RESUMO
Density reversal of senescent red blood cells has been known for a long time, yet the identity of the candidate ion transporter(s) causing the senescent cells to swell is still elusive. While performing fractionation of RBCs from healthy individuals in Percoll density gradient and characterization of the separated fractions, we identified a subpopulation of cells in low-density fraction (1.02% ± 0.47) showing signs of senescence such as loss of membrane surface area associated with a reduction in band 3 protein abundance, and Phosphatidylserine (PS) exposure to the outer membrane. In addition, we found that these cells are overloaded with Na+ and Ca2+. Using a combination of blockers and activators of ion pumps and channels, we revealed reduced activity of Plasma membrane Ca2+ ATPase and an increase in Ca2+ and Na+ leaks through ion channels in senescent-like cells. Our data revealed that Ca2+ overload in these cells is a result of reduced PMCA activity and facilitated Ca2+ uptake via a hyperactive Piezo1 channel. However, we could not exclude the contribution of other Ca2+-permeable ion channels in this scenario. In addition, we found, as a universal mechanism, that an increase in intracellular Ca2+ reduced the initially high selectivity of Piezo1 channel for Ca2+ and allowed higher Na+ uptake, Na+ accumulation, and swelling.
Assuntos
Cálcio , Eritrócitos , Canais Iônicos , Humanos , Eritrócitos/metabolismo , Canais Iônicos/metabolismo , Cálcio/metabolismo , Senescência Celular , Sódio/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismoRESUMO
Adenosine Triphosphatase (ATPase) Phospholipid Transporting 11C gene (ATP11C) encodes the major phosphatidylserine (PS) flippase in human red blood cells (RBCs). Flippases actively transport phospholipids (e.g., PS) from the outer to the inner leaflet to establish and maintain phospholipid asymmetry of the lipid bilayer of cell membranes. This asymmetry is crucial for survival since externalized PS triggers phagocytosis by splenic macrophages. Here we report on pathophysiological consequences of decreased flippase activity, prompted by a patient with hemolytic anemia and hemizygosity for a novel c.2365C > T p.(Leu789Phe) missense variant in ATP11C. ATP11C protein expression was strongly reduced by 58% in patient-derived RBC ghosts. Furthermore, functional characterization showed only 26% PS flippase activity. These results were confirmed by recombinant mutant ATP11C protein expression in HEK293T cells, which was decreased to 27% compared to wild type, whereas PS-stimulated ATPase activity was decreased by 57%. Patient RBCs showed a mild increase in PS surface exposure when compared to control RBCs, which further increased in the most dense RBCs after RBC storage stress. The increase in PS was not due to higher global membrane content of PS or other phospholipids. In contrast, membrane lipid lateral distribution showed increased abundance of cholesterol-enriched domains in RBC low curvature areas. Finally, more dense RBCs and subtle changes in RBC morphology under flow hint toward alterations in flow behavior of ATP11C-deficient RBCs. Altogether, ATP11C deficiency is the likely cause of hemolytic anemia in our patient, thereby underlining the physiological role and relevance of this flippase in human RBCs.
Assuntos
Anemia Hemolítica Congênita , Fosfatidilserinas , Humanos , Fosfatidilserinas/metabolismo , Células HEK293 , Eritrócitos/metabolismo , Anemia Hemolítica Congênita/genética , Anemia Hemolítica Congênita/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Fosfolipídeos/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismoRESUMO
Alternaria blight is a devastating disease that causes significant crop losses in oilseed Brassicas every year. Adoption of conventional breeding to generate disease-resistant varieties has so far been unsuccessful due to the lack of suitable resistant source germplasms of cultivated Brassica spp. A thorough understanding of the molecular basis of resistance, as well as the identification of defense-related genes involved in resistance responses in closely related wild germplasms, would substantially aid in disease management. In the current study, a comparative transcriptome profiling was performed using Illumina based RNA-seq to detect differentially expressed genes (DEGs) specifically modulated in response to Alternaria brassicicola infection in resistant Sinapis alba, a close relative of Brassicas, and the highly susceptible Brassica rapa. The analysis revealed that, at 48 hpi (hours post inoculation), 3396 genes were upregulated and 23239 were downregulated, whereas at 72 hpi, 4023 genes were upregulated and 21116 were downregulated. Furthermore, a large number of defense response genes were detected to be specifically regulated as a result of Alternaria infection. The transcriptome data was validated using qPCR-based expression profiling for selected defense-related DEGs, that revealed significantly higher fold change in gene expression in S. alba when compared to B. rapa. Expression of most of the selected genes was elevated across all the time points under study with significantly higher expression towards the later time point of 72 hpi in the resistant germplasm. S. alba activates a stronger defense response reaction against the disease by deploying an array of genes and transcription factors involved in a wide range of biological processes such as pathogen recognition, signal transduction, cell wall modification, antioxidation, transcription regulation, etc. Overall, the study provides new insights on resistance of S. alba against A. brassicicola, which will aid in devising strategies for breeding resistant varieties of oilseed Brassica.
