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1.
Life Sci ; 243: 117293, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31930971

RESUMO

Ca2+ overload in neurons has been implicated in Alzheimer's Disease (AD). Upregulation of Ca2+ through L-type Ca2+ channels was known to be involved in the neurodegeneration induced by amyloid-ß (Aß) peptides in AD. However, little is known about the mechanism by which upregulation of L-type Ca2+ channel currents is linked to Aß-induced neuronal toxicity. In the present study, we found that the L-type Ca2+ current in transgenic AD mice (Tg2576) neurons is greater than in wild-type (WT) neurons, and this Ca2+ channel current change were rescued in Tg2576/p75NTR+/- (p75 neurotrophin receptor) neurons. We further examined the changes in the gating of L-type Ca2+ channels following Aß42 treatment, and the results showed that the L-type Ca2+ channel current was significantly increased by Aß42 treatment in WT hippocampal neurons. Blocking or decreasing the expression of p75NTR eliminated the influence of Aß42 on the L-type Ca2+ channel current in WT hippocampal neurons. We also evaluated how Aß42 affected the voltage-dependent activation and inactivation of L-type Ca2+ channels in cultured WT neurons. The results indicated that the half-maximal activation voltage (V1/2) was left shifted, and the half-inactivation voltage (V1/2) displayed a right shift in neuron treated by Aß42. Decreasing the expression of p75NTR eliminated the effect of Aß42 on voltage-dependent activation and inactivation of the L-type Ca2+ channel. These results indicate that Aß42 changes L-type Ca2+ channel currents by modulating the channel's activation and inactivation dynamics, while decreasing p75NTR expression can remove this effect.


Assuntos
Peptídeos beta-Amiloides/farmacologia , Canais de Cálcio Tipo L/metabolismo , Neurônios/metabolismo , Receptor de Fator de Crescimento Neural/fisiologia , Peptídeos beta-Amiloides/metabolismo , Animais , Células Cultivadas , Humanos , Ativação do Canal Iônico , Camundongos , Camundongos Transgênicos
2.
Nature ; 577(7792): 695-700, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31969708

RESUMO

Increased cardiac contractility during the fight-or-flight response is caused by ß-adrenergic augmentation of CaV1.2 voltage-gated calcium channels1-4. However, this augmentation persists in transgenic murine hearts expressing mutant CaV1.2 α1C and ß subunits that can no longer be phosphorylated by protein kinase A-an essential downstream mediator of ß-adrenergic signalling-suggesting that non-channel factors are also required. Here we identify the mechanism by which ß-adrenergic agonists stimulate voltage-gated calcium channels. We express α1C or ß2B subunits conjugated to ascorbate peroxidase5 in mouse hearts, and use multiplexed quantitative proteomics6,7 to track hundreds of proteins in the proximity of CaV1.2. We observe that the calcium-channel inhibitor Rad8,9, a monomeric G protein, is enriched in the CaV1.2 microenvironment but is depleted during ß-adrenergic stimulation. Phosphorylation by protein kinase A of specific serine residues on Rad decreases its affinity for ß subunits and relieves constitutive inhibition of CaV1.2, observed as an increase in channel open probability. Expression of Rad or its homologue Rem in HEK293T cells also imparts stimulation of CaV1.3 and CaV2.2 by protein kinase A, revealing an evolutionarily conserved mechanism that confers adrenergic modulation upon voltage-gated calcium channels.


Assuntos
Canais de Cálcio Tipo L/metabolismo , Proteômica , Receptores Adrenérgicos beta/metabolismo , Animais , Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo N/metabolismo , Microambiente Celular , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Feminino , Células HEK293 , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Masculino , Camundongos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Miocárdio/metabolismo , Fosforilação , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Transdução de Sinais , Proteínas ras/química , Proteínas ras/metabolismo
3.
Gene ; 722: 144101, 2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31479714

RESUMO

The catadromous species, eels, invariably exposed to variable Ca2+ concentrations circumstance i.e., lagoon or ocean. They need to maintain Ca2+ homeostasis by exchanging Ca2+ under different culture conditions. To understand the effects of environmental Ca2+ to fish, three types of genes coding for voltage-dependent L-type calcium channels (cacnb1, 2, 3) were cloned by screening an A. marmorata cDNA library. Tissue distribution analysis of Western blot showed that Cacnb1, 2, 3 had a significantly high expression in gill; while mRNA results showed the expressions of cacnb1 and cacnb3 were predominated in skin tissue but only cacnb2 was expressed in intestine. Serum osmolality and Ca2+ concentrations of A.marmorata were increased in a high calcium environment while reduced in a low calcium environment within 7 days; however, they were not significantly different among Ca2+ treatments after the eels were acclimated for 7 days. We also examined the influence of ambient Ca2+ levels on cacnbs expression of eels. With the increasing of exposure time, mRNA and protein expressions of cacnb1 were up-regulated in high level of Ca2+ (10 mM) and down-regulated in deficient Ca2+ (0 mM) compared to the control Ca2+ (2 mM). However, the opposite results were observed in cacnb2 and cacnb3. Notably, the cacnb2 expression was not significant different among Ca2+ treatments on day 7. Our study provided the insightful evidence that cacnbs play important roles in maintaining Ca2+ homeostasis of fish.


