Effect of K+ and K+ gradients on accumulation of sugars by isolated intestinal epithelial cells

Summary The role played by transmembrane K⁺ gradients in providing an energy input for Na⁺-dependent monosaccharide transport systems was evaluated with the use of isolated intestinal epithelial cells. Experimentally imposing a K⁺ gradient in a sense reversed from normal did not lead to extrusion of sugar from cells which had been pre-equilibrated with¹⁴C-3-OMG, even in situations where a reversed Na⁺ gradient was also imposed. Furthermore, cells preloaded with K⁺ have no better ability to accumulate 3-OMG than do cells depleted of K⁺, when the two populations are compared under identical incubation conditions. Fluxes of K⁺ associated with the sugar carrier could not be detected in terms of suspected sensitivity to agents which immobilize the sugar carrier. In addition, fluxes of sugar in response to imposed K⁺ gradients were not demonstrable in cells de-energized by preincubation with DNP, no matter in which direction the K⁺ gradient was imposed. Finally, the severe inhibitory effects of K⁺ on Na⁺-dependent sugar transport by the cells disappears in de-energized cells, despite the fact that Na⁺-dependent carrier-mediated sugar entry still occurs. All of these facts are difficult to reconcile with a significant role for cellular K⁺ gradients in supporting active sugar transport as envisioned by the ion gradient hypothesis. We have suggested instead a fundamental Na⁺-dependent energy transductive event which depends on ATP, and which can generate a membrane-bound energized intermediate which serves to support a variety of active transport events. An analogy is drawn between this concept for animal cell plasma membranes and the better documented phosphotransferase system for sugar transport described for certain microorganisms.     

Interaction between Na+-dependent transport systems for sugars and amino acids. Evidence against a role for the sodium gradient

Summary The concept that interaction between sodium-dependent transport systems represents competition for energy inherent in the transmembrane sodium gradient was examined with the use of isolated intestinal epithelial cells. The isolated cells exhibit transport interactions which are more significant in magnitude than those which have been described for intact tissue preparations. Accumulation of 1mm valine is inhibited 60% by 10mm 3-OMG. Conversely, uptake of 1mm 3-OMG is inhibited only 20% by 10mm valine. These data suggest that 3-OMG must discharge the cellular Na⁺ gradient more effectively than valine, if Na⁺ gradient dissipation can be taken as a basis for the inhibitory interaction. However, entry of 10mm 3-OMG is significantly slower than the entry of 10mm valine. Even if appropriate corrections are made for passive substrate entry and for differences in Na/substrate entry stoichiometry, it appears that valine should be somewhat more effective than 3-OMG in discharging the Na⁺ gradient. In light of these facts, it seems unlikely that the mechanistic basis for interaction between sugar and amino acid transport systems can be related to concomitant co-entry of Na⁺. It is suggested that the interaction results instead from competition for energized intermediates generated at limited rates by basic energy transduction events associated with the cell membrane which serve in support of a variety of active transport systems.     

The effects of potassium and membrane potential on sodium-dependent glutamic acid uptake

The uptake of l-glutamic acid into brush-border membrane vesicles isolated from rat renal proximal tubules is Na⁺-dependent. In contrast to Na⁺-dependent uptake of d-glucose, pre-equilibration of the vesicles with K⁺ stimulates l-glutamic acid uptake. Imposition of a K⁺ gradient ($\text{[}\text{K}{}_{\mathrm{i}}{}^{\mathrm{+}}\text{] > [}\text{K}{}_{\mathrm{o}}{}^{\mathrm{+}}\text{]}$) further enhances Na⁺-dependent l-glutamic acid uptake, but leaves K⁺-dependent glucose transport unchanged. If K⁺ is present only at the outside of the vesicles, transport is inhibited. Intravesicular Rb⁺ and, to a lesser extent, Cs⁺ can replace intravesicular K⁺ to stimulate l-glutamic acid uptake. Changes in membrane potential incurred by the imposition of an H⁺-diffusion potential or anion replacement markedly affect Na⁺-dependent glutamic acid uptake only in the presence of K⁺. Experiments with a potential-sensitive cyanine dye also indicate that, in the presence of intravesicular K⁺ a charge movement is involved in Na⁺-dependent transport of l-glutamic acid.The data indicate that Na⁺-dependent l-glutamic acid transport can be additionally energized by a K⁺ gradient. Furthermore, intravesicular K⁺ renders Na⁺-dependent l-glutamic acid transport sensitive to changes in the transmembrane electrical potential difference.     

