Intracellular Na+:K+ ratios in human cancer cells as revealed by energy dispersive x-ray microanalysis.

Intranuclear sodium, potassium, and chloride contents were measured by energy-dispersive x-ray microanalysis in freeze-fractured, freeze-dried, bulk-tumor samples taken from 10 patients suffering from invasive urogenital cancers. Human biopsies were carried out during the first diagnostic interventions before any cytostatic treatment had been applied. Pathohistological diagnosis established the malignancy in each case. The cancers were classified in three types: keratinizing, transitional cell, and hypernephroid carcinoma. More than 250 cell nuclei were measured from each type of cancer. The results were compared with those obtained in intact human urothelium taken from patients having no malignant processes. Proximal and distal tubular epithelial cell nuclei representing the origin of human hypernephroid cancer were also measured in rat kidney because corresponding healthy human material cannot be obtained. The analyses revealed, in all three types of cancer cells, that the average intranuclear sodium content increased more than three-fold, the potassium content decreased 32, 16, and 13%, respectively; meanwhile the chloride content increased, but to a lesser extent than did the sodium. The intranuclear Na+:K+ ratios were more than five-fold higher in the cancer cells on the average, and their distribution histograms were much broader than in the normal human urothelium and in the tubular cell nuclei of the rat kidney. The results obtained fit well with the theory of Cone, C. D., Jr. 1971. J. Theor. Biol. 30: 151-181 according to which the sustained depolarization of the cell membrane may be of mitogenic effect.

Correlation of malignancy with the intracellular Na+:K+ ratio in human thyroid tumors.

Energy-dispersive X-ray microanalysis was applied on human intraoperative biopsy materials of different thyroid tumors. To ensure suitability of these tissue pieces for quantitative microanalysis in freeze-fractured, freeze-dried bulk specimens, sampling was carried out with strictly defined criteria. Benign adenomas and differentiated and anaplastic carcinomas were selected for the studies on the basis of pathohistological investigations of the same specimen. The results of the tumor cells were compared to those obtained in apparently normal human epithelial cells. The number of normal cells analyzed was 349, whereas in the tumors 408, 423, and 891 cells were measured in the benign, differentiated, and anaplastic groups, respectively. Intracellular monovalent contents were calculated as percentage of cell dry mass; then, Na+:K+ molar ratios were calculated for each cell individually. Due mostly to the increase of Na+ content, the distribution histograms of the Na+:K+ molar ratio show an increase in the number of cells with a higher Na+:K+ ratio with increasing malignancy of the tumors studied. The differences proved to be statistically highly significant by the chi 2 test. Thus, in human thyroid, increasing malignancy is associated with increasing intracellular Na+:K+ ratio. The results give further support to the theory of C. D. Cone (J. Theor. Biol., 30: 151-181, 1971) according to which the sustained depolarization of the cell membrane results in an increased rate of cell division.

The mobility of concanavalin A receptors and surface immunoglobulins on rat hepatocyte plasma membranes.

Lateral mobilities of lectin receptors and surface immunoglobulins were measured in plasma membranes of hepatocytes prepared by smearing small pieces of rat liver tissue and then using the fluorescence recovery after photobleaching (FRAP) technique. Smears were treated with various doses of fluorescein isothiocyanate (FITC) conjugated concanavalin A (ConA), succinylated ConA (SConA), wheat germ agglutinin (WGA), and soybean agglutinin (SBA), as well as with rabbit anti-rat IgG (RARa/IgG) and goat anti-rat IgM(Fc) (GARa/IgM(Fc] antisera. 10 micrograms/ml ConA and SConA concentrations and a 55 X dilution of the GARa/IgM(Fc) antiserum were found to be suitable for measuring the lateral mobilities dependent on age. Diffusion constant and mobile fractions of receptor complexes were measured in different age groups of female Fisher rats (from 1 to 26 month-old). The FRAP measurements revealed that at least two major receptor sites can be distinguished in cell membranes of compact tissue (similar to the cultured and isolated cells), forming a mobile and an immobile fraction. The mobile fractions of both the lectin receptors and the surface immunoglobulins tended to decrease with age, while the age differences of the diffusion constants were not statistically significant. The observed alterations could be due to the covalent crosslinking of the mobile receptors to immobile patches and/or to the retardation of free diffusion by the cytoskeleton, dependent on age.

