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1.
iScience ; 25(1): 103636, 2022 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-35024579

RESUMO

Acidification in intracellular organelles is tightly linked to the influx of Cl- counteracting proton translocation by the electrogenic V-ATPase. We quantified the dynamics of Cl- transfer accompanying cargo incorporation into single phagosomes in alveolar macrophages (AMs). Phagosomal Cl- concentration and acidification magnitude were followed in real time with maximal acidification achieved at levels of approximately 200 mM. Live cell confocal microscopy verified that phagosomal Cl- influx utilized predominantly the Cl- channel CFTR. Relative levels of elemental chlorine (Cl) in hard X-ray fluorescence microprobe (XFM) analysis within single phagosomes validated the increase in Cl- content. XFM revealed the complex interplay between elemental K content inside the phagosome and changes in Cl- during phagosomal particle uptake. Cl- -dependent changes in phagosomal membrane potential were obtained using second harmonic generation (SHG) microscopy. These studies provide a mechanistic insight for screening studies in drug development targeting pulmonary inflammatory disease.

2.
Commun Biol ; 5(1): 13, 2022 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-35013561

RESUMO

Extracellular vesicles (EVs) are cell-derived membranous structures carrying transmembrane proteins and luminal cargo. Their complex cargo requires pH stability in EVs while traversing diverse body fluids. We used a filtration-based platform to capture and stabilize EVs based on their size and studied their pH regulation at the single EV level. Dead-end filtration facilitated EV capture in the pores of an ultrathin (100 nm thick) and nanoporous silicon nitride (NPN) membrane within a custom microfluidic device. Immobilized EVs were rapidly exposed to test solution changes driven across the backside of the membrane using tangential flow without exposing the EVs to fluid shear forces. The epithelial sodium-hydrogen exchanger, NHE1, is a ubiquitous plasma membrane protein tasked with the maintenance of cytoplasmic pH at neutrality. We show that NHE1 identified on the membrane of EVs is functional in the maintenance of pH neutrality within single vesicles. This is the first mechanistic description of EV function on the single vesicle level.


Assuntos
Diagnóstico por Imagem/métodos , Vesículas Extracelulares/fisiologia , Técnicas Analíticas Microfluídicas/métodos , Microfluídica/métodos , Animais , Filtração , Concentração de Íons de Hidrogênio , Camundongos
3.
Methods Mol Biol ; 2169: 71-80, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32548820

RESUMO

The detection of dynamic conformational changes in proteins in live cells is challenging. Live-cell FRET (Förster Resonance Energy Transfer) is an example of a noninvasive technique that can be used to achieve this goal at nanometer resolution. FRET-based assays are dependent on the presence of fluorescent probes, such as CFP- and YFP-conjugated protein pairs. Here, we describe an experimental protocol in which live-cell FRET was used to measure conformational changes in caveolin-1 (Cav-1) oligomers on the surface of plasmalemma vesicles, or caveolae.


Assuntos
Cavéolas/metabolismo , Caveolina 1/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Processamento de Imagem Assistida por Computador/métodos , Caveolina 1/genética , Transferência Ressonante de Energia de Fluorescência/instrumentação , Células HEK293 , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Fosforilação , Transfecção
4.
Mol Biol Cell ; 29(10): 1190-1202, 2018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29563255

