Average Intracellular Concentration Research Articles

Intracellular Ionized Calcium Changes in Squid Giant Axons Monitored by Fura‐2 and Aequorina

Squid giant axons were injected simultaneously with Ca indicators Fura-2 and aequorin. Fura-2 was calibrated in situ by measuring fluorescence at 510 nm upon UV excitation at 340 nm, 360 nm, and 380 nm with a time-sharing multiple wavelength spectrofluorimeter. Limiting values for dye fluorescence were obtained by allowing a massive load of Ca to enter the axon with the aid of procedures such as prolonged depolarization in the presence of CN (for saturation) and by sequestration of all Ca present in the axoplasm accomplished with injection of EGTA into the axon (for a zero-Ca signal). The average intracellular Ca concentration obtained with Fura-2 was 184 nM. The sensitivity of Fura-2 to intracellular Ca is at least as great as that of aequorin, thus permitting its use in the characterization of Ca homeostasis mechanisms such as Na-Ca exchange. It was found, however, that for voltage-clamp experiments requiring an internal current electrode, Fura-2 is not a convenient Ca probe because electrode reactions in the axoplasm denature the dye, thereby restricting its use in characterization of Ca movements associated with electrically induced changes in membrane potential. A comparison of aequorin luminescence with Fura-2 fluorescence demonstrated that light output by aequorin is linear with intracellular Ca concentrations up to values of 750 nM, changing to a square law relationship from 750 nM up to 10 microM Ca.

Radiosensitization by Misonidazole, Pimonidazole and Azomycin and Intracellular Uptake in Human Tumour Cell Lines

The radiosensitization of two human tumour in vitro cell lines, HT-1080 and LoVo, has been compared with that of the Chinese hamster cell line V79-379A. Although the two human tumour cell lines were more radiosensitive than the V79 cell line sensitizer, enhancement ratios for misonidazole, pimonidazole and azomycin were similar (relative to extracellular concentration) for all three cell lines. Average intracellular concentrations of radiosensitizer were measured by high-performance liquid chromatography. In all three cell lines the uptake of misonidazole and azomycin was extremely rapid whereas that of pimonidazole was initially much slower before reaching a plateau. The ratios of intracellular concentration of radiosensitizer to extracellular concentration (Ci to Ce) for misonidazole were 0.8 (HT-1080) and 0.7 (LoVo and V79); for azomycin 0.9 (HT-1080 and LoVo) and 0.8 (V79). In contrast Ci/Ce for pimonidazole varied with cell line, the values being 1.8 (LoVo), 2.6 (HT-1080) and 3.3 (V79). Intracellular amounts of non-protein sulphydryl (NPSH) varied between cell lines by about a factor of three. However, when the average cell volume was taken into consideration the concentrations of NPSH were very similar, being 4.2 (HT-1080), 5.6 (LoVo) and 5.7 (V79) mmol dm-3. NPSH levels expressed as nmol per mg protein were also similar.

Oxygen gradients in CHO cells: Measurement and characterization by electron spin resonance

The concentration of oxygen within cells is important in many physiological and pathological processes, but such oxygen-dependent processes are generally studied as a function of the concentration of extracellular oxygen, due to a lack of suitable methods. Using a newly developed technique based on ESR spectroscopy, we show that respiration stimulation of a cell suspension can result in a significant difference between average intracellular and extracellular concentrations of oxygen. These results indicate that studies of oxygen-dependent phenomena in cells may require measurement of intracellular oxygen concentrations and imply that there are mechanisms in cells that restrict the free diffusion of oxygen.

Radiosensitizer-DNA interactions in relation to intracellular uptake

We have studied the intracellular uptake of a number of neutral, acidic, and basic radiosensitizers. For neutral sensitizers, we observed a correlation between the measured intracellular concentration and sensitization, but for bases, a large change in average intracellular concentration results in only a small change in sensitization. In addition, by modifying the intralysosomal pH, we have altered the measured average intracellular concentration of the weak base pimonidazole by a factor of two, although this had no detectable effect upon sensitization. Using spin filtration of solutions of sensitizers with naked calf thymus DNA or chromatin we have assessed the affinity of DNA for sensitizers with different prototropic and lipophilic properties. We have also shown that this anomalous behavior of the basic sensitizers could be partly explained on the basis of intracellular localization adjacent to the DNA due to ionic interactions. Thus, intracellular localization needs to be considered when interpreting average intracellular uptake data.

