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1.
Mol Pharmacol ; 97(4): 250-258, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32015008

RESUMO

Phenytoin is a hydantoin derivative that is used clinically for the treatment of epilepsy and has been reported to have antiarrhythmic actions on the heart. In a failing heart, the elevated diastolic Ca2+ leak from the sarcoplasmic reticulum can be normalized by the cardiac ryanodine receptor 2 (RyR2) inhibitor, dantrolene, without inhibiting Ca2+ release during systole or affecting Ca2+ release in normal healthy hearts. Unfortunately, dantrolene is hepatotoxic and unsuitable for chronic long-term administration. Because phenytoin and dantrolene belong to the hydantoin class of compounds, we test the hypothesis that dantrolene and phenytoin have similar inhibitory effects on RyR2 using a single-channel recording of RyR2 activity in artificial lipid bilayers. Phenytoin produced a reversible inhibition of RyR2 channels from sheep and human failing hearts. It followed a hyperbolic dose response with maximal inhibition of ∼50%, Hill coefficient ∼1, and IC50 ranging from 10 to 20 µM. It caused inhibition at diastolic cytoplasmic [Ca2+] but not at Ca2+ levels in the dyadic cleft during systole. Notably, phenytoin inhibits RyR2 from failing human heart but not from healthy heart, indicating that phenytoin may selectively target defective RyR2 channels in humans. We conclude that phenytoin could effectively inhibit RyR2-mediated release of Ca2+ in a manner paralleling that of dantrolene. Moreover, the IC50 of phenytoin in RyR2 is at least threefold lower than for other ion channels and clinically used serum levels, pointing to phenytoin as a more human-safe alternative to dantrolene for therapies against heart failure and cardiac arrythmias. SIGNIFICANCE STATEMENT: We show that phenytoin, a Na channel blocker used clinically for treatment of epilepsy, is a diastolic inhibitor of cardiac calcium release channels [cardiac ryanodine receptor 2 (RyR2)] at doses threefold lower than its current therapeutic levels. Phenytoin inhibits RyR2 from failing human heart and not from healthy heart, indicating that phenytoin may selectively target defective RyR2 channels in humans and pointing to phenytoin as a more human-safe alternative to dantrolene for therapies against heart failure and cardiac arrhythmias.


Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Cardiotônicos/farmacologia , Insuficiência Cardíaca/prevenção & controle , Miócitos Cardíacos/efeitos dos fármacos , Fenitoína/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Arritmias Cardíacas/tratamento farmacológico , Arritmias Cardíacas/patologia , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/uso terapêutico , Cardiotônicos/uso terapêutico , Dantroleno/farmacologia , Dantroleno/uso terapêutico , Relação Dose-Resposta a Droga , Vesículas Extracelulares , Insuficiência Cardíaca/patologia , Humanos , Bicamadas Lipídicas , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Fenitoína/uso terapêutico , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/metabolismo , Ovinos
2.
Nat Commun ; 11(1): 922, 2020 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-32066742

RESUMO

Ryanodine receptors (RyR) are ion channels responsible for the release of Ca2+ from the sarco/endoplasmic reticulum and play a crucial role in the precise control of Ca2+ concentration in the cytosol. The detailed permeation mechanism of Ca2+ through RyR is still elusive. By using molecular dynamics simulations with a specially designed Ca2+ model, we show that multiple Ca2+ ions accumulate in the upper selectivity filter of RyR1, but only one Ca2+ can occupy and translocate in the narrow pore at a time, assisted by electrostatic repulsion from the Ca2+ within the upper selectivity filter. The Ca2+ is nearly fully hydrated with the first solvation shell intact during the whole permeation process. These results suggest a remote knock-on permeation mechanism and one-at-a-time occupation pattern for the hydrated Ca2+ within the narrow pore, uncovering the basis underlying the high permeability and low selectivity of the RyR channels.


