Cytoplasmic Ion Concentration Research Articles

Ion Specificity and Ionic Strength Dependence of the Osmoregulatory ABC Transporter OpuA

The ATPase subunit of the osmoregulatory ATP-binding cassette transporter OpuA from Lactococcus lactis has a C-terminal extension, the tandem cystathionine beta-synthase (CBS) domain, which constitutes the sensor that allows the transporter to sense and respond to osmotic stress (Biemans-Oldehinkel, E., Mahmood, N. A. B. N., and Poolman, B. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 10624-10629). C-terminal of the tandem CBS domain is an 18-residue anionic tail (DIPDEDEVEEIEKEEENK). To investigate the ion specificity of the full transporter, we probed the activity of inside-out reconstituted wild-type OpuA and the anionic tail deletion mutant OpuADelta12; these molecules have the tandem CBS domains facing the external medium. At a mole fraction of 40% of anionic lipids in the membrane, the threshold ionic strength for activation of OpuA was approximately 0.15, irrespective of the electrolyte composition of the medium. At equivalent concentrations, bivalent cations (Mg(2+) and Ba(2+)) were more effective in activating OpuA than NH(4)(+), K(+), Na(+), or Li(+), consistent with an ionic strength-based sensing mechanism. Surprisingly, Rb(+) and Cs(+) were potent inhibitors of wild-type OpuA, and 0.1 mM RbCl was sufficient to completely inhibit the transporter even in the presence of 0.2 M KCl. Rb(+) and Cs(+) were no longer inhibitory in OpuADelta12, indicating that the anionic C-terminal tail participates in the formation of a binding site for large alkali metal ions. Compared with OpuADelta12, wild-type OpuA required substantially less potassium ions (the dominant ion under physiological conditions) for activation. Our data lend new support for the contention that the CBS module in OpuA constitutes the ionic strength sensor whose activity is modulated by the C-terminal anionic tail.

Fruit-specific V-ATPase suppression in antisense-transgenic tomato reduces fruit growth and seed formation

The vacuole is a large, multifunctional organelle related to the processes of cell expansion, solute accumulation, regulation of cytoplasmic ion concentrations, pH homeostasis and osmoregulation, which are directly or indirectly achieved by vacuolar H+-pumps: vacuolar H+-ATPase (V-ATPase; EC 3.6.1.3) and vacuolar H+-pyrophosphatase (V-PPase; EC 3.6.1.1). In this study, we produced antisense-transgenic tomatoes (Lycopersicon esculentum L.) of the V-ATPase A subunit, which is under the control of the fruit-specific 2A11 promoter. One beta-glucuronidase (GUS)-transgenic line (GUS control) and seven A subunit antisense-transgenic lines were obtained. The amount of V-ATPase A subunit mRNA in fruit decreased in all antisense-transgenic lines, but in leaves showed no difference compared with the GUS control line and the nontransformant, suggesting that suppression of the V-ATPase A subunit by a 2A11 promoter is limited to fruit. The antisense-transgenic plants had smaller fruits compared with the GUS control line and the nontransformant. Surprisingly, fruits from the antisense-transgenic plants, except the fruit that still had relatively high expression of A subunit mRNA, had few seeds. Sucrose concentration in fruits from the antisense-transgenic plants increased, but glucose and fructose concentrations did not change. These results show the importance of V-ATPase, not only in fruit growth, but also in seed formation and in sugar composition of tomato fruit.

