Changes from High Potassium (HK) to Low Potassium (LK) in Bovine Red Cells

Red cells of newborn calves contain 105–110 mmole K⁺ and 1–5 mmole Na⁺ per liter of cells. As the animals age the K⁺ content decreases to a value of 25–30 mmole/liter of cells after about 60 days. At approximately the same time, the sodium content reaches a value of 60–70 mmole/liter. The time required for half change (t_½) is 35–37 days for both Na⁺ and K⁺. The activity of (Na + K)-adenosine triphosphatase (ATPase) and the influx of K⁴² and Rb⁸⁶ into the red cells are high at birth and are reduced to 5 and 15% of their original values, respectively, in mature animals. t_½ for both is of the order of 30–35 days. The membrane Mg-ATPase activity is also high at birth and is reduced with a t_½ of 28–32 days to a final value of about 20% of its activity at birth. Separation of red cells according to their age showed that, in animals at the age of transition, newly formed red cells contain a higher K/Na ratio and a higher active transport capacity than older red cells of the same animal. It is suggested that the changes observed are a reflection of the average age of the red cell population as the animal grows.     

The permeability of the human erythrocyte to sodium and potassium

Measurements have been made on the permeability of the human erythrocyte to Na and K in vitro, using radioactive tracers to observe the system in the steady state. The average inward K flux is 1.67 m.eq./liter cells hour, and the apparent activation energy is 12,300 ± 1300 calories/mol. The inward K flux is independent of the external K concentration in the range of concentrations studied (4 to 16 m.eq. K/liter plasma). Rb appears to compete with K for transport into the cell, whereas Na and Li do not. The average inward Na flux is 3.08 ± 0.57 m.eq. Na/liter cells hour, and the apparent activation energies are 20,200 ± 2700 calories/mol for inward transport, and 14,900 ± 3,400 calories/mol for outward transport. The inward Na flux is dependent on the external Na concentration, but not in a linear fashion. Li appears to compete with Na for inward transport, whereas K and Rb do not. An approximate maximum estimate shows that the energy required for cation transport is only 8.8 calories/mol liter cells hour of the 110 calories/mol liter cells hour available from the consumption of glucose. A working hypothesis for the transport of Na and K is presented.     

ISOLATION OF CELL NUCLEI FROM THE MAMMALIAN CEREBRAL CORTEX AND THEIR ASSORTMENT ON A MORPHOLOGICAL BASIS

An aqueous method is described for the isolation of highly purified nuclei from the cerebral cortex of adult guinea pigs. Erythrocytes were removed by a short-time perfusion of the brain, myelin fragments by a rapid mechanical method, and blood capillaries by a centrifugal sieving through dense sucrose solutions. The nuclear preparation retained the activity of ATP:NMN adenylyltransferase. Recoveries of DNA in the P4I, P4II, P_L and P_S preparations were 30, 43, 8, and 7%, respectively. Microscopy and phase contrast microscopy showed a satisfactory removal of erythrocytes, myelin fragments, capillaries, and cytoplasmic elements. Biochemical purity of samples was verified by the absence of several cytoplasmic enzyme activities. In the electron microscope, the majority of nuclei showed well-preserved nuclear membranes, with nuclear pores, and were provided with a finely textured nucleoplasm. Occasional contaminants were elements of endoplasmic reticulum and of the endothelium. Assortment of nuclei on a morphological basis showed that 55–65% and 47–53% of nuclei in the P4I and P4II preparations, respectively, consisted of neuronal nuclei. In the P_L preparation, the population of neuronal nuclei ranged between 72 and 83%, while 94–99% of the nuclei in the P_S preparation consisted of smaller nuclei, most likely of oligodendroglial origin.     

