Marker Of Membrane Integrity Research Articles

Validation of Calcein Violet as a New Marker of Semen Membrane Integrity in Domestic Animals.

Many fluorochromes routinely used in semen quality analysis emit in the green and red channels, limiting their possible combination for multiple parameter analysis. The use of fluorophores emitting in different light channels broadens the possibilities of combination to expand the range of simultaneously evaluated criteria. This is of great interest in cases of small ejaculated volumes, such as those naturally occurring in roosters, small dog breeds and drones (Apis mellifera). The purpose of this experiment is to establish Calcein Violet (CaV), a blue fluorochrome, as a marker of viability and acrosomal integrity in domestic animals in order to free the red and green channels. SYBR®14/Propidium Iodide (PI) was used as reference dye, heat-treated samples as negative controls, serial staining combination for validation and epifluorescence microscopy for observation. Dead spermatozoa marked in red with PI showed no blue fluorescence either from the head or the tail. Live spermatozoa showed a decreasing blue emission from head to tail when single stained with CaV. Unreacted acrosomes showed intense blue fluorescence irrespective of plasma membrane integrity. This needs to be further confirmed for species with small and difficult to observe heads. Establishment of CaV as a marker of membrane integrity by fluorescence microscopy is a decisive first step towards further technical development and use with flow cytometry.

Comprehensive evaluation of mitochondrial redox profile, calcium dynamics, membrane integrity and apoptosis markers in a preclinical model of severe penetrating traumatic brain injury.

Traumatic Brain Injury (TBI) is caused by the external physical assaults damages the brain. It is a heterogeneous disorder that remains a leading cause of death and disability in the military and civilian population of the United States. Preclinical investigations of mitochondrial responses in TBI have ascertained that mitochondrial dysfunction is an acute indicator of cellular damage and plays a pivotal role in long-term injury progression through cellular excitotoxicity. The current study was designed to provide an in-depth evaluation of mitochondrial endpoints with respect to redox and calcium homeostasis, and cell death responses following penetrating TBI (PTBI). To evaluate these pathological cascades, anesthetized adult male rats (N=6/group) were subjected to either 10% unilateral PTBI or Sham craniectomy. Animals were euthanized at 24h post-PTBI, and purified mitochondrial fractions were isolated from the brain injury core and perilesional areas. Overall, increased reactive oxygen and nitrogen species (ROS/RNS) production, and elevated oxidative stress markers such as 4-hydroxynonenal (4-HNE), 3-nitrotyrosine (3-NT), and protein carbonyls (PC) were observed in the PTBI group compared to Sham. Mitochondrial antioxidants such as glutathione, peroxiredoxin (PRX-3), thioredoxin (TRX), nicotinamide adenine dinucleotide phosphate (NADPH), superoxide dismutase (SOD), and catalase (CAT) levels were significantly decreased after PTBI. Likewise, PTBI mitochondria displayed significant loss of Ca2+ homeostasis, early opening of mitochondrial permeability transition pore (mPTP), and increased mitochondrial swelling. Both, outer and inner mitochondrial membrane integrity markers, such as voltage-dependent anion channels (VDAC) and cytochrome c (Cyt C) expression were significantly decreased following PTBI. The apoptotic cell death was evidenced by significantly decreased B-cell lymphoma-2 (Bcl-2) and increased glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression after PTBI. Collectively, current results highlight the comprehensive picture of mitochondria-centric acute pathophysiological responses following PTBI, which may be utilized as novel prognostic indicators of disease progression and theragnostic indicators for evaluating neuroprotection therapeutics following TBI.