The Influence of SRC-Family Tyrosine Kinases on Na,K-ATPase Activity in Lens Epithelium

Na,K-adenosine triphosphatase (ATPase) is essential for the regulation of cytoplasmic ion concentrations in lens cells. Earlier studies demonstrated that tyrosine phosphorylation by Lyn kinase, a Src-family member, inhibits Na,K-ATPase activity in porcine lens epithelium. In the present study, experiments were conducted to compare the ability of other Src-family kinases (Fyn, Src, and Lck) and Fes, a non-Src-family tyrosine kinase, to alter Na,K-ATPase activity. Membranes prepared from porcine lens epithelium were incubated with partially purified tyrosine kinases in buffer containing 1 mM adenosine triphosphate (ATP). ATP hydrolysis in the presence and absence of ouabain was used to measure Na,K-ATPase activity. Western blot analysis was used to examine phosphotyrosine-containing proteins and tyrosine kinase expression. Fyn reduced Na,K-ATPase activity by approximately 30%. In contrast, Src caused a approximately 38% increase of Na,K-ATPase activity. Na,K-ATPase activity in membrane material treated with Lck or Fes was not significantly altered, even though Lck and Fes treatment induced robust tyrosine phosphorylation. Added exogenously, each tyrosine kinase induced a different pattern of membrane protein tyrosine phosphorylation. As judged by immunoprecipitation, Src, Fyn, Lyn, and Lck elicited tyrosine phosphorylation of the Na,K-ATPase alpha1 protein. Src, Fyn, Lyn, Lck, and Fes were each detectable in the epithelium by Western blot. The results indicate considerable variation in the Na,K-ATPase activity response of lens epithelium to different tyrosine kinases. This could perhaps explain why inhibition of Na,K-ATPase activity is reported to be caused by tyrosine phosphorylation in some tissues, whereas stimulation of Na,K-ATPase activity is observed in other tissues.

Intracellular Calcium Ion Elevation in Chicken Phagocytes Treated with Activating Agents

Cytoplasmic calcium ion concentrations [Ca2+]i were measured for the first time in chicken heterophils and macrophages stimulated with Leukotriene B4 (LTB4), phorbol myristate acetate (PMA), formyl-methionyl-leucyl-phenylalanine (fMLP) and lipopolysaccharide (LPS) which are known to be activating factors of phagocyte functions. [Ca2+]i in rat neutrophils and macrophages was also measured after stimulation with these activating factors. [Ca2+]i was measured by the Fura-2-AM-loading method using an intracellular ion analyzer. Resting levels of [Ca2+]i in chicken phagocytes were similar to those reported previously in rats. [Ca2+]i of chicken phagocytes was elevated by treatment with LTB4, PMA and LPS, and the patterns and duration times of the elevation of [Ca2+]i by these factors resembled those in rat phagocytes. However, fMLP did not elevate [Ca2+]i in chicken phagocytes, although the agent elevated [Ca2+]i of rat phagocytes. The degree of elevation of [Ca2+]i in chicken heterophils and macrophages stimulated by LTB4, PMA and LPS was significantly higher than that in the same kinds of rat phagocytes (neutrophils and macrophages) stimulated with the corresponding factors. The degree of elevation of [Ca2+]i in granulocytes treated with these factors was significantly higher than that in macrophages in chickens and rats. The reasons for these differences are unclear at the present time. It is valuable to examine the reason for these differences.

Stoichiometry of the Na+/Ca2+ exchanger: models and implications.

We reevaluated the exchange stoichiometry of the Na(+)/Ca(2+) exchange current by measuring its reversal potential. The exchange current was measured from the inside-out macropatch excised from intact sarcolemma of guinea pig ventricular myocytes. This method provides more accurate control of extracellular and cytoplasmic ion concentrations and of membrane potential than is possible with a whole-cell clamped preparation. The exchange current was isolated as exchanger inhibitory peptide (XIP)-sensitive current or as cytoplasmic Na(+)- and Ca(2+)-induced current. The reversal potential of the Na(+)/Ca(2+) exchange current was, for the most part, close to the equilibrium potential of the 4Na(+):1Ca(2+) exchange, although it tended to get closer to that of 3Na(+): 1Ca(2+) exchange at lower Na(+) concentrations. We concluded that the stoichiometry is 4 or that it may vary depending on the cytoplasmic Na(+). The 4Na(+):1Ca(2+) exchange was further studied with computer modeling. A consecutive 4Na(+):1Ca(2+) exchange model with two active states and two inactive states (E2 model) could not well reconstruct the current-voltage relation of the exchanger. However, a consecutive 4Na(+):1Ca(2+) exchange model with 10 active states and 2 inactive states (E10 model), which included voltage-dependent Na(+) and Ca(2+) occlusions, well simulated the current-voltage relation. Implications of 4Na(+):1Ca(2+) exchange is also discussed.

