Energy-dependent endocytosis in erythrocyte ghosts. IV. Effects of Ca2+, Na+ +K+, and 5'-adenylylimidodiphosphate.

The requirement of actual splitting of ATP for endocytosis in erythrocyte ghosts has been confirmed by use of the ATP analog, 5'-adenylylimidodiphosphate, (AMP-P(NH)P). This compound, in which the oxygen connecting the beta and gamma phosphorus atoms was replaced by an NH group, did not cause endocytosis nor was it a substrate for ATPase activity. AMP-P(NH)P was a competitive inhibitor both for the endocytosis and the Mg2+-ATPase activities. The K1 of AMP-P(NH)P for Mg2+ ATPase activity was 2.0 - 10-4 M and, while the Km of ATP for this activity was also 2.0 - 10-4 M indicating nearly identical affinities of ATP and AMP-P(NH)P for the active site. ADP, or ADP plus orthophosphate, did not cause endocytosis, showing that endocytosis was not due to binding of the products of ATP hydrolysis. Sodium or potassium ion or ouabain had no effect on endocytosis, which eliminated the possibility of involvement of the Na+, K+ ATPase in the endocytosis process. Calcium could not be substituted for magnesium; rather it inhibited endocytosis at the concentration of 1 - 10-3 M. EGTA relieved the inhibitory effect of Ca, which indicated that the binding of calcium to the membrane was reversible. These experimental results reaffirm the conclusion that ATP must be split to engender endocytosis under these conditions. Some characteristic parameters of the hemoglobin-free porcine erythrocyte ghosts were studied in order to characterize the system more adequately.     

Evaluation of a role for intracellular Na+, K+, Ca2+, and Mg2+ in hyperthermic cell killing

A dose of heat which renders 98% of a population of Chinese hamster ovary cells reproductively dead has no significant effect on their Na+, K+, or Mg2+ content by 28 h postheat. In contrast, the cellular Ca2+ content increases in a dose-dependent manner as observed at 22 h after heating for 15-35 min at 45 degrees C. However, the rates of both influx and efflux of Ca2+ were reduced by heating. Increasing the cellular Ca2+ content by incubating the cells in high extracellular Ca2+, either at the time of heating or for a period of 22 h following heat, does not potentiate the lethal effect of heat. Completely blocking the heat-induced increase in Ca2+ content by incubating the cells in medium containing a low Ca2+ concentration does not protect the cells. Therefore, we conclude that heat does not produce any significant changes in the Na+, K+, or Mg2+ content of cells and that the heat-induced increase in Ca2+ does not play an important role in hyperthermic cell killing.     

Correlated effects of external magnesium on cation content and DNA synthesis in culture chicken embryo fibroblasts.

Depletion of Mg²⁺ in the growth medium for chicken embryo fibroblasts produces a large decrease in DNA synthesis as measured by ³H-thymidine incorporation, and concomitant decreases in cellular K⁺ and Mg²⁺ and increases in Na⁺ and Ca²⁺. In cells grown in media containing 0.2 mM Ca²⁺, graded reduction of Mg²⁺ from 0.8 mM (control) to 0.016 mM produced graded decreases in DNA synthesis to 10% of control at 0.016 mM Mg²⁺. Concomitantly, cell cations showed graded changes, Na⁺ increasing to 227%, K⁺ decreasing to 52.5%, Mg²⁺ decreasing to 57.5% and Ca²⁺ increasing to 153.5% of control. The effects of Mg²⁺ depletion on DNA synthesis and cell cation content exhibited a dependence on Ca²⁺ concentration, the effects being larger at low Ca²⁺ concentration. Use of inorganic pyrophosphate in the growth medium as a selective complexor of Mg²⁺ caused a marked decrease in DNA synthesis which was accompanied by changes in cellular cation content similar to those produced by direct Mg²⁺ depletion. The effects of Mg²⁺ depletion on cell cation content are explainable in terms of changes in membrane permeability caused by rapid external surface exchange of bound divalent cations. Among the several interpretations of the data in terms of possible mechanisms by which changes in external Mg²⁺ concentration may affect cell metabolism, the most consistent with known properties of the system is the concept of a central role for intracellular free Mg²⁺ in the coordinate control of growth and metabolism in animal cells.     