Assuntos
Anguilla/metabolismo , Canais de Cálcio Tipo L/metabolismo , Cálcio/fisiologia , Aclimatação , Anguilla/sangue , Anguilla/genética , Animais , Cálcio/sangue , Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/genética , Clonagem Molecular , Brânquias/metabolismo , Concentração Osmolar , RNA Mensageiro/metabolismo , Distribuição Tecidual
4.
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
5.
Life Sci ; 238: 116953, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31626793

RESUMO

AIMS: This study focused on investigating whether NS8593 reverses airway smooth muscle (ASM) contraction and the underlying mechanism. MAIN METHODS: ASM contraction in mouse tracheal rings and lung slices was measured. Currents mediated by voltage dependent Ca2+ channels (VDCCs) and ACH-activated channels were measured using the whole-cell patch-clamp technique in single tracheal smooth muscle cells (TSMCs). Intracellular Ca2+ level and cell length were measured using an LSM 700 laser confocal microscope and a Zen 2010 software. Mouse respiratory system resistance (Rrs) was assessed using a FlexiVent FX system. KEY FINDINGS: High K+ (80 mM K+) and ACH induced ASM contraction in mouse tracheal rings and lung slices, which was partially relaxed by nifedipine (blocker of L-type VDCCs, LVDCCs), YM-58483 (blocker of store-operated Ca2+ entry (SOCE), transient receptor potential C3 (TRPC3) and TRPC5 channels), respectively. However, the contraction was completely reversed by NS8593, whereas, slightly relaxed by formoterol. ACH activated inward currents, which displayed linear and reversed around 0 mV, indicating the currents were mediated by non-selective cation channels (NSCCs). Moreover, these currents were blocked by YM-58483. In addition, such currents were abolished by NS8593, implicating that NS8593 inhibits the same channels. Besides, NS8593 inhibited increases of intracellular Ca2+ and the associated cell shortening. Finally, NS8593 inhibited ACH-induced increases of mouse respirator system resistance (Rrs). SIGNIFICANCE: Our results indicate that NS8593 inhibits LVDCCs and NSCCs, resulting in decreases of intracellular Ca2+ and then leading to ASM relaxation. These data suggest that NS8593 might be a new bronchodilator.


Assuntos
1-Naftilamina/análogos & derivados , Asma/tratamento farmacológico , Canais de Cálcio Tipo L/química , Cálcio/metabolismo , Contração Muscular/efeitos dos fármacos , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , 1-Naftilamina/farmacologia , Animais , Antialérgicos/farmacologia , Asma/induzido quimicamente , Asma/patologia , Canais de Cálcio Tipo L/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Músculo Liso/metabolismo , Músculo Liso/patologia , Ovalbumina/toxicidade
6.
Rev Cardiovasc Med ; 20(3): 139-151, 2019 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-31601088

RESUMO

Effective therapy of hypertension represents a key strategy for reducing the burden of cardiovascular disease and its associated mortality. The significance of voltage dependent L-type Ca²âº channels to Ca²âº influx, and of their regulatory mechanisms in the development of heart disease, is well established. A wide variety of L-type Ca²âº channel inhibitors and Ca²âº antagonists have been found to be beneficial not only in the treatment of hypertension, but also in myocardial infarction and heart failure. Over the past two decades, another class of Ca²âº channel - the voltage independent store-operated Ca²âº channel - has been implicated in the regulation and fine tuning of Ca²âº entry in both cardiac and smooth muscle cells. Store-operated Ca²âº channels are activated by the depletion of Ca²âº stores within the endoplasmic/sarcoplasmic reticulum, or by low levels of cytosolic Ca²âº, thereby facilitating agonist-induced Ca²âº influx. Store-operated Ca²âº entry through this pivotal pathway involves both stromal interaction molecule (STIM) and Orai channels. Different degrees of changes in these proteins are considered to promote Ca²âº entry and hence contribute to the pathogenesis of cardiovascular dysfunction. Several blockers of store-operated Ca²âº channels acting at the level of both STIM and Orai channels have been shown to depress Ca²âº influx and lower blood pressure. However, their specificity, safety, and clinical significance remain to be established. Thus, there is an ongoing challenge in the development of selective inhibitors of store-operated Ca²âº channels that act in vascular smooth muscles for the improved treatment of hypertension.


