Phosphate transport by rat intestinal basolateral-membrane vesicles.

The characteristics of phosphate transport across intestinal basolateral membranes of the rat were determined by using enriched preparations in which uphill Na+-dependent D-glucose transport could not be demonstrated, but ATP-dependent Ca2+ transport was present. Phosphate transport was saturable, Na+-dependent and exhibited Michaelis-Menten kinetics. Vmax. was 51.1 +/- 4.2 pmol/10 s per mg of protein and Km was 14 +/- 3.9 microM. The transport process was electroneutral. Tracer-exchange experiments and counter-transport studies confirmed the presence of a Na+-Pi carrier at the basolateral membrane. The presence of inside-positive membrane potential did not enhance phosphate uptake, indicating that the Na+ effect is secondary to the presence of the Na+-Pi carrier rather than an induction of positive membrane potential. The stoichiometry of this carrier at pH 7.4 was 2 Na+:1 phosphate, as shown by direct studies utilizing the static-head method. These studies are the first to determine the presence of a phosphate carrier at the basolateral membrane.     

The response of two rice cultivars to external Na/Ca ratio

The response of the rice cultivars ‘M9’ and ‘M-201’ to nutrient cultures salinated at −0.4 MPa with varying ratios of Na and Ca was studied. Although the dry matter production of both cultivars was sensitive to the Na/Ca ratio, this correlation was significant only for M-201. Calcium nutrition was severely affected by the composition of the external solution, and the laminae exhibited Ca-deficiency symptoms at Na/Ca molar ratios of 78 and 198. Sodium concentration in the shoot decreased as the Na/Ca ratio in the external solution decreased. Patterns of Na and Cl distribution in the shoot tissues were similar; both ions were accumulated preferentially in the tillers and older leaves. The Na-induced inhibition of Ca uptake and transport appears to be more limiting to shoot growth of M9 and M-201 than Na toxicity per se.     

Predicting Cation Ratios in Corn from Saline Solution Composition

The response of Zea mays (L.) cv. Pioneer 3906 to nutrient culturesthat varied in Na/Ca ratios was studied at 5 different salinitylevels; 0, 0.1, –0.2, 0.3 and –0.4 MPa. Principlesof ion exchange theory were used to relate the cation composition(Ca, Mg and Na) in the shoots to activity ratios in the rootmedia. The data are expressed using the Gapon convention wherethe selectivity coefficient is related to the equivalent fractionof the exchange ions. Plots of the cation ratios of Ca/Na, Ca/Mgand Mg/Na can be represented by a single line regardless ofsalinity level. This information supports the concept derivedfrom physiological studies that the uptake of Ca, Mg and Naby salt-stressed plants is passive. The data suggest that theratios of these cations in corn shoots can be predicted forvarious salinities from solution composition of the root mediaand shoot ion concentrations at a single salinity level. Key words: Gapon equation, ion exchange, selectivity, cation uptake     

Supramolecular selective transport of Ca2+/Na+ by dicarboxylic ligands across bulk liquid membranes.

Bulk liquid membrane transport of Ca2+ and Na+ ions was performed with dicarboxylic type ligands containing (S)-phenylalanine (Phe-2-O) and (S)-benzyltyrosine (Bz-Tyr-2-O) in pH gradients. With Bz-Tyr-2-O, calcium was generally favoured at every pH, the transport rate strongly depending upon the ligand concentration. At pH 8, a reversal of selectivity (Na+/Ca2+) was observed for the first time at low ligand concentration, thus suggesting that a supramolecular-type of transport occurs as a consequence of the different assemblages formed by the ligand according to pH and concentration. This is the first case of M2+/M+ selectivity induced by the carrier concentration.     