Na+-coupled Cl− transport in the gastric mucosa of the guinea pig

The Cl^–/HCO ₃ ^– exchange mechanism usually postulated to occur in gastric mucosa cannot account for the Na⁺-dependent electrogenic serosal to mucosal Cl^– transport often observed. It was recently suggested that an additional Cl^– transport mechanism driven by the Na⁺ electrochemical potential gradient may be present on the serosal side of the tissue. To verify this, we have studied Cl^– transport in guinea pig gastric mucosa. Inhibiting the (Na⁺, K⁺) ATPase either by serosal addition of ouabain or by establishing K⁺-free mucosal and serosal conditions abolished net Cl^– transport. Depolarizing the cell membrane potential with triphenylmethylphosphonium (a lipid-soluble cation), and hence reducing both the Na⁺ and Cl^– electrochemical potential gradients, resulted in inhibition of net Cl^– flux. Reduction of short-circuit current on replacing Na⁺ by choline in the serosal bathing solution was shown to be due to inhibition of Cl^– transport. Serosal addition of diisothiocyanodisulfonic acid stilbene (an inhibitor of anion transport systems) abolished net Cl^– flux but not net Na⁺ flux. These results are compatible with the proposed model of a Cl^–/Na⁺ cotransport mechanism governing serosal Cl^– entry into the secreting cells. We suggest that the same mechanism may well facilitate both coupled Cl^–/Na⁺ entry and coupled HCO ₃ ^– /Na⁺ exit on the serosal side of the tissue.     

Transport of selenate and selenite across the brush border membrane of rat and sheep small intestine

Mucosal uptake of⁷⁵Se-labeled selenate and selenite across the brush border was investigated in sheep and rat small intestine, using 3-min mucosal exposures. Uptake of selenate and selenite occurred faster in rat than in sheep small intestine. With the exception of sheep duodenum, mucosal selenate uptake was Na⁺-dependent in sheep and rat small intestine. Mucosal uptake of selenite across the brush border was Na⁺-dependent only in sheep midjejunum, whereas it was Na⁺-independent in sheep duodenum and ileum and the rat whole small intestine. Various anions inhibited selenate transport in the presence of Na⁺ in sheep midjejunum in the order S₂O₂ ^2- = CrO₄ ^2- > MoO₄ ^2- and in rat ileum in the order CrO₄ ^2- = S₂O₃ ^2- > SC₄ ^2- > MoO₄ ^2-. Thiosulfate also inhibited mucosal selenite uptake in the presence of Na⁺ in sheep midjejunum. Preincubation of rat ileum with glutathione (GSH) enhanced mucosal selenite uptake, whereas selenate uptake remained unaffected. These results indicate that selenate transport across the brush border membrane is energized in part by the Na⁺-gradient. Moreover, the Na⁺-dependent transport mechanism for the Se salts apparently has an affinity for other anions (S₂O₃ ^2-, SO₄ ^2-, CrO₄ ^2-, MOo₄ ^2-). The findings further indicate that intracellular GSH plays a role in the absorption of selenite, probably by an increase of intracellular selenite metabolism. The Na⁺-independent mucosal uptake of selenate and selenite probably represents diffusion.     