RESUMO
We employed an Illumina-based high-throughput metagenomics sequencing approach to unveil the rhizosphere and root endosphere microbial community associated with an organically grown Camellia population located at the Experimental Garden for Plantation Crops, Assam (India). The de novo assembled tea root endosphere metagenome contained 24,231 contigs (total 7,771,089 base pairs with an average length of 321 bps), while tea rhizosphere soil metagenome contained 261,965 sequences (total 230,537,174 base pairs, average length 846). The most prominent rhizobacteria belonged to the genera, viz., Bacillus (10.35%), Candidatus Solibacter (6.36%), Burkholderia (5.19%), Pseudomonas (3.9%), Streptomyces (3.52%), and Bradyrhizobium (2.77%), while the root endosphere was dominated by bacterial genera, viz., Serratia (46.64%), Methylobacterium (8.02%), Yersinia (5.97%), Burkholderia (2.05%), etc. The presence of few agronomically important bacterial genera, Bradyrhizobium, Rhizobium (each 0.93%), Sinorhizobium (0.34%), Azorhizobium, and Flavobacterium (0.17% each), was also detected in the root endosphere. KEGG pathway mapping indicated the presence of microbial metabolic pathway genes related to tyrosine metabolism, tryptophan metabolism, glyoxylate, and dicarboxylate metabolism which play important roles in endosphere activities, including survival, growth promotion, and host adaptation. The root endosphere microbiome also contained few important plant growth promoting traits related to phytohormone production, abiotic stress alleviation, mineral solubilization, and plant disease suppression.
Assuntos
Camellia sinensis , Microbiologia do Solo , Raízes de Plantas , Rizosfera , CháRESUMO
The ability of red blood cells (RBCs) to transport gases, their lifespan as well as their rheological properties invariably depend on the deformability, hydration, and membrane stability of these cells, which can be measured by Laser optical rotational red cell analyser (Lorrca® Maxsis, RR Mechatronics). The osmoscan mode of Lorrca is currently used in diagnosis of rare anemias in clinical laboratories. However, a broad range of normal values for healthy subjects reduces the sensitivity of this method for diagnosis of mild disease phenotype. In this pilot study, we explored the impact of age and gender of 45 healthy donors, as well as RBC age on the Lorrca indices. Whereas gender did not affect the Lorrca indices in our study, the age donors had a profound effect on the O_hyper parameter. To study the impact of RBC age on the osmoscan parameters, we have isolated low (L)-, medium (M)-, or high (H)- density fractions enriched with young, mature, and senescent RBCs, respectively, and evaluated the influence of RBC age-related properties, such as density, morphology, and redox state, on the osmoscan indices. As before, O_hyper was the most sensitive parameter, dropping markedly with an increase in RBC density and age. Senescence was associated with a decrease in deformability (EI_max) and tolerability to low and high osmolatites (Area). L-fraction was enriched with reticulocytes and cells with high projected area and EMA staining, but also contained a small number of cells small in projected area and most likely, terminally senescent. L-fraction was on average slightly less deformable than mature cells. The cells from the L-fraction produced more oxidants and NO than all other fractions. However, RBCs from the L-fraction contained maximal levels of reduced thiols compared to other fractions. Our study suggests that reference values for O_hyper should be age-stratified, and, most probably, corrected for the average RBC age. Further multi-center study is required to validate these suggestions before implementing them into clinical practice.