Ion channel diseases.

Ion channels serve many functions apart from electrical signal transduction: chemical signalling (Ca(2+) as a second messenger), transepithelial transport, regulation of cytoplasmic or vesicular ion concentration and pH, and regulation of cell volume. Therefore, ion channel dysfunction can cause diseases in many tissues. The list of human diseases known to be associated with defects in ion channels has grown considerably during the past years. This review gives a short overview of known channelopathies, and focuses in particular on recent findings and on channelopathies that have significantly advanced our physiological insight.

Mitochondrial localization and photodamage during photodynamic therapy with tetraphenylporphines

The subcellular localization sites of TPPS 4 and TPPS 1 and the subsequent cellular site damage during photodynamic therapy were investigated in CT-26 colon carcinoma cells using spectroscopic and electron microscopy techniques. The association of both porphyrins with the mitochondria was investigated and the implications of this association on cellular functions were determined. Spectrofluorescence measurements showed that TPPS 4 favors an aqueous environment, while TPPS 1 interacts with lipophilic complexes. The subcellular localization sites of each sensitizer were determined using spectral imaging. Mitochondrial-CFP transfected cells treated with porphyrins revealed localization of TPPS 1 in the peri-nuclear region, while TPPS 4 localized in the mitochondria, inducing structural damage and swelling upon irradiation, as shown by transmission electron microscopy. TPPS 4 fluorescence was detected in isolated mitochondria following irradiation. The photodamage induced a 38% reduction in mitochondrial activity, a 30% decrease in cellular ATP and a reduction in Na +/K +-ATPase activity. As a result, cytosolic concentrations of Na + and Ca 2+ increased, and the level of K + decreased. In contrast, the lipophilic TPPS 1 did not affect mitochondrial structure or function and ATP content remained unchanged. We conclude that TPPS 4 induces mitochondrial structural and functional photodamage resulting in an altered cytoplasmic ion concentration, while TPPS 1 has no effect on the mitochondria.

Sodium–hydrogen exchange in human platelets exposed to weak acid: comparison between measurements of platelet swelling and cytoplasmic sodium ion concentration

Increased Na + -H + exchange activity has been demonstrated in various pathological states using the rate constant of platelet swelling (k s ). This indirect measure of exchange activity could, however, be influenced by conditions that alter platelet distensibility independent of Na + -H + exchange activity. To determine the validity of k s , the change in light transmission and hence k s , was measured at 24° and 37°C (temperatures associated with alterations in membrane fluidity), and compared with the rate constant (k Na ) for the increase in cytoplasmic sodium ion concentration ([Na + ] i ) and the rate constant of Na + -influx (k ϕ;Na ) under the same conditions. Both k s and k Na were dependent on Na + -H + exchange; the rate constants differed in the order k s <k ϕ;Na <k Na (P<0.01). Neither k Na or k ϕ;Na changed with temperature, whereas k s was significantly lower at 24° than at 37°C (7.49 - 0.31 2 10- 3 s- 1 versus 9.96 - 0.42 2 10- 3 s- 1 , P<0.0001). While both k and k are dependent on Na + -H + exchange, k s may underestimate the activity of the exchanger and may in part, be influenced by membrane fluidity. This could be important in interpreting data where k s has been measured in different groups of subjects.

