RESUMO
This study explored the role of the Na/K-ATPase (NKA) in membrane permeabilization induced by nanosecond electric pulses. Using CRISPR/Cas9 and shRNA, we silenced the ATP1A1 gene, which encodes α1 NKA subunit in U937 human monocytes. Silencing reduced the rate and the cumulative uptake of YoPro-1 dye after electroporation by 300-ns, 7-10 kV/cm pulses, while ouabain, a specific NKA inhibitor, enhanced YoPro-1 entry. We conclude that the α1 subunit supports the electropermeabilized membrane state, by forming or stabilizing electropores or by hindering repair mechanisms, and this role is independent of NKA's ion pump function.
Assuntos
Eletricidade , Eletroporação , Humanos , Permeabilidade da Membrana Celular , Membrana Celular/metabolismo , RNA Interferente Pequeno/metabolismo , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
The study was aimed at identifying endogenous proteins which assist or impede the permeabilized state in the cell membrane disrupted by nsEP (20 or 40 pulses, 300 ns width, 7 kV/cm). We employed a LentiArray CRISPR library to generate knockouts (KOs) of 316 genes encoding for membrane proteins in U937 human monocytes stably expressing Cas9 nuclease. The extent of membrane permeabilization by nsEP was measured by the uptake of Yo-Pro-1 (YP) dye and compared to sham-exposed KOs and control cells transduced with a non-targeting (scrambled) gRNA. Only two KOs, for SCNN1A and CLCA1 genes, showed a statistically significant reduction in YP uptake. The respective proteins could be part of electropermeabilization lesions or increase their lifespan. In contrast, as many as 39 genes were identified as likely hits for the increased YP uptake, meaning that the respective proteins contributed to membrane stability or repair after nsEP. The expression level of eight genes in different types of human cells showed strong correlation (R > 0.9, p < 0.02) with their LD50 for lethal nsEP treatments, and could potentially be used as a criterion for the selectivity and efficiency of hyperplasia ablations with nsEP.
Assuntos
Eletricidade , Eletroporação , Cricetinae , Animais , Humanos , Cricetulus , Permeabilidade da Membrana Celular , Membrana Celular/metabolismo , Transporte BiológicoRESUMO
The principal bioeffect of the nanosecond pulsed electric field (nsPEF) is a lasting cell membrane permeabilization, which is often attributed to the formation of nanometer-sized pores. Such pores may be too small for detection by the uptake of fluorescent dyes. We tested if Ca2+, Cd2+, Zn2+, and Ba2+ ions can be used as nanoporation markers. Time-lapse imaging was performed in CHO, BPAE, and HEK cells loaded with Fluo-4, Calbryte, or Fluo-8 dyes. Ca2+ and Ba2+ did not change fluorescence in intact cells, whereas their entry after nsPEF increased fluorescence within <1 ms. The threshold for one 300-ns pulse was at 1.5-2 kV/cm, much lower than >7 kV/cm for the formation of larger pores that admitted YO-PRO-1, TO-PRO-3, or propidium dye into the cells. Ba2+ entry caused a gradual emission rise, which reached a stable level in 2 min or, with more intense nsPEF, kept rising steadily for at least 30 min. Ca2+ entry could elicit calcium-induced calcium release (CICR) followed by Ca2+ removal from the cytosol, which markedly affected the time course, polarity, amplitude, and the dose-dependence of fluorescence change. Both Ca2+ and Ba2+ proved as sensitive nanoporation markers, with Ba2+ being more reliable for monitoring membrane damage and resealing.
