Studies on the physical state of water in living cells and model systems: IX. Theoretical significance of a straight line relationship between intracellular concentration of a partially excluded solute and its concentration in the bathing medium

The experimentally observed steady-level distribution of Na+ (25 degrees) and of D-glucose (0 degree c) in frog muscle were chosen as examples of solute distribution patterns observed in living cells, for comparison with those predicted by two theoretical models: one derived from the membrane-pump theory and the other from the association-induction (AI) hypothesis. Neither the distribution of Na+ nor that of D-glucose follows the pattern predicted by the membrane-pump models for solutes maintained at lower level than in the external medium, in which the plot of intracellular solute concentration as ordinate against different external concentrations as abscissa bends upward with increasing external solute concentration. Instead, both Na+ and D-glucose exhibit either straight line distribution with unchanging (below unity) slopes, or that of a hyperbola superimposed on such a straight line, both in agreement with the AI hypothesis.     

Basic biological research with the striated muscle by using cryotechniques and electron microscopy.

Several basic mechanisms underlying living phenomena are not really understood. Unequivocal interpretations of data concerning the following phenomena--to name but a few--are missing: cellular accumulation of potassium; cellular exclusion of sodium, cell volume regulation, shape change of cells (e.g. of muscle cells during contraction), electrical potential differences between inside and outside of living cells. The theoretical treatment of these phenomena as found in all current textbooks is based on the membrane-pump theory (MPT) with the following essential features. The bulk of the main cellular cation K+ is freely dissolved in free cellular water and membrane-situated pumps are responsible for the high level of K+ and the low level of Na+ found in virtually all living cells. On the other hand, the above mentioned phenomena are explained by the association-induction hypothesis (AIH) without the proposal of membrane-situated pumps and with the postulations of selective K+ adsorption to cellular proteins and of a specific cell water structure which has a low solvency for Na+ and other solutes. Experimental findings are reviewed which contradict the MPT and support the AIH. In addition, electron microscopic experiments with cryoprocessed striated muscle are reviewed which establish cellular K+ binding (adsorption) and a cellular water structure which is different from that of normal free water. Cryoexperiments with the striated muscle and model systems are proposed which may help to obtain further information on the specific interactions between proteins, ions, and water in living cells.     

Membrane-linked energy transductions. Bioenergetic functions of sodium: H+ is not unique as a coupling ion

The concept is developed according to which Na+, like H+, can play the role of a coupling ion in energy-transducing biomembranes. This idea is based on observations that (i) Na+ can be extruded from the cell by primary pumps (Na-motive NADH-quinone reductase, decarboxylase or ATPase), and (ii) the downhill Na+ flux into the cell can be coupled with the performance of all the three types of membrane-linked work i.e. chemical (ATP synthesis), osmotic (accumulation of solutes) and mechanical (motility). Marine alkalotolerant Vibrio alginolyticus represents the first example of such a complete sodium cycle pattern. Simplified versions of the sodium cycle or some of its constituents are found in the cytoplasmic membrane of a great variety of taxa including anaerobic, aerobic and photosynthetic bacteria, cyanobacteria and animals; this fact indicates that Na+ energetics should be regarded as a common case, rather than a rare exception applied to some natural niches only.     

Cell volumes and water contents of frog muscles in solutions of permeant sugars and sugar alcohols

