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Neurourology and Urodynamics Aug 2020Bladder wall stretch increases tissue tension and releases adenosine 5'-triphosphate (ATP) as part of a transduction process to sense bladder filling. Aging is...
AIMS
Bladder wall stretch increases tissue tension and releases adenosine 5'-triphosphate (ATP) as part of a transduction process to sense bladder filling. Aging is associated with bladder fibrosis to produce a stiffer bladder wall: this may augment ATP release and contribute to age-dependent urgency. Muscarinic agonists also release ATP and present a potential target for antimuscarinic agents, but its age-dependency is unknown. This study aimed, in young and old mice, to: (a) quantify the relationship between bladder wall stiffness and stretch-dependent ATP release and; (b) characterize muscarinic agonist-dependent release.
METHODS
ATP release from young (9-12 weeks) and aged (24 months) mouse bladder wall was measured in vitro, with a luciferin-luciferase assay, after stretch or carbachol exposure. Bladder wall stiffness, measured simultaneously during stretch, was compared to histological proportions of connective tissue and detrusor muscle.
RESULTS
With young mice, stretch-activated ATP release required an intact mucosa and was positively associated with wall stiffness. ATP release by carbachol was about four-fold greater compared to stretch. With aged mice: ATP release varied a hundred-fold and no association with stiffness; carbachol release diminished; connective tissue and mucosa thickness increased.
CONCLUSIONS
With young mice, stretch, or muscarinic agonists potently induce bladder wall ATP release. Stretch-dependent release is proportional to bladder wall stiffness, independent of the extent of stretch. With aged mice dependence of stretch-activated ATP release with stiffness was lost. The huge variability of release suggests that aged mice do not form a homogenous cohort and may underlie the heterogeneity in bladder filling sensations.
Topics: Adenosine Triphosphate; Aging; Animals; Carbachol; Male; Mice; Mucous Membrane; Muscarinic Agonists; Urinary Bladder
PubMed: 32531080
DOI: 10.1002/nau.24426 -
Medical Science Monitor : International... May 2022BACKGROUND Safety concerns about drugs used intracamerally during cataract surgery have been the subject of many studies. In this study, the effect of using intracameral...
BACKGROUND Safety concerns about drugs used intracamerally during cataract surgery have been the subject of many studies. In this study, the effect of using intracameral carbachol and epinephrine on choroidal thickness was evaluated. MATERIAL AND METHODS This prospective interventional study included 81 eyes of 81 patients undergoing cataract surgery. During cataract surgery, intracameral carbachol was administered to 27 eyes, intracameral epinephrine was administered to 20 eyes, and 34 eyes were the control group. Macular choroidal thickness measurement was performed before, 1 day, and 1 week after phacoemulsification surgery in all patients using optical coherence tomography. RESULTS Subfoveal choroidal thickness was significantly reduced at day 1 and week 1 in the group receiving intraoperative carbachol compared with preoperative measurement (P=0.016). In addition, choroidal thickness in the 500 µm nasal fovea was significantly reduced in the carbachol group at 1st week compared to the preoperative measurement (P=0.008). There was no significant difference in postoperative subfoveal thickness in the intraoperative epinephrine group and control group (P=0.179 and P=0.953, respectively). Choroidal thickness at 1000 µm nasal fovea was significantly higher in the epinephrine group at postoperative 1st day than preoperative and postoperative 1st week values (P=0.009). CONCLUSIONS The use of intracameral epinephrine caused an increase in choroidal thickness 1000 µm nasal of the fovea, while intracameral carbachol caused thinning in the subfoveal and 500 µm nasal quadrant. Intracameral drug administration during cataract surgery may be associated with posterior segment complications.
Topics: Carbachol; Cataract; Epinephrine; Humans; Phacoemulsification; Prospective Studies
PubMed: 35491490
DOI: 10.12659/MSM.935315 -
Neurourology and Urodynamics Aug 2021To determine the effect of prostatic radiation therapy (RT) on bladder contractility and morphology, and axon, or neuron profiles within the detrusor and major pelvic...
AIMS
To determine the effect of prostatic radiation therapy (RT) on bladder contractility and morphology, and axon, or neuron profiles within the detrusor and major pelvic ganglia (MPG) in male rats.