Assuntos
Anti-Hipertensivos/uso terapêutico , Pressão Sanguínea/efeitos dos fármacos , Bloqueadores dos Canais de Cálcio/uso terapêutico , Canais de Cálcio Ativados pela Liberação de Cálcio/antagonistas & inibidores , Hipertensão/tratamento farmacológico , Músculo Liso Vascular/efeitos dos fármacos , Moléculas de Interação Estromal/antagonistas & inibidores , Vasodilatadores/uso terapêutico , Animais , Anti-Hipertensivos/efeitos adversos , Bloqueadores dos Canais de Cálcio/efeitos adversos , Canais de Cálcio Tipo L/efeitos dos fármacos , Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio Ativados pela Liberação de Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Humanos , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiopatologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Moléculas de Interação Estromal/metabolismo , Resultado do Tratamento , Vasodilatadores/efeitos adversos
7.
Sheng Li Xue Bao ; 71(5): 717-724, 2019 Oct 25.
Artigo em Chinês | MEDLINE | ID: mdl-31646325

RESUMO

The aim of this study was to investigate the effect of interleukin 6 (IL-6) on the contraction of colon longitudinal muscle strips in rats with acute pancreatitis (AP) and its underlying mechanism. Rat AP model was established by combined injection (i. p.) of ceruletide and lipopolysaccharide. The effect of IL-6 on spontaneous contraction of longitudinal smooth muscle strips of rat colon was observed by biological function experiment system. The level of serum IL-6 was detected by ELISA, the expression and distribution of IL-6 in colon were observed by histochemical staining, and the effect of IL-6 on L-type calcium channel in colon smooth muscle cells was observed by whole cell patch clamp technique. The results showed that, compared with the control group, AP group exhibited reduced contractile amplitude and longer contraction cycle of colon smooth muscle strips. IL-6 prolonged the contraction cycle of colon smooth muscle strips, but did not affect their spontaneous contraction amplitude. Serum IL-6 concentration in AP group was significantly higher than that in control group (P > 0.05). IL-6 was diffusely distributed in the colon of the control group, but the expression of IL-6 was significantly up-regulated in the colon gland, mucosa and submucosa of the AP group. IL-6 significantly decreased the peak current density of L-type calcium channel in rat colon smooth muscle cells. These results suggest that the colon motility of AP rats is weakened, and the mechanism may be that up-regulated IL-6 inactivates L-type voltage-dependent calcium channels, and then inhibits the contraction of colon longitudinal smooth muscle.


Assuntos
Canais de Cálcio Tipo L/metabolismo , Interleucina-6/metabolismo , Contração Muscular , Músculo Liso/fisiopatologia , Pancreatite/fisiopatologia , Animais , Colo , Ratos
8.
Int J Mol Sci ; 20(16)2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31416128

RESUMO

The functional and structural adaptations in cerebral arteries could be one of the fundamental causes in the occurrence of orthostatic intolerance after space flight. In addition, emerging studies have found that many cardiovascular functions exhibit circadian rhythm. Several lines of evidence suggest that space flight might increase an astronaut's cardiovascular risks by disrupting circadian rhythm. However, it remains unknown whether microgravity disrupts the diurnal variation in vascular contractility and whether microgravity impacts on circadian clock system. Sprague-Dawley rats were subjected to 28-day hindlimb-unweighting to simulate the effects of microgravity on vasculature. Cerebrovascular contractility was estimated by investigating vasoconstrictor responsiveness and myogenic tone. The circadian regulation of CaV1.2 channel was determined by recording whole-cell currents, evaluating protein and mRNA expressions. Then the candidate miRNA in relation with Ca2+ signal was screened. Lastly, the underlying pathway involved in circadian regulation of cerebrovascular contractility was determined. The major findings of this study are: (1) The clock gene BMAL1 could induce the expression of miR-103, and in turn modulate the circadian regulation of CaV1.2 channel in rat cerebral arteries at post-transcriptional level; and (2) simulated microgravity disrupted intrinsic diurnal oscillation in rat cerebrovascular contractility by altering circadian regulation of BMAL1/miR-103/CaV1.2 signal pathway.