RESUMO
Parkinson's disease (PD) is a major health problem worldwide affecting millions of people and is a result of neurodegeneration in a small part of the brain known as substantia nigra pars compacta. Aberration in mitochondrial Ca2+ homeostasis plays, among several other factors, an important role for the neuronal loss in PD. Mitochondria are vital for cellular physiology, e.g. for ATP generation, and mitochondrial Ca2+ is a key player in cell functioning and survival. Mitochondrial Ca2+ homeostasis is maintained by a fine balance between the activities of proteins mediating the influx and efflux of Ca2+ across mitochondrial membranes. Malfunctioning of these proteins leading to Ca2+ overload promotes ROS generation, which induces cell death by triggering the opening of mitochondrial permeability transition pore. Till now PD remains incurable and the "gold standard" drug which can only delays the disease progression is l-Dopa from the 1960s and therefore, the situation warrants the search for novel targets for the treatment of the PD patients. In this review, we summarize the current views that suggest mitochondrial Ca2+ regulatory pathways are good candidates for the treatment of PD.
Assuntos
Cálcio/metabolismo , Mitocôndrias/metabolismo , Terapia de Alvo Molecular , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , Animais , Sinalização do Cálcio , Humanos , Modelos BiológicosRESUMO
miRNAs are class of endogenously initiated noncoding RNAs, which are most critical in gene expression and regulation at posttranscriptional level. They do so either by cleavage of the target mRNA or by translational repression. miRNAs are being given enough attention in recent years because of its role in myriad developmental processes including tumorogenesis and host-pathogen interaction. Advent of Next Generation Sequencing (NGS) technology and computational approach made it possible to pinpoint the precise role of miRNA and their target. Identification of miRNAs and their target has several approaches depending on efficiency, cost and time. The present review summarizes the developments in the field of plant miRNA w.r.t. to experimental approaches that are being followed to identify and validate the miRNAs and their targets.
Assuntos
Biologia Computacional/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , MicroRNAs/genética , Plantas/genética , RNA Mensageiro/genética , Análise de Sequência de RNA/métodos , Estudos de Validação como AssuntoRESUMO
BACKGROUND: Topic lifecycle analysis on social networks aims to analyze and track how topics are born from user-generated content, and how they evolve. Twitter researchers have no agreed-upon definition of topics; topics on Twitter are typically derived in the form of (a) frequently used hashtags, or (b) keywords showing sudden trends of large occurrence in a short span of time ("bursty keywords"), or (c) concepts latent within the tweets that are grouped using variations of semantic clustering techniques. METHODS: In the current paper, we jointly model the hashtags present and the semantic concepts embedded in the content, which in turn helps us identify hashtag groups that define a "topic"-a concept space-that are used by a large number of tweets. RESULTS: We observe that different hashtags belonging to a given cluster are more prominent compared to the others, at different times. We further observe that the participation and influence levels of the different users play important roles in determining which hashtag would be more prominent than the others at given times. We thus observe topics to often morph from one to the other (via morphing of dominant hashtags representing the same semantic concept space), rather than becoming extinct outright, which is a novel insight about topic lifecycles. We further present novel observations about the role of users in determining the lifecycle of discussion topics on Twitter. CONCLUSIONS: We infer that topic lifecycles are governed by user interests, and not by user influence, which is a key observation made by our work.
