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Ewing sarcoma (EWS) is a highly aggressive cancer with a survival rate of 70%-80% for patients with localized disease and under 30% for those with metastatic disease. Tumor-infiltrating neutrophils (TIN) can generate extracellular net-like DNA structures known as neutrophil extracellular traps (NETs). However, little is known about the presence and prognostic significance of tumor-infiltrating NETs in EWS. Herein, we investigated 46 patients diagnosed with EWS and treated in the Tel Aviv Medical Center between 2010 and 2021. TINs and NETs were identified in diagnostic biopsies of EWS by immunofluorescence. In addition, NETs were investigated in neutrophils isolated from peripheral blood samples of EWS patients at diagnosis and following neoadjuvant chemotherapy. The relationships between the presence of TINs and NETs, pathological and clinical features, and outcomes were analyzed. Our results demonstrate that TIN and NETs at diagnosis were higher in EWS patients with metastatic disease compared with those with local disease. High NET formation at diagnosis predicted poor response to neoadjuvant chemotherapy, relapse, and death from disease (p < 0.05). NET formation in peripheral blood samples at diagnosis was significantly elevated among patients with EWS compared with pediatric controls and decreased significantly following neoadjuvant chemotherapy. In conclusion, NET formation seems to have a role in the EWS immune microenvironment. Their presence can refine risk stratification, predict chemotherapy resistance and survival, and serve as a therapeutic target in patients with EWS.
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Trampas Extracelulares , Sarcoma de Ewing , Humanos , Niño , Sarcoma de Ewing/genética , Recurrencia Local de Neoplasia , Pronóstico , Neutrófilos/patología , Microambiente TumoralRESUMEN
BACKGROUND: Juvenile idiopathic arthritis (JIA) is the most common chronic rheumatic disease in children, causing significant morbidity. Despite the dramatic improvement in treatment, many patients do not achieve complete remission, and biomarkers for subclinical disease, flares, and response to treatment are lacking. Neutrophils and neutrophil extracellular traps (NETs) play key roles in the pathogenesis of autoimmune and inflammatory conditions. In this study, we characterized neutrophil enzyme activity and NETs formation in oligoarticular and polyarticular JIA and explored their association with disease activity. METHODS: Neutrophils from 6 healthy controls and 7 patients with oligoarticular and polyarticular JIA were freshly isolated at time of diagnosis and after glucocorticoid intra-articular injection. Enzymatic activity of neutrophil granular enzymes was monitored by colorimetry and PMA-activated NETs formation was assessed using fluorescent microscopy. RESULTS: In this pilot and feasibility study, we revealed that NETs were significantly increased in oligoarticular JIA patients at time of diagnosis compared to healthy controls. Anti-inflammatory treatment using intra-articular steroid injection normalized NETs formation in these patients. Correlation between NETs formation and clinical Juvenile Activity Disease Activity Score-10 (cJADAS-10) was linear and significant (P = 0.007) in oligo but not in poly JIA patients. CONCLUSIONS: This is the first study exploring the link of NETs formation with oligo and poly JIA activity. We demonstrated a statistically significant linear correlation between cJADAS-10 and NETs formation in oligo but not in poly JIA patients. Hence, we suggest that NETs may reflect clinical disease activity in JIA, and may serve as a putative biomarker. Further work is needed to validate these initial results and determine the dynamics of NETs formation in JIA.
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Artritis Juvenil , Trampas Extracelulares , Niño , Humanos , Artritis Juvenil/diagnóstico , Artritis Juvenil/tratamiento farmacológico , Neutrófilos , Proyectos Piloto , BiomarcadoresRESUMEN
Neutrophils are central players in the innate immune system. To protect against invading pathogens, neutrophils can externalize chromatin to create neutrophil extracellular traps (NETs). While NETs are critical to host defense, they also have deleterious effects, and dysregulation of NETs formation has been implicated in autoimmune diseases, atherosclerosis and thrombotic conditions, cancer progression and dissemination, and acute respiratory distress syndrome. Here, we report that selinexor, a first-in-class selective inhibitor of nuclear export approved for the treatment of multiple myeloma and diffuse large B-cell lymphoma, markedly suppressed the release of NETs in vitro. Furthermore, we demonstrate a significant inhibitory effect of selinexor on NETs formation, but not on oxidative burst or enzymatic activities central to NETs release such as neutrophil elastase, myeloperoxidase or peptidyl arginine deiminase type IV. The inhibitory effect of selinexor was demonstrated in neutrophils activated by a variety of NETs-inducers, including PMA, TGF-ß, TNF-α and IL-8. Maximal inhibition of NETs formation was observed using TGF-ß, for which selinexor inhibited NETs release by 61.6%. These findings pave the way to the potential use of selinexor in an effort to reduce disease burden by inhibition of NETs.