1. Previous work has suggested that living cells may acquire and then maintain different water contents and hence volume, in solutions containing different concentrations of solutes that are permeant to the cell membrane. Toward better understanding of this phenomenon, two hypotheses were introduced: one hypothesis is based on the membrane-pump theory; another represents an extension of the polarized multilayer theory of cell water, a part of the association-induction (AI) hypothesis. To test the different predictions of these hypotheses, the water contents of frog muscle equilibrated at 25 degrees C in solutions of different concentrations of seven pentoses, seven hexoses, seven dissacharides, two trisaccharides, and six sugar alcohols were determined. 2. The earlier finding of sustained shrinkage of muscle cells in concentrated solutions of permeant solutes was confirmed once more. 3. In equimolal solutions of sugars and sugar alcohols with different steric conformations but the same or closely similar molecular weight(s), muscles had the same or closely similar water content(s). 4. In equimolal solutions of different sugars and sugar alcohols, the equilibrium water contents of the muscles increased with decreasing molecular weights of these solutes. 5. The water contents of muscles, equilibrated in 0.4 M solutions of different sugars and sugar alcohols, are positively correlated with the equilibrium distribution coefficients (or q-values) of the sugar and sugar alcohols in the muscle cell water with a linear correlation coefficient of +0.973. 6. The relationships between the equilibrium water contents of muscles (in solutions containing different concentrations of different sugars and sugar alcohols) and the concentrations of these sugars and sugar alcohols agree in general contours with that predicted by an equation derived on the basis of the polarized multilayer theory of cell water. 7. The experimental findings described above do not agree with the prediction based on the membrane-pump hypothesis; they do agree with all four predictions of the hypothesis based on the polarized multilayer theory of cell water.     

Trimethylamine oxide and the maintenance of volume of dogfish shark rectal gland cells

Determinants of the steady-state vol of the dogfish shark (Squalus acanthias) rectal gland cells were studied. The cellular levels of trimethylamine oxide (TMAO) in fresh tissue and slices incubated aerobically 60 min in standard (TMAO-free) elasmobranch saline were close to those in the plasma (71 +/- 5 mM S.E.M.); therefore, under these conditions, the cell membrane appears to be impermeable to this solute. However, depolarization of the cells in high-K+ media produced a rapid loss of TMAO. Thus, TMAO is a major, effectively impermeant solute in the rectal gland cells. The osmolarity of cell solutes in tissue water (fresh and incubated slices) did not differ significantly from values in the plasma or incubation medium, demonstrating the absence of an osmotic pressure gradient across the cell membrane. An analysis of a simple model of cell solutes under steady-state conditions shows that the presence of an (effectively) impermeant osmolyte decreases the cellular concentration of bulk cations. The analysis is consistent with available observations on the distribution of cell Na+ and K+ in tissues containing high concentrations of (nitrogeneous) osmolytes. One simplifying assumption of the model, i.e., identity (or closeness) of the respective reflection coefficients sigma for Na+ and K+ passage through the cell membranes could not be verified. Compared to available data on the steady-state cellular fluxes of 42K+ in slices of the rectal gland, the uptake of 22Na+ by the tissue was slow (the derived rate constant k' = 0.017 min-1, i.e., about one tenth of that for K+).(ABSTRACT TRUNCATED AT 250 WORDS)     

Exogenously supplied compatible solutes rapidly ameliorate NaCl-induced potassium efflux from barley roots

It has been suggested that the role of compatible solutes in plant stress responses is not limited to conventional osmotic adjustment, but also includes some other regulatory or osmoprotective functions. In this study, we hypothesized that one such function is in maintaining cytosolic K+ homeostasis by preventing NaCl-induced K+ leakage from the cell, a feature that may confer salt tolerance in many species, particularly in barley. This hypothesis was investigated using the non-invasive microelectrode ion flux (MIFE) measuring technique. We show that low (0.5-5 mM) concentrations of exogenously supplied proline or betaine significantly reduced NaCl-induced K+ efflux from barley roots in a dose-response manner. This effect was instantaneous, implying that large intracellular concentrations of compatible solutes are not required for an amelioratory role. Exogenously supplied betaine also significantly enhanced NaCl-induced H+ efflux, but only in pre-incubated roots, implying some alternative mechanism of regulation. Sap K+ and Na+ analysis and membrane potential measurements are also consistent with the model that one function of compatible solutes is in maintaining cytosolic K+ homeostasis by preventing NaCl-induced K+ leakage from the cell, possibly through the enhanced activity of H+-ATPase, controlling voltage-dependent outward-rectifying K+ channels and creating the electrochemical gradient necessary for secondary ion transport processes. These data provide the first direct evidence for regulation of ion fluxes across the plasma membrane by physiologically relevant low concentrations of compatible solutes.     

