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Journal of the American Chemical Society Jul 2020A stereoselective entry to ryanoids is described that culminates in the synthesis of anhydroryanodol and thus the formal total synthesis of ryanodol. The pathway...
A stereoselective entry to ryanoids is described that culminates in the synthesis of anhydroryanodol and thus the formal total synthesis of ryanodol. The pathway described features an annulation reaction conceived to address the uniquely complex and highly oxygenated polycyclic skeleton common to members of this natural product class. It is demonstrated that metallacycle-mediated intramolecular coupling of an alkyne and a 1,3-diketone can proceed with a highly functionalized enyne and with outstanding levels of stereoselection. Furthermore, the first application of this technology in natural product synthesis is demonstrated here. More broadly, the advances described demonstrate the value that programs in natural product total synthesis have in advancing organic chemistry, here through the design and realization of an annulation reaction that accomplishes what previously established reactions do not.
Topics: Biological Products; Cyclization; Molecular Structure; Ryanodine; Stereoisomerism
PubMed: 32609506
DOI: 10.1021/jacs.0c05766 -
The Journal of Physiology Jan 2023Enteroendocrine cells (EECs) are specialized sensors of luminal forces and chemicals in the gastrointestinal (GI) epithelium that respond to stimulation with a release...
Enteroendocrine cells (EECs) are specialized sensors of luminal forces and chemicals in the gastrointestinal (GI) epithelium that respond to stimulation with a release of signalling molecules such as serotonin (5-HT). For mechanosensitive EECs, force activates Piezo2 channels, which generate a very rapidly activating and inactivating (∼10 ms) cationic (Na , K , Ca ) receptor current. Piezo2 receptor currents lead to a large and persistent increase in intracellular calcium (Ca ) that lasts many seconds to sometimes minutes, suggesting signal amplification. However, intracellular calcium dynamics in EEC mechanotransduction remain poorly understood. The aim of this study was to determine the role of Ca stores in EEC mechanotransduction. Mechanical stimulation of a human EEC cell model (QGP-1) resulted in a rapid increase in cytoplasmic Ca and a slower decrease in ER stores Ca , suggesting the involvement of intracellular Ca stores. Comparing murine primary colonic EECs with colonocytes showed expression of intercellular Ca store receptors, a similar expression of IP receptors, but a >30-fold enriched expression of Ryr3 in EECs. In mechanically stimulated primary EECs, Ca responses decreased dramatically by emptying stores and pharmacologically blocking IP and RyR1/3 receptors. RyR3 genetic knockdown by siRNA led to a significant decrease in mechanosensitive Ca responses and 5-HT release. In tissue, pressure-induced increase in the Ussing short circuit current was significantly decreased by ryanodine receptor blockade. Our data show that mechanosensitive EECs use intracellular Ca stores to amplify mechanically induced Ca entry, with RyR3 receptors selectively expressed in EECs and involved in Ca signalling, 5-HT release and epithelial secretion. KEY POINTS: A population of enteroendocrine cells (EECs) are specialized mechanosensors of the gastrointestinal (GI) epithelium that respond to mechanical stimulation with the release of important signalling molecules such as serotonin. Mechanical activation of these EECs leads to an increase in intracellular calcium (Ca ) with a longer duration than the stimulus, suggesting intracellular Ca signal amplification. In this study, we profiled the expression of intracellular Ca store receptors and found an enriched expression of the intracellular Ca receptor Ryr3, which contributed to the mechanically evoked increases in intracellular calcium, 5-HT release and epithelial secretion. Our data suggest that mechanosensitive EECs rely on intracellular Ca stores and are selective in their use of Ryr3 for amplification of intracellular Ca . This work advances our understanding of EEC mechanotransduction and may provide novel diagnostic and therapeutic targets for GI motility disorders.
Topics: Mice; Animals; Humans; Ryanodine Receptor Calcium Release Channel; Ryanodine; Serotonin; Calcium; Receptors, Calcium-Sensing; Mechanotransduction, Cellular; Enteroendocrine Cells
PubMed: 36428286
DOI: 10.1113/JP283383 -
Science (New York, N.Y.) Aug 2016(+)-Ryanodine and (+)-ryanodol are complex diterpenoids that modulate intracellular calcium-ion release at ryanodine receptors, ion channels critical for skeletal and...
