Eduardo Fernández-Segura

Changes in intracellular sodium, chlorine, and potassium concentrations in staurosporine-induced apoptosis

Ion gradients across the plasma membrane, fundamentally K+, play a pivotal role in the execution phase of apoptosis. However, little is known about other monovalent anions (Cl−) or cations (Na+) in apoptosis. In addition, the relationship between changes in total ion composition and morphological and biochemical events are poorly understood. We investigated simultaneous changes in sodium (Na), chlorine (Cl), and potassium (K) concentrations in stauroporine‐induced apoptosis by quantitative electron probe X‐ray microanalysis (EPXMA) in single cells. Apoptotic cells identified unequivocally from the presence of chromatin condensation in backscattered electron images were characterized by an increase in intracellular Na, a decrease in intracellular Cl and K concentrations, and a decrease in K/Na ratio. The ouabain‐sensitive Rb‐uptake assay demonstrated a net decrease in Na+/K+‐ATPase activity, suggesting that increases in Na and decreases in K and the K/Na ratio in apoptotic cells were related with inhibition of the Na+/K+‐ATPase pump. These changes in diffusible elements were associated with externalization of phosphatidyl serine and oligonucleosomal fragmentation of DNA. This alteration in ion homeostasis and morphological hallmarks of apoptosis occur in cells that have lost their inner mitochondrial transmembrane potential and before the plasma membrane becomes permeable.

Biphasic behavior of changes in elemental composition during staurosporine-induced apoptosis

Although the identification of events that occur during apoptosis is a fundamental goal of apoptotic cell death research, little is know about the precise sequence of changes in total elemental composition during apoptosis. We evaluated total elemental composition (Na, Mg, P, Cl, S, and K) in relation to molecular and morphological features in human U937 cells induced to undergo apoptosis with staurosporine, an intrinsic pathway activator. To evaluate total elemental content we used electron probe X-ray microanalysis to measure simultaneously all elements from single, individual cells. We observed two phases in the changes in elemental composition (mainly Na, Cl and K). The early phase was characterized by a decrease in intracellular K (P < 0.001) and Cl (P < 0.001) content concomitant with cell shrinkage, and preceded the increase in proteolytic activity associated with the activation of caspase-3. The later phase started with caspase-3 activation, and was characterized by a decrease in the K/Na ratio (P < 0.001) as a consequence of a significant decrease in K and increase in Na content. The inversion of intracellular K and Na content was related with the inhibition of Na+/K+ ATPase. This later phase was also characterized by a significant increase (P < 0.001) in intracellular Cl with respect to the early phase. In addition, we found a decrease in S content and an increase in the P/S ratio. These distinctive changes coincided with chromatin condensation and DNA fragmentation. Together, these findings support the concept that changes in total elemental composition take place in two phases related with molecular and morphological features during staurosporine-induced apoptosis.

Electron probe X-ray microanalysis for the study of cell physiology.

Of the analytical electron microscopy techniques available, electron probe X-ray microanalysis has been most widely used for the study of biological specimens. This technique is able to identify, localize, and quantify elements both at the whole cell and at the intracellular level. The use SEM or TEM to analyze individual whole cells gives a simple and rapid method to study changes in ion transport after stimulation, whereas the analysis of thin sections of cryoprepared cell sections, although technically more difficult, allows details about ionic content in intracellular compartments, such as mitochondria, ER, and lysosomes, to be obtained. In this chapter the principles underlying X-ray emission are briefly outlined, step-by-step methods for specimen preparation of whole cells and cell sections for microanalysis are given, as are the methods used for deriving quantitative information from spectra. Areas where problems might occur have been highlighted. The different areas in which X-ray microanalysis is being used in the study of cell physiology are briefly reviewed.