Solute transport process in intestinal epithelial cells

In rat small intestine, the active transport of organic solutes results in significant depolarization of the membrane potential measured in an epithelial cell with respect to a grounded mucosal solution and in an increase in the transepithelial potential difference. According to the analysis with an equivalent circuit model for the epithelium, the changes in emf's of mucosal and serosal membranes induced by active solute transport were calculated using the measured conductive parameters. The result indicates that the mucosal cell membrane depolarizes while the serosal cell membrane remarkably hyperpolarizes on the active solute transport. Corresponding results are derived from the calculations of emf's in a variety of intestines, using the data that have hitherto been reported. The hyperpolarization of serosal membrane induced by the active solute transport might be ascribed to activation of the serosal electrogenic sodium pump. In an attempt to determine the causative factors in mucosal membrane depolarization during active solute transport, cell water contents and ion concentrations were measured. The cell water content remarkably increased and, at the same time, intracellular monovalent ion concentrations significantly decreased with glucose transport. Net gain of glucose within the cell was estimated from the restraint of osmotic balance between intracellular and extracellular fluids. In contrast to the apparent decreases in intracellular Na+ and K+ concentrations, significant gains of Na+ and K+ occurred with glucose transport. The quantitative relationships among net gains of Na+, K+ and glucose during active glucose transport suggest that the coupling ratio between glucose and Na+ entry by the carrier mechanism on the mucosal membrane is approximately 1:1 and the coupling ratio between Na+-efflux and K+-influx of the serosal electrogenic sodium pump is approximately 4:3 in rat small intestine. In addition to the electrogenic ternary complex inflow across the mucosal cell membrane, the decreases in intracellular monovalent ion concentrations, the temporary formation of an osmotic pressure gradient across the cell membrane and the streaming potential induced by water inflow through negatively charged pores of the cell membrane in the course of an active solute transport in intestinal epithelial cells are apparently all possible causes of mucosal membrane depolarization.     

Na+K+-ATP酶抑制引起的细胞凋亡和杂合性细胞死亡

Na^ -K^ -ATP酶也称Na^ 泵或Na^ -K^ 泵,是哺乳类细胞膜进行离子转运的跨膜载体蛋白。其基本作用是维持细胞膜内外Na^ -K^ 电化学梯度的平衡。近来研究表明,Na^ -K^ -ATP酶在细胞死亡中起重要作用,细胞K 缺失导致凋亡,在某些类型的细胞中,同一细胞兼具细胞肿胀、细胞器溶解等坏死特征和染色质凝集、DNA梯带、caspase级联反应等凋亡特征,呈现一种特殊的细胞死亡形式,即杂合性细胞死亡。     

Application of the Na+ recirculation theory to ion coupled water transport in low- and high resistance osmoregulatory epithelia

The theory of Na+ recirculation for isosmotic fluid absorption follows logically from Hertz's convection-diffusion equation applied to the exit of water and solutes from the lateral intercellular space. Experimental evidence is discussed indicating Na+ recirculation based upon the following approaches: (i) An isotope tracer method in small intestine. Simultaneous measurement of water flow and ion transport in toad skin epithelium demonstrating, (ii) occasional hyposmotic absorbates, and (iii) reduced fluid absorption in the presence of serosal bumetanide. (iv) Studies of the metabolic cost of net Na+ absorption demonstrating an efficiency that is lower than the 18 Na+ per O2 consumed given by the stoichiometry of the Na+/K+-pump. Mathematical modeling predicts a significant range of observations such as isosmotic transport, hyposmotic transport, solvent drag, anomalous solvent drag, the residual hydraulic permeability in proximal tubule of AQP1(-/-) mice, the adverse relationship between hydraulic permeability and the concentration difference needed to reverse transepithelial water flow, and in a non-contradictory way the wide range of metabolic efficiencies from above to below 18 Na+/O2. Certain types of observations are poorly or not at all reproduced by the model. It is discussed that such lack of agreement between model and experiment is due to cellular regulations of ion permeabilities that are not incorporated in the modeling. Clarification of these problems requires further experimental studies.     