Abstract 12070: Extracellular Vesicles, Derived Early After Myocardial Infarction, Inhibits Staurosporin Induced Cardiac Cell Death

Introduction: Adverse ventricular remodeling may occur after myocardial infarction (MI) and is associated with an increased risk of heart failure and death. Extracellular vesicles (EVs) derived exogenously from pluripotent stem cells exert cardioprotective effects after injury and being tested for therapeutic efficacy for MI. However role of endogenous EVs from MI patients in this regard is not yet addressed. Hypothesis: We aim to characterize EVs derived from MI patients and explore their cardioprotective potential on human primary cardiomyocytes (HCM) upon chemically induced cell death. Methods: The EVs from plasma of MI patients with preserved or reduced left ventricular ejection fraction (LVEF) and healthy controls (HC) were purified and characterized by flow cytometry and transmission electron microscopy (TEM). HCM and human cardiac microvascular endothelial cells (hCMVECs) were used to study functional effects of EVs upon staurosporin (STS), etoposide and rapamycin induced cell death. Results: Higher concentrations of platelet-, leukocyte-, endothelial- and erythrocyte-derived EVs were found in MI patients compared to HC. TEM analysis demonstrated the presence of heterogenous vesicles of different sizes (50-1000-nm) across all tested samples. Human cardiac cells- hCMVECs and HCM when pre-incubated with both preserved and reduced LVEF MI EVs showed significant attenuation to STS-induced caspase 3 activity compared to HC EVs or mock treatment. Lactate dehydrogenase (LDH) release, as a marker for membrane integrity, upon etoposide stimulation was not affected in hCMVECs and partly increased in HCM, whereas pre-incubation with HC EVs or MI EVs did not affect eotoposide treatment outcome by these cells. While rapamycin induced cleavage of LC3-I to LC3-II in hCMVECs, the presence of EVs from either HC or MI patients did not influence autophagic response. Whereas, baseline LC3-II expression in HCM was observed to be too high to be perturbed by the presence of rapamycin. Conclusions: Diverse nature of MI EVs in terms of cells of origin can be further elaborated to consolidate their biomarker potential. Cardioprotective ability of endogenously presented heterogenous mixture of MI EVs opens new avenues to consider in preclinical EV/MI research.

Evaluation of a Product Containing Xyloglucan and Pea Protein on Skin Barrier Permeability

Objective: The skin acts as a mechanical and protective barrier against viral, fungal, and bacterial infections. Skin conditions such as atopic dermatitis and psoriasis are characterized by alterations of the skin barrier, often caused by injury and by bacterial infections. In the last years, non-pharmacological interventions have gained great importance in epidermis-related diseases. Xyloglucan (XG) is a polysaccharide that possesses a “mucin-like” molecular structure that confers mucoadhesive properties, allowing XG-containing formulations to act as a protective barrier for the management of different diseases. Moreover, there is also increasing interest in the use of proteins due to their film-forming features. This study aimed to evaluate the barrier-protective properties of a product containing XG and pea protein (PP) in an in vitro model, assessing its effects on the membrane permeability of keratinocytes infected by Staphylococcus aureus. Methods: HaCaT keratinocytes were pretreated with XG and PP for 3 h and then infected with S. aureus cells (10⁶ bacteria/well) at a multiplicity of infection of 10 for 1 h. The number of bacterial colonies and membrane integrity were measured, respectively. Results: We observed that pretreatment with XG and PP in human HaCaT keratinocytes infected with S. aureus significantly increased trans-epithelial electrical resistance (a marker of skin barrier function) measurement, reduced lucifer yellow (a marker of membrane integrity) permeation across the monolayer, and released lactate dehydrogenase (a marker of tissue damage). Moreover, XG and PP pretreatment was able to reduce bacterial adherence, avoiding S. aureus infection. Conclusion: In summary, we demonstrated that the product containing XG and PP was able to maintain barrier permeability preserving its integrity, and therefore, it can be considered as an interesting approach for the management of epidermis-related diseases.

