ABSTRACT
Transient receptor potential vanilloid 1 (TRPV1) is a calcium-permeable ion channel that is gated by the pungent constituent of red chili pepper, capsaicin, and by related chemicals from the group of vanilloids, in addition to noxious heat. It is expressed mostly in sensory neurons to act as a detector of painful stimuli produced by pungent chemicals and high temperatures. Although TRPV1 is also found outside the sensory nervous system, its expression and function in the bladder detrusor smooth muscle (DSM) remain controversial. Here, by using Ca2+ imaging and patch clamp on isolated rat DSM cells, in addition to tensiometry on multicellular DSM strips, we show that TRPV1 is expressed functionally in only a fraction of DSM cells, in which it acts as an endoplasmic reticulum Ca2+-release channel responsible for the capsaicin-activated [Ca2+]i rise. Carbachol-stimulated contractions of multicellular DSM strips contain a TRPV1-dependent component, which is negligible in the circular DSM but reaches ≤50% in the longitudinal DSM. Activation of TRPV1 in rat DSM during muscarinic cholinergic stimulation is ensured by phospholipase A2-catalysed derivation of arachidonic acid and its conversion by lipoxygenases to eicosanoids, which act as endogenous TRPV1 agonists. Immunofluorescence detection of TRPV1 protein in bladder sections and isolated DSM cells confirmed both its preferential expression in the longitudinal DSM sublayer and its targeting to the endoplasmic reticulum. We conclude that TRPV1 is an essential contributor to the cholinergic contraction of bladder longitudinal DSM, which might be important for producing spatial and/or temporal anisotropy of bladder wall deformation in different regions during parasympathetic stimulation. KEY POINTS: The transient receptor potential vanilloid 1 (TRPV1) heat/capsaicin receptor/channel is localized in the endoplasmic reticulum membrane of detrusor smooth muscle (DSM) cells of the rat bladder, operating as a calcium-release channel. Isolated DSM cells are separated into two nearly equal groups, within which the cells either show or do not show TRPV1-dependent [Ca2+]i rise. Carbachol-stimulated, muscarinic ACh receptor-mediated contractions of multicellular DSM strips contain a TRPV1-dependent component. This component is negligible in the circular DSM but reaches ≤50% in longitudinal DSM. Activation of TRPV1 in rat DSM during cholinergic stimulation involves phospholipase A2-catalysed derivation of arachidonic acid and its conversion by lipoxygenases to eicosanoids, which act as endogenous TRPV1 agonists.
Subject(s)
Muscle Contraction , Muscle, Smooth , TRPV Cation Channels , Urinary Bladder , Animals , TRPV Cation Channels/metabolism , Urinary Bladder/physiology , Urinary Bladder/drug effects , Urinary Bladder/metabolism , Muscle Contraction/physiology , Muscle, Smooth/physiology , Muscle, Smooth/drug effects , Muscle, Smooth/metabolism , Rats , Male , Carbachol/pharmacology , Capsaicin/pharmacology , Calcium/metabolism , Rats, Sprague-Dawley , Rats, WistarABSTRACT
The intestinal epithelium is covered by mucus that protects the tissue from the luminal content. Studies have shown that anion secretion via the cystic fibrosis conductance regulator (Cftr) regulates mucus formation in the small intestine. However, mechanisms regulating mucus formation in the colon are less understood. The aim of this study was to explore the role of anion transport in the regulation of mucus formation during steady state and in response to carbamylcholine (CCh) and prostaglandin E2 (PGE2). The broad-spectrum anion transport inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS), CftrdF508 (CF) mice, and the slc26a3 inhibitor SLC26A3-IN-2 were used to inhibit anion transport. In the distal colon, steady-state mucus expansion was reduced by SLC26A3-IN-2 and normal in CF mice. PGE2 stimulated mucus expansion without de novo mucus release in wild type (WT) and CF colon via slc26a3 sensitive mechanisms, while CCh induced de novo mucus secretion in WT but not in CF colon. However, when added simultaneously, CCh and PGE2 stimulated de novo mucus secretion in the CF colon via DIDS-sensitive pathways. A similar response was observed in CF ileum that responded to CCh and PGE2 with DIDS-sensitive de novo mucus secretion. In conclusion, this study suggests that slc26a3 regulates colonic mucus expansion, while Cftr regulates CCh-induced de novo mucus secretion from ileal and distal colon crypts. Furthermore, these findings demonstrate that in the absence of a functional Cftr channel, parallel stimulation with CCh and PGE2 activates additional anion transport processes that help release mucus from intestinal goblet cells.
