Effect of organic N-halamines on selected membrane functions in intact Staphylococcus aureus cells.

Two N-halamine compounds, 3-chloro-4,4-dimethyl-2-oxazolidinone and 1,3-dichloro-4,4,5,5-tetramethyl-2-imidazolidinone, were compared with free chlorine as to their effects on selected membrane functions of intact Staphylococcus aureus cells. Free chlorine was found to cause a loss of permeability control, as measured by the efflux of potassium from the cells and a dramatic increase in hydrogen ion permeability, and to affect cell respiration in a nonreversible fashion, as measured by oxygen uptake. The two N-halamines were found to have very little effect on permeability to either potassium or hydrogen ions but were both found to dramatically inhibit respiration in a reversible manner. It is proposed that the first step in the disinfection process by these N-halamines is an inhibition of respiratory enzymes that, if not reversed, ultimately leads to a loss of cell viability.     

Effect of chlorine dioxide on selected membrane functions of Escherichia coli

The mode of action of chlorine dioxide on Escherichia coli was assessed by studying outer membrane permeability to macromolecules and potassium, and observing effects on respiration. The results indicate that gross cellular damage involving significant leakage of intracellular macromolecules does not occur. There was a substantial efflux of potassium, however, and respiration was inhibited even at sublethal doses. It was concluded that the inhibition of respiration, which could be due to the damage to the cell envelope, was not the primary lethal event. Observations of the efflux of K+ strongly implicate the loss of permeability control as the primary lethal event at the physiological level, with nonspecific oxidative damage to the outer membrane leading to the destruction of the trans-membrane ionic gradient.     

Effect of chlorine dioxide on selected membrane functions of Escherichia coli

The mode of action of chlorine dioxide on Escherichia coli was assessed by studying outer membrane permeability to macromolecules and potassium, and observing effects on respiration. The results indicate that gross cellular damage involving significant leakage of intracellular macromolecules does not occur. There was a substantial efflux of potassium, however, and respiration was inhibited even at sublethal doses. It was concluded that the inhibition of respiration, which could be due to the damage to the cell envelope, was not the primary lethal event. Observations of the efflux of K⁺ strongly implicate the loss of permeability control as the primary lethal event at the physiological level, with nonspecific oxidative damage to the outer membrane leading to the destruction of the trans-membrane ionic gradient.     

Effect of plasma membrane ion permeability modulators on respiration and heat output of wheat roots

A study was made of changes in the rates of respiration, heat production, and membrane characteristics in cells of excised roots of wheat seedlings under the modulation of plasma membrane ion permeability by two membrane active compounds: valinomycin (20 microM (V50)) and chlorpromazine (50 microM (CP50) and 100 microM (CP100)). Both compounds increased the loss of potassium ions, which correlated with the lowering of membrane potential, rate of respiration, and heat production after a 2 h exposure. The differences in alteration of these parameters were due to specific action of either compound on the membrane and to the extent of ion homeostasis disturbance. V20 had a weak effect on the studied parameters. V50 caused an increase of the rate of respiration and heat production, which enhanced following a prolonged action (5 h) and were associated with ion homeostatis restoration. The extent of alteration of membrane characteristics (an increase of potassium loss by roots, and lowering of cell membrane potential) as well as energy expense under the action of CP50 during the first period were more pronounced than in the presence of V50. During a prolonged action of CP50, the increase of respiration intensity and heat production correlated with partial recovery of ion homeostatis in cells. Essential lowering of membrane potential and substantial loss of potassium by cells, starting from the early stages of their response reaction, were followed by inhibition of respiration rate and heat production. Alterations of the structure and functional characteristics of excised root cells indicate the intensification of the membrane-tropic effect of a prolonged action of CP100, and the lack of cell energy resources.     

Changes in elemental content during apoptotic cell death studied by electron probe X-ray microanalysis

