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1.
PLoS One ; 15(8): e0238155, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32841278

RESUMO

Non-small cell lung cancer (NSCLC), one of the leading causes of cancer-related death, has a low 5-year survival rate owing to the inevitable acquired resistance toward antitumor drugs, platinum-based chemotherapy, and targeted therapy. Epidermal growth factor (EGF)-EGF receptor (EGFR) signaling activates downstream events leading to phospholipase C/inositol trisphosphate (IP3)/Ca2+ release from IP3-sensitive Ca2+ stores to modulate cell proliferation, motility, and invasion. However, the role of EGFR-mediated Ca2+ signaling in acquired drug resistance is not fully understood. Here, we analyzed alterations of intracellular Ca2+ ([Ca2+]i) responses between gefitinib-sensitive NSCLC PC-9 cells and gefitinib-resistant NSCLC PC-9/GR cells, and we found that acute EGF treatment elicited intracellular Ca2+ ([Ca2+]i) oscillations in PC-9 cells but not in PC-9/GR cells. PC-9/GR cells presented a more sustained basal [Ca2+]i level, lower endoplasmic reticulum Ca2+ level, and higher spontaneous extracellular Ca2+ ([Ca2+]e) influx than PC-9 cells. Notably, restricting [Ca2+]e in both cell types induced identical [Ca2+]i oscillations, dependent on phospholipase C and EGFR activation. Consequently, restricting [Ca2+]e in PC-9/GR cells upregulated gefitinib-mediated poly (ADP-ribose) polymerase cleavage, an increase in Bax/Bcl-2 ratio, cytotoxicity, and apoptosis. In addition, nuclear factor of activated T cell (NFAT1) induction in response to EGF was inhibited by gefitinib in PC-9 cells, whereas EGF-mediated NFAT1 induction in PC-9/GR cells was sustained regardless of gefitinib treatment. Restricting [Ca2+]e in PC-9/GR cells significantly reduced EGF-mediated NFAT1 induction. These findings indicate that spontaneous [Ca2+]e influx in NSCLC cells plays a pivotal role in developing acquired drug resistance and suggest that restricting [Ca2+]e may be a potential strategy for modulating drug-sensitivity.


Assuntos
Antineoplásicos/farmacologia , Sinalização do Cálcio , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Gefitinibe/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Estrenos/farmacologia , Humanos , Fatores de Transcrição NFATC/biossíntese , Pirrolidinonas/farmacologia , Fosfolipases Tipo C/antagonistas & inibidores
2.
Nat Commun ; 11(1): 2444, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32415068

RESUMO

The essential role of ORAI1 channels in receptor-evoked Ca2+ signaling is well understood, yet little is known about the physiological activation of the ORAI channel trio natively expressed in all cells. The roles of ORAI2 and ORAI3 have remained obscure. We show that ORAI2 and ORAI3 channels play a critical role in mediating the regenerative Ca2+ oscillations induced by physiological receptor activation, yet ORAI1 is dispensable in generation of oscillations. We reveal that ORAI2 and ORAI3 channels multimerize with ORAI1 to expand the range of sensitivity of receptor-activated Ca2+ signals, reflecting their enhanced basal STIM1-binding and heightened Ca2+-dependent inactivation. This broadened bandwidth of Ca2+ influx is translated by cells into differential activation of NFAT1 and NFAT4 isoforms. Our results uncover a long-sought role for ORAI2 and ORAI3, revealing an intricate control mechanism whereby heteromerization of ORAI channels mediates graded Ca2+ signals that extend the agonist-sensitivity to fine-tune transcriptional control.


Assuntos
Canais de Cálcio Ativados pela Liberação de Cálcio/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Carbacol/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Modelos Biológicos , Fatores de Transcrição NFATC/metabolismo , Proteína ORAI1/metabolismo , Ligação Proteica/efeitos dos fármacos , Isoformas de Proteínas/metabolismo , Multimerização Proteica/efeitos dos fármacos , Molécula 1 de Interação Estromal/metabolismo , Imagem com Lapso de Tempo
3.
Thromb Haemost ; 120(5): 776-792, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32369849

RESUMO

BACKGROUND: Implantable cardiovascular therapeutic devices, while hemodynamically effective, remain limited by thrombosis. A driver of device-associated thrombosis is shear-mediated platelet activation (SMPA). Underlying mechanisms of SMPA, as well as useful biomarkers able to detect and discriminate mechanical versus biochemical platelet activation, are poorly defined. We hypothesized that SMPA induces a differing pattern of biomarkers compared with biochemical agonists. METHODS: Gel-filtered human platelets were subjected to mechanical activation via either uniform constant or dynamic shear; or to biochemical activation by adenosine diphosphate (ADP), thrombin receptor-activating peptide 6 (TRAP-6), thrombin, collagen, epinephrine, or arachidonic acid. Markers of platelet activation (P-selectin, integrin αIIbß3 activation) and apoptosis (mitochondrial membrane potential, caspase 3 activation, and phosphatidylserine externalization [PSE]) were examined using flow cytometry. Platelet procoagulant activity was detected by chromogenic assay measuring thrombin generation. Contribution of platelet calcium flux in SMPA was tested employing calcium chelators, ethylenediaminetetraacetic acid (EDTA), and BAPTA-AM. RESULTS: Platelet exposure to continuous shear stress, but not biochemical agonists, resulted in a dramatic increase of PSE and procoagulant activity, while no integrin αIIbß3 activation occurred, and P-selectin levels remained barely elevated. SMPA was associated with dissipation of mitochondrial membrane potential, but no caspase 3 activation was observed. Shear-mediated PSE was significantly decreased by chelation of extracellular calcium with EDTA, while intracellular calcium depletion with BAPTA-AM had no significant effect. In contrast, biochemical agonists ADP, TRAP-6, arachidonic acid, and thrombin were potent inducers of αIIbß3 activation and/or P-selectin exposure. This differing pattern of biomarkers seen for SMPA for continuous uniform shear was replicated in platelets exposed to dynamic shear stress via circulation through a ventricular assist device-propelled circulatory loop. CONCLUSION: Elevated shear stress, but not biochemical agonists, induces a differing pattern of platelet biomarkers-with enhanced PSE and thrombin generation on the platelet surface. This differential biomarker phenotype of SMPA offers the potential for early detection and discrimination from that mediated by biochemical agonists.