RESUMO

We hypothesized that the maintenance of vascular homeostasis is critically dependent on the expression and reciprocal regulation of caveolin-1 (Cav-1) and endothelial nitric oxide synthase (eNOS) in endothelial cells (ECs). Skeletal muscle biopsies from subjects with type 2 diabetes showed 50% less Cav-1 and eNOS than those from lean healthy controls. The Cav-1:eNOS expression ratio was 200:1 in primary culture human ECs. Cav-1 small interfering RNA (siRNA) reduced eNOS protein and gene expression in association with a twofold increase in eNOS phosphorylation and nitrate production per molecule of eNOS, which was reversed in cells overexpressing Adv-Cav-1-GFP. Upon addition of the Ca2+ ionophore A23187 to activate eNOS, we observed eNOS Ser1177 phosphorylation, its translocation to ß-catenin-positive cell-cell junctions, and increased colocalization of eNOS and Cav-1 within 5 min. We also observed Cav-1 S-nitrosylation and destabilization of Cav-1 oligomers in cells treated with A23187 as well as insulin or albumin, and this could be blocked by L-NAME, PP2, or eNOS siRNA. Finally, caveola-mediated endocytosis of albumin or insulin was reduced by Cav-1 or eNOS siRNA, and the effect of Cav-1 siRNA was rescued by Adv-Cav-1-GFP. Thus, Cav-1 stabilizes eNOS expression and regulates its activity, whereas eNOS-derived NO promotes caveola-mediated endocytosis.


Assuntos
Caveolina 1/metabolismo , Células Endoteliais/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Adulto , Albuminas/metabolismo , Biópsia , Calcimicina/farmacologia , Cálcio/metabolismo , Estudos de Casos e Controles , Membrana Celular/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Células HEK293 , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Insulina/metabolismo , Junções Intercelulares/efeitos dos fármacos , Junções Intercelulares/metabolismo , Ionóforos/farmacologia , Pessoa de Meia-Idade , Peso Molecular , Óxido Nítrico/metabolismo , Nitrosação , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Quinases da Família src/metabolismo
5.
Mol Biol Cell ; 27(13): 2090-106, 2016 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-27170175

RESUMO

Caveolin 1 (Cav1) is a required structural component of caveolae, and its phosphorylation by Src is associated with an increase in caveolae-mediated endocytosis. Here we demonstrate, using quantitative live-cell 4D, TIRF, and FRET imaging, that endocytosis and trafficking of caveolae are associated with a Cav1 Tyr-14 phosphorylation-dependent conformational change, which spatially separates, or loosens, Cav1 molecules within the oligomeric caveolar coat. When tracked by TIRF and spinning-disk microscopy, cells expressing phosphomimicking Cav1 (Y14D) mutant formed vesicles that were greater in number and volume than with Y14F-Cav1-GFP. Furthermore, we observed in HEK cells cotransfected with wild-type, Y14D, or Y14F Cav1-CFP and -YFP constructs that FRET efficiency was greater with Y14F pairs than with Y14D, indicating that pY14-Cav1 regulates the spatial organization of Cav1 molecules within the oligomer. In addition, albumin-induced Src activation or direct activation of Src using a rapamycin-inducible Src construct (RapR-Src) led to an increase in monomeric Cav1 in Western blots, as well as a simultaneous increase in vesicle number and decrease in FRET intensity, indicative of a Src-mediated conformational change in CFP/YFP-tagged WT-Cav1 pairs. We conclude that phosphorylation of Cav1 leads to separation or "spreading" of neighboring negatively charged N-terminal phosphotyrosine residues, promoting swelling of caveolae, followed by their release from the plasma membrane.


Assuntos
Cavéolas/metabolismo , Caveolina 1/genética , Caveolina 1/metabolismo , Animais , Transporte Biológico , Técnicas de Cultura de Células , Membrana Celular/metabolismo , Endocitose/fisiologia , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Fosforilação , Transporte Proteico , Quinases da Família src/metabolismo
6.
PLoS One ; 9(3): e90544, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24614111