ELECTRON PROBE X-RAY MICROANALYSIS OF CULTURED EPITHELIAL TUMOUR CELLS WITH SCANNING ELECTRON MICROSCOPY

Three methods have been used to prepare cultured cells for electron probe X-ray microanalysis (EPXMA): (1) analysis at the subcellular level of freeze-dried ultrathin cryosections with scanning transmission electron microscopy (STEM); (2) analysis at the cellular level of whole freeze-dried cells with STEM; and (3) analysis at the cellular level of whole freeze-dried cells with scanning electron microscopy (SEM) (for a review see: Wroblewski and Roomans, 1984; Wroblewski and Wroblewski, 1993; Warley, 1994). However, EPXMA of whole freeze-dried cells with SEM has some disadvantages. This method requires that cultured cells be adapted to growth on a thick substrate such as plastic or glass coverslips (Zierold and Schafer, 1988), graphite discs (Abraham et al., 1985; Larsson et al., 1986) and microcarrier beads (Hall et al., 1992). In addition, a suitable washing procedure to remove the extracellular medium must be found because the deposition of culture medium on the cell surface after freezing and freeze-drying may interfere with X-ray spectra from the cell. Moreover, it is difficult to perform absolute quantitative analyses of elemental content, since the substrate may contribute to the background, decreasing the peak-to-background (P/B) ratio (Roomans, 1981). We present here a simple method to study the intracellular concentrations of elements in whole cultured epithelial tumour cells by EPXMA with scanning electron microscopy.

Changes in elemental concentrations in K562 target cells after conjugation with human lymphocytes studied by X‐ray microanalysis.

There is considerable interest in determining the mechanisms by which natural killer (NK) cells are able to kill tumor cells. Morphological evidence supports the suggestion that target cell death may occur by either apoptosis or necrosis (I). There have been no studies which describe changes in elemental concentrations in NK cell-mediated cytotoxicity, which would support the mechanism of cell death. Electron probe X-ray microanalysis (EPXMA) should provide the ideal technique for undertaking such studies, since it is the only method which allows elemental analysis of a known (morphologically defined) cell type (2).

Aerobic interval exercise improves parameters of nonalcoholic fatty liver disease (NAFLD) and other alterations of metabolic syndrome in obese Zucker rats

Metabolic syndrome (MS) is a group of metabolic alterations that increase the susceptibility to cardiovascular disease and type 2 diabetes. Nonalcoholic fatty liver disease has been described as the liver manifestation of MS. We aimed to test the beneficial effects of an aerobic interval training (AIT) protocol on different biochemical, microscopic, and functional liver alterations related to the MS in the experimental model of obese Zucker rat. Two groups of lean and obese animals (6 weeks old) followed a protocol of AIT (4 min at 65%-80% of maximal oxygen uptake, followed by 3 min at 50%-65% of maximal oxygen uptake for 45-60 min, 5 days/week, 8 weeks of experimental period), whereas 2 control groups remained sedentary. Obese rats had higher food intake and body weight (P < 0.0001) and suffered significant alterations in plasma lipid profile, area under the curve after oral glucose overload (P < 0.0001), liver histology and functionality, and antioxidant status. The AIT protocol reduced the severity of alterations related to glucose and lipid metabolism and increased the liver protein expression of PPARγ, as well as the gene expression of glutathione peroxidase 4 (P < 0.001). The training protocol also showed significant effects on the activity of hepatic antioxidant enzymes, although this action was greatly influenced by rat phenotype. The present data suggest that AIT protocol is a feasible strategy to improve some of the plasma and liver alterations featured by the MS.

Dynamic reorganization of the alkaline phosphatase-containing compartment during chemotactic peptide stimulation of human neutrophils imaged by backscattered electrons

Scanning electron microscopy (EM) and cytochemical techniques were used to examine the alkaline phosphatase-containing compartment in human neutrophils after stimulation with nanomolar concentrations of N-formylmethionyl-leucyl-phenylalanine (10−8M fMLP). Alkaline phosphatase (AlkPase) activity was demonstrated with a lead-based metal capture cytochemical method. The reaction product was visualized with the backscattered electron imaging mode of scanning EM, and analyzed by electron probe X-ray microanalysis. Alkaline phosphatase activity was detected only in fMLP-stimulated neutrophils; unstimulated neutrophils displayed no activity. Stimulation of human neutrophils with 10−8 M fMLP induced a time-dependent intracellular redistribution of irregular round or tubular granules containing alkaline phosphatase activity, as seen by backscattering. The intracellular redistribution of alkaline phosphatase activity was accompanied by increased cytochemical activity on the cell surface. The reaction product was localized preferentially on ridges and folds of polar neutrophils. Reorganization of the AlkPase-containing compartment correlated with changes induced by fMLP in cell shape, ie, membrane ruffling and front-tail polarity, as observed with the secondary electron image mode of scanning EM. These findings demonstrate the intracellular reorganization, increase, and asymmetric distribution of alkaline phosphatase activity on the plasma membrane of human neutrophils after stimulation by chemotactic peptides.