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Cátions Bivalentes/metabolismo , Simulação de Dinâmica Molecular , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Cálcio/química , Calpaína/metabolismo , Cátions Bivalentes/química , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Eletricidade Estática , Relação Estrutura-Atividade
3.
Bioelectrochemistry ; 132: 107449, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31918058

RESUMO

Dysregulation of the cardiac ryanodine receptor (RYR2) by luminal Ca2+ has been implicated in a life-threatening, stress-induced arrhythmogenic disease. The mechanism of luminal Ca2+-mediated RYR2 regulation is under debate, and it has been attributed to Ca2+ binding on the cytosolic face (the Ca2+ feedthrough mechanism) and/or the luminal face of the RYR2 channel (the true luminal mechanism). The molecular nature and location of the luminal Ca2+ site is unclear. At the single-channel level, we directly probed the RYR2 luminal face by Eu3+, considering the non-permeant nature of trivalent cations and their high binding affinities for Ca2+ sites. Without affecting essential determinants of the Ca2+ feedthrough mechanism, we found that luminal Eu3+ competitively antagonized the activation effect of luminal Ca2+ on RYR2 responsiveness to cytosolic caffeine, and no appreciable effect was observed for luminal Ba2+ (mimicking the absence of luminal Ca2+). Importantly, luminal Eu3+ caused no changes in RYR2 gating. Our results indicate that two distinct Ca2+ sites (available for luminal Ca2+ even when the channel is closed) are likely involved in the true luminal mechanism. One site facing the lumen regulates channel responsiveness to caffeine, while the other site, presumably positioned in the channel pore, governs the gating behavior.


Assuntos
Cálcio/metabolismo , Európio/química , Miocárdio/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais , Cafeína/farmacologia , Humanos , Ativação do Canal Iônico/efeitos dos fármacos
4.
Int Heart J ; 61(1): 145-152, 2020 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-31956148

RESUMO

The aim of this study was to investigate the effect of exendin-4 (Ex-4) on ventricular arrhythmias and calcium sparks-mediated calcium leak in a myocardial infarction-heart failure model.We studied the influence of exendin-4 on ventricular arrhythmogenesis in a rat myocardial infarction-heart failure model. In vivo arrhythmia studies (electrocardiogram [ECG] telemetry studies), ex vivo arrhythmia studies calcium sparks tests, and analysis of total and phosphorylated ryanodine receptor (RyR) 2 and CaMK-II were carried out in sham group, myocardial infarction (MI) group, MI + Ex-4 and MI + Ex-4 + Exendin9-39 (Ex9-39) groups.ECG telemetry studies showed an antiarrhythmic effect of exendin-4 with reduction of spontaneous ventricular arrhythmias. Exendin-4 abbreviated the APD90, which was longer in the heart failure model, and increase the APD alternans thresholds. Exendin-4 also reduced the susceptibility to burst pacing-induced arrhythmia ex vivo. Subcellular sarcoplasmic reticulum (SR) calcium leak characteristics were tested in four groups of rat cardiomyocytes. Exendin-4 reduced calcium spark mass, spark frequency, and calcium leak, which may be due to reduced S2814-RyR2 and CaMK-II phosphorylation. Co-administration of exendin 9-39 with exendin-4 partly abolished the above-mentioned effect of exendin-4.These findings suggest that exendin-4 exerts an antiarrhythmic effect through decreasing SR calcium leak in spontaneous and burst pacing-induced ventricular arrhythmias, which may be due to reduced RyR2 phosphorylation and suppressed CaMK-II activity. Exendin-4 may act as a novel antiarrhythmic strategy in heart failure.


Assuntos
Arritmias Cardíacas/fisiopatologia , Exenatida/metabolismo , Insuficiência Cardíaca/fisiopatologia , Retículo Sarcoplasmático/metabolismo , Animais , Sinalização do Cálcio , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Modelos Animais de Doenças , Eletrocardiografia , Insuficiência Cardíaca/metabolismo , Masculino , Ratos , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo
5.
J Agric Food Chem ; 68(6): 1731-1740, 2020 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-31951399

RESUMO

Diamide insecticides targeting ryanodine receptors (RyRs) are a major class of pesticides used to control a wide range of agricultural pests, but their efficacies have been reduced dramatically by the recent emergence of resistance mutations. There is a pressing need to develop novel insecticides, targeting distinct and novel binding sites within insect RyRs to overcome the resistance crisis; however, the limited structural information on insect RyRs is a major roadblock to our understanding of their molecular mechanisms. Here, we report the crystal structure of the RyR SPRY2 domain from the diamondback moth (DBM), Plutella xylostella, a destructive agricultural pest worldwide that has developed resistance to all classes of insecticide at 2.06 Å resolution. The overall fold of DBM SPRY2 is similar to its mammalian homolog, but it shows distinct conformations in several loops. Docking it into the recently published cryo-electron microscope structure of the full-length RyR reveals that two insect-specific loops interact with the BSol domain from the neighboring subunit. The SPRY2-BSol interface will change the conformation upon channel gating, indicating that it might be a potential targeting site for insect-specific insecticides. Interestingly, several previously identified disease-causing mutations also lie in the same interface, implying that this interface is important for channel gating. Another insect-specific loop located in the SPRY2-SPRY3 interface might indirectly affect another gating interface between SPRY3 and Repeat34.