RESUMO

Pulmonary vascular remodeling and increased arterial wall stiffness are two major causes for the elevated pulmonary vascular resistance and pulmonary arterial pressure in patients and animals with pulmonary hypertension. Cellular copper (Cu) plays an important role in angiogenesis and extracellular matrix remodeling; increased Cu in vascular smooth muscle cells has been demonstrated to be associated with atherosclerosis and hypertension in animal experiments. In this study, we show that the Cu-uptake transporter 1, CTR1, and the Cu-efflux pump, ATP7A, were both upregulated in the lung tissues and pulmonary arteries of mice with hypoxia-induced pulmonary hypertension. Hypoxia also significantly increased expression and activity of lysyl oxidase (LOX), a Cu-dependent enzyme that causes crosslinks of collagen and elastin in the extracellular matrix. In vitro experiments show that exposure to hypoxia or treatment with cobalt (CoCl2) also increased protein expression of CTR1, ATP7A, and LOX in pulmonary arterial smooth muscle cells (PASMC). In PASMC exposed to hypoxia or treated with CoCl2, we also confirmed that the Cu transport is increased using 64Cu uptake assays. Furthermore, hypoxia increased both cell migration and proliferation in a Cu-dependent manner. Downregulation of hypoxia-inducible factor 1α (HIF-1α) with siRNA significantly attenuated hypoxia-mediated upregulation of CTR1 mRNA. In summary, the data from this study indicate that increased Cu transportation due to upregulated CTR1 and ATP7A in pulmonary arteries and PASMC contributes to the development of hypoxia-induced pulmonary hypertension. The increased Cu uptake and elevated ATP7A also facilitate the increase in LOX activity and thus the increase in crosslink of extracellular matrix, and eventually leading to the increase in pulmonary arterial stiffness.


Assuntos
Proteínas de Transporte de Cátions/genética , Cobre/metabolismo , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/genética , Hipóxia/complicações , Regulação para Cima/genética , Animais , Apoptose/efeitos dos fármacos , Proteínas de Transporte de Cátions/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Quelantes/farmacologia , Cobalto/farmacologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Técnicas de Silenciamento de Genes , Humanos , Hipertensão Pulmonar/patologia , Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Pulmão/patologia , Masculino , Camundongos Endogâmicos C57BL , Miócitos de Músculo Liso/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteína-Lisina 6-Oxidase/antagonistas & inibidores , Proteína-Lisina 6-Oxidase/genética , Proteína-Lisina 6-Oxidase/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/metabolismo , Artéria Pulmonar/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Regulação para Cima/efeitos dos fármacos
7.
Hypertens Res ; 37(2): 116-24, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24089267

RESUMO

A recent study from our group demonstrated that the Ca(2+)-sensing receptor (CaSR) was upregulated, and the extracellular Ca(2+)-induced increase in cytosolic Ca(2+) concentration ([Ca(2+)]cyt) was enhanced in pulmonary arterial smooth muscle cells from patients with idiopathic pulmonary arterial hypertension and animals with experimental pulmonary hypertension (PH). However, it is unclear whether CaSR antagonists (for example, NPS2143) rescue the development of experimental PH. We tested the rescue effects of NPS2143 in rats with monocrotaline (MCT)-induced PH and mice with chronic hypoxia-induced PH. For the NPS2143 treatment group, rats and mice were i.p. injected with NPS2143 once per day from days 14 to 24. Four weeks after MCT injection or exposure to normobaric hypoxia, the right ventricular (RV) systolic pressure, right heart hypertrophy (RV/LV+S ratio) and RV myocardial fibrosis were rescued or nearly restored to normal levels by NPS2143 treatment. The rescue effects of NPS2143 on experimental PH further support a critical role for the CaSR in the PH mechanism. Therefore, NPS2143 may be a promising potential treatment for pulmonary arterial hypertension.