Recent data suggest that changes in ionic content, primarily potassium, play a pivotal role in the progression of apoptosis. However, the changes in total element content, i.e., sodium (Na), magnesium (Mg), phosphorous (P), chlorine (Cl), potassium (K), and calcium (Ca), during apoptosis have not been evaluated. Electron probe X-ray microanalysis (EPXMA) was used to measure total element content in U937 cells before and after the induction of apoptosis. As an experimental model we used U937 cells irradiated with ultraviolet (UV) light. Apoptosis was evaluated with phase-contrast microscopy, with scanning and transmission electron microscopy, and with the fluorescent dye bisbenzimide (Hoechst 33342). Plasma membrane permeability as a measure of cell death was determined by trypan blue dye exclusion. To investigate element content with EPXMA, cells were cryoprepared, i.e., cryofixed and freeze-dried, and analyzed as whole cells using a scanning electron microscope. We found that the UV irradiation induced rapid (within 2 h) morphological changes associated with apoptosis, such as plasma membrane blebbing, condensation of the chromatin, and the formation of membrane-bound apoptotic bodies. At this time, 95% of the apoptotic cells excluded trypan blue dye. EPXMA results demonstrated that UV light-irradiated apoptotic cells (cells with membrane-bound apoptotic bodies) had a lower Cl content (P < 0.001) and K content (P < 0.001) and a higher Na content (P < 0.001) in comparison with nonirradiated control cells. Also, P and Ca content was higher in apoptotic cells than in control cells, but this difference did not reach statistical significance. No differences were found in Mg. These data indicated that morphological changes characteristic of apoptotic cell death are related with significant changes in sodium, chlorine, and potassium content. In addition, we demonstrated that these changes in elemental composition were not associated with loss of cell membrane integrity.     

THE COMPOSITION OF THE CELL SAP OF THE PLANT IN RELATION TO THE ABSORPTION OF IONS

1. Chemical examination of the cell sap of Nitella showed that the concentrations of all the principal inorganic elements, K, SO₄, Ca, Mg, PO₄, Cl, and Na, were very much higher than in the water in which the plants were growing. 2. Conductivity measurements and other considerations lead to the conclusion that all or nearly all of the inorganic elements present in the cell sap exist in ionic state. 3. The insoluble or combined elements found in the cell wall or protoplasm included Ca, Mg, S, Si, Fe, and Al. No potassium was present in insoluble form. Calcium was predominant. 4. The hydrogen ion concentration of healthy cells was found to be approximately constant, at pH 5.2. This value was not changed even when the outside solution varied from pH 5.0 to 9.0. 5. The penetration of NO₃ ion into the cell sap from dilute solutions was definitely influenced by the hydrogen ion concentration of the solution. Penetration was much more rapid from a slightly acid solution than from an alkaline one. It is possible that the NO₃ forms a combination with some constituent of the cell wall or of the protoplasm. 6. The exosmosis of chlorine from Nitella cells was found to be a delicate test for injury or altered permeability. 7. Dilute solutions of ammonium salts caused the reaction of the cell sap to increase its pH value. This change was accompanied by injury and exosmosis of chlorine. 8. Apparently the penetration of ions into the cell may take place from a solution of low concentration into a solution of higher concentration. 9. Various comparisons with higher plants are drawn, with reference to buffer systems, solubility of potassium, removal of nitrate from solution, etc.     

An electrogenic NA+/K+ pump in the choroid plexus.

Intracellular electrical potential and potassium activity was measured by means of microelectrodes in the epithelial cells of choroid plexus from bullfrogs (Rana catesbeiana). Ouabain applied from the ventricular side caused an abrupt depolarisation of 10 mV but only a gradual loss of potassium from the cells. Readministration of potassium to the ventricular solution of plexuses which were previously depleted of potassium, caused a hyperpolarisation of about 4 mV. These two experiments are consistent with the notion of an electrogenic Na+/K+ pump situated at the ventricular membrane and which pumps potassium into the cell and sodium into the ventricle. The numerical values obtained suggest that 3 sodium ions are pumped for 2 potassium ions. The permeability coefficient for potassium exit from the cell is calculated to be 1.24 . 10(-5) cm-1 . s-1 expressed per cm2 of flat epithelium.     

Effect of phenoxyethanol on the permeability of Escherichia coli NCTC 5933 to inorganic ions.

Concentrations of phenoxyethanol which retarded the growth rate of Escherichia coli NCTC 5933 in nutrient broth, stimulated the rates of respiration and total oxygen uptake of cell suspensions with glucose as carbon source, and were able to dissipate artificially induced membrane proton gradients. In cells with repressed oxidative phosphorylating activity, no stimulation of respiration was observed. These actions were characteristic of uncouples of oxidative phosphorylation. Similar concentrations of the drug caused additional increased permeability of the cytoplasmic membrane to K+ but not to Li+, Na+, Ca++, Mg++, NO-3, Cl-, So--4, or PO---4. Drug induced permeability of the membrane to protons and potassium ions were not found to be directly coupled.     

