Potential physiological and pathological roles for axonal ryanodine receptors

Clinical disability following trauma or disease to the spinal cord often involves the loss of vital white matter elements including axons and glia.Although excessive Cais an established driver of axonal degeneration,therapeutically targeting externally sourced Cato date has had limited success in both basic and clinical studies.Contributing factors that may underlie this limited success include the complexity of the many potential sources of Caentry and the discovery that axons also contain substantial amounts of stored Cathat if inappropriately released could contribute to axonal demise.Axonal Castorage is largely accomplished by the axoplasmic reticulum that is part of a continuous network of the endoplasmic reticulum that provides a major sink and source of intracellular Cafrom the tips of dendrites to axonal terminals.This“neuron-within-a-neuron”is positioned to rapidly respond to diverse external and internal stimuli by amplifying cytosolic Calevels and generating short and long distance regenerative Cawaves through Cainduced Carelease.This review provides a glimpse into the molecular machinery that has been implicated in regulating ryanodine receptor mediated Carelease in axons and how dysregulation and/or overstimulation of these internodal axonal signaling nanocomplexes may directly contribute to Ca-dependent axonal demise.Neuronal ryanodine receptors expressed in dendrites,soma,and axonal terminals have been implicated in synaptic transmission and synaptic plasticity,but a physiological role for internodal localized ryanodine receptors remains largely obscure.Plausible physiological roles for internodal ryanodine receptors and such an elaborate internodal binary membrane signaling network in axons will also be discussed.

Function of the CaMKII-ryanodine receptor signaling pathway in rabbits with left ventricular hypertrophy and triggered ventricular arrhythmia

BACKGROUND:Calcium calmodulin-dependent kinase II(CaMKII) can be more active in patients with left ventricular hypertrophy(LVH),which in turn causes phosphorylation of ryanodine receptors,resulting in inactivation and the instability of intracellular calcium homeostasis.The present study aimed to determine the effect of CaMKII-ryanodine receptor pathway signaling in rabbits with left ventricular hypertrophy and triggered ventricular arrhythmia.METHODS:Forty New Zealand rabbits were randomized into four groups(10 per group):sham group,LVH group,KN-93 group(LVH+KN-93),and ryanodine group(LVH+ryanodine).Rabbits in the LVH,KN-93,and ryanodine groups were used to establish a left ventricular hypertrophy model by the coarctation of the abdominal aorta,while those in the sham group did not undergo the coarctation.After eight weeks,action potentials(APs) were recorded simultaneously in the endocardium and epicardium,and a transmural electrocardiogram(ECG) was also recorded in the rabbit left ventricular wedge model.Drugs were administered to the animals in the KN-93 and ryanodine groups,and the frequency of triggered APs and ventricular tachycardia was recorded after the rabbits were given isoprenaline(1 μmol/L) and high-frequency stimulation.RESULTS:The frequency(animals/group) of triggered APs was 0/10 in the sham group,10/10 in the LVH group,4/10 in the KN-93 group,and 1/10 in the ryanodine group.The frequencies of ventricular tachycardia were 0/10,9/10,3/10,and 1/10,respectively.The frequencies of polymorphic ventricular tachycardia or ventricular fibrillation were 0/10,7/10,2/10,and 1/10,respectively.The frequencies of triggered ventricular arrhythmias in the KN-93 and ryanodine groups were much lower than those in the LVH group(P<0.05).CONCLUSIONS:KN-93 and ryanodine can effectively reduce the occurrence of triggered ventricular arrhythmia in rabbits with LVH.The CaMKII-ryanodine signaling pathway can be used as a new means of treating ventricular arrhythmia.