The accumulation ratio of K+, Na+, Ca2+ and tetrapropylammonium in steady-state Mitochondria.

The accumulation ratio of a permeant cation under steady-state conditions after active uptake, is defined as: ($\text{{cat}{}_{\mathrm{i}}\text{}}\text{{cat}{}_0\text{}}\text{)}{}^{\mathrm{<mtext>1</mtext><mtext>z</mtext>}}$, where Z is the valence of the cation, and {cat_i} and {cat₀} are the internal and external cation activities, respectively. The electrogenic proton pump predicts that the accumulation ratio should be independent of (i) the chemical structure of the cation and (ii) the degree of permeability of the membrane to cations. Furthermore the accumulation ratio should be the same for all permeant cation species simultaneously present. In the present study it is found that under steady-state conditions: (i) the accumulation ratio is not the same for potassium in the presence of valinomycin, for tetrapropylammonium in the presence of tetraphenylboron, and for calcium in the presence of acetate; (ii) the accumulation ratio is not identical for two cations such as potassium and sodium present simultaneously in the presence of gramicidin; (iii) the accumulation ratio is dependent on the external carrier concentration, such as valinomycin or tetraphenylboron. It is concluded that the cation distribution ratios under steady-state conditions are not compatible with the predictions of the electrogenic proton pump model.     

钠、钾、钙和葡萄糖对白斑狗鱼精子活力的影响

观察了白斑狗鱼精子在0～679．6kPa NaCl、KCl、葡萄糖溶液和0～339．8kPa CaCl2溶液中的活动情况。在NaCl、KCl、葡萄糖溶液中,白斑狗鱼精子快速运动时间和寿命的变化规律基本一致,精子活动最适渗透压介于339．8～453．0kPa。K^ 有延长精子寿命的作用。外源性葡萄糖可被精子利用,增强精子活力．延长精子寿命。56．7kPa CaCl2对精子活动有抑制作用,并引起精子聚集,该效应随着Ca^2 浓度升高而增强。     

Na+,K+-ATPase and Ca2+-ATPase isozymes in malignant neoplasms

A current state of researches on mechanisms of ion homeostasis regulation in the specific conditions of the uncontrolled malignant tumor growth (mainly in carcinomas) concerning the contribution of Na+,K+-ATPase, plasma membrane and sarco(endo)plasmic reticulum Ca2+-ATPases has been reviewed. Particular attention has been focused on the molecular and biochemical links providing the redistribution of the transporting ATPases isozyme pattern for the regulatory requirements of the cell signaling pathways at stable proliferation and viability in malignancy.     