Assuntos
Bário/metabolismo , Cálcio/metabolismo , Permeabilidade da Membrana Celular , Membrana Celular/metabolismo , Animais , Células CHO , Cátions/metabolismo , Linhagem Celular , Cricetinae , Cricetulus , Citosol/metabolismo , Eletroporação/métodos , Corantes Fluorescentes/química , Células HEK293 , Humanos , Microscopia de Fluorescência/métodos , Nanotecnologia/métodos , Imagem com Lapso de Tempo/métodosRESUMO
Intense nanosecond pulsed electric field (nsPEF) is a novel modality for cell activation and nanoelectroporation. Applications of nsPEF in research and therapy are hindered by a high electric field requirement, typically from 1 to over 50â¯kV/cm to elicit any bioeffects. We show how this requirement can be overcome by engaging temporal summation when pulses are compressed into high-rate bursts (up to several MHz). This approach was tested for excitation of ventricular cardiomyocytes and peripheral nerve fibers; for membrane electroporation of cardiomyocytes, CHO, and HEK cells; and for killing EL-4â¯cells. MHz compression of nsPEF bursts (100-1000 pulses) enables excitation at only 0.01-0.15â¯kV/cm and electroporation already at 0.4-0.6â¯kV/cm. Clear separation of excitation and electroporation thresholds allows for multiple excitation cycles without membrane disruption. The efficiency of nsPEF bursts increases with the duty cycle (by increasing either pulse duration or repetition rate) and with increasing the total time "on" (by increasing either pulse duration or number). For some endpoints, the efficiency of nsPEF bursts matches a single "long" pulse whose amplitude and duration equal the time-average amplitude and duration of the bursts. For other endpoints this rule is not valid, presumably because of nsPEF-specific bioeffects and/or possible modification of targets already during the burst. MHz compression of nsPEF bursts is a universal and efficient way to lower excitation thresholds and facilitate electroporation.
Assuntos
Potenciais de Ação/fisiologia , Permeabilidade da Membrana Celular/fisiologia , Eletroporação/métodos , Miócitos Cardíacos/fisiologia , Fibras Nervosas/fisiologia , Animais , Células CHO , Cálcio , Linhagem Celular Tumoral , Células Cultivadas , Cricetulus , Estimulação Elétrica/métodos , Células HEK293 , Humanos , Camundongos Endogâmicos DBA , Miócitos Cardíacos/citologia , Rana catesbeiana/fisiologia , Fatores de TempoRESUMO
Nanosecond pulsed electric fields (nsPEF) are emerging as a novel modality for cell stimulation and tissue ablation. However, the downstream protein effectors responsible for nsPEF bioeffects remain to be established. Here we demonstrate that nsPEF activate TMEM16F (or Anoctamin 6), a protein functioning as a Ca2+-dependent phospholipid scramblase and Ca2+-activated chloride channel. Using confocal microscopy and patch clamp recordings, we investigated the relevance of TMEM16F activation for several bioeffects triggered by nsPEF, including phosphatidylserine (PS) externalization, nanopore-conducted currents, membrane blebbing, and cell death. In HEK 293 cells treated with a single 300-ns pulse of 25.5 kV/cm, Tmem16f expression knockdown and TMEM16F-specific inhibition decreased nsPEF-induced PS exposure by 49 and 42%, respectively. Moreover, the Tmem16f silencing significantly decreased Ca2+-dependent chloride channel currents activated in response to the nanoporation. Tmem16f expression also affected nsPEF-induced cell blebbing, with only 20% of the silenced cells developing blebs compared with 53% of the control cells. This inhibition of cellular blebbing correlated with a 25% decrease in cytosolic free Ca2+ transient at 30 s after nanoporation. Finally, in TMEM16F-overexpressing cells, a train of 120 pulses (300 ns, 20 Hz, 6 kV/cm) decreased cell survival to 34% compared with 51% in control cells (*, p < 0.01). Taken together, these results indicate that TMEM16F activation by nanoporation mediates and enhances the diverse cellular effects of nsPEF.
Assuntos
Anoctaminas/metabolismo , Apoptose/efeitos da radiação , Cálcio/metabolismo , Membrana Celular/fisiologia , Eletricidade , Nanotecnologia , Fosfatidilserinas/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Relação Dose-Resposta à Radiação , Células HEK293 , Humanos , Canais Iônicos/metabolismoRESUMO
Tumor ablation by nanosecond pulsed electric fields (nsPEF) is an emerging therapeutic modality. We compared nsPEF cytotoxicity for human cell lines of cancerous (IMR-32, Hep G2, HT-1080, and HPAF-II) and non-cancerous origin (BJ and MRC-5) under strictly controlled and identical conditions. Adherent cells were uniformly treated by 300-ns PEF (0-2000 pulses, 1.8 kV/cm, 50 Hz) on indium tin oxide-covered glass coverslips, using the same media and serum. Cell survival plotted against the number of pulses displayed three distinct regions (initial resistivity, logarithmic survival decline, and residual resistivity) for all tested cell types, but with differences in LD50 spanning as much as nearly 80-fold. The non-cancerous cells were less sensitive than IMR-32 neuroblastoma cells but more vulnerable than the other cancers tested. The cytotoxic efficiency showed no apparent correlation with cell or nuclear size, cell morphology, metabolism level, or the extent of membrane disruption by nsPEF. Increasing pulse duration to 9 µs (0.75 kV/cm, 5 Hz) produced a different selectivity pattern, suggesting that manipulation of PEF parameters can, at least for certain cancers, overcome their resistance to nsPEF ablation. Identifying mechanisms and cell markers of differential nsPEF susceptibility will critically contribute to the proper choice and outcome of nsPEF ablation therapies.