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OBJECTIVES: Neutrophils and their granular enzymes such as neutrophil elastase (NE) and myeloperoxidase (MPO) play important roles in inflammatory diseases, and might be utilized as biomarkers for disease severity and progression. The aim of this study was to determine reference intervals for NE and MPO activity in healthy volunteers comparing two methods of neutrophil isolation. METHODS: Neutrophils were isolated using ficoll density gradient centrifugation or immunomagnetic negative selection in two separate volunteers' cohorts. Subsequently, cells were lysed and incubated with chromogens for NE and MPO activity measurements, then measured with a microplate reader at 415 or 450 nm respectively. RESULTS: The enzymatic activity of NE and MPO depended on the neutrophil isolation technique. Both enzymatic activities were significantly higher (P < 0.001) after isolating neutrophils with ficoll density gradient centrifugation than using the immunomagnetic negative selection. CONCLUSIONS: We demonstrated that neutrophil isolation is an important factor that influences the outcome of enzymatic activity measurements. Techniques based on immunomagnetic negative selection are favorable, specifically for investigations related to NE and MPO activity. When using NE and MPO activity measurements in clinical practice, care must be taken to interpret the data depending on the applied cell isolation technique.
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Elastasa de Leucocito , Neutrófilos , Biomarcadores , Separación Celular , Ficoll , Humanos , PeroxidasaRESUMEN
Chronic granulomatous disease (CGD) is a rare primary immunodeficiency disorder caused by defects in the NADPH oxidase complex. Mutations in NCF2 encoding the cytosolic factor p67phox result in autosomal recessive CGD. We describe three patients with a novel c.855G>C NCF2 mutation presenting with diverse clinical phenotype. Two siblings were heterozygous for the novel mutation and for a previously described exon 8-9 duplication, while a third unrelated patient was homozygous for the novel mutation. Mutation pathogenicity was confirmed by abnormal DHR123 assay and absent p67phox production and by sequencing of cDNA which showed abnormal RNA splicing. Clinically, the homozygous patient presented with suspected early onset interstitial lung disease and NCF2 mutation was found on genetic testing performed in search for surfactant-related defects. The two siblings also had variable presentation with one having history of severe pneumonia, lymphadenitis, and recurrent skin abscesses and the other presenting in his 30s with discoid lupus erythematosus and without significant infectious history. We therefore identified a novel pathogenic NCF2 mutation causing diverse and unusual clinical phenotype.
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Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Enfermedad Granulomatosa Crónica/genética , Mutación , NADPH Oxidasas/genética , Alelos , Exones , Enfermedad Granulomatosa Crónica/diagnóstico , Homocigoto , Humanos , Neutrófilos/inmunología , Neutrófilos/metabolismo , Fenotipo , HermanosRESUMEN
Impaired neutrophil extracellular trap (NET) formation compromises the host defense after engraftment following hematopoietic stem cell transplantation (HSCT) despite adequate neutrophil counts. The aims of the present study were to determine reference ranges for the activity of key enzymes of NET formation-neutrophil elastase (NE) and myeloperoxidase (MPO)-in a healthy population and to unravel the recovery dynamics of NET formation over time following HSCT, along with NE and MPO enzymatic activities. Reference ranges of NE and MPO activity were derived from 50 healthy volunteers. During 2017 to 2018, 11 consecutive pediatric patients undergoing allogeneic or autologous HSCT were recruited at a single referral center for pediatric hemato-oncology. Patients were followed for up to 1 year following engraftment. The mean reference value was 7.5 ± .4 mU for NE activity and 2.17 ± .4 U for MPO activity in the healthy population, and enzymatic activity of MPO was significantly higher in males. At 3 weeks following neutrophil engraftment, all study participants demonstrated extremely low enzymatic NE activity, whereas MPO activity was above the lower normal reference range at all time points. Reduced NE activity corresponded to the inability to form NETs. Neutrophil function improved over time, but partial impairment persisted for 7 months following transplantation. The ability of neutrophils to form NETs was significantly impaired for 3 weeks after engraftment in the setting of HSCT, exposing patients to bacterial infections. NE activity might serve as a surrogate marker for the capacity of neutrophils to form NETs.