Assuntos
Hipertensão Pulmonar/tratamento farmacológico , Receptores de Detecção de Cálcio/antagonistas & inibidores , Animais , Western Blotting , Cálcio/metabolismo , Fibrose , Hemodinâmica/efeitos dos fármacos , Hipertensão Pulmonar/induzido quimicamente , Hipertensão Pulmonar/fisiopatologia , Hipertrofia Ventricular Direita/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monocrotalina , Miocárdio/patologia , Venenos , Artéria Pulmonar/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Detecção de Cálcio/metabolismo
8.
Am J Physiol Lung Cell Mol Physiol ; 305(2): L154-64, 2013 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-23686856

RESUMO

Hypoxia-induced pulmonary hypertension (HPH) is characterized by sustained pulmonary vasoconstriction and vascular remodeling, both of which are mediated by pulmonary artery smooth muscle cell (PASMC) contraction and proliferation, respectively. An increase in cytosolic Ca²âº concentration ([Ca²âº]cyt) is a major trigger for pulmonary vasoconstriction and an important stimulus for cell proliferation in PASMCs. Ca²âº influx through voltage-dependent Ca²âº channels (VDCC) is an important pathway for the regulation of [Ca²âº]cyt. The potential role for L- and T-type VDCC in the development of HPH is still unclear. Using a hypoxic-induced pulmonary hypertension mouse model, we undertook this study to identify if VDCC in pulmonary artery (PA) are functionally upregulated and determine which type of VDCC are altered in HPH. Mice subjected to chronic hypoxia developed pulmonary hypertension within 4 wk, and high-K⁺- and U-46619-induced contraction of PA was greater in chronic hypoxic mice than that in normoxic control mice. Additionally, we demonstrate that high-K⁺- and U-46619-induced Ca²âº influx in PASMC is significantly increased in the hypoxic group. The VDCC activator, Bay K8864, induced greater contraction of the PA of hypoxic mice than in that of normoxic mice in isometric force measurements. L-type and T-type VDCC blockers significantly attenuated absolute contraction of the PA in hypoxic mice. Chronic hypoxia did not increase high-K⁺- and U-46619-induced contraction of mesenteric artery (MA). Compared with MA, PA displayed higher expression of calcium channel voltage-dependent L-type α1C-subunit (Cav1.2) and T-type α1H-subunit (Cav3.2) upon exposure to chronic hypoxia. In conclusion, both L-type and T-type VDCC were functionally upregulated in PA, but not MA, in HPH mice, which could result from selectively increased expression of Cav1.2 and Cav3.2.


Assuntos
Canais de Cálcio Tipo L/biossíntese , Canais de Cálcio Tipo T/biossíntese , Regulação da Expressão Gênica , Hipóxia/metabolismo , Artéria Pulmonar/metabolismo , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Animais , Cálcio/metabolismo , Agonistas dos Canais de Cálcio/farmacologia , Doença Crônica , Hipóxia/patologia , Masculino , Artérias Mesentéricas/metabolismo , Artérias Mesentéricas/patologia , Camundongos , Potássio/metabolismo , Artéria Pulmonar/patologia , Fatores de Tempo , Vasoconstritores/farmacologia , Vasodilatação/efeitos dos fármacos
9.
Am J Physiol Cell Physiol ; 304(11): C1042-52, 2013 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-23426966

RESUMO

Electromechanical coupling via membrane depolarization-mediated activation of voltage-dependent Ca(2+) channels (VDCC) is an important mechanism in regulating pulmonary vascular tone, while mouse is an animal model often used to study pathogenic mechanisms of pulmonary vascular disease. The function of VDCC in mouse pulmonary artery (PA) smooth muscle cells (PASMC), however, has not been characterized, and their functional role in reactive oxygen species (ROS)-mediated regulation of vascular function remains unclear. In this study, we characterized the electrophysiological and pharmacological properties of VDCC in PASMC and the divergent effects of ROS produced by xanthine oxidase (XO) and hypoxanthine (HX) on VDCC in PA and mesenteric artery (MA). Our data show that removal of extracellular Ca(2+) or application of nifedipine, a dihydropyridine VDCC blocker, both significantly inhibited 80 mM K(+)-mediated PA contraction. In freshly dissociated PASMC, the maximum inward Ca(2+) currents were -2.6 ± 0.2 pA/pF at +10 mV (with a holding potential of -70 mV). Window currents were between -40 and +10 mV with a peak at -15.4 mV. Nifedipine inhibited currents with an IC(50) of 0.023 µM, and 1 µM Bay K8644, a dihydropyridine VDCC agonist, increased the inward currents by 61%. XO/HX attenuated 60 mM K(+)-mediated increase in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) due to Ca(2+) influx through VDCC in PASMC. Exposure to XO/HX caused relaxation in PA preconstricted by 80 mM K(+) but not in aorta and MA. In contrast, H(2)O(2) inhibited high K(+)-mediated increase in [Ca(2+)](cyt) and caused relaxation in both PA and MA. Indeed, RT-PCR and Western blot analysis revealed significantly lower expression of Ca(V)1.3 in MA compared with PA. Thus our study characterized the properties of VDCC and demonstrates that ROS differentially regulate vascular contraction by regulating VDCC in PA and systemic arteries.