Assuntos
Plaquetas/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Mecanotransdução Celular , Ativação Plaquetária/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Biomarcadores/sangue , Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/metabolismo , Plaquetas/patologia , Caspase 3/sangue , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Selectina-P/sangue , Fosfatidilserinas/sangue , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Estresse Mecânico
4.
Nutr Metab Cardiovasc Dis ; 30(6): 1032-1043, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32402583

RESUMO

BACKGROUND AND AIMS: Luteolin is a common flavonoid that is abundantly present in various edible plants, it is known to exhibit beneficial effects on cardiovascular system. However, the mechanisms which underlie the protective effects of luteolin on endothelial cell damage caused by oxidative stress remains unclear. The purpose of this study is to test the hypothesis which states that luteolin protects against H2O2-induced oxidative stress via modulating ROS-mediated P38 MAPK/NF-κB and calcium-evoked mitochondrial apoptotic signalling pathways. METHODS AND RESULTS: Human umbilical vein endothelial cells (HUVECs) were pretreated with luteolin prior to being stimulated by 600 µM H2O2 for another 24 h. The expression of native and phosphorylated-P38, IκB, NF-κB, native eNOS, phosphorylated-eNOS, iNOS and several apoptosis-related proteins were analyzed by Western blot. In addition, intracellular calcium was determined by fura-2 AM and mitochondrial membrane potential was examined by using JC1. Using the data gathered, we found indications that H2O2 induced P38 MAPK/NF-κB activation. H2O2 downregulated the expression of eNOS and upregulated iNOS, which in turn contribute to an elevated NO generation and protein nitrosylation. However, pretreatment with luteolin markedly reversed all of these alterations dose-dependently. Additionally, an intracellular calcium rise and subsequent mitochondrial membrane potential collapse, P53 phosphorylation, reduced BcL-2/Bax ratio in the mitochondrial membrane, release cytochrome c from mitochondria, leading to the subsequent activation of caspase 3 activation by H2O2 were all markedly suppressed in the presence of luteolin. CONCLUSION: Results from this study may provide the possible molecular mechanisms underlying cardiovascular protective effects of luteolin.


Assuntos
Antioxidantes/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Peróxido de Hidrogênio/toxicidade , Luteolina/farmacologia , Mitocôndrias/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Apoptose/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Fosforilação , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
5.
Ecotoxicol Environ Saf ; 194: 110382, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32146195

RESUMO

Deoxynivalenol(DON) has broad toxicity in livestock, but we know little about its neurotoxic mechanisms. We investigated DON neurotoxicity in the cerebral cortex, cerebellum, and hippocampus of "Duroc × Landrace × Yokshire" piglets. Control piglets were fed a basal diet, while those in low- and high-treatment groups were fed diets with 1.3 mg/kg and 2.2 mg/kg DON, respectively. After a 60 d trial, scanning electron microscopy revealed the destruction of hippocampal cell ultrastructure. As DON concentrations increased, oxidative damage also increased in the cerebral cortex, cerebellum, and hippocampus. Norepinephrine and 5-hydroxytryptamine concentrations tended to increase, whereas dopamine and γ-aminobutyric acid concentrations decreased. We also observed an increase in calcium concentration, relative mRNA expression of calcium/calmodulin-dependent protein kinase II (CaMKII), and CaMKII phosphorylation. However, calmodulin (CaM) mRNA and protein content decreased. Overall, our results suggest that DON acts through the Ca2+/CaM/CaMKII signaling pathway to influence cerebral lipid peroxidation and neurotransmitter levels.


Assuntos
Encéfalo/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Síndromes Neurotóxicas/etiologia , Neurotransmissores/metabolismo , Tricotecenos/toxicidade , Animais , Encéfalo/metabolismo , Cálcio/metabolismo , Dieta , Masculino , Síndromes Neurotóxicas/metabolismo , Oxirredução , Suínos , Desmame
6.
Artigo em Inglês | MEDLINE | ID: mdl-32159365