Na+ modulates the K+ permeability and the membrane potential of alkalophilic Bacillus

In the absence of Na+ in the medium, the membrane potential of obligately alkalophilic Bacillus cells was found to be decreased by the addition of K+ to the medium, whereas K+ addition in the presence of Na+ had no effect. Rb+ showed essentially the same effect as K+. The decreased membrane potential was quickly restored by lowering the K+ concentration in the medium or by adding Na+ or Li+ to the medium. Thus, in the absence of Na+, the membrane potential of alkalophilic Bacillus seems to be affected by the concentration difference of K+ between inside and outside of the cell, and Na+ or Li+ in the medium suppresses the K+ effect. An exchange between extracellular Rb+ and intracellular K+ was observed in the absence of Na+. However, the exchange was greatly suppressed by the addition of Na+ or Li+ to the medium, indicating that Na+ in the medium modulates the K+ permeability of the alkalophilic Bacillus cell membrane. The K+-induced decrease in the membrane potential of alkalophilic Bacillus in the absence of Na+ is accounted for by the increased K+-permeability of the cell membrane.     

Distribution of sodium-potassium ATPase in the rat testis and epididymis.

Sodium-potassium ATPase (Na+K(+)-ATPase) is a ubiquitous plasma membrane enzyme which uses the hydrolysis of ATP to regulate cellular Na+ and K+ levels and fluid volume. This ion pumping action is also thought to be involved in fluid movement across certain epithelia. There are several different genes for this enzyme, some of which are tissue specific. Using an antibody specific for the catalytic subunit of canine kidney Na+K(+)-ATPase, we have localized immunoreactivity in the seminiferous and epididymal epithelium of rats of various ages. There was no specific staining of 10-day-old rat testis. Faint staining was detected at 13 days and appeared to be associated with the borders of Sertoli cells. At 16 days prominent apical and lateral staining but no basal staining of Sertoli cell membranes was observed. This type of distribution continued until spermatids were present in the epithelium. In the adult rat testis, specific staining was detected in Sertoli cell crypts associated with elongating spermatids, and on the apical and lateral Sertoli cell membrane. In some instances immunoreactivity was concentrated at presumed sites of junctional specializations. In the excurrent ducts of immature and mature rats, Na+K(+)-ATPase staining was heavy in the efferent ducts and somewhat lighter in the epididymis. In all regions, the staining was basolateral although there were variations in intensity among the different parts of the epididymis. These results show 1) that rat testis and epididymal Na+K(+)-ATPase share some immunological determinants with the canine enzyme; 2) that the epididymal enzyme is located in the conventional basolateral position; and 3) that the distribution of Sertoli cell Na+K(+)-ATPase is probably apical and lateral rather than basal.     

Intracellular activities during volume regulation byNecturus gallbladder

Summary Necturus gallbladder epithelial cells regulate their volume after a change in solution osmolality. We determined the intracellular activities of Na, K and Cl when the mucosal bathing solution osmolality was increased 18% by the addition of mannitol. The gallbladder was mounted in a rapid flow chamber and punctured simultaneously with two single-barrelled microelectrodes. One electrode sensed membrane potential and the other was sensitive to the activity of Na, K or Cl. Cell volume measurements, made in previous studies utilizing quantitative light microscopy, indicated that hypertonicity of the mucosal bath first caused a cell shrinkage of 15% followed by volume readjustment. Some loss of Na, K and Cl was observed during shrinkage; subsequently during volume regulation, the intracellular quantities of all three ions increased. The loss of Na during the initial cell shrinkage could be blocked by ouabain and was therefore due to increased transport. K and Cl losses were probably related to the increase in their concentrations during shrinkage. The gain of Na, K and Cl during volume regulation was similar in magnitude to the loss of these solutes during cell shrinkage. The increase of Na, K and Cl during volume regulation accounted for about 60% of the increase of cell solutes during this period indicating that other solutes also contributed to the volume regulation response.     

Effect of ionophore A23187 on the membrane permeability in mouse fibroblasts.