Transport of H+, K+, Na+ and Ca++ in Streptococcus

Summary The streptococci differ from other bacteria in that cation translocations (with the possible exception of one of the K⁺ uptake systems) occur by primary transport systems, i.e., by cation pumps which use directly the free energy released during hydrolysis of chemical bonds to power transport. Transport systems in other bacteria, especially for Na⁺ and Ca⁺⁺, are often secondary, using the free energy of another ion gradient to drive cation transport. In streptococci H⁺ efflux occurs via the F₁F₀-ATPase. This enzyme is composed of eight distinct subunits. Three of the subunits are embedded in the membrane and form a H⁺ channel; this is called the F₀ portion of the enzyme. The other five subunits form the catalytic part of the enzyme, called F₁, which faces the cytoplasm and can easily be stripped from the membrane. Physiologically, this enzyme functions as a H⁺-ATPase, pumping protons out of the cell to form an electrochemical proton gradient, . The F₁F₀-ATPase, however, is fully reversible and if supplied with P_i, ADP and a + of sufficient magnitude (ca –200 mv) catalyzes the synthesis of ATP. Streptococcus faecalis can accumulate K⁺ and establish a gradient of 50 000:1 (in>out) under some conditions. Uptake occurs by two transport systems. The dominant, constitutive system requires both an electrochemical proton gradient and ATP to operate. The minor, inducible K⁺ transport system, which has many similarities to the K⁺-ATPase of the Kdp transport system found in Escherichia coli, requires only ATP to power K⁺ uptake.Sodium extrusion occurs by a Na⁺/H⁺-ATPase. Exchange is electroneutral and there is no requirement for a . The possibility that the Na⁺/H⁺-ATPase may consist of two parts, a catalytic subunit and a Na⁺/H⁺ antiport subunit, is suggested by the finding that damage to the Na⁺ transport system either through mutation or protease action leads to the appearance of -requiring Na⁺/H⁺ antiporter activity.Ca⁺⁺ like Na⁺ is extruded from metabolizing, intact cells. Transport requires no but does require ATP. Reconstitution of Ca⁺⁺ transport activity with accompanying Ca⁺⁺-stimulated ATPase activity into proteoliposomes suggests that Ca⁺⁺ is transported by a Ca⁺⁺-translocating ATPase.Where respiring organelles and bacteria use secondary transport systems the streptococci have developed cation pumps. The streptococci, which are predominantly glycolyzing bacteria, generate a much inferior to that of respiring organisms and organelles. The cation pumps may have developed simply in response to an inadequate .Abbreviations electrochemical potential of protons - membrane potential - pH pH gradient - p proton-motive force - DCCD N,Na¹-dicyclohexlcarbodiimide - TCS tetrachlorosalicylanilide - FCCP carbonylcyanide-p-trifluoromethylphenylhydrazone - CCCP carbonylcyanie-m-chlorophenylhydrazone - TPMP⁺ triphenylmethyl phosphonium ion - DDA⁺ dibenzyldimethylammonium ion - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - EGTA ethyleneglycol-bis (amino-ethyl-ether)-N,N-tetraacetic acid     

Competitive membrane adsorption of Na+, K+, and Ca2+ in smooth muscle cells

Summary A theory for Na⁺, K⁺ and Ca²⁺ competitive adsorption to a charged membrane is used to explain a number of experimental observations in smooth muscle. Adsorption is described by Langmuir isotherms for mono- and divalent cations which in turn are coupled in a self-consistent way to the bulk solution through the diffuse double layer theory and the Boltzman equations. We found that the dissociation constants for binding of Na⁺, K⁺ and Ca²⁺ in guinea pig taenia coli areca. 0.009, 1.0, and 4×10^–8 m, respectively. Furthermore, the effect of a Ca²⁺ pump that maintains free surface Ca²⁺ concentration constant is investigated. A decrease in intracellular Na⁺ content results in an increased Ca²⁺ uptake; part of this uptake is due to an increase in surface-bound Ca²⁺ in an intracellular compartment which is in contact with the myofilaments. Variations in the amount of charge available to bind Ca²⁺ and the surface charge density are studied and their effect interpreted in terms of different pharmacological agents.     

盐胁迫下杨树根际系统盐分离子分布特性

在温室条件下,采用盆栽根箱培养的方法研究盐胁迫下I 69杨(PopulusdeltoidesBartr.cv.'Lux')和NL 1381杨〔PopulusdeltoidesBartr.cv.'Lux'×P.euramericana(Dode)GeninierCL'I 45 51'〕根际、非根际土壤盐分分布特征。盐处理浓度共设3个水平:CK(NaCl0g kg)、处理A(NaCl1g kg)和处理B(NaCl2g kg),采用完全随机设计。结果表明,2个杨树无性系根际水溶性K+亏缺,水溶性Na+、Ca2+和Mg2+富集。K+的亏缺率及Na+的富集率随NaCl处理浓度的增大而减小,Ca2+和Mg2+的富集率在非盐渍条件下最低,处理A达最高,处理B较处理A略有下降。在盐胁迫下,无性系NL 1381杨根际土壤Na+的浓度和电导率均低于无性系I 69杨,可以有效减轻盐分对根系的渗透胁迫,相对而言具有较强的抗盐性。     