(+)-Ryanodine and (+)-ryanodol are complex diterpenoids that modulate intracellular calcium-ion release at ryanodine receptors, ion channels critical for skeletal and cardiac muscle excitation-contraction coupling and synaptic transmission. Chemical derivatization of these diterpenoids has demonstrated that certain peripheral structural modifications can alter binding affinity and selectivity among ryanodine receptor isoforms. Here, we report a short chemical synthesis of (+)-ryanodol that proceeds in only 15 steps from the commercially available terpene (S)-pulegone. The efficiency of the synthesis derives from the use of a Pauson-Khand reaction to rapidly build the carbon framework and a SeO2-mediated oxidation to install three oxygen atoms in a single step. This work highlights how strategic C-O bond constructions can streamline the synthesis of polyhydroxylated terpenes by minimizing protecting group and redox adjustments.
Topics: Biological Products; Cyclohexane Monoterpenes; Monoterpenes; Oxidation-Reduction; Oxygen; Ryanodine; Ryanodine Receptor Calcium Release Channel; Selenium Oxides
PubMed: 27563092
DOI: 10.1126/science.aag1028 -
Progress in Biophysics and Molecular... 2008To characterize the effects of inhibition of Ryanodine receptor (RyR), TTX-sensitive neuronal Na+ current (iNa), "rapidly activating" delayed rectifier K+ current (iKr)... (Review)
Review
AIMS
To characterize the effects of inhibition of Ryanodine receptor (RyR), TTX-sensitive neuronal Na+ current (iNa), "rapidly activating" delayed rectifier K+ current (iKr) and ultrarapid delayed rectifier potassium current (IKur) on the pacemaker activity of the sinoatrial node (SAN) and the atrioventricular node (AVN) in the mouse.
METHODS
The structure of mouse AVN was studied by histology and immunolabelling of Cx43 and hyperpolarization-activated, cyclic nucleotide-binding channels (HCN). The effects of Ryanodine, TTX, E-4031 and 4-AP on pacemaker activities recorded from mouse intact SAN and AVN preparations have been investigated.
RESULTS
Immuno-histological characterization delineated the structure of the AVN showing the similar molecular phenotype of the SAN. The effects of these inhibitors on the cycle length (CL) of the spontaneous pacemaker activity of the SAN and the AVN were characterized. Inhibition of RyR by 0.2 and 2 microM Ryanodine prolonged CL by 42+/-12.3% and 64+/-18.1% in SAN preparations by 163+/-72.3% and 241+/-91.2% in AVN preparations. Inhibition of TTX-sensitive iNa by 100 nM TTX prolonged CL by 22+/-6.0% in SAN preparations and 53+/-13.6% in the AVN preparations. Block of iKr by E-4031 prolonged CL by 68+/-12.5% in SAN preparations and 28+/-3.4% in AVN preparations. Inhibition of iKur by 50 microM 4-AP prolonged CL by 20+/-3.4% in SAN preparations and 18+/-3.0% in AVN preparations.
CONCLUSION
Mouse SAN and AVN showed distinct different response to the inhibition of RyR, TTX-sensitive INa, IKr and iKur, which reflects the variation in contribution of these currents to the pacemaker function of the cardiac nodes in the mouse. Our data provide valuable information for developing virtual tissue models of mouse SAN and AVN.
Topics: 4-Aminopyridine; Animals; Anti-Arrhythmia Agents; Atrioventricular Node; Mice; Piperidines; Potassium Channel Blockers; Pyridines; Ryanodine; Sinoatrial Node; Tetrodotoxin
PubMed: 17850852
DOI: 10.1016/j.pbiomolbio.2007.07.003 -
Physiological Reviews Oct 2002The ryanodine receptors (RyRs) are a family of Ca2+ release channels found on intracellular Ca2+ storage/release organelles. The RyR channels are ubiquitously expressed... (Review)
Review
The ryanodine receptors (RyRs) are a family of Ca2+ release channels found on intracellular Ca2+ storage/release organelles. The RyR channels are ubiquitously expressed in many types of cells and participate in a variety of important Ca2+ signaling phenomena (neurotransmission, secretion, etc.). In striated muscle, the RyR channels represent the primary pathway for Ca2+ release during the excitation-contraction coupling process. In general, the signals that activate the RyR channels are known (e.g., sarcolemmal Ca2+ influx or depolarization), but the specific mechanisms involved are still being debated. The signals that modulate and/or turn off the RyR channels remain ambiguous and the mechanisms involved unclear. Over the last decade, studies of RyR-mediated Ca2+ release have taken many forms and have steadily advanced our knowledge. This robust field, however, is not without controversial ideas and contradictory results. Controversies surrounding the complex Ca2+ regulation of single RyR channels receive particular attention here. In addition, a large body of information is synthesized into a focused perspective of single RyR channel function. The present status of the single RyR channel field and its likely future directions are also discussed.
