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Background: Although guidelines recommend intracoronary acetylcholine (ACh) and ergonovine (ER) provocation testing for diagnosis of vasospastic angina, the feasibility and safety of sequential (combined) use of both pharmacological agents during the same catheterization session remain unclear., Objectives: In this study, we investigated the feasibility and safety of sequential intracoronary ACh and ER administration for coronary spasm provocation testing., Methods: The study included 235 patients who showed positive results on ACh and ER provocation testing. Initial intracoronary ACh administration was followed by ER administration for left coronary artery (LCA) spasm provocation testing. Subsequently, the right coronary artery (RCA) was subjected to sequential ACh and ER administration for provocation testing. The primary outcome of the study was the safety of sequential intracoronary ACh and ER provocation testing, which was assessed based on a composite of all-cause death, sustained ventricular tachycardia and fibrillation, and cardiogenic shock., Results: Even in patients with negative results on sequential intracoronary ACh and ER provocation testing in the LCA and only ACh administration into the RCA, additional administration of ER into the RCA showed a positive provocation test result in 33 of 235 (14.0%) patients; three (1.3%) patients developed adverse effects (cardiogenic shock occurred in all cases) during LCA provocation testing. We observed no deaths attributable to spasm provocation testing., Conclusion: Sequential administration of intracoronary ACh and ER was associated with a relatively low major complication rate and may be safe and potentially useful for diagnosis of vasospastic angina.

Coronary artery epicardial spasm is involved in the pathogenesis of many cardiac disorders. Vasoreactivity testing, such as intracoronary injection of acetylcholine (ACH) or ergonovine (ER), is the gold standard method for the diagnosis of vasospastic angina. Provoked epicardial spasm phenotypes are classified as focal spasm and diffuse spasm. Multiple factors, including sex, ethnicity, and use of coronary vasoactive stimulators, are related to the provoked phenotypes of epicardial spasm. Diffuse-provoked spasm is often observed in females, where focal-provoked spasm is markedly more common in males. ACH provokes more diffuse and distal spasms, whereas ER induces more focal and proximal spasms. Yellow plaque and coronary thrombi are often observed in lesions with focal spasms, and intimal thickness with a sonolucent zone is significantly more common in lesions with focal spasm. Furthermore, clinical outcomes in patients with focal spasm are unsatisfactory compared with those in patients with diffuse spasm. However, the reproducibility and eternality of provoked spasm phenotypes by vasoreactivity testing is uncertain. Coronary atherosclerosis or endothelial damage may affect coronary vasomotor tone. Although coronary artery spasm may persist in the same coronary artery, provoked coronary spasm phenotypes may exhibit a momentary coronary reaction by intracoronary ACH or ER testing., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest., (Copyright © 2023. Published by Elsevier Ltd.)

Background: Although guidelines recommend intracoronary administration of acetylcholine (ACh) with incremental doses of 20, 50, and 100 μg into the left coronary artery (LCA) during spasm provocation test for diagnosing vasospastic angina, 50 μg of ACh rarely induced significant coronary vasospasm when no vasoconstriction was observed with 20 μg of ACh in a previous report. The aim of this study was to evaluate the safety and feasibility of omitting 50 μg according to the vasoreactivity by 20 μg of ACh in the LCA., Methods: A total of 556 patients undergoing ACh provocation test with 20 μg followed by 50 and/or 100 μg were retrospectively included. Injection of 50 μg of ACh was primarily omitted when vasoconstriction <25 % was observed with 20 μg, which was left to operator's discretion. Adverse events were defined as a composite of ventricular fibrillation, sustained ventricular tachycardia, and cardiogenic shock during ACh test in the LCA., Results: Positive ACh test in the LCA was observed in 245 (44.1 %) patients. Overall, patients with LCA constriction <25 % by 20 μg of ACh had a lower rate of positive ACh test than their counterpart (24.0 % vs. 88.4 %, p < 0.001). In patients with LCA constriction ≥25 % by 20 μg, the incidence of adverse events was significantly higher than in those with LCA constriction <25 % during the provocation test at doses of 50 and 100 μg (2.3 % vs. 0 %, p = 0.009)., Conclusions: Omitting 50 μg of ACh in the LCA may be safe and feasible when little vasoconstriction was observed with preceding injection of 20 μg of ACh during spasm provocation test for diagnosing vasospastic angina. However, we believe that 50 μg of ACh should not be omitted when 20 μg of ACh induced LCA constriction ≥25 %., Competing Interests: Declaration of competing interest Yoshio Kobayashi is a member of the editorial team of Journal of Cardiology., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Purpose: Intravenous anesthetics have excellent analgesic activity without inducing the side effect in the respiratory system. The aim and objective of the current experimental study was to access the neuroprotective effect of sevoflurane against isoflurane induced cognitive dysfunction in rats., Methods: Isoflurane was used for induction the neurodysfunction in the rats, and rats received the oral administration of sevoflurane (2.5, 5 and 10 mg/kg). Morris water test was carried out for the estimation of cognitive function. Neurochemical parameters, antioxidant parameters and pro-inflammatory cytokines were also estimated., Results: Sevoflurane significantly (P < 0.001) altered the neurochemical parameters such as anti-choline acetyltransferase, acetylcholine esterase, acetylcholine, protein carbonyl, choline brain-derived neurotrophic factor, and amyloid β; antioxidant parameters such as glutathione, superoxide dismutase, and malondialdehyde; pro-inflammatory cytokines include interleukin (IL-2, IL-10, IL-4, IL-6, IL-10, IL-1β), and tumor necrosis factor-α. Sevoflurane significantly reduced the activity of caspase-3., Conclusions: Sevoflurane exhibited the neuroprotection against the cognitive dysfunction in rats via anti-inflammatory and antioxidant mechanism.

Background: J-waves may be observed during coronary angiography (CAG) or intracoronary acetylcholine (ACh) administration, but their significance is unknown., Methods: Forty-nine patients, 59.1 ± 11.5 years old and 59% male, were studied on suspicion of vasospastic angina, and J wave dynamicity was compared between CAG and Ach administration., Results: Diagnostic (≥0.1 mV) or nondiagnostic (<0.1 mV) J waves in 9 and 3 patients, respectively, were augmented, and J waves were newly observed in 2 patients during CAG and Ach administration. Similar changes in the J-wave amplitude were observed: from 0.10 ± 0.09 mV to 0.20 ± 0.15 mV (p < .002) and from 0.10 ± 0.10 mV to 0.20 ± 0.16 mV (p < .001) during CAG and Ach administration, respectively. J waves were located in the inferior leads and changed only during the right coronary interventions. In the remaining 35 patients, J waves were absent before and during the coronary interventions. Augmentation of J waves was found when the RR interval was shortened in some patients. Injection of anoxic media into the coronary artery might induce a conduction delay from myocardial ischemia that manifests as augmentation or new occurrence of J waves., Conclusions: Both CAG and intracoronary Ach administration affected J waves similarly in the same individuals. A myocardial ischemia-induced conduction delay may be responsible for the changes in J waves, but further studies are needed., (© 2023 Wiley Periodicals LLC.)

Objectives: In this study, we determined the gastroprotective and ulcer-healing effects of extracts (aqueous and methanolic) of Nauclea pobeguinii stem-back., Methods: Gastroprotective and healing activity were evaluated following a HCl/ethanol and an indomethacin-induced acute ulcers models; acetic acid, pylorus-ligature, pylorus ligature/histamine and pylorus ligature/acetylcholine-induced chronic ulcers models., Results: It emerges from this study that, at 100, 200 and 400 mg/kg, the extracts significantly reduced the various ulceration parameters. Compared to negative control male rats, the aqueous (100 mg/kg) and methanolic (400 mg/kg) extracts of Nauclea pobeguinii inhibited the ulcers induced by HCl/ethanol by 80.76 % and 100 % respectively, as well as ulcers induced by indomethacin by 88.28 % and 93.47 % respectively. Animals that received 200 mg/kg of both extracts showed a significant reduction in the levels of monocytes, lymphocytes, nitric oxide, MDA and a significant increase in the activities of SOD and catalase. Histological analysis showed repaired mucous epithelium at all doses of both extracts. Aqueous and methanol extracts inhibited ulceration indices by 89.33 % and 88.53 % for pylorus ligature, 83.81 % and 61.07 % for pylorus ligature/acetylcholine and 87.29 % and 99.63 % for pylorus ligature/histamine respectively. Both extracts protected the stomach lining with percentages inhibition of 79.49 % and 81.73 %, respectively in the ethanol test. The extracts induced a significant increase in mucus mass (p<0.001)., Conclusions: The aqueous and methanol extracts of Nauclea pobeguinii healed ulcers thanks to their anti-inflammatory, anti-oxidant, anti-secretory and cytoprotective properties., (© 2023 Walter de Gruyter GmbH, Berlin/Boston.)