Assuntos
Diamida/química , Proteínas de Insetos/química , Inseticidas/química , Mariposas/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Animais , Diamida/farmacologia , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Inseticidas/farmacologia , Simulação de Acoplamento Molecular , Mariposas/química , Mariposas/efeitos dos fármacos , Mariposas/genética , Domínios Proteicos , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo
6.
Life Sci ; 242: 117158, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31837328

RESUMO

AIMS: Pediatric heart failure is a common cardiovascular disease in clinical pediatrics. CCCTC-binding factor (CTCF), a novel transcriptional repressor, was reported to participate in the occurrence of various cardiovascular diseases. The present study focuses on exploring the effects of CTCF on tunicamycin (TM)-induced endoplasmic reticulum (ER) stress, and investigating the underlying mechanisms. MATERIALS AND METHOD: Expression of CTCF in blood samples of heart failure children and TM-induced cardiomyocytes were evaluated by real-time quantitative PCR (RT-qPCR). Apoptotic rate of cardiomyocytes was detected by Annexin v assay. Western blotting and enzyme-linked immunosorbent assay (ELISA) were applied to examine the effect of CTCF on ER stress. Co-immunoprecipitation and western blotting were devoted to explore the mechanism by which CTCF contributes to ER stress. KEY FINDINGS: We proved that CTCF was lowly expressed in blood samples of heart failure children and TM-induced cardiomyocytes, and overexpression of CTCF weaken the TM-induced ER stress. Using co-immunoprecipitation and protein blots, we demonstrated that CTCF upregulates RYR2 by inhibiting S100A1, thus mediating the PERK signaling pathway and regulating ER stress. SIGNIFICANCE: Our data revealed that CTCF protects cardiomyocytes from ER stress through S100A1-RYR2 axis, and can be applied as a therapeutic target for the treatment of pediatric heart failure in future.


Assuntos
Apoptose/fisiologia , Fator de Ligação a CCCTC/fisiologia , Estresse do Retículo Endoplasmático/fisiologia , Miócitos Cardíacos/efeitos dos fármacos , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Proteínas S100/metabolismo , Adolescente , Animais , Western Blotting , Fator de Ligação a CCCTC/metabolismo , Criança , Pré-Escolar , Ensaio de Imunoadsorção Enzimática , Feminino , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Humanos , Imunoprecipitação , Masculino , Camundongos , Miócitos Cardíacos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia , Proteínas S100/fisiologia , Regulação para Cima
7.
Sci Total Environ ; 701: 134901, 2020 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-31710906

RESUMO

Heavy metal lead (Pb) is widely distributed in the environment and can induce neurodegeneration. Accumulating evidence has shown that ryanodine receptors (RyRs) play vital roles in neurodegenerative brain. However, whether aberrant RyRs levels contribute to Pb-induced neurodegeneration has largely remained unknown. In the present study, we report the important role of elevated levels of RyRs in Pb-induced neurodegeneration. Pb was found to upregulate the levels of RyRs in the rat hippocampal tissues and rat pheochromocytoma (PC12) cells. Furthermore, exposure to Pb induced neurodegenerative cognitive impairment in rats, depressed the long-term potentiation (LTP) in the rat brain slices, increased the neuronal intracellular free calcium concentration ([Ca2+]i), inhibited the phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and cyclic adenosine 3',5'-monophosphate (cAMP) response element binding protein (CREB) as well as the expression of anti-apoptotic protein B-cell lymphoma 2 (Bcl2), and activated the phosphorylation of extracellular regulated protein kinases (Erk) protein both in vitro and in vivo. In addition, the knockdown of RyR3 in PC12 cells significantly decreased the [Ca2+]i levels, increased the CaMKIIα and CREB phosphorylation, decrease the phosphorylation of Erk, and elongated the cognitive function-related neurite outgrowth after exposure to Pb. Moreover, treatment with a RyRs agonist showed the involvement of RyRs in Pb-induced depression in LTP in the rat brain slices. In summary, we determined that Pb-mediated upregulation of RyRs led to neurodegeneration via high levels of free calcium, depression of the calcium-dependent CaMKIIα/CREB mnemonic signaling pathway, and activation of the calcium-dependent Erk/Bcl2 apoptotic signaling pathway. These findings on the impact of Pb on the levels of RyRs could further improve our understanding of Pb-induced neurotoxicity and provide a promising molecular target to antagonize Pb-induced neurodegenerative diseases.


