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The Journal of Biological Chemistry Sep 1982The role of protein phosphorylation in the regulation of pancreatic function by carbachol, cholecystokinin-octapeptide (CCK8), and insulin was investigated using...
The role of protein phosphorylation in the regulation of pancreatic function by carbachol, cholecystokinin-octapeptide (CCK8), and insulin was investigated using isolated pancreatic acini of the mouse. Carbachol and CCK8 increased the phosphorylation of a Mr = 32,500 particulate protein and Mr = 16,000 and 23,000 soluble proteins. These agents also caused the dephosphorylation of Mr = 21,000 and 20,500 soluble proteins. Alterations in phosphorylation produced by carbachol were dose-dependent (maximal at 1-3 microM) and consistent with the dose-response relationship for carbachol-induced amylase secretion. Maximal dephosphorylation of the Mr = 21,000 and 20,500 proteins occurred within 1 min of addition of carbachol and was concurrent with the first detectable stimulation of amylase cyclase by carbachol. Moreover, atropine inhibition of carbachol-induced amylase secretion was concurrent with reversal of dephosphorylation of these two proteins. The calcium ionophore A23187, which mimicks the actions of carbachol and CCK8 on the pancreas, also mimicked the effects of these agents on protein phosphorylation, suggesting that pancreatic protein phosphorylation is regulated by calcium. Insulin, which enhances many of the actions of carbachol and CCK8 on the pancreas, alone increased the phosphorylation of the Mr = 16,000, 23,000, and 32,500 proteins and enhanced in an additive manner the effects of carbachol and CCK8 on the latter two proteins. By contrast, phosphorylation of the Mr = 21,000 or 20,500 proteins was not altered by insulin either in the absence or presence of carbachol or CCK8. In conclusion, the results suggest that alterations in phosphorylation of specific acinar cell proteins may provide a mechanism by which hormones and neurotransmitters interact in the regulation of pancreatic function.
Topics: Animals; Atropine; Carbachol; Cholecystokinin; Insulin; Kinetics; Male; Mice; Molecular Weight; Pancreas; Phosphoproteins; Phosphorylation; Proteins
PubMed: 7050109
DOI: No ID Found -
Neuropharmacology Jun 2021The medial septum/diagonal band of Broca (MS/DBB) receives direct GABAergic input from the hippocampus via hippocamposeptal (HS) projection neurons as part of a...
The medial septum/diagonal band of Broca (MS/DBB) receives direct GABAergic input from the hippocampus via hippocamposeptal (HS) projection neurons as part of a reciprocal loop that mediates cognition and is altered in Alzheimer's disease. Cholinergic and GABAergic interactions occur throughout the MS/DBB, but it is not known how HS GABA release is impacted by these circuits. Most HS neurons contain somatostatin (SST), so to evoke HS GABA release we expressed Cre-dependent mCherry/channelrhodopisin-2 (ChR2) in the hippocampi of SST-IRES-Cre mice and then used optogenetics to stimulate HS fibers while performing whole-cell patch clamp recordings from MS/DBB neurons in acute slices. We found that the acetylcholine receptor (AChR) agonist carbachol and the GABA receptor (GABAR) agonist baclofen significantly decreased HS GABA release in the MS/DBB. Carbachol's effects were blocked by eliminating local GABAergic activity or inhibiting GABARs, indicating that it was indirectly decreasing HS GABA release by increasing GABAergic tone. There was no effect of acute exposure to amyloid-β on HS GABA release. Repetitive stimulation of HS fibers increased spontaneous GABA release in the MS/DBB, revealing that HS projections can modulate local GABAergic tone. These results show that HS GABA release has far-reaching impacts on overall levels of inhibition in the MS/DBB and is under regulatory control by cholinergic and GABAergic activity. This bidirectional modulation of GABA release from local and HS projections in the MS/DBB will likely have profound impact not only on activity within the MS/DBB, but also on output to the hippocampus and hippocampal-dependent learning and memory.
Topics: Animals; Baclofen; Carbachol; Diagonal Band of Broca; Female; Hippocampus; Male; Mice; Mice, Transgenic; Neural Pathways; Optogenetics; Receptors, GABA-B; Septum of Brain; Synaptic Transmission; gamma-Aminobutyric Acid
PubMed: 33933476
DOI: 10.1016/j.neuropharm.2021.108589 -
Biomedicine & Pharmacotherapy =... Dec 2023In intestinal smooth muscle cells, receptor-operated TRPC4 are responsible for the majority of muscarinic receptor cation current (mI), which initiates cholinergic...