Effects of the cationic protein poly-L-arginine on airway epithelial cells in vitro

BACKGROUND: Allergic asthma is associated with an increased number of eosinophils in the airway wall. Eosinophils secrete cationic proteins, particularly major basic protein (MBP). AIM: To investigate the effect of synthetic cationic polypeptides such as poly-L-arginine, which can mimic the effect of MBP, on airway epithelial cells. METHODS: Cultured airway epithelial cells were exposed to poly-L-arginine, and effects were determined by light and electron microscopy. RESULTS: Poly-L-arginine induced apoptosis and necrosis. Transmission electron microscopy showed mitochondrial damage and changes in the nucleus. The tight junctions were damaged, as evidenced by penetration of lanthanum. Scanning electron microscopy showed a damaged cell membrane with many pores. Microanalysis showed a significant decrease in the cellular content of magnesium, phosphorus, sodium, potassium and chlorine, and an increase in calcium. Plakoglobin immunoreactivity in the cell membrane was decreased, indicating a decrease in the number of desmosomes CONCLUSIONS: The results point to poly-L-arginine induced membrane damage, resulting in increased permeability, loss of cell-cell contacts and generalized cell damage.     

Chlorination and ozonation of waste-water: comparative analysis of efficacy through the effect on Escherichia coli membranes

The effect of chlorine and ozone on Escherichia coli cells resuspended in waste-water was compared. Selected chlorination and ozonation conditions produced a similar decrease in culturability (2-2.5 log). Under these conditions, differences in membrane permeability and cell surface hydrophobicity, depending on the disinfectant tested, were detected. After ozonation, while no changes in cell surface hydrophobicity were observed, approximately 95.5% of cells showed altered membrane permeability. The effect of chlorine was not linked to changes in membrane permeability. After chlorination, E. coli cells showed a tendancy to aggregate. The possibility that aggregation of cells could interfere with conventional colony counts is discussed. The degree of toxicity (Microtox assay) was unrelated to the effect on cellular activity.     

An electrogenic Na+/K+ pump in the choroid plexus

Intracellular electrical potential and potassium activity was measured by means of microelectrodes in the epithelial cells of choroid plexus from bullfrogs (Rana catesbeiana). Ouabain applied from the ventricular side caused an abrupt depolarisation of 10 mV but only a gradual loss of potassium from the cells. Readministration of potassium to the ventricular solution of plexuses which were previously depleted of potassium, caused a hyperpolarisation of about 4 mV. These two experiments are consistent with the notion of an electrogenic Na⁺/K⁺ pump situated at the ventricular membrane and which pumps potassium into the cell and sodium into the ventricle. The numerical values obtained suggest that 3 sodium ions are pumped for 2 potassium ions. The permeability coefficient for potassium exit from the cell is calculated to be 1.24 · 10^?5 cm^?1 · s^?1 expressed per cm² of flat epithelium.     

X-ray microanalysis of cockroach fat body in relation to ion and water regulation

X-Ray microanalysis of fat body from the cockroach (Periplaneta americana) showed that the crystals in the urate cells sequestered potassium and sodium during water deprivation. Postassium sequestration appeared to occur by the formation of additional crystals whereas sodium appeared to be taken up by pre-existing crystals of the “amorphous granule” type. Urate cells increased in cytoplasmic density i.e. mass per unit volume as a result of water deprivation and lost potassium from the cytoplasm but not from the nucleus. Trophocyte cells also showed an increase in density and a loss of potassium from the cytoplasm. The nucleus in trophocyte cells however, did not increase in density and showed an increase in chlorine concentration. A previously undescribed type of crystal in the trophocyte cell cytoplasm also increased markedly in chlorine concentration. The mycetocyte cells did not appear to increase in density but chlorine concentration was elevated in symbionts, cytoplasm and nucleus. An increase in density is considered to be a consequence of a reduction in volume due to water loss. It is confirmed that the fat body plays a major role in the maintenance of ion balance during water deprivation.     

Prehemolytic effects of hydrogen peroxide and t-butylhydroperoxide on selected red cell properties

To provide further understanding of how oxidative damage affects red cell membrane function, the effects of low levels of two different types of oxidants on selected red cell properties have been studied. Hydrogen peroxide (H2O2), an example of a water soluble oxidant, and t-butylhydroperoxide (tBHP), a hydrophobic hydroperoxide, were compared with respect to their effects on membrane permeability, membrane mechanical properties and binding of autologous serum antibodies to the cell surface. Whereas H2O2 treatment resulted in a dose-dependent increase in membrane permeability to potassium that was evident after one hour of oxidant exposure, cells treated with tBHP at doses up to 5 mumol/ml cells showed no immediate change in cation permeability. H2O2 also caused a marked decrease in membrane deformability, whereas tBHP-treated cells showed minimal loss of deformability. However, tBHP treatment did result in a dose-dependent increase in the susceptibility of the membrane to fragmentation under high shear stress. With exclusion of treated samples that bound excess rabbit anti-spectrin antibody, indicating exposure of intracellular components, neither agent promoted the binding of autologous serum antibody in amounts comparable to that found in vivo on high density or some pathologic red cells. Taken together, the results suggest that tBHP and H2O2 cause damage to human red cells by distinct oxidative mechanisms which do not lead directly to substantive generation of binding sites for autologous serum antibodies.     