Ca2+-induced down-regulation of Na+ channels in toad bladder epithelium

Regulation of epithelial Na+ channels was investigated by measuring the amiloride-blockable 22Na+ fluxes in apical membrane vesicles, derived from cells exposed to various treatments. Maximal amiloride-blockable 22Na+ uptake into vesicles was obtained if the cells were preincubated at 25 degrees C in a Ca2+-free [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) solution. Including 10(-5) M Ca2+ in the cell incubating medium blocked nearly all of the amiloride-sensitive flux in vesicles, even though the Ca2+ was removed before homogenization of the cells. This Ca2+-dependent inhibition of Na+ channels could be induced in whole cells only; incubating cell homogenates with Ca2+ had no effect on the transport in vesicles. The dose-response relationships of this effect were measured by equilibrating cell aliquots with various Ca2+-EGTA buffers, preparing membrane vesicles (in the absence of Ca2+ ions), and assaying them for amiloride-sensitive Na+ permeability. It was found that the Ca2+ blockage is highly cooperative (Hill coefficient of nearly 4) and is characterized by an inhibition constant which varies between 6.4 X 10(-8) to 8.15 X 10(-6)M Ca2+. Thus, it is likely that the above process is involved in the physiological control of Na+ transport. The Ca2+-dependent transport changes were not affected by the calmodulin inhibitor trifluoperasine, vanadate (VO3-), phorbol ester, colchicine, cytochalasin B, 3-deazaadenosine, and 8-bromo-cAMP. Vanadyl (VO2+) ions, on the other hand, produced a "Ca2+-like" inhibition of transport.     

A mathematical model for spreading cortical depression.

A mathematical model is derived from physiological considerations for slow potential waves (called spreading depression) in cortical neuronal structures. The variables taken into account are the intra- and extracellular concentrations of Na+, Cl-, K+, and Ca++, together with excitatory and inhibitor transmitter substances. The general model includes conductance changes for these various ions, which may occur at nonsynaptic and synaptic membrane together with active transport mechanisms (pumps). A detailed consideration of only the conductance changes due to transmitter release leads to a system of nonlinear diffusion equations coupled with a system or ordinary differential equations. We obtain numerical solutions of a set of simplified model equations involving only K+ and Ca++ concentrations. The solutions agree qualitatively with experimentally obtained time-courses of these two ionic concentrations during spreading depression. The numerical solutions exhibit the observed phenomena of solitary waves and annihilation of colliding waves.     

Effect of sodium on cellular calcium transport in rat kidney

Summary The influence of extracellular Na (Na _o ) on cellular Ca transport and distribution was studied in rat kidney slices. Calcium efflux from prelabeled slices was depressed when Na _o was completely replaced by choline or tetraethylammonium (TEA) ions and it was markedly stimulated when Na was reintroduced in a Na-free medium. However, reducing Na _o (with choline or TEA as substituting ions) did not increase the total slice⁴⁰Ca, their total exchangeable Ca pool, or the⁴⁰Ca or⁴⁵Ca of mitochondria isolated from these slices. Kinetic analyses of steady-state⁴⁵Ca desaturation curves showed that reducing Na _o depressed the exchange of Ca across the plasma membrane, slightly decreased the cytosolic Ca pool, but did not significantly affect the mitochondrial Ca pool and Ca cycling. Ouabain (10^–3 m) which should reduce the Na gradient across the plasma membrane had no effect on calcium distribution and transport. These results suggest that in kidney cells low Na _o depresses Ca influx as well as Ca efflux; there may be an interaction between Na and Ca at a possible carrier located in the plasma membrane, but there is no Na/Ca exchange as described in several excitable tissues.     

Kinetics of sulfate transport by Penicillium notatum. Interactions of sulfate, protons, and calcium.

The active transport of inorganic sulfate by an ATP sulfurylase-negative strain of Penicillium notatum is promoted by H+ ions and metal ions (divalent metal ions being more effective than monovalent metal ions). Initial velocity studies suggest that H+ and SO4(2-) add to the carrier in an ordered sequence (H+ before SO4(2-)), with H+ at equilibrium with free carrier and carrier-H+ complex. The linear reciprocal plots and replots suggest a 1:1 stoichiometry between H+ and SO4(2-). Ca2+ and other divalent metal ions stimulate sulfate transport markedly in buffered suspensions of low ionic strength. The kinetics of the Ca2+/SO4(2-) interaction suggest that Ca2+ (like H+) adds to the carrier before SO4(2-) and is at equilibrium with free carrier and carrier-Ca2+ complex. The linear reciprocal plots and replots indicate a 1:1 stoichiometry between Ca2+ and SO4(2-). Thus the fully loaded carrier-SO4(2-) -Ca2+ -H+ complex has a net positive charge relative to that of the free carrier, a fact consistent with the chemiosmotic hypothesis of membrane transport. The kinetics of the H+/Ca2+ interaction point to a random A-B (rapid equilibrium), ordered C sequence with A = H+, B = Ca2+, and C = SO4(2-). Selenate (an alternate substrate competitive with sulfate) is an uncompetitive inhibitor with respect to Ca2+, in agreement with the suggested mechanism. Internal charge balance is not accomplished by a stoichiometric coaccumulation of Ca2+ and SO4(2-). Sulfate transport does, however, promote 45Ca2+ uptake. A significant fraction of the added Ca2+ is bound by the mycelial surface. Binding is extremely rapid, but reversible.     