P0821OBESITY, FAT TISSUE, CELL MEMBRANE INTEGRITY PARAMETERS AND ARTERIAL STIFFNESS IN RENAL TRANSPLANT RECIPIENTS

Abstract Background and Aims Arterial stiffness, altered body composition (increased body fat mass and decreased soft lean mass) and cell membrane integrity (Phase Angle) are acknowledged risk factors for adverse outcomes after kidney transplantation related to cardiovascular diseases. Phase angle is a linear method of measuring the relationship between electric resistance and reactance. Taking the arc tangent value of the ratio of reactance versus electric resistance provides us with the phase angle value. Lower phase angles appear to be consistent with low reactance and equals either cell death or a breakdown in the selective permeability of the cell membrane. The aim of the study was the assessment of the relationship between arterial stiffness and fat tissue parameters, cell membrane integrity marker in renal transplants recipients (RTRs). Method 344 stable RTRs with the mean age 52.7 years, (62.5% M) transplanted between 1994 and 2018 were randomly enrolled to the study. The following parameters of arterial stiffness were measured: brachial-ankle and carotid-femoral pulse wave velocities (baPWV left and right, cfPWV). The obesity and fat tissue (body mass index (BMI), waist to hip ratio (WHR), body fat mass (BFM), fat free mass (FFM), percent fat of the body (PBF), trunk segmental fat analysis (TSFA) and visceral fat area (VFA)) parameters and cell membrane integrity phase angle (PhA) were assessed with InBody 170. Results The median time of dialysis and after kidney transplantation was 58.5 and 78 months respectively. The obesity according to BMI, WHR and VFA was diagnosed in 49.7%, 45% and 44.5% of patients, respectively. The median value of BFM, FFM, VFA, TSFA, PhA and the mean value of PBF were 19.3kg, 55kg, 93.2cm2, 24.9 kg, 4.9 and 27.3%, respectively. We found significant positive correlations between WHR, VFA and baPWV right, baPWV left and cfPWV. PhA correlated with arterial stiffness in univariate (r=-0.34) and multivariate analysis (R=-0.23), negatively (p<0.05). Conclusion 1. Obesity, visceral fat tissue, cell membrane integrity marker influence on arterial stiffness. 2. The analysis of magnitude of obesity, body fat tissue and cell membrane integrity parameters can be used as an additional cardiovascular risk factor in RTRs. 3. The cell membrane integrity impairment correlated with arterial stiffness.

Mechanically Induced Bacterial Death Imaged in Real Time: A Simultaneous Nanoindentation and Fluorescence Microscopy Study.

Mechano-bactericidal nanomaterials rely on their mechanical or physical interactions with bacteria and are promising antimicrobial strategies that overcome bacterial resistance. However, the real effect of mechanical versus chemical action on their activity is under debate. In this paper, we quantify the forces necessary to produce critical damage to the bacterial cell wall by performing simultaneous nanoindentation and fluorescence imaging of single bacterial cells. Our experimental setup allows puncturing the cell wall of an immobilized bacterium with the tip of an atomic force microscope (AFM) and following in real time the increase in the fluorescence signal from a cell membrane integrity marker. We correlate the forces exerted by the AFM tip with the fluorescence dynamics for tens of cells, and we find that forces above 20 nN are necessary to exert critical damage. Moreover, a similar experiment is performed in which bacterial viability is assessed through physiological activity, in order to gain a more complete view of the effect of mechanical forces on bacteria. Our results contribute to the quantitative understanding of the interaction between bacteria and nanomaterials.

Suitability of Artificial Membranes in Lipolysis-Permeation Assays of Oral Lipid-Based Formulations