Transient Cytoplasmic pH Change and Ion Fluxes through the Plasma Memberan in Suspension-Cultured Rice Cells Triggered by N-Acetylchitooligosaccharide Elicitor

N-Acetylchitooligosaccharides, fragments of a main backbone polymer of fungal ceil wall, elicit defense responses including phytoalexin production in suspension-cultured rice cells. The purified oligosaccharide triggers rapid, transient membrane depolarization. Ion fluxes induced by the oligosaccharides were analyzed by using ion-selective electrodes. Treatment of the cells with the oligosaccharides induced transient efflux of K+ and influx of H+ immediately after the elicitation. To monitor the pH values of the cytoplasm and the vacuoles noninvasively under a physiological condition, in vivo 31P-nuclear magnetic resonance spectroscopy was applied to the cells to which oxygenated growth medium was perfused continuously. The cytoplasmic pH showed significant transient decrease, correspondingly. Only the N-acetylchitooligosaccharides with a degree of polymerization higher than 5 were active, whereas deacetylated chitosan oligomers caused no effect. Less than 1 nM of N-acetylchitoheptaose was sufficient to induce rapid flux of ions. Such strict structural requirements for the induction of ion fluxes were similar to those of specific binding to the putative plasma membrane receptor as well as a series of signaling events specifically induced by the oligosaccharides, suggesting the involvement of transient changes in cytoplasmic ion concentration in oligosaccharide signaling for defense responses.

Ion Gradients Across Contractile Vacuole Membranes of Amoebae Grown Under Different Osmotic Conditions

The contractile vacuole (CV) regulates internal osmolality of free-living protozoa such as amoebae. The CV membrane is known to contain a V-H+ ATPase but the other ion transporters involved in the water pumping mechanism are not yet established. Here we continue our earlier investigation by analytical electron microscopy to measure vacuolar and cytoplasmic ion concentrations inAcanthamoeba castellanii. The present aim is to determine concentrations of ions transported across the CV membrane of amoebae grown under different osmotic conditions.Subcellular ion concentrations in freeze-dried cryosections can generally be determined from x-ray microanalysis using the Hall method.4However, this approach is not suitable for measuring ion concentrations in the CV, which contains mainly inorganic salts and little, if any, organic compounds. A new method based on electron scattering measurements was therefore devised to deal with this highly aqueous compartment. This method involves: (i) measuring the volume of water in the hydrated cryosectioned vacuole with parallel electron energy loss spectroscopy (PEELS); (ii) measuring the mass of vacuolar salt in the freeze-dried cryosection by dark-field scanning transmission electron microscopy (STEM); and (iii) measuring the ratios of ions by energy-dispersive x-ray spectroscopy (EDXS).

Acquisition and analysis of fluorescence ratio images using a multispectral microanalytical imaging program

Any study regarding the influence of physiological, pathological, or pharmacological/toxicological factors on cellular ion homeostasis requires knowledge of multiple parameters including cytoplasmic ion concentrations, ion fluxes, and subcellular elemental distribution so that an accurate assessment of the state of ion homeostasis can be obtained. These multiple parameters are often obtained by using different types of microscopy and microanalysis. For example, cytoplasmic ion concentrations are measured by fluorescence digital imaging microscopy using various ion indicators, while cellular and subcellular element contents are determined by electron probe X-ray microanalysis (EPXMA) digital imaging. For correlating information from various microscopies, it would be advantageous to have one software package and image format that operates on the same computer platform. Here, we describe the modification of an existing software (ImagNSpect) developed for EPXMA digital imaging for use in analyzing fluorescence digital images.Fluorescence images (490 and 440 nm excitation, 540 nm emission) of BCECF-loaded cultured rat hepatocytes (Fig. 1a, b) were obtained on an inverted fluorescence microscope (Carl Zeiss, Inc., Thorn wood, NY) equipped with a rotating excitation filter wheel, a CCD camera (Photometries, Ltd., Tucson, AZ), a controller box, and a Macintosh Quadra 800 computer (Apple Computer Corp., Cupertino, CA).