Assuntos
Chumbo/toxicidade , Doenças Neurodegenerativas/induzido quimicamente , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais , Sinalização do Cálcio/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , Ratos
8.
Adv Exp Med Biol ; 1131: 281-320, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646515

RESUMO

In mammalian cardiomyocytes, Ca2+ influx through L-type voltage-gated Ca2+ channels (VGCCs) is amplified by release of Ca2+ via type 2 ryanodine receptors (RyR2) in the sarcoplasmic reticulum (SR): a process termed Ca2+-induced Ca2+-release (CICR). In mammalian skeletal muscles, VGCCs play a distinct role as voltage-sensors, physically interacting with RyR1 channels to initiate Ca2+ release in a mechanism termed depolarisation-induced Ca2+-release (DICR). In the current study, we surveyed the genomes of animals and their close relatives, to explore the evolutionary history of genes encoding three proteins pivotal for ECC: L-type VGCCs; RyRs; and a protein family that anchors intracellular organelles to plasma membranes, namely junctophilins (JPHs). In agreement with earlier studies, we find that non-vertebrate eukaryotes either lack VGCCs, RyRs and JPHs; or contain a single homologue of each protein. Furthermore, the molecular features of these proteins thought to be essential for DICR are only detectable within vertebrates and not in any other taxonomic group. Consistent with earlier physiological and ultrastructural observations, this suggests that CICR is the most basal form of ECC and that DICR is a vertebrate innovation. This development was accompanied by the appearance of multiple homologues of RyRs, VGCCs and junctophilins in vertebrates, thought to have arisen by 'whole genome replication' mechanisms. Subsequent gene duplications and losses have resulted in distinct assemblies of ECC components in different vertebrate clades, with striking examples being the apparent absence of RyR2 from amphibians, and additional duplication events for all three ECC proteins in teleost fish. This is consistent with teleosts possessing the most derived mode of DICR, with their Cav1.1 VGCCs completely lacking in Ca2+ channel activity.


Assuntos
Canais de Cálcio Tipo L , Evolução Molecular , Acoplamento Excitação-Contração , Canal de Liberação de Cálcio do Receptor de Rianodina , Animais , Canais de Cálcio Tipo L/metabolismo , Acoplamento Excitação-Contração/genética , Peixes/genética , Peixes/metabolismo , Genoma/genética , Músculo Esquelético/fisiologia , Miócitos Cardíacos/fisiologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/fisiologia
9.
Adv Exp Med Biol ; 1131: 321-336, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646516

RESUMO

Ryanodine receptor calcium release channels (RyRs) play central roles in controlling intracellular calcium concentrations in excitable and non-excitable cells. RyRs are located in the sarcoplasmic or endoplasmic reticulum, intracellular Ca2+ storage compartment, and release Ca2+ during cellular action potentials or in response to other cellular stimuli. Mammalian cells express three structurally related isoforms of RyR. RyR1 and RyR2 are the major RyR isoforms in skeletal and cardiac muscle, respectively, and RyR3 is expressed in various tissues along with the other two isoforms. A prominent feature of RyRs is that the Ca2+ release channel activities of RyRs are regulated by calcium ions; therefore, intracellular Ca2+ release controls positive- and negative-feedback phenomena through the RyRs. RyR channel activities are also regulated by Ca2+ indirectly, i.e. through Ca2+ binding proteins at both cytosolic and sarco/endoplasmic reticulum luminal sides. Here, I summarize Ca2+-dependent feedback regulation of RyRs including recent progress in the structure/function aspects.