METHODS
Male Sprague-Dawley rats (8 weeks) received a single dose of prostatic RT (0 or 22 Gy). Bladders and MPG were collected 2- and 10-weeks post-RT. Detrusor contractile responses to carbachol and electrical field stimulation (EFS) were measured. Bladders were stained with Masson's trichrome, and antibodies for nonspecific neuronal marker, cholinergic nerve marker choline acetyltransferase (ChAT), and alpha-smooth muscle actin. MPG gene expression was assessed by quantitative polymerase chain reaction for ubiquitin carboxy-terminal hydrolase L1 (Uchl1) and Chat.
RESULTS
At 2 weeks post-RT, bladder smooth muscle, detrusor cholinergic axon profiles, and MPG Chat gene expression were increased (p < .05), while carbachol and EFS-mediated contractions were decreased (p < .05). In contrast, at 10 weeks post-RT, nerve-mediated contractions were increased compared with control (p < .05), while bladder smooth muscle, detrusor cholinergic axon profiles, MPG Chat expression, and carbachol contractions had normalized. At both 2- and 10-weeks post-RT, there was no change in detrusor nonspecific axon profiles and MPG Uchl1 expression.
CONCLUSION
In a rat model, RT of the prostate and MPG was associated with early changes in MPG Chat gene expression, and bladder cholinergic axon profiles and smooth muscle content which resolved over time. After RT recovery, bladder contractility decreased early and increased by 10 weeks. Long-term changes to the MPG and increased bladder cholinergic axons may contribute to RT-induced bladder dysfunction in prostate cancer survivors.
Topics: Animals; Carbachol; Male; Muscle Contraction; Muscle, Smooth; Rats; Rats, Sprague-Dawley; Urinary Bladder
PubMed: 34015163
DOI: 10.1002/nau.24705 -
Purinergic Signalling Mar 2015The purpose of this study is to investigate if the cholinergic stimulation by carbachol on tear secretion is a direct process or if it is also mediated by purinergic...
The purpose of this study is to investigate if the cholinergic stimulation by carbachol on tear secretion is a direct process or if it is also mediated by purinergic mechanisms. Experiments were performed in New Zealand male rabbits. The amount of tear secretion was measured with Schirmer's test and then analyzed by a HPLC protocol in order to study the nucleotide levels. Animal eyes were instilled with carbachol (a cholinergic agonist), pirenzepine, gallamine and 4-DAMP (muscarinic antagonists), PPADS, suramin and reactive blue 2 (purinergic antagonists), and a P2Y2 receptor small interfering RNA (siRNA). Tear secretion increased with the instillation of carbachol, approximately 84 % over control values 20 min after the instillation and so did Ap4A and ATP release. When we applied carbachol in the presence of muscarinic antagonists, tear volume only increased to 4 % with atropine, 12 % in the case of pirenzepine, 3 % with gallamine, and 8 % with 4-DAMP. In the presence of carbachol and purinergic antagonists, tear secretion was increased to 12 % (all values compared to basal tear secretion). By analyzing tear secretion induced with carbachol in presence of a P2Y2 receptor siRNA, we found that tear secretion was diminished to 60 %. The inhibition of tear secretion in the presence of carbachol and purinergic antagonists or P2Y2 siRNA occurred with no apparent change in the tear amount of Ap4A. These experiments demonstrated the participation of Ap4A in lacrimal secretion process.
Topics: Animals; Atropine; Carbachol; Cholinergic Agonists; Dinucleoside Phosphates; Male; Muscarinic Antagonists; Rabbits; Tears
PubMed: 25398705
DOI: 10.1007/s11302-014-9434-3 -
Anatomical Record (Hoboken, N.J. : 2007) Dec 2021Among the pathologies affecting the salivary glands, the Sjögren's syndrome (SS), an autoimmune disease, causes progressive destruction of the glandular tissue. The...