Identification and conformational changes of the intestinal proline carrier

Fluorescein isothiocyanate (FITC) was used to selectively label the rabbit intestinal brush-border imino carrier, identify the binding protein on SDS-polyacrylamide gel electrophoresis, and monitor the effect of ions on fluorescein quenching. FITC inhibits Na+-dependent L-proline transport irreversibly, but transport is protected by physiological concentrations of Na+ and L-proline. About 1 nmol of FITC/mg of protein binds specifically to the transporter, which was identified by SDS-polyacrylamide gel electrophoresis as a 100 +/- 5-kDa peptide. Na+ produced a specific, saturable quench in the fluorescence of FITC bound to the proline carrier. Both transport and FITC quenching are inhibited by n-acetylimidazole, and membranes are protected from acetylation by Na+. We conclude that Na+ binds to the proline carrier (100-kDa peptide) to produce a change in conformation that results in an increase in the affinity of the carrier for proline.     

Stoichiometry of the Cardiac Na+/Ca2+ exchanger NCX1.1 measured in transfected HEK cells

The stoichiometry with which the Na+/Ca2+ exchanger, NCX1, binds and transports Na+ and Ca2+ has dramatic consequences for ionic homeostasis and cellular function of heart mycocytes and brain neurons, where the exchanger is highly expressed. Previous studies have examined this question using native NCX1 in its endogenous environment. We describe here whole-cell voltage clamp studies using recombinant rat heart NCX1.1 expressed heterologously in HEK-293 cells. This system provides the advantages of a high level of NCX1 protein expression, very low background ion transport levels, and excellent control over clamped voltage and ionic composition. Using ionic conditions that allowed bi-directional currents, voltage ramps were employed to determine the reversal potential for NCX1.1-mediated currents. Analysis of the relation between reversal potential and external [Na+] or [Ca2+], under a variety of intracellular conditions, yielded coupling ratios for Na+ of 1.9-2.3 ions per net charge and for Ca2+ of 0.45 +/- 0.03 ions per net charge. These data are consistent with a stoichiometry for the NCX1.1 protein of 4 Na+ to 1 Ca2+ to 2 charges moved per transport cycle.     

Stoichiometry of the retinal cone Na/Ca-K exchanger heterologously expressed in insect cells: comparison with the bovine heart Na/Ca exchanger

The transport stoichiometry is an essential property of antiporter and symporter transport proteins. In this study, we determined the transport stoichiometry of the retinal cone potassium-dependent Na/Ca exchanger (NCKX) expressed in sodium-loaded cultured insect cells. The Na/Ca and Rb/Ca coupling ratios were obtained by direct measurements of the levels of (86)Rb and (45)Ca uptake and sodium release associated with reverse Na/Ca exchange. Rb/Ca coupling ratios of 0.98 [standard deviation (SD) of 0.12, 15 observations] and 0.92 (SD of 0.12, 13 observations) were obtained for the chicken and human retinal cone NCKX, respectively. Na/Ca coupling ratios of 4.11 (SD of 0.24, 10 observations) and 3.98 (SD of 0.34, 15 observations) were obtained for the chicken and human retinal cone NCKX, respectively, whereas a lower average coupling ratio of 3.11 (SD of 0.34, 10 observations) was obtained with cells expressing the bovine Na/Ca exchanger (NCX1). These results are consistent with a 4Na/1Ca + 1K stoichiometry for retinal cone NCKX. High Five cells expressing full-length dolphin rod NCKX, Caenorhabditis elegans NCKX, or bovine rod NCKX from which the two large hydrophilic loops were removed all showed a significant calcium-dependent (86)Rb uptake, whereas no calcium-dependent (86)Rb uptake was observed in cells expressing bovine NCX1. The calcium dependence of (45)Ca uptake yielded values between 1 and 2.5 microM for the external calcium dissociation constant of the different NCKX proteins studied here.     