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Infecciones Bacterianas/sangre , Trampas Extracelulares/metabolismo , Trasplante de Células Madre Hematopoyéticas , Elastasa de Leucocito/sangre , Neutrófilos/metabolismo , Adolescente , Adulto , Aloinjertos , Biomarcadores/sangre , Niño , Preescolar , Femenino , Estudios de Seguimiento , Humanos , Masculino , Peroxidasa/sangreRESUMEN
BACKGROUND: Fatty acid synthase 1 (FAS I) from Mycobacterium tuberculosis (Mtb) is an essential protein and a promising drug target. FAS I is a multi-functional, multi-domain protein that is organized as a large (1.9 MDa) homohexameric complex. Acyl intermediates produced during fatty acid elongation are attached covalently to an acyl carrier protein (ACP) domain. This domain is activated by the transfer of a 4'-Phosphopantetheine (4'-PP, also termed P-pant) group from CoA to ACP catalyzed by a 4'-PP transferase, termed acyl carrier protein synthase (AcpS). METHODS: In order to obtain an activated FAS I in E. coli, we transformed E. coli with tagged Mtb fas1 and acpS genes encoded by a separate plasmid. We induced the expression of Mtb FAS I following induction of AcpS expression. FAS I was purified by Strep-Tactin affinity chromatography. RESULTS: Activation of Mtb FAS I was confirmed by the identification of a bound P-pant group on serine at position 1808 by mass spectrometry. The purified FAS I displayed biochemical activity shown by spectrophotometric analysis of NADPH oxidation and by CoA production, using the Ellman reaction. The purified Mtb FAS I forms a hexameric complex shown by negative staining and cryo-EM. CONCLUSION: Purified hexameric and active Mtb FAS I is required for binding and drug inhibition studies and for structure-function analysis of this enzyme. This relatively simple and short procedure for Mtb FAS I production should facilitate studies of this enzyme.
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Proteínas Bacterianas/metabolismo , Escherichia coli/enzimología , Ácido Graso Sintasas/metabolismo , Mycobacterium tuberculosis/enzimología , Proteínas Recombinantes/metabolismo , Antituberculosos , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/ultraestructura , Descubrimiento de Drogas , Escherichia coli/genética , Ácido Graso Sintasas/genética , Ácido Graso Sintasas/aislamiento & purificación , Ácido Graso Sintasas/ultraestructura , Vectores Genéticos , Mycobacterium tuberculosis/genética , Unión Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/ultraestructura , Transformación BacterianaRESUMEN
Tuberculosis (TB) is a devastating and rapidly spreading disease caused by Mycobacterium tuberculosis (Mtb). Therapy requires prolonged treatment with a combination of multiple agents and interruptions in the treatment regimen result in emergence and spread of multi-drug resistant (MDR) Mtb strains. MDR Mtb poses a significant global health problem, calling for urgent development of novel drugs to combat TB. Here, we report the 3.3 Å resolution structure of the ~2 MDa type-I fatty acid synthase (FAS-I) from Mtb, determined by single particle cryo-EM. Mtb FAS-I is an essential enzymatic complex that contributes to the virulence of Mtb, and thus a prime target for anti-TB drugs. The structural information for Mtb FAS-I we have obtained enables computer-based drug discovery approaches, and the resolution achieved by cryo-EM is sufficient for elucidating inhibition mechanisms by putative small molecular weight inhibitors.