Ionic and Metabolic Requirements for High-Affinity Choline Uptake and Acetylcholine Synthesis in Nerve Terminals at a Neuromuscular Junction

Abstract: We have shown previously that in the chick ciliary nerve-iris muscle preparation Na⁺-dependent high-affinity choline uptake was confined to the nerve terminals. In this paper the sodium-dependent high-affinity choline uptake (SDHACU), which is coupled to acetylcholine (ACh) synthesis, was further characterized by measuring uptake of [³H]choline and its conversion to [³hjach under a variety of ionic and metabolic perturbations. Mannitol equilibration with the extracellular space was found to occur in less than 1 min in this preparation. Na⁺-dependent choline (Ch⁺) uptake was shown to be linear for 16 min and to reach an equilibrium before Na⁺-independent Ch⁺ uptake, which continued to increase for 60 min. Elevated [K⁺]₀ concentrations inhibited Ch⁺ uptake and ACh synthesis. Glycolytic and respiratory inhibitors also reduced both processes, as did ouabain and omission of [K⁺]₀. Incubation conditions that reduce transmitter release had no effect on inhibition by high [K⁺]₀. Reduction of SDHACU and sodium-dependent ACh synthesis by depolarization with high [K⁺]₀ or by inhibition of Na, K-ATPase implies that the electrochemical gradients for Ch⁺ and Na⁺ are important in providing a driving force for high-affinity Ch⁺ uptake. The inhibition by metabolic blockers suggests active transport, but the effects may be indirect, caused by reduced Na, K-ATPase activity and alterations in membrane potential. While most metabolic inhibitors exerted parallel effects on both Ch⁺ uptake and ACh synthesis, in some cases Ch⁺ uptake was more strongly inhibited than ACh synthesis. This occurred in preparations incubated with high [K⁺]₀ and ouabain. Na⁺-dependent Ch⁺ uptake and ACh synthesis were found to be temperature-dependent with a Q₁₀ (20–30°) of 3.6 and 6.6, respectively and a Q₁₀ (30–40°) of 1.3 and 1.0, respectively. Inhibition of acetylcholinesterase by paraoxon increases to 92% the proportion of the Ch⁺ taken up which is converted to ACh. ACh did not reduce Ch⁺ transport when present at 100 μM.     

Stimulation of Na+-dependent amino acid uptake by activation of the Ca2+-dependent K+ channel in the Ehrlich ascites tumor cell

The activation of Ca²⁺-dependent K⁺ channel by propranolol or by ascorbate-phenazine methosulphate stimulates Na⁺-dependent transport of α-aminoisobutyric acid. This stimulation arises from a membrane hyperpolarization due to the specific increase of membrane K⁺ conductance. The same treatment does not modify the Na⁺-independent uptake of the norbornane amino acid.     

[3H]2-deoxyglucose transport by slices of cerebral cortex: Apparent dependence upon mitochondrial energy

The transport of [³H]2-deoxyglucose by brain slices was studied. Cerebral cortex slices were incubated in vitro in the presence of [³H]2-deoxyglucose, orl-[³H] glucose as a marker for diffusion. Transport was defined as the difference between [³H]2DG uptake andl-[³H]glucose uptake. Half-maximal velocity was seen at 2.0 mM 2DG and [³H]2DG transport was not inhibited by 20-fold higher concentrations ofl-glucose. Net [³H]2DG transport was unchanged in media deficient in Na⁺, K⁺, Mg²⁺, Ca²⁺ or Cl^–. Uptake was significantly inhibited by 1.0 mM 2,4-DNP and a suggestion of inhibition by azide was seen. These data are consistent with a hypothesis that hexose transport in the brain depends to some extent upon mitochondrial energy.     

Activation of Rb+ and Na+ uptake into yeast by monovalent cations

⁸⁶Rb⁺ uptake by yeast was not only stimulated by Rb⁺ or K⁺ but also by Na⁺. The uptake of ²²Na⁺ was enhanced by both Rb⁺ and K⁺, but not by Na⁺, which was inhibitory at all concentrations applied. Inhibition of ²²Na⁺ uptake by inactive Na⁺ occurred in two phases: one phase refers to inhibition at low Na⁺ concentrations and the other to inhibition at high Na⁺ concentrations. Our results can be qualitatively described by a two-site transport mechanism, having two cation binding sites, which must be occupied with monovalent cations before transport can occur.     