Among the pathologies affecting the salivary glands, the Sjögren's syndrome (SS), an autoimmune disease, causes progressive destruction of the glandular tissue. The effect of SS is particularly evident on the labial glands and the morphological analysis of these minor glands is considered useful for diagnosis. Cevimeline hydrochloride (SNI), a selective muscarinic agonist drug, is one of the elective treatments for the hyposalivation due to SS, acting not only on major salivary glands, but also on labial glands since their secretion is primarily under parasympathetic control. Aim of this study is to describe the morphology of human labial glands treated with SNI by light, transmission, and high-resolution scanning electron microscopy. Moreover, a morphometric analysis was applied to the light and transmission electron microscopy micrographs to obtain data that were then compared with analogous data collected on control and carbachol-treated labial glands. Following SNI administration, the mucous tubules exhibited enlarged lumina, which were filled with a dense mucous secretion. Occasionally, small broken debris of the cells were retrieved into the lumen. In the mucous secretory cells, some mucous droplets fused to form a large vacuole-like structure. Similarly, the seromucous acini showed both dilated lumina and canaliculi. These above reported signs of secretion were confirmed through morphometric analysis and a milder action of SNI than carbachol on labial parenchyma was observed. This study confirmed that SNI also evoked secretion on labial glands and that its effect is more physiologic than that of the pan-muscarinic agonists.
Topics: Carbachol; Humans; Lip; Quinuclidines; Salivary Glands; Sjogren's Syndrome; Thiophenes
PubMed: 33704905
DOI: 10.1002/ar.24617 -
British Journal of Pharmacology May 2018The aim of this study was to develop potent and long-acting antagonists of muscarinic ACh receptors. The 4-hexyloxy and 4-butyloxy derivatives of...
BACKGROUND AND PURPOSE
The aim of this study was to develop potent and long-acting antagonists of muscarinic ACh receptors. The 4-hexyloxy and 4-butyloxy derivatives of 1-[2-(4-oxidobenzoyloxy)ethyl]-1,2,3,6-tetrahydropyridin-1-ium were synthesized and tested for biological activity. Antagonists with long-residence time at receptors are therapeutic targets for the treatment of several neurological and psychiatric human diseases. Their long-acting effects allow for reduced daily doses and adverse effects.
EXPERIMENTAL APPROACH
The binding and antagonism of functional responses to the agonist carbachol mediated by 4-hexyloxy compounds were investigated in CHO cells expressing individual subtypes of muscarinic receptors and compared with 4-butyloxy analogues.
KEY RESULTS
The 4-hexyloxy derivatives were found to bind muscarinic receptors with micromolar affinity and antagonized the functional response to carbachol with a potency ranging from 30 nM at M to 4 μM at M receptors. Under washing conditions to reverse antagonism, the half-life of their antagonistic action ranged from 1.7 h at M to 5 h at M receptors.
CONCLUSIONS AND IMPLICATIONS
The 4-hexyloxy derivatives were found to be potent long-acting M -preferring antagonists. In view of current literature, M -selective antagonists may have therapeutic potential for striatal cholinergic dystonia, delaying epileptic seizure after organophosphate intoxication or relieving depression. These compounds may also serve as a tool for research into cognitive deficits.
Topics: Animals; CHO Cells; Carbachol; Cells, Cultured; Cricetulus; Dose-Response Relationship, Drug; Molecular Structure; Muscarinic Antagonists; Pyridines; Receptors, Muscarinic; Structure-Activity Relationship
PubMed: 29498041
DOI: 10.1111/bph.14187 -
Stem Cell Research & Therapy Jul 2023Atrial fibrillation is the most common arrhythmia syndrome and causes significant morbidity and mortality. Current therapeutics, however, have limited efficacy. Notably,...
BACKGROUND
Atrial fibrillation is the most common arrhythmia syndrome and causes significant morbidity and mortality. Current therapeutics, however, have limited efficacy. Notably, many therapeutics shown to be efficacious in animal models have not proved effective in humans. Thus, there is a need for a drug screening platform based on human tissue. The aim of this study was to develop a robust protocol for generating atrial cardiomyocytes from human-induced pluripotent stem cells.
METHODS
A novel protocol for atrial differentiation, with optimized timing of retinoic acid during mesoderm formation, was compared to two previously published methods. Each differentiation method was assessed for successful formation of a contractile syncytium, electrical properties assayed by optical action potential recordings and multi-electrode array electrophysiology, and response to the G-protein-gated potassium channel activator, carbamylcholine. Atrial myocyte monolayers, derived using the new differentiation protocol, were further assessed for cardiomyocyte purity, gene expression, and the ability to form arrhythmic rotors in response to burst pacing.