Assuntos
Canais de Cálcio/metabolismo , Contração Muscular/fisiologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Artéria Pulmonar/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Western Blotting , Camundongos , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
10.
Circ Res ; 112(4): 640-50, 2013 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-23300272

RESUMO

RATIONALE: An increase in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) in pulmonary arterial smooth muscle cells (PASMC) is a major trigger for pulmonary vasoconstriction and an important stimulus for PASMC proliferation and pulmonary vascular remodeling. The dihydropyridine Ca(2+) channel blockers, such as nifedipine, have been used for treatment of idiopathic pulmonary arterial hypertension (IPAH). OBJECTIVE: Our previous study demonstrated that the Ca(2+)-sensing receptor (CaSR) was upregulated and the extracellular Ca(2+)-induced increase in [Ca(2+)](cyt) was enhanced in PASMC from patients with IPAH and animals with experimental pulmonary hypertension. Here, we report that the dihydropyridines (eg, nifedipine) increase [Ca(2+)](cyt) by activating CaSR in PASMC from IPAH patients (in which CaSR is upregulated), but not in normal PASMC. METHODS AND RESULTS: The nifedipine-mediated increase in [Ca(2+)](cyt) in IPAH-PASMC was concentration dependent with a half maximal effective concentration of 0.20 µmol/L. Knockdown of CaSR with siRNA in IPAH-PASMC significantly inhibited the nifedipine-induced increase in [Ca(2+)](cyt), whereas overexpression of CaSR in normal PASMC conferred the nifedipine-induced rise in [Ca(2+)](cyt). Other dihydropyridines, nicardipine and Bay K8644, had similar augmenting effects on the CaSR-mediated increase in [Ca(2+)](cyt) in IPAH-PASMC; however, the nondihydropyridine blockers, such as diltiazem and verapamil, had no effect on the CaSR-mediated rise in [Ca(2+)](cyt). CONCLUSIONS: The dihydropyridine derivatives increase [Ca(2+)](cyt) by potentiating the activity of CaSR in PASMC independently of their blocking (or activating) effect on Ca(2+) channels; therefore, it is possible that the use of dihydropyridine Ca(2+) channel blockers (eg, nifedipine) to treat IPAH patients with upregulated CaSR in PASMC may exacerbate pulmonary hypertension.


Assuntos
Bloqueadores dos Canais de Cálcio/efeitos adversos , Canais de Cálcio Tipo L/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Hipertensão Pulmonar/induzido quimicamente , Miócitos de Músculo Liso/efeitos dos fármacos , Nifedipino/efeitos adversos , Artéria Pulmonar/citologia , Receptores de Detecção de Cálcio/efeitos dos fármacos , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Sinalização do Cálcio/fisiologia , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Células Cultivadas/ultraestrutura , Citosol/metabolismo , Progressão da Doença , Humanos , Hipertensão Pulmonar/tratamento farmacológico , Hipertensão Pulmonar/fisiopatologia , Fosfatos de Inositol/fisiologia , Masculino , Monocrotalina/toxicidade , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/ultraestrutura , Naftalenos/farmacologia , Naftalenos/uso terapêutico , Nifedipino/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de Detecção de Cálcio/genética , Receptores de Detecção de Cálcio/fisiologia , Proteínas Recombinantes de Fusão/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Transfecção , Regulação para Cima/efeitos dos fármacos , Vasoconstrição/efeitos dos fármacos
11.
Circ Res ; 111(4): 469-81, 2012 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-22730443

