Photobleaching measurements of diffusion in cell membranes and aqueous cell compartments.

This commentary unit discusses in great detail the theoretical nature of fluorescence recovery after photobleaching (FRAP). This information is crucial to an understanding of how and why FRAP works in a cell system. Further, understanding how to interpret the data sets requires a sound knowledge of the processes involved. Of primary importance are the nature of membrane diffusion and the nature of the multiple compartments into which fluorescent dyes can enter. The unit provides a complete discussion of all aspects of FRAP from the perspective of cellular measurements.

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.

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 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.

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.

Aging and the mobilization of intracellular calcium by phytohemagglutinin in human T cells.

Studies of T cell surface marker expression have shown that the diminished response of peripheral blood T cells from healthy older subjects to mitogens is not due to decreased expression of the CD3/Ti antigen receptor complex or imbalance in regulatory subsets. To determine if there is a defect in transmembrane signaling we have followed kinetics of increase in intracellular free calcium with Indo-1 after addition of phytohemagglutinin (PHA) to cells from healthy old and young adults. Using both flow cytometry and spectrofluorimetry, we find no differences in increase in mean calcium concentration and only a small decrease in number of cells with increased free calcium very early (1 minute after PHA) in the response of the older subjects' cells. It appears that the major age-associated defect(s) in signal transduction in human T cells may involve either early biochemical steps parallel to calcium mobilization or steps subsequent to transmembrane signaling.

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 dehydration of postmitotic cells as measured by X-ray microanalysis of bulk specimens.

In this paper we give a brief outline of our bulk specimen technique developed to measure intracellular water concentration in frozen-hydrated biological specimens by means of energy dispersive X-ray microanalysis. Fractured surface of the deep-frozen tissue samples is analyzed in an electron microscope (a specimen area of 15 x 11.5 micron is scanned) using 20 kV accelerating voltage and 1-5 pA effective beam current (measured in the specimen). Strong electric charging, which is the main problem associated with the low temperature X-ray microanalysis of frozen-hydrated specimens, is reduced by choosing optimum temperature range for the measurements (170-185 K) and by etching a thin surface layer on specimen surface. The main advantage of the method over other X-ray microanalytical techniques using sections and bulk specimens for water and dry-mass content determinations in cells (which are shortly reviewed) is the simple specimen preparation, the easy sample handling and the good stability of specimen during measurements. The main disadvantage is the poor spatial resolution as compared to the analysis of sections. Measurements with our method provided meaningful results of the change in intracellular water contents in various postmitotic cells of rats dependent on age. The observed decline of the intracellular water contents results in increased ionic strength and slower diffusion in old cells than in young ones. These effects may be implicated in senescent deterioration of cell metabolism.

Energy dispersive X-ray microanalysis of sulfated glycosaminoglycans in cartilage matrix stained with alcian blue 8GX.

X-ray spectra were recorded from 400-700 nm matrix areas of 0.5 micron sections prepared from the articular cartilages of 15- and 23-year-old human cadavers. The X-ray microanalysis was carried out (i) on untreated material; (ii) after removing sulfate group by a methylation procedure; (iii) after staining with a copper containing cationic phatolcyanin dye, alcian blue 8GX, preceded by carboxymethylation. K alpha peaks of sulphur could be detected in methylated (i.e. desulfated) samples. These peaks probably indicated the presence of sulphur-containing amino acids in different matrix proteins. Consequently, the measurements of sulphur despite its general use cannot be recommended for the X-ray microanalysis of sulfated glycosaminoglycans of cartilage matrix. K alpha peaks of copper could be identified after carboxymethylation and staining with alcian blue. After carboxymethylation, alcian blue can only be bound to the dissociated sulfate groups of glycosaminoglycans in the cartilage matrix. According to our spectrophotometric studies, approximately one molecule of alcian blue combined with one sulfate group. These data suggested that this technique could be used for semiquantitative estimation of sulfated glycosaminoglycans in small areas of the cartilage matrix. Using this method, we found a higher occurrence of sulfated glycosaminoglycans in the territorial matrix than in the interterritorial matrix of the intermediate and deep zones of the human articular cartilage.

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.

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.

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 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.

Alterations of the intracellular water and ion concentrations in brain and liver cells during aging as revealed by energy dispersive X-ray microanalysis of bulk specimens.

Age dependence of the intracellular concentrations of monovalent ions (Na+, K+ and Cl-) was examined in 1, 11 and 25-month-old rat brain and liver cells by using energy dispersive X-ray microanalysis. The in vivo concentrations of Na+, K+ and Cl- ions were calculated from two different measurements: The elemental concentrations were measured in freeze-dried tissue pieces, and the intracellular water content was determined by means of a recently developed X-ray microanalytic method, using frozen-hydrated and fractured bulk specimens as well as subsequent freeze-drying. All the single monovalent ion concentrations and consequently, also the total monovalent ion content showed statistically significant increases during aging in brain cortical neurons. A 3-6% loss of the intracellular water content was accompanied by a 25-45% increase of the monovalent ionic strengths by the age of 25 months. A membrane protective OH radical scavenger (centrophenoxine) reversed the dehydration in the nerve cells of old animals, resulting in a decrease of the intracellular ion concentrations. Aging has a less prominent effect on the water and ion contents of the hepatocytes. The degree of water loss of cytoplasm exceeds that of the nuclei in the liver, suggesting that dominantly the translational steps can be involved in the general age altered slowing down of the protein synthetic machinery, predicted by the membrane hypothesis of aging (Zs.-Nagy, 1978).

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.

Ion content and the response of L5178Y-R and L5178Y-S cells to X rays and ionophore A23187.

We examined the response of L5178Y-S (radiosensitive, LY-S) and L5178Y-R (radioresistant, LY-R) lymphoblasts to X-irradiation with concomittant treatment with divalent cation ionophore, A23187 (3 h or 5 h, 5 micrograms/ml). Cells treated with A23187 alone progressed through the cell cycle more slowly than the untreated cells and their cloning efficiency was reduced. In both cell strains the ionophore prolonged duration of the postirradiation mitotic delay. Radiation-induced inhibition of DNA synthesis was reversed by A23187 in LY-S but not in LY-R cells. Cells subjected to the ionophore treatment survived X-irradiation in almost the same way as untreated cells, as if the effect of A23187 treatment were reversed by irradiation. There was also a reversion in the ion content: A23187 caused a marked increase in Na+ content and a decrease in K+ content, irradiation itself did not change the ion content, whereas in the A23187-treated cells it restored almost the same pattern as that found in the control cells. We found less Mg2+ ions in LY-S cells after treatment with A23187 and A23187 + X than in LY-R cells, in relation to untreated (control) cells. These observations point to the possible importance of ion transport for recovery from radiation damage.