Na+-dependent transport of glycine in renal brush border membrane vesicles: Evidence for a single specific transport system

The uptake of glycine in rabbit renal brush border membrane vesicles was shown to consist of glycine transport into an intravesicular space. An Na⁺ electrochemical gradient (extravesicular>intravesicular) stimulated the initial rate of glycine uptake and effected a transient accumulation of intravesicular glycine above the steady-state value. This stimulation could not be induced by the imposition of a K⁺, Li⁺ or choline⁺ gradient and was enhanced as extravesicular Na⁺ was increased from 10 mM to 100 mM. Dissipation of the Na⁺ gradient by the ionophore gramicidin D resulted in diminished Na⁺-stimulated glycine uptake. Na⁺-stimulated uptake of glycine was electrogenic. Substrate-velocity analysis of Na⁺-dependent glycine uptake over the range of amino acid concentrations from 25 μM to 10 mM demonstrated a single saturable transport system with apparent K_m = 996 μM and V_max = 348 pmol glycine/mg protein per min. Inhibition observed when the Na⁺-dependent uptake of 25 μM glycine was inhibited by 5 mM extravesicular test amino acid segregated dibasic amino acids, which did not inhibit glycine uptake, from all other amino acid groups. The amino acids d-alanine, d-glutamic acid, and d-proline inhibited similarly to their l counterparts. Accelerative exchange of extravesicular [³H]glycine was demonstrated when brush border vesicles were preloaded with glycine, but not when they were preloaded with l-alanine, l-glutamic acid, or with l-proline. It is concluded that a single transport system exists at the level of the rabbit renal brush border membrane that functions to reabsorb glycine independently from other groups of amino acids.     

Sodium transport measured in plasma membrane vesicles isolated from wheat genotypes with differing K+/Na+ discrimination traits

Right-side-out plasma membrane vesicles were isolated from wheat roots using an aqueous polymer two-phase system. The purity and orientation of the vesicles were confirmed by marker enzyme analysis. Membrane potential (Ψ)-dependent ²²Na⁺ influx and sodium/proton (Na⁺/ H⁺) antiport-mediated efflux across the plasma membrane were studied using these vesicles. Membrane potentials were imposed on the vesicles using either K⁺ gradients in the presence of valinomycin or H⁺ gradients. The ΔΨ was quantified by the uptake of the lipophilic cation tetraphenylphosphonium. Uptake of Na⁺ into the vesicles was stimulated by a negative ΔΨ and had a K_m for extrav-esicular Na⁺ of 34.8 ± 5.9 mol m³. The ΔΨ-dependent uptake of Na⁺ was similar in vesicles from roots of hexaploid (cv. Troy) and tetraploid (cv. Langdon) wheat differing in a K⁺/Na⁺ discrimination trait, and was also unaffected by growth in 50 mol m^?3 NaCl. Inhibition of ΔΨ-dependent Na⁺ uptake by Ca²⁺ was greater in the hexaploid than in the tetraploid. Sodium/proton antiport was measured as Na⁺-dependent, amiloride-inhibited pH gradient formation in the vesicles. Acidification of the vesicle interior was measured by the uptake of ¹⁴C-methylamine. The Na⁺/H⁺ antiport had a K_m, for intravesicular Na⁺ of between 13 and 19 mol m^?3. In the hexaploid, Na⁺/H⁺ antiport activity was greater when roots were grown in the presence of 50 mol m^?3NaCl, and was also greater than the activity in salt-grown tetraploid wheat roots. Antiport activity was not increased in a Langdon 4D chromosome substitution line which carries a trait for K⁺/Na⁺ discrimination. It is concluded that neither of the transport processes measured is responsible for the Na⁺/K⁺ discrimination trait located on the 4D chromosome of wheat.     