Assuntos
Cálcio , Regulação da Expressão Gênica , Canal de Liberação de Cálcio do Receptor de Rianodina , Animais , Cálcio/metabolismo , Citosol/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Relação Estrutura-Atividade
10.
Adv Exp Med Biol ; 1131: 337-370, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646517

RESUMO

The sarcoplasmic/endoplasmic reticulum (SR/ER) is the main intracellular calcium (Ca2+) pool in muscle and non-muscle eukaryotic cells, respectively. The reticulum accumulates Ca2+ against its electrochemical gradient by the action of sarco/endoplasmic reticulum calcium ATPases (SERCA pumps), and the capacity of this Ca2+ store is increased by the presence of Ca2+ binding proteins in the lumen of the reticulum. A diversity of physical and chemical signals, activate the main Ca2+ release channels, i.e. ryanodine receptors (RyRs) and inositol (1, 4, 5) trisphosphate receptors (IP3Rs), to produce transient elevations of the cytoplasmic calcium concentration ([Ca2+]i) while the reticulum is being depleted of Ca2+. This picture is incomplete because it implies that the elements involved in the Ca2+ release process are acting alone and independently of each other. However, it appears that the Ca2+ released by RyRs and IP3Rs is trapped in luminal Ca2+ binding proteins (Ca2+ lattice), which are associated with these release channels, and the activation of these channels appears to facilitate that the trapped Ca2+ ions become available for release. This situation makes the initial stage of the Ca2+ release process a highly efficient one; accordingly, there is a large increase in the [Ca2+]i with minimal reductions in the bulk of the free luminal SR/ER [Ca2+] ([Ca2+]SR/ER). Additionally, it has been shown that active SERCA pumps are required for attaining this highly efficient Ca2+ release process. All these data indicate that Ca2+ release by the SR/ER is a highly regulated event and not just Ca2+ coming down its electrochemical gradient via the open release channels. One obvious advantage of this sophisticated Ca2+ release process is to avoid depletion of the ER Ca2+ store and accordingly, to prevent the activation of ER stress during each Ca2+ release event.


Assuntos
Cálcio , Retículo Endoplasmático , Retículo Sarcoplasmático , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Retículo Endoplasmático/metabolismo , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo
11.
Adv Exp Med Biol ; 1131: 371-394, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646518

RESUMO

Ca2+ signals are probably the most common intracellular signaling cellular events, controlling an extensive range of responses in virtually all cells. Many cellular stimuli, often acting at cell surface receptors, evoke Ca2+ signals by mobilizing Ca2+ from intracellular stores. Inositol trisphosphate (IP3) was the first messenger shown to link events at the plasma membrane to release Ca2+ from the endoplasmic reticulum (ER), through the activation of IP3-gated Ca2+ release channels (IP3 receptors). Subsequently, two additional Ca2+ mobilizing messengers were discovered, cADPR and NAADP. Both are metabolites of pyridine nucleotides, and may be produced by the same class of enzymes, ADP-ribosyl cyclases, such as CD38. Whilst cADPR mobilizes Ca2+ from the ER by activation of ryanodine receptors (RyRs), NAADP releases Ca2+ from acidic stores by a mechanism involving the activation of two pore channels (TPCs). In addition, other pyridine nucleotides have emerged as intracellular messengers. ADP-ribose and 2'-deoxy-ADPR both activate TRPM2 channels which are expressed at the plasma membrane and in lysosomes.


Assuntos
Cálcio , ADP-Ribose Cíclica , Piridinas , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Retículo Endoplasmático/metabolismo , Humanos , Espaço Intracelular/metabolismo , NADP/metabolismo , Piridinas/química , Piridinas/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo
12.
J Agric Food Chem ; 67(49): 13751-13757, 2019 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-31721577

RESUMO

Although the action site of wilforine is located in the muscle tissue of insects, the insecticidal mechanism of wilforine is not yet clear. This research explored the effects of wilforine on the calcium signaling pathway using the calcium imaging technique to reveal the insecticidal mechanism. It was confirmed that wilforine had strong cytotoxicity to Mythimna separata myocytes with the IC50 values of 25.14 and 19.65 mg/L using CCK-8 and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide methods, respectively. The morphological development of M. separata myocytes was also affected. The calcium imaging technique showed that the intracellular calcium ion concentration ([Ca2+]i) increased by 23.45% of the initial value after being treated with 100 nM wilforine. However, wilforine did not increase [Ca2+]i after the myocytes were preincubated with thapsigargin, and the [Ca2+]i could not be decreased by 50 µM ryanodine after being treated with 100 nM wilforine. These results indicated that the targets of wilforine are located in the sarcoplasmic reticulum, and ryanodine receptor (RyR) is an important action target of wilforine. Furthermore, wilforine can also activate the inositol triphosphate receptor (IP3R), which was confirmed through the use of 2-aminoethyl diphenylborinate, an inhibitor of IP3R. Connected with previous research studies, it can be concluded that wilforine affects the calcium signaling pathway by combining with RyR and IP3R, causing calcium dyshomeostasis, which results in insect paralysis and death.


Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Inseticidas/toxicidade , Lactonas/toxicidade , Mariposas/efeitos dos fármacos , Piridinas/toxicidade , Animais , Cálcio/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Mariposas/genética , Mariposas/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo
13.
Int J Mol Sci ; 20(16)2019 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-31426283

RESUMO

Arrhythmogenic cardiomyopathy (ACM) is an inherited heart disease characterized by sudden death in young people and featured by fibro-adipose myocardium replacement, malignant arrhythmias, and heart failure. To date, no etiological therapies are available. Mutations in desmosomal genes cause abnormal mechanical coupling, trigger pro-apoptotic signaling pathways, and induce fibro-adipose replacement. Here, we discuss the hypothesis that the ACM causative mechanism involves a defect in the expression and/or activity of the cardiac Ca2+ handling machinery, focusing on the available data supporting this hypothesis. The Ca2+ toolkit is heavily remodeled in cardiomyocytes derived from a mouse model of ACM defective of the desmosomal protein plakophilin-2. Furthermore, ACM-related mutations were found in genes encoding for proteins involved in excitation‒contraction coupling, e.g., type 2 ryanodine receptor and phospholamban. As a consequence, the sarcoplasmic reticulum becomes more eager to release Ca2+, thereby inducing delayed afterdepolarizations and impairing cardiac contractility. These data are supported by preliminary observations from patient induced pluripotent stem-cell-derived cardiomyocytes. Assessing the involvement of Ca2+ signaling in the pathogenesis of ACM could be beneficial in the treatment of this life-threatening disease.


Assuntos
Arritmias Cardíacas/patologia , Cálcio/metabolismo , Cardiomiopatias/patologia , Desmossomos/patologia , Miócitos Cardíacos/patologia , Animais , Arritmias Cardíacas/metabolismo , Sinalização do Cálcio , Cardiomiopatias/metabolismo , Desmossomos/metabolismo , Humanos , Miócitos Cardíacos/metabolismo , Placofilinas/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo
14.
Mar Drugs ; 17(8)2019 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-31426471

RESUMO

Herein, we report on the synthesis of a small set of linear precursors of an inosine analogue of cyclic ADP-ribose (cADPR), a second messenger involved in Ca2+ mobilization from ryanodine receptor stores firstly isolated from sea urchin eggs extracts. The synthesized compounds were obtained starting from inosine and are characterized by an N1-alkyl chain replacing the "northern" ribose and a phosphate group attached at the end of the N1-alkyl chain and/or 5'-sugar positions. Preliminary Ca2+ mobilization assays, performed on differentiated C2C12 cells, are reported as well.


Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Cálcio/metabolismo , ADP-Ribose Cíclica/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Ouriços-do-Mar/química , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Ovos , Relação Estrutura-Atividade
15.
Mol Pharmacol ; 96(3): 401-407, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31337666

RESUMO

Dantrolene is a ryanodine receptor (RyR) inhibitor, which is used to relax muscles in malignant hyperthermia syndrome. Although dantrolene binds to the RyR protein, its mechanism of action is unknown, mainly because of the controversial results showing that dantrolene inhibited Ca2+ release from intact fibers and sarcoplasmic reticulum (SR) vesicles, but failed to inhibit single RyR channel currents in bilayers. Accordingly, it was concluded that an important factor for dantrolene's action was lost during the purification procedure of RyR. Recently, Mg2+ was demonstrated to be the essential factor for dantrolene to inhibit Ca2+ release in skinned muscle fibers. The aim of the present study was to confirm these results in Ca2+ release and bilayer experiments, using SR vesicles and solubilized channels, respectively. Our Ca2+ release experiments demonstrated that the effect of dantrolene and Mg2+ was cooperative and that ATP enhanced the inhibiting effect of dantrolene. Namely, 10 µM dantrolene reduced RyR channel open probability by ∼50% in the presence of 3 mM free Mg2+ and 1 mM ATP, whereas channel activity further decreased to ∼20% of control when [ATP] was increased to 2 mM. Our data provide important complementary information that supports the direct, Mg2+-dependent mechanism of dantrolene's action and suggests that dantrolene also requires ATP to inhibit RyR.