Assuntos
Eletricidade , Eletroporação/métodos , Morte Celular , Linhagem Celular , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular , Núcleo Celular/metabolismo , Proliferação de Células , Forma Celular , Tamanho Celular , Sobrevivência Celular , Humanos , Fatores de TempoRESUMO
Mycoplasma pneumoniae (Mp) infections cause tracheobronchitis and "walking" pneumonia, and are linked to asthma and other reactive airway diseases. As part of the infectious process, the bacterium expresses a 591-aa virulence factor with both mono-ADP ribosyltransferase (mART) and vacuolating activities known as Community-Acquired Respiratory Distress Syndrome Toxin (CARDS TX). CARDS TX binds to human surfactant protein A and annexin A2 on airway epithelial cells and is internalized, leading to a range of pathogenetic events. Here we present the structure of CARDS TX, a triangular molecule in which N-terminal mART and C-terminal tandem ß-trefoil domains associate to form an overall architecture distinct from other well-recognized ADP-ribosylating bacterial toxins. We demonstrate that CARDS TX binds phosphatidylcholine and sphingomyelin specifically over other membrane lipids, and that cell surface binding and internalization activities are housed within the C-terminal ß-trefoil domain. The results enhance our understanding of Mp pathogenicity and suggest a novel avenue for the development of therapies to treat Mp-associated asthma and other acute and chronic airway diseases.
Assuntos
Proteínas de Bactérias/química , Toxinas Bacterianas/química , Citotoxinas/química , Mycoplasma pneumoniae/metabolismo , Vacúolos/metabolismo , 1,2-Dipalmitoilfosfatidilcolina/metabolismo , ADP Ribose Transferases/química , ADP Ribose Transferases/metabolismo , Adenosina Difosfato Ribose/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Domínio Catalítico , Citotoxinas/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Fosfatidilcolinas/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Esfingomielinas/metabolismo , Relação Estrutura-AtividadeRESUMO
Electric pulses of nanosecond duration (nsEP) are emerging as a new modality for tissue ablation. Plasma membrane permeabilization by nsEP may cause osmotic imbalance, water uptake, cell swelling, and eventual membrane rupture. The present study was aimed to increase the cytotoxicity of nsEP by fostering water uptake and cell swelling. This aim was accomplished by lowering temperature after nsEP application, which delayed the membrane resealing and/or suppressed the cell volume mechanisms. The cell diameter in U-937 monocytes exposed to a train of 50, 300-ns pulses (100 Hz, 7 kV/cm) at room temperature and then incubated on ice for 30 min increased by 5.6 +/- 0.7 µm (40-50%), which contrasted little or no changes (1 +/- 0.3 µm, <10%) if the incubation was at 37 °C. Neither this nsEP dose nor the 30-min cooling caused cell death when applied separately; however, their combination reduced cell survival to about 60% in 1.5-3 h. Isosmotic addition of a pore-impermeable solute (sucrose) to the extracellular medium blocked cell swelling and rescued the cells, thereby pointing to swelling as a primary cause of membrane rupture and cell death. Cooling after nsEP exposure can potentially be employed in medical practice to assist tissue and tumor ablation.