Molecular characterization of the ryanodine receptor from Adoxophyes orana and its response to lethal and sublethal doses of chlorantraniliprole

The insect ryanodine receptor(RyR)is a novel target of the anthranilic and phthalic insecticides,which have high activity against lepidopteran insects.Several diamide insecticides have been used to control pests in orchards in China.To enhance our understanding of the effects of diamides on RyRs,full-length cDNAs were isolated and characterized from the summer fruit tortrix moth,Adoxophyes orana,which is the most severe pest of stone and pome trees worldwide.In addition,the modulation of AoRyRmRNA expression by diamide insecticides was investigated.The AoRyRmRNA obtained had an open reading frame(ORF)of 15402 bp nucleotides encoding 5113 amino acids,and shared high and low identity with its orthologs in other insects and mammals of 77–92 and 45–47%identity,respectively.One alternative splice site with two exclusive exons was revealed in AoRyR(a/b).The usage of exon was more frequent in eggs and larvae than in pupae and adults.Quantitative real-time reverse transcription PCR(qRT-PCR)showed that AoRyRmRNA was expressed at all developmental stages,especially in eggs,male pupae and male adults.The expression levels of AoRyRmRNA in the whole body were up-regulated markedly after 3 rd instar larvae were treated with chlorantraniliprole at LC_(10),LC_(20)and LC_(50)dosages.The results could provide the basis for further functional studies of Ao Ry R and for the development of new chemicals with selective activity against insects.

Remodeling of the ryanodine receptor isoform 1 channel regulates the sweet and umami taste perception of Rattus norvegicus

Sweet and umami tastes are elicited by sweet and umami receptors on the tongue and palate epithelium,respectively.However,the molecular machinery allowing the taste reaction remains incompletely understood.Through a phosphoproteomic approach,we identified the key proteins that trigger taste mechanisms based on phosphorylation cascades.Ryanodine receptor isoform 1(RYR1)was further verified by sensory and behavioral assays.We propose a model of RYR1-mediated sweet/umami signaling in which the RYR1 channel,which mediates Ca^(2+)release from the endoplasmic reticulum,is closed by dephosphorylation in bud tissue after sweet/umami treatment.The alteration in Ca^(2+)content in the cytosol induces transient membrane depolarization and generates a cell current for taste signal transduction.We demonstrate that RYR1 is a new channel involved in the regulation of sweet/umami signal transduction and propose a“metabolic clock”notion based on sweet/umami sensing.Our study provides a valuable foundation for a system-level understanding of the taste perception mechanism.

Expression and reconstitution of the bioluminescent Ca2+reporter aequorin in human embryonic stem cells,and exploration of the presence of functional IP3 and ryanodine receptors during the early stages of their differentiation into cardiomyocytes

In order to develop a novel method of visualizing possible Ca^(2+) signaling during the early differentiation of h ESCs into cardiomyocytes and avoid some of the inherent problems associated with using fluorescent reporters, we expressed the bioluminescent Ca^(2+) reporter, apo-aequorin, in HES2 cells and then reconstituted active holo-aequorin by incubation with f-coelenterazine. The temporal nature of the Ca^(2+) signals generated by the holo-f-aequorin-expressing HES2 cells during the earliest stages of differentiation into cardiomyocytes was then investigated. Our data show that no endogenous Ca^(2+) transients(generated by release from intracellular stores) were detected in 1–12-day-old cardiospheres but transients were generated in cardiospheres following stimulation with KCl or Ca Cl_2, indicating that holo-f-aequorin was functional in these cells. Furthermore, following the addition of exogenous ATP, an inositol trisphosphate receptor(IP_3R) agonist, small Ca^(2+) transients were generated from day 1 onward. That ATP was inducing Ca^(2+) release from functional IP_3 Rs was demonstrated by treatment with 2-APB, a known IP_3 R antagonist. In contrast, following treatment with caffeine, a ryanodine receptor(Ry R) agonist, a minimal Ca^(2+) response was observed at day 8 of differentiation only. Thus, our data indicate that unlike Ry Rs, IP_3 Rs are present and continually functional at these early stages of cardiomyocyte differentiation.