Assuntos
Fatores de Transcrição ARNTL/genética , Canais de Cálcio Tipo L/metabolismo , Circulação Cerebrovascular/genética , Ritmo Circadiano , MicroRNAs/genética , Vasoconstrição/genética , Ausência de Peso , Fatores de Transcrição ARNTL/metabolismo , Animais , Linhagem Celular , Regulação da Expressão Gênica , Masculino , Modelos Biológicos , Ratos , Transdução de Sinais
9.
Nutrients ; 11(9)2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31443528

RESUMO

Severe food restriction (FR) impairs cardiac performance, although the causative mechanisms remain elusive. Since proteins associated with calcium handling may contribute to cardiac dysfunction, this study aimed to evaluate whether severe FR results in alterations in the expression and activity of Ca2+-handling proteins that contribute to impaired myocardial performance. Male 60-day-old Wistar-Kyoto rats were fed a control or restricted diet (50% reduction in the food consumed by the control group) for 90 days. Body weight, body fat pads, adiposity index, as well as the weights of the soleus muscle and lung, were obtained. Cardiac remodeling was assessed by morphological measures. The myocardial contractile performance was analyzed in isolated papillary muscles during the administration of extracellular Ca2+ and in the absence or presence of a sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) specific blocker. The expression of Ca2+-handling regulatory proteins was analyzed via Western Blot. Severe FR resulted in a 50% decrease in body weight and adiposity measures. Cardiac morphometry was substantially altered, as heart weights were nearly twofold lower in FR rats. Papillary muscles isolated from FR hearts displayed mechanical dysfunction, including decreased developed tension and reduced contractility and relaxation. The administration of a SERCA2a blocker led to further decrements in contractile function in FR hearts, suggesting impaired SERCA2a activity. Moreover, the FR rats presented a lower expression of L-type Ca2+ channels. Therefore, myocardial dysfunction induced by severe food restriction is associated with changes in the calcium-handling properties in rats.


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Restrição Calórica , Cardiopatias/etiologia , Desnutrição/complicações , Mitocôndrias Cardíacas/metabolismo , Contração Miocárdica , Miócitos Cardíacos/metabolismo , Músculos Papilares/metabolismo , Adiposidade , Animais , Canais de Cálcio Tipo L/metabolismo , Modelos Animais de Doenças , Cardiopatias/metabolismo , Cardiopatias/patologia , Cardiopatias/fisiopatologia , Masculino , Desnutrição/metabolismo , Desnutrição/patologia , Desnutrição/fisiopatologia , Mitocôndrias Cardíacas/patologia , Miócitos Cardíacos/patologia , Músculos Papilares/patologia , Músculos Papilares/fisiopatologia , Ratos Endogâmicos WKY , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Perda de Peso
10.
Invest Ophthalmol Vis Sci ; 60(8): 3150-3161, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31335952

RESUMO

Purpose: Cav1.4 is a voltage-gated calcium channel clustered at the presynaptic active zones of photoreceptors. Cav1.4 functions in communication by mediating the Ca2+ influx that triggers neurotransmitter release. It also aids in development since rod ribbon synapses do not form in Cav1.4 knock-out mice. Here we used a rescue strategy to investigate the ability of Cav1.4 to trigger synaptogenesis in both immature and mature mouse rods. Methods: In vivo electroporation was used to transiently express Cav α1F or tamoxifen-inducible Cav α1F in a subset of Cav1.4 knock-out mouse rods. Synaptogenesis was assayed using morphologic markers and a vision-guided water maze. Results: We found that introduction of Cav α1F to knock-out terminals rescued synaptic development as indicated by PSD-95 expression and elongated ribbons. When expression of Cav α1F was induced in mature animals, we again found restoration of PSD-95 and elongated ribbons. However, the induced expression of Cav α1F led to diffuse distribution of Cav α1F in the terminal instead of being clustered beneath the ribbon. Approximately a quarter of treated animals passed the water maze test, suggesting the rescue of retinal signaling in these mice. Conclusions: These data confirm that Cav α1F expression is necessary for rod synaptic terminal development and demonstrate that rescue is robust even in adult animals with late stages of synaptic disease. The degree of rod synaptic plasticity seen here should be sufficient to support future vision-restoring treatments such as gene or cell replacement that will require photoreceptor synaptic rewiring.


Assuntos
Canais de Cálcio Tipo L/genética , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Transmissão Sináptica/genética , Animais , Animais Recém-Nascidos , Canais de Cálcio Tipo L/metabolismo , Feminino , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Terminações Pré-Sinápticas/metabolismo , Sinapses/metabolismo
11.
Eur J Pharmacol ; 859: 172488, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31233746

RESUMO

Cardiac arrhythmias are among the most important pathologies that lead to sudden death. The discovery of new therapeutic options against arrhythmias with low adverse effects is of paramount importance. Farnesol is found in essential oils with antioxidant, anti-inflammatory and cardioprotective properties. The aim of this work was to investigate the effects of farnesol on the contractile and electrophysiological properties in rat heart and evaluate its antiarrhythmic action. It was evaluated farnesol effects on the left ventricular developed pressure, ECG, potassium (Ik) and L-type Ca2+ currents (ICa,L), action potential, intracellular Ca2+ transient, Ca2+ sparks and waves and reactive oxygen species production. Antiarrhythmic activity of farnesol was determined in vivo and ex vivo. The results showed that 50 µM farnesol did not alter left ventricular developed pressure, heart rate, ECG parameters and intracellular Ca2+ transient but reduced ICa,L. Farnesol reduced action potential duration at 90% repolarization. Notably, farnesol improved arrhythmia score and the incidence of the most severe arrhythmias. Farnesol attenuated the generation of reactive oxygen species, Ca2+ sparks and waves in isolated cardiomyocytes submitted to Ca2+ overload. In conclusion, farnesol has antiarrhythmic effect mediated by reducing of ICa,L and IK along with a decrease of reactive oxygen species production and normalized Ca2+ sparks and waves.


