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1.
Aquat Toxicol ; 226: 105568, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32791376

RESUMO

An emerging Multi-Ion Toxicity (MIT) model for assessment of environmental salt pollution is based on the premise that major ion toxicity to aquatic organisms is related to a critical disturbance of the trans-epithelial potential across the gills (ΔTEP), which can be predicted by electrochemical theory. However, the model has never been evaluated physiologically. We directly tested key assumptions by examining the individual effects of eight different salts (NaCl, Na2SO4, MgCl2, MgSO4, KCl, K2SO4, CaCl2, and CaSO4) on measured TEP in three different fish species (fathead minnow, Pimephales promelas = FHM; channel catfish, Ictalurus punctatus = CC; bluegill, Lepomis macrochirus = BG). A geometric concentration series based on previously reported 96-h LC50 values for FHM was used. All salts caused concentration-dependent increases in TEP to less negative/more positive values in a pattern well-described by the Michaelis-Menten equation. The ΔTEP responses for different salts were similar to one another within each species when concentrations were expressed as a percentage of the FHM LC50. A plateau was reached at or before 100 % of the LC50 where the ΔTEP values were remarkably consistent, with only 1.4 to 2.2-fold variation. This relative uniformity in the ΔTEP responses contrasts with 28-fold variation in salt concentration (in mmol L-1), 9.6-fold in total dissolved solids, and 7.9-fold in conductivity at the LC50. The Michaelis-Menten Km values (salt concentrations causing 50 % of the ΔTEPmax) were positively related to the 96-h LC50 values. ΔTEP responses were not a direct effect of osmolarity in all species and were related to specific cation rather than specific anion concentrations in FHM. These responses were stable for up to 24 h in CC. The results provide strong physiological support for the assumptions of the MIT model, are coherent with electrochemical theory, and point to areas for future research.


Assuntos
Cyprinidae/fisiologia , Epitélio/fisiologia , Brânquias/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Perciformes/fisiologia , Sais/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Eletrodos , Brânquias/fisiologia , Concentração Osmolar
2.
Proc Natl Acad Sci U S A ; 117(30): 18079-18090, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32647060

RESUMO

Ion channels in excitable cells function in macromolecular complexes in which auxiliary proteins modulate the biophysical properties of the pore-forming subunits. Hyperpolarization-activated, cyclic nucleotide-sensitive HCN4 channels are critical determinants of membrane excitability in cells throughout the body, including thalamocortical neurons and cardiac pacemaker cells. We previously showed that the properties of HCN4 channels differ dramatically in different cell types, possibly due to the endogenous expression of auxiliary proteins. Here, we report the discovery of a family of endoplasmic reticulum (ER) transmembrane proteins that associate with and modulate HCN4. Lymphoid-restricted membrane protein (LRMP, Jaw1) and inositol trisphosphate receptor-associated guanylate kinase substrate (IRAG, Mrvi1, and Jaw1L) are homologous proteins with small ER luminal domains and large cytoplasmic domains. Despite their homology, LRMP and IRAG have distinct effects on HCN4. LRMP is a loss-of-function modulator that inhibits the canonical depolarizing shift in the voltage dependence of HCN4 in response to the binding of cAMP. In contrast, IRAG causes a gain of HCN4 function by depolarizing the basal voltage dependence in the absence of cAMP. The mechanisms of action of LRMP and IRAG are independent of trafficking and cAMP binding, and they are specific to the HCN4 isoform. We also found that IRAG is highly expressed in the mouse sinoatrial node where computer modeling predicts that its presence increases HCN4 current. Our results suggest important roles for LRMP and IRAG in the regulation of cellular excitability, as tools for advancing mechanistic understanding of HCN4 channel function, and as possible scaffolds for coordination of signaling pathways.


Assuntos
Retículo Endoplasmático/metabolismo , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Animais , Células CHO , Linhagem Celular , Cricetulus , AMP Cíclico/metabolismo , Regulação da Expressão Gênica , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/química , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Masculino , Potenciais da Membrana/efeitos dos fármacos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Modelos Biológicos , Família Multigênica , Miócitos Cardíacos/metabolismo , Fosfoproteínas/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas , Nó Sinoatrial/fisiologia , Nó Sinoatrial/fisiopatologia
3.
PLoS One ; 15(7): e0236373, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32702063

RESUMO

The diagnosis of patients with malignancies relies on the results of a clinical cytological examination. To enhance the diagnostic qualities of cytological examinations, it is important to have a detailed analysis of the cell's characteristics. There is, therefore, a need for developing a new auxiliary method for cytological diagnosis. In this study, we focused on studying the charge of the cell membrane surface of fixed cells, which is one of important cell's characteristics. Although fixed cells lose membrane potential which is observed in living cells owing to ion dynamics, we hypothesized that fixed cells still have a cell membrane surface charge due to cell membrane components and structure. We used 5 cell lines in this study (ARO, C32TG, RT4, TK, UM-UC-14). After fixation with CytoRich Red, we measured the cell membrane surface charge of fixed cells in solution using zeta potential measurements and fixed cells on glass slides, visualizing it using antibody-labeled beads and positively-charged beads. Furthermore, we measured the cell membrane surface charge of fixed cells under different conditions, such as different solution of fixative, ion concentration, pH, and pepsin treatments. The zeta potential measurements and visualization using the beads indicated that the cell membrane surface of fixed cells was negatively charged, and also that the charge varied among fixed cells. The charge state was affected by the different treatments. Moreover, the number of cell-bound beads was small in interphase, anaphase, and apoptotic cells. We concluded that the negative cell membrane surface charge was influenced by the three-dimensional structure of proteins as well as the different types of amino acids and lipids on the cell membrane. Thus, cell surface charge visualization can be applied as a new auxiliary method for clinical cytological diagnosis. This is the first systematic report of the cell membrane surface charge of fixed cells.