Assuntos
Temperatura Baixa , Eletroporação , Morte Celular/fisiologia , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/fisiologia , Tamanho Celular , Sobrevivência Celular/fisiologia , HumanosRESUMO
Transforming growth factor (TGF) ß1, ß2, and ß3 (TGF-ß1-TGF-ß3, respectively) are small secreted signaling proteins that each signal through the TGF-ß type I and type II receptors (TßRI and TßRII, respectively). However, TGF-ß2, which is well-known to bind TßRII several hundred-fold more weakly than TGF-ß1 and TGF-ß3, has an additional requirement for betaglycan, a membrane-anchored nonsignaling receptor. Betaglycan has two domains that bind TGF-ß2 at independent sites, but how it binds TGF-ß2 to potentiate TßRII binding and how the complex with TGF-ß, TßRII, and betaglycan undergoes the transition to the signaling complex with TGF-ß, TßRII, and TßRI are not understood. To investigate the mechanism, the binding of the TGF-ßs to the betaglycan extracellular domain, as well as its two independent binding domains, either directly or in combination with the TßRI and TßRII ectodomains, was studied using surface plasmon resonance, isothermal titration calorimetry, and size-exclusion chromatography. These studies show that betaglycan binds TGF-ß homodimers with a 1:1 stoichiometry in a manner that allows one molecule of TßRII to bind. These studies further show that betaglycan modestly potentiates the binding of TßRII and must be displaced to allow TßRI to bind. These findings suggest that betaglycan functions to bind and concentrate TGF-ß2 on the cell surface and thus promote the binding of TßRII by both membrane-localization effects and allostery. These studies further suggest that the transition to the signaling complex is mediated by the recruitment of TßRI, which simultaneously displaces betaglycan and stabilizes the bound TßRII by direct receptor-receptor contact.
Assuntos
Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais , Animais , Sítios de Ligação , Células CHO , Calorimetria , Cricetinae , Cricetulus , Ressonância de Plasmônio de SuperfícieRESUMO
Ca2+ activation and membrane electroporation by 10-ns and 4-ms electric pulses (nsEP and msEP) were compared in rat embryonic cardiomyocytes. The lowest electric field which triggered Ca2+ transients was expectedly higher for nsEP (36 kV/cm) than for msEP (0.09 kV/cm) but the respective doses were similar (190 and 460 mJ/g). At higher intensities, both stimuli triggered prolonged firing in quiescent cells. An increase of basal Ca2+ level by >10 nM in cells with blocked voltage-gated Ca2+ channels and depleted Ca2+ depot occurred at 63 kV/cm (nsEP) or 0.14 kV/cm (msEP) and was regarded as electroporation threshold. These electric field values were at 150-230% of stimulation thresholds for both msEP and nsEP, notwithstanding a 400,000-fold difference in pulse duration. For comparable levels of electroporative Ca2+ uptake, msEP caused at least 10-fold greater uptake of propidium than nsEP, suggesting increased yield of larger pores. Electroporation by msEP started Ca2+ entry abruptly and locally at the electrode-facing poles of cell, followed by a slow diffusion to the center. In a stark contrast, nsEP evoked a "supra-electroporation" pattern of slower but spatially uniform Ca2+ entry. Thus nsEP and msEP had comparable dose efficiency, but differed profoundly in the size and localization of electropores.
Assuntos
Permeabilidade da Membrana Celular/fisiologia , Eletroporação/métodos , Miócitos Cardíacos/fisiologia , Miócitos Cardíacos/efeitos da radiação , Propídio/farmacocinética , Animais , Permeabilidade da Membrana Celular/efeitos da radiação , Células Cultivadas , Relação Dose-Resposta à Radiação , Taxa de Depuração Metabólica/efeitos da radiação , Doses de Radiação , Ratos , Eletricidade EstáticaRESUMO
Exposure to intense, nanosecond-duration electric pulses (nsEP) opens small but long-lived pores in the plasma membrane. We quantified the cell uptake of two membrane integrity marker dyes, YO-PRO-1 (YP) and propidium (Pr) in order to test whether the pore size is affected by the number of nsEP. The fluorescence of the dyes was calibrated against their concentrations by confocal imaging of stained homogenates of the cells. The calibrations revealed a two-phase dependence of Pr emission on the concentration (with a slower rise at<4µM) and a linear dependence for YP. CHO cells were exposed to nsEP trains (1 to 100 pulses, 60ns, 13.2kV/cm, 10Hz) with Pr and YP in the medium, and the uptake of the dyes was monitored by time-lapse imaging for 3min. Even a single nsEP triggered a modest but detectable entry of both dyes, which increased linearly when more pulses were applied. The influx of Pr per pulse was constant and independent of the pulse number. The influx of YP per pulse was highest with 1- and 2-pulse exposures, decreasing to about twice the Pr level for trains from 5 to 100 pulses. The constant YP/Pr influx ratio for trains of 5 to 100 pulses suggests that increasing the number of pulses permeabilizes cells to a greater extent by increasing the pore number and not the pore diameter.