SIMS Investigation of the Distribution of K+, Na+, Mg2+ and Ca2+ Cations in Birch Pollen

Two microanalytical techniques were used to investigate the inorganic cation content and distributions in birch (Betula verrucosa Ehrh.) pollen. With intact pollen grains. X-ray microanalysis (EDX) could only give a mean ionic composition. Secondary Ion Microscopy and Spectrometry (SIMS) appeared to be a more suitable technique to image ion distributions in the different pollen structures. This was carried out with samples prepared using a new vapour phase technique designed to improve ion retention. Transmission electron microscopy (TEM)showed good structural preservation of the samples. Monovalent ion (K⁺, Na⁺) distribution showed features different from those of the divalent cations (Ca²⁺, Mg²⁺). In the vegetative cell, the alkaline cations were mainly distributed in the most internal part of the cytoplasm and they were probably associated with starch grains or concentrated in dry vacuoles. Calcium distribution correlated well with the areas in the cytoplasm of the vegetative cell containing a dense network of mitochondria and endoplasmic reticulum. Within the pollen grain, the sperm cell appeared to contain the most calcium. Calcium was also abundant in the sporoderm. These results reveal the potential of SIMS for pollen studies that include germination, the monitoring of air pollutants and the allergens-ion interactions.     

Effects of intracellular and extracellular concentrations of Ca2+, K+, and Cl- on the Na+-dependent Mg2+ efflux in rat ventricular myocytes

Intracellular Mg2+ concentration ([Mg2+]i) was measured in rat ventricular myocytes with the fluorescent indicator furaptra (25 degrees C). After the myocytes were loaded with Mg2+, the initial rate of decrease in [Mg2+]i (initial Delta[Mg2+]i/Deltat) was estimated upon introduction of extracellular Na+, as an index of the rate of Na+-dependent Mg2+ efflux. The initial Delta[Mg2+]i/Deltat values with 140 mM [Na+]o were essentially unchanged by the addition of extracellular Ca2+ up to 1 mM (107.3+/-8.7% of the control value measured at 0 mM [Ca2+]o in the presence of 0.1 mM EGTA, n=5). Intracellular loading of a Ca2+ chelator, either BAPTA or dimethyl BAPTA, by incubation with its acetoxymethyl ester form (5 microM for 3.5 h) did not significantly change the initial Delta[Mg2+]i/Deltat: 115.2+/-7.5% (seven BAPTA-loaded cells) and 109.5+/-10.9% (four dimethyl BAPTA loaded cells) of the control values measured in the absence of an intracellular chelator. Extracellular and/or intracellular concentrations of K+ and Cl- were modified under constant [Na+]o (70 mM), [Ca2+]o (0 mM with 0.1 mM EGTA), and membrane potential (-13 mV with the amphotericin-B-perforated patch-clamp technique). None of the following conditions significantly changed the initial Delta[Mg2+]i/Deltat: 1), changes in [K+]o between 0 mM and 75 mM (65.6+/-5.0% (n=11) and 79.0+/-6.0% (n=8), respectively, of the control values measured at 140 mM [Na+]o without any modification of extracellular and intracellular K+ and Cl-); 2), intracellular perfusion with K+-free (Cs+-substituted) solution from the patch pipette in combination with removal of extracellular K+ (77.7+/-8.2%, n=8); and 3), extracellular and intracellular perfusion with K+-free and Cl--free solutions (71.6+/-5.1%, n=5). These results suggest that Mg2+ is transported in exchange with Na+, but not with Ca2+, K+, or Cl-, in cardiac myocytes.     