Evidence Relating Cessation of Respiration, Cell Envelope Changes, and Death in Ultraviolet-Irradiated Escherichia coli B/r Cells

Ionic and nonionic detergents have little effect on respiring bacteria, but in cultures poisoned with KCN rapid solubilization of the cell membrane, as indicated by turbidity losses, takes place. Ultraviolet radiations cause Escherichia coli cells grown in minimal medium with glycerol as a carbon source to cease respiring and growing about 1 h after irradiation. We tested the effect of the nonionic detergent Triton X-100 on growth and cell membrane dissolution (both measured by turbidity changes), respiration, and viability of unirradiated and irradiated E. coli B/r cells. When the detergent was added to cells immediately after irradiation, a decrease in turbidity occurred only when respiration was about to cease; when it was added after cessation of respiration, the turbidity loss was immediate. In both cases the turbidity loss was about 60%, and disintegration of the cell walls did not take place. 5-Fluorouracil (FU) and thermal (42 C) treatments cause respiration of irradiated cells to be maintained and also cause viability increases. Irradiated cells treated with FU and detergent show no turbidity loss just prior to the time respiration normally ceases, but a loss does occur in irradiated cells incubated with detergent at 42 C. We conclude that FU maintains respiration for all of the cells, but that thermal treatment maintains respiration for only part of the cells. In all cases the detergent had only a negligible effect on the respiration and viability of unirradiated and irradiated cells. We conclude that Triton X-100 causes solubilization of cell membranes of only nonrespiring cells that are not destined to survive.     

Role of AQP2 during apoptosis in cortical collecting duct cells

Background information. A major hallmark of apoptosis is cell shrinkage, termed apoptotic volume decrease, due to the cellular outflow of potassium and chloride ions, followed by osmotically obliged water. In many cells, the ionic pathways triggered during the apoptotic volume decrease may be similar to that observed during a regulatory volume decrease response under hypotonic conditions. However, the pathways involved in water loss during apoptosis have been largely ignored. It was recently reported that in some systems this water movement is mediated via specific water channels (aquaporins). Nevertheless, it is important to identify whether this is a ubiquitous aspect of apoptosis as well as to define the mechanisms involved. The aim of the present work was to investigate the role of aquaporin‐2 during apoptosis in renal‐collecting duct cells. We evaluated the putative relationship between aquaporin‐2 expression and the activation of the ionic pathways involved in the regulatory volume response. Results. Apoptosis was induced by incubating cells with a hypertonic solution or with cycloheximide in two cortical collecting duct cell lines: one not expressing aquaporins and the other stably transfected with aquaporin‐2. Typical features of apoptosis were evaluated with different approaches and the water permeability was measured by fluorescence videomicroscopy. Our results show that the rate of apoptosis is significantly increased in aquaporin‐2 cells and it is linked to the rapid activation of volume‐regulatory potassium and chloride channels. Furthermore, the water permeability of cells expressing aquaporin‐2 was strongly reduced during the apoptotic process and it occurs before DNA degradation. Conclusions. These results let us propose that under apoptotic stimulation aquaporin‐2 would act as a sensor leading to a co‐ordinated activation of specific ionic channels for potassium and chloride efflux, resulting in both more rapid cell shrinkage and more rapid achievement of adequate levels of ions necessary to activate the enzymatic apoptotic cascade.     

The effects of cells on oxygen transfer in bioreactors

Cells may affect oxygen transfer rates by three mechanisms: respiration of cells accumulated at the gas/liquid interface, physical presence of cells as solid particles, and modification of the medium by cells. These effects were studied experimentally in bubble-aerated bioreactors using baker's yeast at different cell concentrations, agitation speeds, aeration rates, and specific oxygen uptake rates. The overall effect of cells was to enhance oxygen transfer rates. The physical presence of cells as solid particles was found to retard oxygen transfer, presumably due to the lower oxygen permeability in the cell layer accumulated near the bubble surfaces. Cell respiration and medium modification, on the other hand, enhanced oxygen transfer rates. The retardation by nonrespiring cells and the enhancement due to cell respiration were found stronger at higher agitation speeds and lower aeration rates employed. This was attributed to the higher interfacial cell accumulation associated with the smaller bubbles produced under these conditions in the systems studied.     