Assuntos
Permeabilidade da Membrana Celular/fisiologia , Membrana Celular/metabolismo , Estimulação Elétrica/métodos , Campos Eletromagnéticos , Eletroporação/métodos , Nanoporos , Animais , Benzoxazóis/química , Células CHO , Membrana Celular/efeitos da radiação , Permeabilidade da Membrana Celular/efeitos da radiação , Cricetinae , Cricetulus , Propídio/química , Pulso Arterial , Compostos de Quinolínio/química , Imagem com Lapso de TempoRESUMO
Opening of long-lived pores in the cell membrane is the principal primary effect of intense, nanosecond pulsed electric field (nsPEF). Here we demonstrate that the evolution of pores, cell survival, the time and the mode of cell death (necrotic or apoptotic) are determined by the level of external Ca(2+) after nsPEF. We also introduce a novel, minimally disruptive technique for nsEP exposure of adherent cells on indium tin oxide (ITO)-coated glass coverslips, which does not require cell detachment and enables fast exchanges of bath media. Increasing the Ca(2+) level from the nominal 2-5µM to 2mM for the first 60-90min after permeabilization by 300-nsPEF increased the early (necrotic) death in U937, CHO, and BPAE cells. With nominal Ca(2+), the inhibition of osmotic swelling rescued cells from the early necrosis and increased caspase 3/7 activation later on. However, the inhibition of swelling had a modest or no protective effect with 2mM Ca(2+) in the medium. With the nominal Ca(2+), most cells displayed gradual increase in YO-PRO-1 and propidium (Pr) uptake. With 2mM Ca(2+), the initially lower Pr uptake was eventually replaced by a massive and abrupt Pr entry (necrotic death). It was accompanied by a transient acceleration of the growth of membrane blebs due to the increase of the intracellular osmotic pressure. We conclude that the high-Ca(2+)-dependent necrotic death in nsPEF-treated cells is effected by a delayed, sudden, and osmotically-independent pore expansion (or de novo formation of larger pores), but not by the membrane rupture.
Assuntos
Cálcio/metabolismo , Caspase 3/metabolismo , Caspase 7/metabolismo , Eletroporação , Pressão Osmótica , Animais , Células CHO , Bovinos , Cricetinae , Cricetulus , Humanos , Necrose/metabolismo , Células U937RESUMO
Community-acquired respiratory distress syndrome (CARDS) toxin from Mycoplasma pneumoniae is a 591-amino-acid virulence factor with ADP-ribosyltransferase (ADPRT) and vacuolating activities. It is expressed at low levels during in vitro growth and at high levels during colonization of the lung. Exposure of experimental animals to purified recombinant CARDS toxin alone is sufficient to recapitulate the cytopathology and inflammatory responses associated with M. pneumoniae infection in humans and animals. Here, by molecular modelling, serial truncations and site-directed mutagenesis, we show that the N-terminal region is essential for ADP-ribosylating activity. Also, by systematic truncation and limited proteolysis experiments we identified a portion of the C-terminal region that mediates toxin binding to mammalian cell surfaces and subsequent internalization. In addition, the C-terminal region alone induces vacuolization in a manner similar to full-length toxin. Together, these data suggest that CARDS toxin has a unique architecture with functionally separable N-terminal and C-terminal domains.