Solute Accumulation in Tobacco Cells Adapted to NaCl

Cells of Nicotiana tabacum L. var Wisconsin 38 adapted to NaCl (up to 428 millimolar) which have undergone extensive osmotic adjustment accumulated Na(+) and Cl(-) as principal solutes for this adjustment. Although the intracellular concentrations of Na(+) and Cl(-) correlated well with the level of adaptation, these ions apparently did not contribute to the osmotic adjustment which occurred during a culture growth cycle, because the concentrations of Na(+) and Cl(-) did not increase during the period of most active osmotic adjustment. The average intracellular concentrations of soluble sugars and total free amino acids increased as a function of the level of adaptation; however, the levels of these solutes did not approach those observed for Na(+) and Cl(-). The concentration of proline was positively correlated with cell osmotic potential, accumulating to an average concentration of 129 millimolar in cells adapted to 428 millimolar NaCl and representing about 80% of the total free amino acid pool as compared to an average of 0.29 millimolar and about 4% of the pool in unadapted cells. These results indicate that although Na(+) and Cl(-) are principal components of osmotic adjustment, organic solutes also may make significant contributions.

Measurement of intracellular oxygen concentration using the spin label TEMPOL.

We have developed a noninvasive method with general applicability for measuring intracellular oxygen using the spin label TEMPOL (2,2,6,6,-tetramethypiperidine-N-oxyl-4-ol) which has superhyperfine structure in its electron spin resonance spectra that is broadened in the presence of oxygen. This broadening is linear over a range of 1 to 6 ppm oxygen which covers the important physiological range of oxygen concentration. Viscosity, TEMPOL concentration, and instrument modulation intensity also can affect superhyperfine structure but the contributions from these effects can be determined. The TEMPOL distributes equally into the intra- and extracellular compartments but its intracellular signal can be studied selectively by addition of transition metal ions such as potassium ferricyanide and potassium tris(oxalato)chromiate, which broaden away the signal from extracellular TEMPOL and do not cross the cell membrane to affect the intracellular TEMPOL. Results with a cell culture line (mouse thymus-bone marrow) indicate that under our experimental conditions these cells may maintain an average intracellular oxygen concentration lower than the extracellular oxygen concentration, and that there is not a constant relationship between extracellular and intracellular oxygen concentrations.

Cellular uptake of misonidazole and analogues with acidic or basic functions.

Average intracellular concentrations of five radiosensitizers in hamster fibroblast-like V79-379A cells in vitro were measured by high performance liquid chromatography, varying the extracellular pH (pHe) and estimating the apparent intracellular pH from the distribution of 5,5-dimethyloxazolidine-2,4-dione. The intracellular: extracellular concentration ratio for the 2-nitroimidazole, misonidazole was constant at about 0.7 for pHe = 6.6-7.6, whereas the weak base, Ro 03-8799 (1-(2-nitro-1-imidazolyl)-3-N-piperidino-2-propanol) was concentrated intracellularly at pHe = 7.3-7.4 by a factor of 3.3, the factor increasing from about 0.8 at pHe = 6.0, to 7.5 at pHe = 7.85. The weak acid, azomycin (2-nitroimidazole) showed approximately constant uptake (factor 1.1) between pHe = 6.0-7.0, decreasing to 0.8 at pHe = 7.3 and 0.4 at pHe = 7.8. Measurements of intracellular uptake of Ro 31-0052 (the more hydrophilic and less basic 3'-hydroxypiperidino analogue of Ro 03-8799) and of Ro 31-0258 (3-(2-nitro-1-imidazolyl)propionic acid, a stronger acid than azomycin) were made for comparison. The results were compared with theoretical calculations of pH-induced concentration gradients; the time dependence of the uptake of the bases is not at present clearly understood. These measurements of uptake are broadly consistent with the distribution of misonidazole and Ro 03-8799 in human and animal tissues and provide a useful insight into the likely intracellular concentrations in the clinical use of Ro 03-8799 or other basic radiosensitizers. The measurements also resolve the apparent discrepancy in radiosensitizer efficiency for weak bases in vitro and in vivo which has been previously noted.