Subject(s)
Carbachol , Colon , Cystic Fibrosis Transmembrane Conductance Regulator , Dinoprostone , Mucus , Sulfate Transporters , Animals , Dinoprostone/metabolism , Dinoprostone/pharmacology , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Sulfate Transporters/metabolism , Sulfate Transporters/genetics , Mice , Colon/metabolism , Colon/drug effects , Mucus/metabolism , Mucus/drug effects , Carbachol/pharmacology , Mice, Inbred C57BL , 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid/pharmacology , Antiporters/metabolism , Intestinal Mucosa/metabolism , Intestinal Mucosa/drug effects , MaleABSTRACT
Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) have found utility for conducting in vitro drug screening and disease modelling to gain crucial insights into pharmacology or disease phenotype. However, diseases such as atrial fibrillation, affecting >33 M people worldwide, demonstrate the need for cardiac subtype-specific cells. Here, we sought to investigate the base characteristics and pharmacological differences between commercially available chamber-specific atrial or ventricular hiPSC-CMs seeded onto ultra-thin, flexible PDMS membranes to simultaneously measure contractility in a 96 multi-well format. We investigated the effects of GPCR agonists (acetylcholine and carbachol), a Ca2+ channel agonist (S-Bay K8644), an HCN channel antagonist (ivabradine) and K+ channel antagonists (4-AP and vernakalant). We observed differential effects between atrial and ventricular hiPSC-CMs on contractile properties including beat rate, beat duration, contractile force and evidence of arrhythmias at a range of concentrations. As an excerpt of the compound analysis, S-Bay K8644 treatment showed an induced concentration-dependent transient increase in beat duration of atrial hiPSC-CMs, whereas ventricular cells showed a physiological increase in beat rate over time. Carbachol treatment produced marked effects on atrial cells, such as increased beat duration alongside a decrease in beat rate over time, but only minimal effects on ventricular cardiomyocytes. In the context of this chamber-specific pharmacology, we not only add to contractile characterization of hiPSC-CMs but propose a multi-well platform for medium-throughput early compound screening. Overall, these insights illustrate the key pharmacological differences between chamber-specific cardiomyocytes and their application on a multi-well contractility platform to gain insights for in vitro cardiac liability studies and disease modelling.
Subject(s)
Heart Atria , Heart Ventricles , Induced Pluripotent Stem Cells , Myocardial Contraction , Myocytes, Cardiac , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/physiology , Humans , Induced Pluripotent Stem Cells/drug effects , Heart Atria/drug effects , Heart Atria/cytology , Myocardial Contraction/drug effects , Myocardial Contraction/physiology , Heart Ventricles/drug effects , Heart Ventricles/cytology , Receptors, G-Protein-Coupled/metabolism , Receptors, G-Protein-Coupled/agonists , Drug Development/methods , Ion Channels/drug effects , Cells, Cultured , Drug Evaluation, Preclinical/methods , Carbachol/pharmacology , Microphysiological SystemsABSTRACT
We studied the inhibitory actions of docosahexaenoic acid (DHA) on the contractions induced by carbachol (CCh), angiotensin II (Ang II), and bradykinin (BK) in guinea pig (GP) gastric fundus smooth muscle (GFSM), particularly focusing on the possible inhibition of store-operated Ca2+ channels (SOCCs). DHA significantly suppressed the contractions induced by CCh, Ang II, and BK; the inhibition of BK-induced contractions was the strongest. Although all contractions were greatly dependent on external Ca2+, more than 80% of BK-induced contractions remained even in the presence of verapamil, a voltage-dependent Ca2+ channel inhibitor. BK-induced contractions in the presence of verapamil were not suppressed by LOE-908 (a receptor-operated Ca2+ channel (ROCC) inhibitor) but were suppressed by SKF-96365 (an SOCC and ROCC inhibitor). BK-induced contractions in the presence of verapamil plus LOE-908 were strongly inhibited by DHA. Furthermore, DHA inhibited GFSM contractions induced by cyclopiazonic acid (CPA) in the presence of verapamil plus LOE-908 and inhibited the intracellular Ca2+ increase due to Ca2+ addition in CPA-treated 293T cells. These findings indicate that Ca2+ influx through SOCCs plays a crucial role in BK-induced contraction in GP GFSM and that this inhibition by DHA is a new mechanism by which this fatty acid inhibits GFSM contractions.