Dramatic magnesium efflux induced by high potassium in rat thymocytes

When incubated in 150 mM KCl, rat thymocytes exhibited a very important magnesium efflux (11.4 +/- 0.7 mmoles/liter cells/20 min, n = 29), about 90 times higher than the physiological magnesium efflux catalyzed by the Na-Mg exchanger (0.126 +/- 0.093 mmoles/liter cells/20 min). Cells remained viable (trypan blue test) and membrane integrity was shown by the absence of an increase in sodium permeability. K(+)-induced magnesium efflux exhibited the following properties: (i) it required the presence of external chloride; (ii) it was fully blocked by DIOA, a selective KCl-cotransporter inhibitor (IC(50) = 35 microm); and (iii) it was associated to a progressive increase in cell volume via the DIOA-sensitive K-Cl cotransporter. Such cell swelling seems to play a causal role, because (i) hypertonic media (+400 mM sucrose) abolished K(+)-induced magnesium efflux and (ii) hypotonic Ringer media (205 mOsm) increased both cell volume and magnesium efflux (from a basal value of 0.35 +/- 0.03 mmoles/liter cells/20 min up to 1.44 +/- 0.24 mmoles/liter cells/20 min), even in the presence of DIOA. In conclusion, high potassium induced a dramatic release of intracellular magnesium from rat thymocytes. Such a phenomenon was, at least in part, caused by cell swelling via the DIOA-sensitive K-Cl cotransporter. The nature of the magnesium transport mechanism and its role in the transduction signal of K-Cl cotransporter activation by cell swelling deserve further investigation.     

MAINTENANCE OF IMAGINAL DISCS OF DROSOPHILA MELANOGASTER IN CHEMICALLY DEFINED MEDIA

A phosphate-buffered saline and a chemically defined synthetic medium for in vitro maintenance of imaginal discs of Drosophila melanogaster were developed. The composition of the chemically defined medium was varied in order to optimize the incorporation of tritiated uridine into RNA and tritiated amino acids into acid-insoluble protein. The optimal ranges obtained were: pH, 6.75–7.35; osmolarity, 285–345 milliosmoles/liter; sodium concentration, 40–60 mM/liter; potassium concentration, 40–60 mM/liter; magnesium concentration, 0.5–3.5 mM/liter; calcium concentration, 0.3–1.5 mM/liter; and inorganic phosphate concentration, 1.5–4.0 mM/liter. The phosphate-buffered saline is superior to a commonly used insect Ringer solution in maintaining total RNA and acid-insoluble protein synthesis in culture. The chemically defined synthetic medium permits linear total RNA and acid-insoluble protein synthesis for more than 48 hr, DNA synthesis for several hours, normal differentiation to occur after 74 hr in vitro, and trypsinization of imaginal discs into single cell suspensions without developmental damage.     

PROTEINS IN NUCLEOCYTOPLASMIC INTERACTIONS : II. Turnover and Changes in Nuclear Protein Distribution with Time and Growth

In previous studies, we showed that essentially all the proteins of the Amoeba proteus nucleus could be classified either as Rapidly Migrating Proteins (RMP), which shuttle between nucleus and cytoplasm continuously at a relatively rapid rate during interphase, or as Slow Turnover Proteins (STP), which seem to move hardly at all during interphase. In this paper, we report on the kinetics and direction of the movement of both classes of protein, as well as on aspects of their localization, with and without growth. The effects of growth were observed with and without cell division. These nuclear proteins have been studied in several ways: by transplantation of labeled nuclei into unlabeled cells and noting the rate of distribution to cytoplasm and host cell nuclei; by repeated amputation of cytoplasm from labeled cells—with and without initially labeled cytoplasm—each amputation being followed by refeeding on unlabeled food; by noting the redistribution of the various protein classes following growth and cell division. The data show (a) labeled RMP equilibrate between a grafted labeled nucleus and an unlabeled host nucleus in ca. 3 hr, but are detectable in the latter less than 30 min after the operation; (b) STP label does, indeed, leave the nucleus and does so at a rate of ca. 25% of the nuclear total per cell generation (ca. 36–40 hr at 23°C); (c) the cytoplasm appears to have a reserve of material that is converted to RMP; (d) when labeled cells are amputated just before they would have divided and are refed unlabeled food after each amputation, there is a loss of 20–25% of the nuclear protein label with each amputation; (e) under the latter circumstances, an essentially complete turnover of all nuclear protein can be demonstrated.     