Modulation of free Ca oscillations in single hepatocytes by changes in extracellular K+, Na+ and Ca2+

Single rat hepatocytes, microinjected with the calcium-sensitive photoprotein aequorin, when stimulated with either phenylephrine or arg8-vasopressin exhibit agonist-specific oscillations in cytosolic free calcium levels (free Ca). In the majority of the cells examined adding excess potassium chloride, sodium chloride or choline chloride abolished transient behaviour. However, in cells that continued to oscillate the transient parameters were subtly modified by these treatments. In experiments using phenylephrine as the agonist, adding excess potassium chloride to the superfusate significantly reduced transient length, increased the rate of transient rise and reduced the smoothed peak free Ca level without significantly altering the intertransient resting free Ca level or the falling time constant. The possible mechanisms by which these alterations may occur are discussed.     

Regulation of Na+,K+ pump activity by nerve growth factor in chick embryo dorsal root ganglion cells

Nerve growth factor (NGF) is required for the growth and development of sensory and sympathetic neurons. Incubation of chick dorsal root ganglionic cells without NGF resulted in a decrease of active (Na+,K+-pump-mediated) K+ influx over a period of several hours. Addition of NGF to NGF-deprived cells caused 1) a return of the active K+ influx to the values occurring in cells continuously exposed to NGF, preceded by 2) a very rapid, but transient overstimulation of the Na+,K+-pump-mediated K+ influx. Restoration of normal Na+,K+-pump activity occurred at NGF concentrations of 1 biological unit/ml or greater, whereas the NGF concentration in the 1-100 biological unit/ml range affected the rapidity with which the pump restoration took place. The transient pump behavior was only observed in NGF-deprived cells and could not be elicited in NGF-supported steady-state cells or in cells having already received delayed NGF once. This transient Na+,K+-pump behavior was exclusively displayed in conjunction with a high intracellular Na+ concentration. Decreasing the external Na+ concentration below 70 mM reduced the hyperstimulation response to NGF, until at 10 mM Na+ the delayed presentation of NGF caused no overshoot at all. The effect of NGF on the Na+,K+-pump was specific for the NGF molecule and could not be mimicked by other proteins.     

Ionophore-mediated Ca2+ countertransport: role of Na+, Li+ or H+ gradient

Summary In an artificial system, the ionophore A23187, which transports Ca²⁺ but not Na⁺, is able to mediate the uphill translocation of Ca²⁺ from one aqueous medium to another across an organic immiscible phase, provided that a Na⁺, Li⁺ or H⁺ gradient is imposed on the system. Therefore, in the process known as Na-Ca countertransport, the downhill influx of Na⁺ may not be necessary for causing Ca²⁺ extrusion against its electrochemical gradient.     

Effects of lectins on cytoskeleton and morphology or cultured chick embryo fibroblasts

The microfilaments and microtubules of cultured chick embryo skin fibroblasts were studied in the presence of exogenous lectins by an indirect immunofluorescence technique. Lectin treatment induced modifications in the arrangement of myosin, actin and tubulin, determined depolymerization of the proteins and caused changes in cell shape and size. The results suggest that the interaction between lectins and their specific membrane receptors triggers a series of changes in the cytoskeletal pattern via transmembrana as yet unknown mechanisms and that these are responsible for the alterations in cell shape and size.     

Effects of K+, Ba2+ and Ca2+ on the excitable membrane in Paramecium tetraurelia

• 1.1. The effects of Ba²⁺ and K⁺ ions on the membrane currents of Paramecium tetraurelia under a voltage clamp were investigated.
• 2.2. External Ba²⁺ suppresses the inward-going K-current and the Ca-induced K-outward current and changes the activation and inactivation kinetics of transient inward current through the Ca-channel.
• 3.3. K⁺ increases the Ca-induced K-conductances but little affects the leakage conductance.
• 4.4. The resting potentials by changing those ionic concentrations shift the voltage sensitivities of all voltage sensitive channels, simultaneously.
• 5.5. The competition between ions to the channel responses was discussed.