Taurine transport by brush border membrane vesicles isolated from the flounder kidney

Summary Taurine transport was investigated in brush border membrane vesicles isolated from renal tubules of the winter flounder (Pseudopleuronectes americanus). Taurine uptake by the vesicles was greater in the presence of NaCl as compared to uptake in KCl. The Na⁺-dependent taurine transport was electrogenic and demonstrated tracer replacement and inhibition by -alanine and HgCl₂, indicating the presence of Na⁺-dependent, carrier-mediated taurine transport. In contrast to Na⁺-dependent taurine transport across the basolateral membrane, there was not a specific Cl^– dependency for transport in the brush border membrane. No evidence was obtained for Na⁺-independent carrier-mediated taurine transport. The possible involvement of the brush border Na⁺-dependent transport system in the net secretion of taurine from blood to tubular lumen in vivo (Schrock et al. 1982) is discussed.     

Electrogenic transport of 5-oxoproline in rabbit renal brush-border membrane vesicles Effect of intravesicular potassium

The Na⁺-dependent transport of 5-oxoproline into rabbit renal brush-border vesicles was stimulated by a K⁺ diffusion potential (interior-negative) induced by valinomycin. Na⁺ salts of two anions of different epithelial permeabilities also affected 5-oxoproline transport. These results show that the Na⁺-dependent 5-oxoproline transport in renal brush-border vesicles is an electrogenic process which results in a net transfer of positive charge. Maximum transport of 5-oxoproline occurred at an extravesicular pH of 6.0 to 8.0 and over that pH range, 5-oxoproline exists completely as an anion with a negative charge. The simplest stoichiometry consistent with this process is, therefore, the cotransport of one 5-oxoproline anion with two sodium ions. The presence of K⁺ inside the vesicles stimulated the Na⁺-dependent transport of 5-oxoproline. This stimulatory effect was specific for K⁺ and required the presence of Na⁺. The presence of Na⁺ gradient was not mandatory for the K⁺ action. The stimulation by the intravesicular K⁺ was seen in the presence as well as in the absence of a K⁺ gradient. Therefore, the increased influx of 5-oxoproline was not coupled to the simultaneous efflux of K⁺. The presence of K⁺ in the extravesicular medium alone did not affect the Na⁺-dependent transport of 5-oxoproline, showing that the site of K⁺ action was intravesicular. Glutamate did not interact with the Na⁺-dependent 5-oxoproline transport even in the presence of an outward K⁺ gradient.     

Neutral amino acid transport by marine fish intestine: role of the side chain

This work was devoted to the study of the structure-affinity relationships in neutral amino acid transport by intestinal brush border of marine fish (Dicentrarchus labrax). The effects of the length of the side chain on kinetics of glycine, alanine, methionine and amino isobutyric acid were investigated. In the presence of K⁺ two components were characterized: one is saturable by increased substrate concentrations, whereas the other can be described by simple diffusion mechanism. Simple diffusion, a passive, non-saturable, Na⁺-independent route, contributes largely to the transport of methionine and to a much lesser extend to alanine, glycine or alphaaminoisobutyric acid uptakes. If a branched chain is present, as in the case of amino isobutyric acid, diffusion is low. A Na⁺-independent, saturable system has been fully characterized for methionine, but not for branched amino acids such as amino isobutyric acid. In the presence of Na⁺ saturable components were shown. Two distinct Na⁺-dependent pathways have been characterized for glycine uptake, with low and high affinities. For alanine and methionine only one Na⁺-dependent high affinity system exists with the same half-saturation concentration and the same maximum uptake at saturable concentrations. Glycine high affinity system has the same half-saturation concentration as methionine or alanine uptake, whereas maximum uptake is lower. The substitution of the hydrogen by a methyl group results in a severe decrease of uptake (aminoisobutyric acid). Mutual inhibition experiments indicate that the same carriers could be responsible for methionine and alanine uptakes and probably glycine Na⁺-dependent uptake. The influence of Na⁺ concentrations (100^-1 mol·l^-1) on amino acid uptake was examined. Glycine, alanine, methionine and amino isobutyric acid transport can be described by a hyperbolic function, with a saturation uptake which is highly increased for methionine. However, the half-saturation concentration does not seem to be strongly affected by the amino acid structure. The effect of Na⁺ concentration (25 and 100 mmol·l^-1) on the kinetics of methionine uptake have been also examined. The maximum uptake of the saturable system clearly shows a typical relationship with concentration.Abbreviations [AA] amino acid concentration - AIB aminoisobutyric acid - [I] Inhibitor amino acid concentration - J _i uptake in the presence of inhibitor - J _o uptake without inhibitor - K _d passive diffusion constant - K _i inhibitor constant - K _t concentration of test amino acid for half-maximal flux - MES 2[N-morpholino]ethanesulphonic acid - V _max maximum uptake at saturable amino acid concentrations - V _tot total amino acid uptake     