Assuntos
Trifosfato de Adenosina/metabolismo , Dantroleno/farmacologia , Magnésio/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais , Sítios de Ligação , Cálcio/metabolismo , Dantroleno/química , Masculino , Modelos Moleculares , Conformação Molecular , Músculo Esquelético/metabolismo , Ligação Proteica , Coelhos , Canal de Liberação de Cálcio do Receptor de Rianodina/química
16.
Eur J Med Chem ; 179: 837-848, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31299492

RESUMO

Type-1 ryanodine receptor (RyR1) is a calcium-release channel localized on sarcoplasmic reticulum (SR) of the skeletal muscle, and mediates muscle contraction by releasing Ca2+ from the SR. Genetic mutations of RyR1 are associated with skeletal muscle diseases such as malignant hyperthermia and central core diseases, in which over-activation of RyR1 causes leakage of Ca2+ from the SR. We recently developed an efficient high-throughput screening system based on the measurement of Ca2+ in endoplasmic reticulum, and used it to identify oxolinic acid (1) as a novel RyR1 channel inhibitor. Here, we designed and synthesized a series of quinolone derivatives based on 1 as a lead compound. Derivatives bearing a long alkyl chain at the nitrogen atom of the quinolone ring and having a suitable substituent at the 7-position of quinolone exhibited potent RyR1 channel-inhibitory activity. Among the synthesized compounds, 14h showed more potent activity than dantrolene, a known RyR1 inhibitor, and exhibited high RyR1 selectivity over RyR2 and RyR3. These compounds may be promising leads for clinically applicable RyR1 channel inhibitors.


Assuntos
Cálcio/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Quinolonas/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Estrutura Molecular , Quinolonas/síntese química , Quinolonas/química , Relação Estrutura-Atividade
17.
Nat Commun ; 10(1): 3223, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31324793

RESUMO

It is widely assumed that inositol trisphosphate (IP3) and ryanodine (Ry) receptors share the same Ca2+ pool in central mammalian neurons. We now demonstrate that in hippocampal CA1 pyramidal neurons IP3- and Ry-receptors are associated with two functionally distinct intracellular Ca2+ stores, respectively. While the IP3-sensitive Ca2+ store refilling requires Orai2 channels, Ry-sensitive Ca2+ store refilling involves voltage-gated Ca2+ channels (VGCCs). Our findings have direct implications for the understanding of function and plasticity in these central mammalian neurons.


Assuntos
Cálcio/metabolismo , Hipocampo/metabolismo , Proteína ORAI2/metabolismo , Células Piramidais/metabolismo , Animais , Canais de Cálcio , Regulação da Expressão Gênica , Fosfatos de Inositol/metabolismo , Íons , Camundongos , Camundongos Knockout , Modelos Animais , Proteína ORAI2/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo
18.
Eur Biophys J ; 48(6): 579-584, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31236612

RESUMO

Calcium release sites (CRSs) play a key role in excitation-contraction coupling of cardiac myocytes. Recent studies based on electron tomography and super-resolution imaging revealed that CRSs are not completely filled with ryanodine receptors (RyRs) and that the spatial arrangement of RyRs is neither uniform nor static. In this work, we studied the effect of spatial arrangement of RyRs on RyR activation using simulations based on Monte Carlo (MC) and mean-field (MF) approaches. Both approaches showed that activation of RyRs is sensitive to the arrangement of RyRs in the CRS. However, the MF simulations did not reproduce results of MC simulations for non-compact CRSs, suggesting that the approximations used in the MF approach are not suitable for simulation studies of RyRs arrangements observed experimentally. MC simulations revealed the importance of realistic spatial arrangement of RyRs for adequate modelling of calcium release in cardiac myocytes.