Assuntos
Antiarrítmicos/farmacologia , Arritmias Cardíacas/tratamento farmacológico , Arritmias Cardíacas/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Cálcio/metabolismo , Farneseno Álcool/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Antiarrítmicos/uso terapêutico , Arritmias Cardíacas/patologia , Arritmias Cardíacas/fisiopatologia , Bloqueadores dos Canais de Cálcio/uso terapêutico , Canais de Cálcio Tipo L/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Eletrocardiografia/efeitos dos fármacos , Farneseno Álcool/uso terapêutico , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Masculino , Contração Miocárdica/efeitos dos fármacos , Oxigênio/metabolismo , Potássio/metabolismo , Ratos , Ratos Wistar , Disfunção Ventricular Esquerda/tratamento farmacológico
12.
Life Sci ; 231: 116576, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31211998

RESUMO

AIMS: Studies suggest that cardiovascular function in offspring can be epigenetically programmed by environmental changes during pregnancy. CaV1.2 channel plays a major role in the regulation of the vascular tone. This study investigated the effects and underlying mechanisms of exercise during pregnancy on CaV1.2 channel functional remodeling in hypertensive offspring. MAIN METHODS: Exercise groups were subjected to swimming at the first day of pregnancy and on a regular schedule thereafter for 3 weeks. Their offspring (6-month-old, male) were tested for baseline blood pressure, cardiovascular response, and vascular tone of the mesenteric artery. Mesenteric artery smooth muscle cells were taken to study the whole-cell current of the CaV1.2 channel. Western blotting, RT-PCR and DNA bisulfite sequencing PCR were performed to study the protein, mRNA expression and DNA methylation of the CaV1.2 channel α1C subunit. KEY FINDINGS: Exercise during pregnancy reduced the pressor response to norepinephrine and Bay K8644, and the depressor response to nifedipine in offspring of hypertensive rats. The level of the CaV1.2 channel in norepinephrine-induced vasoconstrictions decreased, and the whole-cell current of the CaV1.2 channel declined in the SHR-EX group. Further studies found that exercise during pregnancy reduced the protein and mRNA expression of the CaV1.2 channel α1C subunit and upregulated DNA methylation of the Cacna1c gene promoter region in the hypertensive offspring. SIGNIFICANCE: These data suggest that exercise during pregnancy improves vascular functional remodeling in offspring of hypertensive rats, downregulating the CaV1.2 channel function and protein expression, a change that is most likely caused by DNA methylation.


Assuntos
Canais de Cálcio Tipo L/fisiologia , Hipertensão/fisiopatologia , Condicionamento Físico Animal/fisiologia , Animais , Pressão Sanguínea/efeitos dos fármacos , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Metilação de DNA/efeitos dos fármacos , Regulação para Baixo , Epigênese Genética/genética , Repressão Epigenética , Feminino , Masculino , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Gravidez , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Regulação para Cima , Vasoconstrição/efeitos dos fármacos
13.
Cells ; 8(6)2019 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-31185584

RESUMO

Safety is one of the most important and critical issues in drug development. Many drugs were abandoned in clinical trials and retracted from the market because of unknown side effects. Cardiotoxicity is one of the most common reasons for drug retraction due to its potential side effects, i.e., inducing either tachycardia, bradycardia or arrhythmia. The zebrafish model could be used to screen drug libraries with potential cardiotoxicity in a high-throughput manner. In addition, the fundamental principles of replacement, reduction, and refinement of laboratory animal usage, 3R, could be achieved by using zebrafish as an alternative to animal models. In this study, we used a simple ImageJ-based method to evaluate and screen 70 ion channel ligands and successfully identify six compounds with strong cardiotoxicity in vivo. Next, we conducted an in silico-based molecular docking simulation to elucidate five identified compounds that might interact with domain III or domain IV of the Danio rerio L-type calcium channel (LTCC), a known pharmaceutically important target for arrhythmia. In conclusion, in this study, we provide a web lab and dry lab combinatorial approach to perform in vivo cardiotoxicity drug screening and in silico mechanistic studies.