Subject(s)
Angiotensin II , Bradykinin , Carbachol , Docosahexaenoic Acids , Gastric Fundus , Muscle Contraction , Muscle, Smooth , Animals , Guinea Pigs , Docosahexaenoic Acids/pharmacology , Bradykinin/pharmacology , Muscle, Smooth/drug effects , Muscle, Smooth/physiology , Muscle, Smooth/metabolism , Carbachol/pharmacology , Muscle Contraction/drug effects , Angiotensin II/pharmacology , Gastric Fundus/drug effects , Gastric Fundus/physiology , Gastric Fundus/metabolism , Verapamil/pharmacology , Calcium/metabolism , Male , Humans , Calcium Channels/metabolism , HEK293 Cells , Calcium Channel Blockers/pharmacology , Imidazoles/pharmacologyABSTRACT
High serum estrogen concentrations are associated with asthma development and severity, suggesting a link between estradiol and airway hyperresponsiveness (AHR). 17ß-estradiol (E2) has non-genomic effects via Ca2+ regulatory mechanisms; however, its effect on the plasma membrane Ca2+-ATPases (PMCA1 and 4) and sarcoplasmic reticulum Ca2+-ATPase (SERCA) is unknown. Hence, in the present study, we aim to demonstrate if E2 favors AHR by increasing intracellular Ca2+ concentrations in guinea pig airway smooth muscle (ASM) through a mechanism involving Ca2+-ATPases. In guinea pig ASM, Ca2+ microfluorometry, muscle contraction, and Western blot were evaluated. Then, we performed molecular docking analysis between the estrogens and Ca2+ ATPases. In tracheal rings, E2 produced AHR to carbachol. In guinea pig myocytes, acute exposure to physiological levels of E2 modified the transient Ca2+ peak induced by caffeine to a Ca2+ plateau. The incubation with PMCA inhibitors (lanthanum and carboxyeosin, CE) partially reversed the E2-induced sustained plateau in the caffeine response. In contrast, cyclopiazonic acid (SERCA inhibitor), U-0126 (an inhibitor of ERK 1/2), and choline chloride did not modify the Ca2+ plateau produced by E2. The mitochondrial uniporter activity and the capacitative Ca2+ entry were unaffected by E2. In guinea pig ASM, Western blot analysis demonstrated PMCA1 and PMCA4 expression. The results from the docking modeling demonstrate that E2 binds to both plasma membrane ATPases. In guinea pig tracheal smooth muscle, inhibiting the PMCA with CE, induced hyperresponsiveness to carbachol. 17ß-estradiol produces hyperresponsiveness by inhibiting the PMCA in the ASM and could be one of the mechanisms responsible for the increase in asthmatic crisis in women.
Subject(s)
Calcium , Estradiol , Molecular Docking Simulation , Plasma Membrane Calcium-Transporting ATPases , Animals , Guinea Pigs , Estradiol/pharmacology , Plasma Membrane Calcium-Transporting ATPases/metabolism , Calcium/metabolism , Muscle, Smooth/drug effects , Muscle, Smooth/metabolism , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Male , Trachea/drug effects , Trachea/metabolism , Muscle Contraction/drug effects , Respiratory Hypersensitivity/chemically induced , Respiratory Hypersensitivity/metabolism , Cell Membrane/drug effects , Cell Membrane/metabolism , Carbachol/pharmacology , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/metabolismABSTRACT
Muscarinic neurotransmission is fundamentally involved in supporting several brain functions by modulating flow of information in brain neural circuits including the hippocampus which displays a remarkable functional segregation along its longitudinal axis. However, how muscarinic neuromodulation contributes to the functional segregation along the hippocampus remains unclear. In this study we show that the nonselective muscarinic receptor agonist carbachol similarly suppresses basal synaptic transmission in the dorsal and ventral CA1 hippocampal field, in a concentration-depended manner. Furthermore, using a ten-pulse stimulation train of varying frequency we found that carbachol changes the frequency filtering properties more in ventral than dorsal hippocampus by facilitating synaptic inputs at a wide range of input frequencies in the ventral compared with dorsal hippocampus. Using the M2 receptor antagonist gallamine and the M4 receptor antagonist tropicamide, we found that M2 receptors are involved in controlling basal synaptic transmission and short-term synaptic plasticity (STSP) in the ventral but not the dorsal hippocampus, while M4 receptors participate in modulating basal synaptic transmission and STSP in both segments of the hippocampus. These results were corroborated by the higher protein expression levels of M2 receptors in the ventral compared with dorsal hippocampus. We conclude that muscarinic transmission modulates excitatory synaptic transmission and short-term synaptic plasticity along the entire rat hippocampus by acting through M4 receptors and recruiting M2 receptors only in the ventral hippocampus. Furthermore, M4 receptors appear to exert a permissive role on the actions of M2 receptors on STSP in the ventral hippocampus. This dorsoventral differentiation of muscarinic modulation is expected to have important implications in information processing along the endogenous hippocampal circuitry.
Subject(s)
Hippocampus , Neuronal Plasticity , Synaptic Transmission , Animals , Neuronal Plasticity/physiology , Neuronal Plasticity/drug effects , Synaptic Transmission/physiology , Synaptic Transmission/drug effects , Rats , Hippocampus/metabolism , Hippocampus/drug effects , Hippocampus/physiology , Male , Carbachol/pharmacology , Receptor, Muscarinic M2/metabolism , Receptors, Muscarinic/metabolism , Rats, Wistar , Muscarinic Antagonists/pharmacology , Receptor, Muscarinic M4/metabolism , Muscarinic Agonists/pharmacology , Excitatory Postsynaptic Potentials/physiology , Excitatory Postsynaptic Potentials/drug effectsABSTRACT
Selective activation of individual subtypes of muscarinic receptors is a promising way to safely alleviate a wide range of pathological conditions in the central nervous system and the periphery as well. The flexible G-protein interface of muscarinic receptors allows them to interact with several G-proteins with various efficacy, potency, and kinetics. Agonists biased to the particular G-protein mediated pathway may result in selectivity among muscarinic subtypes and, due to the non-uniform expression of individual G-protein alpha subunits, possibly achieve tissue specificity. Here, we demonstrate that novel tetrahydropyridine-based agonists exert specific signalling profiles in coupling with individual G-protein α subunits. These signalling profiles profoundly differ from the reference agonist carbachol. Moreover, coupling with individual Gα induced by these novel agonists varies among subtypes of muscarinic receptors which may lead to subtype selectivity. Thus, the novel tetrahydropyridine-based agonist can contribute to the elucidation of the mechanism of pathway-specific activation of muscarinic receptors and serve as a starting point for the development of desired selective muscarinic agonists.