RESUMO
FXYD5 is a Na+-K+-ATPase regulator, expressed in a variety of normal epithelia. In parallel, it has been found to be associated with several types of cancer and effect lethal outcome by promoting metastasis. However, the molecular mechanism underlying FXYD5 mediated invasion has not yet been identified. In this study, using in vivo 4T1 murine breast cancer model, we found that FXYD5-specific shRNA significantly inhibited lung cancer metastasis, without having a substantial effect on primary tumor growth. Our study reveals that FXYD5 participates in multiple stages of metastatic development and exhibits more than one mode of E-cadherin regulation. We provide the first evidence that FXYD5-related morphological changes are mediated through its interaction with Na+-K+-ATPase. Experiments in cultured 4T1 cells have indicated that FXYD5 expression may downregulate the ß1 isoform of the pump. This behavior could have implications on both transcellular interactions and intracellular events. Further studies suggest that differential localization of the adaptor protein Annexin A2 in FXYD5-expressing cells may correlate with matrix metalloproteinase 9 secretion and adhesion changes in 4T1 wild-type cells.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Neoplasias Mamárias Experimentais/genética , Proteínas de Membrana/genética , Neoplasias Lipomatosas/genética , ATPase Trocadora de Sódio-Potássio/genética , Animais , Anexina A2/genética , Anexina A2/metabolismo , Caderinas/genética , Caderinas/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Modelos Animais de Doenças , Feminino , Canais Iônicos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/patologia , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas dos Microfilamentos , Neoplasias Lipomatosas/metabolismo , Neoplasias Lipomatosas/patologia , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
BACKGROUND: The Na+/Ca2+/Li+ exchanger (NCLX) is a member of the Na+/Ca2+ exchanger family. NCLX is unique in its capacity to transport both Na+ and Li+, unlike other members, which are Na+ selective. The major aim of this study was twofold, i.e., to identify NCLX residues that confer Li+ or Na+ selective Ca2+ transport and map their putative location on NCLX cation transport site. METHOD: We combined molecular modeling to map transport site of NCLX with euryarchaeal H+/Ca2+ exchanger, CAX_Af, and fluorescence analysis to monitor Li+ versus Na+ dependent mitochondrial Ca2+ efflux of transport site mutants of NCLX in permeabilized cells. RESULT: Mutation of Asn149, Pro152, Asp153, Gly176, Asn467, Ser468, Gly494 and Asn498 partially or strongly abolished mitochondrial Ca2+ exchange activity in intact cells. In permeabilized cells, N149A, P152A, D153A, N467Q, S468T and G494S demonstrated normal Li+/Ca2+ exchange activity but a reduced Na+/Ca2+ exchange activity. On the other hand, D471A showed dramatically reduced Li+/Ca2+ exchange, but Na+/Ca2+ exchange activity was unaffected. Finally, simultaneous mutation of four putative Ca2+ binding residues was required to completely abolish both Na+/Ca2+ and Li+/Ca2+ exchange activities. CONCLUSIONS: We identified distinct Na+ and Li+ selective residues in the NCLX transport site. We propose that functional segregation in Li+ and Na+ sites reflects the functional properties of NCLX required for Ca2+ exchange under the unique membrane potential and ion gradient across the inner mitochondrial membrane. GENERAL SIGNIFICANCE: The results of this study provide functional insights into the unique Li+ and Na+ selectivity of the mitochondrial exchanger. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.
Assuntos
Cálcio/metabolismo , Lítio/metabolismo , Mitocôndrias/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Sódio/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Transporte Biológico , Células HEK293 , Humanos , Proteínas Mitocondriais , Mutação , Homologia de Sequência de Aminoácidos , Trocador de Sódio e Cálcio/químicaRESUMO
The role of angiotensin II in regulating Na+/K(+)-ATPase activity has been investigated in bovine pulmonary artery smooth muscle cells (BPASMCs). Our study reveals that angiotensin II inhibits the Na+/K+ATPase activity via glutathionylation of the pump with the involvement of an increase in NADPH oxidase-derived O2*-. Additionally, angiotensin II treatment to the cells increases the inhibitory potency of the 15.6 kDa inhibitor towards the Na+/K+ATPase activity.