Background: The primary phytoconstituents reported to have neuroprotective effects are flavonoids and phenolic compounds. Aerva persica roots are reported to be rich in flavonoids and phenolic compounds. Therefore, this study aimed to explore the nootropic potential of Aerva persica roots., Objective: The objective of this study was to evaluate the nootropic potential of Aerva persica roots against D-galactose-induced memory impairment., Methods: In this study, the roots of Aerva persica were extracted with 70% ethanol. The obtained extract was evaluated for total phenolic content using the Folin-Ciocalteu method and total flavonoid content using the aluminium chloride colorimetric assay. Afterward, the acute oral toxicity of the extract was determined following the Organisation for Economic Co-operation and Development (OECD) guideline 423. Additionally, two doses of Aerva persica (100 and 200 mg/kg body weight (BW)) were evaluated for their nootropic potential against D-galactose-induced memory impairment. The nootropic potential of the crude extract was assessed through a behavioural study and brain neurochemical analysis. Behavioural studies involved the evaluation of spatial reference- working memory using the radial arm maze test and the Y-maze test. Neurochemical analysis was performed to determine the brain's acetylcholine, acetylcholinesterase, glutathione (GSH), and malondialdehyde (MDA) levels., Results: The total phenolic content and total flavonoid content were found to be 179.14 ± 2.08 μg GAE/mg and 273.72 ± 3.94 μg QE/mg, respectively. The Aerva persica extract was found to be safe up to 2000 mg/kg BW. Following the safety assessment, the experimental mice received various treatments for 14 days. The behavioural analysis using the radial maze test showed that the extract at both doses significantly improved spatial reference-working memory and reduced the number of total errors compared to disease control groups. Similarly, in the Y-maze test, both doses significantly increased the alteration percentage and the percentage of novel arm entry (both indicative of intact spatial memory) compared to disease control. In neurochemical analysis, Aerva persica at 200 mg/kg significantly normalised the acetylcholine level (p<0.0001) and GSH level (p<0.01) compared to disease control. However, the same effect was not observed with Aerva persica at 100 mg/kg. Additionally, Aerva persica at 200mg/kg BW significantly decreased the acetylcholinesterase level (p<0.0001) and decreased the brain's MDA level (p<0.01) compared to the disease control, whereas the effect of Aerva persica at 100 mg/kg BW in reducing acetylcholinesterase was non-significant., Conclusion: Based on the results, it can be concluded that the nootropic potential of Aerva persica was comparable to that of the standard drug, Donepezil, and the effect might be attributed to the higher content of flavonoids and phenolic compounds., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: Intracoronary provocation testing with acetylcholine (ACh) is crucial for the diagnosis of functional coronary alterations in patients with suspected myocardial ischaemia and non-obstructive coronary arteries., Aims: Our intention was to assess the safety and predictive value for major adverse cardiovascular and cerebrovascular events (MACCE) in patients presenting with ischaemia with non-obstructive coronary arteries (INOCA) or with myocardial infarction with non-obstructive coronary arteries (MINOCA)., Methods: We prospectively enrolled consecutive INOCA or MINOCA patients undergoing intracoronary ACh provocation testing., Results: A total of 317 patients were enrolled: 174 (54.9%) with INOCA and 143 (45.1%) with MINOCA. Of these, 185 patients (58.4%) had a positive response to the ACh test. Complications during ACh provocative testing were all mild and transient and occurred in 29 (9.1%) patients, with no difference between patients with positive or negative responses to ACh testing, nor between INOCA and MINOCA patients. A history of paroxysmal atrial fibrillation, moderate/severe diastolic dysfunction and a higher QT dispersion at baseline electrocardiogram were independent predictors of complications. MACCE occurred in 30 patients (9.5%) during a median follow-up of 22 months. The incidence of MACCE was higher among patients with a positive ACh test (24 [13.0%] vs 6 [4.5%], p=0.017), and a positive ACh test was an independent predictor of MACCE., Conclusions: ACh provocation testing is associated with a low risk of mild and transient complications, with a similar prevalence in both INOCA and MINOCA patients. Importantly, ACh provocation testing can help to identify patients at higher risk of future clinical events, suggesting a net clinical benefit derived from its use in this clinical setting.

Purpose: Extracellular acidification is a major component of tissue inflammation, including airway inflammation. The extracellular proton-sensing mechanisms are inherent in various cells including airway structural cells, although their physiological and pathophysiological roles in bronchial smooth muscles (BSMs) are not fully understood. In the present study, to explore the functional role of extracellular acidification on the BSM contraction, the isolated mouse BSMs were exposed to acidic pH under contractile stimulation., Methods and Results: The RT-PCR analyses revealed that the proton-sensing G protein-coupled receptors were expressed both in mouse BSMs and cultured human BSM cells. In the mouse BSMs, change in the extracellular pH from 8.0 to 6.8 caused an augmentation of contraction induced by acetylcholine. Interestingly, the acidic pH-induced BSM hyper-contraction was further augmented in the mice that were sensitized and repeatedly challenged with ovalbumin antigen. In this animal model of asthma, upregulations of G protein-coupled receptor 68 (GPR68) and GPR65, that were believed to be coupled with Gq and Gs proteins respectively, were observed, indicating that the acidic pH could cause hyper-contraction probably via an activation of GPR68. However, psychosine, a putative antagonist for GPR68, failed to block the acidic pH-induced responses., Conclusion: These findings suggest that extracellular acidification contributes to the airway hyperresponsiveness, a characteristic feature of bronchial asthma. Further studies are required to identify the receptor(s) responsible for sensing extracellular protons in BSM cells., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Heterogeneity in diagnostic criteria and provocation protocols has posed challenges in understanding the safety of coronary provocation testing with intracoronary acetylcholine (ACh) for the contemporary diagnosis of epicardial and microvascular spasm., Objectives: We examined the safety of testing and subgroup differences in procedural risks based on ethnicity, diagnostic criteria, and provocation protocols., Methods: PubMed and Embase were searched in November 2021 to identify original articles reporting procedural complications associated with intracoronary ACh administration. The primary outcome was the pooled estimate of the incidence of major complications including death, myocardial infarction, ventricular tachycardia/fibrillation, and shock., Results: A total of 16 studies with 12,585 patients were included in the meta-analysis. The overall pooled estimate of the incidence of major complications was 0.5% (95% CI: 0.0%-1.3%) without any reports of death. Exploratory subgroup analyses revealed that the pooled incidence of major complications was significantly higher in the studies that followed the contemporary diagnosis criteria for epicardial spasm defined as ≥90% diameter reduction (1.0%; 95% CI: 0.3%-2.0%) but significantly lower in Western populations (0.0%; 95% CI: 0.0%-0.45%). The rate of positive epicardial spasm and the incidence of major complications were similar between provocation protocols using the maximum ACh doses of 100 μg and 200 μg., Conclusions: Intracoronary ACh administration for the contemporary diagnosis of epicardial and microvascular spasm is a safe procedure. Moreover, excellent safety records are observed in Western populations primarily presenting with myocardial ischemia and/or infarction with nonobstructive coronary arteries. This study will help standardize ACh testing to improve clinical diagnosis and ensure procedural safety., Competing Interests: Funding Support and Author Disclosures Dr Samuels has a consulting agreement with Abbott Vascular; and serves as a consultant and on the Speakers Bureau for Abbott Vascular and Philips. Dr Parikh has a consulting agreement with Abbott Vascular; and serves on the advisory boards of Abbott Vascular, Boston Scientific, and Medtronic. Dr Wei has a consulting agreement with and serves on the advisory board of Abbott Vascular. Drs Moses, Fearon, Henry, Tremmel, and Kobayashi have consulting agreements with Abbott Vascular. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose., (Copyright © 2022 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Background: It has been reported that significant endothelial dysfunction or clinically evident vasospasm can be associated with drug-eluting stents (DESs). However, the impact of DES associated coronary artery spasm (CAS) on long-term clinical outcomes has not been fully elucidated as compared with those of patients with vasospastic angina., Methods: A total of 2797 consecutive patients without significant coronary artery lesion (<70%), who underwent the Acetylcholine (Ach) provocation test, were enrolled between Nov 2004 and Oct 2010. DES-associated spasm was defined as significant CAS in proximal or distal to previously implanted DES site at follow-up angiography with Ach test. Patients were divided into two groups (DES-CAS; n = 108, CAS; n = 1878). For adjustment, propensity score matching (PSM) was done (C-statistics = 0.766, DES-CAS; n = 102, CAS; n = 102). SPSS 20 (Inc., Chicago, Illinois) was used to analyze this data., Results: Baseline characteristics were worse in the DES-CAS group. After PSM, both baseline characteristics and the Ach test results were balanced except higher incidence of diffuse CAS and ECG change in the DES-CAS group. During Ach test, the incidence of diffuse spasm (93.1% vs. 81.3%, p = 0.012) and ST-T change (10.7% vs. 1.9%, p = 0.010) were higher in the DES-CAS group. At 3-year, before and after adjustment, the DES-CAS group showed a higher incidence of coronary revascularization (9.8% vs. 0.0%, p = 0.001), recurrent chest pain requiring follow up coronary angiography (CAG, 24.5% vs. 7.8%, p = 0.001) and major adverse cardiac events (MACEs, 9.8% vs. 0.9%, p < 0.005)., Conclusion: In this study, DES associated CAS was associated with higher incidence of diffuse spasm, ST-T change and adverse 3-year clinical outcomes. Special caution should be exercised in this particular subset of patients., Competing Interests: Declaration of competing interest Nil., (Copyright © 2022. Published by Elsevier, a division of RELX India, Pvt. Ltd.)