Subject(s)
Muscarinic Agonists , Receptors, Muscarinic , Muscarinic Agonists/pharmacology , Receptors, Muscarinic/metabolism , Animals , Signal Transduction/drug effects , Humans , Pyridines/pharmacology , Carbachol/pharmacology , CHO Cells , Cricetulus , GTP-Binding Proteins/metabolism , GTP-Binding Protein alpha Subunits/metabolism , GTP-Binding Protein alpha Subunits/geneticsABSTRACT
INTRODUCTION: Hyoscine butylbromide (HBB) is one of the most used antispasmodics in clinical practice. Recent translational consensus has demonstrated a similarity between human colonic motor patterns studied ex vivo and in vivo, suggesting ex vivo can predict in vivo results. It is unclear whether the mechanism of action of antispasmodics can predict different use in clinical practice. The aim of the present study is to bridge this gap dissecting HBB's role in excitatory and inhibitory neural pathways. METHODS: 309 colon samples from 48 patients were studied in muscle bath experiments. HBB was tested on: 1-spontaneous phasic contractions (SPCs); 2-carbachol-induced contractility; electrical field stimulation (EFS)-induced selective stimulation of 3-excitatory and 4-inhibitory pathways and 5- SPCs and EFS-induced contractions enhanced by neostigmine. Atropine, AF-DX116 (M2 blocker) and DAU-5884 (M3 blocker) were used as comparators. RESULTS: In the presence of tetrodotoxin (TTX), HBB and atropine 1 µM reduced SPCs. HBB and atropine concentration-dependently reduced carbachol- and EFS-induced contractions. Inhibitory effects of DAU-5884 on EFS-induced contractions were more potent than of AF-DX116. HBB did not affect the off-response associated to neural inhibitory responses. Neostigmine enhanced both SPCs and EFS-induced contractions. In the presence of TTX and ω-conotoxin (GVIA), neostigmine still enhanced SPCs. Addition of HBB and atropine reduced these responses. CONCLUSIONS: This study demonstrates that HBB inhibits neural cholinergic contractions associated to muscarinic (mainly M3) receptors. HBB has a potential role in reducing colonic spasm induced by the release of acetylcholine from enteric motor neurons and from an atypical source including a potential non-neuronal origin.
Subject(s)
Butylscopolammonium Bromide , Colon , Muscle Contraction , Humans , Butylscopolammonium Bromide/pharmacology , Colon/drug effects , Colon/physiology , Male , Female , Muscle Contraction/drug effects , Middle Aged , Aged , Electric Stimulation , Adult , Carbachol/pharmacology , Parasympatholytics/pharmacology , Aged, 80 and over , In Vitro TechniquesABSTRACT
OBJECTIVES: Local anesthetics act on G protein-coupled receptors (GPCRs); thus, their potential as allosteric modulators of GPCRs has attracted attention. Intracellular signaling via GPCRs involves both G-protein- and ß-arrestin-mediated pathways. To determine the effects of local anesthetics on muscarinic acetylcholine receptors (mAChR), a family of GPCRs, we analyzed the effects of local anesthetics on mAChR-mediated Ca2+ responses and formation of receptor-ß-arrestin complexes in the HSY human parotid cell line. METHODS: Ca2+ responses were monitored by fura-2 spectrofluorimetry. Ligand-induced interactions between mAChR and ß-arrestin were examined using a ß-arrestin GPCR assay kit. RESULTS: Lidocaine reduced mAChR-mediated Ca2+ responses but did not change the intracellular Ca2+ concentration in non-stimulated cells. The membrane-impermeant lidocaine analog QX314 and procaine inhibited mAChR-mediated Ca2+ responses, with EC50 values of 48.0 and 20.4 µM, respectively, for 50 µM carbachol-stimulated Ca2+ responses. In the absence of extracellular Ca2+, the pretreatment of cells with QX314 reduced carbachol-induced Ca2+ release, indicating that QX314 reduced Ca2+ release from intracellular stores. Lidocaine and QX314 did not affect store-operated Ca2+ entry as they did not alter the thapsigargin-induced Ca2+ response. QX314 and procaine reduced the carbachol-mediated recruitment of ß-arrestin, and administration of procaine suppressed pilocarpine-induced salivary secretion in mice. CONCLUSION: Local anesthetics, including QX314, act on mAChR to reduce carbachol-induced Ca2+ release from intracellular stores and the recruitment of ß-arrestin. These findings support the notion that local anesthetics and their derivatives are starting points for the development of functional allosteric modulators of mAChR.