Assuntos
Angiotensina II/fisiologia , Glutationa/metabolismo , Músculo Liso Vascular/enzimologia , Artéria Pulmonar/enzimologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Bovinos , Células Cultivadas , Músculo Liso Vascular/citologia , Artéria Pulmonar/citologiaRESUMO
We have identified a novel endogenous low mol wt. (15.6 kDa) protein inhibitor of Na(+)/K(+)-ATPase in cytosolic fraction of bovine pulmonary artery smooth muscle cells. The inhibitor showed different affinities toward the α2ß1 and α1ß1 isozymes of Na(+)/K(+)-ATPase, where α2 is more sensitive than α1. The inhibitor interacted reversibly to the E1 site of the enzyme and blocked the phosphorylated intermediate formation. Circular dichroism study suggests that the inhibitor causes an alteration in the confirmation of the enzyme.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/isolamento & purificação , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Animais , Bovinos , Dicroísmo Circular , Citosol/enzimologia , Peso Molecular , Miócitos de Músculo Liso/enzimologia , Artéria Pulmonar/enzimologia , ATPase Trocadora de Sódio-Potássio/químicaRESUMO
We investigated the mechanism by which TxA2 mimetic, U46619, activates proMMP-2 in bovine pulmonary artery smooth muscle cells. Our study showed that treatment of the cells with U46619 caused an increase in the expression and subsequently activation of proMMP-2 in the cells. Pretreatment with p(38)MAPK inhibitor, SB203580; and NF-κB inhibitor, Bay11-7082 inhibited the expression and activation of proMMP-2 induced by U46619. U46619 also induced increase in MT1-MMP expression, which was inhibited upon pretreatment with SB203580 and Bay11-7082. U46619 treatment to the cells stimulated p(38)MAPK activity as well as NF-κB activation by IκB-α phosphorylation, translocation of NF-κBp65 subunit from cytosol to nucleus and subsequently, by increasing its DNA-binding activity. Induction of NF-κB activation seems to be mediated through IKK, as transfection of cells with either IKKα or IKKß siRNA prevented U46619-induced phosphorylation of IκB-α and NF-κBp65 DNA-binding activity. U46619 treatment to the cells also downregulated the TIMP-2 level. Pretreatment of the cells with SB203580 and Bay11-7082 did not show any discernible change in TIMP-2 level by U46619. Overall, U46619-induced activation of proMMP-2 is mediated via involvement of p(38)MAPK-NFκB-MT1MMP signaling pathway with concomitant downregulation of TIMP-2 expression in bovine pulmonary artery smooth muscle cells.
Assuntos
Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Precursores Enzimáticos/metabolismo , Gelatinases/metabolismo , Metaloproteinase 14 da Matriz/metabolismo , Miócitos de Músculo Liso/enzimologia , NF-kappa B/metabolismo , Artéria Pulmonar/citologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Bovinos , Regulação para Baixo/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Proteínas I-kappa B/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Inibidor de NF-kappaB alfa , Fosforilação/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Treatment of bovine pulmonary smooth muscle cells with U46619 inhibited the Na(+)/K(+) ATPase activity in two parallel pathways: one of which is mediated via glutathionylation of the pump and the other by augmenting the inhibitory activity of the 70kDa inhibitor protein of Na(+)/K(+) ATPase. Although phospholemman deglutathionylates the pump leading to its activation, the inhibitor is responsible for irreversible inhibition of Na(+)/K(+) ATPase in an isoform specific manner during treatment of the cells with U46619.
Assuntos
Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Proteínas de Membrana/metabolismo , Miócitos de Músculo Liso/metabolismo , Fosfoproteínas/metabolismo , Artéria Pulmonar/enzimologia , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/metabolismo , Vasoconstritores/farmacologia , Animais , Bovinos , Isoformas de Proteínas/metabolismo , Artéria Pulmonar/metabolismoRESUMO
We have recently reported that treatment of bovine pulmonary artery smooth muscle cells with the thromboxane A(2) mimetic, U46619 stimulated NADPH oxidase derived O(2)(·-) level, which subsequently caused marked increase in [Ca(2+)](i)[17]. Herein, we demonstrated that O(2)(·-)-mediated increase in [Ca(2+)](i) stimulates an aprotinin sensitive proteinase activity, which proteolytically activates PKC-α under U46619 treatment to the cells. The activated PKC-α then phosphorylates p(38)MAPK and that subsequently caused G(i)α phosphorylation leading to stimulation of cPLA(2) activity in the cell membrane.
