Changes in intracellular cations during the cell cycle in HeLa cells

Intracellular Na+, K+, and Mg2+ concentrations have been measured during the HeLa cell cycle and compared with changes in oxygen utilization and macromolecular synthesis. Cell water content remains relatively constant at 79 +/- 1% during the cell cycle. A biphasic change in intracellular Na+ occurs with low values as cells reach peak S phase and again in early G1. The decrease in S coincides with an increase in cell volume during increased macromolecular synthesis. The fall in intracellular Na+ during mitosis/early G1 coincides with decreased energy utilization as macromolecular synthesis decreases with a continued decrease in [Na+]i in G1 corresponding to a period of increasing cell volume and an increase in protein synthesis. Intracellular Na+ is relatively high during late S/G2 when phosphate incorporation into protein and phospholipid is maximal. Intracellular K+ concentrations largely parallel intracellular Na+ levels although the intracellular K+:Na+ ratio is significantly lower as the cell volume increases during late G2/mitosis. Additions of a Na+-pump inhibitor (strophanthidin) not only caused a rise in [Na+]i and fall in [K+]i but also inhibited protein synthesis. Conversely, addition of a protein synthesis inhibitor (cycloheximide) blocked amino acid incorporation and produces a fall in intracellular Na+ levels. These findings indicate that intracellular Na+ and K+ play an important role in regulating cell hydration during the cell cycle and that changes in Na+, K+-ATPase activity, synthesis and/or utilization of high energy phosphate compounds, fluid phase turnover (endocytosis), Na+:H+ exchange (pHi), Donnan forces, and ionic adsorption may all be involved.     

Membrane regulation of the Na+,K+-ATPase during the neuroblastoma cell cycle: correlation with protein lateral mobility

The pumping activity of the plasma membrane-bound Na+,K+-ATPase shows considerable variation during the cell cycle of mouse neuroblastoma Neuro-2A cells. Addition of external ATP at millimolar concentrations, which selectively enhances the plasma membrane permeability of Neuro-2A cells for sodium ions, stimulates the Na+,K+-ATPase pumping activity at all phases of the cell cycle from a factor of 1.05 in mitosis up to 2.2 in G1 phase. Determination of the number of Na+,K+-ATPase copies per cell by direct 3H-ouabain binding studies in the presence of external ATP shows a gradual increase in the number of pump sites on passing from mitosis to the late S/G2-phase by approximately a factor of 2. From these data the pumping activity per copy of Na+,K+-ATPase, optimally stimulated with respect to its various substrate ions, has been determined during the various phases of the cell cycle. This optimally stimulated pumping activity per enzyme copy, which is a reflection of the physicochemical state of the plasma membrane, is high in mitosis, almost twofold lower in early G1 phase, and increases gradually again during the other phases of the cell cycle. This shows that the observed regulation of Na+,K+-ATPase activity during the cell cycle is caused by a combination of three independent factors--namely variation in intracellular substrate availability (Na+), changes in number of enzyme copies per cell, and modulation of the plasma membrane environment of the protein molecules. The modulation of the optimal pumping activity per enzyme copy shows a good correlation (rho = 0.96) with the known modulation of protein lateral mobility during the cell cycle, such that a high protein lateral mobility correlates with a low enzyme activity. It is concluded that changes in plasma membrane properties take place during the Neuro-2A cell cycle that result in changes in the rate of protein lateral diffusion and Na+,K+-ATPase activity in directly correlated way.     

Properties of a Sodium Channel (Nax) Activated by Strong Depolarization of Xenopus Oocytes

Short (<1 sec) duration depolarization of Xenopus laevis oocytes to voltages greater than +40 mV activates a sodium-selective channel (Na(x)) with sodium permeability five to six times greater than the permeability of other monovalent cations examined, including K+, Rb+, Cs+, TMA+, and Choline+. The permeability to Li+ is about equal to that of Na+. This channel was present in all oocytes examined. The kinetics, voltage dependence and pharmacology of Na(x)distinguish it from TTX-sensitive or epithelial sodium channels. It is also different from the sodium channel of Xenopus oocytes activated by prolonged depolarization, which is more highly selective for Na+, requires prolonged depolarization to be activated, and is blocked by Li+. Intracellular Mg2+ reversibly inhibits Na(x), whereas extracellular Mg2+ does not have an inhibitory effect. Intracellular Mg2+ inhibition of Na(x), is voltage dependent, suggesting that Mg2+ binding occurs within the membrane field. Eosin is also a reversible voltage-dependent intracellular inhibitor of Na(x), suggesting that a P-type ATPase may mediate the current. An additional cytoplasmic factor is involved in maintaining Na(x) since the current runs down in internally perfused oocytes and excised membrane patches. The rundown is reversible by reintroduction of the membrane patch into oocyte cytoplasm. The cytoplasmic factor is not ATP, because ATP has no effect on Na(x) current magnitude in either cut-open or inside-out patch preparations. Extracellular Gd3+ is also an inhibitor of Na(x). Na(x) activation follows a sigmoid time course. Its half-maximal activation potential is +100 mV and the effective valence estimated from the steepness of conductance activation is 1.0. Na(x) deactivates monoexponentially upon return to the holding potential (-40 mV). The deactivation rate is voltage dependent, increasing at more negative membrane potentials.     