In vivo effects of vitamin E deficiency on the intracellular monovalent electrolyte concentrations in brain and liver of rat. An energy dispersive X-ray microanalytic study.

Vitamin E is known to play a protective role for cell membranes against free-radical attacks. Vitamin E deficiency causes a rapid macroscopic ageing of rats. On the other hand, during normal ageing, cell membranes undergo functional alterations resulting in an increased intracellular potassium concentration in brain and liver cells. Therefore, is was of interest to study whether vitamin E deficiency produces similar alterations in young rats. Female Wistar rats were fed with a vitamin E deficient diet from 1 month of age for 10 months. The parietal brain cortex and the liver were analyzed by means of a quantitative energy dispersive X-ray microanalytic method using a JEOL JSM-35C-EDAX-711-NOVA-3 system. Monovalent electrolyte contents as well as the water content of the cells were determined in 5 treated and 5 control animals. Water content was measured by analyzing the potassium content in aqueous, frozen state, and again in the dry mass of the cells. On the basis of these data, a computer program calculated the water proportions. Average values for 200 or more cells of each organ per group revealed a significant increase in the intracellular potassium content of the brain cells, whereas the sodium and chloride contents increased to a much lower extent. There was a 2.6% loss of intracellular water in the brain cells in the vitamin E deficient group. The liver monovalent ions and water content remained unchanged. The results obtained are discussed in terms of the membrane hypothesis of ageing.

Microfluorimetric and X-ray microanalytic studies on the DNA content and Na+:K+ ratios of the cell nuclei in various types of thyroid tumors.

Parallel studies were performed using microfluorimetric DNA determination and X-ray microanalysis on the same thyroid biopsy material to compare the intranuclear DNA and monovalent electrolyte contents (Na+, K+, Cl-). Samples were taken from apparently healthy, adenomatous, and cancerous parts of human thyroid glands removed surgically. The time interval was less than 1 min. The tissues were classified by the pathologist into four main classes: 1) Normal thyroid tissue; 2) benign adenomas; 3) differentiated (follicular and papillary) carcinomas; and 4) anaplastic cancers. The results revealed that the level of aneuploidization showed an increase parallel with the malignancy of the studied tumor. At the same time, a similar tendency was found in the average values of the intranuclear Na+:K+ ratios. The results obtained in this way confirm the possibility that the electric properties of the cell membrane, that is the sustained membrane depolarization, may have a role in the regulation of DNA synthesis and in mitogenesis. These results may offer new diagnostic and/or therapeutic possibilities.

Characterization of intracellular calcium oscillations induced by extracellular nucleotides in HEp-2 cells.

The effect of extracellular nucleotides on the cytosolic calcium concentration of fluo-3-loaded HEp-2 cells was examined using confocal microscopy. Extracellular ATP and UTP at micromolar concentration induced cytosolic calcium oscillations in 42-66% of the cells. Oscillations were usually sinusoid and their frequency depended only slightly on agonist concentration. Oscillations developed in calcium-free medium but were diminished by depletion of intracellular calcium stores with thapsigargin, indicating periodic calcium release from internal stores. Inhibition of phospholipase C with U73122 prevented the development of oscillations, while ryanodine did not abolish the response to extracellular nucleotides. Activation of protein kinase C with 4beta-phorbol 12-myristate 13-acetate also prevented the development of oscillations. These results indicate that extracellular nucleotides induce periodic calcium release from inositol 1,4,5-trisphosphate-sensitive pools in HEp-2 cells and that the inhibitory effect of protein kinase C on the phosphatidylinositol signaling pathway can contribute to the development of intracellular calcium oscillations.

Intracellular water and dry mass content as measured in bulk specimens by energy-dispersive X-ray microanalysis.

Intracellular water content (IWC) was measured in freeze-fractured biological bulk specimens by means of energy-dispersive X-ray microanalysis. The method is based on the concentration differences of certain elements (potassium and phosphorus) between frozen-hydrated and frozen-dried states of the tissues as applied formerly to sectioned material by others. A new mathematical formula has been derived giving rather precise figures for IWC. No elemental standards are necessary for the measurement: one has to obtain only the peak to background ratios in wet and dry states of the cells. the method is sensitive enough to reveal age-dependent as well as drug-induced changes of IWC in liver and brain cells. The values obtained are quite comparable with the theoretically expected one. Technical problems of the application of this method are discussed in detail.