RESUMO

RATIONALE: A rise in cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) in pulmonary arterial smooth muscle cells (PASMC) is an important stimulus for pulmonary vasoconstriction and vascular remodeling. Increased resting [Ca(2+)](cyt) and enhanced Ca(2+) influx have been implicated in PASMC from patients with idiopathic pulmonary arterial hypertension (IPAH). OBJECTIVE: We examined whether the extracellular Ca(2+)-sensing receptor (CaSR) is involved in the enhanced Ca(2+) influx and proliferation in IPAH-PASMC and whether blockade of CaSR inhibits experimental pulmonary hypertension. METHODS AND RESULTS: In normal PASMC superfused with Ca(2+)-free solution, addition of 2.2 mmol/L Ca(2+) to the perfusate had little effect on [Ca(2+)](cyt). In IPAH-PASMC, however, restoration of extracellular Ca(2+) induced a significant increase in [Ca(2+)](cyt). Extracellular application of spermine also markedly raised [Ca(2+)](cyt) in IPAH-PASMC but not in normal PASMC. The calcimimetic R568 enhanced, whereas the calcilytic NPS 2143 attenuated, the extracellular Ca(2+)-induced [Ca(2+)](cyt) rise in IPAH-PASMC. Furthermore, the protein expression level of CaSR in IPAH-PASMC was greater than in normal PASMC; knockdown of CaSR in IPAH-PASMC with siRNA attenuated the extracellular Ca(2+)-mediated [Ca(2+)](cyt) increase and inhibited IPAH-PASMC proliferation. Using animal models of pulmonary hypertension, our data showed that CaSR expression and function were both enhanced in PASMC, whereas intraperitoneal injection of the calcilytic NPS 2143 prevented the development of pulmonary hypertension and right ventricular hypertrophy in rats injected with monocrotaline and mice exposed to hypoxia. CONCLUSIONS: The extracellular Ca(2+)-induced increase in [Ca(2+)](cyt) due to upregulated CaSR is a novel pathogenic mechanism contributing to the augmented Ca(2+) influx and excessive PASMC proliferation in patients and animals with pulmonary arterial hypertension.


Assuntos
Sinalização do Cálcio , Hipertensão Pulmonar/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Receptores de Detecção de Cálcio/metabolismo , Vasoconstrição , Compostos de Anilina/farmacologia , Animais , Calcimiméticos/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Hipertensão Pulmonar Primária Familiar , Humanos , Hipertensão Pulmonar/induzido quimicamente , Hipertensão Pulmonar/patologia , Hipertensão Pulmonar/fisiopatologia , Hipertensão Pulmonar/prevenção & controle , Hipertrofia Ventricular Direita/etiologia , Hipertrofia Ventricular Direita/metabolismo , Hipertrofia Ventricular Direita/patologia , Hipertrofia Ventricular Direita/prevenção & controle , Hipóxia/complicações , Hipóxia/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monocrotalina , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/patologia , Músculo Liso Vascular/fisiopatologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/patologia , Naftalenos/farmacologia , Fenetilaminas , Propilaminas , Artéria Pulmonar/metabolismo , Artéria Pulmonar/patologia , Artéria Pulmonar/fisiopatologia , Interferência de RNA , Ratos , Ratos Sprague-Dawley , Receptores de Detecção de Cálcio/efeitos dos fármacos , Receptores de Detecção de Cálcio/genética , Espermina/farmacologia , Fatores de Tempo , Transfecção , Vasoconstrição/efeitos dos fármacos
12.
Am J Physiol Cell Physiol ; 300(3): C588-99, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21191107