Intracoronary acetylcholine (ACh) testing has become popular in the world as a spasm provocation test as well as an ergonovine test. Intracoronary ACh test based on the Japanese Circulation Society guidelines is necessary to insert a temporary pace maker (PM). We analyzed the ACh spasm provocation test procedures retrospectively. We performed 1829 ACh spasm provocation testing during 28 years. We investigated the procedural approach sites of artery and vein. Femoral artery and vein approach, brachial artery and femoral vein approach, brachial artery and vein approach, radial artery and brachial vein approach, radial artery and femoral vein approach were performed in 292 patients (16.0%), 498 patients (27.2%), 589 patients (32.2%), 252 patients (13.8%), and 175 patients (9.6%), respectively. We could perform the ACh testing by the femoral artery and brachial artery in all patients, while the success rate of radial artery approach was 97.1%. We could also insert the temporary PM by the brachial vein in 94.8% (841/887) of the study patients, whereas we could insert the temporary PM in all femoral vein approach [100% (965/965)]. We experienced the pulmonary embolism by the femoral artery and vein approach in two patients, while we also had the arterio-venous fistula necessary for surgical repair in two patients by the brachial artery and vein approach. Although there was no difference about the procedure-related major complications among the various procedures, we had no pulmonary embolism or arterio-venous fistula by the radial artery and brachial vein approach. Considering the disinfection with povidone iodine, procedural performance or procedure-related complications by the ACh testing, we recommend that radial artery and brachial vein approach is more comfortable method of the future ACh testing not only for patients but also for operators.

Pharmacological spasm provocation tests such as acetylcholine (ACh) and ergonovine (ER) had been performed in the clinic. We retrospectively analyzed the incidence of provoked spasm, complications during testing and the cardiac events after these tests. From January 1991 and October 2018, we performed pharmacological spasm provocation tests in 2500 patients: 1810 ACh tests, 1232 ER tests, 542 both tests, and 310 ACh added after ER tests. ACh was injected in incremental doses of 20/50/100/200 μg into the LCA and 20/50/80 μg into the RCA. ER was administered as a total dose of 64 μg into the LCA and 40 μg into the RCA. When adding ACh after ER, the total dose was 50/80 μg into the RCA and 100/200 μg into the LCA. Positive spasm was defined as ≥ 90% stenosis and usual chest pain or ischemic ECG changes. Mean follow-up duration was 47.5 ± 29.9 months. Overall, provoked positive spasm was found in 1095 patients (43.8%). The incidence of positive provoked spasm during ACh testing was significantly higher than that during other tests (ACh: 48.7% vs. ER: 28.9%, Both: 24%, ACh added after ER: 33.5%, p < 0.001). Multiple spasms were remarkably more frequent during ACh testing compared with the other 3 types of testing (ACh: 28.2% vs. ER: 7.4%, Both: 4.1%, ACh added after ER: 13.2%, p < 0.001). No death or acute myocardial infarction was observed, while major complications during ACh testing were significantly more frequent than during ER testing. Readmission due to recurrent angina pectoris in spasm-positive patients was remarkably more frequent than in spasm-negative patients. The incidence of sudden cardiac death, ventricular fibrillation, and acute coronary syndrome were not different between the spasm-positive and spasm-negative groups during the follow-up periods. We could perform all spasm provocation tests without any irreversible complications. All sequential spasm provocation tests were useful for documenting coronary spasm.

The objective of this project was to find a bronchodilatory compound from herbs and clarify the mechanism. We found that the ethanol extract of Folium Sennae (EEFS) can relax airway smooth muscle (ASM). EEFS inhibited ASM contraction, induced by acetylcholine, in mouse tracheal rings and lung slices. High-performance liquid chromatography assay showed that EEFS contained emodin. Emodin had a similar reversal action. Acetylcholine-evoked contraction was also partially reduced by nifedipine (a selective inhibitor of L-type voltage-dependent Ca 2+ channels, LVDCCs), YM-58483 (a selective inhibitor of store-operated Ca 2+ entry, SOCE), as well as Y-27632 (an inhibitor of Rho-associated protein kinase). In addition, LVDCC- and SOCE-mediated currents and cytosolic Ca 2+ elevations were inhibited by emodin. Emodin reversed acetylcholine-caused increases in phosphorylation of myosin phosphatase target subunit 1. Furthermore, emodin, in vivo, inhibited acetylcholine-induced respiratory system resistance in mice. These results indicate that EEFS-induced relaxation results from emodin inhibiting LVDCC, SOCE, and Ca 2+ sensitization. These findings suggest that Folium Sennae and emodin may be new sources of bronchodilators., (© 2020 International Federation for Cell Biology.)

Background: Among patients with angina and nonobstructive coronary artery disease, those with coronary microvascular dysfunction have a poor outcome. Coronary microvascular dysfunction is usually diagnosed by assessing flow reserve with an endothelium-independent vasodilator like adenosine, but the optimal diagnostic threshold is unclear. Furthermore, the incremental value of testing endothelial function has never been assessed before. We sought to determine what pharmacological thresholds correspond to exercise pathophysiology and myocardial ischemia in patients with coronary microvascular dysfunction., Methods: Patients with angina and nonobstructive coronary artery disease underwent simultaneous acquisition of coronary pressure and flow during rest, supine bicycle exercise, and pharmacological vasodilatation with adenosine and acetylcholine. Adenosine and acetylcholine coronary flow reserve were calculated as vasodilator/resting coronary blood flow (CFR and AchFR, respectively). Coronary wave intensity analysis was used to quantify the proportion of accelerating wave energy; a normal exercise response was defined as an increase in accelerating wave energy from rest to peak exercise. Ischemia was assessed by quantitative 3-Tesla stress perfusion cardiac magnetic resonance imaging and dichotomously defined by a hyperemic endo-epicardial gradient <1.0., Results: Ninety patients were enrolled (58±10 years, 77% female). Area under the curve using receiver-operating characteristic analysis demonstrated optimal CFR and AchFR thresholds for identifying exercise pathophysiology and ischemia as 2.6 and 1.5, with positive and negative predictive values of 91% and 86%, respectively. Fifty-eight percent had an abnormal CFR (of which 96% also had an abnormal AchFR). Of those with a normal CFR, 53% had an abnormal AchFR, and 47% had a normal AchFR; ischemia rates were 83%, 63%, and 14%, respectively., Conclusions: The optimal CFR and AchFR diagnostic thresholds are 2.6 and 1.5, with high-positive and negative predictive values, respectively. A normal CFR value should prompt the measurement of AchFR. A stepwise algorithm incorporating both vasodilators can accurately identify an ischemic cause in patients with nonobstructive coronary artery disease.