In intestinal smooth muscle cells, receptor-operated TRPC4 are responsible for the majority of muscarinic receptor cation current (mI), which initiates cholinergic excitation-contraction coupling. Our aim was to examine the effects of the TRPC4 inhibitor Pico145 on mI and Ca signalling in mouse ileal myocytes, and on intestinal motility. Ileal myocytes freshly isolated from two month-old male BALB/c mice were used for patch-clamp recordings of whole-cell currents and for intracellular Ca imaging using Fura-2. Functional assessment of Pico145's effects was carried out by standard in vitro tensiometry, ex vivo video recordings and in vivo postprandial intestinal transit measurements using carmine red. Carbachol (50 µM)-induced mI was strongly inhibited by Pico145 starting from 1 pM. The IC value for the inhibitory effect of Pico145 on this current evoked by intracellularly applied GTPγS (200 µM), and thus lacking desensitisation, was found to be 3.1 pM, while carbachol-induced intracellular Ca rises were inhibited with IC of 2.7 pM. In contrast, the current activated by direct TRPC4 agonist (-)-englerin A was less sensitive to the action of Pico145 that caused only ∼43 % current inhibition at 100 pM. The inhibitory effect developed rather slowly and it was potentiated by membrane depolarisation. In functional assays, Pico145 produced concentration-dependent suppression of both spontaneous and carbachol-evoked intestinal smooth muscle contractions and delayed postprandial intestinal transit. Thus, Pico145 is a potent GI-active small-molecule which completely inhibits mI at picomolar concentrations and which is as effective as trpc4 gene deficiency in in vivo intestinal motility tests.
Topics: Animals; Male; Mice; Carbachol; Gastrointestinal Motility; Myocytes, Smooth Muscle; Receptors, Muscarinic; TRPC Cation Channels
PubMed: 37857250
DOI: 10.1016/j.biopha.2023.115672 -
Anesthesiology Jun 2000Neuronal excitability is in part determined by Ca2+ availability that is controlled by regulatory mechanisms of cytosolic Ca2+ ([Ca2+]cyt). Alteration of any of those...
BACKGROUND
Neuronal excitability is in part determined by Ca2+ availability that is controlled by regulatory mechanisms of cytosolic Ca2+ ([Ca2+]cyt). Alteration of any of those mechanisms by volatile anesthetics (VAs) may lead to a change in presynaptic transmission and postsynaptic excitability. Using a human neuroblastoma cell line, the effects of halothane and isoflurane on cytosolic Ca2+ concentration ([Ca2+]cyt) in response to K+ and carbachol stimulation were investigated.
METHODS
Volatile anesthetic (0.05-1 mm) action on stimulated [Ca2+]cyt transients were monitored in suspensions of SH-SY5Y cells loaded with fura-2. Potassium chloride (KCl; 100 mm) was used to depolarize and activate Ca2+ entry through voltage-dependent calcium channels; 1 mm carbachol was used to activate muscarinic receptor-mediated inositol triphosphate (IP3)-dependent intracellular Ca2+ release. Sequential stimulations, KCl followed by carbachol and vice versa, were used to investigate interactions between intracellular Ca2+ stores.
RESULTS
Halothane and isoflurane in clinically relevant concentrations enhanced the K+-evoked [Ca2+]cyt transient whether intracellular Ca2+ stores were full or partially depleted. In contrast, halothane and isoflurane reduced the carbachol-evoked [Ca2+]cyt transient when the intracellular Ca2+ stores were full but had no effect when the Ca2+ stores were partially depleted by KCl stimulation.
CONCLUSIONS
Volatile anesthetics acted on sites that differently affect the K+- and carbachol-evoked [Ca2+]cyt transients. These data suggest the involvement of an intracellular Ca2+ translocation from the caffeine-sensitive Ca2+ store to the inositol triphosphate-sensitive Ca2+ store that was altered by halothane and isoflurane.
Topics: Anesthetics, Inhalation; Calcium; Carbachol; Cytosol; Electric Stimulation; Electrophysiology; Fluorescent Dyes; Fura-2; Halothane; Humans; Isoflurane; Membrane Potentials; Muscarinic Agonists; Potassium Chloride; Tumor Cells, Cultured
PubMed: 10839927
DOI: 10.1097/00000542-200006000-00035 -
Physiological Reports Mar 2020This study associates cholinergic stimulation of the pancreatic β-cell electrical activity with a short-term memory phenomenon. Glucose pulses applied to a basal...