Binding of radiolabeled [formula omitted] to aspartate transcarbamylase from ehrlich ascites tumor cells

Binding of N-( phosphonacetyl)-[ 3 H] L - aspartate (PALA) to aspartate transcarbamylase (ATCase, EC 2.1.3.1) in crude extracts from Ehrlich ascites tumor cells was examined. At pH 7.4, the dissociation constant was 1.39 ± 0.22 nM; the maximal binding capacity indicated an average intracellular ATCase concentration of 0.13 μM. The presence of phosphate, MgCl 2, or CaCl 2 increased the apparent dissociation constant for [ 3H]PALA without altering the maximal binding capacity. Phosphate, a product of the ATCase reaction, probably acts as a competitor for the PALA binding site; Mg 2+ and Ca 2+ may inhibit [ 3H]PALA binding by forming a chelate which reduces the effective concentration of the free [ 3H]PALA. Carbamyl phosphate was a relatively weak inhibitor of [ 3H]PALA binding in N-2-hydroxyethylpiperazine- N'-2-ethanesulfonic acid (HEPES) buffer alone. Addition of NaF, an inhibitor of nonspecific phosphatases, decreased the carbamyi phosphate “ K i ” as an inhibitor of [ 3H]PALA binding to 7 μM, a value close to the K m . NaF appears to act as an inhibitor of carbamyi phosphatase activity present in the cell extract. A first-order dissociation rate constant of 0.050 ± 0.004 min −1 ( T 1 2 = 14 min ) was determined by following displacement of [ 3H]PALA with excess unlabeled PALA. The dissociation rate was strongly temperature dependent. A second-order rate constant of 3.6 × 10 7 liters mol −1 min −1 was calculated from this rate constant and the dissociation constant. Using these kinetic constants, a simple computer model predicted that PALA binding to ATCase is 95% complete within 14 min under the conditions of the assay; at intracellular ATCase concentrations, binding is slightly faster. These results are discussed in the context of both the kinetics of inhibition and the reversal of inhibition of pyrimidine synthesis within the intact cell.

The Ionic Basis of the Resting Potential in a Cross-Striated Muscle of the Aquatic Snail Lymnaea Stagnalis

ABSTRACT The mean resting potential in the heart ventricle muscle cells of the freshwater snail Lymnaea stagnalis was found to be – 61·2±3·5(..)mV (ranging from –56mV to –68mV). The average intracellular potassium concentration was estimated to be 51·5 ± 14·6 (..) m (ranging from 27·8 m to 77·3 m). The membrane of the heart ventricle muscle cells appears to be permeable to both potassium and chloride, as changes in the extracellular concentration of either of these ions resulted in a change in the membrane potential. A ten-fold change in the extracellular potassium concentration was associated with a 50·4 ± 3·8 (..) mV slope when the potassium concentration was above about 6 m. Deviations from the straight-line relation predicted for a potassium electrode could be accounted for by introducing a term for sodium permeability. The ionic basis of the membrane potential in these cells can be described by a modified form of the Goldman-Hodgkin-Katz equation.

Control of cell mobility by cyclic AMP

Cyclic AMP concentrations have been measured in a number of different cell types under a variety of culture conditions in an attempt to define the relationship between the endogenous concentration of cyclic AMP and cell mobility. In previous work it was shown that agents that increase the intracellular concentration of cyclic AMP can effectively suppress cell movement. In Balb/c 3T3 cells, which have a very low mobility in cellular aggregates, the intracellular concentration of cyclic AMP was elevated only transiently soon after the formation of the three-dimensional cell masses. In contrast, in the highly mobile virally transformed counterpart of Balb/c 3T3 cells, called SVT-2, the concentration of cyclic AMP was relatively low soon after the cell masses were formed, but later rose to a level that was higher than that in Balb/c 3T3 cells. Using NIL B cells, SV40-transformed NIL B cells, and several lines of tumour cells derived from NIL B cells, it was found that the average intracellular concentration of cyclic AMP did not vary significantly from one population of cells to another. Finally, the intracellular concentration of cyclic AMP was measured in chick embryo ventricle cells. The mobility of these cells had previously been found to decrease as embryonic development progressed; furthermore, it had been shown that dibutyryl cyclic AMP plus theophylline produced nearly complete inhibition of their movement in cell masses. In the series of experiments reported here we found that the endogenous concentration of cyclic AMP in aggregates and fragments of chick embryo ventricle cells decreases as development proceeds; these data are consistent with preliminary experiments reported by other investigators. In a separate set of experiments, the intracellular concentration of cyclic AMP was measured in cells that had been cultured in a medium containing 1.2 mM-dibutyryl cyclic AMP plus 1.0 mM-theophylline. This drug treatment has previously been shown to inhibit the movement of cells both in aggregates and in monolayers; it also produces striking effects on cell shape and ultrastructure. In aggregates of chick embryo ventricle cells, treatment with these drugs resulted in increases in the intracellular concentrations of cyclic AMP from approximately 10 picomol/mg protein to approximately 500 picomol/mg protein. In Balb/c 3T3 and SVT-2 cells this treatment increased cyclic AMP concentrations from 3.7 to 160 and from 6.4 to 470 picomol/mg protein, respectively.