The spasm provocation test (SPT) is important for diagnosing vasospastic angina (VSA), and acetylcholine (ACh) is usually used for this test in Japan. However, some patients with VSA have negative SPT results with the use of the standard ACh regimen alone. We herein report two cases in which VSA was diagnosed by the SPT with the combined use of ACh and ergonovine (EM). VSA could not be diagnosed in either case by the SPT using ACh infusions alone. For patients with negative SPT results, cardiologists should consider performing the SPT using a combination of ACh and EM.

Background: Temporary pace maker is necessary because of transient block or bradycardia during the intracoronary acetylcholine spasm provocation tests based on the Japanese Circulation Society guidelines., Objectives: We examined the feasibility and safety of the acetylcholine spasm provocation test via the radial artery and brachial vein approach., Methods: We tried to perform the acetylcholine spasm provocation tests in 252 patients via the radial artery and brachial vein approach procedures during 5 years. Acetylcholine was injected in incremental doses of 20/50/80 μg into the right coronary artery (RCA) and 20/50/100/200 μg into the left coronary artery (LCA). Back-up pacing rate was set at 40 beats/min. Positive spasm was defined as transient ≥90% luminal narrowing and ischemic electrocardiographic change or usual chest pain., Results: The procedure success of radial artery and brachial vein access was 94.4% (238/252) and 93.3% (235/252), respectively. We performed 221 patients (87.7%) with acetylcholine tests by radial artery and brachial vein approach. We changed to the brachial approach due to the failures of radial artery access in 14 patients. We also changed to the femoral vein in 11 patients and internal jugular vein in two patients. Back-up pace maker rhythm was observed in 92.1% (232/252) of all study patients, while it was significantly higher in the RCA testing than that in the LCA tests (84.9% (191/225) vs. 52.2% (131/251), P < 0.001). No irreversible complication was found., Conclusions: We recommend the radial artery and brachial vein approach for safety and convenience when performing the acetylcholine spasm provocation tests., (© 2018 Wiley Periodicals, Inc.)

Postprandial hyperglycemia in diabetic and nondiabetic subjects is associated with endothelial dysfunction. Evidence shows that high glucose generates oxidative stress and a pro-inflammatory state promoting the development of cardiovascular diseases. trans-Resveratrol (t-RV) has been shown to reduce cardiovascular risk. To determine whether t-RV acts as a protector against acute high glucose (AHG)-induced damage, two in vitro models, rat aortic rings (RAR) and human umbilical vein endothelial cells (HUVEC) were used. RAR pretreated with AHG (25 mM D-glucose) for 3 h dramatically decreased the endothelium-dependent relaxation (EDR) induced by acetylcholine in phenylephrine (PE)-precontracted vessels. However, coincubation with t-RV significantly mitigated the damage induced by AHG on EDR. Pretreatment with AHG did not affect the vasodilation induced by sodium nitroprusside. HUVEC treated with t-RV decreased cytotoxicity and reduced radical oxygen species production induced by AHG. Taken together, these results suggest that t-RV can mitigate the AHG-induced EDR damage through a mechanism involving ROS scavenging and probably an increase in the bioavailability of NO.

Background: Coronary artery vasospasm (CS) can be identified as either a diffuse type or focal type; however, the difference in endothelial characteristics between these spasm types remains unclear. The features of coronary intima associated with diffuse spasm and focal spasm using coronary angioscopy (CAS) were evaluated and the optical coherence tomography (OCT) findings were compared., Methods: CAS and/or OCT observational analysis was performed in 55 patients (mean age: 61.4 years, 31 men) who had acetylcholine-provoked CS (diffuse CS, 31 patients; focal CS, 24 patients). The yellowness of the intima, presence of thrombus in CAS, and intimal characteristics based on the OCT results were evaluated., Results: CAS showed more atherosclerotic yellow plaques at the focal spasm segment than at the diffuse spasm segment (p=0.032). Moreover, there were more thrombi at the focal spasm segment (p=0.039). In addition, OCT results revealed that the intima area, maximum intima thickness, and lipid content in the focal CS group were larger than the diffuse CS group (4.22±1.67mm 2 vs. 3.45±2.36mm 2 ; 0.71±0.29mm vs. 0.53±0.30mm; 55.9% vs. 32.0%, p<0.001, respectively)., Conclusions: These results indicate that the presence of atherosclerotic plaques at the spasm site is likely to be related to the occurrence of a focal vasospasm. This may support the difference of features between focal CS and diffuse CS and contribute to precise treatment for each spasm type., (Copyright © 2018 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.)

Rationale: Augmented smooth muscle contractility of the airways is one of the causes of airway hyperresponsiveness in asthmatics. However, the mechanism of the altered properties of airway smooth muscle cells is not well understood., Objectives: To identify differentially expressed genes (DEGs) related to the bronchial smooth muscle (BSM) hyper-contractility in a murine asthma model., Methods: The ovalbumin (OA)-sensitized mice were repeatedly challenged with aerosolized OA to induce asthmatic reaction. Transcriptomic profiles were generated by microarray analysis of BSM tissues from the OA-challenged and control animals, and KEGG (Kyoto Encyclopedia of Genes and Genomes) Pathway Analysis was applied., Measurements and Main Results: Tension study showed a BSM hyperresponsiveness to acetylcholine (ACh) in the OA-challenged mice. A total of 770 genes were differentially expressed between the OA-challenged and control animals. Pathway analysis showed a significant change in arachidonic acid (AA) metabolism pathway in BSM tissues of the OA-challenged mice. Validation of DEGs by quantitative RT-PCR showed a significant increase in PLA2 group 4c (Pla2g4c)/COX-2 (Ptgs2)/PGD2 synthase 2 (Hpgds) axis. PGD2 level in bronchoalveolar fluids of the OA-challenged mice was significantly increased. A 24-h incubation of BSM tissues with PGD2 caused a hyperresponsiveness to ACh in naive control mice., Conclusions: AA metabolism is shifted towards PGD2 production in BSM tissues of asthma. Increased PGD2 level in the airways might be a cause of the BSM hyperresponsiveness in asthma., Competing Interests: The authors have declared that no competing interests exist.

Background: The prevalence of coronary artery spasm (CAS) inducible by intracoronary injection of acetylcholine (ACh) is high in survivors of acute myocardial infarction (AMI). Although there is a potential risk of sudden cardiac death in patients with CAS, the prognostic value of CAS was not clear. Thus, this study examined the effect of CAS on long-term prognosis in survivors of AMI in a prospective manner., Methods: The study included a total of 437 patients with AMI who underwent a CAS provocation test using ACh. All patients were followed prospectively for 5years or until the occurrence of the primary composite endpoint that consisted of cardiac death and acute coronary syndrome (ACS)., Results: CAS was induced in 195 (45%) of the study patients. During the follow-up period, 30 patients had a recurrent event (4 had cardiac death and 26 had ACS). Kaplan-Meier estimates in time-to-first-event analysis demonstrated a similar probability of the primary endpoint in patients with and without inducible CAS (p=0.13, log-rank test). The rate of each component of the composite endpoint was also comparable between the 2 patient groups. In Cox proportional hazards risk analysis, treatment with calcium channel blockers (CCBs) negatively predicted the primary endpoints in patients with inducible CAS (HR, 0.21; 95% CI, 0.08-0.55, p<0.01)., Conclusions: The presence of inducible CAS did not increase the incidence of the cardiac events in AMI survivors. Treatment with CCBs may improve outcomes in AMI survivors with inducible CAS., Clinical Trial Registration: URL: https://upload.umin.ac.jp/cgi-open-bin/ctr&#95;e/ctr&#95;view.cgi?recptno=R000021340, unique identifier: UMIN000018432., (Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.)