Measurement of the Cytoplasmic and Vacuolar Buffer Capacities in Chara corallina

The cytoplasm and the vacuole were isolated from internodal cells of Chara corallina by using the intracellular perfusion technique, and their buffer capacities (β_i) were determined from the titration curves. The pH of the isolated vacuolar sap was 5.19 ± 0.029 (mean ± standard error). At this pH, β_i was minimal and amounted to 0.933 ± 0.11 millimoles H⁺/pH unit/liter vacuolar sap. The pH of isolated cytoplasm was 7.22 ± 0.028. β_i was minimal in this pH region and amounted to 14.2 ± 0.80 millimoles H⁺/pH unit/liter cytoplasm. When 1% (volume/volume) Triton X-100 was added to the cytoplasmic solution to permeabilize the subcellular organelles, the cytoplasmic pH increased to 7.32 ± 0.026, where β_i was 20.35 ± 2.66 millimoles H⁺/pH unit/liter cytoplasm. This shows that alkaline subcellular compartments exist in the cytoplasm and also that the cytoplasmic pH before adding Triton X-100 may represent the cytosolic pH. These data indicate that the pH values of the cytoplasm and the vacuole are regulated at the values where the β_i values are minimal. This suggests that ATP- and inorganic pyrophosphate-dependent H⁺ pumps in the plasma membrane and the tonoplast could efficiently regulate the pH of both cytoplasm and vacuole in Chara internodal cells.     

Cat Heart Muscle in Vitro : VIII. Active transport of sodium in papillary muscles

The cells of cat right ventricular papillary muscles were depleted of K and caused to accumulate Na and water by preincubation at 2–3°C. The time courses of changes in cellular ion content and volume and of the resting membrane potential (V_m) were then followed after abrupt rewarming to 27–28°C. At physiological external K concentration ([K]_o = 5.32 mM) recovery of cellular ion and water contents was complete within 30 minutes, the maximal observable rates of K uptake and Na extrusion (Δmmol cell ion/(kg dry weight) (min.)) being 3.4 and 3.6, respectively. The recovery rate was markedly slowed at [K]_o = 1.0 mM. Rewarming caused V_m measured in cells at the muscle surface to recover within from <1 to 9 minutes, but only slight restoration of cellular ion contents (measured in whole muscles) had occurred after 10 minutes. Studies of recovery in NaCl-free sucrose Ringer''s solution made it possible to separate the ouabain-insensitive outward diffusion of Na as a salt from a simultaneous ouabain-sensitive Na extrusion which is associated with a net cellular K uptake. A hypothesis consistent with these observations is that rewarming may activate a ouabain-sensitive "electrogenic" mechanism, most probably the net active transport of Na out of the cell, from which net K uptake may then follow passively.     

The transport of sodium into human erythrocytes in vivo

The relative Na²⁴ specific activity of red cells and plasma was measured at periods up to 30 hours following a single intravenous injection of Na²⁴ in normal healthy young adults. The average specific activity of the red cells relative to that of the plasma at 24 hours and beyond was found to average 0.83 ± 0.05 in a series of five normal individuals, significantly different from 1.0. This indicates that all the intracellular Na is not exchangeable in 24 hours, and confirms earlier in vitro results. The red cell Na concentration in man was shown to be 12.1 ± 1.1 m.eq. Na/liter red cell, as measured in a series of nineteen normal healthy young adults. A theoretical analysis of the data on exchangeable cell Na suggests that the red cell Na (5.3 m.eq. Na/liter blood) is divided into a fast compartment comprising 4.25 m.eq. Na/liter blood, and a slow compartment comprising 1.07 m.eq. Na/liter blood. If these compartments are arranged in parallel, the flux between plasma and fast compartment is 1.32 m.eq. Na/liter blood hour, and that between plasma and slow compartment is 0.016 m.eq. Na/liter blood hour. Results of experiments on two patients with congenital hemolytic jaundice suggest that the fraction of slowly exchanging Na may increase with the age of the red cell.     

Sodium absorption by intact sugar beet plants

Sodium absorption by intact sugar beet plants (Beta vulgaris) was found to be mediated by at least two distinct mechanisms when uptake was studied over a wide range of Na and K concentrations. The first mechanism operates at low Na concentrations (<1 milliequivalent per liter); presence of K completely blocks this mechanism for Na. The second mechanism operates at high Na concentrations (>1 milliequivalent per liter), transporting Na as well as K; but apparently this mechanism is not active for Na absorption in young sugar beet plants up to the 10-leaf stage.     