Addition of the divalent cation ionophore A23187 to transformed mouse fibroblasts (3T6) resulted in an increase in the cell membrane permeability to normally impermeant solutes (e.g., nucleotides). The membrane permeability was assessed by following the efflux of prelabeled adenine nucleotides, the influx of p-nitrophenyl phosphate in cells attached to plastic dishes and reconstitution of intracellular protein synthesis in the presence of exogenously added normally impermeant factors required for macromolecular synthesis. The permeability change of 3T6 cells was found to be dependent on the specific presence of external calcium ion. The permeabilization was found to occur preferably in alkaline pH and specific to certain transformed cells. It is preceded by rapid efflux of K+, influx of Na+ and partial hydrolysis of cellular nucleotides in 3T6 cells. Similar ion fluxes were previously found to precede cell permeabilization by electrogenic ionophores for monovalent ions and by exogenous ATP. Our data suggest that a calcium dependent process caused the K+ release and excess Na+ entry, causing dissipation of the membrane potential and subsequent formation of aqueous channels.     

Human and dog erythrocytes: relationship between cellular ATP levels, ATP consumption and potassium concentrations.

The intracellular K+/Na+ ratio of various mammalian cell types are known to differ remarkably. Particularly noteworthy is the fact that erythrocytes of different mammalian species contain entirely different potassium and sodium concentrations. The human erythrocyte is an example of the supposedly "normal" high potassium cell, while the dog erythrocyte contains ten times more sodium than potassium ions (Table I). Furthermore, this difference is sustained despite the plasma sodium and potassium concentrations being almost identical in both species (high Na+ and low K+). In spite of these inorganic ion differences, both human and dog erythrocytes contain 33% dry material (mostly Hb) and 67% water. Conventional cell theory would couple cellular volume regulation with Na+ and K+ dependent ATPase activity which is believed to control intracellular Na+/K+ concentrations. Since the high Na+ and low K+ contents of dog erythrocytes are believed to be due to the lack of the postulated Na/K-ATPase enzyme, they must presumably have an alternative mechanism of volume regulation, otherwise current ideas of membrane ATPase activity coupled volume regulation need serious reconsideration. The object of our investigation was to explore the relationship between ATPase activity, ATP levels and the Na+/K+ concentrations in human and dog erythrocytes. Our results indicate that the intracellular ATP level in erythrocytes correspond with their K+, Na+ content. They are discussed in relation to conventional membrane transport theory and also to Ling's "association-induction hypothesis", the latter proving to be a more useful basis on which to interpret results.     

The influence of cellular amino acids and the Na+ : K+ pump on the membrane potential of the Ehrlich ascites tumor cell.

The membrane potential of the Ehrlich ascites tumor cell was shown to be influenced by its amino acid content and the activity of the Na+ :K+ pump. The membrane potential (monitored by the fluorescent dye, 3,3'-dipropylthiodicarbocyanine iodide) varied with the size of the endogenous amino acid pool and with the concentration of accumulated 2-aminoisobutyrate. When cellular amino acid content was high, the cells were hyperpolarized; as the pool declined in size, the cells were depolarized. The hyperpolarization seen with cellular amino acid required cellular Na+ but not cellular ATP. Na+ efflux was more rapid from cells containing 2-aminoisobutyrate than from cells low in internal amino acids. These observations indicate that the hyperpolarization recorded in cells with high cellular amino acid content resulted from the electrogenic co-efflux of Na+ and amino acids. Cellular ATP levels were found to decline rapidly in the presence of the dye and hence the influence of the pump was seen only if glucose was added to the cells. When the cells contained normal Na+ (approx. 30mM), the Na+ :K+ pump was shown to have little effect on the membrane potential (the addition of ouabain had little effect on the potential). When cellular Na+ was raised to 60mM, the activity of the pump changed the membrane potential from the range -25 to -30 mV to -44 to -63 mV. This hyperpolarization required external K+ and was inhibited by ouabain.     

Capacity of the outer membrane of a gram-negative marine bacterium in the presence of cations to prevent lysis by Triton X-100.