Aims: Functional alterations of epicardial coronary arteries or coronary microcirculation represent a frequent cause of myocardial infarction and non-obstructive coronary arteries (MINOCA). We aimed at assessing the prognostic value of intracoronary provocative tests in patients presenting with MINOCA and in which other causes of MINOCA have been excluded., Methods and Results: We prospectively evaluated patients with a diagnosis of MINOCA, excluding patients with aetiologies other than suspected coronary vasomotor abnormalities. Immediately after coronary angiography, an invasive provocative test using acetylcholine or ergonovine was performed. The incidence of death from any cause, cardiac death, and recurrence of acute coronary syndrome (ACS) was assessed at follow-up. We also assessed angina status using Seattle Angina Questionnaires (SAQ). We enrolled 80 consecutive patients [mean age 63.0 ± 10.7 years, 40 (50%) male]. Provocative test was positive in 37 (46.2%) patients without any complication. Among patients with a positive test, epicardial spasm was detected in 24 (64.9%) patients and microvascular spasm in 13 (35.1%) patients. After a median follow-up of 36.0 (range 12.0-60.0) months, patients with a positive test had a significantly higher occurrence of death from any cause [12 (32.4%) vs. 2 (4.7%); P = 0.002], cardiac death [7 (18.9%) vs. 0 (0.0%); P = 0.005], and readmission for ACS [10 (27.0%) vs. 3 (7.0%); P = 0.015] as well as a worse angina status as assessed by SAQ [Seattle score: 88.0 (33.0-100.0) vs. 100.0 (44.0-100.0); P = 0.001] when compared with patients with a negative test., Conclusions: We demonstrate that in patients presenting with MINOCA and suspected coronary vasomotor abnormalities, a positive provocative test for spasm is safe and identifies a high-risk subset of patients., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2017. For permissions, please email: journals.permissions@oup.com.)

Aims: Systematic review of literature to evaluate safety of intracoronary (i.c.) pharmacologic testing with acetylcholine (ACh), or ergonovine (ERGO), to induce coronary artery spasm., Methods and Results: Review of all relevant publications using MEDLINE and EMBASE databases yielded 10 publications, totalling 9,444 patients. Prevalence of provoked spasm varied from 2.3% to 54.7% of patients tested in the selected studies. The wide variability in prevalence was due to heterogeneity of study populations and provocation protocols. No deaths were reported. Overall occurrence of major (0.8%) and minor (4.7%) complications for i.c. pharmacologic testing was low. Compared to ERGO, ACh showed significantly higher rate of major (1.09% vs 0.15%; p<0.001) and minor complications (5.87% vs 2.36%; p<0.001)., Conclusion: Provocative testing with i.c. ACh or ERGO are safe and can facilitate the diagnosis of inducible coronary artery spasm during diagnostic coronary angiography. These tests should be part of the routine armamentarium of interventional cardiologists., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Coronary artery spasm (CAS) and significant coronary stenosis are known to be major causes of myocardial ischemia. However, their association and the impact of insignificant coronary stenosis (ICS) on long-term clinical outcomes of CAS patients are largely unknown., Methods: A total of 2797 patients without significant coronary artery disease (CAD) who underwent the acetylcholine (ACH) provocation test between November 2004 and October 2010 were enrolled. Significant CAS was defined as having ≥70% of temporary narrowing by ACH test and ICS as having <70% of fixed stenosis on angiography. Patients were divided into two groups: ICS group (n=764) and non-ICS group (n=845). To adjust potential confounders, a propensity score matching (PSM) analysis was performed using the logistic regression model. Primary endpoint was the composite of total death, myocardial infraction (MI), de novo percutaneous coronary intervention (PCI), and cerebrovascular accidents (CVA). Secondary endpoint was the incidence of recurrent angina requiring repeat coronary angiography (CAG) at 3years., Results: After PSM analysis, two well-balanced groups (548 pairs, total=1096) were generated. The baseline clinical characteristics were similar between the two groups. During the ACH test, compared with the non-ICS group, the ICS group had smaller spastic narrowing diameter (0.69±0.35 vs. 0.73±0.37, P=0.039) and incidence of ST-segment depression (4.0% vs. 0.9%, P=0.001). The incidence of primary and secondary endpoints was similar between the two groups up to 3years., Conclusions: Although, the ICS group was expected to have more adverse long-term clinical outcomes, it was not associated with the increased incidence of major adverse clinical outcomes compared with the non-ICS group up to 3years. Longer term follow-up studies are needed., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Intracoronary acetylcholine (ACh) provocation test is useful to diagnose vasospastic angina. However, paroxysmal atrial fibrillation (AF) often occurs during intracoronary ACh provocation test, leading to disabling symptoms. The aim of this study was to investigate the incidence and predictors of paroxysmal AF during the test. A total of 377 patients without persistent AF who underwent intracoronary ACh provocation test were included. Paroxysmal AF during ACh provocation test was defined as documented AF on electrocardiogram during the procedure. There were 31 patients (8%) with paroxysmal AF during the test. Of these, 11 (35%) required antiarrhythmic drugs, but none received electrical cardioversion. All of them recovered sinus rhythm within 48 h. At procedure, paroxysmal AF occurred mostly during provocation for the right coronary artery (RCA) rather than for the left coronary artery (LCA) (90 vs. 10%). Multivariate logistic regression analysis demonstrated that a history of paroxysmal AF (OR 4.38 CI 1.42-13.51, p = 0.01) and body mass index (OR 0.88 CI 0.78-0.99, p = 0.03) were independent predictors for occurrence of paroxysmal AF during intracoronary ACh provocation test. In conclusions, paroxysmal AF mostly occurs during ACh provocation test for the RCA, especially in patients with a history of paroxysmal AF and lower body mass index. It may be better to initially administer intracoronary ACh in the LCA when the provocation test is performed.

Pharmacological spasm provocation tests are invasive methods and we always have the potential to encounter complications when performing these tests. In 1980, Buxton et al. reported three deaths when they performed intravenous ergonovine testing. However, we now employ the intracoronary ergonovine test instead of the intravenous injection of ergonovine from a safety procedure point of view. Past serious major complications of intravenous ergonovine tests, intracoronary ergonovine tests, and intracoronary acetylcholine tests were 0.31% (26/8419), 0.51% (11/2173), and 0.95% (148/15,527), respectively. Selective intracoronary testing had the serious major complications in 0.89% of patients including just one death (0.006%) and two acute myocardial infarctions (0.01%). Selective spasm provocation tests had no additional risks compared with performing diagnostic coronary angiography alone. In the Western countries, the pharmacological spasm provocation tests are not familiar in the clinic except for some specialized institutions. We need international clinical studies using the same protocol of spasm provocation tests to compare the frequency, clinical features, and prognosis of acetylcholine- or ergonovine-provoked coronary spasm between Western and Asian countries. And we hope that Western guidelines give spasm provocation testing a class I indication similar to Japanese Circulation Society guidelines because coronary artery spasm may have fewer racial differences and borders., (Copyright © 2016 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.)

Angiotensin converting enzyme (ACE) inhibitors therapy is aassociated with bothersome dry cough as an adverse effect. The mechanisms underlying this adverse effect are not clear. Therefore, influence of captopril (an ACE inhibitor) on acetylcholine (ACh)-induced bronchial smooth muscle contractions was investigated. Further, the mechanisms underlying the captopril-induced changes were also explored. In vitro contractions of rat bronchial smooth muscle to cumulative concentrations of ACh were recorded before and after exposure to captopril. Further, the involvement of kinin and inositol triphosphate (IP₃) pathways for captopril-induced alterations were explored. ACh produced concentration-dependent (5-500 µM) increase in bronchial smooth muscle contractions. Pre-treatment with captopril augmented the ACh-induced contractions at each concentration significantly. Pre-treatment with aprotinin (kinin synthesis inhibitor) or heparin (inositol triphosphate, IP₃-inhibitor), blocked the captopril-induced augmentation of bronchial smooth muscle contractions evoked by ACh. Further, captopril-induced augmentation was absent in calcium-free medium. These results suggest that captopril sensitizes bronchial smooth muscles to ACh-induced contractions. This sensitization may be responsible for dry cough associated with captopril therapy.

Background: The parasympathetic nervous system (PNS), via neurotransmitter acetylcholine (ACh), modulates fibrogenesis in animal models. However, the role of ACh in human hepatic fibrogenesis is unclear., Aims: We aimed to determine the fibrogenic responses of human hepatic stellate cells (hHSC) to ACh and the relevance of the PNS in hepatic fibrosis in patients with non-alcoholic steatohepatitis (NASH)., Methods: Primary hHSC were analyzed for synthesis of endogenous ACh and acetylcholinesterase and gene expression of choline acetyltransferase and muscarinic ACh receptors (mAChR). Cell proliferation and fibrogenic markers were analyzed in hHSC exposed to ACh, atropine, mecamylamine, methoctramine, and 4-diphenylacetoxy-N-methylpiperidine methiodide. mAChR expression was analyzed in human NASH scored for fibrosis., Results: We observed that hHSC synthesize ACh and acetylcholinesterase and express choline acetyltransferase and M1-M5 mAChR. We also show that M2 was increased during NASH progression, while both M2 and M3 were found upregulated in activated hHSC. Furthermore, endogenous ACh is required for hHSC basal growth. Exogenous ACh resulted in hHSC hyperproliferation via mAChR and phosphoinositide 3-kinase and Mitogen-activated protein kinase kinase (MEK) signaling pathways, as well as increased fibrogenic markers., Conclusion: We show that ACh regulates hHSC activation via M2 and M3 mAChR involving the phosphoinositide 3-kinase and MEK pathways in vitro. Finally, we provide evidence that the PNS may be involved in human NASH fibrosis., (© 2015 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.)