RESUMO

Cardiac inflammation has been proposed as one of the primary mechanisms of anthracycline-induced acute cardiotoxicity. A reduction in cardiac inflammation might also reduce cardiotoxicity. This study aimed to evaluate the potential of estrogen therapy and regular exercise on attenuating cardiac inflammation in the context of doxorubicin-induced cardiomyopathy. Ovariectomized rats were randomly allocated into estrogen supplementation, exercise training, and mast cell stabilizer treatment groups. Eight weeks after ovariectomy, rats received six cumulative doses of doxorubicin for two weeks. Echocardiography demonstrated a progressive decrease in ejection fraction in doxorubicin-treated rats without hypertrophic effect. This systolic defect was completely prevented by either estrogen supplementation or mast cell stabilizer treatment but not by regular exercise. As a heart disease indicator, increased ß-myosin heavy chain expression induced by doxorubicin could only be prevented by estrogen supplementation. Decrease in shortening and intracellular Ca2+ transients of cardiomyocytes were due to absence of female sex hormones without further effects of doxorubicin. Again, estrogen supplementation and mast cell stabilizer treatment prevented these changes but exercise training did not. Histological analysis indicated that the hyperactivation of cardiac mast cells in ovariectomized rats was augmented by doxorubicin. Estrogen supplementation and mast cell stabilizer treatment completely prevented both increases in mast cell density and degranulation, whereas exercise training partially attenuated the hyperactivation. Our results, therefore, suggest that estrogen supplementation acts similarly to mast cell stabilizers in attenuating the effects of doxorubicin. Ineffectiveness of regular exercise in preventing the acute cardiotoxicity of doxorubicin might be due to a lesser effect on preventing cardiac inflammation.


Assuntos
Degranulação Celular/efeitos dos fármacos , Doxorrubicina , Estradiol/administração & dosagem , Terapia de Reposição de Estrogênios , Terapia por Exercício , Mastócitos/efeitos dos fármacos , Contração Miocárdica/efeitos dos fármacos , Disfunção Ventricular Esquerda/prevenção & controle , Função Ventricular Esquerda/efeitos dos fármacos , Animais , Sinalização do Cálcio/efeitos dos fármacos , Cardiotoxicidade , Modelos Animais de Doenças , Feminino , Mediadores da Inflamação/metabolismo , Mastócitos/metabolismo , Mastócitos/patologia , Miocárdio/metabolismo , Miocárdio/patologia , Ovariectomia , Estresse Oxidativo/efeitos dos fármacos , Ratos Sprague-Dawley , Disfunção Ventricular Esquerda/induzido quimicamente , Disfunção Ventricular Esquerda/patologia , Disfunção Ventricular Esquerda/fisiopatologia
7.
Cardiovasc Drugs Ther ; 34(2): 153-164, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32146638

RESUMO

PURPOSE: Vepoloxamer (VEPO), a rheologic agent, repairs damaged cell membranes, thus inhibiting unregulated Ca2+ entry into cardiomyocytes. This study examined the effects of i.v. infusion of VEPO on LV function in dogs with coronary microembolization-induced heart failure (HF) (LV ejection fraction, EF ~ 30%). METHODS: Thirty-five HF dogs were studied. Study 1: 21 of 35 dogs were randomized to 2-h infusion of VEPO at dose of 450 mg/kg (n = 7) or VEPO at 225 mg/kg (n = 7) or normal saline (control, n = 7). Hemodynamics were measured at 2 h, 24 h, 1 week, and 2 weeks after infusion. Study 2: 14 HF dogs were randomized to 2-h infusions of VEPO (450 mg/kg, n = 7) or normal saline (control, n = 7). Each dog received 2 infusions of VEPO or saline (pulsed therapy) 3 weeks apart and hemodynamics measured at 24 h, and 1, 2, and 3 weeks after each infusion. In both studies, the change between pre-infusion measures and measures at other time points (treatment effect, Δ) was calculated. RESULTS: Study 1: compared to pre-infusion, high dose VEPO increased LVEF by 11 ± 2% at 2 h, 8 ± 2% at 24 h (p < 0.05), 8 ± 2% at 1 week (p < 0.05), and 4 ± 2% at 2 weeks. LV EF also increased with low-dose VEPO but not with saline. Study 2: VEPO but not saline significantly increased LVEF by 6.0 ± 0.7% at 2 h (p < 0.05); 7.0 ± 0.7%% at 1 week (p < 0.05); 1.0 ± 0.6% at 3 weeks; 6.0 ± 1.3% at 4 weeks (p < 0.05); and 5.9 ± 1.3% at 6 weeks (p < 0.05). CONCLUSIONS: Intravenous VEPO improves LV function for at least 1 week after infusion. The benefits can be extended with pulsed VEPO therapy. The results support development of VEPO for treating patients with acute on chronic HF.


Assuntos
Fármacos Cardiovasculares/administração & dosagem , Insuficiência Cardíaca/tratamento farmacológico , Poloxâmero/administração & dosagem , Volume Sistólico/efeitos dos fármacos , Disfunção Ventricular Esquerda/tratamento farmacológico , Função Ventricular Esquerda/efeitos dos fármacos , Animais , Sinalização do Cálcio/efeitos dos fármacos , Modelos Animais de Doenças , Cães , Relação Dose-Resposta a Droga , Insuficiência Cardíaca/diagnóstico por imagem , Insuficiência Cardíaca/fisiopatologia , Infusões Intravenosas , Disfunção Ventricular Esquerda/diagnóstico por imagem , Disfunção Ventricular Esquerda/fisiopatologia
8.
Cardiovasc Diabetol ; 19(1): 13, 2020 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-32035482