Assuntos
Linhagem Celular/ultraestrutura , Membrana Celular/ultraestrutura , Células Cultivadas/ultraestrutura , Citodiagnóstico , Anáfase/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Fixadores/farmacologia , Humanos , Potenciais da Membrana/efeitos dos fármacos , Pepsina A/farmacologia , Propriedades de Superfície
4.
Eur J Pharmacol ; 882: 173237, 2020 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-32525005

RESUMO

Pirfenidone (PFD), a pyridone compound, is well recognized as an antifibrotic agent tailored for the treatment of idiopathic pulmonary fibrosis. Recently, through its anti-inflammatory and anti-oxidant effects, PFD based clinical trial has also been launched for the treatment of coronavirus disease (COVID-19). To what extent this drug can perturb membrane ion currents remains largely unknown. Herein, the exposure to PFD was observed to depress the amplitude of hyperpolarization-activated cation current (Ih) in combination with a considerable slowing in the activation time of the current in pituitary GH3 cells. In the continued presence of ivabradine or zatebradine, subsequent application of PFD decreased Ih amplitude further. The presence of PFD resulted in a leftward shift in Ih activation curve without changes in the gating charge. The addition of this compound also led to a reduction in area of voltage-dependent hysteresis evoked by long-lasting inverted triangular (downsloping and upsloping) ramp pulse. Neither the amplitude of M-type nor erg-mediated K+ current was altered by its presence. In whole-cell potential recordings, addition of PFD reduced the firing frequency, and this effect was accompanied by the depression in the amplitude of sag voltage elicited by hyperpolarizing current stimulus. Overall, this study highlights evidence that PFD is capable of perturbing specific ionic currents, revealing a potential additional impact on functional activities of different excitable cells.


Assuntos
Membrana Celular/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , Piridonas/farmacologia , Animais , Betacoronavirus/metabolismo , Cátions/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Infecções por Coronavirus/virologia , Humanos , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/metabolismo , Transporte de Íons/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Pandemias , Pneumonia Viral/virologia , Potássio/metabolismo , Piridonas/uso terapêutico , Ratos , Sódio/metabolismo
5.
PLoS One ; 15(6): e0234114, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32516325

RESUMO

We previously reported that a single 5 ns high intensity electric pulse (NEP) caused an E-field-dependent decrease in peak inward voltage-gated Na+ current (INa) in isolated bovine adrenal chromaffin cells. This study explored the effects of a pair of 5 ns pulses on INa recorded in the same cell type, and how varying the E-field amplitude and interval between the pulses altered its response. Regardless of the E-field strength (5 to 10 MV/m), twin NEPs having interpulse intervals ≥ than 5 s caused the inhibition of TTX-sensitive INa to approximately double relative to that produced by a single pulse. However, reducing the interval from 1 s to 10 ms between twin NEPs at E-fields of 5 and 8 MV/m but not 10 MV/m decreased the magnitude of the additive inhibitory effect by the second pulse in a pair on INa. The enhanced inhibitory effects of twin vs single NEPs on INa were not due to a shift in the voltage-dependence of steady-state activation and inactivation but were associated with a reduction in maximal Na+ conductance. Paradoxically, reducing the interval between twin NEPs at 5 or 8 MV/m but not 10 MV/m led to a progressive interval-dependent recovery of INa, which after 9 min exceeded the level of INa reached following the application of a single NEP. Disrupting lipid rafts by depleting membrane cholesterol with methyl-ß-cyclodextrin enhanced the inhibitory effects of twin NEPs on INa and ablated the progressive recovery of this current at short twin pulse intervals, suggesting a complete dissociation of the inhibitory effects of twin NEPs on this current from their ability to stimulate its recovery. Our results suggest that in contrast to a single NEP, twin NEPs may influence membrane lipid rafts in a manner that enhances the trafficking of newly synthesized and/or recycling of endocytosed voltage-gated Na+ channels, thereby pointing to novel means to regulate ion channels in excitable cells.


Assuntos
Células Cromafins/fisiologia , Eletricidade , Glândulas Suprarrenais/citologia , Animais , Bovinos , Células Cultivadas , Células Cromafins/citologia , Potenciais da Membrana/efeitos dos fármacos , Técnicas de Patch-Clamp , Canais de Sódio Disparados por Voltagem/metabolismo , beta-Ciclodextrinas/farmacologia
6.
PLoS One ; 15(5): e0233591, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32453773