We examined the safety of acetylcholine (ACh) and ergonovine (ER) tests retrospectively and investigated the optimal protocol of provocation test for diagnosis of multivessel coronary spasm. We performed 1546 ACh tests and 1114 ER tests during 23 years. ACh was injected in incremental doses of 20/50/80 μg into the right coronary artery (RCA) and of 20/50/100/200 μg into the left coronary artery (LCA) over 20 s. ER was administered in total doses of 40 μg into the RCA and of 64 μg into the LCA over 2-4 min. When a coronary spasm was induced and did not resolve spontaneously within 3 min after the completion of ACh/ER injection, or when hemodynamic instability due to coronary spasms occurred, 2.5-5.0 mg of nitrate was administered into the responsible vessel. To relive provoked spasm, it is necessary to administer nitrate in 31 cases by ACh and in 76 cases by ER (2.0 vs. 6.8 %, p < 0.01) before another vessel attempts. Multivessel spasms were often observed in LCA testing than in RCA testing on both agents [ACh: 78.6 % (11/14) vs. 11.8 % (2/17), p < 0.001, ER: 37.8 % (14/37) vs. 20.5 % (8/39), ns]. Even after the administration of nitrates, positive coronary spasm was obtained in 21.1 % by ACh and 52.9 % by ER tests on another coronary artery. No irreversible complications were recognized on both tests. We should firstly perform spasm provocation tests in the LCA and we may be able to diagnose another vessel spasm by performing the complete spasm provocation tests after the administration of nitrates to relieve provoked spasm in the first attempt.

As a spasm provocation test of acetylcholine (ACH), incremental dose up (20/50/100 μg) into the left coronary artery (LCA) is recommended in the guidelines established by Japanese Circulation Society. Recently, Ong et al. reported the ACOVA study which maximal ACH dose was 200 μg in the LCA. We compared the angiographic findings between ACH 100 μg and ACH 200 μg in the LCA and also examined the usefulness and safety of ACH 200 μg in Japanese patients without variant angina. As a spasm provocation test, we performed intracoronary injection of ACH 200 μg after ACH 100 μg in 88 patients (55 males, 68.4 ± 11.7 years old) including 59 ischemic heart disease (IHD) patients and 29 non-IHD patients. Positive spasm was defined as >99 % transient stenosis (focal spasm) or 90 % severe diffuse vasoconstriction (diffuse spasm). Positive spasm by ACH 200 μg was significantly higher than that by ACH 100 μg (36 pts: 40.9 % vs. 17 pts: 19.3 %, p < 0.01). Diffuse distal spasm on the left anterior descending artery was more recognized in ACH 200 μg than in ACH 100 μg (30.7 vs. 13.6 %, p < 0.01). In 29 rest angina patients, positive spasm by ACH 200 μg (19 pts) was significantly higher than that by ACH 100 μg (7 pts) (65.5 vs. 24.1 %, p < 0.01). No serious irreversible complications were found during ACH 200 μg. Administration of ACH 200 μg into the LCA was safe and useful. We may reexamine the maximal ACH dose into the LCA.

Aims: This study hypothesized that activation of endothelial nucleotide oligomerization domain-like receptor protein with pyrin domain containing 3 (Nlrp3) inflammasomes directly produces endothelial dysfunction during hypercholesterolemia, which is distinct from its canonical roles in inflammation., Results: Acute hypercholesterolemia in mice was induced by intraperitoneal administration of poloxamer 407 (0.5 g/kg) for 24 h. Endothelial dysfunction was assessed by evaluating endothelium-dependent vasodilation in isolated, perfused, and pressurized coronary arteries in response to bradykinin (10(-10)-10(-6) M) and acetylcholine (10(-9)-10(-5) M). Impaired endothelium-dependent vasodilation was observed in Nlrp3(+/+) mice with acute hypercholesterolemia, which was markedly ameliorated in Nlrp3(-/-) mice. Treatment of mice with inhibitors for caspase-1 or high mobility group box 1 (HMGB1) significantly restored endothelium-dependent vasodilation in Nlrp3(+/+) mice with acute hypercholesterolemia. Confocal microscopic analysis demonstrated that hypercholesterolemia markedly increased caspase-1 activity and HMGB1 expression in coronary arterial endothelium of Nlrp3(+/+) mice, which was absent in Nlrp3-deficient mice. Further, recombinant HMGB1 directly induced endothelial dysfunction in normal Nlrp3(+/+) coronary arteries. In vitro, Nlrp3 inflammasome formation and its activity were instigated in cultured endothelial cells by cholesterol crystal, a danger factor associated with hypercholesterolemia. Moreover, cholesterol crystals directly induced endothelial dysfunction in coronary arteries from Nlrp3(+/+) mice, which was attenuated in Nlrp3(-/-) arteries. Such cholesterol crystal-induced impairment was associated with enhanced superoxide production, downregulation of endothelial nitric oxide synthase activity, and pyroptosis., Innovation and Conclusion: Our data provide the first evidence that activation of endothelial Nlrp3 inflammasome directly impairs endothelial function beyond its canonical inflammatory actions. This novel non-canonical action of Nlrp3 inflammasomes may initiate or exacerbate vascular injury during hypercholesterolemia.

Background: Previous studies on complications with coronary spasm provocation tests were based on small sample sizes or were limited to high-volume centers. The risk of provocation tests using acetylcholine (ACH) or ergonovine (ER) remains to be fully examined by a large-scale multicenter study., Hypothesis: ACH provocation tests are associated with a higher rate of serious cardiac complications than ER tests., Methods: Using the Diagnosis Procedure Combination database in Japan, we identified patients aged ≥20 years who underwent a pharmacological provocation test during coronary angiography. We assessed the composite outcome of cardiac complications requiring urgent procedures (defibrillation, chest compression, intra-aortic balloon pumping, or extracorporeal membrane oxygenation) or death on the day of the provocation test, and compared the outcome between ACH and ER tests., Results: Of 21 512 eligible patients in 602 hospitals, 10 628 (49.4%) underwent an ACH test and 10 884 (50.6%) underwent an ER test. The composite outcome occurred in 141 (0.7%) patients. The ACH group was significantly more likely to have the composite outcome than the ER group (0.9% vs 0.4%, P < 0.001). The propensity-score analyses showed consistent results (propensity score-matched, 0.9% vs 0.4%, P = 0.003; inverse probability-weighted, 0.8% vs 0.4%, P < 0.001). In a multivariable logistic regression analysis, ACH tests were significantly associated with a higher rate of the composite outcome than ER tests (odds ratio: 1.75, 95% confidence interval: 1.13-2.69, P = 0.011)., Conclusions: This retrospective cohort study suggested that ACH tests were associated with a higher rate of cardiac complications than ER tests., (© 2015 Wiley Periodicals, Inc.)

Myrrh is the oleo-gum resin of mainly Commiphora molmol and as a powdered substance, one compound in the traditional medicinal product Myrrhinil-Intest®, which has been used for the treatment of unspecific, inflammatory intestinal disorders. The aim of the present study was to evaluate the antispasmodic effect of myrrh under healthy and inflamed conditions, and to evaluate a calcium-antagonistic effect as a possible mode of action. Therefore, an ethanolic myrrh extract was tested for its effects on muscle tone and acetylcholine-induced contractions in untreated and inflamed rat ileum/jejunum preparations. Inflammation was experimentally induced by 2,4,6-trinitrobenzene sulfonic acid (10 mM, 30 min). Additionally, the effect of the calcium channel agonist Bay K8644 in the presence of varying myrrh extract concentrations was examined. Myrrh extract (0.99 mg/mL) suppressed the acetylcholine-induced contraction down to 25.8 % in untreated and 15.2 % in inflamed preparations. Myrrh extract (0.15; 0.25 and 0.35 mg/mL) induced a concentration-dependent rightward shift of the Bay K8644 concentration-response curve in untreated and inflamed preparations with a significant EC50 shift. Schild analysis resulted in a pA2 value of 0.93 for untreated preparations. Increasing myrrh extract concentrations induced a concentration-dependent decrease of the agonistic maximum effect in untreated and inflamed preparations down to 15.8 % and 25.8 %, respectively, for the highest concentration leading to a pD2 value of 0.58. Myrrh extract reduced intestinal muscle tone and acetylcholine-induced contraction of untreated and inflamed ileum/jejunum preparations based on dual calcium antagonism characterized by a right shift of the agonistic dose-response curve and a depression of the maximum effect. The resulting reduction of intestinal motility and spasmolytic effects provide a rationale for the symptom treatment of intestinal disorders such as irritable bowel syndrome., (Georg Thieme Verlag KG Stuttgart · New York.)