RESUMO

BACKGROUND AND AIMS: Sodium-glucose linked cotransporter-2 (SGLT2) inhibitors reduce the likelihood of hospitalization for heart failure and cardiovascular death in both diabetic and non-diabetic individuals with reduced ejection fraction heart failure. Because SGLT2 inhibitors lead to volume contraction with reductions in both preload and afterload, these load-dependent factors are thought to be major contributors to the cardioprotective effects of the drug class. Beyond these effects, we hypothesized that SGLT2 inhibitors may also improve intrinsic cardiac function, independent of loading conditions. METHODS: Pressure-volume (P-V) relationship analysis was used to elucidate changes in intrinsic cardiac function, independent of alterations in loading conditions in animals with experimental myocardial infarction, a well-established model of HFrEF. Ten-week old, non-diabetic Fischer F344 rats underwent ligation of the left anterior descending (LAD) coronary artery to induce myocardial infarction (MI) of the left ventricle (LV). Following confirmation of infarct size with echocardiography 1-week post MI, animals were randomized to receive vehicle, or the SGLT2 inhibitor, empagliflozin. Cardiac function was assessed by conductance catheterization just prior to termination 6 weeks later. RESULTS: The circumferential extent of MI in animals that were subsequently randomized to vehicle or empagliflozin groups was similar. Empagliflozin did not affect fractional shortening (FS) as assessed by echocardiography. In contrast, load-insensitive measures of cardiac function were substantially improved with empagliflozin. Load-independent measures of cardiac contractility, preload recruitable stroke work (PRSW) and end-systolic pressure volume relationship (ESPVR) were higher in rats that had received empagliflozin. Consistent with enhanced cardiac performance in the heart failure setting, systolic blood pressure (SBP) was higher in rats that had received empagliflozin despite its diuretic effects. A trend to improved diastolic function, as evidenced by reduction in left ventricular end-diastolic pressure (LVEDP) was also seen with empagliflozin. MI animals treated with vehicle demonstrated myocyte hypertrophy, interstitial fibrosis and evidence for changes in key calcium handling proteins (all p < 0.05) that were not affected by empagliflozin therapy. CONCLUSION: Empagliflozin therapy improves cardiac function independent of loading conditions. These findings suggest that its salutary effects are, at least in part, due to actions beyond a direct effect of reduced preload and afterload.


Assuntos
Compostos Benzidrílicos/farmacologia , Glucosídeos/farmacologia , Insuficiência Cardíaca/tratamento farmacológico , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Volume Sistólico/efeitos dos fármacos , Função Ventricular Esquerda/efeitos dos fármacos , Pressão Ventricular/efeitos dos fármacos , Animais , Sinalização do Cálcio/efeitos dos fármacos , Modelos Animais de Doenças , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Infarto do Miocárdio/complicações , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/fisiopatologia , Miocárdio/metabolismo , Miocárdio/patologia , Ratos Endogâmicos F344
9.
J Neurosci ; 40(11): 2189-2199, 2020 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-32019828

RESUMO

The interaction between the immune system and the nervous system has been at the center of multiple research studies in recent years. Whereas the role played by cytokines as neuronal mediators is no longer contested, the mechanisms by which cytokines modulate pain processing remain to be elucidated. In this study, we have analyzed the involvement of granulocyte-macrophage colony stimulating factor (GM-CSF) in nociceptor activation in male and female mice. Previous studies have suggested GM-CSF might directly activate neurons. However, here we established the absence of a functional GM-CSF receptor in murine nociceptors, and suggest an indirect mechanism of action, via immune cells. We report that GM-CSF applied directly to magnetically purified nociceptors does not induce any transcriptional changes in nociceptive genes. In contrast, conditioned medium from GM-CSF-treated murine macrophages was able to drive nociceptor transcription. We also found that conditioned medium from nociceptors treated with the well established pain mediator, nerve growth factor, could also modify macrophage gene transcription, providing further evidence for a bidirectional crosstalk.SIGNIFICANCE STATEMENT The interaction of the immune system and the nervous system is known to play an important role in the development and maintenance of chronic pain disorders. Elucidating the mechanisms of these interactions is an important step toward understanding, and therefore treating, chronic pain disorders. This study provides evidence for a two-way crosstalk between macrophages and nociceptors in the peripheral nervous system, which may contribute to the sensitization of nociceptors by cytokines in pain development.


Assuntos
Dor Crônica/fisiopatologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/fisiologia , Nociceptores/efeitos dos fármacos , Animais , Sinalização do Cálcio/efeitos dos fármacos , Comunicação Celular , Células Cultivadas , Dor Crônica/induzido quimicamente , Meios de Cultivo Condicionados/farmacologia , Feminino , Gânglios Espinais/citologia , Regulação da Expressão Gênica/efeitos dos fármacos , Inflamação/induzido quimicamente , Inflamação/fisiopatologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator de Crescimento Neural/farmacologia , Nociceptores/fisiologia , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/efeitos dos fármacos , Fator de Transcrição STAT5/fisiologia , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/metabolismo , Transcrição Genética/efeitos dos fármacos
10.
Am J Physiol Renal Physiol ; 318(2): F496-F505, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31904286