Uptake of latex particles by macrophages: characterization using flow cytometry.

Flow cytometry can be used to characterize the uptake of particles by small samples of pulmonary macrophages both quickly and accurately. We found that there was a linear relationship between the number of fluorescent latex particles and the fluorescent intensity associated with each cell up to 47 particles/cell. Macrophages lavaged from the lungs of Syrian golden hamsters were metabolically and functionally stable for 3 h of incubation; the average intracellular concentrations of Na+ and K+ were 29 +/- 2 and 158 +/- 6 mM, respectively. Particle uptake was significantly inhibited by removing divalent cations with ethylenediaminetetraacetic acid and lowering the incubation temperature (37 degrees C) to 24 and 3 degrees C. The cell-to-cell variability in the uptake of particles was greater than the Poisson distribution would predict based on the mean number of particles per cell.

Polyamines: A Dual Role in the Modulation of Cellular Sensitivity to Heat

The organic polycations known as polyamines occur at average intracellular concentrations (as determined by acid extraction) approaching the millimolar level. Positively charged at normal intracellular pH's, they have long been suspected of ionic interaction with cellular macromolecules of functional importance (1). The chain structure of the polyamines (see Table I for spermidine and spermine) distinguishes these polycations from the monoand divalent metal ions in a variety of biochemical interactions. The degree of specificity of polyamine interactions is, however, unclear and it may be that these polycations fulfill general stabilizing and charge-buffering functions within the cell [for general review, see Ref. (2)]. This report will discuss a small area within the vast polyamine literature, that of the relationship between these compounds and the responses of cell populations to elevated temperatures, or hyperthermia. The review will be divided into what have become two major areas of interest. The first involves the interaction of exogenous polyamines with what we believe to be membrane targets. In this mode, we believe the polyamines are acting directly, or immediately upon interaction with some membrane component, as heat sensitizers. The second and recently emerging body of work centers on the use of polyamine biosynthesis inhibitors to deplete critical intracellular target(s) of polyamines in such a way as to achieve delayed sensitivity to heat.

Are DNA precursors concentrated at replication sites?

We have asked whether the effective concentrations of deoxyribonucleotide 5'-triphosphates (dNTPs) at sites of DNA replication in vivo might be higher than the concentrations of dNTPs averaged over the entire cell volume. The approach involved determination of the dependence of DNA replication rate upon thymidine triphosphate concentration, both in vivo and in vitro system that closely approximates the intracellular replication apparatus. In T4 phage-infected Escherichia coli maximal rates of DNA synthesis were attained with dTTP pools of approximately 1.2 x 10(5) molecules per cell, corresponding to an average intracellular concentration of about 65 microM. When DNA synthesis was measured in the T4 purified protein system [Sinha, N. K., Morris, C. F. & Alberts, B. M. (1980) J. Biol. Chem. 255 4290--4303], maximal rates were observed at dTTP concentrations of 200--240 microM. This represents a minimal estimate, therefore, of dTTP concentration at replication sites and suggests that at least a 3- to 4-fold concentration gradient exists near these sites. We discuss why such concentration gradients might be needed and how they might be generated. We also discuss the implications of these results for understanding the relationship between intracellular dNTP pools and mutation rates. A by-product of our study was the finding that exogenous thymidine is used for T4 DNA synthesis in preference to endogenous pathways to thymidine nucleotides; at high thymidine concentrations in vivo the endogenous pathways can be completely bypassed.