PurposeTo evaluate the performance of artificial membranes in in vitro lipolysis-permeation assays useful for absorption studies of drugs loaded in lipid-based formulations (LBFs).MethodsPolycarbonate as well as PVDF filters were treated with hexadecane, or lecithin in n-dodecane solution (LiDo) to form artificial membranes. They were thereafter used as absorption membranes separating two compartments mimicking the luminal and serosal side of the intestine in vitro. Membranes were subjected to dispersions of an LBF that had been digested by porcine pancreatin and spiked with the membrane integrity marker Lucifer Yellow (LY). Three fenofibrate-loaded LBFs were used to explore the in vivo relevance of the assay.ResultsOf the explored artificial membranes, only LiDo applied to PVDF was compatible with lipolysis by porcine pancreatin. Formulation ranking based on mass transfer in the LiDo model exposed was the same as drug release in single-compartment lipolysis. Ranking based on observed apparent permeability coefficients of fenofibrate with different LBFs were the same as those obtained in a cell-based model.ConclusionsThe LiDo membrane was able to withstand lipolysis for a sufficient assay period. However, the assay with porcine pancreatin as digestive agent did not predict the in vivo ranking of the assayed formulations better than existing methods. Comparison with a Caco-2 based assay method nonetheless indicates that the in vitro in vivo relationship of this cell-free model could be improved with alternative digestive agents.

Polymorphonuclear neutrophil integrity and functionality are preserved when exposed to saliva

ObjectivePolymorphonuclear neutrophils (PMNs) are the most abundant innate immune cells and are also important effectors in the maintenance of oral health. However, little is known about the effects of saliva on the PMN. We therefore aimed to investigate the effect of saliva on the PMNs’ morphology and functioning. DesignEffect of saliva on the membrane integrity of PMNs isolated from blood was evaluated with FACS using Annexin V (apoptosis marker) and propidum iodide (membrane integrity marker). The effect on cell morphology was examined using transmission electron imaging. Binding and phagocytosis of the oral bacterium Fusobacterium nucleatum by PMNs was analysed by FACS. Reactive oxygen species (ROS) production was measured with chemiluminescence. ResultsIncubation with saliva for 60 min had no detectable effects on the membrane integrity or the morphology of PMNs. In contrast, preincubation of F. nucleatum with saliva inhibited its subsequent interaction with PMNs, resulting in a diminished production of ROS. ConclusionsSaliva does not impair the function of PMNs. However, interaction of salivary components with F. nucleatum may affect their recognition by PMNs resulting in a diminished functional response.

Antiproliferative and Apoptotic Potential of Cyanidin-Based Anthocyanins on Melanoma Cells.

Elderberries are known for their high anthocyanins content, which have been shown to possess anti-proliferative and anti-cancer effects. Anthocyanins enriched extract (AEE) was obtained from elderberries and was characterized by LC/DAD/ESI-MS analysis. Five cyanidin-based anthocyanins were identified, among which Cy-3-O-samb was the major compound (51%). The total anthocyanins content of AEE was 495 mg Cy-3-O-samb/100 g FW. AEE inhibited proliferation of metastatic B16-F10 murine melanoma cells, in a concentration-dependent manner, with an IC50 of 264.3 μg/mL. LDH (lactate dehydrogenase), as a marker of membrane integrity, increased 74% in B16-F10 cells treated with 250 μg/mL AEE, compared to control. It was observed that apoptosis is the mechanism of melanoma cell death after AEE treatment, confirmed morphologically by acridine orange/ethidium bromide double staining and TUNEL analysis. These results indicate that elderberry-derived anthocyanins might be utilized in future applications as topical adjuvant in skin cancer therapy.

Nutritional management of search and rescue dogs.

Dogs used for search and rescue (SAR) may experience continuous micro-traumas that predispose them to skeletal disorders. The aim of the present study was to evaluate the effect of diet on osteo-articular apparatus in healthy SAR dogs. A total of sixteen SAR dogs were divided into two groups (low supplementation (LS) and high supplementation (HS)) and were fed for 3 months with two experimental diets, characterised by the same protein and energy density, but different in n-3 PUFA (6·2 v. 8·4 % of metabolisable energy), chondroitin sulfate (219·8 v. 989·0 mg/kg DM) and glucosamine (769·2 v. 1318·7 mg/kg DM) in the LS and HS groups, respectively. At recruitment all dogs showed no joint inflammation signs, except four that showed mild symptoms. Haematology and serum biochemistry were performed every 30 d. Joint status was scored by physical and lameness evaluations. The sampling effect analysis showed potential beneficial effects by a decrease in a specific marker of membrane integrity (creatine kinase; CK). Comparing groups, glucose was significantly higher and CK was significantly lower in the HS group; however, in both cases the levels of these parameters fell in the normal range. At the end of the experiment, erythrocytes, Hb and packed cell volume were significantly higher in the HS compared with the LS group. These could result in an improvement in dogs' performance, even if this aspect was not investigated in the present study. Concerning joint evaluation (pain on manipulation, lameness and range of motion), no statistically significant differences were detected between the groups and within the experimental period.