RESUMO

Copper is an essential micronutrient in humans and is required for a wide range of physiological processes, including neurotransmitter biosynthesis, oxidative metabolism, protection against reactive oxygen species, and angiogenesis. The first step in the acquisition of dietary copper is absorption from the intestinal lumen. The major human high-affinity copper uptake protein, human copper transporter hCTR1, was recently shown to be at the basolateral or blood side of both intestinal and renal epithelial cell lines and thus does not play a direct role in this initial step. We sought to functionally identify the major transport pathways available for the absorption of dietary copper across the apical intestinal membrane using Caco2 cells, a well-established model for human enterocytes. The initial rate of apical copper uptake into confluent monolayers of Caco2 cells is greatly elevated if amino acids and serum proteins are removed from the growth media. Uptake from buffered saline solutions at neutral pH (but not at lower pH) is inhibited by either d- or l-histidine, unaltered by the removal of sodium ions, and inhibited by ∼90% when chloride ions are replaced by gluconate or sulfate. Chloride-dependent copper uptake occurs with Cu(II) or Cu(I), although Cu(I) uptake is not inhibited by histidine, nor by silver ions. A well-characterized inhibitor of anion exchange systems, DIDS, inhibited apical copper uptake by 60-70%, while the addition of Mn(II) or Fe(II), competitive substrates for the divalent metal transporter DMT1, had no effect on copper uptake. We propose that anion exchangers play an unexpected role in copper absorption, utilizing copper-chloride complexes as pseudo-substrates. This pathway is also observed in mouse embryonic fibroblasts, human embryonic kidney cells, and Cos-7 cells. The special environment of low pH, low concentration of protein, and protonation of amino acids in the early intestinal lumen make this pathway especially important in dietary copper acquisition.


Assuntos
Proteínas de Transporte de Ânions/metabolismo , Cobre/metabolismo , Células Epiteliais/metabolismo , Absorção Intestinal/fisiologia , Mucosa Intestinal/metabolismo , Animais , Células COS , Células CACO-2 , Polaridade Celular/fisiologia , Chlorocebus aethiops , Células Epiteliais/ultraestrutura , Fibroblastos , Células HEK293 , Humanos , Mucosa Intestinal/ultraestrutura , Camundongos , Camundongos Knockout , Microvilosidades/metabolismo
13.
Am J Physiol Renal Physiol ; 294(1): F53-61, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17928409

RESUMO

Kidneys regulate their copper content more effectively than many other organs in diseases of copper deficiency or excess. We demonstrate that two copper-transporting ATPases, ATP7A and ATP7B, contribute to this regulation. ATP7A is expressed, to a variable degree, throughout the kidney and shows age-dependent intracellular localization. In 2-wk-old mice, ATP7A is located in the vicinity of the basolateral membrane, whereas in 20-wk-old mice, ATP7A is predominantly in intracellular vesicles. Acute elevation of serum copper, via intraperitoneal injection, results in the in vivo redistribution of ATP7A from intracellular compartments toward the basolateral membrane, illustrating a role for ATP7A in renal response to changes in copper load. Renal copper homeostasis also requires functional ATP7B, which is coexpressed with ATP7A in renal cells of proximal and distal origin. The kidneys of Atp7b(-/-) mice, an animal model of Wilson disease, show metabolic alterations manifested by the appearance of highly fluorescent deposits; however, in marked contrast to the liver, renal copper is not significantly elevated. The lack of notable copper accumulation in the Atp7b(-/-) kidney is likely due to the compensatory export of copper by ATP7A. This interpretation is supported by the predominant localization of ATP7A at the basolateral membrane of Atp7b(-/-) cortical tubules. Our results suggest that both Cu-ATPases regulate renal copper, with ATP7A playing a major role in exporting copper via basolateral membranes and protecting renal tissue against copper overload.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Membrana Celular/enzimologia , Rim/enzimologia , Adenosina Trifosfatases/genética , Envelhecimento/metabolismo , Envelhecimento/patologia , Animais , Proteínas de Transporte de Cátions/genética , Membrana Celular/patologia , Cobre/sangue , ATPases Transportadoras de Cobre , Modelos Animais de Doenças , Feminino , Degeneração Hepatolenticular/enzimologia , Degeneração Hepatolenticular/patologia , Rim/patologia , Túbulos Renais Distais/enzimologia , Túbulos Renais Distais/patologia , Túbulos Renais Proximais/enzimologia , Túbulos Renais Proximais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
14.
J Biol Chem ; 282(36): 26471-80, 2007 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-17627945