Multienzyme complexes in DNA precursor biosynthesis

Several features of DNA replication suggest the involvement of multienzyme complexes in deoxyribonucleotide biosynthesis. Such complexes could generate intracellular concentration gradients of DNA precursors, with high levels being maintained at their sites of utilization, namely, replication forks. Relevant facts or observations include the following. (1) Deoxyribonucleotides are highly specialized metabolites, whose sole evident function is to serve as DNA precursors; for the most part they are found only in cells which are synthesizing DNA. (2) Although average intracellular deoxyribonucleoside triphosphate concentrations are low, most in vitro systems for DNA synthesis require rather high triphosphate concentrations to maintain maximal incorporation rates; thus, the effective intracellular levels are probably much higher than the average concentrations; (3) DNA chain growth rates in vivo are one to two orders of magnitude higher than the corresponding rates for RNA chain growth; thus, DNA precursor supplies must be replenished very rapidly at the replication fork. Data on deoxyribonucleotide pool labeling kinetics indicate that DNA precursor pools are compartmentalized. Even in the absence of physically separated compartments, one can visualize a functional compartmentation in which the separate pools are kinetically distinct. In bacteria, for example, one could envisage a small, rapidly turning over pool of triphosphates near the replication forks, and a larger, more slowly replenished pool which is more evenly distributed through the cell and which is used primarily in DNA repair. A complex of deoxyribonucleotide-synthesizing enzymes, juxtaposed with the replication apparatus, could serve to maintain high local concentrations of triphosphates at the replication forks. Using T4 phage-infected E. coli as a model system, our laboratory has obtained considerable evidence supporting the above model. The evidence is derived from deoxyribonucleotide pool kinetic studies, experiments on DNA synthesis in vitro, and properties of an isolated multienzyme aggregate. The evidence indicates that effective DNA precursor concentrations at the replication fork are quite different from average intracellular concentrations as estimated from pool measurements. The desirability of being able to determine effective concentrations is emphasized.

Pyrophosphate:d-Fructose 6-Phosphate 1-Phosphotransferase: A NEW ENZYME WITH THE GLYCOLYTIC FUNCTION OF 6-PHOSPHOFRUCTOKINASE

Abstract The major enzyme of Entamoeba histolytica which produces fructose 1,6-diphosphate from fructose 6-phosphate utilizes inorganic pyrophosphate as phosphate donor. In its reverse reaction the enzyme utilizes orthophosphate and fructose diphosphate to yield inorganic pyrophosphate and fructose 6-phosphate. The enzyme has been purified 100-fold from an amebal homogenate. At pH 7 with 2.5 mm MgCl the following Km values were observed: pyrophosphate, 14 µm; fructose 6-phosphate, 38 µm; fructose 1,6-diphosphate, 18 µm; and orthophosphate, 800 µm. At this pH and magnesium ion concentration the maximum velocity of the reaction was approximately the same in each direction. The average intracellular concentration of pyrophosphate in the ameba is 0.18 mm, a value 13-fold greater than the pyrophosphate Km for the new enzyme. This enzyme raises to three the number of known enzymes in the glycolytic pathway of E. histolytica which utilize or produce pyrophosphate. The trivial name proposed for the new enzyme is 6-phosphofructokinase (PPi).

Continuous culture of Rhodotorula rubra: kinetics of phosphate-arsenate uptake, inhibition, and phosphate-limited growth.

The pink yeast Rhodotorula rubra of marine origin was found to be capable of extended growth at very low phosphate concentrations (K(0.5) = 10.8 nm). Average intracellular phosphate concentrations, based on isotope exchange techniques, were 15 to 200 nm, giving concentration gradients across the cell envelope of about 10(6). Sensitivity to metabolic inhibitors occurred at micromolar concentrations. Inability of the phosphate transport system, K(s) = 0.5 to 2.8 mum, V(max) = 55 mumoles per g of cells per min, to discriminate against arsenate transport led to arsenate toxicity at 1 to 10 nm, whereas environmental arsenate levels are reportedly much higher. Phosphate competitively prevented arsenate toxicity. The K(i) for phosphate inhibition of arsenate uptake was 0.7 to 1.2 mum. Phosphate uptake experiments showed that maximal growth rates could be achieved with approximately 4% of the total phosphate-arsenate transport system. Organisms adapted to a range both of concentration of NaCl and of pH. Maximal affinity for phosphate occurred at pH 4 and at low concentrations of NaCl; however, V(max) for phosphate transport was little affected. Maximal specific growth rates on minimal medium were consistent in batch culture but gradually increased to the much higher rates found with yeast extract media when the population was subjected to long-term continuous culture with gradually increasing dilution rates. Phosphate initial uptake rates that were in agreement with the steady-state flux in continuous culture were obtained by using organisms and medium directly from continuous culture. This procedure resulted in rates about 500 times greater than one in which harvested batch-grown cells were used. Discrepancies between values found and those reported in the literature for other organisms were even larger. Growth could not be sustained below a threshold phosphate concentration of 3.4 nm. Such thresholds are explained in terms of a system where growth rate is set by intracellular nutrient concentrations. Threshold concentrations occur in response to nutrient sinks not related to growth, such as efflux and endogenous metabolism. Equations are presented for evaluation of growth rate-limiting substrate concentrations in the presence of background substrate and for evaluating low inhibitor concentration inhibition mechanisms by substrate prevention of inhibitor flux.