RESUMO

The heart is critically dependent on mitochondrial respiration for energy supply. Ischemia decreases oxygen availability, with catastrophic consequences for cellular energy systems. After a few minutes of ischemia, the mitochondrial respiratory chain halts, ATP levels drop and ion gradients across cell membranes collapse. Activation of cellular proteases and generation of reactive oxygen species by mitochondria during ischemia alter mitochondrial membrane permeability, causing mitochondrial swelling and fragmentation and eventually cell death. The mitochondria, therefore, are important targets of cardioprotection against ischemic injury. We have previously shown that ixazomib (IXA), a proteasome inhibitor used for treating multiple myeloma, effectively reduced the size of the infarct produced by global ischemia in isolated rat hearts and prevented degradation of the sarcoplasmic reticulum calcium release channel RyR2. The aim of this work was to further characterize the protective effect of IXA by determining its effect on mitochondrial morphology and function after ischemia. We also quantified the effect of IXA on levels of mitofusin-2, a protein involved in maintaining mitochondrial morphology and mitochondria-SR communication. We found that mitochondria were significantly preserved and functional parameters such as oxygen consumption, the ability to generate a membrane potential, and glutathione content were improved in mitochondria isolated from hearts perfused with IXA prior to ischemia. IXA also blocked the release of cytochrome c observed in ischemia and significantly preserved mitofusin-2 integrity. These beneficial effects resulted in a significant decrease in the left ventricular end diastolic pressure upon reperfusion and a smaller infarct in isolated hearts.


Assuntos
Compostos de Boro/farmacologia , Glicina/análogos & derivados , Coração/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Isquemia Miocárdica/tratamento farmacológico , Animais , Quimotripsina/farmacologia , Modelos Animais de Doenças , Glutationa/genética , Glutationa/metabolismo , Glicina/farmacologia , Coração/fisiopatologia , Humanos , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/genética , Isquemia Miocárdica/genética , Isquemia Miocárdica/fisiopatologia , Consumo de Oxigênio/genética , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Inibidores de Proteassoma/farmacologia , Ratos
7.
PLoS One ; 15(5): e0232759, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32453737

RESUMO

SUMMARY: Reprogramming autologous adult cells to pluripotent cells allows for relatively safe cell replacement therapy. This can be achieved by nuclear transfer, cell fusion, or induced pluripotent stem cell technology However, the epigenetic memory of the cell is considered as a great challenge facing the complete reprograming of cells by these methods. Introducing oocyte-specific factors into differentiated cells may present a promising approach by mimicking cellular reprogramming during fertilization. METHODS: Human bone marrow mesenchymal stromal cells (hBM-MSCs) were cultured with different concentrations of human metaphase II (M II) oocyte extract (0.1, 1, 5, 10, 30 ng/µl). Reprogramming was assessed at various exposure times (1, 4, 7 days). Cells were tested for their proliferation rate, morphological changes, expression of pluripotency markers, expression of mesenchymal to epithelial transition markers, and mitochondrial rejuvenation. (mitochondrial localization, morphological changes, bioenergetics, transmembrane potential, and levels of reactive oxygen species, ROS). RESULTS: Treatment of human BM-MSCs with 10 ng/µl oocyte extract resulted in increased cell proliferation, which was associated with the upregulation of the pluripotency genes OCT-4, NANOG, and SOX-2 and a concomitant downregulation of mesenchymal-specific genes. MSCs exhibited small, immature round mitochondria with few swollen cristae localized proximal to the cell nucleus. This was accompanied by morphological cell changes, a metabolic shift towards oxidative phosphorylation, a high mitochondrial membrane potential, and increased ROS production. CONCLUSION: These data show that treatment with 10 ng/µl human MII-phase oocyte extract induced genetic and mitochondrial reprogramming of human BM-MSCs to a more embryonic phenotype.


Assuntos
Extratos Celulares/farmacologia , Reprogramação Celular/genética , Células-Tronco Mesenquimais/metabolismo , Mitocôndrias/metabolismo , Oócitos/metabolismo , Biomarcadores/metabolismo , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Células Cultivadas , Reprogramação Celular/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Potenciais da Membrana/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Oócitos/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Fatores de Tempo
8.
Mol Pharmacol ; 98(1): 61-71, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32358165

RESUMO

The blood-brain barrier (BBB) is essential for the maintenance of homeostasis in the brain. Brain capillary endothelial cells (BCECs) comprise the BBB, and thus a delicate balance between their proliferation and death is required. Although the activity of ion channels in BCECs is involved in BBB functions, the underlying molecular mechanisms remain unclear. In the present study, the molecular components of Ca2+-activated Cl- (ClCa) channels and their physiological roles were examined using mouse BCECs (mBCECs) and a cell line derived from bovine BCECs, t-BBEC117. Expression analyses revealed that TMEM16A was strongly expressed in mBCECs and t-BBEC117 cells. In t-BBEC117 cells, whole-cell Cl- currents were sensitive to the ClCa channel blockers, 100 µM niflumic acid and 10 µM T16Ainh-A01, and were also reduced markedly by small-interfering RNA (siRNA) knockdown of TMEM16A. Importantly, block of ClCa currents with ClCa channel blockers or TMEM16A siRNA induced membrane hyperpolarization. Moreover, treatment with TMEM16A siRNA caused an increase in resting cytosolic Ca2+ concentration ([Ca2+]cyt). T16Ainh-A01 reduced cell viability in a concentration-dependent manner. Either ClCa channel blockers or TMEM16A siRNA also curtailed cell proliferation and migration. Furthermore, ClCa channel blockers attenuated the trans-endothelial permeability. In combination, these results strongly suggest that TMEM16A contributes to ClCa channel conductance and can regulate both the resting membrane potential and [Ca2+]cyt in BCECs. Our data also reveal how these BCECs may be involved in the maintenance of BBB functions, as both the proliferation and migration are altered following changes in channel activity. SIGNIFICANCE STATEMENT: In brain capillary endothelial cells (BCECs) of the blood-brain barrier (BBB), TMEM16A is responsible for Ca2+-activated Cl- channels and can regulate both the resting membrane potential and cytosolic Ca2+ concentration, contributing to the proliferation and migration of BCECs. The present study provides novel information on the molecular mechanisms underlying the physiological functions of BCECs in the BBB and a novel target for therapeutic drugs for disorders associated with dysfunctions in the BBB.