Proteins in nucleocytoplasmic interactions. I. The fundamental characteristics of the rapidly migrating proteins and the slow turnover proteins of the Amoeba proteus nucleus

By the transplantation of amino acid-³H-labeled nuclei between cells and the subsequent isolation of nuclei for quantitative assay, we have confirmed that all the nuclear proteins of Amoeba proteus are divisible into two classes that are sharply defined by their physiological behavior. About 40% of the proteins in the nucleus rapidly migrates back and forth between the nucleus and the cytoplasm. These rapidly migrating proteins (RMP) are 25–50 times more concentrated in the nucleus than in the cytoplasm, and migration into the nucleus therefore occurs against a high concentration differential. The remaining 60% of nuclear proteins has been classified as slow turnover proteins (STP) since (as reported in a following paper) virtually all of them ultimately undergo turnover. Turnover in this context means loss of label from the nucleus, by either protein breakdown or protein migration to the cytoplasm. Isolation of nuclei in the detergent Triton X-100 results in a 20% loss of nuclear proteins but conclusions about RMP and STP were not found to be significantly affected by this loss.     

The microbiology and biogeochemistry of the Dead Sea

The Dead Sea is a hypersaline water body. Its total dissolved salts content is on the average 322.6 gm/liter. The dominant cation is Mg (40.7 gm/liter), followed by Na (39.2 gm/liter), Ca (17 gm/liter) and K (7 gm/liter). The major anion is Cl (212 gm/liter), followed by Br (5 gm/liter); SO₄ and HCO₃, are very minor. The lake contains a limited variety of microorganisms and no higher organisms. The number of recorded species is very low, but the total biomass is reasonably high (about 10⁵ bacteria/ml and 10⁴ algal cells/ml). The indigenous flora is comprised mainly of obligate halophylic bacteria, such as the pink, pleomorphicHalobacterium sp., aSarcina-like coccus, and the facultative halophilic green alga,Dunaliella. Sulfate reducers can be isolated from bottom sediments. Recently a unique obligate magnesiophile bacteria was isolated from Dead Sea sediment. Several of the Dead Sea organisms possess unusual properties. TheHalobacterium sp. has extremely high intercellular K⁺ concentration (up to 4.8M) and extraordinary specificity for K⁺ over Na. TheDunaliella has very high intracellular concentration of glycerol (up to 2.1M). The microorganisms exert marked influence on some biogeochemical processes occurring in the lake, such as the control of the sulfur cycle and the formation and diagenesis of organic matter in the sediments. The Dead Sea is an excellent example of the development of two different mechanisms for adjusting to a hostile environment. The algae adjust to the high salinity by developing a mechanism for the exclusion of salts from the intracellular fluid and using glycerol for osmotic regulation. On the other hand, the bacteria adapt to the environment by adjusting their internal inorganic ionic strength, but not composition, to that of the medium. The problem of population dynamics and limiting factors for algal and bacterial productivity are discussed in view of the total absence of zooplankton and other consumers other than bacteria.     

Lysosome-mediated processing of chromatin in senescence

Cellular senescence is a stable proliferation arrest, a potent tumor suppressor mechanism, and a likely contributor to tissue aging. Cellular senescence involves extensive cellular remodeling, including of chromatin structure. Autophagy and lysosomes are important for recycling of cellular constituents and cell remodeling. Here we show that an autophagy/lysosomal pathway processes chromatin in senescent cells. In senescent cells, lamin A/C–negative, but strongly γ-H2AX–positive and H3K27me3-positive, cytoplasmic chromatin fragments (CCFs) budded off nuclei, and this was associated with lamin B1 down-regulation and the loss of nuclear envelope integrity. In the cytoplasm, CCFs were targeted by the autophagy machinery. Senescent cells exhibited markers of lysosomal-mediated proteolytic processing of histones and were progressively depleted of total histone content in a lysosome-dependent manner. In vivo, depletion of histones correlated with nevus maturation, an established histopathologic parameter associated with proliferation arrest and clinical benignancy. We conclude that senescent cells process their chromatin via an autophagy/lysosomal pathway and that this might contribute to stability of senescence and tumor suppression.     

