Effects of high hydrostatic pressures on living cells: a consequence of the properties of macromolecules and macromolecule-associated water.

Sixty percent of the Earth's biomass is found in the sea, at depths greater than 1000 m, i.e., at hydrostatic pressures higher than 100 atm. Still more surprising is the fact that living cells can reversibly withstand pressure shifts of 1000 atm. One explanation lies in the properties of cellular water. Water forms a very thin film around macromolecules, with a heterogeneous structure that is an image of the heterogeneity of the macromolecular surface. The density of water in contact with macromolecules reflects the physical properties of their different domains. Therefore, any macromolecular shape variations involving the reorganization of water and concomitant density changes are sensitive to pressure (Le Chatelier's principle). Most of the pressure-induced changes to macromolecules are reversible up to 2000 atm. Both the effects of pressure shifts on living cells and the characteristics of pressure-adapted species are opening new perspectives on fundamental problems such as regulation and adaptation.

Intracellular pH in one-cell mouse embryo differs between subcellular compartments and between interphase and mitosis.

The pH-sensitive dual-emission fluorophore SNARF-1 coupled with a laser confocal microspectrofluorimeter was used to measure the internal pH (pHi) in different subcellular and subnuclear compartments of early mouse embryos. By this method we analysed the first cell cycle of naturally fertilised embryos in order to detect possible pHi changes correlated to cellular events, particularly the onset of replication or transcription and the first mitosis. Throughout interphase, significant differences of pHi were observed between cytoplasm and pronuclei, and, even more striking, between these compartments and nucleolus precursor bodies, whose pHi was systematically lower. We could detect significant pHi change neither during the replication phase nor at the onset of zygotic transcription, but pHi increased at the end of the one-cell stage in both cytoplasm and chromatin regions, a process that seemed specifically correlated with mitosis.

An unexpected effect of an ouabain-sensitive ATPase activity on the amount of antigen-antibody complexes formed in situ.

A classical method of indirect immunofluorescence was applied on various kinds of lightly fixed and permeabilized cells to analyze the formation of the complexes between a nuclear antigen and its antibody (AAC). The amount of AAC decreased dramatically when the incubation with the first antibody was realized in the presence of ATP in a sodium-rich medium with 0.5 mM KCl. Addition of sodium vanadate, a general inhibitor of ATPases, ouabain or tetrabutylammonium ion, specific inhibitors of the Na+,K+-ATPase, prevented this effect. The established role of this enzyme is to increase free-K+ concentration and decrease free Na+ concentration in the cell. It is not surprising to find an ATPase still active since light fixation and permeabilization do not destroy phosphatases. But it is rather surprising to find something looking like Na+,K+-ATPase activity in permeabilized cells. The importance of potassium in this puzzling result is suggested by the fact that appearance of ACC was equally suppressed if the incubation was made in the absence of ATP in a potassium-rich medium without sodium. Results are discussed, taking into account the properties of cell-associated water and recently found interrelation between Na+,K+-ATPase and tubulin. In any case, the results seem interesting in the field of immunocytochemistry.

Pressure-sensitivity of endoplasmic reticulum membrane and nucleolus as revealed by electron microscopy.

Murine erythroleukemia cells submitted to high hydrostatic pressure (up to 110 MPa, 17 min. at room temperature) remain viable (Mentré et al., 1997) but present, at the ultrastructure level, perturbations which are documented in this work. In cells submitted to 50 MPa, endoplasmic reticulum membranes displayed a characteristic rigid aspect, whereas plasma membrane remained unaffected up to 110 MPa. This result is in agreement with: i) the liquid-crystalline --> gel state transition undergone by phospholipid bilayers under pressure, which involves the structuration of water at the polar-apolar interface; ii) the low concentration in cholesterol of endoplasmic reticulum membranes compared to plasma membrane, and iii) the ability of cholesterol to protect membranes against the effects of high hydrostatic pressure. Nucleoli displayed a remarkable compact aspect above 80 MPa, involving the disappearance of vacuoles and the diminution of fibrillar component, but the retention of granular component. Pressure inhibition of translation is advanced as a possible cause of this perturbation.

Divalent cation changes in cerebellar Purkinje cells after climbing fiber deafferentation.

A secondary ion mass spectrometry (SIMS) microscope was used to detect intracellular stores of calcium, magnesium, sodium and potassium. Measurements were made in semithin sections of fixed tissues of normal and climbing fiber deafferented cerebellar cortex. Quantitative data were collected from 150 microns diameter image fields in the molecular and granule layers. The results indicate smaller quantities of both calcium and magnesium in the deafferented cerebellar cortex compared to the normals, the molecular as well as the granule layer being affected. The results are discussed in terms of the usefulness and limitations of the SIMS microscope for histological preparations.

Preservation of the diffusible cations for secondary ion mass spectrometry. II. Artefacts in material embedded in araldite or melamine.

Flotation on hot water (about 60 degrees C) which is frequently employed to stretch semithin sections on substrates for SIMS (secondary ion mass spectrometry) microscopy, is the cause of numerous artefacts. In the case of epoxy resin-embedded tissue, one observes loss of potassium and sodium and accumulation of calcium. The relative contrast of cell nuclei in the ionic images, is rapidly affected by these ion migrations. After prolonged contact with hot water, tissue becomes uniformly emissive. In the case of hydrosoluble resin-embedded tissue, potassium and sodium do not appear to be affected by the action of water, which suggests that they are covalently bound with chelating sites buried beneath the layer of water bound to the surface of the macromolecules. Calcium accumulates, probably on widely exposed anionic sites. Moreover, the domains observed in hydrosoluble resin-embedded tissue shrink differently according to the proportion of water removed by melamine; this can provide interesting information on the initial equilibrium between water, ion sand macromolecules. Our results seem to support the assumption that bound water should play an important role in the preservation of both macromolecular architecture and ion distributions.