Rabbit esophageal cells possess an Na+,H+ antiport

The development of esophagitis is the result of hydrogen ion diffusion into the mucosa leading to cellular acidification and necrosis. In these studies, whether esophageal cells possess transport system(s) that can respond to cytoplasmic acidification was assessed; specifically, whether esophageal cells possess an Na+,H+ antiport was determined. Nucleated esophageal cells were isolated from rabbit esophagi using a trypsin-digestion technique that yielded 5–8 × 106 cells per esophagus, of which 74% ± 3% were basal and 26% ±8% were squamous. Trypan blue was excluded by 95% ± 2% of the cells. Cytoplasmic pH (pHi) was measured using the pH-sensitive fluorescence dye 2′,7′-bis(2-carboxyethyl)-5 (and -6) carboxyfluorescein acetoxymethyl ester. Cells were acidified to the desired pHi by suspension in solutions with varying external pH (pHo) in the presence of nigericin. When cells acidified to pHi 6.3 were suspended in a choline chloride solution (pHo 7.4), cytoplasmic pHi did not increase. In contrast, Nao+ caused a concentration-dependent increase in the rate of cytoplasmic alkalinization with saturation occurring above 50 mmol/L Nao+. The transporter behaved according to first-order Michaelis-Menten type kinetics with respect to external Na+ and had an apparent Km for Nao+ of 38.4 mmol/L. In contrast, the transporter behaved with greater than first-order kinetics with respect to cytoplasmic hydrogen ion concentration. Amiloride (10−4 mol/L) caused a reversible inhibition of Na+-dependent alkalinization. Amiloride-sensitive cytoplasmic alkalinization was not observed when either cholineo or Ko+ was substituted for Nao+, while Lio+ resulted in alkalinization that was 60% ± 8% of that seen with equimolar concentrations of Nao+. The basal pHi of cells suspended in a bicarbonate-free 130 mmol/L NaCl solution (pHo 7.4) averaged 7.42 ± 0.03 (n = 10); amiloride (10−4 mmol/L caused the basal pHi to decrease to 7.26 ± 0.05 (n = 10; P < 0.0025). When cells were suspended in a choline chloride (pHo 7.4) solution, pHi averaged 7.29 ± 0.06 (n = 10) (P < 0.0025 compared with Nao+). These studies indicate that nucleated esophageal cells obtained from rabbits possess an amiloride-sensitive Na+,H+ antiport that functions to regulate basal pHi and responds to intracellular acidification.

External Potassium Supply is not Required for Root Growth in Saline Conditions: Experiments withRicinus communisL. Grown in a Reciprocal Split-Root System

Jeschke, W. D. and Wolf, 0.1988. External potassium supply is not required for root growth in saline conditions: experiments with Ricinus communis L. grown in a reciprocal split-root system.—J. exp. Bot. 39: 1149-1167. Ricinus communis L. (castor bean) plants were grown in the absence (control) and in the presence of 100 mol m3 NaCl with a reciprocal split-root system, in which K+ was supplied to one and NO3 to the other part of the root system. In these plants shoot and, to a lesser extent, total root growth were inhibited compared to plants with non-split roots. Without and with NaCl, growth of roots receiving NO J but no K+ ('minus K/plus N-roots') was substantially more vigorous than under the reverse conditions ('plus K/minus N-roots'). 100 mol m-3 NaCl inhibited growth of'minus K/plus N-roots' to the same extent as that of non-split roots, indicating that externally supplied K + was not required for root growth under saline conditions. In growth media without added K+ the root depleted the external low K + levels resulting from chemicals down to a minimum value Cmin ( 1 0 to 1 -4 mmol m 3); in the presence of 100 mol m-3 NaCl, Cmin was higher (10-18 mmol m ~3) and resulted from an initial net loss of K + . Cmin was pH-dependent. TThe distribution of K + , Na+ and Mg2+ along the root was measured. In meristematic root tissues, K+ concentrations were scarcely affected by external K+ or by NaCl, where Na + concentrations were low, but somewhat elevated at low external K + and/or high NaCl. In differentiated, vacuolated tissues K+ concentrations were low and Na+ concentrations high, if K+ was not supplied externally and/or NaCl was present. The longitudinal distribution of ions within the root was used to estimate cytoplasmic and vacuolar ion concentrations. These data showed a narrow homoeostasis of cytoplasmic K+ concentrations (100-140 mol m-3) independent of external K + supply even in the presence of 100 mol m ~3 NaCl. Cytoplasmic Na+ concentrations were maintained at remarkably low levels. Hence, external K+ concentrations above Cmin were not required for maintaining K/Na selectivity, i.e. for controlling Na+ entry. The results are discussed with regard to mechanisms of K/Na selectivity and to the importance of phloem import of K + for salt tolerance of roots and for cytoplasmic K+ homoeostasis.