Subject(s)
Anesthetics, Local , Calcium , Lidocaine , Parotid Gland , Receptors, Muscarinic , beta-Arrestins , Humans , Anesthetics, Local/pharmacology , beta-Arrestins/metabolism , Calcium/metabolism , Receptors, Muscarinic/metabolism , Receptors, Muscarinic/drug effects , Animals , Mice , Parotid Gland/drug effects , Parotid Gland/metabolism , Lidocaine/pharmacology , Lidocaine/analogs & derivatives , Cell Line , Carbachol/pharmacology , Calcium Signaling/drug effects , Procaine/pharmacologyABSTRACT
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.
Subject(s)
Shock, Hemorrhagic , Animals , Dogs , Female , Carbachol/pharmacology , Hemorrhage , Iloprost/therapeutic use , Microcirculation , Nitroglycerin/pharmacology , Nitroglycerin/therapeutic use , Shock, Hemorrhagic/drug therapyABSTRACT
OBJECTIVES: Neuropathic pain is characterized by long-lasting, intractable pain. Sciatic nerve ligation is often used as an animal model of neuropathic pain, and the spared nerve injury (SNI) model, in which the common peroneal nerve (CPN) and tibial nerve (TN) are ligated, is widely used. In the present study, we evaluated the analgesic effect of a cholinergic agonist, carbachol, on a neuropathic pain model prepared by sural nerve (SN) ligation in mice. METHODS: The SN was tightly ligated as a branch of the sciatic nerve. Mechanical and thermal allodynia, and hyperalgesia were assessed using von Frey filaments and heat from a hot plate. The analgesic effects of intracerebroventricularly-administered morphine and carbachol were compared. RESULTS: SN ligation resulted in a significant decrease in pain threshold for mechanical stimulation 1 day after ligation. In response to thermal stimulation, allodynia was observed at 50°C and hyperalgesia at 53 and 56°C 3 days after ligation. Content of thiobarbituric acid reactive substances (TBARS) in the spinal cord increased significantly at 6 and 12 h after ligation. Acetylcholine content of the spinal cord also increased at 5 and 7 days after ligation. Intracerebroventricular administration of carbachol at 7 days after ligation produced a marked analgesic effect against mechanical and thermal stimuli, which was stronger and longer-lasting than morphine at all experimental time points. CONCLUSION: These findings suggest that cholinergic nerves are involved in allodynia and hyperalgesia of the SN ligation neuropathic pain model.
Subject(s)
Carbachol , Disease Models, Animal , Hyperalgesia , Neuralgia , Sural Nerve , Animals , Hyperalgesia/drug therapy , Male , Neuralgia/drug therapy , Neuralgia/etiology , Carbachol/pharmacology , Ligation , Mice , Sural Nerve/drug effects , Cholinergic Agonists/pharmacology , Pain Threshold/drug effects , Morphine/pharmacology , Analgesics/pharmacology , Pain Measurement , Spinal Cord/drug effects , Acetylcholine/metabolismABSTRACT
Gonadotropin-releasing hormone (GnRH)-synthesizing neurons orchestrate reproduction centrally. Early studies have proposed the contribution of acetylcholine (ACh) to hypothalamic control of reproduction, although the causal mechanisms have not been clarified. Here, we report that in vivo pharmacogenetic activation of the cholinergic system increased the secretion of luteinizing hormone (LH) in orchidectomized mice. 3DISCO immunocytochemistry and electron microscopy revealed the innervation of GnRH neurons by cholinergic axons. Retrograde viral labeling initiated from GnRH-Cre neurons identified the medial septum and the diagonal band of Broca as exclusive sites of origin for cholinergic afferents of GnRH neurons. In acute brain slices, ACh and carbachol evoked a biphasic effect on the firing rate in GnRH neurons, first increasing and then diminishing it. In the presence of tetrodotoxin, carbachol induced an inward current, followed by a decline in the frequency of miniature postsynaptic currents (mPSCs), indicating a direct influence on GnRH cells. RT-PCR and whole-cell patch-clamp studies revealed that GnRH neurons expressed both nicotinic (α4ß2, α3ß4, and α7) and muscarinic (M1-M5) AChRs. The nicotinic AChRs contributed to the nicotine-elicited inward current and the rise in firing rate. Muscarine via M1 and M3 receptors increased, while via M2 and M4 reduced the frequency of both mPSCs and firing. Optogenetic activation of channelrhodopsin-2-tagged cholinergic axons modified GnRH neuronal activity and evoked cotransmission of ACh and GABA from a subpopulation of boutons. These findings confirm that the central cholinergic system regulates GnRH neurons and activates the pituitary-gonadal axis via ACh and ACh/GABA neurotransmissions in male mice.