Preservation of the diffusible cations for SIMS microscopy. I. A problem related to the state of water in the cell.

The notion of diffusible ions is reviewed in the light of recent knowledge on the stage of water in biological matrices. It appears that ion distributions would be little affected as long as water-macromolecular equilibrium is maintained, but they risk to be significantly modified during dehydration, because the transformation of bound water into highly solvating free water can produce ion displacements. In addition, differently hydrated areas may undergo unequal volume variations. The principal modes of preparing material for SIMS (secondary ion mass spectrometry) microscopy are envisaged from this viewpoint.

Application of the pyroantimonate method and electron probe microanalysis to the study of glycogen metabolism in liver.

Glycogen distribution in the liver of mouse under different metabolic conditions was studied by the pyroantimonate (PA) method combined with semi-quantitative electron probe microanalysis (EPMA). In the liver of animals subjected to a sugar-rich diet, glycogen granules were abundant and electron transparent. In fasted animals, they were less numerous and stained by PA, which indicates the presence of a complexed cation. This cation was identified as calcium by EPMA. In both cases, adjacent cytoplasmic areas contained "masked" calcium not revealed by PA but detected by EPMA, which is characteristic of a neutral complexed form; but in the case of the fasted animals, the calcium concentration was significantly lower. If the liver of fasted animals was dissected in 0.2% glucose-containing medium, the glycogen areas dramatically released calcium and lost their stainability by PA, whereas mitochondria and adjacent cytoplasm contained many PA precipitates rich in calcium and sodium, suggesting a sudden increase of intracellular [Ca2+]. In mitochondria, the sodium:calcium ratio was relatively constant, which suggests a process involving a coupling between these two elements. Our results could be explained in the light of physiological and biochemical data. We particularly noted that diffusible cations as calcium and sodium did not appear to be displaced over long distances from their likely source. This observation agrees with recent theories on the state of water and ion mobility in the cell.

Cytochemical study of abnormal intranuclear structures rich in beryllium.

During prolonged intoxication with beryllium sulphate, intranuclear beryllium-rich structures (IBRS) develop mainly in the cells of the convoluted tubules of the kidney. These structures are constituted by the accumulation of dense granules approximately 20 nm in diameter. The present work shows: 1) by electron probe microanalysis that IBRS are rich in phosphorus and calcium, and 2) by high resolution ion microanalysis that the granules are rich in beryllium and proteins. Staining with thallium alcoholate and regressive staining with ethylenediaminetetraacetate (EDTA) seem to demonstrate the presence of ribonucleoproteins in the granules. But the richness in calcium and phosphorus makes it difficult to interprete cytochemical reactions based on thallium and lead because complexes can be formed between calcium and thallium or lead, and between phosphorus and lead. Extraction with EDTA and digestion with RNase carried out on floating slices fixed with glutaraldehyde and embedded in glycol methacrylate show that: 1) the positive response of IBRS to cytochemical techniques used seems due solely to calcium; 2) the RNase forms a stable complex with a constituent of the granules that could be the highly phosphorylated acidic protein that binds preferentially to beryllium described by Parker and Stevens.

Localization of cations by pyroantimonate. I. Influence of fixation on distribution of calcium and sodium. An approach by analytical ion microscopy.

A modification of the potassium pyroantimonate (PA) method for localization of calcium and sodium was tested using skeletal muscle of mouse. Massive diffusion of these cations, depending on the method of fixation, was demonstrated by analytical ion microscopy (AIM) images on the optical microscopy level. Rapid penetration of the fixative appeared to be the principal condition that reduced diffusion of Ca2+ and Na+. Paraformaldehyde (2%) appeared more efficient than glutaraldehyde (1%) for preserving metal composition. Addition of 1% phenol strikingly improved the quality of the AIM images. Supersaturated PA (4%) appeared to retain about 10 times more sodium in the tissue than insaturated PA (2%). The role of different buffers is also discussed, particularly collidine, which permitted better preservation of sodium. Fixation with this buffer should be very useful for study by AIM of large-scale distribution of sodium. These results are analyzed at the ultrastructural level in the accompanying report.

Localization of cations by pyroantimonate. II. Electron probe microanalysis of calcium and sodium in skeletal muscle of mouse.

A new formulation of the pyroantimonate (PA) method for localization of calcium and sodium is proposed and evaluated in mouse skeletal muscle. This study, performed at the ultrastructural level by means of transmission electron microscopy (TEM) and electron probe microanalysis (EPMA), completes a previous work done at the optical level with analytical ion microscopy (AIM), which enabled us to define the appropriate composition of fixatives. In our present experiments, calcium and sodium were shown localized in various cell structures, e.g., T-tubules, glycogen, granules, nuclei. For AIM, the best fixatives were characterized by PA supersaturation, which resulted in smaller crystals and a high rate of penetration in the presence of paraformaldehyde and either phenol or collidine. Contrary to the findings at the optical level, collidine did not give satisfactory results at the ultrastructural level. The method of floating sections on the microtome trough was an important cause of cation displacement. We found that alkalinization of the floating medium significantly decreased ion loss. The technique also provided an indication of the form of these elements: free or easily liberated cations were precipitated into coarse PA deposits; electron-positive chelates were "stained" by PA; neutral chelates were not stained, but some of them could be detected by EPMA. This PA method should make possible more precise localization of cellular calcium, such as in glycogen metabolism, and perhaps detection of movements of cytoplasmic calcium and sodium.