The influence of cytoplasmic sodium concentration on the stoichiometry of the sodium pump

Using inside-out vesicles of human red cell membranes, the effects of cytoplasmic Na+ in the range 0-5 mM on ATP-dependent 22Na+ influx (normal efflux) and 86Rb+ efflux (normal influx) were tested. The sodium pump stoichiometry, i.e. the ratio of net 22Na+ influx:86Rb+ efflux was reduced markedly when the cytoplasmic Na+ was reduced to less than 1 mM. Reduction in cytoplasmic Na+ concentration was associated also with a decreased sensitivity of the pump to effects of extracellular Rb+. Thus, extracellular (intravesicular) Rb+ stimulation observed at high ATP concentration and inhibition observed at low ATP concentration were not observed when the cytoplasmic (extravesicular) Na+ concentration was reduced to less than or equal to 0.2 mM. It is suggested that at low cytoplasmic Na+, the pump can operate with less than maximal sites filled with Na+ ions. Under this condition, it is likely that an enzymic step associated with either the ion translocation step or the enzyme's conformational transition becomes rate-limiting.     

Kinetics of the Mechanism of Action of Monoamine Oxidase in the Regulation of Na+, K+-ATPase Activity in Rat Brain

Kinetic studies on the action of monoamine oxidase (MAO) in the regulation of Na+,K+-ATPase were performed using 3-methoxy-4-hydroxybenzaldehyde (MHB), which is an analogue of 3-methoxy-4-hydroxy-phenylacetylaldehyde (product of MAO-catalysed reaction with dopamine as substrate). It was observed that at 2.6 microM MHB, the activation of Na+,K+-ATPase may be the result of the removal of the inhibitory Ca2+, thereby increasing the Vmax. Double-reciprocal plots of Pi versus MHB showed that Ca2+ counteracted the effect of the aldehyde not by changing the Km, but be decreasing the Vmax of the Na+,K+-ATPase stimulation. The removal of 3',5'-cyclic AMP-dependent protein kinase from the microsomes by sodium dodecyl sulphate treatment abolished the activation and/or inhibition of the Na+,K+-ATPase by aldehyde; it can therefore be inferred that 3',5'-cyclic AMP-dependent protein kinase is involved in the regulation of Na+,K+-ATPase.     

Comparative study of the effects of amphotericin B on the glucose metabolism in Saccharomyces cerevisiae in K(+)- and Na(+)-rich media.

In order to elucidate the effects of amphotericin B (AMB) on the glycolytic pathway, the metabolism of [1-13C]glucose in glucose-grown repressed Saccharomyces cerevisiae was studied. The cells were aerobically suspended in pyrophosphate solutions of high potassium concentration with or without 10(-6) M amphotericin B and measurements were made using 1H-, 13C-NMR spectroscopy and biochemical methods. The results were compared with those obtained under the same experimental conditions but in a medium rich in sodium salts containing the same antibiotic concentration. In general the presence of 10(-6) M AMB reduces the glucose consumption and the ethanol production while favouring the glycerol and trehalose formation. These effects are greatly reduced when a high K+ concentration was used. The AMB effects on the glucose consumption and the production of ethanol, glycerol and trehalose, observed in a suspension rich in Na+, can be fairly well explained by the leakage of K+ through AMB membrane channels. This outflux induces a substantial decrease in the activity of some K(+)-dependent enzymes, such as aldolase, phosphofructokinase and pyruvate kinase. The intensities of the glutamate C2 and C4 signals are higher with a suspension rich in Na+ than with a suspension rich in K+, suggesting that the Krebs cycle operates more effectively in a solution rich in Na+. In the absence of AMB, the passive diffusion of glycerol through the cell membrane is relatively slow and apparently depends on the ionic external medium: it is more efficient in solutions with a high K+ than with a high Na+ concentration. In the presence of 10(-6) M AMB, the glycerol C1,3 resonance drastically decreases at 20 min and then disappears in the noise. This rapid disappearance suggests that glycerol can easily pass through the pores arising from the interaction of AMB with the membrane sterols. However, the rate of pore formation is slow, independent of the external medium (Na+ or K+) and this process is not completed within 20 min.     