Identification of the ryanodine receptor mutation I4743M and its contribution to diamide insecticide resistance in Spodoptera exigua (Lepidoptera: Noctuidae)

Insect ryanodine receptors(RyRs)are the targets of diamide insecticides.Two point mutations G4946E and I4790M(numbering according to Plutella xylostella,PxRyR)in the transmembrane domain of the insect RyRs associated with diamide resistance have so far been identified in three lepidopteran pests,P.xylostella,Tuta absoluta and Chilo suppressalis.In this study,we identified one of the known RyR target site resistance mutations(I4790M)in a field-collected population of Spodoptera exigua.The field-collected WF population of S.exigua exhibited 154 fold resistance to chlorantraniliprole when compared with the susceptible WH-S strain.Sequencing the transmembrane domains of S.exigua RyR(SeRyR)revealed that the resistant WF strain was homozygous for the 14743M mutation(corresponding to I4790M in PxRyR),whereas the G4900E allele(corresponding to G4946E of PxRyR)was not detected.The 4743M allele was introgressed into the susceptible WH-S strain by crossing WF with WH-S,followed by three rounds of backcrossing with WH-S.The introgressed strain 4743M was homozygous for the mutant 4743M allele and shared about 94%of its genetic background with that of the recipient WH-S strain.Compared with WH-S,the near-isogenic 4743M strain showed moderate levels of resistance to chlorantraniliprole(21 fold),cyantraniliprole(25 fold)and flubendiamide(22 fold),suggesting that the I4743M mutation confers medium levels of resistance to all three diamides.Genetic analysis showed diamide resistance in the 4743M strain was inherited as an autosomal and recessive trait.Results from this study have direct implications for the design of appropriate resistance monitoring and management practices to sustainably control S.exigua.

Cardiac ryanodine receptor gene(hRyR2) mutation underlying catecholaminergic polymorphic ventricular tachycardia in a Chinese adolescent presenting with sudden cardiac arrest and cardiac syncope

Sudden cardiac death （SCD） in children and adolescents is uncommon and yet it is devastating for both victim＇s family and the society. Recently, it was increasingly recognized that SCD in young patients with structurally normal heart may be caused by inheritable primary electrical diseases due to the malfunction of cardiac ion channels, a disease entity known as the ion channelopathies. Catecholaminergic polymorphic ventricular tachycardia （CPVT） is a specific form of ion channelopathy which can cause cardiac syncope or SCD in young patients by producing catecholamine-induced bi-directional ventricular tachycardia （BiVT）, polymorphic VT and ventricular fibrillation （VF） during physical exertion or emotion. We reported here an index case of CPVT caused by cardiac ryanodine receptor gene （hRyR2） mutation which presented as cardiac syncope and sudden cardiac arrest in a Chinese adolescent female.

Design, Synthesis, Biological Evaluation and SARs of Novel N-Substituted Sulfoximfnes as Potential Ryanodine Receptor Modulators

The sulfoximine group has been evaluated as a pharmacophore. Introducing the sulfoximine structure into medicinal compounds as exciting motifs has brought opportunities in drug discovery. In order to develop new ryanodine receptor （RyR） modulators, a series of phthalamides containing sulfoximine derivatives were designed, synthesized, and evaluated against oriental armyworm and diamondback moth for their insecticidal activities. These studies helped to elucidate the electronic and structural requirements around the sulfoximine motif for insecticidal activity. All new structures were synthesized and characterized by ^1H NMR, ^13C NMR, HRMS and bioassay and a preliminary structure-activity relationship （SAR） was discussed. The biological assessment indicated that most title compounds showed good to excellent larvicidal activities. Compounds la, le, and If gave excellent insecticidal activity against oriental armyworm, which showed 100% larvicidal activity at 0.5 mg/L. All compounds showed 100% larvicidal activity at 0.1 mg/L against diamondback moth. In particular, the larvicidal activities of le, If, and lh at 0.0001 mg/L were 50%, 20%, and 40%, respectively, reaching an activity as high as that of the commercial flubendiamide （40%, 0.0001mg/L）. Therefore, Ia, Ie, If and Ih could be considered as new lead structures for the development of new ryanodine receptor （RyR） modulators.