The effect of primycin on the intracellular monovalent ion and water contents of rat hepatocytes as revealed by energy dispersive X-ray microanalysis and interference microscopy.

Using energy-dispersive X-ray microanalytic and interference microscopic techniques, the intracellular concentration of the monovalent ions (Na+, K+, Cl+) as well as the intracytoplasmic and intracellular water contents were studied in normal and adrenalectomized rat hepatocytes with and without primycin treatment. Although primycin influenced significantly only the intracellular potassium content of the adrenalectomized group, it exerted a marked influence on the intranuclear water content in both the normal and adrenalectomized rats. The intranuclear water content increased significantly in the primycin-treated animals. The conclusion is drawn that the increased level of hydration of the nuclear substances reflects a 'decondensation' of the chromatin which on the other hand, may represent the basis for the various effects of primycin on the induction of certain hepatic enzymes.

The lack of age-pigments and the alterations in intracellular monovalent electrolytes in spontaneously hypertensive, stroke-prone (SHRsp) rats as revealed by electron microscopy and X-ray microanalysis.

Male, spontaneously hypertensive, stroke-prone (SHRsp) rats established by Okamoto et al. (1974) were studied. About 80% of the males of this strain have a particularly short life span (33-41 weeks); they display a considerable hypertension (above 220 mmHg) and a tendency for plurifocal brain strokes. Hypertension and strokes can be provoked in an accelerated and synchronized fashion by supplementing 1% NaCl into their drinking water. Symptoms of the appearance of brain strokes can be judged from characteristic signs of motor disorders, and can be established also by pathohistology. Since hypertension and arteriosclerosis are frequently involved in aging, the question we intended to answer was whether these animals may represent a model of the normal aging process or not. Two approaches are described: (1) Accumulation of lipofuscin granules in their brain, liver and myocardium was followed by transmission electron microscopy before and after the appearance of strokes. It has been established that these tissues do not show any typical accumulation of lipofuscin granules, although submicroscopic signs of an enhanced damage of cell organelles (especially of mitochondria in liver and brain cells, but not in myocardium) were encountered. (2) The intracellular monovalent composition in the brain and liver was measured by using bulk-specimen X-ray microanalysis. The intracellular Na-content (mEq/kg water) was significantly higher (170-200%) in both the brain and liver cells, whereas the K-content increased only moderately (118-130%). The results suggest that although the SHRsp rats do not represent a direct model for the normal aging process from the point of view of lipofuscin accumulation, the shifts of the monovalent electrolyte contents in the brain and liver cells observed already in the youngest ages, are similar to those observed in aged normal rats. The theoretical consequences of such a conclusion are discussed.

Age-dependent alterations of the intracellular water and electrolyte content of heart and muscle cells.

Age-dependent alterations in intracellular concentrations of monovalent ions (Na+, K+ and Cl-) were measured in heart and muscle cells of rats using energy dispersive X-ray microanalysis of bulk specimens. Separate measurements were performed in order to get the elemental concentrations in the dry mass of the cells, and to determine the intracellular water and dry-mass content. The in vivo concentrations were calculated from these two measurements assuming that the monovalent ions were dissolved in the cell water. A statistically significant decrease was measured in the water content of the myocytes of old rats, suggesting an increase in density and viscosity of the cytoplasmic colloid during aging. This loss of cellular water was accompanied by a significant increase in both the single ion concentrations and the total monovalent ion content of the intracellular water. These age-dependent alterations in heart and muscle cells are similar to those demonstrated previously in neurons and hepatocytes.

Verification of the membrane hypothesis of aging on the identified giant neurons of the snail Lymnaea stagnalis L. (Gastropoda, Pulmonata) by a combined application of intracellular electrophysiology and X-ray microanalysis.