UPTAKE OF 3-O-METHYL-d-GLUCOSE INTO CULTURED HUMAN GLIOMA CELLS

Human glioma cells (138 MG) were found to take up 3-O-methyl-d -glucose (3-OMG) by a saturable low affinity transport system with a K_m of 20 mm and a V_max of 500 nmol/mg protein/min. About 20 per cent of the total uptake was due to passive diffusion. d -Glucose was a competitive inhibitor with a K_i of 10 mm . Follow-up experiments indicated that the same transport mechanism is involved in the uptake of n-glucose and 3-OMG. Phloretin (0·02 mm ) and cytochalasin B (0·002 mm ) strongly inhibited the uptake of 3-OMG, whereas phlorizin (0·02 mm ), ouabain (0·1 mm ), NaCN (0·5 mm ) and iodoacetic acid (1·0 mm ) had no effect. The data suggest that 3-OMG and d -glucose enter 138 MG cells mainly by a Na⁺-independent passive carrier-mediated transport system. Serum-deprivation doubled the population doubling time (T_d) without affecting the total uptake of 3-OMG. An increase in the non-specific (diffusional) uptake was balanced by a decrease in the specific (carrier-medíated) uptake. After addition of dibutyryl cyclic AMP (dbcAMP, 0·25 mm ) the cells attained a morphology characteristic of differentiated glia cells. T_d was maintained unchanged. The non-specific uptake of 3-OMG was not affected in cells grown in serum-containing medium plus dbcAMP, whereas the specific uptake increased by 40 per cent and there-fore also the total uptake. Similar, but more pronounced, changes were observed if serum-deprived cells were treated with dbcAMP.     

Reconstitution and characterization of a sodium-stimulated active aminoisobutyric acid transport system derived from partially purified plasma membranes from mouse fibroblasts transformed by simian virus 40: comparison of reconstituted vesicles with native membrane vesicles

A Na⁺-specific and Na⁺-stimulated active α-aminoisobutyric acid transport system was reconstituted from plasma membranes isolated from mouse fibroblast BALB/c 3T3 cells transformed by simian virus 40. The plasma membranes were treated with dimethylmaleic anhydride and then extracted with 2% cholate. The cholate-solubilized supernatant proteins were combined with exogenous phospholipids and eluted through a Sephadex G-50 column. This yielded reconstituted vesicles which in the presence of Na⁺ could actively transport α-aminoisobutyric acid as shown by the transient accumulation above the equilibrium level (overshoot). The overshoot was not obtained with other monovalent cations such as K⁺, Li⁺, and choline⁺. The electrochemical effect of the lipophilic anion, SCN^?, led to greater α-aminoisobutyric acid uptake as compared to that observed with Cl^? or SO₄^2?. The Na⁺-stimulated transport of a-aminoisobutyric acid was a saturable process with an apparent K_m of 2 mm. Studies of the inhibition of α-aminoisobutyric acid transport by other amino acids showed that methylaminoisobutyric acid [specifically transported by A system (alanine preferring)]had a pronounced inhibitory effect on a-aminoisobutyric acid uptake in contrast to the slight inhibitory effect produced by phenylalanine [primarily transported by L system (leucine preferring)]. The results show that the reconstituted vesicles, prepared from partially purified membrane proteins and exogenous phospholipids, regained the same important transport properties of native membrane vesicles, i.e., Na⁺-specific and Na⁺-stimulated concentrative α-aminoisobutyric acid uptake.     