Dynamic Coupling between Ryanodine Receptors and its Role in Cacium Release

The calcium release channels/ryanodine receptors (RyRs) usually form 2-D regular lattice in the endoplasmic/sarcoplasmic reticulum membranes. Several inter-RyR coupling

Dissociation of FK506 Binding Protein 12.6 from Ryanodine Receptor Type 2 Is Regulated by cADPR but not β-Adrenergic Stimulation in Mouse Cardiomyocytes

AIMS: β-adrenergic augmentation of Ca2+ sparks and cardiac contractility has been functionally linked to phosphorylation-dependent dissociation of FK506 binding protein 12.

INTRACELLULAR SOLITARY PULSE CALCIUM WAVES IN FROG SYMPATHETIC NEURONS

In certain extracellular environments, there would appear a kind of solitary pulse calcium waves in Rana pipiens sympathetic neurons, propagating inwards along the radial direction from the plasma membrane. To gain a deeper insight into the waves, a model describing intracellular calcium waves in frog sympathetic neurons was established. In the piecewise linear approximation, the present model is identical to the Sneyd model. Thus, with (Sneyd's) method, analytical expressions for the wave speed and profiles of 1-D solitary pulse wave were obtained. A wave speed of (21.5 μm/s) was deduced, which agrees rather well with experimental data.

The current status of malignant hyperthermia

Malignant hyperthermia(MH) is a rare and life-threatening pharmacogenetic disorder triggered by volatile anesthetics, the depolarizing muscle relaxant succinylcholine, and rarely by strenuous exercise or environmental heat. The exact prevalence of MH is unknown, and it varies from 1:16 000 in Denmark to 1:100 000 in New York State. The underlying mechanism of MH is excessive calcium release from the sarcoplasmic reticulum(SR),leading to uncontrolled skeletal muscle hyper-metabolism. Genetic mutations in ryanodine receptor type 1(RYR1)and CACNA1 S have been identified in approximately 50% to 86% and 1% of MH-susceptible(MHS) individuals,respectively. Classic clinical symptoms of MH include hypercarbia, sinus tachycardia, masseter spasm,hyperthermia, acidosis, muscle rigidity, hyperkalemia, myoglobinuria, and etc. There are two types of testing for MH: a genetic test and a contracture test. Contracture testing is still being considered as the gold standard for MH diagnosis. Dantrolene is the only available drug approved for the treatment of MH through suppressing the calcium release from SR. Since clinical symptoms of MH are highly variable, it can be difficult to establish a diagnosis of MH. Nevertheless, prompt diagnosis and treatments are crucial to avoid a fatal outcome. Therefore, it is very important for anesthesiologists to raise awareness and understand the characteristics of MH. This review summarizes epidemiology, clinical symptoms, diagnosis and treatments of MH and any new developments.

Design, Syntheses and Biological Activities of Novel Anthranilic Diamide Insecticides Containing N-Pyridylpyrazole

In search of environmentally benign insecticides with high activity, low toxicity and low residue, a series of novel anthranilic diamide derivatives containing N-pyridylpyrazole was designed and synthesized. All the compounds were characterized by H NMR spectroscopy and elemental analysis. The single crystal structure of com pound 8j was determined by X-ray diffraction. The insecticidal activities of the new compounds were evaluated. The results show that some compounds exhibited moderate insecticidal activities against Lepidoptera pests. Among this series of compounds, compounds 80 and 8p showed 100% larvicidal activity against Mythimna separate Walker, Plutella xylostella Linnaeus and Laphygma exigua Hubner at a test concentration of 200 mg/kg, which is equal to the commercial chlorantraniliprole.

From contraction to gene expression:nanojunctions of the sarco/endoplasmic reticulum deliver site-and function-specific calcium signals