Assuntos
Anoctamina-1/metabolismo , Barreira Hematoencefálica/metabolismo , Encéfalo/citologia , Cálcio/metabolismo , Canais de Cloreto/metabolismo , Animais , Anoctamina-1/antagonistas & inibidores , Barreira Hematoencefálica/citologia , Barreira Hematoencefálica/diagnóstico por imagem , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Bovinos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células HEK293 , Humanos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Ácido Niflúmico/farmacologia , Pirimidinas/farmacologia , Tiazóis/farmacologia
9.
Nat Commun ; 11(1): 2188, 2020 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-32366818

RESUMO

Olfactory receptor neurons (ORNs) use odour-induced intracellular cAMP surge to gate cyclic nucleotide-gated nonselective cation (CNG) channels in cilia. Prolonged exposure to cAMP causes calmodulin-dependent feedback-adaptation of CNG channels and attenuates neural responses. On the other hand, the odour-source searching behaviour requires ORNs to be sensitive to odours when approaching targets. How ORNs accommodate these conflicting aspects of cAMP responses remains unknown. Here, we discover that olfactory marker protein (OMP) is a major cAMP buffer that maintains the sensitivity of ORNs. Upon the application of sensory stimuli, OMP directly captured and swiftly reduced freely available cAMP, which transiently uncoupled downstream CNG channel activity and prevented persistent depolarization. Under repetitive stimulation, OMP-/- ORNs were immediately silenced after burst firing due to sustained depolarization and inactivated firing machinery. Consequently, OMP-/- mice showed serious impairment in odour-source searching tasks. Therefore, cAMP buffering by OMP maintains the resilient firing of ORNs.


Assuntos
AMP Cíclico/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Proteína de Marcador Olfatório/metabolismo , Neurônios Receptores Olfatórios/metabolismo , Animais , Butorfanol/farmacologia , Cílios/metabolismo , Células HEK293 , Humanos , Masculino , Medetomidina/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Midazolam/farmacologia , Odorantes , Proteína de Marcador Olfatório/genética , Mucosa Olfatória/citologia , Mucosa Olfatória/efeitos dos fármacos , Mucosa Olfatória/fisiologia , Neurônios Receptores Olfatórios/citologia , Neurônios Receptores Olfatórios/fisiologia , Técnicas de Patch-Clamp
10.
Medicine (Baltimore) ; 99(17): e19848, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32332640

RESUMO

Xiakemycin A (XKA), a new antibiotic in the pyranonaphthoquinone family, shows antitumor activity. However, the type of cell death induced by XKA remains elusive. In this study, we aim to investigate the type of death induced by XKA in hepatic cancer.The apoptotic features, such as chromatic agglutination, reactive oxygen species generation and membrane potential of mitochondria, in HepG2 cells treated by XKA were measured by Hoechst 33342 staining and flow cytometry. Apoptosis of HepG2 cells treated with XKA was determined by Annexin V-FITC/propidium iodide double staining and Western blot analysis, respectively.XKA had a significant dose-dependent elevation of chromatic agglutination, reactive oxygen species generation, Annexin V and propidium iodide staining, decrease of membrane potential. Meanwhile, in apoptotic HepG2 cells induced by XKA, robust increment was noticed in p53 expression, cleavage of PARP, caspase-3, and caspase-9.XKA showed potent inhibitory effects on the proliferation of HepG2 cells. Such phenomenon may be related to activation of the apoptotic pathway.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/patologia , Naftoquinonas/farmacologia , Anexina A5/metabolismo , Carcinoma Hepatocelular/tratamento farmacológico , Caspase 3/metabolismo , Caspase 9/metabolismo , Dano ao DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células Hep G2 , Humanos , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias Hepáticas/fisiologia , Poli(ADP-Ribose) Polimerase-1/metabolismo , Propídio/metabolismo , Espécies Reativas de Oxigênio/metabolismo
11.
Artigo em Inglês | MEDLINE | ID: mdl-32186932