Electroporation of mammalian cells by nanosecond electric field oscillations and its inhibition by the electric field reversal.

The present study compared electroporation efficiency of bipolar and unipolar nanosecond electric field oscillations (NEFO). Bipolar NEFO was a damped sine wave with 140 ns first phase duration at 50% height; the peak amplitude of phases 2–4 decreased to 35%, 12%, and 7% of the first phase. This waveform was rectified to produce unipolar NEFO by cutting off phases 2 and 4. Membrane permeabilization was quantified in CHO and GH3 cells by uptake of a membrane integrity marker dye YO-PRO-1 (YP) and by the membrane conductance increase measured by patch clamp. For treatments with 1–20 unipolar NEFO, at 9.6–24 kV/cm, 10 Hz, the rate and amount of YP uptake were consistently 2-3-fold higher than after bipolar NEFO treatments, despite delivering less energy. However, the threshold amplitude was about 7 kV/cm for both NEFO waveforms. A single 14.4 kV/cm unipolar NEFO caused a 1.5–2 times greater increase in membrane conductance (p < 0.05) than bipolar NEFO, along with a longer and less frequent recovery. The lower efficiency of bipolar NEFO was preserved in Ca2+-free conditions and thus cannot be explained by the reversal of electrophoretic flows of Ca2+. Instead, the data indicate that the electric field polarity reversals reduced the pore yield.

Multiple nanosecond electric pulses increase the number but not the size of long-lived nanopores in the cell membrane

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.

Quantifying Molecular Transport through Lipid Electropores Induced by Nanosecond Pulsed Electric Fields

High amplitude, nanosecond-duration, pulsed electric fields (nsPEFs) induce the formation of nanometer-diameter pores in cell membranes. These transient lipid electropores facilitate the transport of normally impermeant ions and small molecules across the membrane. Quantitative measurements of molecular transport through lipid electropores are essential for the validation of electroporation models and for optimizing the nsPEF dose for application-specific responses. We calibrated the fluorescence intensity of the membrane integrity marker dye YO-PRO-1 in dense cell lysates using a fast confocal microscope in order to quantify the time-dependent molecular influx of YO-PRO-1 into living cells after nsPEF exposure. Using this method we were able to measure the number of YO-PRO-1 molecules that enter U-937 human histiocytic lymphoma cells following electropermeabilization by a single 6 ns, 20 MV/m pulse.

Effect of Ichnocarpus frutescens (L.) R. Br. hexane extract on preadipocytes viability and lipid accumulation in 3T3-L1 cells

ObjectiveTo investigate the crude extracts of Ichnocarpus frutescens (I. frutescens) for antiobesity effect. MethodsLeaves of I. frutescens were sequentially extracted with hexane, ethyl acetate, and methanol and their effect on viability of 3T3-L1 preadipocytes were evaluated. Based on this the apoptosis on preadipocytes was confirmed by DNA fragmentation and LDH (Lactate dehydrogenase) leakage assays. Anti-adipogenesis was performed by oil red O (ORO) staining and free glycerol release in the medium of differentiated adipocytes. ResultsThe hexane extract of I. frutescens (IFHE) inhibited cell viability in a time- and dose-related manner. An increased release of LDH, as a marker of membrane integrity, was observed at a dose of 200 μ g/mL. The discontinuous DNA fragments on agarose gel electrophoresis showed the apoptotic effect of the IFHE. Morphological observations of cells stained with ORO showed a decrease in cellular lipid content at the concentrations tested compared to the induced control cells. In the experiment of lipolytic activity, treatment with IFHE enhanced glycerol secretion with the rates of approximately 28%, 55%, and 46% at the concentrations of 100, 200 and 300 μ g/mL, respectively. ConclusionsThe observed properties clearly revealed the medicinal property of I. frutescens in the treatment of obesity.