Subject(s)
Acetylcholine , Gonadotropin-Releasing Hormone , Mice , Animals , Male , Acetylcholine/pharmacology , Carbachol/pharmacology , Neurons/physiology , Cholinergic Agents/pharmacology , Nicotine/pharmacology , Luteinizing Hormone , gamma-Aminobutyric Acid/pharmacologyABSTRACT
In salivary acinar cells, cholinergic stimulation induces elevations of cytosolic [Ca2+]i to activate the apical exit of Cl- through TMEM16A Cl- channels, which acts as a driving force for fluid secretion. To sustain the Cl- secretion, [Cl-]i must be maintained to levels that are greater than the electrochemical equilibrium mainly by Na+-K+-2Cl- cotransporter-mediated Cl- entry in basolateral membrane. Glucose transporters carry glucose into the cytoplasm, enabling the cells to produce ATP to maintain Cl- and fluid secretion. Sodium-glucose cotransporter-1 is a glucose transporter highly expressed in acinar cells. The salivary flow is suppressed by the sodium-glucose cotransporter-1 inhibitor phlorizin. However, it remains elusive how sodium-glucose cotransporter-1 contributes to maintaining salivary fluid secretion. To examine if sodium-glucose cotransporter-1 activity is required for sustaining Cl- secretion to drive fluid secretion, we analyzed the Cl- currents activated by the cholinergic agonist, carbachol, in submandibular acinar cells while comparing the effect of phlorizin on the currents between the whole-cell patch and the gramicidin-perforated patch configurations. Phlorizin suppressed carbachol-induced oscillatory Cl- currents by reducing the Cl- efflux dependent on the Na+-K+-2Cl- cotransporter-mediated Cl- entry in addition to affecting TMEM16A activity. Our results suggest that the sodium-glucose cotransporter-1 activity is necessary for maintaining the oscillatory Cl- secretion supported by the Na+-K+-2Cl- cotransporter activity in real time to drive fluid secretion. The concerted effort of sodium-glucose cotransporter-1, Na+-K+-2Cl- cotransporter, and apically located Cl- channels might underlie the efficient driving of Cl- secretion in different secretory epithelia from a variety of animal species.
Subject(s)
Acinar Cells , Phlorhizin , Animals , Mice , Acinar Cells/metabolism , Carbachol/pharmacology , Chlorides/metabolism , Glucose , Phlorhizin/pharmacology , Sodium/metabolism , Sodium-Potassium-Chloride SymportersABSTRACT
OBJECTIVES: Typical agonists of G protein-coupled receptors (GPCRs), including muscarinic acetylcholine receptors (mAChRs), activate both G-protein and ß-arrestin signaling systems, and are termed balanced agonists. In contrast, biased agonists selectively activate a single pathway, thereby offering therapeutic potential for the specific activation of that pathway. The mAChR agonists carbachol and pilocarpine are known to induce phosphorylation of extracellular signal-regulated kinase-1/2 (ERK1/2) via G-protein-dependent and -independent pathways, respectively. We investigated the involvement of ß-arrestin and its downstream mechanisms in the ERK1/2 phosphorylation induced by carbachol and pilocarpine in the human salivary ductal cell line, HSY cells. METHODS: HSY cells were stimulated with pilocarpine or carbachol, with or without various inhibitors. The cell lysates were analyzed by western blotting using the antibodies p44/p42MAPK and phosphor-p44/p42MAPK. RESULTS: Western blot analysis revealed that carbachol elicited greater stimulation of ERK1/2 phosphorylation compared to pilocarpine. ERK1/2 phosphorylation was inhibited by atropine and gefitinib, suggesting that mAChR activation induces transactivation of epidermal growth factor receptors (EGFR). Moreover, inhibition of carbachol-mediated ERK1/2 phosphorylation was achieved by GF-109203X (a PKC inhibitor), a ßARK1/GRK2 inhibitor, barbadin (a ß-arrestin inhibitor), pitstop 2 (a clathrin inhibitor), and dynole 34-2 (a dynamin inhibitor). In contrast, pilocarpine-mediated ERK1/2 phosphorylation was only inhibited by barbadin (a ß-arrestin inhibitor) and PP2 (a Src inhibitor). CONCLUSION: Carbachol activates both G-protein and ß-arrestin pathways, whereas pilocarpine exclusively activates the ß-arrestin pathway. Additionally, downstream of ß-arrestin, carbachol activates clathrin-dependent internalization, while pilocarpine activates Src.