RESUMO

The increase in cytosolic Ca2+ concentration ([Ca2+]cyt) and upregulation of calcium-sensing receptor (CaSR) and stromal interaction molecule 2 (STIM2) along with inhibition of voltage-gated K+ (KV) channels in pulmonary arterial smooth muscle cells (PASMC) have been implicated in the development of pulmonary arterial hypertension; however, the precise upstream mechanisms remain elusive. Activation of CaSR, a G protein-coupled receptor (GPCR), results in Ca2+ release from the endoplasmic/sarcoplasmic reticulum (ER/SR) and Ca2+ influx through receptor-operated and store-operated Ca2+ channels (SOC). Upon Ca2+ depletion from the SR, STIM forms clusters to mediate store-operated Ca2+ entry. Activity of KV channels, like KCNA5/KV1.5 and KCNA2/KV1.2, contributes to regulating membrane potential, and inhibition of KV channels results in membrane depolarization that increases [Ca2+]cyt by opening voltage-dependent Ca2+ channels. In this study, we show that activation of Notch by its ligand Jag-1 promotes the clustering of STIM2, and clustered STIM2 subsequently enhances the CaSR-induced Ca2+ influx through SOC channels. Extracellular Ca2+-mediated activation of CaSR increases [Ca2+]cyt in CASR-transfected HEK293 cells. Treatment of CASR-transfected cells with Jag-1 further enhances CaSR-mediated increase in [Ca2+]cyt. Moreover, CaSR-mediated increase in [Ca2+]cyt was significantly augmented in cells co-transfected with CASR and STIM2. CaSR activation results in STIM2 clustering in CASR/STIM2-cotransfected cells. Notch activation also induces significant clustering of STIM2. Furthermore, activation of Notch attenuates whole cell K+ currents in KCNA5- and KCNA2-transfected cells. Together, these results suggest that Notch activation enhances CaSR-mediated increases in [Ca2+]cyt by enhancing store-operated Ca2+ entry and inhibits KCNA5/KV1.5 and KCNA2/KV1.2, ultimately leading to voltage-activated Ca2+ entry.


Assuntos
Canal de Potássio Kv1.2/genética , Canal de Potássio Kv1.5/genética , Hipertensão Arterial Pulmonar/genética , Receptores de Detecção de Cálcio/genética , Molécula 2 de Interação Estromal/genética , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/genética , Sinalização do Cálcio/genética , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Estrenos/farmacologia , Células HEK293 , Humanos , Indóis/farmacologia , Proteína Jagged-1/genética , Potenciais da Membrana/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Hipertensão Arterial Pulmonar/metabolismo , Hipertensão Arterial Pulmonar/patologia , Artéria Pulmonar/metabolismo , Artéria Pulmonar/patologia , Pirrolidinonas/farmacologia , Receptores de Detecção de Cálcio/efeitos dos fármacos , Receptores Notch/genética , Análise de Célula Única
12.
Biochim Biophys Acta Rev Cancer ; 1873(2): 188355, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32135169

RESUMO

The human ether-à-go-go related gene (HERG) encodes the alpha subunit of Kv11.1, which is a voltage-gated K+ channel protein mainly expressed in heart and brain tissue. HERG plays critical role in cardiac repolarization, and mutations in HERG can cause long QT syndrome. More recently, evidence has emerged that HERG channels are aberrantly expressed in many kinds of cancer cells and play important roles in cancer progression. HERG could therefore be a potential biomarker for cancer and a possible molecular target for anticancer drug design. HERG affects a number of cellular processes, including cell proliferation, apoptosis, angiogenesis and migration, any of which could be affected by dysregulation of HERG. This review provides an overview of available information on HERG channel as it relates to cancer, with focus on the mechanism by which HERG influences cancer progression. Molecular docking attempts suggest two possible protein-protein interactions of HERG with the ß1-integrin receptor and the transcription factor STAT-1 as novel HERG-directed therapeutic targeting which avoids possible cardiotoxicity. The role of epigenetics in regulating HERG channel expression and activity in cancer will also be discussed. Finally, given its inherent extracellular accessibility as an ion channel, we discuss regulatory roles of this molecule in cancer physiology and therapeutic potential. Future research should be directed to explore the possibilities of therapeutic interventions targeting HERG channels while minding possible complications.


Assuntos
Carcinogênese/patologia , Canal de Potássio ERG1/metabolismo , Integrina beta1/metabolismo , Neoplasias/patologia , Fator de Transcrição STAT1/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Benzimidazóis/farmacologia , Benzimidazóis/uso terapêutico , Carcinogênese/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Canal de Potássio ERG1/antagonistas & inibidores , Canal de Potássio ERG1/química , Canal de Potássio ERG1/genética , Epigênese Genética/efeitos dos fármacos , Fluoxetina/farmacologia , Fluoxetina/uso terapêutico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Síndrome do QT Longo/genética , Potenciais da Membrana/efeitos dos fármacos , Simulação de Acoplamento Molecular , Mutação , Miócitos Cardíacos/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética , Piperidinas/farmacologia , Piperidinas/uso terapêutico , Conformação Proteica em alfa-Hélice , Mapeamento de Interação de Proteínas , Estrutura Quaternária de Proteína , Piridinas/farmacologia , Piridinas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Sulfanilamidas/farmacologia , Sulfanilamidas/uso terapêutico
13.
Brain ; 143(3): 771-782, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-32011655

RESUMO

Small fibre neuropathy is a common pain disorder, which in many cases fails to respond to treatment with existing medications. Gain-of-function mutations of voltage-gated sodium channel Nav1.7 underlie dorsal root ganglion neuronal hyperexcitability and pain in a subset of patients with small fibre neuropathy. Recent clinical studies have demonstrated that lacosamide, which blocks sodium channels in a use-dependent manner, attenuates pain in some patients with Nav1.7 mutations; however, only a subgroup of these patients responded to the drug. Here, we used voltage-clamp recordings to evaluate the effects of lacosamide on five Nav1.7 variants from patients who were responsive or non-responsive to treatment. We show that, at the clinically achievable concentration of 30 µM, lacosamide acts as a potent sodium channel inhibitor of Nav1.7 variants carried by responsive patients, via a hyperpolarizing shift of voltage-dependence of both fast and slow inactivation and enhancement of use-dependent inhibition. By contrast, the effects of lacosamide on slow inactivation and use-dependence in Nav1.7 variants from non-responsive patients were less robust. Importantly, we found that lacosamide selectively enhances fast inactivation only in variants from responders. Taken together, these findings begin to unravel biophysical underpinnings that contribute to responsiveness to lacosamide in patients with small fibre neuropathy carrying select Nav1.7 variants.