Dielectric evolution of mammalian cell membranes after exposure to pulsed electric fields

We have investigated the temporal evolution of the dielectric properties of Jurkat cells, a malignant human T-cell line, after exposure to microsecond and nanosecond pulsed electric fields. Suspensions with a 10% volume fraction of Jurkat cells were exposed to either 8 consecutive 1.1 kV/cm electroporation pulses of 100 μs, or 8 consecutive 20-kV/cm pulses of 300 ns, applied with a repetition rate of about 1 Hz. The dielectric spectrum of the cell suspensions before and after exposure was determined by means of time domain reflectometry dielectric spectroscopy. After correction for electrode polarization, the conductivity and permittivity of the cell membrane was derived by fitting the suspension spectrum to a combination of Maxwell-Wagner mixture model and single-shell cell model. Both exposure regimens lead to an increase in the conductivities of suspending medium and cell membrane indicating that membrane poration had occurred. This was confirmed by membrane integrity markers. Significantly higher membrane conductivities without uptake of membrane integrity marker suggest different pore formation mechanisms. Accordingly, dielectric measurements allow us to assess conformational and possibly induced functional changes of cells after exposure on biologically relevant time scales, and in turn, evaluate and compare the efficacy of chosen pulse parameters.

Measurement of Cell Death in Mammalian Cells

Methods for assessing mammalian cell death are presented in this unit. The unit is divided into six sections: (1) a brief overview of cytotoxicity and pathways of cell death, (2) a method to measure cell death using lactate dehydrogenase (LDH) release as a marker of membrane integrity, (3) a flow cytometry method that simultaneously measures two types of cell death, necrosis, and apoptosis, (4) use of fluorescence microscopy and nuclear morphology to assess apoptosis and necrosis, (5) the use of multi-well plates and high-content analysis imaging systems to assess nuclear morphology, and (6) a discussion of the use of cytotoxicity assays to determine the mechanisms of cell death.

Simultaneous quantification of d- vs. l-serine, taurine, kynurenate, phosphoethanolamine and diverse amino acids in frontocortical dialysates of freely-moving rats: Differential modulation by N-methyl- d-aspartate (NMDA) and other pharmacological agents

This study describes a novel analytical method permitting simultaneous HPLC-fluorimetric quantification of multiple (15) d- and l-amino acids, kynurenate, taurine and phosphoethanolamine (a marker of membrane integrity) in microdialysates of prefrontal cortex of freely-moving rats. Levels of GABA were elevated by the transporter inhibitor, nipecotic acid, and by the transaminase inhibitor, vigabatrine. Supporting a neuronal origin, they were decreased by the GABA B autoreceptor agonist, baclofen, yet unaffected by fluoroacetate which disrupts glial metabolism. Glutamate levels were elevated by the transporter inhibitor, l-trans-PDC, and mainly neuronal since they were not decreased by fluoroacetate, yet reduced by baclofen (which recruits GABA B receptors on glutamatergic terminals) and elevated by the NMDA receptor antagonist, dizocilpine. By contrast, levels of glutamine were reduced by l-trans-PDC. Consistent with glial origin, they were unaffected by baclofen, yet reduced by fluoroacetate. Administration of d-serine selectively increased its levels over l-serine, and vice versa. d-serine modestly decreased levels of glycine, which were enhanced by administration of glycine itself and of the glycine transporter-1 inhibitor, sarcosine. Kynurenate levels were increased by its precursor, kynurenine, an effect abolished by the amino-transferase inhibitor, amino-oxyacetate. Taurine and the energy drink, Red Bull ®, selectively elevated levels of taurine, which were only slightly reduced by fluoroacetate. Finally, administration of NMDA increased levels of taurine, kynenurate and phosphoethanolamine, while reducing d-serine. These actions were abolished by the competitive NMDA receptor antagonist, CPP, which was inactive alone. This broad-based dialysis system should prove instructive for exploring actions of psychotropic drugs, and for characterising animal models of CNS disorders.