RESULTS
Application of retinoic acid at day 1 of mesoderm formation resulted in a robust differentiation of atrial myocytes with contractile syncytium forming in 16/18 differentiations across two cell lines. Atrial-like myocytes produced have shortened action potentials and field potentials, when compared to standard application of retinoic acid at the cardiac mesoderm stage. Day 1 retinoic acid produced atrial cardiomyocytes are also carbamylcholine sensitive, indicative of active I currents, which was distinct from ventricular myocytes and standard retinoic addition in matched differentiations. A current protocol utilizing reduced Activin A and BMP4 can produce atrial cardiomyocytes with equivalent functionality but with reduced robustness of differentiation; only 8/17 differentiations produced a contractile syncytium. The day 1 retinoic acid protocol was successfully applied to 6 iPSC lines (3 male and 3 female) without additional optimization or modification. Atrial myocytes produced could also generate syncytia with rapid conduction velocities, > 40 cm s, and form rotor style arrhythmia in response to burst pacing.
CONCLUSIONS
This method combines an enhanced atrial-like phenotype with robustness of differentiation, which will facilitate further research in human atrial arrhythmia and myopathies, while being economically viable for larger anti-arrhythmic drug screens.
Topics: Animals; Female; Male; Humans; Induced Pluripotent Stem Cells; Atrial Fibrillation; Myocytes, Cardiac; Carbachol; Cell Differentiation; Action Potentials; Tretinoin
PubMed: 37501071
DOI: 10.1186/s13287-023-03405-5 -
Naunyn-Schmiedeberg's Archives of... Apr 2024Carbachol, an agonist at muscarinic receptors, exerts a negative inotropic effect in human atrium. Carbachol can activate protein phosphatases (PP1 or PP2A). We...
Carbachol, an agonist at muscarinic receptors, exerts a negative inotropic effect in human atrium. Carbachol can activate protein phosphatases (PP1 or PP2A). We hypothesized that cantharidin or sodium fluoride, inhibitors of PP1 and PP2A, may attenuate a negative inotropic effect of carbachol. During bypass-surgery trabeculae carneae of human atrial preparations (HAP) were obtained. These trabeculae were mounted in organ baths and electrically stimulated (1 Hz). Force of contraction was measured under isometric conditions. For comparison, we studied isolated electrically stimulated left atrial preparations (LA) from mice. Cantharidin (100 µM) and sodium fluoride (3 mM) increased force of contraction in LA (n = 5-8, p < 0.05) by 113% ± 24.5% and by 100% ± 38.2% and in HAP (n = 13-15, p < 0.05) by 625% ± 169% and by 196% ± 23.5%, respectively. Carbachol (1 µM) alone exerted a rapid transient maximum negative inotropic effect in LA (n = 6) and HAP (n = 14) to 46.9% ± 3.63% and 19.4% ± 3.74%, respectively (p < 0.05). These negative inotropic effects were smaller in LA (n = 4-6) and HAP (n = 9-12) pretreated with 100 µM cantharidin and amounted to 58.0% ± 2.27% and 59.2% ± 6.19% or 3 mM sodium fluoride to 63.7% ± 9.84% and 46.3% ± 5.69%, (p < 0.05). We suggest that carbachol, at least in part, exerts a negative inotropic effect in the human atrium by stimulating the enzymatic activity of PP1 and/or PP2A.
Topics: Humans; Mice; Animals; Carbachol; Cantharidin; Sodium Fluoride; Myocardial Contraction; Heart Atria
PubMed: 37801145
DOI: 10.1007/s00210-023-02747-4 -
The Journal of Physiology Jan 19881. Fura2 was loaded by permeation and hydrolysis of the acetoxymethyl ester into smooth muscle cells of intact thin sheets of the longitudinal layer of the small...