A Comparison of the Methods of X-ray Microanalysis, Compartmental Analysis and Longitudional Ion Profiles to Estimate Cytoplasmic Ion Concentrations in Two Maize Varieties

Hajibagheri, M. A., Flowers, T. J., Collins, J. C. and Yeo, A. R. 1988. A comparison of the methods of X-ray microanalysis, compartmental analysis and longitudinal ion profiles to estimate cytoplasnuc ion concentrations in two maize varieties.—J. exp. Bot. 39: 279-290. The ion content of compartments within plant root cells has been studied by three different methods; flux analysis using radioactive isotopes, longitudinal ion profiles and X-ray microanalysis. The data provide estimates of the concentrations of K+ and CI− in the cytoplasm of roots of culture solution and salt grown maize by three independent methods. In the cultivar LG11 grown in 50 mol m−3 NaCl X-ray microanalysis, compartmental analysis and longitudinal profiles yielded approximately agreeing values for cytoplasmic K+ of 90, 70 and 62 mol m−33 of tissue volume respectively. However, the methods disagreed on cytoplasmic Cl− where the value obtained by compartmental analysis was about four times that from X-ray microanalysis and longitudinal profiles. Possible reasons for this are discussed.

Depolarization- and transmitter-induced changes in intracellular Ca2+ of rat cerebellar granule cells in explant cultures

Digital imaging of the Ca indicator fura-2 has been used to study the responses of developing granule cells in culture to depolarization and transmitter action. Unstimulated cells bathed in Krebs saline exhibited cytoplasmic Ca ion concentrations, [Ca2+], that were generally in the 30-60 nM range. Exposure of cells to high-potassium (25 mM) saline depolarized the membrane potential and produced an immediate rise in [Ca2+] that recovered within 2-3 min in normal saline. The response grew progressively larger over the first 20 d in culture. Transient increases in [Ca2+] to levels greater than 1 microM were observed after 12-14 d in vitro, at which time the cells displayed intense electrical activity when exposed to high K. At this stage, the increases were attenuated by blocking action potential activity with TTX. In TTX-treated or immature cells, in which the transient phase of the Ca change was relatively small, a second exposure to high K typically produced a much larger Ca response that the initial exposure. The duration of this facilitation of the response persisted for periods longer than 5 min. Application of the neurotransmitter GABA induced a transient increase in membrane conductance, with a reversal potential near resting potential (approx. -60 mV), and caused an intracellular Ca2+ increase that outlasted the exposure to GABA by several minutes. Glutamate, or kainate, induced an increase in membrane conductance but with a reversal potential more positive than spike threshold. These agents also elevated intracellular Ca2+, but unlike the case with GABA, this Ca response reversed rapidly upon removal of the transmitter. The facilitatory effect of repeated exposures to high-K saline, as well as the persistent Ca elevation following a brief GABA application, suggests that granule cells possess the capability of displaying activity-dependent changes in Ca levels in culture.