Exchange transport between various amino acids and alanine in isolated intestinal epithelial cells

The characteristics of exchange transport between various amino acids and alanine were investigated in epithelial cells isolated from rat intestine. Cells preloaded with alanine (average intracellular concentration 5.1 mM) showed a 6–36% increase in the uptake of 1 mM neutral amino acids and an 80–102% increase in the uptake of 1 mM basic amino acids as compared to control cells. The magnitude of these increases was at least as great when the studies were performed in a Na +-free medium. Extra-cellular lysine accelerated the movement of alanine out of alanine-preloaded cells but extracellular alanine had no effect on the movement of lysine out of lysine-preloaded cells. The presence of 1 mM alanine in the incubation media inhibited the uptake of 1 mM neutral amino acids by 20–30%, but increased the uptake of 1 mM basic amino acids by 47–81%. These results indicate the presence of two distinct alanine-amino acid exchange transport pathways: (1) a comparatively low level competitive exchange with neutral amino acids mediated by the neutral amino acid transport pathway; (2) a strongly accelerating hetero-exchange with basic amino acids mediated by at least two different transport pathways.

Sugar and electrolyte absorption in the rat intestine perfused "in vivo".

1. Studies are reported of rat intestinal perfusion “in vivo” in which data relating to the transport of sugars, sodium, fluid as well as intracellular concentrations of sugars and electrolytes have been obtained. 2. A direct linear relationship with a zero intercept between net sodium and net sugar transport has been observed. 3. The average intracellular concentration of sugars is always lower than in the luminal fluid and blood; this fact is consistent with the existence of a sugar pump located at the serosal pole of the absorbing cell. 4. Intracellular sodium concentration is lower and intracellular potassium concentration is higher than in “in vitro” experiments. 5. Cell sodium concentration and content as well as cell water content are increased by raising the luminal glucose concentration. In the same situation cell potassium concentration decreases while cell potassium content remains unchanged. 6. Transport phenomena are more efficient in “in vivo” than in “in vitro” experiments.

An application of the constant-field theory to the behaviour of giant neurones of the snail, Helix aspersa.

ABSTRACT The resting potentials of giant neurones in the abdominal ganglia of the snail, Helix aspersa, have been measured, and their dependence on the extracellular concentration of potassium ions has been investigated. The behaviour of the resting potentials differs considerably from that of a potassium electrode, as given by the Nemst equation. A modification of the equation derived from the ‘constant-field’ theory is described, which transforms the equation for the resting potential into a linear relationship ; it is found that the experimental results can be fitted quite well by this equation, for potassium concentrations up to 12 mM. This is taken as evidence that the relative permeabilities of the cell membrane to potassium and sodium ions are independent of the external potassium concentration over the range of concentrations used. By fitting ‘best straight lines’ to the experimental data, estimates can be obtained from the ‘constant-field’ equation of the intracellular potassium concentration, and the ratio of permeabilities of the cell membrane to sodium and potassium ions, PNa/PK-The average results from experiments on fifty-seven neurones are : the error in each case being one standard deviation, calculated from the spread of the results. The average intracellular potassium concentrations of snail neurones show a distinct seasonal variation, being highest in the spring and lowest in the autumn. These changes are accompanied by changes in the selectivity of the cell membranes, as characterized by the ratio of permeabilities to sodium and potassium ions, so that the resting potentials of the cells are comparatively little affected. The changes could be connected with changes in the pattern of activity of the animals.