Cell shrinkage and apoptosis: a role for potassium and sodium ion efflux

In this study we have shown that redistribution of the lipid composition of the external and internal leaflets of the PM during apoptosis results in two main alterations in the cell surface, externalisation of PS, and a looser packing of the lipid hydrophobic head groups in the external leaflet. Significantly, neither of these alterations can be directly implicated in the mechanism of apoptotic cell shrinkage, however they do have functions in other phases of the apoptotic process. Progressional studies involving morphological and flow cytometric evaluation, and DNA gel electrophoresis revealed that apoptotic cell shrinkage is associated with a decrease in [Na+]i and [K+]i which occurs after visualisation of chromatin condensation and internucleosomal DNA fragmentation, and prior to apoptotic body formation. When apoptotic cultures were supplemented with inhibitors of the Na+, K+-ATPase pump or the Ca2+-dependent K+ channel, essentially all of the respective Na+ or K+ efflux during apoptosis can be inhibited, suggesting that essentially all of the Na+ and K+ efflux can be ascribed to active pumping via the Na+, K+-ATPase pump and the Ca2+-dependent K+ channel.     

The effects of cyanide on intracellular ionic exchange in ferret and rat ventricular myocardium

The effects of cyanide on Ca2+ exchange in isolated ventricular myocytes and on the intracellular concentrations of Ca2+, Na+ and H+ have been investigated to assess the contribution that mitochondria might play in cellular Ca2+ metabolism. Ionic levels were measured with ion-selective electrodes. KCN (2.5 mM) inhibited a component of Ca2+ exchange in myocytes that could be attributed to mitochondrial exchange, but was without effect on non-mitochondrial Ca2+ exchange. NaCN (2.5 mM) caused a transient reduction of [H+]i, [Na+]i and [Ca2+]i when applied to the superfusate bathing ventricular trabeculae or papillary muscles. The transient changes of [Na+]i were accentuated when the preparation was exposed to a solution which would be expected to increase the cellular calcium content. The reduction of [Na+]i which accompanies a reduction of the extracellular sodium concentration, [Na]o, was attenuated in the presence of NaCN, but the intracellular acidosis resulting from a reduction of [Na]o was unaffected by NaCN. A small, but significant, rise of [Ca2+]i accompanied a reduction of [Na]o but only when NaCN was present in the superfusate. It is concluded that cyanide ions have a reasonably specific action on cardiac cellular ionic metabolism. Its primary action is to prevent mitochondrial Ca2+ sequestration. It is postulated that a Na+/H+ exchange, possibly at the sarcolemma, could account for some of the changes to sarcoplasmic ionic levels observed. In a solution of low [Na]o, it is concluded that mitochondria could sequester at least 30% of the calcium accumulated by the cell even though the sarcoplasmic [Ca2+] does not exceed 0.3 microM.     

Effect of amiloride on the activity of membrane-bound and soluble Na+-,K+-ATPase preparations

Amiloride (8 X 10(-4), an inhibitor of sodium channels of nonexcited membranes, inhibits the activity of Na+,K+-ATPase in the kidney cortex homogenate as well as that of the partially purified membrane-bound and lubrol-soluble Na+,K+-ATPase preparations from the cattle brain. Inhibition of Na+,K+-ATPase from different organs of various animals by amiloride, a blocker of sodium channels, indicates similarity of the molecular organization of the Na+-recognizing component both of sodium channels and sodium centres of Na+,K+-ATPase.     

Intracellular acidification-induced alkali metal cation/H+ exchange in human neutrophils

Pretreatment of isolated human neutrophils (resting pHi congruent to 7.25 at pHo 7.40) with 30 mM NH4Cl for 30 min leads to an intracellular acidification (pHi congruen to 6.60) when the NH4Cl prepulse is removed. Thereafter, in 140 mM Na+ medium, pHi recovers exponentially with time (initial rate, approximately 0.12 pH/min) to reach the normal resting pHi by approximately 20 min, a process that is accomplished mainly, if not exclusively, though an exchange of internal H+ for external Na+. This Na+/H+ countertransport is stimulated by external Na+ (Km congruent to 21 mM) and by external Li+ (Km congruent to 14 mM), though the maximal transport rate for Na+ is about twice that for Li+. Both Na+ and Li+ compete as substrates for the same translocation sites on the exchange carrier. Other alkali metal cations, such as K+, Rb+, or Cs+, do not promote pHi recovery, owing to an apparent lack of affinity for the carrier. The exchange system is unaffected by ouabain or furosemide, but can be competitively inhibited by the diuretic amiloride (Ki congruent to 8 microM). The influx of Na+ or Li+ is accompanied by an equivalent counter-reflux of H+, indicating a 1:1 stoichiometry for the exchange reaction, a finding consistent with the lack of voltage sensitivity (i.e., electroneutrality) of pHi recovery. These studies indicate that the predominant mechanism in human neutrophils for pHi regulation after intracellular acidification is an amiloride-sensitive alkali metal cation/H+ exchange that shares a number of important features with similar recovery processes in a variety of other mammalian cell types.     