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Proteínas Bacterianas/química , Descubrimiento de Drogas/métodos , Ácido Graso Sintasas/química , Mycobacterium tuberculosis/química , Antituberculosos/farmacología , Antituberculosos/uso terapéutico , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/aislamiento & purificación , Dominio Catalítico , Microscopía por Crioelectrón , Ácido Graso Sintasas/antagonistas & inhibidores , Ácido Graso Sintasas/aislamiento & purificación , Modelos Moleculares , Mycobacterium tuberculosis/patogenicidad , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiología , VirulenciaRESUMEN
Recent studies demonstrated the dependence of speed adaptation in Escherichia coli on acetylation of the chemotaxis signaling molecule CheY. Here, we examined whether CheY acetylation is involved in chemotactic adaptation. A mutant lacking the acetylating enzyme acetyl-CoA synthetase (Acs) requires more time to adapt to attractant stimulation, and vice versa to repellent stimulation. This effect is avoided by conditions that favor production of acetyl-CoA, thus enabling Acs-independent CheY autoacetylation, or reversed by expressing Acs from a plasmid. These findings suggest that CheY should be acetylated for ordinary adaptation time, and that the function of this acetylation in adaptation is to enable the motor to shift its rotation to clockwise. We further identify the enzyme phosphotransacetylase as a third deacetylase of CheY in E. coli.
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Adaptación Fisiológica , Quimiotaxis , Escherichia coli/citología , Escherichia coli/metabolismo , Proteínas Quimiotácticas Aceptoras de Metilo/metabolismo , Acetilcoenzima A/metabolismo , Acetilación , Escherichia coli/fisiología , Proteínas de Escherichia coliRESUMEN
Chemoreceptor methylation and demethylation has been shown to be at the core of the adaptation mechanism in Escherichia coli chemotaxis. Nevertheless, mutants lacking the methylation machinery can adapt to some extent. Here we carried out an extensive quantitative analysis of chemotactic and chemokinetic methylation-independent adaptation. We show that partial or complete adaptation of the direction of flagellar rotation and the swimming speed in the absence of the methylation machinery each occurs in a small fraction of cells. Furthermore, deletion of the main enzyme responsible for acetylation of the signaling molecule CheY prevented speed adaptation but not adaptation of the direction of rotation. These results suggest that methylation-independent adaptation in bacterial chemotaxis involves chemokinetic adaptation, which is dependent on CheY acetylation.
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Adaptación Fisiológica , Quimiotaxis , Escherichia coli/citología , Escherichia coli/fisiología , Acetilación/efectos de los fármacos , Adaptación Fisiológica/efectos de los fármacos , Quimiotaxis/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Flagelos/efectos de los fármacos , Flagelos/metabolismo , Metilación/efectos de los fármacos , Movimiento/efectos de los fármacos , N-Metilaspartato/farmacologíaRESUMEN
Presenilin (PSEN) deficiency is accompanied by accumulation of endosomes and autophagosomes, likely caused by impaired endo-lysosomal fusion. Recently, Lee et al. (2010. Cell. doi: http://dx.doi.org/10.1016/j.cell.2010.05.008) attributed this phenomenon to PSEN1 enabling the transport of mature V0a1 subunits of the vacuolar ATPase (V-ATPase) to lysosomes. In their view, PSEN1 mediates the N-glycosylation of V0a1 in the endoplasmic reticulum (ER); consequently, PSEN deficiency prevents V0a1 glycosylation, compromising the delivery of unglycosylated V0a1 to lysosomes, ultimately impairing V-ATPase function and lysosomal acidification. We show here that N-glycosylation is not a prerequisite for proper targeting and function of this V-ATPase subunit both in vitro and in vivo in Drosophila melanogaster. We conclude that endo-lysosomal dysfunction in PSEN(-/-) cells is not a consequence of failed N-glycosylation of V0a1, or compromised lysosomal acidification. Instead, lysosomal calcium storage/release is significantly altered in PSEN(-/-) cells and neurons, thus providing an alternative hypothesis that accounts for the impaired lysosomal fusion capacity and accumulation of endomembranes that accompanies PSEN deficiency.