Topics: Adaptation, Physiological; Animals; Calcium; Calcium Channels; Calcium Signaling; Feedback; Humans; Ryanodine; Ryanodine Receptor Calcium Release Channel
PubMed: 12270947
DOI: 10.1152/physrev.00013.2002 -
The Journal of General Physiology Sep 2022Flecainide, a cardiac class 1C blocker of the surface membrane sodium channel (NaV1.5), has also been reported to reduce cardiac ryanodine receptor (RyR2)-mediated...
Flecainide, a cardiac class 1C blocker of the surface membrane sodium channel (NaV1.5), has also been reported to reduce cardiac ryanodine receptor (RyR2)-mediated sarcoplasmic reticulum (SR) Ca2+ release. It has been introduced as a clinical antiarrhythmic agent for catecholaminergic polymorphic ventricular tachycardia (CPVT), a condition most commonly associated with gain-of-function RyR2 mutations. Current debate concerns both cellular mechanisms of its antiarrhythmic action and molecular mechanisms of its RyR2 actions. At the cellular level, it targets NaV1.5, RyR2, Na+/Ca2+ exchange (NCX), and additional proteins involved in excitation-contraction (EC) coupling and potentially contribute to the CPVT phenotype. This Viewpoint primarily addresses the various direct molecular actions of flecainide on isolated RyR2 channels in artificial lipid bilayers. Such studies demonstrate different, multifarious, flecainide binding sites on RyR2, with voltage-dependent binding in the channel pore or voltage-independent binding at distant peripheral sites. In contrast to its single NaV1.5 pore binding site, flecainide may bind to at least four separate inhibitory sites on RyR2 and one activation site. None of these binding sites have been specifically located in the linear RyR2 sequence or high-resolution structure. Furthermore, it is not clear which of the inhibitory sites contribute to flecainide's reduction of spontaneous Ca2+ release in cellular studies. A confounding observation is that flecainide binding to voltage-dependent inhibition sites reduces cation fluxes in a direction opposite to physiological Ca2+ flow from SR lumen to cytosol. This may suggest that, rather than directly blocking Ca2+ efflux, flecainide can reduce Ca2+ efflux by blocking counter currents through the pore which otherwise limit SR membrane potential change during systolic Ca2+ efflux. In summary, the antiarrhythmic effects of flecainide in CPVT seem to involve multiple components of EC coupling and multiple actions on RyR2. Their clarification may identify novel specific drug targets and facilitate flecainide's clinical utilization in CPVT.