Subject(s)
Carbachol , Muscarinic Agonists , Pilocarpine , Receptors, Muscarinic , Signal Transduction , Humans , Phosphorylation/drug effects , Receptors, Muscarinic/metabolism , Pilocarpine/pharmacology , Carbachol/pharmacology , Muscarinic Agonists/pharmacology , Signal Transduction/drug effects , Salivary Ducts/metabolism , beta-Arrestins/metabolism , Cell Line , Extracellular Signal-Regulated MAP Kinases/metabolism , Blotting, Western , Arrestins/metabolismABSTRACT
The vagal regulation of cardiac function involves acetylcholine (ACh) receptor activation followed by negative chronotropic and negative as well as positive inotropic effects. The resulting signaling pathways may include Gi/o protein-coupled reduction in adenylyl cyclase (AC) activity, direct Gi/o protein-coupled activation of ACh-activated potassium current (IKACh), inhibition of L-type calcium ion channels, and/or the activation of protein phosphatases. Here, we studied the role of the protein phosphatases 1 (PP1) and 2A (PP2A) for muscarinic receptor signaling in isolated atrial preparations of transgenic mice with cardiomyocyte-specific overexpression of either the catalytic subunit of PP2A (PP2A-TG) or the inhibitor-2 (I2) of PP1 (I2-TG) or in double transgenic mice overexpressing both PP2A and I2 (DT). In mouse left atrial preparations, carbachol (CCh), cumulatively applied (1 nM-10 µM), exerted at low concentrations a negative inotropic effect followed by a positive inotropic effect at higher concentrations. This biphasic effect was noted with CCh alone as well as when CCh was added after ß-adrenergic pre-stimulation with isoprenaline (1 µM). Whereas the response to stimulation of ß-adrenoceptors or adenosine receptors (used as controls) was changed in PP2A-TG, the response to CCh was unaffected in atrial preparations from all transgenic models studied here. Therefore, the present data tentatively indicate that neither PP2A nor PP1, but possibly other protein phosphatases, is involved in the muscarinic receptor-induced inotropic and chronotropic effects in the mouse heart.
Subject(s)
Carbachol , Heart Atria , Mice, Transgenic , Protein Phosphatase 1 , Protein Phosphatase 2 , Receptors, Muscarinic , Signal Transduction , Animals , Receptors, Muscarinic/metabolism , Heart Atria/metabolism , Heart Atria/drug effects , Protein Phosphatase 2/metabolism , Carbachol/pharmacology , Protein Phosphatase 1/metabolism , Mice , Male , Myocardial Contraction/drug effects , Myocardial Contraction/physiology , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Isoproterenol/pharmacology , Phosphoprotein Phosphatases/metabolism , Phosphoprotein Phosphatases/geneticsABSTRACT
ABSTRACT: Chronic stress induces a group of unrecognized cardiovascular impairments, including elevated hemodynamic variables and vascular dysfunction. Moreover, hydrogen sulfide (H 2 S), a gasotransmitter that regulates the cardiovascular system decreases under chronic stress. Thus, this study assessed the impact of sodium hydrosulfide (NaHS) (H 2 S donor) on chronic restraint stress (CRS)-induced cardiovascular changes. For that purpose, male Wistar rats were restrained for 2 hours a day in a transparent acrylic tube over 8 weeks. Then, body weight, relative adrenal gland weight, serum corticosterone, H 2 S-synthesizing enzymes, endothelial nitric oxide synthetize expression, reactive oxygen species levels, lipid peroxidation, and reduced glutathione-to-oxidized glutathione (GSH 2 :GSSG) ratio were determined in the thoracic aorta. The hemodynamic variables were measured in vivo by the plethysmograph method. The vascular function was evaluated in vitro as vasorelaxant responses induced by carbachol or sodium nitroprusside, and norepinephrine (NE)-mediated vasocontractile responses in the thoracic aorta. CRS increased (1) relative adrenal gland weight; (2) hemodynamic variables; (3) vasoconstrictor responses induced by NE, (4) reactive oxygen species levels, and (5) lipid peroxidation in the thoracic aorta. In addition, CRS decreased (1) body weight; (2) vasorelaxant responses induced by carbachol; (3) GSH content, and (4) GSH 2 :GSSG ratio. Notably, NaHS administration (5.6 mg/kg) restored hemodynamic variables and lipid peroxidation and attenuated the vasoconstrictor responses induced by NE in the thoracic aorta. In addition, NaHS treatment increased relative adrenal gland weight and the GSH 2 :GSSG ratio. Taken together, our results demonstrate that NaHS alleviates CRS-induced hypertension by reducing oxidative stress and restoring vascular function in the thoracic aorta.
Subject(s)
Hydrogen Sulfide , Sulfides , Rats , Animals , Male , Reactive Oxygen Species/metabolism , Glutathione Disulfide/metabolism , Glutathione Disulfide/pharmacology , Carbachol/pharmacology , Rats, Wistar , Hydrogen Sulfide/metabolism , Oxidative Stress , Vasoconstrictor Agents/pharmacology , Vasodilator Agents/pharmacology , Body WeightABSTRACT
Cholinergic innervation of the hippocampus correlates with memory formation. In a well-established animal model of type 1 diabetes mellitus, obtained by injecting young adult rats with streptozotocin (STZ), reductions have been reported in the expression of acetylcholine receptors and choline acetyltransferase. In this study, we showed that long-term synaptic depression (LTD) induced by carbachol (CCh), a nonselective cholinergic receptor agonist, at Schaffer collateral-CA1 synapses in hippocampal slices was significantly weaker in streptozotocin-induced diabetic rats (STZ rats) than in age-matched control rats. No significant change was observed in the paired-pulse ratio between before and 80 min after the application of CCh in control and STZ rats. Moreover, CCh-induced LTD in control and STZ rats was not affected by an NMDA receptor antagonist. Although the application of CCh down-regulated the surface expression of GluA2 in the hippocampus of control rats, but not STZ rats. Therefore, the present results suggest that acetylcholine receptor-mediated LTD in STZ rats requires the internalization of AMPA receptors on the postsynaptic surface and their intracellular effects in the hippocampus.