Na-NMR Studies of the Intracellular Sodium Ion Concentration in the Halotolerant Alga Dunaliella salina

The Intracellular Na⁺ concentration in the halotolerant alga Dunaliella salina was measured in intact cells by ²³Na-NMR spectroscopy, utilizing the dysprosium tripolyphosphate complex as a sodium shift reagent, and was found to be 88 ± 28 millimolar. Intracellular sodium ion content and intracellular volume were the same, within the experimental error, in cells adapted to grow in media containing between 0.1 and 4.0 molar NaCl. These values assume extracellular and intracellular NMR visibilities of the ²³Na nuclei of 100 and 40%, respectively. The relaxation rate of intracellular sodium was enhanced with increasing salinity of the growth medium, in parallel to the intracellular osmosity due to the presence of glycerol, indicating that Na⁺ ions and glycerol are codistribbuted within the cell volume.     

Ion metabolism in a Halobacterium. I. Influence of age of culture on intracellular concentrations

Work is described on the changes in cell ions during growth of cultures of a species of Halobacterium isolated from the Dead Sea. Cell K concentration fell from 5.5 to 3.8 moles per kg cell water during the logarithmic phase of growth and maintained the latter value during the stationary phase (initial medium concentration, 7 mM). Cell Na and Cl followed a complex series of roughly parallel changes. The logarithmic phase ion concentrations were: Na, 1.0–2.3 moles/kg cell water; Cl, 2.3–3.7 moles/kg cell water. The final stationary phase values were: Na, 0.5 moles/kg cell water; Cl, 2.3–2.9 moles/kg cell water (medium NaCl concentration, 3.9 Molal). It is suggested that most of the K⁺ is bound within the cytoplasm.     

X-ray microanalysis of ion distribution within root cortical cells of the halophyte Suaeda maritima (L.) Dum.

The ion content of compartments within cortical cells of mature roots of the halophyte Suaeda maritima grown at 200 mol·m^-3 NaCl has been studied by X-ray microanalysis of freeze-substituted thin sections. Sodium and Cl were found in the vacuoles at about four-times the concentration in the cytoplasm or cell walls, whereas K was more concentrated in the cell walls and cytoplasm than in vacuoles. The vacuolar Na concentration was 12- to 13-times higher than that of K. The Na concentration of cell walls of cortical cells was about 95 mol·m^-3 of analysed volume. The cytoplasmic K concentration within the mature cortical cells was estimated to be 55 mol·m^-3 of analysed volume.     

Distribution of proteins between nucleus and cytoplasm of amoeba proteus

By transplanting nuclei between labeled and unlabeled cells, we determined the localization of the major proteins of amebas and described certain features of their intracellular distributon. We identified approximately 130 cellular proteins by fluorography of one-dimensional polyacrylamide electrophoretic gels and found that slightly less than half of them (designated NP, for nuclear proteins) are almost exclusively nuclear. About 95 percent of the other proteins (designated CP for cytoplamsic proteins) are roughly equally concentrated in nucleus and cytoplasm, but—because the cytoplasm is 50 times larger than the nucleus—about 98 percent of each of the latter is in the cytoplasm. Of the CP, roughly 5 percent are not detectable in the nucleus. Assuming that these are restricted to the cytoplasm only because, for example, they are in structures too large to enter the nucleus and labeled CP readily exit a nucleus introduced into unlabeled cytoplasm, we conclude that the nuclear envelope does not limit the movement of any nonstructural cellular protein in either direction between the two compartments. Some NP are not found in the cytoplasm (although ostensibly synthesized there) presumably because of preferential binding within the nucleus. Almost one half of the protein mass in nuclei in vivo is CP and apparently only proteins of that group are lost from nuclei when cells are lysed. Thus, while an extracellular environment allows CP to exit isolated nuclei, the nuclear binding affinities for NP are retained. Further examination of NP distribution shows that many NP species are, in fact, detectable in the cytoplasm (although at only about 1/300 the nuclear concentration), apparently because the nuclear affinity is relatively low. These proteins are electrophoretically distinguishable from the high-affinity NP not found in the cytoplasm. New experiments show that an earlier suggestion that the nuclear transplantation operation causes an artifactual release of NP to the cytoplasm is largely incorrect. Moreover, we show that cytoplasmic “contamination” of nuclear preparations is not a factor in classifying proteins by these nuclear transplantation experiments. We speculate the no mechanism has evolved to confine most CP to the cytoplasm (where they presumably function exclusively) because the cytoplasm’s large volume ensures that CP will be abundant there. Extending Bonner’s idea of “quasi-functional nuclear binding sites” for NP, we suggest that a subset of NP usually have a low affinity for available intranuclear sites because their main function(s) occurs at other intranuclear sites to which they bind tightly only when particular metabolic conditions demand. The other NP (those completely absent from cytoplasm) presumable always are bound with high affinity at their primary functional sites.     