Topics: Anti-Arrhythmia Agents; Calcium; Flecainide; Humans; Myocytes, Cardiac; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sodium; Tachycardia, Ventricular
PubMed: 35713932
DOI: 10.1085/jgp.202213089 -
The Journal of Physiology 19911. Charge movement and myoplasmic calcium transients were simultaneously recorded from frog skeletal muscle fibres by using the double-seal Vaseline-gap technique.... (Comparative Study)
Comparative Study
1. Charge movement and myoplasmic calcium transients were simultaneously recorded from frog skeletal muscle fibres by using the double-seal Vaseline-gap technique. Calcium transients were monitored with the fluorescent indicator Rhod-2. 2. Ryanodine modified the kinetics and the total amount of charge moved during depolarizing pulses (Q(on)), while it did not significantly modify the charge after repolarization (Q(off)). The extracellular application of 100 microM-ryanodine elicited a temporary initial increase of the delayed component of charge movement (Q gamma) and the calcium transient. Both phenomena were later blocked with the same temporal course and to the same extent. 3. The blockade of Q gamma and the calcium transient was also observed with ryanodine concentrations of 1-10 microM. For membrane potentials positive to -10mV, the Qon measured was larger in the presence of ryanodine; Qoff was not modified. 4. Tetracaine (400-500 microM) blocked a similar delayed component of Qon, identified as Q gamma, as well as the calcium transient monitored simultaneously. This effect was observed in the first minutes after the addition of tetracaine to the extracellular solution. 5. Tetracaine blocked a faster initial component of Qon for voltages positive to -10 mV, corresponding to the voltage range of activation of the calcium current. At these same membrane potentials, Qoff was also reduced to a similar extent to Qon. 6. Ryanodine and tetracaine showed different effects on calcium current. Whereas the slow calcium current was not modified upon the addition of ryanodine, it was completely blocked in the presence of tetracaine. The blockade of the slow calcium current made evident the fast calcium current. The effects of tetracaine on the charge movement, the calcium transient and the slow calcium current were reversible. 7. These results suggest that ryanodine and tetracaine may act at different sites. Ryanodine exerts its effect on the sarcoplasmic reticulum ryanodine receptor, blocking calcium release and Q gamma, while tetracaine at these concentrations may affect the release channel and the dihydropyridine receptor, causing a blockade of the charge movement, calcium transient and calcium current.
Topics: Animals; Calcium; Calcium Channels; Electrophysiology; In Vitro Techniques; Ion Channel Gating; Muscles; Rana pipiens; Ryanodine; Tetracaine; Time Factors
PubMed: 1666652
DOI: 10.1113/jphysiol.1991.sp018715 -
Experimental Biology and Medicine... Dec 2023The mammalian target of rapamycin (mTOR) inhibitors, everolimus (but not dactolisib), is frequently associated with lung injury in clinical therapies. However, the...
The mammalian target of rapamycin (mTOR) inhibitors, everolimus (but not dactolisib), is frequently associated with lung injury in clinical therapies. However, the underlying mechanisms remain unclear. Endothelial cell barrier dysfunction plays a major role in the pathogenesis of the lung injury. This study hypothesizes that everolimus increases pulmonary endothelial permeability, which leads to lung injury. We tested the effects of everolimus on human pulmonary microvascular endothelial cell (HPMEC) permeability and a mouse model of intraperitoneal injection of everolimus was established to investigate the effect of everolimus on pulmonary vascular permeability. Our data showed that everolimus increased human pulmonary microvascular endothelial cell (HPMEC) permeability which was associated with MLC phosphorylation and F-actin stress fiber formation. Furthermore, everolimus induced an increasing concentration of intracellular calcium Ca leakage in HPMECs and this was normalized with ryanodine pretreatment. In addition, ryanodine decreased everolimus-induced phosphorylation of PKCα and MLC, and barrier disruption in HPMECs. Consistent with data, everolimus treatment caused a visible lung-vascular barrier dysfunction, including an increase in protein in BALF and lung capillary-endothelial permeability, which was significantly attenuated by pretreatment with an inhibitor of PKCα, MLCK, and ryanodine. This study shows that everolimus induced pulmonary endothelial hyper-permeability, at least partly, in an MLC phosphorylation-mediated EC contraction which is influenced in a Ca-dependent manner and can lead to lung injury through mTOR-independent mechanisms.
Topics: Animals; Mice; Humans; Endothelial Cells; Everolimus; Lung Injury; Endothelium, Vascular; Protein Kinase C-alpha; Ryanodine; Lung; Phosphorylation; Cells, Cultured; TOR Serine-Threonine Kinases; Mammals
PubMed: 38158699
DOI: 10.1177/15353702231220672 -
Frontiers in Immunology 2023Mast cell (MC) activation is implicated in the pathogenesis of multiple immunodysregulatory skin disorders. Activation of an IgE-independent pseudo-allergic route has...