Assuntos
Lacosamida/farmacologia , Potenciais da Membrana/fisiologia , Canal de Sódio Disparado por Voltagem NAV1.7/fisiologia , Neuropatia de Pequenas Fibras/fisiopatologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Células Cultivadas , Humanos , Lacosamida/uso terapêutico , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Pessoa de Meia-Idade , Mutação , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Dor/complicações , Dor/tratamento farmacológico , Medição da Dor/efeitos dos fármacos , Técnicas de Patch-Clamp , Neuropatia de Pequenas Fibras/tratamento farmacológico , Bloqueadores dos Canais de Sódio/farmacologia , Bloqueadores dos Canais de Sódio/uso terapêutico , Resultado do Tratamento , Adulto Jovem
14.
Environ Toxicol ; 35(7): 774-782, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32061153

RESUMO

This study aims to investigate the protective effects of the Bauhinia championii (BC) against ischemia/reperfusion (I/R)-induced injury in an isolated heart model. Langendorff-perfused C57BL/6JNarl mice hearts were performed with 30 minutes ischemia and 60 minutes reperfusion by left anterior descending artery ligation. Before reperfusion, boiling water extracts of BC (10 mg/L) was pretreated for 15 minutes. During reperfusion, BC significantly decreased the occurrence of ventricular arrhythmias by lead II electrocardiogram (ECG). Electrophysiological effect of BC was further determined in isolated ventricular myocytes by whole-cell patch clamp technique. The underlying mechanism may result from its Na+ channel blocking activity characterized with reduced rise slope of action potential and Na+ current density. Moreover, BC dramatically reduced I/R-caused infarct size, which was accessed by 2,3,5-triphenyltetrazolium chloride (TTC) assay. Since BC decreased I/R-induced myoglobin release and oxidation of Ca2+ -calmodulin-dependent protein kinase, inhibition of myocardial necroptosis may account for the protective effects of BC on myocytes lose. This study indicated that BC may prevent I/R induced ventricular arrhythmias and myocyte death by blocking Na+ channels and decreasing necroptosis, respectively. Since most of the available antiarrhythmic remedies have unwanted adverse actions, BC could be a novel candidate for the treatment of myocardial infarction and ventricular arrhythmia.


Assuntos
Bauhinia/química , Coração/efeitos dos fármacos , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Extratos Vegetais/farmacologia , Bloqueadores dos Canais de Sódio/farmacologia , Animais , Eletrocardiografia , Técnicas In Vitro , Masculino , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Necroptose/efeitos dos fármacos , Técnicas de Patch-Clamp , Componentes Aéreos da Planta/química , Extratos Vegetais/isolamento & purificação , Bloqueadores dos Canais de Sódio/isolamento & purificação , Canais de Sódio/metabolismo
15.
J Toxicol Sci ; 45(1): 15-23, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31932554

RESUMO

Eupalinolide J (EJ) is a new sesquiterpene lactone isolated from Eupatorium lindleyanum DC. In the present study, we investigated the anti-cancer activity of EJ on cell proliferation in human prostate cancer cells. The MTT results indicated that EJ showed marked anti-proliferative activity in PC-3 and DU-145 cells in a dose- and time-dependent manner. DAPI staining analysis demonstrated that this effect was mediated by induction of cell apoptosis. Flow cytometric analysis indicated a significant increase in apoptotic cells, cell cycle arrest at G0/G1 phase and disruption of mitochondrial membrane potential (MMP) after EJ treatment. Meanwhile, the activation of caspase-3 and caspase-9 was visibly observed. Furthermore, our results demonstrated that the expression levels of γH2AX, p-Chk1 and p-Chk2 were significantly up-regulated, suggesting the induction of DNA damage responses in EJ-treated prostate cancer cells. The above results indicated that EJ exhibited effective anti-cancer activity in vitro. It could be a promising candidate agent for the clinical treatment of prostate cancer.