Membrane surface charge dictates the structure and function of the epithelial Na+/H+exchanger

The Na(+)/H(+) exchanger NHE3 plays a central role in intravascular volume and acid-base homeostasis. Ion exchange activity is conferred by its transmembrane domain, while regulation of the rate of transport by a variety of stimuli is dependent on its cytosolic C-terminal region. Liposome- and cell-based assays employing synthetic or recombinant segments of the cytosolic tail demonstrated preferential association with anionic membranes, which was abrogated by perturbations that interfere with electrostatic interactions. Resonance energy transfer measurements indicated that segments of the C-terminal domain approach the bilayer. In intact cells, neutralization of basic residues in the cytosolic tail by mutagenesis or disruption of electrostatic interactions inhibited Na(+)/H(+) exchange activity. An electrostatic switch model is proposed to account for multiple aspects of the regulation of NHE3 activity.

Acute Depression of Energy Metabolism after Microdialysis Probe Implantation is Distinct from Ischemia-Induced Changes in Mouse Brain

The current study used measurements of metabolites and markers of membrane integrity to determine the most suitable time point for microdialysis experiments following probe implantation. Leakage of Evans blue and sodium fluorescein indicated increased BBB permeability only immediately (15 min), but not 1.5 and 24 h following probe implantation. Acute implantation decreased glucose and lactate levels relative to the levels after 24 h (to 13-37% and 25-60%, respectively). No change in extracellular levels of glutamate or glycerol was seen. In comparison to acute probe implantation, the pattern of damage under brain ischemia (middle cerebral artery occlusion) differed: While glucose levels dropped, lactate levels rose after ischemia, and glutamate (tenfold) and glycerol (eightfold) increased sharply. In conclusion, acute implantation of a microdialysis probe causes transient depression of the energy metabolites, glucose and lactate, likely due to injury-induced hypermetabolism. However, no massive tissue damage or severe ischemic conditions around the probe occur.

Effects of Perfluorocarbons on surfactant exocytosis and membrane properties in isolated alveolar type II cells

BackgroundPerfluorocarbons (PFC) are used to improve gas exchange in diseased lungs. PFC have been shown to affect various cell types. Thus, effects on alveolar type II (ATII) cells and surfactant metabolism can be expected, data, however, are controversial.ObjectiveThe study was performed to test two hypotheses: (I) the effects of PFC on surfactant exocytosis depend on their respective vapor pressures; (II) different pathways of surfactant exocytosis are affected differently by PFC.MethodsIsolated ATII cells were exposed to two PFC with different vapor pressures and spontaneous surfactant exocytosis was measured. Furthermore, surfactant exocytosis was stimulated by either ATP, PMA or Ionomycin. The effects of PFC on cell morphology, cellular viability, endocytosis, membrane permeability and fluidity were determined.ResultsThe spontaneous exocytosis was reduced by PFC, however, the ATP and PMA stimulated exocytosis was slightly increased by PFC with high vapor pressure. In contrast, Ionomycin-induced exocytosis was decreased by PFC with low vapor pressure. Cellular uptake of FM 1-43 - a marker of membrane integrity - was increased. However, membrane fluidity, endocytosis and viability were not affected by PFC incubation.ConclusionsWe conclude that PFC effects can be explained by modest, unspecific interactions with the plasma membrane rather than by specific interactions with intracellular targets.