1. Fura2 was loaded by permeation and hydrolysis of the acetoxymethyl ester into smooth muscle cells of intact thin sheets of the longitudinal layer of the small intestine of the guinea-pig, to record Ca2+ transients during contraction. 2. Cytoplasmic Ca2+ ([Ca2+]i) was monitored by computing the ratio of the fluorescence signal excited at 340 and 380 nm wavelengths. The dye loading and the exposure to UV light required for the experiments had no significant effect on the contractile parameters observed. 3. Spontaneous, rhythmic increases in [Ca2+]i were often observed, preceding the onset of force. Removal of extracellular Ca2+ caused a very transient increase in [Ca2+]i accompanied by a phasic force transient; this was followed by a decline in [Ca2+]i and tension below control levels. Elevated Ca2+ from 1.2 to 15 mM also caused a fall in [Ca2+]i and a relaxation of basal tension. 4. Elevation of [K+]o increased [Ca2+]i. Graded concentrations of K+ caused graded changes in both fluorescence ratio and tension. 5. Carbachol evoked a transient increase in [Ca2+]i and contraction. Thereafter, in spite of the continued presence of the drug, both signals declined, presumably as the result of cholinergic desensitization. The initial phasic force response to carbachol was usually followed by an 'after-contraction', that was only occasionally accompanied by a similar (small) secondary rise in the fluorescence signal. 6. In depolarized smooth muscle, both in the presence and in the absence of extracellular Ca2+, carbachol induced a transient increase in [Ca2+]i, indicating that Ca2+ release from intracellular stores is a major mechanism of pharmacomechanical coupling. 7. In some preparations an applied stretch caused, after a few seconds, a rise in [Ca2+]i and force development.
Topics: Animals; Calcium; Carbachol; Egtazic Acid; Female; Fluorescent Dyes; Guinea Pigs; In Vitro Techniques; Male; Muscle Contraction; Muscle, Smooth; Osmolar Concentration; Potassium; Verapamil
PubMed: 3137325
DOI: 10.1113/jphysiol.1988.sp016932 -
Frontiers in Immunology 2024Hemorrhagic shock is characterized by derangements of the gastrointestinal microcirculation. Topical therapy with nitroglycerine or iloprost improves gastric tissue...
INTRODUCTION
Hemorrhagic shock is characterized by derangements of the gastrointestinal microcirculation. Topical therapy with nitroglycerine or iloprost improves gastric tissue oxygenation but not regional perfusion, probably due to precapillary adrenergic innervation. Therefore, this study was designed to investigate the local effect of the parasympathomimetic carbachol alone and in combination with either nitroglycerine or iloprost on gastric and oral microcirculation during hemorrhagic shock.
METHODS
In a cross-over design five female foxhounds were repeatedly randomized into six experimental groups. Carbachol, or carbachol in combination with either nitroglycerine or iloprost were applied topically to the oral and gastric mucosa. Saline, nitroglycerine, or iloprost application alone served as control groups. Then, a fixed-volume hemorrhage was induced by arterial blood withdrawal followed by blood retransfusion after 1h of shock. Gastric and oral microcirculation was determined using reflectance spectrophotometry and laser Doppler flowmetry. Oral microcirculation was visualized with videomicroscopy. Statistics: 2-way-ANOVA for repeated measurements and Bonferroni post-hoc analysis (mean ± SEM; p < 0.05).
RESULTS
The induction of hemorrhage led to a decrease of gastric and oral tissue oxygenation, that was ameliorated by local carbachol and nitroglycerine application at the gastric mucosa. The sole use of local iloprost did not improve gastric tissue oxygenation but could be supplemented by local carbachol treatment. Adding carbachol to nitroglycerine did not further increase gastric tissue oxygenation. Gastric microvascular blood flow remained unchanged in all experimental groups. Oral microvascular blood flow, microvascular flow index and total vessel density decreased during shock. Local carbachol supply improved oral vessel density during shock and oral microvascular flow index in the late course of hemorrhage.
CONCLUSION
The specific effect of shifting the autonomous balance by local carbachol treatment on microcirculatory variables varies between parts of the gastrointestinal tract. Contrary to our expectations, the improvement of gastric tissue oxygenation by local carbachol or nitroglycerine application was not related to increased microvascular perfusion. When carbachol is used in combination with local vasodilators, the additional effect on gastric tissue oxygenation depends on the specific drug combination. Therefore, modulation of tissue oxygen consumption, mitochondrial function or alterations in regional blood flow distribution should be investigated.
Topics: Dogs; Female; Animals; Shock, Hemorrhagic; Carbachol; Iloprost; Microcirculation; Hemorrhage; Nitroglycerin
PubMed: 38566995
DOI: 10.3389/fimmu.2024.1369617