Assuntos
Cálcio/metabolismo , Modelos Biológicos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Método de Monte Carlo , Processos Estocásticos
19.
Environ Health Perspect ; 127(6): 67003, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31166131

RESUMO

BACKGROUND: The pyrethroid deltamethrin (DM) is broadly used for insect control. Although DM hyperexcites neuronal networks by delaying inactivation of axonal voltage-dependent [Formula: see text] channels, this mechanism is unlikely to mediate neurotoxicity at lower exposure levels during critical perinatal periods in mammals. OBJECTIVES: We aimed to identify mechanisms by which acute and subchronic DM altered axonal and dendritic growth, patterns of synchronous [Formula: see text] oscillations (SCOs), and electrical spike activity (ESA) functions critical to neuronal network formation. METHODS: Measurements of SCOs using [Formula: see text] imaging, ESA using microelectrode array (MEA) technology, and dendritic complexity using Sholl analysis were performed in primary murine cortical neurons from wild-type (WT) and/or ryanodine receptor 1 ([Formula: see text]) mice between 5 and 14 d in vitro (DIV). [Formula: see text] binding analysis and a single-channel voltage clamp were utilized to measure engagement of RyRs as a direct target of DM. RESULTS: Neuronal networks responded to DM ([Formula: see text]) as early as 5 DIV, reducing SCO amplitude and depressing ESA and burst frequencies by 60-70%. DM ([Formula: see text]) enhanced axonal growth in a nonmonotonic manner. [Formula: see text] enhanced dendritic complexity. DM stabilized channel open states of RyR1, RyR2, and cortical preparations expressing all three isoforms. DM ([Formula: see text]) altered gating kinetics of RyR1 channels, increasing mean open time, decreasing mean closed time, and thereby enhancing overall open probability. SCO patterns from cortical networks expressing [Formula: see text] were more responsive to DM than WT. [Formula: see text] neurons showed inherently longer axonal lengths than WT neurons and maintained less length-promoting responses to nanomolar DM. CONCLUSIONS: Our findings suggested that RyRs were sensitive molecular targets of DM with functional consequences likely relevant for mediating abnormal neuronal network connectivity in vitro. https://doi.org/10.1289/EHP4583.


Assuntos
Neurônios/efeitos dos fármacos , Nitrilos/toxicidade , Piretrinas/toxicidade , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais , Axônios/efeitos dos fármacos , Cálcio/metabolismo , Células Cultivadas , Feminino , Inseticidas/toxicidade , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Plasticidade Neuronal/efeitos dos fármacos , Canal de Liberação de Cálcio do Receptor de Rianodina/genética
20.
Int J Mol Sci ; 20(12)2019 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-31207916

RESUMO

Cardiomyocytes and myocardial sleeves dissociated from pulmonary veins (PVs) potentially generate ectopic automaticity in response to noradrenaline (NA), and thereby trigger atrial fibrillation. We developed a mathematical model of rat PV cardiomyocytes (PVC) based on experimental data that incorporates the microscopic framework of the local control theory of Ca2+ release from the sarcoplasmic reticulum (SR), which can generate rhythmic Ca2+ release (limit cycle revealed by the bifurcation analysis) when total Ca2+ within the cell increased. Ca2+ overload in SR increased resting Ca2+ efflux through the type II inositol 1,4,5-trisphosphate (IP3) receptors (InsP3R) as well as ryanodine receptors (RyRs), which finally triggered massive Ca2+ release through activation of RyRs via local Ca2+ accumulation in the vicinity of RyRs. The new PVC model exhibited a resting potential of -68 mV. Under NA effects, repetitive Ca2+ release from SR triggered spontaneous action potentials (APs) by evoking transient depolarizations (TDs) through Na+/Ca2+ exchanger (APTDs). Marked and variable latencies initiating APTDs could be explained by the time courses of the α1- and ß1-adrenergic influence on the regulation of intracellular Ca2+ content and random occurrences of spontaneous TD activating the first APTD. Positive and negative feedback relations were clarified under APTD generation.


Assuntos
Potenciais de Ação , Catecolaminas/farmacologia , Modelos Teóricos , Miócitos Cardíacos/metabolismo , Veias Pulmonares/metabolismo , Animais , Sinalização do Cálcio , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Veias Pulmonares/citologia , Veias Pulmonares/efeitos dos fármacos , Veias Pulmonares/fisiologia , Ratos , Receptores Adrenérgicos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Trocador de Sódio e Cálcio/metabolismo
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