Interaction of dichlone with human erythrocytes. I. Changes in cell permeability

The uptake of the fungicide dichlone (2,3-dichloro-1,4-naphthoquinone) by human erythrocytes was extremely rapid, reaching a maximum within 5 min of treatment. Most of the dichlone taken up was present in the interior of the cell; only a small fraction of the pesticide (less than 5%) was bound to the cell membrane. Dichlone (3 · 10^?5M-10^?4M) induced a rapid loss of intracellular potassium from the erythrocytes; the leakage of K⁺ varied with the fungicide concentration as well as with cell concentration. Pretreatment of the cells with glutathione was able to reduce potassium loss. Cells exposed to dichlone showed increased osmotic fragility. Dichlone also inhibited Na⁺-K⁺ ATPase, which is associated with active ion transport. However, the leakage of potassium in dichlone-treated cells does not appear to be related to the interference with active ion transport. An extensive loss of potassium within a relatively short time after treatment suggests that dichlone produces its effect by increasing passive cation permeability, probably as a result of direct action on the membrane structure. Dichlone was able to induce hemolysis, but only at concentrations higher than those which resulted in K⁺ loss. The loss of hemoglobin appeared to be mainly due to osmotic swelling of the treated cells. Exposure of red cells to dichlone also resulted in a rapid and extensive formation of methemoglobin as well as a denaturation of hemoglobin. Thus, dichlone not only may be capable of lowering the capacity of erythrocytes to transport oxygen but also alters their permeability.     

The action of gamma-ray-irradiated medium on bacteria: relation to the electron transport system

Gamma-ray-irradiated medium is found to produce an immediate slowing of respiration in cells of Escherichia coli 15 T-L-. It also introduces a lag in cell division that can be shortened by the presence of methylene blue and potassium cyanide. The lag is absent if the cells are grown anaerobically. These results suggest that the peroxides produced by irradiating medium block the electron transport system. Since cells concentrate peroxides, the loss of this function induced by bombardment was used to deduce the target molecular weight of the enzyme responsible. It is 73,000.     

The diversity of sulfhydryl groups in the human erythrocyte membrane

Human bank blood erythrocytes were exposed to the mercurials p-chloromercuribenzoate (PCMB), chlormerodrin (CM), p-chloromercuribenzenesulfonate (PCMBS), and 1-bromomercuri-2-hydroxypropane (BMHP) for different time intervals, at different concentrations and in combination with n-ethylmaleimide (NEM) added before, and 2-mercaptoethylguanidine (MEG) and reduced glutathione (GSH) added after the mercurial. Binding patterns of the mercurials to the cells and effects on permeability of the cells were measured. The results indicate that the erythrocyte membrane contains multiple classes of sulfhydryl groups, alteration of which has a variety of effects on cell permeability. PCMB, chlormerodrin and PCMBS react with at least three classes of sulfhydryls, two of which are associated with the sodium-potassium barrier and, when altered, result in potassium loss, sodium accumulation and hemolysis. BMHP reacts with at least two classes of sulfhydryls, one of which is associated with permeability, and, when altered, results in hemolysis in isotonic solutions of choline chloride or lactose. The results provide additional insight into the structure and function of the erythrocyte membrane.     

Divalent cation chelators citrate and EDTA unmask an intrinsic uncoupling pathway in isolated mitochondria

We demonstrate a suppression of ROS production and uncoupling of mitochondria by exogenous citrate in Mg²⁺ free medium. Exogenous citrate suppressed H₂O₂ emission and depolarized mitochondria. The depolarization was paralleled by the stimulation of respiration of mitochondria. The uncoupling action of citrate was independent of the presence of sodium, potassium, or chlorine ions, and it was not mediated by the changes in permeability of the inner mitochondrial membrane to solutes. The citrate transporter was not involved in the citrate effect. Inhibitory analysis data indicated that several well described mitochondria carriers and channels (ATPase, IMAC, ADP/ATP translocase, mPTP, mKATP) were not involved in citrate’s effect. Exogenous MgCl₂ strongly inhibited citrate-induced depolarization. The uncoupling effect of citrate was demonstrated in rat brain, mouse brain, mouse liver, and human melanoma cells mitochondria. We interpreted the data as an evidence to the existence of a hitherto undescribed putative inner mitochondrial membrane channel that is regulated by extramitochondrial Mg²⁺ or other divalent cations.