RESUMO

Urethral smooth muscle (USM) generates tone to prevent urine leakage from the bladder during filling. USM tone has been thought to be a voltage-dependent process, relying on Ca2+ influx via voltage-dependent Ca2+ channels in USM cells, modulated by the activation of Ca2+-activated Cl- channels encoded by Ano1. However, recent findings in the mouse have suggested that USM tone is voltage independent, relying on Ca2+ influx through Orai channels via store-operated Ca2+ entry (SOCE). We explored if this pathway also occurred in the pig using isometric tension recordings of USM tone. Pig USM strips generated myogenic tone, which was nearly abolished by the Cav1.2 channel antagonist nifedipine and the ATP-dependent K+ channel agonist pinacidil. Pig USM tone was reduced by the Orai channel blocker GSK-7975A. Electrical field stimulation (EFS) led to phentolamine-sensitive contractions of USM strips. Contractions of pig USM were also induced by phenylephrine. Phenylephrine-evoked and EFS-evoked contractions of pig USM were reduced by ~50-75% by nifedipine and ~30% by GSK-7975A. Inhibition of Ano1 channels had no effect on tone or EFS-evoked contractions of pig USM. In conclusion, unlike the mouse, pig USM exhibited voltage-dependent tone and agonist/EFS-evoked contractions. Whereas SOCE plays a role in generating tone and agonist/neural-evoked contractions in both species, this dominates in the mouse. Tone and agonist/EFS-evoked contractions of pig USM are the result of Ca2+ influx primarily through Cav1.2 channels, and no evidence was found supporting a role of Ano1 channels in modulating these mechanisms.


Assuntos
Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio Ativados pela Liberação de Cálcio/metabolismo , Sinalização do Cálcio , Contração Isométrica , Músculo Liso/metabolismo , Uretra/metabolismo , Animais , Benzamidas/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/efeitos dos fármacos , Canais de Cálcio Ativados pela Liberação de Cálcio/antagonistas & inibidores , Sinalização do Cálcio/efeitos dos fármacos , Estimulação Elétrica , Feminino , Técnicas In Vitro , Contração Isométrica/efeitos dos fármacos , Masculino , Músculo Liso/efeitos dos fármacos , Nifedipino/farmacologia , Fenilefrina/farmacologia , Pirazóis/farmacologia , Sus scrofa , Uretra/efeitos dos fármacos
11.
Am J Respir Crit Care Med ; 201(8): 946-954, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-31898911

RESUMO

Rationale: Enhancing non-CFTR (cystic fibrosis transmembrane conductance regulator)-mediated anion secretion is an attractive therapeutic approach for the treatment of cystic fibrosis (CF) and other mucoobstructive diseases.Objectives: To determine the effects of TMEM16A potentiation on epithelial fluid secretion and mucociliary clearance.Methods: The effects of a novel low-molecular-weight TMEM16A potentiator (ETX001) were evaluated in human cell and animal models of airway epithelial function and mucus transport.Measurements and Main Results: Potentiating the activity of TMEM16A with ETX001 increased the Ca2+-activated Cl- channel activity and anion secretion in human bronchial epithelial (HBE) cells from patients with CF without impacting calcium signaling. ETX001 rapidly increased fluid secretion and airway surface liquid height in CF-HBE cells under both static conditions and conditions designed to mimic the shear stress associated with tidal breathing. In ovine models of mucus clearance (tracheal mucus velocity and mucociliary clearance), inhaled ETX001 was able to accelerate clearance both when CFTR function was reduced by administration of a pharmacological blocker and when CFTR was fully functional.Conclusions: Enhancing the activity of TMEM16A increases epithelial fluid secretion and enhances mucus clearance independent of CFTR function. TMEM16A potentiation is a novel approach for the treatment of patients with CF and non-CF mucoobstructive diseases.


Assuntos
Anoctamina-1/efeitos dos fármacos , Fibrose Cística/metabolismo , Células Epiteliais/efeitos dos fármacos , Moduladores de Transporte de Membrana/farmacologia , Depuração Mucociliar/efeitos dos fármacos , Muco/efeitos dos fármacos , Administração por Inalação , Animais , Anoctamina-1/metabolismo , Brônquios/citologia , Sinalização do Cálcio/efeitos dos fármacos , Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Humanos , Transporte de Íons/efeitos dos fármacos , Técnicas de Patch-Clamp , Respiração , Mucosa Respiratória/citologia , Ovinos , Traqueia/efeitos dos fármacos , Traqueia/metabolismo
12.
Mol Cell Biochem ; 465(1-2): 65-73, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31894530

RESUMO

Curcumin (Cur) is widely used as an anti-inflammation agent and has anti-depression potential. Neuroinflammation mediated by Ca2+ channel activation is closely associated with the progression of post-stroke depression (PSD). In the current study, the role of P2X7 receptor (P2X7R) in the anti-PSD function of Cur was explored. Rats were subjected to middle cerebral artery occlusion (MCAO) surgery and chronic mild stress administration to induce PSD symptoms and then treated with Cur. The behaviors of rats were assessed with sucrose preference and forced swim tests. The accumulation of Ca2+ and the systemic inflammatory response in rats were detected. To determine the role of P2X7R in the anti-PSD function of curcumin, the PSD mice were further administrated with P2X7R agonist and antagonist. The administration of Cur attenuated behavior disorders associated with PSD. Moreover, the Ca2+ accumulation and the inflammatory response associated with PSD were also blocked by Cur. Cur also inhibited the activation of Ca2+ channel. The induced activity of P2X7R blocked the function of Cur by maintaining the symptoms of PSD in Cur-treated rats. Collectively, the anti-PSD function of Cur was dependent on the inhibition of P2X7R, which then deactivated Ca2+ channel-mediated inflammatory response associated with PSD progression.


Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Cálcio/metabolismo , Curcumina/farmacologia , Microglia/metabolismo , Agonistas do Receptor Purinérgico P2X/farmacologia , Receptores Purinérgicos P2X7/metabolismo , Animais , Depressão/metabolismo , Depressão/patologia , Masculino , Microglia/patologia , Antagonistas do Receptor Purinérgico P2X/farmacologia , Ratos , Ratos Sprague-Dawley , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologia
13.
Cardiovasc Diabetol ; 19(1): 7, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31924211

RESUMO

BACKGROUND: Diabetes mellitus type 2 (DM2) is a risk factor for developing heart failure but there is no specific therapy for diabetic heart disease. Sodium glucose transporter 2 inhibitors (SGLT2I) are recently developed diabetic drugs that primarily work on the kidney. Clinical data describing the cardiovascular benefits of SGLT2Is highlight the potential therapeutic benefit of these drugs in the prevention of cardiovascular events and heart failure. However, the underlying mechanism of protection remains unclear. We investigated the effect of Dapagliflozin-SGLT2I, on diabetic cardiomyopathy in a mouse model of DM2. METHODS: Cardiomyopathy was induced in diabetic mice (db/db) by subcutaneous infusion of angiotensin II (ATII) for 30 days using an osmotic pump. Dapagliflozin (1.5 mg/kg/day) was administered concomitantly in drinking water. Male homozygous, 12-14 weeks old WT or db/db mice (n = 4-8/group), were used for the experiments. Isolated cardiomyocytes were exposed to glucose (17.5-33 mM) and treated with Dapagliflozin in vitro. Intracellular calcium transients were measured using a fluorescent indicator indo-1. RESULTS: Angiotensin II infusion induced cardiomyopathy in db/db mice, manifested by cardiac hypertrophy, myocardial fibrosis and inflammation (TNFα, TLR4). Dapagliflozin decreased blood glucose (874 ± 111 to 556 ± 57 mg/dl, p < 0.05). In addition it attenuated fibrosis and inflammation and increased the left ventricular fractional shortening in ATII treated db/db mice. In isolated cardiomyocytes Dapagliflozin decreased intracellular calcium transients, inflammation and ROS production. Finally, voltage-dependent L-type calcium channel (CACNA1C), the sodium-calcium exchanger (NCX) and the sodium-hydrogen exchanger 1 (NHE) membrane transporters expression was reduced following Dapagliflozin treatment. CONCLUSION: Dapagliflozin was cardioprotective in ATII-stressed diabetic mice. It reduced oxygen radicals, as well the activity of membrane channels related to calcium transport. The cardioprotective effect manifested by decreased fibrosis, reduced inflammation and improved systolic function. The clinical implication of our results suggest a novel pharmacologic approach for the treatment of diabetic cardiomyopathy through modulation of ion homeostasis.


Assuntos
Compostos Benzidrílicos/farmacologia , Glicemia/efeitos dos fármacos , Diabetes Mellitus/tratamento farmacológico , Cardiomiopatias Diabéticas/prevenção & controle , Glucosídeos/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Função Ventricular Esquerda/efeitos dos fármacos , Angiotensina II , Animais , Biomarcadores/sangue , Glicemia/metabolismo , Canais de Cálcio Tipo L/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Diabetes Mellitus/sangue , Cardiomiopatias Diabéticas/induzido quimicamente , Cardiomiopatias Diabéticas/metabolismo , Cardiomiopatias Diabéticas/fisiopatologia , Modelos Animais de Doenças , Fibrose , Mediadores da Inflamação/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Ratos Sprague-Dawley , Trocador de Sódio e Cálcio/metabolismo , Trocador 1 de Sódio-Hidrogênio/metabolismo
14.
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
15.
J Biochem Mol Toxicol ; 34(3): e22440, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31926057

RESUMO

Although vitamin D (VD) and calcium (Ca) attenuate cadmium (Cd) metabolism, their combined antioxidant and anti-inflammatory actions against Cd toxicity have not been previously explored. Hence, this study measured the protective effects of VD ± Ca supplements against Cd hepatotoxicity. Forty adult male rats were distributed to: negative controls (NCs), positive controls (PCs), VD, Ca, and VD3 and Ca (VDC) groups. All groups, except NC, received CdCl2 in drinking water (44 mg/L) for 4 weeks individually or concurrently with intramuscular VD3 (600 IU/kg; three times per week) and/or oral Ca (100 mg/kg; five times per week). The PC group showed abnormal hepatic biochemical parameters and increase in cellular cytochrome C, caspase-9, and caspase-3 alongside the apoptotic/necrotic cell numbers by terminal deoxynucleotidyl transferase dUTP nick end labeling technique. The PC hepatic tissue also had substantially elevated pro-oxidants (malondialdehyde [MDA]/H2 O2 /protein carbonyls) and inflammatory cytokines (interleukin 1ß [IL-1ß]/IL-6/IL17A/tumor necrosis factor-α), whereas the anti-inflammatory (IL-10/IL-22) and antioxidants (glutathione [GSH]/GPx/catalase enzyme [CAT]) markers declined. Hypovitaminosis D, low hepatic tissue Ca, aberrant hepatic expression of VD-metabolizing enzymes (Cyp2R1/Cyp27a1/cyp24a1), receptor and binding protein alongside Ca-membrane (CaV 1.1/CaV 3.1), and store-operated (RyR1/ITPR1) channels, and Ca-binding proteins (CAM/CAMKIIA/S100A1/S100B) were observed in the PC group. Both monotherapies decreased serum, but not tissue Cd levels, restored the targeted hepatic VD/Ca molecules' expression. However, these effects were more prominent in the VD group than the Ca group. The VDC group, contrariwise, disclosed the greatest alleviations on serum and tissue Cd, inflammatory and oxidative markers, the VD/Ca molecules and tissue integrity. In conclusion, this report is the first to reveal boosted protection for cosupplementing VD and Ca against Cd hepatotoxicity that could be due to enhanced antioxidative, anti-inflammatory, and modulation of the Ca pathways.