This study associates cholinergic stimulation of the pancreatic β-cell electrical activity with a short-term memory phenomenon. Glucose pulses applied to a basal glucose concentration induce depolarizing waves which are used to estimate the evolution of the β-cell glucose sensitivity. Exposure to carbamoylcholine (carbachol) increases the size of the glucose-induced depolarizing waves. This change appears after carbachol withdrawal and implies a temporal potentiation of sensitivity (TPS) lasting up to one hour. TPS induction requires the simultaneous action of carbachol and glucose. The substitution of glucose with the secretagogues glyceraldehyde or 2-ketoisocaproate mimics glucose-induced TPS, while palmitate does not. TPS is not produced if the membrane is kept hyperpolarized by diazoxide. Glucose can be replaced by tolbutamide, suggesting a role of depolarization and a subsequent increase in intracellular calcium concentration. A role for kinases is suggested because staurosporine prevents TPS induction. Cycloheximide does not impair TPS induction, indicating that de novo protein synthesis is not required. The fact that the two inputs acting simultaneously produce an effect that lasts up to one hour without requiring de novo protein synthesis suggests that TPS constitutes a case of short-term associative conditioning in non-neural tissue. The convergence of basal glucose levels and muscarinic activation happens physiologically during the cephalic phase of digestion, in order to later absorb incoming fuels. Our data reveals that the role of the cephalic phase may be extended, increasing nutrient sensitivity during meals while remaining low between them.
Topics: Adaptation, Physiological; Animals; Carbachol; Cholinergic Agonists; Glucose; Insulin-Secreting Cells; Membrane Potentials; Mice
PubMed: 32232927
DOI: 10.14814/phy2.14403 -
Cell Calcium May 2022PGE is a potent bronchodilator, but the mechanisms underlying this effect have not been fully elucidated. Acetylcholine-induced contractions of airway smooth muscle...
PGE is a potent bronchodilator, but the mechanisms underlying this effect have not been fully elucidated. Acetylcholine-induced contractions of airway smooth muscle (ASM) are associated with the generation of repetitive Ca oscillations in airway smooth muscle cells (ASMC) and the force of contraction is positively correlated with the frequency of the underlying Ca oscillations. The purpose of the present study was to examine if carbachol-evoked Ca oscillations in isolated ASMC were inhibited by PGE. Isolated murine ASMC loaded with fluo4-AM were imaged with a Nipkow spinning disk confocal microscope. Cells responded to application of CCh (1 μM) by generating an initial Ca transient followed by a series of Ca oscillations. This activity was abolished by PGE (300 nM) and the EPR agonist (R)-butaprost (3 μM) and the inhibitory effects of PGE were reversed by application of the EPR antagonist PF-04418948 (100 nM). Activation of adenylate cyclase using forskolin (1 μM) mimicked the effects of PGE. The PKA activator, 6-MB-cAMP (300 μM) reduced the frequency of CCh-induced Ca oscillations by 33% and the PKA inhibitor Rp-8-CPT-cAMPs partially reversed the inhibitory effects of PGE. The EPAC activator 007-AM (10 μM) reduced the frequency of the oscillations by 60% and joint application of 007-AM and 6-MB-cAMP reduced oscillation frequency by ∼85%. CCh-induced Ca oscillations were inhibited by 2-APB and tetracaine, but caffeine-evoked Ca transients were resistant to PGE. These data suggest that PGE inhibits CCh-induced Ca oscillations in murine ASMC via stimulation of EPRs and a mechanism involving activation of PKA and EPAC.
Topics: Animals; Calcium; Carbachol; Colforsin; Dinoprostone; Mice; Muscle Contraction; Muscle, Smooth; Myocytes, Smooth Muscle
PubMed: 35134593
DOI: 10.1016/j.ceca.2022.102547 -
BMC Developmental Biology Oct 2008The hypothalamic-neurohypophysial system plays a fundamental role in the maintenance of body fluid homeostasis by secreting arginine vasopressin (AVP) and oxytocin (OT)...
BACKGROUND
The hypothalamic-neurohypophysial system plays a fundamental role in the maintenance of body fluid homeostasis by secreting arginine vasopressin (AVP) and oxytocin (OT) in response to a variety of signals, including osmotic and nonosmotic stimuli. It is well established that central cholinergic mechanisms are critical in the regulation of cardiovascular responses and maintenance of body fluid homeostasis in adults. Our recent study demonstrated that intracerebroventricular (i.c.v.) injection of carbachol elicited an increase of blood pressure in the near-term ovine fetuses. However, in utero development of brain cholinergic mechanisms in the regulation of the hypothalamic neuropeptides is largely unknown. This study investigated AVP and OT neural activation in the fetal hypothalamus induced by central carbachol.