Assuntos
Frequência Cardíaca , Canais Iônicos/metabolismo , Ligantes , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Sítios de Ligação , Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/metabolismo , Embrião não Mamífero/metabolismo , Frequência Cardíaca/efeitos dos fármacos , Ligações de Hidrogênio , Canais Iônicos/química , Simulação de Acoplamento Molecular , Estrutura Terciária de Proteína , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/química
14.
Chem Biol Interact ; 309: 108710, 2019 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-31199930

RESUMO

Formic acid is a common organic acid used in many industrial processes. There is a paucity of research on the direct toxicity of formic acid and how it might affect the cardiovascular system. This study aimed to understand the effect of formic acid on vascular tension in an animal model and the underlying mechanism. Results found that the vasodilation induced by formic acid was related to the endothelium. When the dosage of formic acid was 1 mM or 5 mM, the vasodilation of endothelium-intact rings was partially suppressed by l-NAME, NS-2028 and nifedipine, and vasoconstriction caused by CaCl2 was inhibited, and the mRNA levels of eNOS, the activity of NOS (tNOS, iNOS and cNOS) and the level of NO and cGMP were significantly increased. Results also found that eNOS protein expression was significantly enhanced by 5 mM of formic acid. These results suggest formic acid can relax the aortic vessels of rats in a dose-dependent pattern. Further, the mechanism of the formic acid-induced vasodilatation likely involved the NO/cGMP pathway. Finally, the current study has revealed that vasodilation induced by high concentrations of formaldehyde may be the effect of the metabolite formic acid. This study will help further inform the etiologies of formic acid-related angiocardiopathies.


Assuntos
Endotélio Vascular/efeitos dos fármacos , Formiatos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Animais , Canais de Cálcio Tipo L/metabolismo , Cloreto de Cálcio/farmacologia , GMP Cíclico/metabolismo , Endotélio Vascular/fisiologia , Expressão Gênica/efeitos dos fármacos , Masculino , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Ratos , Ratos Wistar , Vasoconstrição/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos
15.
Cell Physiol Biochem ; 53(1): 36-48, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31169990

RESUMO

BACKGROUND/AIMS: Ivabradine lowers the heart rate by inhibition of hyperpolarisation-activated cyclic nucleotide-gated (HCN) channels mediating the 'funny' pacemaker current If in the sinoatrial node. It is clinically approved for the treatment of heart failure and angina pectoris. Due to its proposed high selectivity for If administration of ivabradine is not associated with the side effects commonly observed following the application of other heart rate lowering agents. Recent evidence, however, has shown significant affinity of ivabradine towards Kv11.1 (ether-a-go-go related gene, ERG) potassium channels. Despite the inhibition of Kv11.1 channels by ivabradine, cardiac action potential (AP) duration and heart rate corrected QT interval (QTc) of the human electrocardiogram (ECG) were not prolonged. We thus surmised that compensatory mechanisms might counteract the drug's inhibitory action on Kv11.1. METHODS: The effects of ivabradine on human Kv11.1 and Kv7.1 potassium, Cav1.2 calcium, and Nav1.5 sodium channels, heterologously expressed in tsA-201 cells, were studied in the voltage-clamp mode of the whole cell patch clamp technique. In addition, changes in action potential parameters of human induced pluripotent stem cell (iPSC) derived cardiomyocytes upon application of ivabradine were studied with current-clamp experiments. RESULTS: Here we show that ivabradine exhibits significant affinity towards cardiac ion channels other than HCN. We demonstrate for the first time inhibition of human voltage-gated Nav1.5 sodium channels at therapeutically relevant concentrations. Within this study we also confirm recent findings of human Kv11.1 inhibition by low µM concentrations of ivabradine and observed no prolongation of ventricular-like APs in cardiomyocytes derived from iPSCs. CONCLUSION: Our results provide an explanation why ivabradine, despite its affinity for Kv11.1 channels, does not prolong the cardiac AP and QTc interval. Furthermore, our results suggest the inhibition of voltage-gated Nav1.5 sodium channels to underlie the recent observations of slowed atrioventricular conduction by increased atrial-His bundle intervals upon administration of ivabradine.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Fármacos Cardiovasculares/farmacologia , Canais Iônicos/metabolismo , Ivabradina/farmacologia , Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/metabolismo , Linhagem Celular , Canal de Potássio ERG1/antagonistas & inibidores , Canal de Potássio ERG1/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Canais Iônicos/antagonistas & inibidores , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/química , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Técnicas de Patch-Clamp
16.
Eur J Pharmacol ; 858: 172455, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31202801