The validity of the membrane hypothesis of aging (Zs.-Nagy, 1978) was tested on identified giant neurons of the snail Lymnaea stagnalis L. by using a combination of intracellular microelectrophysiology and X-ray microanalysis of the intracellular water and electrolyte concentrations on the very same cells. The snails were taken from an inbred stock and divided into young, adult and old age groups (3, 12 and 24 mth, respectively). The giant neuron called LPa-2 from the left parietal ganglion was selected for the studies. The resting potential of the cell membrane was recorded by means of intracellular microelectrode technique. The very same cells were then explored by freeze fracture and analyzed by an energy dispersive bulk specimen method of X-ray microanalysis. The resting membrane potential displayed an age-dependent hyperpolarization, the intracellular water content decreased considerably and the intracellular potassium concentration increased almost 90% by old age. The relative passive permeability ratio for potassium (PK) and chloride (PCl) was calculated from the measured data by means of the Goldman-Hodgkin-Katz equation. Such calculations revealed that PK decreases nearly 50% with age causing the increase of the intracellular potassium content, and this is accompanied also by a significant decrease of the PCl. The results support the validity of the membrane hypothesis of aging and are in agreement with the general knowledge regarding the electrophysiological behaviour of the giant neurons of Gastropode snails.

Effects of idebenone on mitogen-induced proliferation of human lymphocytes.

In vitro effect of idebenone on human lymphocytes isolated from old and young donors was determined. The effects of drug were the same with the old and young donors cells. At concentrations of 2 microg/ml (6 microM) or less in the culture medium, idebenone showed no effect on phytohemagglutinin (PHA)-induced proliferation and protein synthesis, or on cell viability measured by Trypan Blue exclusion. Concentrations of 25 and 50 microg/ml showed dose-dependent suppression of the proliferation and protein synthesis which was associated with significant cytotoxicity. At concentrations of 8-10 microg/ml the compound appears to have just detectable effect on lymphocyte viability or ability to respond to PHA stimulation. It seems clear that such in vivo concentrations which would be associated with lymphopenia and immunologic suppression are not achieved with therapeutic doses of idebenone. The pattern of protein bands observed on fluorograms of sodium dodecyl sulfate-polyacrylamide gels of cells incubated with [(3)H]leucine and [(35)S]methionine was similar in control and idebenone-treated samples, consistent with a slight, nonspecific inhibitory effect on protein synthesis in cultures with higher doses of the compound. At these concentrations, idebenone induced a slight, but detectable, enhancement of the intracellular stress proteins, HSP70 and HSP90.

Loss of water from heart muscle cells during aging of rats as measured by X-ray microanalysis.

Age-dependent changes of the intracellular water content (IWC) of the rat myocardium have been measured by X-ray microanalysis of deep-frozen bulk specimens (Zs.-Nagy et al., 1982), using a slow warming up and drying of a very superficial layer of the sample, in order to minimize space charging effects. These results were compared with those calculated from the conventionally measured tissue water contents (TWC) and data from literature of morphologically estimated volume density of the extracellular space (David et al., 1981). The IWC values obtained from these two independent methods are in very good agreement, showing that the etching of the surface during the bulk specimen analysis is sufficiently small, i.e., it does not result in any considerable error in the quantitative X-ray microanalysis. The IWC of heart muscle cells decreases significantly with advancing age (from about 80% by weight at the age of 14 days to 71% by the age of 24 months). This observation is consistent with the membrane hypothesis of aging (Zs.-Nagy, 1978). Comparison of the IWC with TWC of the heart muscle shows that the increase of the volume density of the extracellular space during aging balances the age-dependent loss of myocytes to a certain extent.

Age-dependent changes in the osmotic behavior of rat brain and liver.

Osmotic potential of liver and brain tissue has been determined by measuring swelling or shrinkage of the tissues in anoxic Ringer solution the osmotic concentration of which was rendered hypo- or hyperosmotic by dilution or addition of polyethylene glycol (PEG 6000), respectively. The percentages of volume change were fitted to an exponential equation permitting the calculation of the initial speed of volume change. The age-dependent increase of the intracellular dry mass content was also taken into consideration. It has been established that initial velocity of the volume change displays an age-dependent decline in all kinds of media tested; however, the slope of this decrease is steeper in the diluted Ringer solution than in the other ones. Calculations show that the colloid osmotic pressure (mmHg) of the intracellular mass decreases between 1 and 26 months of age from 810 to 596 and from 2,477 to 904 in the brain and liver, respectively. The results may be interpreted as consequences of a decreased colloid dispersity which may be related to an oxygen free radical induced intermolecular cross-linking.