ATP-dependent Na+ transport in cardiac sarcolemmal vesicles

Although the enzyme (Na⁺ + K⁺)-ATPase has been extensively characterized, few studies of its major role, ATP-dependent Na⁺ pumping, have been reported in vesicular preparations. This is because it is extremely difficult to determine fluxes of isotopic Na⁺ accurately in most isolated membrane systems. Using highly purified cardiac sarcolemmal vesicles, we have developed a new technique to detect relative rates of ATP-dependent Na⁺ transport sensitively. This technique relies on the presence of Na⁺-Ca²⁺ exchange and ATP-driven Na⁺ pump activities on the same inside-out sarcolemmal vesicles. ATP-dependent Na⁺ uptake is monitored by a subsequent Na_i⁺-dependent Ca²⁺ uptake reaction (Na⁺-Ca²⁺ exchange) using ⁴⁵Ca²⁺. We present evidence that the Na⁺-Ca²⁺ exchange will be linearly related to the prior active Na⁺ uptake. Although this method is indirect, it is much more sensitive than a direct approach using Na⁺ isotopes. Applying this method, we measure cardiac ATP-dependent Na⁺ transport and (Na⁺ + K⁺)-ATPase activities in identical ionic media. We find that the (Na⁺ + K⁺)-ATPase and the Na⁺ pump have identical dependencies on both Na⁺ and ATP. The dependence on [Na⁺] is sigmoidal, with a Hill coefficient of 2.8. Na⁺ pumping is half-maximal at [Na⁺] = 9 mM. The K_m for ATP is 0.21 mM. ADP competitively inhibits ATP-dependent Na⁺ pumping. This approach should allow other new investigations on on ATP-dependent Na⁺ transport across cardiac sarcolemma.     

Inhibition of the (Na+ + K+)-dependent ATPase by inorganic phosphate

Inhibition of the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-dependent}$ ATPase by inorganic phosphate, P_i, was examined in terms of product inhibition of the various activities catalyzed by an enzyme preparation from rat brain, and considered in terms of the specific transport processes of the membrane Na⁺,K⁺-pump that these activities reflect. The K⁺-dependent phosphatase activity of the enzyme was most sensitive to P_i, and inhibition was competitive toward the substrate, nitrophenyl phosphate, as would be expected if P_i were released from the same enzyme form that bound substrate. However, this enzymatic activity does not seem to represent a transport process, and thus a cyclical discharge of K⁺ may not be involved. The Na⁺-dependent exchange activity was unaffected by P_i, in accord with the absence of P_i release in the reaction sequence. For the corresponding Na⁺/Na⁺ exchange function of the pump, which reportedly does not involve ATP hydrolysis either, prior release of P_i obviously cannot be required for Na⁺ discharge. With the Na⁺-dependent ATPase activity, measured using micromolar concentrations of ATP, P_i inhibited, but far less than with the phosphatase activity, and inhibition was not competitive toward ATP. Moreover, inhibition decreased as the Na⁺ concentration was raised from 10 to 100 mM. This elevated concentration of Na⁺ also led to substrate inhibition. For this ATPase activity, and the corresponding transport process, uncoupled Na⁺ efflux, the findings suggest that Na⁺ discharge follows P_i release, in contrast to Na⁺/Na⁺ exchange. The $\text{(}\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-dependent}$ ATPase activity, measured with millimolar concentrations of ATP and reflecting the coupled Na⁺,K⁺-transport function, was similarly sensitive to P_i, and again inhibition was not competitive toward ATP. However, in this case inhibition did not increase as the Na⁺ concentration was lowered. For this activity, and the associated transport process, the site of Na⁺ discharge in the overall reaction sequence remains unresolved.     