Mast cell (MC) activation is implicated in the pathogenesis of multiple immunodysregulatory skin disorders. Activation of an IgE-independent pseudo-allergic route has been recently found to be mainly mediated Mas-Related G protein-coupled receptor X2 (MRGPRX2). Ryanodine receptor (RYR) regulates intracellular calcium liberation. Calcium mobilization is critical in the regulation of MC functional programs. However, the role of RYR in MRGPRX2-mediated pseudo-allergic skin reaction has not been fully addressed. To study the role of RYR , we established a murine skin pseudo-allergic reaction model. RYR inhibitor attenuated MRGPRX2 ligand substance P (SP)-induced vascular permeability and neutrophil recruitment. Then, we confirmed the role of RYR in an MC line (LAD2 cells) and primary human skin-derived MCs. In LAD2 cells, RYR inhibitor pretreatment dampened MC degranulation (detected by β-hexosaminidase retlease), calcium mobilization, IL-13, TNF-α, CCL-1, CCL-2 mRNA, and protein expression activated by MRGPRX2 ligands, namely, compound 48/80 (c48/80) and SP. Moreover, the inhibition effect of c48/80 by RYR inhibitor was verified in skin MCs. After the confirmation of RYR2 and RYR3 expression, the isoforms were silenced by siRNA-mediated knockdown. MRGPRX2-induced LAD2 cell exocytosis and cytokine generation were substantially inhibited by RYR3 knockdown, while RYR2 had less contribution. Collectively, our finding suggests that RYR activation contributes to MRGPRX2-triggered pseudo-allergic dermatitis, and provides a potential approach for MRGPRX2-mediated disorders.
Topics: Humans; Animals; Mice; Calcium; Ryanodine; Ryanodine Receptor Calcium Release Channel; Mast Cells; Receptors, G-Protein-Coupled; Dermatitis, Atopic; Nerve Tissue Proteins; Receptors, Neuropeptide
PubMed: 37404822
DOI: 10.3389/fimmu.2023.1207249 -
The Biochemical Journal Sep 1997Dantrolene inhibits and ryanodine stimulates calcium release from skeletal-muscle sarcoplasmic reticulum (SR), the former by an unknown mechanism, and the latter by...
Dantrolene inhibits and ryanodine stimulates calcium release from skeletal-muscle sarcoplasmic reticulum (SR), the former by an unknown mechanism, and the latter by activating the ryanodine receptor (RyR), the primary Ca2+-release channel of SR. Dantrolene is used to treat malignant hyperthermia (MH), a genetic predisposition to excessive intracellular Ca2+ release upon exposure to volatile anaesthetics. Porcine MH results from a point mutation in the SR RyR that alters the open probability of the channel, and is reflected in altered [3H]ryanodine binding parameters. Specific binding sites for [3H]dantrolene and [3H]ryanodine co-distribute on SR that has been isolated by discontinuous sucrose gradient centrifugation. If the two drug-binding sites are functionally linked, [3H]dantrolene binding might be affected both by pharmacological and by genetic modulators of the functional state of the RyR. Accordingly, we compared the characteristics of [3H]dantrolene binding to porcine malignant-hyperthermia-susceptible and normal-skeletal-muscle SR, and examined the effects of RyR modulators on [3H]dantrolene binding to these membranes. Additionally, the feasibility of separating the SR binding sites for [3H]dantrolene and [3H]ryanodine was investigated. No significant differences in [3H]dantrolene binding characteristics to SR membranes from the two muscle types were detected, and the Bmax ratio for [3H]dantrolene/[3H]ryanodine was 1.4(+/-0.1):1 in both muscle types. [3H]Dantrolene binding is unaffected by the RyR modulators caffeine, ryanodine, Ruthenium Red and calmodulin, and neither dantrolene nor azumolene have any effect on [3H]ryanodine binding. Additionally, distinct peaks of [3H]dantrolene and [3H]ryanodine binding are detected in SR membranes fractionated by linear sucrose centrifugation, although no differences in protein patterns are detected by SDS/PAGE or Western-blot analysis. We suggest that the binding sites for these two drugs are pharmacologically distinct, and may exist on separate molecules.
Topics: Animals; Binding Sites; Cell Membrane; Dantrolene; Malignant Hyperthermia; Muscle Relaxants, Central; Muscle, Skeletal; Osmolar Concentration; Radioligand Assay; Ryanodine; Swine
PubMed: 9307036
DOI: 10.1042/bj3260847