RESUMO

Hydrogen sulfide (H2S) exerts different effects on the cardiovascular system by modulating ion channels. The present study was to ascertain whether H2S affects L-type calcium (Ca2+) channels in vascular smooth muscle cells (VSMCs) and the subsequent signaling pathways. Here, CaV1.2 L-type Ca2+ currents (ICa, L) were inhibited by sodium hydrosulfide (NaHS, an H2S donor) in A7r5 cell lines using the whole-cell patch-clamp technique. Then NaHS significantly reduced intracellular Ca2+ concentration ([Ca2+]i) in Bayk8644-stimulated CaV1.2-HEK293 cells by using flow cytometry. However, NaHS did not affect the ryanodine-induced elevation of [Ca2+]i by means of confocal microscopy, ruling out its influence on the intracellular Ca2+ release. In the following, the sulfhydration of L-type Ca2+ channels was determined by Ellman's Test. The results showed that NaHS decreased the number of free sulfhydryls, which was further strengthened by the oxidant sulfhydryl modifier diamide (DM) and significantly counteracted by the reductant sulfhydryl modifier dithiothreitol (DTT). DTT also partly reversed the NaHS-reduced [Ca2+]i in CaV1.2-HEK293 cells. Additionally, NaHS did not change CaV1.2 expression. Furthermore, NaHS increased phosphorylation of PKC and ERK in both a concentration- and a time-dependent manner in VSMCs. Isradipine, L-type Ca2+ channel specific blocker, further increased H2S-induced phosphorylation of PKC and ERK, showing an additive effect with H2S. Therefore, our results suggest that H2S reduced ICa, L & [Ca2+]i and hence influenced the downstream PKC/ERK pathway, which was likely through regulating the sulfhydration of L-type Ca2+ channels in VSMCs.


Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/metabolismo , Sulfeto de Hidrogênio/farmacologia , Músculo Liso Vascular/citologia , Animais , Relação Dose-Resposta a Droga , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HEK293 , Humanos , Músculo Liso Vascular/metabolismo , Proteína Quinase C/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo
17.
Life Sci ; 231: 116555, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31194991

RESUMO

AIMS: Caffeine is a methylxanthine with multiple actions in vascular smooth muscle cells (VSMCs), including the increase in the intracellular Ca2+ (iCa2+) concentration by the activation of ryanodine receptors (RyRs). The present study aimed at investigating the participation of Ca2+-influx through different Ca2+-channels on the transient contraction (TC) induced by caffeine in mice mesenteric arteries. MAIN METHODS: Second-order of mesenteric arteries was isolated from male Swiss mice. Vessels without functional endothelium were stimulated with caffeine (10 mM). The caffeine-induced TC was evaluated after the incubation of artery rings for 30 min with the following drugs: nifedipine (10 µM), a Cav1.2 blocker; 2-aminoethoxydiphenyl borate (2-APB; 10 µM) and ruthenium red (RuR; 10 µM), transient receptor potential (TRPs) channels blockers; capsazepine (10 µM) and HC067047 (10 µM), TRPV1 and TRPV4 antagonists, respectively; paxilline (1 µM), a selective BKCa blocker; and SKF-96365 (30 µM), an Orai blocker. Ca2+-fluorescence measurements were also performed on the investigated arteries. KEY FINDINGS: The TC induced by caffeine was partially dependent on Ca2+-influx. However, the blockage of Cav1.2 increased the TC while reduced the iCa2+ signal. Similar results were observed after the blockage of TRPs or BKCa. Therefore, caffeine promoted Ca2+-influx via TRPs and Cav1.2, and hyperpolarization through the activation of BKCa, inducing negative feedback of TC. SIGNIFICANCE: Our results indicate an alternative mechanism for the control of VSMCs contraction in resistance arteries. The evidence of the negative feedback of contraction via TRP-Cav1.2-BKCa provides a new perspective for understanding the mechanism involved in the vascular responses triggered by caffeine.


Assuntos
Cafeína/farmacologia , Canais de Cálcio Tipo L/metabolismo , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Artérias Mesentéricas/efeitos dos fármacos , Animais , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Masculino , Artérias Mesentéricas/metabolismo , Camundongos , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Canais de Cátion TRPV/metabolismo , Vasoconstrição/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia
18.
Cell Prolif ; 52(4): e12623, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31115100

RESUMO

L-type voltage-gated calcium ion channels (L-VGCCs) have been demonstrated to be the mediator of several significant intracellular activities in excitable cells, such as neurons, chromaffin cells and myocytes. Recently, an increasing number of studies have investigated the function of L-VGCCs in non-excitable cells, particularly stem cells. However, there appear to be no systematic reviews of the relationship between L-VGCCs and stem cells, and filling this gap is prescient considering the contribution of L-VGCCs to the proliferation and differentiation of several types of stem cells. This review will discuss the possible involvement of L-VGCCs in stem cells, mainly focusing on osteogenesis mediated by mesenchymal stem cells (MSCs) from different tissues and neurogenesis mediated by neural stem/progenitor cells (NSCs). Additionally, advanced applications that use these channels as the target for tissue engineering, which may offer the hope of tissue regeneration in the future, will also be explored.