Age-dependent decrease of the passive Rb+ and K+ permeability of the nerve cell membranes in rat brain cortex as revealed by in vivo measurement of the Rb+ discrimination ratio.

Young, adult and old male CFY rats (2, 12 and 24 mth of age, respectively) were treated with a daily dose of 30 mg RbCl/100 g body weight, in form of aqueous solution injected intraperitoneally for 14 days. A considerable part of the intracellular K+-content of the body was replaced by Rb+ during this treatment. After cessation of the RbCl injections, a relative steady state came into being in each age group, called Rb+-release period. During this period Rb+ and K+ contents of the blood serum and the cisternal CSF were measured by atomic absorption spectrophotometry, and of the intracellular space of brain cortical cells by energy-dispersive X-ray microanalysis. Ultrastructural features of the brain cortex were also checked by transmission electron microscopy. For X-ray microanalysis, the L-line of Rb at 1.694 keV energy was used at 10 kV accelerating voltage in a scanning electron microscope equipped with an EDAX System F. Rb+ and K+ concentrations were obtained for the cellular dry mass and converted into wet concentrations on the basis of intracellular water contents known from former experiments. Rb+-replacement of K+ did not cause any ultrastructural alteration in the brain cortex. However, the Rb+ accumulation displayed a very significant age-dependent increase: at the beginning of release, adult and old rats had 32.6 and 44.7 mM Rb+ in their intracellular water as against the 8.6 mM found in the young group, and similar proportional difference persisted during 20 days of the release. Rb+ discrimination ratios (DR) calculated either for the blood or the CSF displayed very considerable age-dependent increase: the values of the adult and old groups were 191 and 242% of the young one, indicating that the passive Rb+ (and K+) permeability of the nerve cell membrane decreases throughout the life span of rats. These results give further support to the membrane hypothesis of aging.

The effect of idebenone on the passive K+ and Rb+ permeability of the brain cell membranes of CFY rats as revealed by the Rb+-discrimination ratio.

Rubidium (Rb(+)) uptake and release of brain cortical neurons of adult (12-15 month old), normal, female CFY rats were studied in placebo- and idebenone-treated animals. Treatment period was up to 5 weeks, with 50 mg/kg bw/day idebenone (oxidized form) suspended in 5% gum arabic (verum-group), or only with the latter solvent (placebo-group). Rb(+) can replace up to 60% of the intracellular K(+), and can be used as a tracer of the K(+) movement across the cell membrane. Loading with RbCI was performed from the 3rd week of the idebenone treatment by daily intraperitoneal injections of a dose of 300 mg/kg bw, for 14 days. During the subsequent Rb(+)-release period, the so-called Rb(+)/K(+) discrimination ratio (DR) (Relman et al., 1957, J. Clin. Invest., 36, 1249) was determined on the 3rd and 8th days. Rb(+) and K(+) contents were measured by means of bulk specimen X-ray microanalysis in the intracellular water of brain cells and by atomic absorption spectrophotometry in the serum, in 3-4 animals per group, whereas these concentrations in the cerebrospinal fluid were calculated on the basis of known serum/liquor distribution factors. Normal aging causes a marked increase of DR in brain and liver cells. The values of DR obtained in both placebo and verum groups were identical with those of the age-matched, completely untreated controls. It is important to stress that the subacute idebenone treatment did not cause any deterioration of this parameter, i.e., under the given conditions idebenone does not affect the cell membrane passive Rb(+) and K(+) permeability characteristics in the neurons of adult, normal, female CFY rats.

Effects of idebenone on the intracellular water and dry mass content as well as of the intracellular monovalent ion concentrations of brain cortical cells of SHRsp rats as revealed by X-ray microanalysis.