     

Dependence of GnRH action on Na+, K+, and Ca2+ in the frog, Rana pipiens, pituitary

The roles of K+, Ca2+, and Na+ ions in the mechanism of gonadotropin releasing hormone (GnRH) action on frog (Rana pipiens) hemipituitaries were studied using an in vitro superfusion system. The effects of elevated K+ alone or in combination with Ca2+-depleted medium, tetrodotoxin (TTX), or with 100 ng/ml GnRH were examined. The involvement of K+ was also studied indirectly through the use of tetraethyl ammonium chloride (TEA). The importance of Ca2+ was established by the loss of responsiveness to GnRH in Ca2+-depleted medium, or in the presence of the Ca2+ competitor CoCl2. The absence of a major dependence of GnRH on Na+ was revealed by the continued gonadotropin secretion after addition of 1 microM TTX to medium containing GnRH or 36.3 mM KCl, or by replacement of NaCL with choline chloride. High (10 X normal) KCl (36.3 mM) stimulated gonadotropin--both LH and FSH--secretion, but the response was more gradual than for GnRH. The inclusion of TEA (to block K+ efflux) in medium with GnRH accentuated the effect of GnRH, and the effects of elevated (36.3 mM) KCl and 100 ng/ml GnRH (a relatively high dose) were additive. Responses to high K+, like GnRH, were abolished by removal of Ca2+ from the medium. Overall, the roles of K+, Ca2+, and Na+ ions in the mechanism of GnRH action are very similar between mammals and frogs; Ca2+ apparently serves a critical function in the mechanism of GnRH action, while Na+ appears not to be involved. K+ can induce gonadotropin secretion, but it is not clear that it plays a direct role in the mediation of the action of GnRH.     

Unidirectional Na+, Ca2+, and K+ fluxes through the bovine rod outer segment Na-Ca-K exchanger

The properties of the Na-Ca exchanger in the plasma membrane of rod outer segments isolated from bovine retinas (ROS) were studied. Unidirectional Ca2+, Na+, and K+ fluxes were measured with radioisotopes and atomic absorption spectroscopy. We measured K+ fluxes associated with the Ca-Ca self-exchange mode of the Na-Ca exchanger to corroborate our previous conclusion that the ROS Na-Ca exchanger differs from Na-Ca exchangers in other tissues by its ability to transport K+ (Schnetkamp, P. P. M., Basu, D. K. & Szerencsei, R. T. (1989) Am. J. Physiol. 257, C153-C157). The Na-Ca-K exchanger was the only functional cation transporter in the plasma membrane of bovine ROS with an upper limit of a flux of 10(5) cations/ROS/s or a current of 0.01 pA contributed by other cation channels, pumps, or carriers; cation fluxes via the Na-Ca-K exchanger amounted to 5 x 10(6) cations/ROS/s or a current of 1 pA. Ca2+ efflux via the forward mode of the Na-Ca-K exchanger did not operate with a fixed single stoichiometry. 1) The Na/Ca coupling ratio was increased from three to four when ionophores were added that could provide electrical compensation for the inward Na-Ca exchange current. 2) The K/Ca coupling ratio could vary by at least 2-fold as a function of the external Na+ and K+ concentration. The results are interpreted in terms of a model that can account for the variable Ca/K coupling ratio: we conclude that the Ca2+ site of the exchanger can translocate independent of translocation of the K+ site, whereas translocation of the K+ site requires occupation of the Ca2+ site, but not its translocation. The results are discussed with respect to the physiological role of Na-Ca-K exchange in rod photoreceptors.