Subject(s)
Acetylcholine , Diabetes Mellitus, Experimental , Rats , Animals , Streptozocin , Acetylcholine/pharmacology , Receptors, Cholinergic , Depression , Hippocampus , Synapses , Long-Term Synaptic Depression , Carbachol/pharmacology , Long-Term PotentiationABSTRACT
AIM: Inositol 1,4,5-trisphosphate receptors (IP3 Rs) are intracellular Ca2+ -release channels with crucial roles in cell function. Current IP3 R inhibitors suffer from off-target effects and poor selectivity towards the three distinct IP3 R subtypes. We developed a novel peptide inhibitor of IP3 Rs and determined its effect on connexin-43 (Cx43) hemichannels, which are co-activated by IP3 R stimulation. METHODS: IP3RPEP6 was developed by in silico molecular docking studies and characterized by on-nucleus patch-clamp experiments of IP3 R2 channels and carbachol-induced IP3 -mediated Ca2+ responses in IP3 R1, 2 or 3 expressing cells, triple IP3 R KO cells and astrocytes. Cx43 hemichannels were studied by patch-clamp and ATP-release approaches, and by inhibition with Gap19 peptide. IP3RPEP6 interactions with IP3 Rs were verified by co-immunoprecipitation and affinity pull-down assays. RESULTS: IP3RPEP6 concentration-dependently reduced the open probability of IP3 R2 channels and competitively inhibited IP3 Rs in an IC50 order of IP3 R2 (~3.9 µM) < IP3 R3 (~4.3 µM) < IP3 R1 (~9.0 µM), without affecting Cx43 hemichannels or ryanodine receptors. IP3RPEP6 co-immunoprecipitated with IP3 R2 but not with IP3 R1; interaction with IP3 R3 varied between cell types. The IC50 of IP3RPEP6 inhibition of carbachol-induced Ca2+ responses decreased with increasing cellular Cx43 expression. Moreover, Gap19-inhibition of Cx43 hemichannels significantly reduced the amplitude of the IP3 -Ca2+ responses and strongly increased the EC50 of these responses. Finally, we identified palmitoyl-8G-IP3RPEP6 as a membrane-permeable IP3RPEP6 version allowing extracellular application of the IP3 R-inhibiting peptide. CONCLUSION: IP3RPEP6 inhibits IP3 R2/R3 at concentrations that have limited effects on IP3 R1. IP3 R activation triggers hemichannel opening, which strongly affects the amplitude and concentration-dependence of IP3 -triggered Ca2+ responses.
Subject(s)
Connexin 43 , Peptides , Molecular Docking Simulation , Carbachol/pharmacology , Peptides/pharmacology , Peptides/metabolism , Astrocytes/metabolismABSTRACT
Stress-induced antinociception (SIA) is due to the activation of several neural pathways and neurotransmitters that often suppress pain perception. Studies have shown that the orexin neuropeptide system is essential in pain modulation. Therefore, this study aimed to investigate the role of orexinergic receptors in the hippocampal CA1 region in modulating SIA response during the formalin test as an animal model of inflammatory pain. The orexin-1 receptor (OX1r) antagonist, SB334867, at 1, 3, 10, and 30 nmol or TCS OX2 29 as an orexin-2 receptor (OX2r) antagonist at the same doses were microinjected into the CA1 region in rats. Five minutes later, rats were exposed to restraint stress (RS) for 3â h, and pain-related behaviors were monitored in 5-min blocks for the 60-min test period in the formalin test. Results showed that applying RS for 3â h reduced pain responses in the early and late phases of the formalin test. The main findings showed that intra-CA1 injection of orexin receptor antagonists reduced the antinociception caused by stress in both phases of the formalin test. In addition, the contribution of OX2r in mediating the antinociceptive effect of stress was more prominent than that of OX1r in the early phase of the formalin test. However, in the late phase, both receptors worked similarly. Accordingly, the orexin system and its two receptors in the CA1 region of the hippocampus regulate SIA response to this animal model of pain in formalin test.
Subject(s)
CA1 Region, Hippocampal , Pain , Rats , Animals , Orexins/metabolism , Orexin Receptors/metabolism , Rats, Wistar , Pain Measurement , Carbachol/pharmacology , Pain/drug therapy , Pain/metabolism , CA1 Region, Hippocampal/metabolism , Orexin Receptor Antagonists/pharmacologyABSTRACT
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.