Cells of marine pseudomonad B-16 (ATCC 19855) washed with a solution containing 0.3 M NaCl, 50 mM MgCl2, and 10 mM KCl (complete salts) could be protected from lysis in a hypotonic environment if the suspending medium contained either 20 mM Mg2+, 40 mM Na+, or 300 mM K+. When the outer double-track layer (the outer membrane) of the cell envelope was removed to yield mureinoplasts, the Mg2+, Na+ or K+, requirements to prevent lysis were raised to 80, 210, and 400 mM, respectively. In the presence of 0.1% Triton X-100, 220, 320, and 360 mM Mg2+, Na+ or K+, respectively, prevented lysis of the normal cells. Mureinoplasts and protoplasts, however, lysed instantly in the presence of the detergent at all concentrations of Mg2+, Na+, or K+ tested up to 1.2 M. Thus, the structure of the outer membrane appears to be maintained by appropriate concentrations of Mg2+ or Na+ in a form preventing the penetration of Triton X-100 and thereby protecting the cytoplasmic membrane from dissolution by the detergent. K+ was effective in this capacity with cells washed with complete salts solution but not with cells washed with a solution of NaCl, suggesting that bound Mg2+ was required in the cell wall membrane for K+ to be effective in preventing lysis by the detergent. At high concentrations (1 M) K+ and Mg2+, but not Na+, appeared to destabilize the structure of the outer membrane in the presence of Triton X-100.     

Membrane domains of intestinal epithelial cells: distribution of Na+,K+- ATPase and the membrane skeleton in adult rat intestine during fetal development and after epithelial isolation

The organization of the basolateral membrane domain of highly polarized intestinal absorptive cells was studied in adult rat intestinal mucosa, during development of polarity in fetal intestine, and in isolated epithelial sheets. Semi-thin frozen sections of these tissues were stained with a monoclonal antibody (mAb 4C4) directed against Na+,K+-ATPase, and with other reagents to visualize distributions of the membrane skeleton (fodrin), an epithelial cell adhesion molecule (uvomorulin), an apical membrane enzyme (aminopeptidase), and filamentous actin. In intact adult epithelium, Na+,K+-ATPase, membrane-associated fodrin, and uvomorulin were concentrated in the lateral, but not basal, subdomain. In the stratified epithelium of fetal intestine, both fodrin and uvomorulin were localized in areas of cell-cell contact at 16 and 17 d gestation, a stage when Na+,K+-ATPase was not yet expressed. These molecules were excluded from apical domains and from cell surfaces in contact with basal lamina. When Na+,K+-ATPase appeared at 18-19 d, it was codistributed with fodrin. Detachment of epithelial sheets from adult intestinal mucosa did not disrupt intercellular junctions or lateral cell contacts, but cytoplasmic blebs appeared at basal cell surfaces, and a diffuse pool of fodrin and actin accumulated in them. At the same time, Na+,K+-ATPase moved into the basal membrane subdomain, and extensive endocytosis of basolateral membrane, including Na+,K+-ATPase, occurred. Endocytosis of uvomorulin was not detected and no fodrin was associated with endocytic vesicles. Uvomorulin, along with some membrane-associated fodrin and some Na+,K+-ATPase, remained in the lateral membrane as long as intercellular contacts were maintained. Thus, in this polarized epithelium, interaction of lateral cell-cell adhesion molecules as well as basal cell-substrate interactions are required for maintaining the stability of the lateral membrane skeleton and the position of resident membrane proteins concentrated in the lateral membrane domain.     