RESUMO

Copper is essential for human growth and survival. Enterocytes mediate the absorption of dietary copper from the intestinal lumen into blood as well as utilizing copper for their biosynthetic needs. Currently, the pathways for copper entry into enterocytes remain poorly understood. We demonstrate that the basolateral copper uptake into intestinal cells greatly exceeds the apical uptake. The basolateral but not apical transport is mediated by the high affinity copper transporter hCTR1. This unanticipated conclusion is supported by cell surface biotinylation and confocal microscopy of endogenous hCTR1 in Caco2 cells as well as copper influx measurements that show saturable high affinity uptake at the basolateral but not the apical membrane. Basolateral localization of hCTR1 and polarized copper uptake are also conserved in T84 cells, models for intestinal crypt cells. The lateral localization of hCTR1 seen in intestinal cell lines is recapitulated in immunohistochemical staining of mouse intestinal sections. Biochemical and functional assays reveal the basolateral localization of hCTR1 also in renal Madin-Darby canine kidney cells and opossum kidney cells. Overexpression of hCTR1 in Madin-Darby canine kidney cells results in both apical and basolateral delivery of the overexpressed protein and greatly enhanced copper uptake at both cell surfaces. We propose a model of intestinal copper uptake in which basolateral hCTR1 plays a key role in the physiologically important delivery of copper from blood to intracellular proteins, whereas its role in the initial apical uptake of dietary copper is indirect.


Assuntos
Proteínas de Transporte de Cátions/biossíntese , Cobre/metabolismo , Enterócitos/metabolismo , Homeostase/fisiologia , Rim/metabolismo , Animais , Células CACO-2 , Proteínas de Transporte de Cátions/genética , Transportador de Cobre 1 , Cães , Enterócitos/citologia , Expressão Gênica , Humanos , Transporte de Íons/fisiologia , Rim/citologia , Gambás , Especificidade de Órgãos/fisiologia , Transporte Proteico/fisiologia
15.
Biometals ; 20(3-4): 355-64, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17211679

RESUMO

In this brief review we summarize what is known about the role of hCTR1 in mediating the entry of copper into human cells. There is a body of information that clearly identifies this protein as being a major source (though not the only source) of copper entry into human cells, and thus a crucial element of copper homeostasis. However, much remains that is poorly understood and key aspects of the physiological roles of hCTR1 and its regulation are only superficially appreciated. The particular characteristics of a transport process that in vivo involves the binding, transmembrane transport and release of a substrate that is not present in a free form in the intracellular or extracellular compartments poses particular challenges that are not encountered in the transport of more familiar physiologically important metal cations. Thus much of what we have learned about the more commonly encountered transported ions provides an inadequate model for studies of copper homeostasis. In this article we review progress made and identify the major questions that need to be resolved before an adequate description is attained of how copper entry into human cells is mediated and regulated by hCTR1.


Assuntos
Transporte Biológico , Proteínas de Transporte de Cátions/metabolismo , Cobre/metabolismo , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Transportador de Cobre 1 , Humanos , Oxirredução , Processamento de Proteína Pós-Traducional
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