Assuntos
Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Cálcio/farmacologia , Doença Hepática Induzida por Substâncias e Drogas , Colecalciferol/farmacologia , Fígado , Animais , Cádmio/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Ratos , Ratos Wistar
16.
Biochem Biophys Res Commun ; 524(2): 340-345, 2020 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-31996305

RESUMO

The brain-gut axis which is an interaction between recognition and emotion and the gut sensory system for food and microbiota is important for health. However, there is no real-time monitoring system of the brain and the gut simultaneously so far. We attempted to establish a dual real-time monitoring system for the brain-gut axis by a combination of intravital Ca2+ imaging of the gut and electroencephalogram. Using a conditional Yellow Cameleon 3.60 expression mouse line, we performed intravital imaging of the gut, electrophysiological recordings of the vagus nerve, and electroencephalogram recordings of the various cortical regions simultaneously upon capsaicin stimuli as a positive control. Upon capsaicin administration into the small intestinal lumen, a simultaneous response of Ca2+ signal in the enteric nervous system and cortical local field potentials (LFPs) was successfully observed. Both of them responded immediately upon capsaicin stimuli. Capsaicin triggered a significant increase in the frequency of vagus nerve spikes and a significant decrease in the slow-wave power of cortical LFPs. Furthermore, capsaicin induced delayed and sustained Ca2+ signal in intestinal epithelial cells and then suppressed intestinal motility. The dual real-time monitoring system of the brain and the gut enables to dissect the interaction between the brain and the gut over time with precision.


Assuntos
Encéfalo/fisiologia , Sistema Nervoso Entérico/fisiologia , Trato Gastrointestinal/fisiologia , Nervo Vago/fisiologia , Animais , Encéfalo/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Capsaicina/farmacologia , Linhagem Celular , Células Cultivadas , Fenômenos Eletrofisiológicos , Sistema Nervoso Entérico/efeitos dos fármacos , Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/inervação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monitorização Fisiológica , Fármacos do Sistema Sensorial/farmacologia , Nervo Vago/efeitos dos fármacos
17.
Artigo em Inglês | MEDLINE | ID: mdl-31913694

RESUMO

We studied the mechanisms by which carotid body glomus (type 1) cells produce spontaneous Ca2+ oscillations in normoxia and hypoxia. In cells perfused with normoxic solution at 37°C, we observed relatively uniform, low-frequency Ca2+ oscillations in >60% of cells, with each cell showing its own intrinsic frequency and amplitude. The mean frequency and amplitude of Ca2+ oscillations were 0.6 ± 0.1 Hz and 180 ± 42 nM, respectively. The duration of each Ca2+ oscillation ranged from 14 to 26 s (mean of ∼20 s). Inhibition of inositol (1,4,5)-trisphosphate receptor and store-operated Ca2+ entry (SOCE) using 2-APB abolished Ca2+ oscillations. Inhibition of endoplasmic reticulum Ca2+-ATPase (SERCA) using thapsigargin abolished Ca2+ oscillations. ML-9, an inhibitor of STIM1 translocation, also strongly reduced Ca2+ oscillations. Inhibitors of L- and T-type Ca2+ channels (Cav; verapamil>nifedipine>TTA-P2) markedly reduced the frequency of Ca2+ oscillations. Thus, Ca2+ oscillations observed in normoxia were caused by cyclical Ca2+ fluxes at the ER, which was supported by Ca2+ influx via Ca2+ channels. Hypoxia (2-5% O2) increased the frequency and amplitude of Ca2+ oscillations, and Cav inhibitors (verapamil>nifedipine>>TTA-P2) reduced these effects of hypoxia. Our study shows that Ca2+ oscillations represent the basic Ca2+ signaling mechanism in normoxia and hypoxia in CB glomus cells.


Assuntos
Cálcio/metabolismo , Corpo Carotídeo/metabolismo , Hipóxia/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Corpo Carotídeo/efeitos dos fármacos , Linhagem Celular , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Feminino , Masculino , Nifedipino/farmacologia , Ratos , Ratos Sprague-Dawley , Molécula 1 de Interação Estromal/metabolismo , Tapsigargina/farmacologia
18.
Aquat Toxicol ; 218: 105352, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31790938

RESUMO

Cadmium (Cd) affects plants and animal health seriously. Ca2+ signals in plant cells are important for adaptive responses to environmental stresses. Here we showed that 50 µM Cd shock stimulated the Ca2+ signal via modifying the instantaneous Ca2+ flux from influx of 17 pmol·cm-2·s-1 to the efflux of 240 pmol·cm-2·s-1 at 100 µm from rhizoid tip. And the Ca2+ signal transferred to the vein and mesophyll cell. The Ca addition decreased the accumulation of Cd. The gene expression of glutamate receptor-like (GLR) proteins, which is activated by Glu and triggers Ca2+ flux, was increased significantly by 24 h Cd stress. Glu content was increased under Cd stress and exogenous Glu triggered the Ca2+ signal in duckweed, while Ca2+ addition caused no influence to Glu content. GABA, which is synthesized from Glu and acts as an inhibitory neurotransmitter, has been decreased with 24 h Cd treatment. GABA addition increased the abscission rate and Glu addition decreased the abscission rate during Cd stress, suggesting that the Glu/GABA ratio is important for responding to Cd. This research shows the sight of the Glu, Ca2+, GABA signaling networks during Cd stress.