Calcium signals determine, for example, smooth muscle contraction and changes in gene expression. How calcium signals select for these processes is enigmatic. We build on the "panjunctional sarcoplasmic reticulum" hypothesis, describing our view that different calcium pumps and release channels, with different kinetics and affinities for calcium, are strategically positioned within nanojunctions of the SR and help demarcate their respective cytoplasmic nanodomains. SERCA2 b and Ry R1 are preferentially targeted to the sarcoplasmic reticulum(SR) proximal to the plasma membrane(PM), i.e., to the superficial buffer barrier formed by PM-SR nanojunctions, and support vasodilation. In marked contrast, SERCA2 a may be entirely restricted to the deep, perinuclear SR and may supply calcium to this sub-compartment in support of vasoconstriction. Ry R3 is also preferentially targeted to the perinuclear SR, where its clusters associate with lysosome-SR nanojunctions. The distribution of Ry R2 is more widespread and extends from this region to the wider cell. Therefore, perinuclear Ry R3 s most likely support the initiation of global calcium waves at L-SR junctions, which subsequently propagate by calcium-induced calcium release via Ry R2 in order to elicit contraction. Data also suggest that unique SERCA and Ry R are preferentially targeted to invaginations of the nuclear membrane. Site- and function-specific calcium signals may thus arise to modulate stimulus-response coupling and transcriptional cascades.

Synthesis, insecticidal activities and SAR studies of novel anthranilic diamides containing trifluoroethoxyl substituent and chiral amino acid moieties

Ryanodine receptors（RyRs） activator has become one class of popular insecticide because of its unique mode of action. In order to find more new RyRs activators as insecticidal agents, a series of 18 novel chiral anthranilic diamides were designed by introducing the D-alanine acid and D-serine acid esters as well as trifluoroethoxyl group into the anthranilic diamide skeleton and synthesized successfully based on anthranilic diamide and FKI-1033 structures. The structures of the title compounds Ia–i and IIa–i were confirmed by melting points,~1H NMR,^（13）C NMR, elemental analysis and specific optical rotation analysis.The preliminary bioassay results indicated that most of the title compounds exhibited considerable larvicidal activities against oriental armyworm at 10 mg/L, especially Ib, Ie and IIh showed remarkable insecticidal activities at 0.5 mg/L. The larvicidal activity against diamondback moth of Ia and IId were 80%and 90% respectively at 0.0001 mg/L, which was similar to that of chlorantraniliprole. The relationship between structure and insecticidal activity was analyzed to reveal a possible co-regulated effect of the chiral amino acid ester, halogen atom or cyano group, and trifluoroethyloxyl group of the skeleton structures of the title compounds, which will provide useful information for guiding the design and discovery of new RyRs activators and insecticidal agrochemicals.

Ginsenoside Rb1 Pretreatment Attenuates Myocardial Ischemia by Reducing Calcium/Calmodulin-Dependent Protein Kinase Ⅱ-Medicated Calcium Release

Objective:The aim of this study was to investigate the protective effects of ginsenoside Rb1 and assess whether these protective effects are related to calcium/calmodulin-dependent protein kinaseⅡ(Ca MKⅡ).Methods:A myocardial ischemia(IS)rat.model and a myocardial H9 C2 cell hypoxia model were established.MI was induced by occluding the left anterior descending artery for 120 min.Ginsenoside Rb1(10 mg/kg)was administered 30 min before ischemia induction,and the treatment continued for 7 days.Results:In the rat IS injury model,ginsenoside Rb1 reduced myocardial infarct size,mean left ventricular diastolic pressure,incidence of arrhythmia,and levels of serum creatine kinase,lactate dehydrogenase,and malondialdehyde.However,the mean left ventricular systolic pressure,and maximal rising and falling rates of ventricular pressure(±dp/dtmax)increased.In the myocardial H9 C2 cell hypoxia model,ginsenoside Rb1 reduced intracellular calcium concentrations([Ca2+]i)during hypoxia,and markedly reversed the hypoxia-induced decrease in cell survival.Ginsenoside Rb1 was involved in the downregulation of CaMKⅡand the ryanodine receptor,as well as hypoxia-induced H9 C2 cell survival.Conclusion:The findings of the present study suggest that ginsenoside Rb1 attenuates MI injury in rats,partially through the downregulation of CaMKⅡexpression.

Effect of metoprolol on sarcoplasmic reticulum Ca^2＋ leak in a rabbit model of heart failure

Background Studies have shown that B-blockers can improve cardiac performance in heart failure （HF） by reversing protein kinase A （PKA）-mediated sarcoplasmic reticulum （SR） Ca^2＋ leak. However, it is being strongly questioned as to whether the PKA-mediated ryanodine receptor （RyR2） hyper-phosphorylation is a critical regulator of SR Ca^2＋ leak. In this study, we used a rabbit HF model to investigate whether β-blockers affect SR Ca^2＋ leak by other potential mechanisms.