Kinetics of hyperosmotically stimulated Na-K-2Cl cotransporter in Xenopus laevis oocytes

A detailed study of hypertonically stimulated Na-K-2Cl cotransport (NKCC1) in Xenopus laevis oocytes was carried out to better understand the 1 K(+):1 Cl(-) stoichiometry of transport that was previously observed. In this study, we derived the velocity equations for K(+) influx under both rapid equilibrium assumptions and combined equilibrium and steady-state assumptions and demonstrate that the behavior of the equations and curves in Lineweaver-Burke plots are consistent with a model where Cl(-) binds first, followed by Na(+), a second Cl(-), and then K(+). We further demonstrate that stimulation of K(+) movement by K(+) on the trans side is an intrinsic property of a carrier that transports multiple substrates. We also demonstrate that K(+) movement through NKCC1 is strictly dependent upon the presence of external Na(+), even though only a fraction of Na(+) is in fact transported. Finally, we propose that the larger transport of K(+), as compared with Na(+), is a result of the return of partially unloaded carriers, which masks the net 1Na(+):1K(+):2Cl(-) stoichiometry of NKCC1. These data have profound implications for the physiology of Na-K-2Cl cotransport, since transport of K-Cl in some conditions seems to be uncoupled from the transport of Na-Cl.     

The cycling of calcium, sodium, and protons across the inner membrane of cardiac mitochondria.

A method is described that permits simultaneous determination of the net charge transfer associated with Ca2+ transport by the ruthenium-red-sensitive carrier and the ionized internal [Ca2+] in heart mitochondria. The data indicate that this carrier catalyses a charge-uncompensated flux of Ca2+. Full charge compensation for Ca2+ influx is provided by the respiration-dependent efflux of H+. The net efflux of Ca2+ induced by Na+ is analysed in terms of two other carriers, a Na+-Ca2+ antiporter and a Na+-H+ antiporter. Evidence is presented that these two carriers are separate and that the Na+-H+ exchange is the more rapid. The fluxes of Ca2+, Na+ and H+ during the Na+-induced efflux of Ca2+ support a series of events in which the Na+-H+ exchange enables unidirectional Ca2+ fluxes via the uniport and antiport systems to be integrated into a cycle.     

Complexation and ionophoric properties of taxol and colchicine: complex formation and transport of sodium, potassium, magnesium and calcium ions across a liquid membrane

We report the activities of taxol (an anticancer drug) and colchicine, which are inhibitors of microtubule organization, on the complexation and transport of Na+, K+, Mg2+ and Ca2+ ions across a liquid membrane, using a spectrophotometric procedure. Taxol, a diterpenoid compound, that has been demonstrated to possess a potent antitumour activity, is shown to extract Na+, K+, Mg2+ and Ca2+ ions from the aqueous solution to the organic phase with preference for Ca2+ ions. A kinetic study of the transport and complexation of Na+, K+, Mg2+ and Ca2+ ions through a liquid membrane revealed that the K+ ion is more rapidly transported and the Ca2+ ion is more rapidly complexed than other ions. However, colchicine, another alkaloid compound, extracted and transported only the divalent ions tested, Mg2+ and Ca2+. In both complexation and transport, the flux of the ions increases with the concentration of taxol or colchicine. Complexation and ionophoric properties of taxol and colchicine sheds new lights on therapeutic properties of these drugs. The treatment of disease states by the administration of these drugs to alter membrane permeability will prove to be a valuable therapeutic concept.     

Characteristics of a rat liver cellular extract protein which mediates transport of various phospholipids from liposomes to mitochondria]

The effect of pH, Ca++ and Na+ concentration on the phosphatidylethanolamine transport from liposomes to mitochondria by an aspecific soluble rat liver protein has been studied. Results obtained indicate that at pH higher than 6.5 and Ca++ ion concentrations from 5 mM on the transport is strongly inhibited. Preliminary data with ESR spectrometry concerning the nature of lipoprotein association between phosphatidylethanolamine and carrier protein during the transport are also presented.     

Relationship between ionic surroundings and insulin actions on glucose transport and Na,K-pump in muscles

1. It is well known that insulin has various effects on glucose transport and the Na,K-pump in muscles. It is also known to have some effects on the membrane potential--in general, insulin induces a hyperpolarization of the membrane in muscles. Furthermore, it is suggested that the actions of insulin are modified by changes in ionic surroundings. 2. In this review article, the actions of ionic surroundings and insulin on glucose transport in muscles are discussed; in particular, the effects of changes in extracellular and/or intracellular concentrations of Na, K, Ca and H ions will be mentioned. 3. The actions of ionic surroundings and insulin on the Na,K-pump in muscles are discussed; in particular, the effects of changes in extracellular an/or intracellular concentrations of Na, K, Ca and H ions will be examined. 4. The relationship between the actions of ionic surroundings and insulin are discussed. 5. In particular, the effects of changes in ionic surroundings on the insulin-induced hyperpolarization of the membrane are discussed by relating it to the Na,K-pump function. The relationship between the insulin-induced change in membrane potential and glucose transport will be also mentioned.     