Assuntos
Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/enzimologia , NADPH Oxidases/metabolismo , Fosfolipases A2 Citosólicas/metabolismo , Artéria Pulmonar/citologia , Superóxidos/metabolismo , Animais , Materiais Biomiméticos/farmacologia , Cálcio/metabolismo , Bovinos , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Ativação Enzimática/efeitos dos fármacos , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Proteína Quinase C-alfa/genética , Proteína Quinase C-alfa/metabolismo , Tromboxano A2/metabolismo , Vasoconstritores/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
We have recently reported that α(2)ß(1) and α(1)ß(1) isozymes of Na(+)/K(+)-ATPase (NKA) are localized in the caveolae whereas only the α(1)ß(1) isozyme of NKA is localized in the non-caveolae fraction of pulmonary artery smooth muscle cell membrane. It is well known that different isoforms of NKA are regulated differentially by PKA and PKC, but the mechanism is not known in the caveolae of pulmonary artery smooth muscle cells. Herein, we examined whether this regulation occurs through phospholemman (PLM) in the caveolae. Our results suggest that PKC mediated phosphorylation of PLM occurs only when it is associated with the α(2) isoform of NKA, whereas phosphorylation of PLM by PKA occurs when it is associated with the α(1) isoform of NKA. To investigate the mechanism of regulation of α(2) isoform of NKA by PKC-mediated phosphorylation of PLM, we have purified PLM from the caveolae and reconstituted into the liposomes. Our result revealed that (i) in the reconstituted liposomes phosphorylated PLM (PKC mediated) stimulate NKA activity, which appears to be due to an increase in the turnover number of the enzyme; (ii) phosphorylated PLM did not change the affinity of the pump for Na(+); and (iii) even after phosphorylation by PKC, PLM still remains associated with the α(2) isoform of NKA.
Assuntos
Cavéolas/enzimologia , Proteínas de Membrana/metabolismo , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/enzimologia , Fosfoproteínas/metabolismo , Proteína Quinase C/fisiologia , Artéria Pulmonar/citologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Cavéolas/metabolismo , Cavéolas/ultraestrutura , Células Cultivadas , Cromatografia de Afinidade , Humanos , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Lipossomos/metabolismo , Proteínas de Membrana/isolamento & purificação , Dados de Sequência Molecular , Miócitos de Músculo Liso/metabolismo , Fosfoproteínas/isolamento & purificação , Fosforilação , Ligação Proteica , Proteína Quinase C/metabolismo , Estabilidade Proteica , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/isolamento & purificaçãoRESUMO
AIMS: We sought to identify, purify and partially characterize a protein inhibitor of Na(+)/K(+)-ATPase in cytosol of pulmonary artery smooth muscle. MAIN METHODS: (i) By spectrophotometric assay, we identified an inhibitor of Na(+)/K(+)-ATPase in cytosolic fraction of pulmonary artery smooth muscle; (ii) the inhibitor was purified by a combination of ammonium sulfate precipitation, diethylaminoethyl (DEAE) cellulose chromatography, hydroxyapatite chromatography and gel filtration chromatography; (iii) additionally, we have also purified Na(+)/K(+)-ATPase alpha(2)beta(1) and alpha(1)beta(1) isozymes for determining some characteristics of the inhibitor. KEY FINDINGS: We identified a novel endogenous protein inhibitor of Na(+)/K(+)-ATPase having an apparent mol mass of approximately 70kDa in the cytosolic fraction of the smooth muscle. The IC(50) value of the inhibitor towards the enzyme was determined to be in the nanomolar range. Important characteristics of the inhibitor are as follows: (i) it showed different affinities toward the alpha(2)beta(1) and alpha(1)beta(1) isozymes of the Na(+)/K(+)-ATPase; (ii) it interacted reversibly to the E(1) site of the enzyme; (iii) the inhibitor blocked the phosphorylated intermediate formation; and (iv) it competitively inhibited the enzyme with respect to ATP. CD studies indicated that the inhibitor causes an alteration of the conformation of the enzyme. The inhibition study also suggested that the DHPC solubilized Na(+)/K(+)-ATPase exists as (alphabeta)(2) diprotomer. SIGNIFICANCE: The inhibitor binds to the Na(+)/K(+)-ATPase at a site different from the ouabain binding site. The novelty of the inhibitor is that it acts in an isoform specific manner on the enzyme, where alpha(2) is more sensitive than alpha(1).