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Calcio/metabolismo , Proteínas de Drosophila/metabolismo , Homeostasis/fisiología , Lisosomas/metabolismo , Presenilina-1/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , Animales , Transporte Biológico , Línea Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/enzimología , Fibroblastos/metabolismo , Glicosilación , Hipocampo/metabolismo , Humanos , Ratones , Ratones Noqueados , Neuronas/metabolismo , ATPasas de Translocación de Protón Vacuolares/genéticaRESUMEN
Lipid rafts are often considered as microdomains enriched in sphingomyelin and cholesterol, predominantly residing in the plasma membrane but which originate in earlier compartments of the cellular secretory pathway. Within this pathway, the membranes of the Golgi complex represent a transition stage between the cholesterol-poor membranes of the endoplasmic reticulum (ER) and the cholesterol-rich plasma membrane. The rafts are related to detergent-resistant membranes, which because of their ordered structure are poorly penetrated by cold non-ionic detergents and float in density gradient centrifugation. In this study the microdomain niche of the Golgi-resident SPCA Ca(2+)/Mn(2+) pumps was investigated in HT29 cells by Triton X-100 detergent extraction and density-gradient centrifugation. Similarly to cholesterol and the raft-resident flotillin-2, SPCA1 was found mainly in detergent-resistant fractions, while SERCA3 was detergent-soluble. Furthermore, cholesterol depletion of cells resulted in redistribution of flotillin-2 and SPCA1 to the detergent-soluble fractions of the density gradient. Additionally, the time course of solubilization by Triton X-100 was investigated in live COS-1 and HT29 cells expressing fluorescent SERCA2b, SPCA1d or SPCA2. In both cell types, the ER-resident SERCA2b protein was gradually solubilized, while SPCA1d resisted to detergent solubilization. SPCA2 was more sensitive to detergent extraction than SPCA1d. To investigate the functional impact of cholesterol on SPCA1, ATPase activity was monitored. Depletion of cholesterol inhibited the activity of SPCA1d, while SERCA2b function was not altered. From these results we conclude that SPCA1 is associated with cholesterol-rich domains of HT29 cells and that the cholesterol-rich environment is essential for the functioning of the pump.
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Adenocarcinoma/metabolismo , ATPasas Transportadoras de Calcio/química , ATPasas Transportadoras de Calcio/metabolismo , Colesterol/química , Colesterol/metabolismo , Neoplasias del Colon/metabolismo , Microdominios de Membrana/química , Microdominios de Membrana/metabolismo , Animales , Secuencia de Bases , Células COS , ATPasas Transportadoras de Calcio/genética , Línea Celular Tumoral , Chlorocebus aethiops , Cartilla de ADN/genética , Aparato de Golgi/metabolismo , Humanos , Inmunohistoquímica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismoRESUMEN
Detailed practical information is provided with emphasis on mapping cytosolic and mitochondrial pH, mitochondrial Na(+), and briefly also aspects related to mitochondrial Ca(2+) measurements in living cells, as grown on (un)coated glass coverslips. This chapter lists (laser scanning confocal) microscope instrumentation and setup requirements for proper imaging conditions, cell holders, and an easy-to-use incubator stage. For the daily routine of preparing buffer and calibration solutions, extensive annotated protocols are provided. In addition, detailed measurement and image analysis protocols are given to routinely obtain optimum results with confidence, while avoiding a number of typical pitfalls.
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Calcio/metabolismo , Mitocondrias/metabolismo , Sodio/metabolismo , Animales , Calibración , Línea Celular , Núcleo Celular/metabolismo , Supervivencia Celular , Citosol/metabolismo , Perros , Colorantes Fluorescentes/metabolismo , Vidrio , Concentración de Iones de Hidrógeno , Indicadores y Reactivos/metabolismo , Microscopía Confocal , Imagen Molecular , Movimiento , Programas InformáticosRESUMEN
The neuropeptide-like precursor 1 (NPLP1) was first identified in a peptidomics experiment on Drosophila melanogaster. Limited data on this novel neuropeptide precursor suggest a role in the regulation of ecdysis in holometabolous larvae. In this study, we characterized the NPLP1 precursor in the gray flesh fly, Neobellieria bullata, which is an excellent model for physiological assays and hence to discover the role of the NPLP1 peptides. Antisera against three of the D. melanogaster NPLP1 neuropeptides stained an identical set of neurons in the central nervous system of N. bullata compared to D. melanogaster. A novel approach was applied to identify the N. bullata NPLP1 orthologs. Using a combination of affinity chromatography, mass spectrometry, cDNA cloning and RACE experiments, we obtained almost the complete coding sequence of the NPLP1 mRNA. Three encoded NPLP1 peptides were identified in central nervous system extracts by mass spectrometry. Neither doses of 25-250pmol of synthetic Neb-MGYamide and Neb-PQNamide peptides, nor the NPLP1 antisera did affect the speed of retraction, contraction and tanning in the pupariation bioassay.