Assuntos
Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Eupatorium/química , Humanos , Masculino , Células Tumorais Cultivadas
16.
Toxicol Appl Pharmacol ; 390: 114883, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31981640

RESUMO

Human-based in silico models are emerging as important tools to study the effects of integrating inward and outward ion channel currents to predict clinical proarrhythmic risk. The aims of this study were 2-fold: 1) Evaluate the capacity of an in silico model to predict QTc interval prolongation in the in vivo anesthetized cardiovascular guinea pig (CVGP) assay for new chemical entities (NCEs) and; 2) Determine if a translational pharmacokinetic/pharmacodynamic (tPKPD) model can improve the predictive capacity. In silico simulations for NCEs were performed using a population of human ventricular action potential (AP) models. PatchXpress® (PX) or high throughput screening (HTS) ion channel data from respectively n = 73 and n = 51 NCEs were used as inputs for the in silico population. These NCEs were also tested in the CVGP (n = 73). An M5 pruned decision tree-based regression tPKPD model was used to evaluate the concentration at which an NCE is liable to prolong the QTc interval in the CVGP. In silico results successfully predicted the QTc interval prolongation outcome observed in the CVGP with an accuracy/specificity of 85%/73% and 75%/77%, when using PX and HTS ion channel data, respectively. Considering the tPKPD predicted concentration resulting in QTc prolongation (EC5%) increased accuracy/specificity to 97%/95% using PX and 88%/97% when using HTS. Our results support that human-based in silico simulations in combination with tPKPD modeling can provide correlative results with a commonly used early in vivo safety assay, suggesting a path toward more rapid NCE assessment with reduced resources, cycle time, and animal use.


Assuntos
Antiarrítmicos/farmacologia , Arritmias Cardíacas , Simulação por Computador , Técnicas Eletrofisiológicas Cardíacas , Modelos Biológicos , Animais , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Linhagem Celular , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Cobaias , Células HEK293 , Humanos , Potenciais da Membrana/efeitos dos fármacos , Modelos Químicos
17.
J Med Chem ; 63(4): 1709-1716, 2020 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-31999455

RESUMO

A hybrid of dearomatized isoprenylated acylphloroglucinol (DIAP) and monoterpenoid, hypatone A (1), together with its biosynthetic analogues 2-4 is characterized from Hypericum patulum. Structurally, 1 possesses an unprecedented spiro[bicyclo[3.2.1]octane-6,1'-cyclohexan]-2',4',6'-trione core as elucidated by extensive spectroscopic and X-ray crystallographic analyses. Biological studies reveal that compounds 1 and 2-4 produce opposite effects on Cav3.1 low voltage-gated Ca2+ channel, with 1 and 4, respectively, being the most potent Cav3.1 agonist and antagonist from natural products. Further studies suggest that compound 1 and its biogenetical precursor, 2, have the same binding site on Cav3.1 and that the rigid cagelike moiety at C-5 and C-6 is a key structural feature responsible for 1 being an agonist. Furthermore, 1 can normalize the pathological gating of a mutant Cav3.1 channel found in spinocerebellar ataxia 42 (SCA42), a hereditary neurodegenerative disorder with no available therapy. Collectively, our findings provide valuable tools for future studies on Cav3.1 physiology and pathophysiology, as well as afford possible leads for developing new drugs against SCA42, epilepsy, and pain.


Assuntos
Agonistas dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo T/metabolismo , Monoterpenos/farmacologia , Floroglucinol/análogos & derivados , Floroglucinol/farmacologia , Animais , Agonistas dos Canais de Cálcio/isolamento & purificação , Canais de Cálcio Tipo T/genética , Células HEK293 , Humanos , Hypericum/química , Ativação do Canal Iônico/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Monoterpenos/isolamento & purificação , Mutação , Floroglucinol/isolamento & purificação
18.
Aquat Toxicol ; 220: 105398, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31891816

RESUMO

Recent studies suggest that 2,4-DABA, a neurotoxic excitatory amino acid present in virtually all environments, but predominantly in aquatic ecosystems may be a risk factor for development of neurodegenerative diseases in animals and humans. Despite its neurotoxicity and potential environmental importance, mechanisms underlying the excitatory and putative excitotoxic action of 2,4-DABA in neurons are still unexplored. We previously reported on extensive two-stage membrane depolarization and functional disturbances in leech Retzius neurons induced by 2,4-DABA. Current study presents the first detailed look into the electrophysiological processes leading to this depolarization. Intracellular recordings were performed on Retzius neurons of the leech Haemopis sanguisuga using glass microelectrodes and input membrane resistance (IMR) was measured by injecting hyperpolarizing current pulses through these electrodes. Results show that the excitatory effect 2,4-DABA elicits on neurons' membrane potential is dependent on sodium ions. Depolarizing effect of 5·10-3 mol/L 2,4-DABA in sodium-free solution was significantly diminished by 91% reducing it to 3.26 ±â€¯0.62 mV and its two-stage nature was abrogated. In addition to being sodium-dependent, the depolarization of membrane potential induced by this amino acid is coupled with an increase of membrane permeability, as 2,4-DABA decreases IMR by 8.27 ±â€¯1.47 MΩ (67.60%). Since present results highlight the role of sodium ions, we investigated the role of two putative sodium-dependent mechanisms in 2,4-DABA-induced excitatory effect - activation of ionotropic glutamate receptors and the electrogenic transporter for neutral amino acids. Excitatory effect of 5·10-3 mol/L 2,4-DABA was partially blocked by 10-5 mol/L 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) a non-NMDA receptor antagonist as the first stage of membrane depolarization was significantly reduced by 2.59 ±â€¯0.98 mV (40%), whilst second stage remained unaltered. Moreover, involvement of the sodium-dependent transport system for neutral amino acids was investigated by equimolar co-application of 5·10-3 mol/L 2,4-DABA and L-alanine, a competitive inhibitor of this transporter. Although L-alanine exhibited no effect on the first stage of membrane depolarization elicited by 2,4-DABA, it substantially reduced the second stage (the overall membrane depolarization) from 39.63 ±â€¯2.22 mV to 16.28 ±â€¯2.58 mV, by 58.92%. We therefore propose that the electrophysiological effect of 2,4-DABA on Retzius neurons is mediated by two distinct mechanisms, i.e. by activation of ionotropic glutamate receptor that initiates the first stage of membrane depolarization followed by the stimulation of an electrogenic sodium-dependent neutral amino acid transporter, leading to additional influx of positive charge into the cell and the second stage of depolarization.