Assuntos
ADP Ribose Transferases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Mycoplasma pneumoniae , ADP Ribose Transferases/química , ADP Ribose Transferases/genética , Motivos de Aminoácidos , Animais , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Células HeLa , Humanos , Modelos Moleculares , NAD/metabolismo , Estrutura Terciária de Proteína , Proteólise , Fatores de Virulência/química , Fatores de Virulência/metabolismoRESUMO
Nanoelectroporation of biomembranes is an effect of high-voltage, nanosecond-duration electric pulses (nsEP). It occurs both in the plasma membrane and inside the cell, and nanoporated membranes are distinguished by ion-selective and potential-sensitive permeability. Here we report a novel phenomenon of bioeffects cancellation that puts nsEP cardinally apart from the conventional electroporation and electrostimulation by milli- and microsecond pulses. We compared the effects of 60- and 300-ns monopolar, nearly rectangular nsEP on intracellular Ca(2+) mobilization and cell survival with those of bipolar 60 + 60 and 300 + 300 ns pulses. For diverse endpoints, exposure conditions, pulse numbers (1-60), and amplitudes (15-60 kV/cm), the addition of the second phase cancelled the effects of the first phase. The overall effect of bipolar pulses was profoundly reduced, despite delivering twofold more energy. Cancellation also took place when two phases were separated into two independent nsEP of opposite polarities; it gradually tapered out as the interval between two nsEP increased, but was still present even at a 10-µs interval. The phenomenon of cancellation is unique for nsEP and has not been predicted by the equivalent circuit, transport lattice, and molecular dynamics models of electroporation. The existing paradigms of membrane permeabilization by nsEP will need to be modified. Here we discuss the possible involvement of the assisted membrane discharge, two-step oxidation of membrane phospholipids, and reverse transmembrane ion transport mechanisms. Cancellation impacts nsEP applications in cancer therapy, electrostimulation, and biotechnology, and provides new insights into effects of more complex waveforms, including pulsed electromagnetic emissions.
Assuntos
Polaridade Celular/fisiologia , Eletroporação , Nanotecnologia , Animais , Células CHO , Cálcio/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular , Cricetinae , Cricetulus , Humanos , Espécies Reativas de Oxigênio/metabolismo , Fatores de TempoRESUMO
Multiple studies have shown that bipolar (BP) electric pulses in the microsecond range are more effective at permeabilizing cells while maintaining similar cell survival rates as compared to monopolar (MP) pulse equivalents. In this paper, we investigated whether the same advantage existed for BP nanosecond-pulsed electric fields (nsPEF) as compared to MP nsPEF. To study permeabilization effectiveness, MP or BP pulses were delivered to single Chinese hamster ovary (CHO) cells and the response of three dyes, Calcium Green-1, propidium iodide (PI), and FM1-43, was measured by confocal microscopy. Results show that BP pulses were less effective at increasing intracellular calcium concentration or PI uptake and cause less membrane reorganization (FM1-43) than MP pulses. Twenty-four hour survival was measured in three cell lines (Jurkat, U937, CHO) and over ten times more BP pulses were required to induce death as compared to MP pulses of similar magnitude and duration. Flow cytometry analysis of CHO cells after exposure (at 15 min) revealed that to achieve positive FITC-Annexin V and PI expression, ten times more BP pulses were required than MP pulses. Overall, unlike longer pulse exposures, BP nsPEF exposures proved far less effective at both membrane permeabilization and cell killing than MP nsPEF.
Assuntos
Apoptose/efeitos da radiação , Permeabilidade da Membrana Celular/fisiologia , Permeabilidade da Membrana Celular/efeitos da radiação , Estimulação Elétrica/métodos , Eletroporação/métodos , Animais , Células CHO , Cricetinae , Cricetulus , Relação Dose-Resposta à Radiação , Campos Eletromagnéticos , Humanos , Células Jurkat , Doses de RadiaçãoRESUMO
Exceeding physiological limits of the cell membrane potential compromises structural integrity, enabling the passage of normally impermeant solutes and disrupting cell function. Electropermeabilization has been studied extensively at the cellular scale, but not at the individual membrane lesion level. We employed fast total internal reflection fluorescence (TIRF) imaging of Ca2+ entry transients to discern individual lesions in a hyperpolarized cell membrane and characterize their focality, thresholds, electrical conductance, and the lifecycle. A diffuse and momentary membrane permeabilization without a distinct pore formation was observed already at a -100 mV threshold. Polarizing down to -200 mV created focal pores with a low 50- to 300-pS conductance, which disappeared instantly once the hyperpolarization was removed. Charging to -240 mV created high-conductance (> 1 nS) pores which persisted for seconds even at zero membrane potential. With incremental hyperpolarization steps, persistent pores often emerged at locations different from those where the short-lived, low-conductance pores or diffuse permeabilization were previously observed. Attempts to polarize membrane beyond the threshold for the formation of persistent pores increased their conductance adaptively, preventing further potential build-up and "clamping" it at a certain limit (-270 ± 6 mV in HEK cells, -284 ± 5 mV in CHO cells, and -243 ± 9 mV in neurons). The data suggest a previously unknown role of electroporative lesions as a protective mechanism against a potentially fatal membrane overcharging and cell disintegration.