Distribution pattern of acid phosphatase activity in human peripheral blood leukocytes: a cytochemical scanning electron microscopy study

SummaryThe distribution patterns of acid phosphatase hydrolytic activity were studied in human peripheral blood cells with enzymocytochemical techniques together with light and scanning electron microscopy in the secondary and backscattered electron imaging modes. The acid phosphatase reaction product was seen in three different patterns of distribution: focal, granular and diffuse. These patterns were correlated with similar findings obtained with light microscopy. Acid phosphatase distribution patterns seen with SEM in the BEI mode were also correlated with the surface morphology of peripheral blood cells seen in the SEI mode. Cells exhibiting the focal pattern were smooth-surfaced with few microvilli; cells showing a granular pattern presented microvilli and microridges; ruffles were characteristic of cells with a diffuse pattern of activity. No reaction product was seen in cells bearing microvilli or ridges. Our findings demonstrate the correlation between acid phosphatase activity patterns and surface features in different subpopulations of peripheral blood cells.

Changes in Intracellular Sodium, Chlorine, and Potassium Content in Hematopoietic Cells after Hypotermic Storage

Hematopoietic precursor cells (HPCs) hold tremendous potential in the emerging field of cell-based therapies. The purpose of this therapy is to replace, repair or enhance the biological function of damaged tissue or organs. This can be achieved by the transplantation of cells in sufficient number and quality to restore function. Transplantation of HPCs requires the ability to preserve cells. Cell preservation technology involves both hypothermic methods for short-term storage and cryopreservation for long-term storage. However, both methods they are not exempt to induce cell damage and therefore to diminish the engraftment kinetics. Oxidative stress, mechanical injury due to ice crystal formation, altered physical properties of cellular structures, osmotic injury, and disturbed ion homeostasis are responsible for cell damage. However, in spite of the not clear correlation with capacity of engraftment, quality control assays currently used are based on methods revealing the loss of integrity of plasma membrane or ex vivo expansion capacity through clonogenic assays. Based on these premises, we propose to utilize the elemental composition as an indicator of cell viability and quality previous to cell transplantation.

Ultrastructural and Intracellular Elemental Composition Analysis of Human Hematopoietic Cells During Cold Storage in Preservation Solutions

Hematopoietic cell transplantation is a medical therapeutic procedure which aims to reconstitute the hematopoietic activity of bone marrow. Stem cells from bone marrow, mobilized peripheral blood or umbilical cord blood cells are used for these transplants. These transplant modalities require hypothermic storage and cryopreservation. However, cellular injury still occurs after extended cold storage. Despite large number of studies, however, optimal non-frozen clinical storage conditions for hematopoietic cells have not been established. Optimization of cell preservation protocols to maintain the viability and quality of hematopoietic progenitor cells has been an important task for tissue banks, and several attempts have been made to create alternative solutions for preservation of cells for transplantation. Evaluation of the storage solutions has been carried out using morphological techniques, studies of cell viability, functional studies as cellular ATP levels as well as by clinical assessment. However, few studies determining changes in electrolyte composition of cells during cold storage have been carried out [1]. In this study, we propose electron probe X-ray microanalysis (EPXMA) as a gold standard method for evaluation of the effect of preservation solutions on the intracellular elemental composition of cells for transplantation. For this, we evaluated the time-related elemental changes during cold storage in tissue culture medium in comparison with EuroCollins and lactated Ringer’s solutions. In addition, we examined the time-related ultrastructural changes of hematopoietic cells in preservation solutions.