Assuntos
Canais de Cálcio Tipo L/metabolismo , Cálcio/metabolismo , Células-Tronco/metabolismo , Animais , Sinalização do Cálcio/fisiologia , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Humanos , Osteogênese/fisiologia , Engenharia Tecidual/métodos
19.
Mol Med Rep ; 19(6): 5185-5194, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31059080

RESUMO

Sphincter of Oddi dysfunction (SOD) is a benign obstructive disorder predominantly resulting from spasms of the SO. Pharmacological therapies aim to induce SO relaxation; the hypercholesterolemic (HC) rabbit is the only SOD model available for study. In the present study, SO muscle strips, intracellular calcium ion concentrations and the mRNA expression levels of the α1C subunit of the L­type calcium channel in the SO muscle cells of HC rabbits were employed to investigate the effects of paeoniflorin (PF). Alterations in L­type calcium channel α subunit 1C mRNA and protein expression in SO cells with HC following the application of different concentrations of PF were determined by reverse transcription­quantitative polymerase chain reaction and western blotting. The whole cell patch clamp technique was used to observe the effects of different concentrations of paeoniflorin on L­type calcium channel current. The results of the present study demonstrated that PF induced the relaxation of SO muscle strips and reduced the intracellular calcium concentration in the SO muscle cells of HC rabbits. In addition, PF decreased the mRNA expression levels of the α1C subunit of the L­type calcium channel and reduced the L­type calcium channel current in SO cells. These results suggested that the mechanism underlying the relaxation of the SO muscle by PF may be associated with the reduction of calcium ion influx via L­type calcium channels.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Canais de Cálcio Tipo L/metabolismo , Glucosídeos/farmacologia , Hipercolesterolemia/patologia , Monoterpenos/farmacologia , Músculos/efeitos dos fármacos , Esfíncter da Ampola Hepatopancreática/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Anti-Inflamatórios não Esteroides/uso terapêutico , Cálcio/metabolismo , Canais de Cálcio Tipo L/genética , Colesterol/sangue , Modelos Animais de Doenças , Feminino , Glucosídeos/uso terapêutico , Hipercolesterolemia/tratamento farmacológico , Hipercolesterolemia/metabolismo , Masculino , Monoterpenos/uso terapêutico , Tono Muscular/efeitos dos fármacos , Músculos/fisiologia , Técnicas de Patch-Clamp , Coelhos
20.
Mol Biol Cell ; 30(14): 1743-1756, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31091162

RESUMO

In neurons, regulation of activity-dependent transcription by the nuclear factor of activated T-cells (NFAT) depends upon Ca2+ influx through voltage-gated L-type calcium channels (LTCC) and NFAT translocation to the nucleus following its dephosphorylation by the Ca2+-dependent phosphatase calcineurin (CaN). CaN is recruited to the channel by A-kinase anchoring protein (AKAP) 79/150, which binds to the LTCC C-terminus via a modified leucine-zipper (LZ) interaction. Here we sought to gain new insights into how LTCCs and signaling to NFAT are regulated by this LZ interaction. RNA interference-mediated knockdown of endogenous AKAP150 and replacement with human AKAP79 lacking its C-terminal LZ domain resulted in loss of depolarization-stimulated NFAT signaling in rat hippocampal neurons. However, the LZ mutation had little impact on the AKAP-LTCC interaction or LTCC function, as measured by Förster resonance energy transfer, Ca2+ imaging, and electrophysiological recordings. AKAP79 and NFAT coimmunoprecipitated when coexpressed in heterologous cells, and the LZ mutation disrupted this association. Critically, measurements of NFAT mobility in neurons employing fluorescence recovery after photobleaching and fluorescence correlation spectroscopy provided further evidence for an AKAP79 LZ interaction with NFAT. These findings suggest that the AKAP79/150 LZ motif functions to recruit NFAT to the LTCC signaling complex to promote its activation by AKAP-anchored calcineurin.


Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Canais de Cálcio Tipo L/metabolismo , Núcleo Celular/metabolismo , Fatores de Transcrição NFATC/metabolismo , Neurônios/metabolismo , Transdução de Sinais , Proteínas de Ancoragem à Quinase A/química , Motivos de Aminoácidos , Animais , Calcineurina/metabolismo , Sinalização do Cálcio , Linhagem Celular , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Espinhas Dendríticas/metabolismo , Hipocampo/citologia , Modelos Biológicos , Ligação Proteica , Transporte Proteico , Ratos Sprague-Dawley , Transcrição Genética
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