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Proteínas de Drosophila/genética , Neuropéptidos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bioensayo , Cromatografía de Afinidad , Cromatografía Líquida de Alta Presión , Clonación Molecular , Dípteros/metabolismo , Datos de Secuencia Molecular , Neuropéptidos/aislamiento & purificación , Pupa/fisiología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Extracellular agonists increase the cytosolic free Ca2+ concentration ([Ca2+]c) by Ca2+ influx and by stimulating Ca2+ release from intracellular stores, mainly the endoplasmic reticulum and to a lesser extent also later compartments of the secretory pathway, particularly the Golgi. The Golgi takes up Ca2+ via Sarco/Endoplasmic Reticulum Ca2+ATPases (SERCAs) and the Secretory-Pathway Ca2+ATPases (SPCAs). The endogenous expression of SERCAs and SPCAs neutrophils was demonstrated by Western blotting and immunocytochemistry. Up till now, all cytosolic Ca2+ transients due to intracellular Ca2+ release have been found to originate from SERCA-dependent stores. We found that human neutrophils also present Ca2+ release from a SERCA-independent store. Changes in [Ca2+]c of neutrophils were investigated during chemokinesis induced by chemotactic factors in Ca2+-free solution with and without the SERCA-specific inhibitor thapsigargin. Using N-formyl-methionyl-leucyl-phenylalanine or interleukin-8 as agonists, Ca2+ release from intracellular stores was observed in respectively about 40% and 20% of the neutrophils pre-treated with Ca2+-free solution and thapsigargin. In the latter condition, 20-30% of the cells preserved migratory behaviour. These results indicate that both SERCA-dependent and SERCA-independent (presumably SPCA-dependent) intracellular Ca2+ stores contribute to Ca2+ signaling during chemokinesis of human neutrophil granulocytes.
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Señalización del Calcio/fisiología , Calcio/metabolismo , Movimiento Celular/fisiología , Granulocitos , Neutrófilos , Animales , Células Cultivadas , Citoesqueleto/metabolismo , Exocitosis/fisiología , Aparato de Golgi/metabolismo , Granulocitos/citología , Granulocitos/metabolismo , Humanos , Neutrófilos/citología , Neutrófilos/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Vías Secretoras/fisiologíaRESUMEN
In ischemic or hypoxic tissues, elevated cytosolic calcium levels can induce lethal processes. Mitochondria, besides the endoplasmic reticulum, play a key role in clearing excessive cytosolic Ca2+. In a previous study, it was suggested that the clearance of cytosolic Ca2+, after approximately 18 min of metabolic inhibition (MI) in renal epithelial cells, occurs via the reverse action of the mitochondrial Na+/Ca2+ exchanger (NCX). For further investigating the underlying mechanism, changes in the mitochondrial Na+ concentration ([Na+](m)) were monitored in metabolically inhibited MDCK cells. CoroNa Red, a sodium-sensitive fluorescence probe, was used to monitor [Na+]m. In the first 15 min of MI, a twofold increase of [Na+]m was observed reaching 113 +/- 7 mM, whereas the cytosolic Na+ concentration ([Na+]c) elevated threefold, to a level of 65 +/- 6 mM. In the next 45 min of MI, [Na+]m dropped to 91 +/- 7 mM, whereas [Na+]c further increased to 91 +/- 4 mM. The striking rise in [Na+]m is likely sufficient to sustain the driving force for mitochondrial Ca2+ uptake via the NCX. Furthermore, when CGP-37157, a specific inhibitor of the mitochondrial NCX, was applied during MI, the second-phase drop of [Na+]m was completely abolished. The obtained results support the hypothesis that the mitochondrial NCX reverses after approximately 15 min of MI. Moreover, because the cellular homeostasis can recover after MI, the mitochondria likely protect MDCK cells from injury during MI by the reversal of the mitochondrial NCX. This study is the first to report [Na+]m measurements in nonpermeabilized living cells.