RESULTS
Chronically prepared near-term ovine fetuses (0.9 gestation) received an i.c.v. carbachol (3 microg/kg). Fetal blood samples were collected for AVP and OT assay, and brains were used for c-fos mapping studies. I.c.v. carbachol significantly increased fetal plasma AVP and OT concentrations. Intense FOS immunoreactivity (FOS-ir) was observed in the fetal supraoptic nuclei (SON) and paraventricular nuclei (PVN) in the hypothalamus. Double labeling demonstrated that a number of AVP- and OT-containing neurons in the fetal SON and PVN were expressing c-fos in response to central carbachol.
CONCLUSION
The results indicate that the central cholinergic mechanism is established and functional in the regulation of the hypothalamic neuropeptides during the final trimester of pregnancy. This provides evidence for a functional link between the development of central cholinergic mechanisms and hypothalamic neuropeptide systems in the fetus.
Topics: Acetylcholine; Animals; Arginine Vasopressin; Carbachol; Cholinergic Agonists; Female; Fetus; Injections, Intraventricular; Oxytocin; Pregnancy; Sheep; Signal Transduction
PubMed: 18828925
DOI: 10.1186/1471-213X-8-95 -
Journal of Applied Physiology... Apr 2019We tested the hypothesis that oscillatory airway smooth muscle (ASM) mechanics is governed by mechanosensitive energy loss and energy release elements that can be...
We tested the hypothesis that oscillatory airway smooth muscle (ASM) mechanics is governed by mechanosensitive energy loss and energy release elements that can be recruited by prestrain and cholinergic stimulation. We measured mechanical energy loss and mechanical energy release in unstimulated and carbachol-stimulated bovine ASM held at prestrains ranging from 0.3 to 1.0 L (reference length) and subjected to sinusoidal length oscillation at 1 hz with oscillatory strain amplitudes ranging from 0.1 to 1.5% L. We found that oscillatory ASM mechanics during sinusoidal length oscillation is governed predominantly by one class of nonlinear mechanosensitive energy loss element and one class of nonlinear mechanosensitive energy release element with differential mechanosensitivities to oscillatory strain amplitude. The greater mechanosensitivity of the energy loss element than energy release element may explain the bronchodilatory effect of deep inspiration. Prestrain, an important determinant of ASM responsiveness, differentially increased energy loss and energy release in unstimulated and carbachol-stimulated ASM. Cholinergic stimulation, an important cause of bronchoconstriction and airway inflammation, also differentially increased energy loss and energy release. When prestrain and cholinergic stimulation were combined, we found that prestrain and cholinergic stimulation synergistically increased energy loss and energy release by ASM. The relationship between recruitment of energy loss elements and recruitment of energy release elements was nonlinear, suggesting that energy loss and energy release elements are not coupled in ASM cells. These findings imply that large lung volume and cholinergic ASM activation would synergistically increase mechanical energy expenditure during inspiration and mechanical recoil of ASM during expiration. NEW & NOTEWORTHY We report for the first time that oscillatory airway smooth muscle mechanics is governed predominantly by one class of nonlinear mechanosensitive energy loss element and one class of nonlinear mechanosensitive energy release element with differential mechanosensitivities to oscillatory strain amplitude. Prestrain and cholinergic stimulation synergistically and differentially recruit energy loss and energy release elements. The greater mechanosensitivity of the energy loss element than the energy release element may explain the bronchodilatory effect of deep inspiration.
Topics: Animals; Bronchoconstriction; Carbachol; Cattle; Cholinergic Agents; Lung; Muscle Contraction; Muscle, Smooth; Receptors, Cholinergic; Trachea
PubMed: 30653417
DOI: 10.1152/japplphysiol.01008.2018 -
Cell Biology and Toxicology Aug 2023Awareness is growing that, besides several neurotoxic effects, cholinomimetic drugs able to interfere the cholinergic neurotransmitter system may exert a teratogen...