Measurement of cytoplasmic Ca ion concentration in hippocampal brain slice by using Ca 2+-sensitive fluorescent dye fura-2

Measurement of cytoplasmic Ca ion concentration in hippocampal brain slice by using Ca 2+-sensitive fluorescent dye fura-2

Cytokinin activation and redistribution of plasma-membrane ion channels in Funaria

I have investigated changes in electrical current across the plasma membrane that occur during cytokinin-induced bud formation in Funaria hygrometrica Hedw., using a non-intrusive vibrating microelectrode. Before cytokinin treatment the target caulonema cells have maximal inward current at the nuclear region. After cytokinin treatment inward current increases twofold along the length of the cell. Within minutes, however, current decreases at both the nuclear zone and the proximal end while increasing at the distal end of target cells, preceding and predicting the presumptive division site. Inward current at the distal end falls to resting levels after establishment of a bulging growth zone, and remains low around developing buds. This current is blocked by gadolinium nitrate, a Ca(2+)-uptake inhibitor, indicating a Ca(2+) component of the current. The polarity of the target cells can be disrupted by microfilament inhibitors and cytokinin-induced buds form over the nucleus, halfway along the length of the cell. I suggest that cytokinin activates plasma-membrane ion channels which are subsequently redistributed to the distal ends of target cells by a microfilament-dependent process. Cytokinin-induced concentration of ion channels over presumptive bud sites may be envisioned to exert spatial control of cytoplasmic ion concentrations and stimulate bud formation by establishing a new growth zone, directing nuclear migration, and stimulating cell division.

Intracellular compartmentation of Na+, K+ and Cl- in the Ehrlich ascites tumor cell: correlation with the membrane potential.

The intracellular distribution of Na+, K+, Cl- and water has been studied in the Ehrlich ascites tumor cell. Comparison of the ion and water contents of whole cells with those of cells exposed to La3+ and mechanical stress indicated that La3+ treatment results in selective damage to the cell membrane and permits evaluation of cytoplasmic and nuclear ion concentrations. The results show that Na+ is sequestered within the nucleus, while K+ and Cl- are more highly concentrated in the cell cytoplasm. Reduction of the [Na+] of the incubation medium by replacement with K+ results in reduced cytoplasmic [Na+], increased [Cl-] and no change in [K+]. Nuclear concentrations of these ions are virtually insensitive to the cation composition of the medium. Concomitant measurements of the membrane potential were made. The potential in control cells was -13.7 mV. Reduction of [Na+] in the medium caused significant depolarization. The measured potential is describable by the Cl- equilibrium potential and can be accounted for in terms of cation distributions and permeabilities. The energetic implications of the intracellular compartmentation of ions are discussed.

Intracellular sodium ion concentration changes in the early amphibian embryo and the influence on nuclear metabolism

During early cleavage the total sodium and potassium concentrations of the R. pipiens embryo remain relatively constant at 70 m M and 100 m M, respectively, per kg dry weight. As the blastocoel forms, intracellular sodium decreases to about 40 m M with no significant change in intracellular potassium. The sodium which leaves the cells can be recovered in the blastocoel fluid. As a result of the redistribution of sodium, the Na K ratio in the blastocoel fluid resembles that of the adult plasma. Thus, prior to gastrulation, the cells lining the blastocoel become exposed to the medium in which differentiated cells normally function, and the intracellular Na K ratio approaches adult cell values. Studies with isolated late blastula stage nuclei indicate that 14C-labelled tryptophan uptake into both the protein and acid-soluble pool is maximal at extranuclear sodium concentrations of about 40 m M but markedly reduced at 70 m M sodium concentrations. One of the factors permitting nucleo-cytoplasmic interactions at the beginning of gastrulation may be a decrease in the cytoplasmic sodium ion concentration.