Evidence for Na-Independent HCO(3) Uptake by the Cyanobacterium Synechococcus leopoliensis

At low levels of dissolved inorganic carbon (DIC) and alkaline pH the rate of photosynthesis by air-grown cells of Synechococcus leopoliensis (UTEX 625) was enhanced 7- to 10-fold by 20 millimolar Na⁺. The rate of photosynthesis greatly exceeded the CO₂ supply rate and indicated that HCO₃⁻ was taken up by a Na⁺-dependent mechanism. In contrast, photosynthesis by Synechococcus grown in standing culture proceeded rapidly in the absence of Na⁺ and exceeded the CO₂ supply rate by 8 to 45 times. The apparent photosynthetic affinity (K_½) for DIC was high (6-40 micromolar) and was not markedly affected by Na⁺ concentration, whereas with air-grown cells K_½ (DIC) decreased by more than an order of magnitude in the presence of Na⁺. Lithium, which inhibited Na⁺-dependent HCO₃⁻ uptake in air-grown cells, had little effect on Na⁺-independent HCO₃⁻ uptake by standing culture cells. A component of total HCO₃⁻ uptake in standing culture cells was also Na⁺-dependent with a K_½ (Na⁺) of 4.8 millimolar and was inhibited by lithium. Analysis of ¹⁴C-fixation during isotopic disequilibrium indicated that standing culture cells also possessed a Na⁺-independent CO₂ transport system. The conversion from Na⁺-independent to Na⁺-dependent HCO₃⁻ uptake was readily accomplished by transferring cells grown in standing to growth in cultures bubbled with air. These results demonstrated that the conditions experienced during growth influenced the mode by which Ssynechococcus acquired HCO₃⁻ for subsequent photosynthetic fixation.     

Cation flux in the ehrlich ascites tumor cell evidence for Na-for-Na and K-for-K exchange diffusion

In a previous study, evidence was presented for an external Na⁺-dependent, ouabain-insensitive component of Na⁺ efflux and an external K⁺-dependent component of K⁺ efflux in the Ehrlich ascites tumor cell. Evidence is now presented that these components are inhibited by the diuretic furosemide and that under conditions of normal extracellular Na⁺ and K⁺ they represent Na⁺-for-Na⁺ and K-⁺for-K⁺ exchange mechanisms. Using ⁸⁶Rb to monitor K⁺ movements, furosemide is shown to inhibit an ouabain-insensitive component of Rb⁺ influx and a component of Rb⁺ efflux, both representing approx. 30% of the total fux. Inhibition of Rb⁺ efflux is greatly reduced by removal of extracellular K⁺. Furosemide does not alter steady-state levels of intracellular K⁺ and it does not prevent cells depleted of K⁺ by incubation in the cold from regaining K⁺ upon warming. Using ²²Na to monitor Na⁺ movements, furosemide is shown to inhibit an ouabain-insensitive component of unidirectional Na⁺ efflux which represents approx. 22% of total Na⁺ efflux. Furosemide does not alter steady-state levels of intracellular Na⁺ and does not prevent removal of intracellular Na⁺ upon warming from cells loaded with Na⁺ by preincubation in the cold. The ability of furosemide to affect unidirectional Na⁺ and K⁺ fluxes but not net fluxes is consistent with the conclusion that these components of cation movement across the cell membrane represent one-for-one exchange mechanisms. Data are also presented which demonstrate that the uptake of α-aminoisobutyrate is not affected by furosemide. This indicates that these components of cation flux are not directly involved in the Na⁺-dependent amino acid transport system A.