Assuntos
Araceae/efeitos dos fármacos , Cádmio/toxicidade , Sinalização do Cálcio/efeitos dos fármacos , Ácido Glutâmico/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Ácido gama-Aminobutírico/metabolismo , Animais , Araceae/metabolismo , Cádmio/metabolismo , Poluentes Químicos da Água/metabolismo
19.
Am J Physiol Heart Circ Physiol ; 318(2): H264-H282, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31834834

RESUMO

The ß1-adrenergic regulation of cardiac myocyte contraction plays an important role in regulating heart function. Activation of this system leads to an increased heart rate and stronger myocyte contraction. However, chronic stimulation of the ß1-adrenergic signaling system can lead to cardiac hypertrophy and heart failure. To understand the mechanisms of action of ß1-adrenoceptors, a mathematical model of cardiac myocyte contraction that includes the ß1-adrenergic system was developed and studied. The model was able to simulate major experimental protocols for measurements of steady-state force-calcium relationships, cross-bridge release rate and force development rate, force-velocity relationship, and force redevelopment rate. It also reproduced quite well frequency and isoproterenol dependencies for intracellular Ca2+ concentration ([Ca2+]i) transients, total contraction force, and sarcomere shortening. The mathematical model suggested the mechanisms of increased contraction force and myocyte shortening on stimulation of ß1-adrenergic receptors is due to phosphorylation of troponin I and myosin-binding protein C and increased [Ca2+]i transient resulting from activation of the ß1-adrenergic signaling system. The model was used to simulate work-loop contractions and estimate the power during the cardiac cycle as well as the effects of 4-aminopyridine and tedisamil on the myocyte contraction. The developed mathematical model can be used further for simulations of contraction of ventricular myocytes from genetically modified mice and myocytes from mice with chronic cardiac diseases.NEW & NOTEWORTHY A new mathematical model of mouse ventricular myocyte contraction that includes the ß1-adrenergic system was developed. The model simulated major experimental protocols for myocyte contraction and predicted the effects of 4-aminopyridine and tedisamil on the myocyte contraction. The model also allowed for simulations of work-loop contractions and estimation of the power during the cardiac cycle.


Assuntos
Ventrículos do Coração , Contração Miocárdica/fisiologia , Miócitos Cardíacos/fisiologia , Receptores Adrenérgicos beta 1/fisiologia , Algoritmos , Animais , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Cardiotônicos/farmacologia , Proteínas de Transporte/metabolismo , Simulação por Computador , Frequência Cardíaca/fisiologia , Ventrículos do Coração/citologia , Ventrículos do Coração/efeitos dos fármacos , Camundongos , Modelos Teóricos , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Fosforilação , Bloqueadores dos Canais de Potássio/farmacologia , Receptores Adrenérgicos beta 1/efeitos dos fármacos , Sarcômeros/fisiologia , Troponina I/metabolismo , Troponina I/fisiologia
20.
Proc Natl Acad Sci U S A ; 117(3): 1711-1721, 2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31811023

RESUMO

Drug resistance in fungal pathogens has risen steadily over the past decades due to long-term azole therapy or triazole usage in agriculture. Modification of the drug target protein to prevent drug binding is a major recognized route to induce drug resistance. However, mechanisms for nondrug target-induced resistance remain only loosely defined. Here, we explore the molecular mechanisms of multidrug resistance resulted from an efficient adaptation strategy for survival in drug environments in the human pathogen Aspergillus fumigatus We show that mutants conferring multidrug resistance are linked with mitochondrial dysfunction induced by defects in heme A biosynthesis. Comparison of the gene expression profiles between the drug-resistant mutants and the parental wild-type strain shows that multidrug-resistant transporters, chitin synthases, and calcium-signaling-related genes are significantly up-regulated, while scavenging mitochondrial reactive oxygen species (ROS)-related genes are significantly down-regulated. The up-regulated-expression genes share consensus calcium-dependent serine threonine phosphatase-dependent response elements (the binding sites of calcium-signaling transcription factor CrzA). Accordingly, drug-resistant mutants show enhanced cytosolic Ca2+ transients and persistent nuclear localization of CrzA. In comparison, calcium chelators significantly restore drug susceptibility and increase azole efficacy either in laboratory-derived or in clinic-isolated A. fumigatus strains. Thus, the mitochondrial dysfunction as a fitness cost can trigger calcium signaling and, therefore, globally up-regulate a series of embedding calcineurin-dependent-response-element genes, leading to antifungal resistance. These findings illuminate how fitness cost affects drug resistance and suggest that disruption of calcium signaling might be a promising therapeutic strategy to fight against nondrug target-induced drug resistance.


Assuntos
Aspergillus fumigatus/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Farmacorresistência Fúngica/efeitos dos fármacos , Farmacorresistência Fúngica/fisiologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Doenças Mitocondriais/metabolismo , Alquil e Aril Transferases/genética , Antifúngicos/farmacologia , Aspergillus fumigatus/genética , Quelantes de Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Quitina Sintase/genética , Farmacorresistência Fúngica/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Heme/análogos & derivados , Heme/biossíntese , Humanos , Proteínas de Membrana/genética , Mitocôndrias/genética , Doenças Mitocondriais/genética , Fosfoproteínas Fosfatases/metabolismo , Espécies Reativas de Oxigênio , Fatores de Transcrição/metabolismo , Triazóis/farmacologia
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