RESUMO
Cell permeabilization by electric pulses (EP), or electroporation, is widely used for intracellular delivery of drugs and plasmids, as well as for tumour and tissue ablation. We found that cells pre-treated with 100-µs EP develop delayed hypersensitivity to subsequent EP applications. Sensitizing B16 and CHO cells by splitting a single train of eight 100-µs EP into two trains of four EP each (with 5-min. interval) decreased the LD(50) 1.5-2 times. Sensitization profoundly enhanced the electroporation-assisted uptake of bleomycin, a cell-impermeable cytotoxic agent accepted for killing tumours by electrochemotherapy. EP exposures that were not lethal per se caused cell death in the presence of bleomycin and proportionally to its concentration. Sensitizing cells by a split-dose EP exposure increased bleomycin-mediated lethality to the same extent as a 10-fold increase in bleomycin concentration when using a single EP dose. Likewise, sensitization by a split-dose EP exposure (without changing the overall dose, pulse number, or amplitude) enhanced the electroporative uptake of propidium up to fivefold. Enhancement of the electroporative uptake appears a key mechanism of electrosensitization and may benefit electrochemotherapy and numerous applications that employ EP for cell permeabilization.
Assuntos
Bleomicina/metabolismo , Permeabilidade da Membrana Celular , Citotoxinas/metabolismo , Eletroporação/métodos , Propídio/metabolismo , Animais , Transporte Biológico , Bleomicina/farmacologia , Células CHO , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Citotoxinas/farmacologia , Melanoma Experimental , Camundongos , Propídio/farmacologiaRESUMO
The quest for safe and effective ablation resulted in the development of nanosecond pulsed electric fields (nsPEF) technology for tumor treatment. For future applications of nsPEF in urothelial cancer treatment, we evaluated the effect of urine presence at the ablation site. We prepared artificial urine (AU) with compounds commonly present in the healthy human urine at physiological concentrations. We compared nsPEF cytotoxicity for cancerous (T24) and non-cancerous (SV-HUC-1) human urothelial cell lines pulsed either in the AU or in a physiological solution (PS). Cell monolayers were exposed to trains of 300-ns, 10-Hz pulses using a two-needle electrode assembly placed orthogonal to the monolayer. The assembly produced the electric field gradually weakening with the distance from the electrodes. The electric field which killed 50 % of cells (LD50) was measured by staining with propidium iodide and matching the stained area with the simulated electric field strength. nsPEF exposure in PS was more cytotoxic to cancer cells. The AU protected both healthy and cancer urothelial cells, increasing their LD50 1.4 and 1.6 times, respectively. Omitting urea from the AU reduced the LD50 for healthy and cancer urothelial cells. Testing the role of other AU components, we found that it was the high concentration of phosphates what also rendered the protective effect of the AU. Our findings suggest that the nsPEF ablation of bladder cancer will be less efficient if the bladder is filled with urine.
Assuntos
Eletricidade , Humanos , Linhagem Celular , PropídioRESUMO
Intense nanosecond-duration electric pulses (nsEP) open stable nanopores in the cell membrane, followed by cell volume changes due to water uptake or expulsion, as regulated by the osmolality balance of pore-impermeable solutes inside and outside the cell. The size of pores opened by either fifty 60-ns EP (~13 kV/cm) or five, 600-ns EP (~6 kV/cm) in GH3 cells was estimated by isoosmotic replacement of bath NaCl with polyethylene glycols and sugars. Such replacement reduced cell swelling or resulted in transient or sustained cell shrinking in response to EP. depending on the availability of pores permeable to the test solute. Unexpectedly, solute substitutions showed that for the same integral area of pores opened by 60- and 600-ns treatments (as estimated by cell volume changes), the pore sizes were similar. However, the 600-ns exposure triggered significantly higher cell uptake of propidium. We concluded that 600-ns EP opened a greater number of larger (propidium-permeable pores), but the fraction of the larger pores in the entire pore population was insufficient to contribute to cell volume changes. For both the 60- and 600-ns exposures, cell volume changes were determined by pores smaller than 0.9 nm in diameter; however, the diameter increased with increasing the nsEP intensity.