Awareness is growing that, besides several neurotoxic effects, cholinomimetic drugs able to interfere the cholinergic neurotransmitter system may exert a teratogen effect in developing embryos of vertebrate and invertebrate organisms. Cholinomimetic substances exert their toxic activity on organisms as they inhibit the functionality of the cholinergic system by completely or partially replacing the ACh molecule both at the level of the AChE active site and at the level of acetylcholine receptors. In this work, we focused the attention on the effects of muscarinic antagonist (atropine) and agonist (carbachol) drugs during the early development and ontogenesis of chick embryos. An unsteady-state mathematical model of the drug release and fate was developed, to synchronize exposure to a gradient of drug concentrations with the different developmental events. Since concentration measures in time and space cannot be taken without damaging the embryo itself, the diffusion model was the only way to establish at each time-step the exact concentration of drug at the different points of the embryo body (considered two-dimensional up to the 50 h stage). This concentration depends on the distance and position of the embryo with respect to the releasing source. The exposure to carbachol generally enhanced dimensions and stages of the embryos, while atropine mainly caused delay in development and small size of the embryos. Both the drugs were able to cause developmental anomalies, depending on the moment of development, in a time- and dose-dependent way, regardless the expression of genes driving each event. 1. Early chick embryos were exposed to muscarinic drugs in a spatial-temporal context. 2. Effects were stage-(time) dependent, according to distance and position of the source. 3. Atropine inhibited growth, mainly interfering with the cephalic process formation and heart differentiation; carbachol increased growth reducing differentiation. 4. Interferences may be exerted by alteration of calcium responses to naturally occurring morphogen-driven mechanisms.
Topics: Animals; Chick Embryo; Carbachol; Receptors, Muscarinic; Cholinergic Agents; Atropine; Models, Theoretical
PubMed: 36098822
DOI: 10.1007/s10565-022-09770-w -
World Journal of Gastroenterology Apr 2011To investigate the effect of carbachol on gastrointestinal function in a dog model of oral resuscitation for burn shock.
AIM
To investigate the effect of carbachol on gastrointestinal function in a dog model of oral resuscitation for burn shock.
METHODS
Twenty Beagle dogs with intubation of the carotid artery, jugular vein and jejunum for 24 h were subjected to 35% total body surface area full-thickness burns, and were divided into three groups: no fluid resuscitation (NR, n = 10), in which animals did not receive fluid by any means in the first 24 h post-burn; oral fluid resuscitation (OR, n = 8), in which dogs were gavaged with glucose-electrolyte solution (GES) with volume and rate consistent with the Parkland formula; and oral fluid with carbachol group (OR/CAR, n = 8), in which dogs were gavaged with GES containing carbachol (20 μg/kg), with the same volume and rate as the OR group. Twenty-four hours after burns, all animals were given intravenous fluid replacement, and 72 h after injury, they received nutritional support. Hemodynamic and gastrointestinal parameters were measured serially with animals in conscious and cooperative state.
RESULTS
The mean arterial pressure, cardiac output and plasma volume dropped markedly, and gastrointestinal tissue perfusion was reduced obviously after the burn injury in all the three groups. Hemodynamic parameters and gastrointestinal tissue perfusion in the OR and OR/CAR groups were promoted to pre-injury level at 48 and 72 h, respectively, while hemodynamic parameters in the NR group did not return to pre-injury level till 72 h, and gastrointestinal tissue perfusion remained lower than pre-injury level until 120 h post-burn. CO(2) of the gastric mucosa and intestinal mucosa blood flow of OR/CAR groups were 56.4 ± 4.7 mmHg and 157.7 ± 17.7 blood perfusion units (BPU) at 24 h post-burn, respectively, which were significantly superior to those in the OR group (65.8 ± 5.8 mmHg and 127.7 ± 11.9 BPU, respectively, all P < 0.05). Gastric emptying and intestinal absorption rates of GES were significantly reduced to the lowest level (52.8% and 23.7% of pre-injury levels) in the OR group at about 2 and 4 h post-burn, and did not return to 80% of pre-injury level until 24 h. In the first 24 h post-burn, the rate of gastric emptying and intestinal water absorption were elevated by a mean 15.7% and 11.5%, respectively, in the OR/CAR group compared with the OR group. At 5 days, the mortality in the NR group was 30% (3/10), 12.5% in the OR group (1/8), and none in the OR/CAR group.
CONCLUSION
Carbachol had a beneficial effect on oral resuscitation of burn shock by promoting gastric emptying and intestinal absorption in our canine model.
Topics: Animals; Burns; Carbachol; Cholinergic Agonists; Dogs; Fluid Therapy; Gastrointestinal Tract; Hemodynamics; Intestinal Absorption; Models, Animal; Resuscitation; Shock
PubMed: 21483636
DOI: 10.3748/wjg.v17.i13.1746