The cerebral cortex of ten male SHRsp rats kept in conventional housing conditions were studied. Starting from the age of 3 months, five animals received placebo (5% gum arabic solution) and five other rats received 50 mg/kg idebenone suspended in the gum arabic, through a gastric tube for 4 weeks (except Sundays). During the last 2 weeks of treatment, 0.9% NaCl was added to the drinking water. Several completely untreated SHRsp rats of various ages were also involved in these studies. Blood pressure was measured weekly on the tail by means of an appropriate instrument. Serious hypertension was observed already by the end of the second week of treatment, displaying values of 250-260 mm Hg and increased further by about 20 mm Hg during the last 2 weeks of treatment. Intracellular water, dry mass and monovalent electrolyte concentrations were measured by means of a bulk specimen X-ray microanalytic method. The brain cells contained 77+/-1% water and 23% dry mass by weight in both placebo and verum-treated groups. The intracellular Na(+) content of all the male SHRsp rats was found to be significantly higher (180-200%) in the brain cells, whereas K(+) content increased only moderately, when expressed as percent of the intracellular dry mass. Idebenone treatment, however, lowered the intracellular Na(+) content of the brain cells to a significant extent (about 20%), i.e., it improved the Na(+) tolerance of the SHRsp rats, but did not alter the blood pressure.

The effect of idebenone on the total content and solubility characteristics of proteins and osmometric behavior of the intracellular mass in brain of CFY and SHRsp rats.

Female CFY rats (21 months old) and male spontaneously hypertensive, stroke-prone (SHRsp) rats (3 months old), in conventional housing conditions, received placebo (5% gum arabic solution) or 50 mg/kg bw/day idebenone suspended in 5% gum arabic, through a gastric tube for 5 weeks; then their brains were elaborated as follows: (1) Total proteins as well as water-soluble and water-insoluble proteins (WSP and WIP, respectively) were separated from the brain homogenate by centrifugation at 500 X g. The WIP fractions were tested also in vitro by heat denaturation at 64 degrees C (10 min) and by 3 M urea treatment. In the placebo group of CFY rats the total protein content was 113.9 mg per g fresh weight. WIP amounted to 27.2% of the total proteins. Idebenone-treatment did not alter the protein composition in these old rats. In the SHRsp rats the total protein content of the brain cortex was almost identical with that of the normal, Wistar-derived CFY rats of much more advanced age (about 2 years). The idebenone-treatment did not alter the protein content of the brain cortex, although the WIP content and the heat-resistant fraction of it increased significantly in this strain. (2) The osmotic potential of brain tissue was determined by measuring swelling or shrinkage velocities in Ringer solution, the osmotic concentration of which was rendered hypo- or hyperosmotic by dilution or addition of polythylene glycol (PEG 6000), respectively. Idebenone treatment exerted no effect on the osmometric properties of the brain tissue in either the normal old or the SHRsp rats.

Effects of preliminary culture on the membrane microviscosity of lymphocytes from young and old donors. Microviscosity correlates with mitogenic response.

Membrane microviscosity was assessed by a fluorescence polarization technique in fresh and precultured human peripheral blood lymphocytes of young and old subjects. Membrane microviscosity was significantly higher in fresh, non-treated cells of old donors as compared to young adults. Preincubation of cells in culture medium supplemented with pooled human serum diminishes the original microviscosity difference between the age groups. The observed increase in membrane fluidity correlates with the improvement of the mitogen-induced proliferative response due to preculturing cells from aged subjects. The results support the suggestion that membrane microviscosity can affect the proliferative response of lymphocytes, and it may play a role in the decline of the immune responsiveness in the elderly.

Experimental glomerular lesions induced by chronic immune complex formation. I. Formation and elimination of the immune complex (relationship between the immune status and the glomerular changes).

Chronic immune complex formation was induced in rabbits by daily administration of 12.5 g bovine serum. In good antibody producer animals immediate immune complex production and elimination from the circulation were demonstrable. This was followed within a few minutes by the appearance of free 125I in fairly large amounts in blood, as a sign of immediate phagocytosis and disintegration of the 125I-labelled immune complexes. Phagocytic activity decreased in the host animal during chronic heteroprotein administration in every case. The earliest glomerular changes were those of exudative glomerulonephritis, the extent of which depended on the antibody productivity of the animal. Persistent immunocomplexaemia induced by administration of the antigen over 60 and 100 days, respectively, resulted in mesangioproliferative glomerulonephritis in 7, in membranoproliferative glomerulonephritis in 3, and in membraneous glomerulonephritis in 1 out of 11 laboratory animals.