Sodium-calcium exchange and calcium-calcium exchange in internally dialyzed squid giant axons.

The influx and efflux of calcium (as 45Ca) and influx of sodium (as 24Na) were studied in internally dialyzed squid giant axons. The axons were poisoned with cyanide and ATP was omitted from the dialysis fluid. The internal ionized Ca2+ concentration ([Ca2+]i) was controlled with Ca-EGTA buffers. With [Ca2+]i greater than 0.5 muM, 45Ca efflux was largely dependent upon external Na and Ca. The Nao-dependent Ca efflux into Ca-free media appeared to saturate as [Ca2+]i was increased to 160 muM; the half-saturation concentration was about 8 muM Ca2+. In two experiments 24Na influx was measured; when [Ca2+]i was decreased from 160 muM to less than 0.5 muM, Na influx declined by about 5 pmoles/cm2 sec. The Nao-dependent Ca efflux averaged 1.6 pmoles/cm2 sec in axons with a [Ca2+]i of 160 muM, and was negligible in axons with a [Ca2+]i of less than 0.5 muM. Taken together, the Na influx and Ca efflux data may indicate that the fluxes are coupled with a stoichiometry of about 3 Na+-to-1 Ca2+. Ca efflux into Na-free media required the presence of both Ca and an alkali metal ion (but not Cs) in the external medium. Ca influx from Li-containing media was greatly reduced when [Ca2+]i was decreased from 160 to 0.23 muM, or when external Li was replaced by choline. These data provide evidence for a Ca-Ca exchange mechanism which is activated by certain alkali metal ions. The observations are consistent with a mobile carrier mechanism which can exchange Ca2+ ions from the axoplasm for either 3 Na+ ions, or one Ca2+ and an alkali metal ion (but not Cs) from the external medium. This mechanism may utilize energy from the Na electrochemical gradient to help extrude Ca against an electrochemical gradient.     

Contributions of secondary active transport processes to membrane potentials

Summary Equations are developed to examine the effects of secondary active transport processes on the steady-state membrane potential of symmetrical cells. It is shown that, with suitable modifications, equations of the type developed by Goldman, Hodgkin and Katz may be derived to accommodate the contributions to the membrane potential of both electroneutral and electrogenic transporters. Where the membrane potential is function of the dominant medium ions (Na, K, and Cl), other contributions can come only from an electrogenic Na pump and from neutral co- and counter-transporters if, and only if, these involve the dominant ions. Experimental approaches to measure the parameters necessary to solve the equations developed here are discussed.     

Effects of adrenergic agonists and mitochondrial energy state on the Ca2+ transport systems of mitochondria

This study investigates the effects of adrenergic agonists and mitochondrial energy state on the activities of the Ca2+ transport systems of female rat liver mitochondria. Tissue perfusion with the alpha-adrenergic agonist phenylephrine and with adrenaline, but not with the beta-adrenergic agonist isoprenaline, induced significant activation of the uniporter and the respiratory chain. Uniporter activation was evident under two sets of experimental conditions that excluded influences of delta psi, i.e., at high delta psi, where uniporter activity was delta psi independent, and at low delta psi, where uniporter conductance was measured. Preincubation of mitochondria with extracts from phenylephrine-perfused tissue quantitatively reproduced uniporter activation when comparison was made with mitochondria treated similarly with extracts from tissue perfused without agonist. Similar, but more extensive, data were obtained with heart mitochondria pretreated with extracts from hearts perfused with the alpha-adrenergic agonist methoxamine. Phenylephrine did not affect Ca2+ efflux mediated by the Na+-Ca2+ carrier or the Na+-independent system. In contrast, the liver mitochondrial Na+-Ca2+ carrier was activated by tissue perfusion with isoprenaline; the Na+-independent system was unaffected. Na+-Ca2+ carrier activation was not associated with any change in a number of basic bioenergetic parameters. It is concluded that the Ca2+ transport systems of liver mitochondria may be controlled in an opposing manner by alpha-adrenergic agonists (promotion of Ca2+ influx) and beta-adrenergic agonists (promotion of Ca2+ efflux). At delta psi values greater than 110 mV, the Na+-independent system was activated by increase in delta psi; the uniporter and Na+-Ca2+ carrier activities were insensitive to delta psi changes in this range.(ABSTRACT TRUNCATED AT 250 WORDS)