Background: Coronary spasm can cause myocardial ischemia and angina in patients with and those without obstructive coronary artery disease. However, provocation tests using intracoronary acetylcholine administration are rarely performed in clinical routine in the United States and Europe. Thus, we assessed the clinical usefulness, angiographic characteristics, and safety of intracoronary acetylcholine provocation testing in white patients with unobstructed coronary arteries., Methods and Results: From September 2007 to June 2010, a total of 921 consecutive patients (362 men, mean age 62±12years) who underwent diagnostic angiography for suspected myocardial ischemia and were found to have unobstructed coronary arteries (no stenosis ≥50%) were enrolled. The intracoronary acetylcholine provocation testing was performed directly after angiography according to a standardized protocol. Three hundred forty-six patients (35%) reported chest pain at rest, 222 (22%) reported chest pain on exertion, 238 (24%) reported a combination of effort and resting chest pain, and 41 (4%) presented with troponin-positive acute coronary syndrome. The overall frequency of epicardial spasm (>75% diameter reduction with angina and ischemic ECG shifts) was 33.4%, and the overall frequency of microvascular spasm (angina and ischemic ECG shifts without epicardial spasm) was 24.2%. Epicardial spasm was most often diffuse and located in the distal coronary segments (P<0.01). No fatal or irreversible nonfatal complications occurred. However, 9 patients (1%) had minor complications (nonsustained ventricular tachycardia [n=1], fast paroxysmal atrial fibrillation [n=1], symptomatic bradycardia [n=6], and catheter-induced spasm [n=1])., Conclusions: Epicardial and microvascular spasm are frequently found in white patients with unobstructed coronary arteries. Epicardial spasm is most often diffuse and located in the distal coronary segments. The intracoronary acetylcholine provocation test is a safe technique to assess coronary vasomotor function.

Objectives: To determine the response of nebulized magnesium sulfate on the lung function of acetylcholine-induced asthma children., Methods: Three hundred and thirty children of asthma with positive bronchial provocation test were randomly divided into three groups: magnesium sulfate, albuterol, and a combination of magnesium sulfate and albuterol. Lung function was compared between the three groups., Results: Forced expiratory volume in one second (FEV1) and peak expiratory flow (PEF) as percentage over predicted at 10 min and 20 min in albuterol and combination group were significantly improved when compared to magnesium group. The changes in FEV1 and PEF expressed as absolute and percentage over predicted was not statistically significant from baseline to 20 min in magnesium, albuterol, and combination of magnesium sulfate and albuterol. There was no significant adverse effect observed during the present study., Conclusion: Nebulized magnesium sulfate alone has a bronchodilatory effect in Ach-induced asthmatic children. The combination of MgSO4 and albuterol did not has a synergistic effect.

Introduction: Fluoxetine is an open channel blocker of fetal muscle acetylcholine (ACh) receptor (AChR) and slow-channel mutant AChRs. It is used commonly to treat patients with slow-channel congenital myasthenic syndromes. Fluoxetine effects on adult wild-type endplate AChR are less characterized, although muscle AChR isoforms are differentially modulated by some drugs., Methods: Excitotoxicity assays and patch clamp recordings were performed in human embryonic kidney 293 (HEK) cells expressing wild-type or slow-channel mutant human AChRs., Results: Fluoxetine (2-10 μM) abolished ACh-induced death and decreased ACh-activated whole-cell currents in cells expressing all AChR types. In outside-out patches, fluoxetine rapidly curtailed ACh evoked unitary activity and macroscopic currents. The effect was increased if fluoxetine was applied before ACh., Conclusions: Fluoxetine is an open channel blocker, but it also affects AChR in the closed state. AChR blockade likely underlies the rescue of HEK cells from ACh-induced death., (Copyright © 2013 Wiley Periodicals, Inc.)

Venom (10-100 μg/ml) from Bothrops alcatraz, a pitviper from the Alcatrazes Archipelago off the coast of southeastern Brazil, caused progressive, irreversible neuromuscular blockade in chick isolated biventer cervicis preparations. The venom also inhibited contractures to exogenous ACh (110 μM) and KCl (20 mM), caused myofiber damage and increased creatine kinase release. Commercial bothropic antivenom raised against mainland Bothrops species neutralized the neuromuscular activity, depending on the venom concentration., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The p21-activated protein kinases (Paks) have been implicated in the regulation of smooth muscle contractility, but the physiologic effects of Pak activation on airway reactivity in vivo are unknown. A mouse model with a genetic deletion of Pak1 (Pak1(-/-)) was used to determine the role of Pak in the response of the airways in vivo to challenge with inhaled or intravenous acetylcholine (ACh). Pulmonary resistance was measured in anesthetized mechanically ventilated Pak1(-/-) and wild type mice. Pak1(-/-) mice exhibited lower airway reactivity to ACh compared with wild type mice. Tracheal segments dissected from Pak1(-/-) mice and studied in vitro also exhibited reduced responsiveness to ACh compared with tracheas from wild type mice. Morphometric assessment and pulmonary function analysis revealed no differences in the structure of the airways or lung parenchyma, suggesting that that the reduced airway responsiveness did not result from structural abnormalities in the lungs or airways due to Pak1 deletion. Inhalation of the small molecule synthetic Pak1 inhibitor, IPA3, also significantly reduced in vivo airway responsiveness to ACh and 5-hydroxytryptamine (5-Ht) in wild type mice. IPA3 inhibited the contractility of isolated human bronchial tissues to ACh, confirming that this inhibitor is also effective in human airway smooth muscle tissue. The results demonstrate that Pak is a critical component of the contractile activation process in airway smooth muscle, and suggest that Pak inhibition could provide a novel strategy for reducing airway hyperresponsiveness.

Objective: To discuss phytopharmacological potential and anti-asthmatic activity of Ficus religiosa (F. religiosa) (L.)., Methods: Fresh leaves of F. religiosa were obtained from Vastrapur Lake, Ahmedabad, and dried to obtain powder. Histamine and acetylcholine were used to guinea pigs to establish bronchospasm model. In in vivo study, the aqueous extract of F. religiosa leaves (AEFR) at doses of 150 and 300 mg/kg was administrated to guinea pigs, and the broncho-protective activity of AEFR was compared with aminophylline at 25 mg/kg. While in in vitro study, and 10 g/mL, 20 g/mL, 30 g/mL of AEFRL was administrated to guinea pigs, respectively, and mast cell stabilizing activity of AEFR was compared with ketotifen at 10 g/mL., Results: In the in-vivo model, pre-treatment with aminophylline (25 mg/kg, ip.) could significantly delay the onset of histamine induced pre-convulsive dyspnea, compared with vehicle control. Administration of AEFRL (150 and 300 mg/kg, ip.) also produced significant effect on latency to develop histamine & acetylcholine induced pre-convulsive dyspnea. In the mast cell stabilizing model, AEFRL at 10, 20 and 30 μg/mL could significantly increase the number of intact cells., Conclusions: It can be concluded that AEFRL is effective on histamine & acetylcholine induced bronchospasm in guinea pigs. In addition, AEFRL can potentiate the number of intact cells in the mast cell stabilizing model., (Copyright © 2011 Hainan Medical College. Published by Elsevier B.V. All rights reserved.)

Butorphanol tartrate (BT) was injected into mice before injection with acetylcholine in a murine model infected latently with pseudorabies virus. The analgesic effect and its influence on virus reactivation were observed. Mice preinjected with BT showed suppression of screaming, moving and excitation and the same level of movement after excitation as mice injected with PBS. In the group injected with BT i.p., one mouse died and another developed diarrhea with increased virus excretion. These results showed that BT has analgesic effects by both injection routes, s.c. and i.p.; however, BT induced death as a side effect, especially with i.p. injection. The injection route for BT should therefore be investigated further.