Assuntos
Aminobutiratos/toxicidade , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Sanguessugas/fisiologia , Neurônios/efeitos dos fármacos , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Alanina/farmacologia , Sistema A de Transporte de Aminoácidos/antagonistas & inibidores , Animais , Ácido Glutâmico/metabolismo , Sanguessugas/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Neurônios/fisiologia
19.
Am J Physiol Renal Physiol ; 318(4): F870-F877, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-31984792

RESUMO

Adenosine plays an important role in various aspects of kidney physiology, but the specific targets and mechanisms of actions are not completely understood. The collecting duct has the highest expression of adenosine receptors, particularly adenosine A1 receptors (A1Rs). Interstitial adenosine levels are greatly increased up to a micromolar range in response to dietary salt loading. We have previously shown that the basolateral membrane of principal cells has primarily K+ conductance mediated by Kir4.1/5.1 channels to mediate K+ recycling and to set up a favorable driving force for Na+/K+ exchange (47). Intercalated cells express the Cl- ClC-K2/b channel mediating transcellular Cl- reabsorption. Using patch-clamp electrophysiology in freshly isolated mouse collecting ducts, we found that acute application of adenosine reversely inhibits ClC-K2/b open probability from 0.31 ± 0.04 to 0.17 ± 0.06 and to 0.10 ± 0.05 for 1 and 10 µM, respectively. In contrast, adenosine (10 µM) had no measureable effect on Kir4.1/5.1 channel activity in principal cells. The inhibitory effect of adenosine on ClC-K2/b was abolished in the presence of the A1R blocker 8-cyclopentyl-1,3-dipropylxanthine (10 µM). Consistently, application of the A1R agonist N6-cyclohexyladenosine (1 µM) recapitulated the inhibitory action of adenosine on ClC-K2/b open probability. The effects of adenosine signaling in the collecting duct were independent from its purinergic counterpartner, ATP, having no measurable actions on ClC-K2/b and Kir4.1/5.1. Overall, we demonstrated that adenosine selectively inhibits ClC-K2/b activity in intercalated cells by targeting A1Rs. We propose that inhibition of transcellular Cl- reabsorption in the collecting duct by adenosine would aid in augmenting NaCl excretion during high salt intake.


Assuntos
Agonistas do Receptor A1 de Adenosina/farmacologia , Adenosina/farmacologia , Proteínas de Transporte de Ânions/antagonistas & inibidores , Canais de Cloreto/antagonistas & inibidores , Cloretos/metabolismo , Túbulos Renais Coletores/efeitos dos fármacos , Receptor A1 de Adenosina/efeitos dos fármacos , Reabsorção Renal/efeitos dos fármacos , Animais , Proteínas de Transporte de Ânions/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Canais de Cloreto/metabolismo , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/metabolismo , Masculino , Potenciais da Membrana/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Receptor A1 de Adenosina/metabolismo
20.
Chin J Integr Med ; 26(4): 277-282, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31227963

RESUMO

OBJECTIVE: To study the antimalarial effects and mechanisms of artemisinin (Qinghaosu in Chinese, QHS) on mitochondria in mice infected with Plasmodium berghei. METHODS: A total of 108 C57 mice infected with Plasmodium berghei were randomly divided into 3 groups by weight: the control group, 200 and 400 mg/kg QHS groups. The two QHS treatment groups were further divided into 4 sub-groups with 12 animals each time according to the treatment time, 0.5, 1, 2, and 4 h. Normal saline was intragastrically (i.g.) administered to the control group. The other two groups received different doses of QHS by i.g. administration. Animals were treated once with QHS for different detection time as follows: 0.5, 1, 2, and 4 h. The mitochondrial energy metabolism, oxidative damage, membrane potential, and membrane permeability and other indexes were detected. RESULTS: After administration of 200 and 400 mg/kg QHS, adenosine triphosphate (ATP) levels in Plasmodium and its mitochondria were reduced (P<0.05), the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) were increased (P<0.05), and the activity of superoxide dismutase (SOD) was also increased (P<0.05). At the same time, the membrane potential of the mitochondria was reduced and the degree to which the membrane permeability transition pore was opened was irreversibly increased (P<0.05). CONCLUSIONS: Mitochondria in Plasmodium were the targets of QHS, which can adversely affect mitochondrial energy metabolism, oxidative damage, membrane potential, and membrane opening, and ultimately exert an antimalarial effect.


Assuntos
Antimaláricos/farmacologia , Artemisininas/farmacologia , Plasmodium berghei/efeitos dos fármacos , Animais , Metabolismo Energético/efeitos dos fármacos , Malária Falciparum , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Estresse Oxidativo , Espécies Reativas de Oxigênio , Superóxido Dismutase
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