Cat Heart Muscle in Vitro : I. Cell volumes and intracellular concentrations in papillary muscle

Methods have been developed for the simultaneous determination of total water, inulin space, and K and Na content in muscles of 0.5 to 10 mg. wet weight. These methods have been used to define steady state conditions with respect to intracellular K concentration in papillary muscles from cat hearts perfused and contracting isometrically at 27–28°C. and at 37–38°C. Cell volumes and intracellular ionic concentrations have been followed as a function of the external K concentration and compared with values predicted on the basis of electroneutrality and osmotic equilibrium.     

Outward sodium and potassium cotransport in human red cells

Summary This paper reports some kinetic properties of Na–K cotransport in human red cells. All fluxes were measured in the presence of 10^–4 M ouabain. We measured Na and K efflux from cells loaded by the PCMBS method to contain different concentrations of these ions into a medium that contained neither Na nor K (MgCl₂-sucrose substitution) in the absence and presence of furosemide. Furosemide inhibited 30–60% of the total efflux depending on the internal ion concentration and the individual subject. We took the furosemide-sensitive fluxes to be a measure of Na–K cotransport. The ratio of Na to K cotransport was 1 over the entire range of internal Na and K concentrations studied. When Na was substituted for K as the only internal cation, cotransport was maximally activated when the Na and K concentrations were between 20 and 90 mmol/liter cells. The concentration of internal Na required to produce half-maximal cotransport was about 13±4 mmol/liter cells (n=4), while the comparable concentration of K was somewhat lower. The activation curve was definitely sigmoid in character, suggesting that at least two Na ions are involved in the transport process. The maximum of Na–K cotransport was about 0.5±0.15 mmol/liter cells × hr (n=5); it had a flat maximum in the medium at about pH 7.0, decreasing in both the acid and alkaline sides. furosemide-resistant effluxes were found to be linear functions of internal Na and K concentrations and to yield rate coefficients of 0.019±0.002 hr^–1 and 0.014±0.002 hr^–1 (n=7), respectively. These values are of the same order of magnitude expected of ions moving across phospholipid bilayers.Charge de Recherches CNRS.     

The Effect of Ouabain and External Potassium on the Ion Transport of Rabbit Red Cells

Na efflux of rabbit RBC is approximately 10 mmoles/kg wet weight. hr. One-half of this consists of a ouabain-insensitive exchange diffusion component. Ouabain inhibits 2.5 mmoles/kg.hr of Na efflux. K influx is 3.0 mmoles/kg.hr; 2.2 mmoles/kg.hr are inhibited by ouabain. In contrast with human RBC, ouabain inhibition of Na efflux and K influx of rabbit RBC is easily reversible. After 2 hr, ouabain inhibition of Na efflux is completely compensated for by increased internal Na concentration and Na efflux returns to initial levels. Removal of ouabain at this stage results in stimulation of the efflux by 4.3 mmoles/kg.hr. Na influx is initially not affected by ouabain but is increased by 2.4 mmoles/kg.hr after 2 hr incubation with the drug. Removal of K from normal Ringer does not affect Na efflux and increases Na influx by 1.6 mmoles/kg.hr. Addition of ouabain to K-free Ringer inhibits Na efflux and influx to the same extent (1.6 mmoles/kg.hr). Removal of Na from K-free Ringer has an inhibitory effect on efflux similar to that of ouabain. These findings suggest that the fraction of Na efflux inhibited by removal of external K is completely replaced by a new, ouabain-sensitive exchange diffusion of Na ions.