Unlabelled: Recent studies have shown that drug-eluting stents (DES) induce vascular endothelial dysfunction in both Cypher and Taxus stents. These studies evaluated coronary vasomotion in the peristent coronary segment for 1 lesion with 1 DES. The angiographic and clinical characteristics of real-world patients with coronary spasm following DES implantation have not been well documented., Methods and Results: All patients at our hospital who underwent coronary angiography at follow up after DES deployment (Cypher and Taxus stents) between July 2007 and March 2009 were included. We performed an acetylcholine (ACh) provocation test for diagnosing coronary vasospasm in 3 vessels, except in patients with significant stenosis or contraindications to ACh administration. ACh provocation test was positive in 36/55 of the coronary arteries (65.4%) and in 30/42 of the patients with DES (71.4%). There was no difference in the positive rate between patients with and without symptoms. A total of 13/20 asymptomatic patients (65.0%) also showed positive results. In patients with positive results in the ACh provocation test, vasoconstriction at segments distal to the stent was exaggerated compared with corresponding segments in non-stented vessels (0.46 ± 0.27 versus 0.31 ± 0.20, respectively; p = 0.008). Vessels with positive results had a longer stent length compared with those with negative results (31.6 ± 13.6 mm versus 24.2 ± 11.2 mm, respectively; p = 0.049)., Conclusion: Coronary vasoconstriction was exaggerated at distal segments in DES-implanted vessels compared to non-stented vessel segments and stent length was longer in the ACh provocation test positive group.

The aim of this study was to investigate the microvascular responses in the skin, to local heat, iontophoretically administered acetylcholine and to sodium nitroprusside in relation to cardiovascular damage in patients with systemic lupus erythematosus (SLE) and matched controls. We also wanted to examine if the ongoing medication in SLE patients influenced this vascular response. We investigated 30 women with SLE and compared them with 20 age and sex-matched controls. The cutaneous blood flow response to local heat (+44°C), iontophoretically administered endothelium-dependent (acetylcholine), as well as independent (sodium nitroprusside) vasodilatation, was measured by laser Doppler flowmetry. Clinical data and medication were retrieved from the clinical database and patient records. The cutaneous microvascular reactivity did not differ between SLE patients and a group of matched controls nor did it correlate with cardiovascular damage [assessed by Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index (SLICC/ACR-DI)]. However, patients on antimalarial drugs (hydroxychloroquine n = 8 and chloroquine diphosphate n = 3) responded more strongly to sodium nitroprusside (endothelium-independent vasodilatation) compared with those without antimalarial drugs (p < 0.01). The response to acetylcholine was higher among patients on warfarin compared with those without (p < 0.05), whereas glucocorticoid use (≥5 mg daily) was associated with reduced response to acetylcholine (p < 0.05). Smokers in general tended to have a lower response to acetylcholine (p = 0.064). Smoking SLE patients versus non-smoking SLE patients had a significantly lower response to acetylcholine (p = 0.01). Medication with antimalarial drugs-enhanced endothelium-independent vasodilatation, while glucocorticoid use was associated with reduction and warfarin-treatment with enhancement of endothelium-dependent vasodilatation. Therefore, despite there is no difference in microvascular endothelium-dependent vasodilatation, other factors such as medication and smoking may affect vasodilatation in SLE patients., (© 2010 The Authors. Basic & Clinical Pharmacology & Toxicology © 2010 Nordic Pharmacological Society.)

Recent studies revealed an involvement of RhoA/Rho-kinase signaling in the agonist-induced Ca(2+) sensitization of bronchial smooth muscle contraction, and the pathway has now been proposed as a new target for the treatment of airway obstructive diseases, such as asthma. On the other hand, volatile anesthetics such as isoflurane are traditionally used to treat status asthmaticus. In the present study, the effect of inhibition of Rho-kinase on the isoflurane-induced relaxation of bronchial smooth muscle was investigated. Smooth muscle strips of intrapulmonary bronchi obtained from Wistar rats were used. Application of isoflurane (0.5-4.0%, generated by a calibrated vaporizer) to the acetylcholine (30 microM)-precontracted rat bronchial smooth muscles caused a concentration-dependent relaxation. Interestingly, the isoflurane-induced relaxation was significantly augmented by the pretreatment with subthreshold concentration of Y-27632, a Rho-kinase inhibitor. Thus, the combined use of Y-27632 and isoflurane might be useful for treatment of severe airway blockade, such as status asthmaticus.

Objectives: We examined some recommendations for performing acetylcholine (ACh) tests safely., Methods and Results: We performed 1000 ACh tests from 1991 to December 2004. ACh was injected in incremental doses of 20/50/80 microg into the RCA and of 20/50/100 microg into the LCA. During these periods, we encountered various major/minor complications; 12 ventricular tachycardia (1.2%) necessary one dc, one ventricular fibrillation (0.1%) necessary dc, 3 shock like the left main stem spasm (0.3%), one cardiac tamponade necessary surgical drainage (0.1%), and 164 Paf (164/959:17.1%) necessary administration of antiarrhythmic agents to sinus rhythm in about one third patients (31.7%). We did not experience irreversible severe complications, such as acute myocardial infarction or death., Recommendations: (1) Stand by direct current with pasting, (2) Thump version when ventricular tachycardia or fibrillation occurred, (3) Over infusion to avoid hypovolemia, (4) Perform angiography before complete spasm provocation if a severe spasm, (5) Drainage if cardiac tamponade occurred, (6) Cibenzoline or disopyramid administration when ACh induced paroxysmal atrial fibrillation, (7) Incremental ACh dose up should be performed, (8) Administer small amount of noradrenaline if shock observed and (9) Test shot should be performed before 1-min angiography., Conclusions: We recommend STOP DCIAT for performing ACh tests safely.

The comparative long-term antianginal efficacy of long-acting nitrates versus calcium channel antagonists remains unclear. The goal of the present study was to compare the coronary endothelial cell function and coronary artery vasoconstriction between patients with normal or mildly diseased coronary arteries treated with long-acting nitrates or calcium channel antagonists. Forty-two patients suspected to have angina pectoris and with normal or mildly diseased coronary arteries underwent Doppler flow study of the left anterior descending coronary artery. All patients were suspected to have angina pectoris and were receiving either long-acting nitrates (n = 18; Nitrates group) or calcium channel antagonists (n = 24; Ca-antagonists group) for at least 1 year. Vascular reactivity was assessed by intracoronary administration of papaverine, acetylcholine (Ach), and nitroglycerin using a Doppler guidewire. Segments that showed the greatest constrictive response to Ach were used for assessment of vasoconstriction. The percent increase in coronary blood flow (CBF) and coronary artery diameter (CAD) induced by Ach was significantly smaller in the Nitrates group than in the Ca-antagonists group (33% +/- 74% vs 83% +/- 77%, P < 0.05; -3% +/- 16% vs 11% +/- 12%, P < 0.01, respectively). The percent diameter reduction in the region of greatest constrictive response to Ach was significantly greater in the Nitrates group than in the Ca-antagonists group (44% +/- 39% vs 15% +/- 32%, P < 0.02). Long-term treatment with long-acting nitrates may produce less favorable effects on coronary endothelial function and the constrictive response to Ach when compared with long-acting calcium channel antagonists in patients with normal or mildly diseased coronary arteries.

Corticosteroids are known to inhibit bronchial hyperresponsiveness (BHR) and allergic inflammation but there is little information on its dose-dependence. We examined the effect of different doses of the glucocorticosteroid budesonide in an allergic model. Brown-Norway rats were sensitised to ovalbumin (OVA) and pretreated with an intra-gastric dose of budesonide (0.1, 1.0, or 10 mgkg(-1)). Exposure to OVA induced BHR, accumulation of eosinophils in the bronchoalveolar lavage (BAL) fluid and in the airways submucosa. Budesonide dose-dependently inhibited BAL fluid influx of lymphocytes, eosinophils and neutrophils, tissue eosinophils and lymphocytes and BHR. At 0.1 mgkg(-1), budesonide did not inhibit these parameters but at 1 mgkg(-1), BAL fluid eosinophils and T-cells, and submucosal T-cells were significantly reduced. At 10 mgkg(-1), budesonide suppressed BHR, BAL fluid inflammatory cells numbers and tissue eosinophilia. T-cell numbers were more related to BHR than eosinophil numbers. Budesonide inhibited both airway inflammation and BHR, but BAL fluid eosinophil cell counts may be dissociated from BHR.