Assuntos
Citosol/metabolismo , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/metabolismo , Ouabaína/isolamento & purificação , Ouabaína/metabolismo , 4-Nitrofenilfosfatase/metabolismo , Animais , Bovinos , Citosol/enzimologia , Concentração de Íons de Hidrogênio , Concentração Inibidora 50 , Músculo Liso/citologia , Músculo Liso/enzimologia , Músculo Liso/metabolismo , Isoformas de Proteínas , Artéria Pulmonar/citologia , Artéria Pulmonar/enzimologia , Artéria Pulmonar/metabolismo , ATPase Trocadora de Sódio-Potássio/isolamento & purificação , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
We identified alpha(2), alpha(1), and beta(1) isoforms of Na(+)/K(+)-ATPase in caveolae vesicles of bovine pulmonary smooth muscle plasma membrane. The biochemical and biophysical characteristics of the alpha(2)beta(1) isozyme of Na(+)/K(+)-ATPase from caveolae vesicles were studied during solubilization and purification using the detergents 1,2-heptanoyl-sn-phosphatidylcholine (DHPC), poly(oxy-ethylene)8-lauryl ether (C(12)E(8)), and Triton X-100, and reconstitution with the phospholipid dioleoyl-phosphatidylcholine (DOPC). DHPC was determined to be superior to C(12)E(8), whereas C(12)E(8) was better than Triton X-100 in the active enzyme yields and specific activity. Fluorescence studies with DHPC-purified alpha(2)beta(1) isozyme of Na(+)/K(+)-ATPase elicited higher E1Na-E2 K transition compared with that of the C(12)E(8)- and Triton X-100-purified enzyme. The rate of Na(+) efflux in DHPC-DOPC-reconstituted isozyme was higher compared to the C(12)E(8)-DOPC- and Triton X100-DOPC-reconstituted enzyme. Circular dichroism analysis suggests that the DHPC-purified alpha(2)beta(1) isozyme of Na(+)/K(+)-ATPase possessed more organized secondary structure compared to the C(12)E(8)- and Triton X-100-purified isozyme.
Assuntos
Cavéolas/enzimologia , Detergentes/química , Isoenzimas/metabolismo , Pulmão/enzimologia , Músculo Liso Vascular/enzimologia , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Bovinos , Cavéolas/química , Isoenzimas/química , Lipossomos/química , Lipossomos/metabolismo , Pulmão/anatomia & histologia , Músculo Liso Vascular/citologia , Octoxinol/química , Fosfatidilcolinas/química , Éteres Fosfolipídicos/química , Conformação ProteicaRESUMO
AIMS: We sought to determine the mechanisms of an increase in Ca(2+) level in caveolae vesicles in pulmonary smooth muscle plasma membrane during Na(+)/K(+)-ATPase inhibition by ouabain. MAIN METHODS: The caveolae vesicles isolated by density gradient centrifugation were characterized by electron microscopic and immunologic studies and determined ouabain induced increase in Na(+) and Ca(2+) levels in the vesicles with fluorescent probes, SBFI-AM and Fura2-AM, respectively. KEY FINDINGS: We identified the alpha(2)beta(1) and alpha(1)beta(1) isozymes of Na(+)/K(+)-ATPase in caveolae vesicles, and only the alpha(1)beta(1) isozyme in noncaveolae fraction of the plasma membrane. The alpha(2)-isoform contributes solely to the enzyme inhibition in the caveolae vesicles at 40 nM ouabain. Methylisobutylamiloride (Na(+)/H(+)-exchange inhibitor) and tetrodotoxin (voltage-gated Na(+)-channel inhibitor) pretreatment prevented ouabain induced increase in Na(+) and Ca(2+) levels. Ouabain induced increase in Ca(2+) level was markedly, but not completely, inhibited by KB-R7943 (reverse-mode Na(+)/Ca(2+)-exchange inhibitor) and verapamil (L-type Ca(2+)-channel inhibitor). However, pretreatment with tetrodotoxin in conjunction with KB-R7943 and verapamil blunted ouabain induced increase in Ca(2+) level in the caveolae vesicles, indicating that apart from Na(+)/Ca(+)-exchanger and L-type Ca(2+)-channels, "slip-mode conductance" of Na(+) channels could also be involved in this scenario. SIGNIFICANCE: Inhibition of alpha(2) isoform of Na(+)/K(+)-ATPase by ouabain plays a crucial role in modulating the Ca(2+) influx regulatory components in the caveolae microdomain for marked increase in (Ca(2+))(i) in the smooth muscle, which could be important for the manifestation of pulmonary hypertension.