Cardioprotective Effects of Qishen Granule(芪参颗粒)on Sarcoplasmic Reticulum Ca^2＋ Handling in Heart Failure Rats

Objective：To assess the effects of Qishen Granule（芪参颗粒, QSG） on sarcoplasmic reticulum（SR） Ca^2＋ handling in heart failure（HF） model of rats and to explore the underlying molecular mechanisms. Methods：HF rat models were induced by left anterior descending coronary artery ligation surgery and high-fat diet feeding. Rats were randomly divided into sham（n=10）, model（n=10）, QSG（n=12, 2.2 g/kg daily） and metoprolol groups（n=12, 10.5 mg/kg daily）. The therapeutic effects of QSG were evaluated by echocardiography and blood lipid testing. Intracellular Ca^2＋ concentration and sarco-endoplasmic reticulum ATPase 2a（SERCA2a） activity were detected by specific assay kits. Expressions of the critical regulators in SR Ca^2＋ handling were evaluated by Western blot and real-time quantitative polymerase chain reaction. Results：HF model of rats developed ventricular remodeling accompanied with calcium overload and defective Ca^2＋ releaseuptake cycling in cardiomyocytes. Treatment with QSG improved contractive function, attenuated ventricular remodeling and reduced the basal intracellular Ca^2＋ level. QSG prevented defective Ca^2＋ leak by attenuating hyperphosphorylation of ryanodine receptor 2, inhibiting expression of protein kinase A and up-regulating transcriptional expression of protein phosphatase 1. QSG also restored Ca^2＋ uptake by up-regulating expression and activity of SERCA2 a and promoting phosphorylation of phospholamban. Conclusion：QSG restored SR Ca^2＋cycling in HF rats and served as an ideal alternative drug for treating HF.

Design, Synthesis, Structure-Activity Relationship and Insecticidal Activities of Trifluoromethyl-Containing Sulfiliminyl and Sulfoximinyl Phthalic Acid Diamide Structure

Due to a novel mode action,low toxicity to mammals and low residue characteristics,phthalic acid diamides have aroused considerable interests in agricultural chemistry.With introduction of N-cyano,N-trifluoroacetyl,N-carbamoylsulfiliminyl and sulfoximinyl substituents into phthalamides,12 novel derivatives containing trifluoromethyl moiety were designed and synthesized.All title compounds were characterized by ^(1)H NMR and high-resolution mass spectrometry.The preliminary results of biological activity assessment indicated that some title compounds exhibited moderate to good insecticidal activities against oriental armyworm(Pseudaletia separata Walker).In particular,Va gave higher activity against oriental armyworm and diamondback moth.The present work reported that the new trifluoromethylated diamides incorporating N-trifluoroacetylsulfoximinyl moieties are potential lead compounds for further structure optimization,providing some insight into the relating structure-activity relationship.

Design,synthesis and insecticidal activities of novel anthranilic diamides containing polyfluoroalkyl pyrazole moiety

In order to discover new molecules with good insecticidal activities, a series of anthranilic diamides containing polyfluoroalkyl pyrazole were designed and synthesized, and their structures were characterized by1 H NMR and HRMS. Bioassays demonstrated that some of the title compound exhibited excellent insecticidal activities. The larvicidal activities of compound 8a, 8c, 8g, 8k and 8l against Mythimna separata Walker were 100% at 0.8 mg/L. The insecticidal activities of compound 8a, 8c,8e, 8g, 8k and 8l against Plutella xylostella Linnaeus were 100% at 0.4 mg/L. Surprisingly compounds 8a and 8c still showed 100% larvicidal activities against Plutella xylostella Linnaeus at 0.08 mg/L comparable to the commercialized Chlorantraniliprole. The LC_（50） of compound 8a and 8c against M. separata is 0.048 and 0.043 mg/L respectively.