Kidney Na+,K(+)-ATPase is associated with moesin

Na+,K(+)-ATPase is a ubiquitous plasmalemmal membrane protein essential for generation and maintenance of transmembrane Na+ and K+ gradients in virtually all animal cell types. Activity and polarized distribution of renal Na+,(+)-ATPase appears to depend on connection of ankyrin to the spectrin-based membrane cytoskeleton as well as on association with actin filaments. In a previous study we showed copurification and codistribution of renal Na+,K(+)-ATPase not only with ankyrin, spectrin and actin, but also with two further peripheral membrane proteins, pasin 1 and pasin 2. In this paper we show by sequence analysis through mass spectrometry as well as by immunoblotting that pasin 2 is identical to moesin, a member of the FERM (protein 4.1, ezrin, radixin, moesin) protein family, all members of which have been shown to serve as cytoskeletal adaptor molecules. Moreover, we show that recombinant full-length moesin as well as its FERM domain bind to Na+,K(+)-ATPase and that this binding can be inhibited by an antibody specific for the ATPase activity-containing cytoplasmic loop (domain 3) of the Na+,K(+)-ATPase alpha-subunit. This loop has been previously shown to be a site essential for ankyrin binding. These observations indicate that moesin might not only serve as direct linker molecule of Na+,K(+)-ATPase to actin filaments but also modify ankyrin binding at domain 3 of Na+,K(+)-ATPase in a way similar to protein 4.1 modifying the binding of ankyrin to the cytoplasmic domain of the erythrocyte anion exchanger (AE1).     

A unitary cause for the exclusion of Na+ and other solutes from living cells, suggested by effluxes of Na+, D-arabinose, and sucrose from normal, dying, and dead muscles

1. The effluxes of labeled Na+, D-arabinose, and sucrose from normal muscle and muscle poisoned with low concentrations of iodoacetate were studied. The procedure involved repeated loading with isotope, followed by washing of the same muscle while still normal and at different states of dying. 2. The rates of Na+ efflux in both the fast and slow fraction remained either quite constant or showed some unpredictable, minor fluctuations. This was true for both Na+ and the two sugars studied, confirming earlier conclusions that the steady levels of these solutes were not maintained by pumps. 3. In all cases studied, the efflux curves showed at least two fractions. It is the fast-exchanging fraction that steadily and consistently increased in magnitude as the muscles were dying, until finally the concentration of solute in this fraction reached and sometimes surpassed the labeled solute concentrations in the original labeled solutions in which the muscles were equilibrated. The slow fractions showed only a transient increase or none at all. These observations show that it is the fast fraction that represents solute dissolved in cell water and rate-limited by passage through the cell surface and that the partial exclusion of Na+ and the sugars have a unitary cause--a reduced solubility in the cell water which in the presence of ATP exists in the state of polarized multilayers.     

Membrane regulation of the Na+,K+-ATPase during the neuroblastoma cell cycle: correlation with protein lateral mobility

The pumping activity of the plasma membrane-bound Na+,K+-ATPase shows considerable variation during the cell cycle of mouse neuroblastoma Neuro-2A cells. Addition of external ATP at millimolar concentrations, which selectively enhances the plasma membrane permeability of Neuro-2A cells for sodium ions, stimulates the Na+,K+-ATPase pumping activity at all phases of the cell cycle from a factor of 1.05 in mitosis up to 2.2 in G1 phase. Determination of the number of Na+,K+-ATPase copies per cell by direct 3H-ouabain binding studies in the presence of external ATP shows a gradual increase in the number of pump sites on passing from mitosis to the late S/G2-phase by approximately a factor of 2. From these data the pumping activity per copy of Na+,K+-ATPase, optimally stimulated with respect to its various substrate ions, has been determined during the various phases of the cell cycle. This optimally stimulated pumping activity per enzyme copy, which is a reflection of the physicochemical state of the plasma membrane, is high in mitosis, almost twofold lower in early G1 phase, and increases gradually again during the other phases of the cell cycle. This shows that the observed regulation of Na+,K+-ATPase activity during the cell cycle is caused by a combination of three independent factors--namely variation in intracellular substrate availability (Na+), changes in number of enzyme copies per cell, and modulation of the plasma membrane environment of the protein molecules. The modulation of the optimal pumping activity per enzyme copy shows a good correlation (rho = 0.96) with the known modulation of protein lateral mobility during the cell cycle, such that a high protein lateral mobility correlates with a low enzyme activity. It is concluded that changes in plasma membrane properties take place during the Neuro-2A cell cycle that result in changes in the rate of protein lateral diffusion and Na+,K+-ATPase activity in directly correlated way.