreasing seizure score and frequency in comparison to sulindac. This suggests that both GSK-3β and β-catenin are potential and novel drug targets for acute phase of TLE, and treatment options targeting these proteins could be beneficial in successfully managing acute epilepsy. Further evaluation of 6-BIO to explore its therapeutic potential in other models of epilepsy should be conducted.
Topics: Rats; Animals; Pilocarpine; Wnt Signaling Pathway; Lithium; Glycogen Synthase Kinase 3 beta; beta Catenin; Sulindac; Hippocampus; Status Epilepticus; Seizures; Epilepsy, Temporal Lobe
PubMed: 38307381
DOI: 10.1016/j.ejphar.2024.176375 -
Clinical Science (London, England :... Feb 2024Hypercholesterolemia in pregnancy is a physiological process required for normal fetal development. In contrast, excessive pregnancy-specific hypercholesterolemia...
Hypercholesterolemia in pregnancy is a physiological process required for normal fetal development. In contrast, excessive pregnancy-specific hypercholesterolemia increases the risk of complications, such as preeclampsia. However, the underlying mechanisms are unclear. Toll-like receptor 4 (TLR4) is a membrane receptor modulated by high cholesterol levels, leading to endothelial dysfunction; but whether excessive hypercholesterolemia in pregnancy activates TLR4 is not known. We hypothesized that a high cholesterol diet (HCD) during pregnancy increases TLR4 activity in uterine arteries, leading to uterine artery dysfunction. Sprague Dawley rats were fed a control diet (n=12) or HCD (n=12) during pregnancy (gestational day 6-20). Vascular function was assessed in main uterine arteries using wire myography (vasodilation to methacholine and vasoconstriction to phenylephrine; with and without inhibitors for mechanistic pathways) and pressure myography (biomechanical properties). Exposure to a HCD during pregnancy increased maternal blood pressure, induced proteinuria, and reduced the fetal-to-placental weight ratio for both sexes. Excessive hypercholesterolemia in pregnancy also impaired vasodilation to methacholine in uterine arteries, whereby at higher doses, methacholine caused vasoconstriction instead of vasodilation in only the HCD group, which was prevented by inhibition of TLR4 or prostaglandin H synthase 1. Endothelial nitric oxide synthase expression and nitric oxide levels were reduced in HCD compared with control dams. Vasoconstriction to phenylephrine and biomechanical properties were similar between groups. In summary, excessive hypercholesterolemia in pregnancy impairs uterine artery function, with TLR4 activation as a key mechanism. Thus, TLR4 may be a target for therapy development to prevent adverse perinatal outcomes in complicated pregnancies.
Topics: Animals; Female; Male; Pregnancy; Rats; Hypercholesterolemia; Hyperlipidemias; Methacholine Chloride; Phenylephrine; Placenta; Rats, Sprague-Dawley; Toll-Like Receptor 4; Uterine Artery; Vasodilation
PubMed: 38299431
DOI: 10.1042/CS20231442 -
Neurochemistry International Mar 2024It is widely acknowledged that epilepsy is a neurological disorder characterized by recurrent and atypical neuronal discharges, resulting in transient dysfunction within...
It is widely acknowledged that epilepsy is a neurological disorder characterized by recurrent and atypical neuronal discharges, resulting in transient dysfunction within the brain. The protective role of hydrogen sulfide (HS) in epilepsy has been elucidated by recent studies, but the underlying mechanisms remain poorly understood. To investigate this, the concentration of HS was measured by spectrophotometry and a fluorescent probe in LiCl/Pilocarpine (LiCl/Pilo)-induced seizures in rats. The localization of proteins was examined using immunofluorescence. Electroencephalogram and behavioral tests were employed to evaluate the occurrence of seizures. Neuropathological changes in the hippocampus were examined by hematoxylin-eosin staining, Nissl staining, and transmission electron microscopy. Through proteomics and bioinformatics analysis, we identified the differential proteins in the hippocampus of rats following HS intervention. Protein changes were detected through western blotting. The results showed that HS treatment significantly alleviated seizures and minimized post-seizures neurological damage in rats. Proteomics analysis revealed adenylate cyclase 3 (AC3) as a protein potentially targeted by HS. Moreover, the AC3 activator forskolin reversed the downregulation effect of HS on the AC3/cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)/transient receptor potential vanilloid 2 (TRPV2) signaling pathway. In conclusion, HS targets and downregulates the expression of AC3, thereby modulating the AC3/cAMP/PKA signaling pathway to regulate the expression of TRPV2 in LiCl/Pilo-induced seizures, ultimately leading to seizure inhibition and neuroprotection.
Topics: Rats; Animals; Pilocarpine; Neuroprotection; Cyclic AMP-Dependent Protein Kinases; Seizures; Cyclic AMP; Epilepsy; Adenylyl Cyclases
PubMed: 38290616
DOI: 10.1016/j.neuint.2024.105677 -
Neurobiology of Disease Mar 2024Managing refractory epilepsy presents a significant a substantial clinical challenge. Deep brain stimulation (DBS) has emerged as a promising avenue for addressing...
BACKGROUND
Managing refractory epilepsy presents a significant a substantial clinical challenge. Deep brain stimulation (DBS) has emerged as a promising avenue for addressing refractory epilepsy. However, the optimal stimulation targets and effective parameters of DBS to reduce seizures remian unidentified.
OBJECTIVES
This study endeavors to scrutinize the therapeutic potential of DBS within the zona incerta (ZI) across diverse seizure models and elucidate the associated underlying mechanisms.
METHODS
We evaluated the therapeutic potential of DBS with different frequencies in the ZI on kainic acid (KA)-induced TLE model or M1-cortical seizures model, pilocarpine-induced M1-cortical seizure models, and KA-induced epilepsy model. Further, employing calcium fiber photometry combined with cell-specific ablation, we sought to clarified the causal role of ZI GABAergic neurons in mediating the therapeutic effects of DBS.
RESULTS
Our findings reveal that DBS in the ZI alleviated the severity of seizure activities in the KA-induced TLE model. Meanwhile, DBS attenuated seizure activities in KA- or pilocarpine-induced M1-cortical seizure model. In addition, DBS exerts a mitigating influence on KA induced epilepsy model. DBS in the ZI showed anti-seizure effects at low frequency spectrum, with 5 Hz exhibiting optimal efficacy. The low-frequency DBS significantly increased the calcium activities of ZI GABAergic neurons. Furthermore, selective ablation of ZI GABAergic neurons with taCasp3 blocked the anti-seizure effect of low-frequency DBS, indicating the anti-seizure effect of DBS is mediated by the activation of ZI GABAergic neurons.
CONCLUSION
Our results demonstrate that low-frequency DBS in the ZI attenuates seizure via driving GABAergic neuronal activity. This suggests that the ZI represents a potential DBS target for treating both hippocampal and cortical seizure through the activation of GABAergic neurons, thereby holding therapeutic significance for seizure treatment.
Topics: Humans; Drug Resistant Epilepsy; Zona Incerta; Pilocarpine; Calcium; Deep Brain Stimulation; GABAergic Neurons; Epilepsy; Kainic Acid; Seizures
PubMed: 38290566
DOI: 10.1016/j.nbd.2024.106424