Induction of differentiation of murine embryonal carcinoma cells by ouabain

Murine embryonal carcinoma cells (EC) can be induced to differentiate by a variety of chemical agents, including retinoid acid (RA) and dimethyl acetamide (DMA). However, it is not known how these agents exert their effects. In this study we demonstrate that murine EC cells can also be induced to differentiate by ouabain at concentrations which inhibit Na+, K+-ATPase activity as measured by inhibition of 86Rb+ uptake. Since the pharmacologic action of ouabain is thought to be specific, we investigated the role of Na+, K+-ATPase inhibition and specific metabolic consequences of this inhibition in the induction of EC differentiation, and explored whether this might be a common mode of action for a variety of structurally diverse inducers. Although the Na+, K+-ATPase maintains ion gradients in cells, our studies failed to demonstrate a consistent role for alterations of ion flux or ion concentration on the differentiation process. Ouabain inhibited cell growth, but a direct correlation between the degree of growth inhibition and the extent of differentiation could not be demonstrated. There was also no evidence that RA or DMA induces differentiation by inhibiting the Na+, K+-ATPase. The mechanism of ouabain induction may be mediated by some alternative consequence of Na+, K+-ATPase inhibition, but it appears to be specific for that inducer and cannot be generalized to that of other inducers of EC differentiation.     

Conformational transitions between Na+-bound and K+-bound forms of (Na+ + K+)-ATPase, studied with formycin nucleotides.

1. Fluorescence measurements have shown that formycin triphosphate (FTP) or formycin diphosphate (FDP) bound to (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) in Na+-containing media can be displaced by the following ions (listed in order of effectiveness): Tl+, K+, Rb+, NH4+, Cs+. 2. The differences between the nucleotide affinities displayed by the enzyme in predominantly Na+ and predominantly K+ media in the absence of phosphorylation, are thought to reflect changes in enzyme conformation. These changes can therefore be monitored by observing the changes in fluorescence that accompany net binding or net release of formycin nucleotides. 3. The transition from a K+-bound form (E2-(K)) to an Na+-bound form (E1-Na) is remarkably slow at low nucleotide concentrations, but is accelerated if the nucleotide concentration is increased. This suggests that the binding of nucleotide to a low-affinity site on E2-(K) accelerates its conversion to E1-Na; it supports the hypothesis that during the normal working of the pump, ATP, acting at a low affinity site, accelerates the conversion of dephosphoenzyme, newly formed by K+-catalysed hydrolysis of E2P, to a form in which it can be phosphorylated in the presence of Na+. 4. The rate of the reverse transformation, E1-Na to E2-(K), varies roughly linearly with the K+ concentration up to the highest concentration at which the rate can be measured (15 mM). Since much lower concentrations of K+ are sufficient to displace the equilibrium to the K-form, we suggest that the sequence of events is: (i) combination of K+ with low affinity (probably internal) binding sites, followed by (ii) spontaneous conversion of the enzyme to a form, E2-(K), containing occluded K+. 5. Mg2+ or oligomycin slows the rate of conversion of E1-Na to E2-(K) but does not significantly affect the rate of conversion of E2-(K) to E1-Na. 6. In the light of these and previous findings, we propose a model for the sodium pump in which conformational changes alternate with trans-phosphorylations, and the inward and outward fluxes of both Na+ and K+ each involve the transfer of a phosphoryl group as well as a change in conformation between E1 and E2 forms of the enzyme or phosphoenzyme.     

Sodium-coupled ATP synthesis in the bacterium Vitreoscilla.

The bacterium Vitreoscilla generates an electrical potential gradient due to sodium ion (delta psi Na+) across its membrane via respiratory-driven primary Na+ pump(s). The role of the delta psi Na+ as a driving force for ATP synthesis was, therefore, investigated. In respiring starved cells pulsed with 100 mM external Na+ [( Na+]o) there was a 167% net increase in cellular ATP concentration over basal levels compared with 0, 56, 78, and 78% for no addition, choline, Li+, and K+ controls, respectively. Doubling the [Na+]o to 200 mM boosted the net increase to 244% but a similar doubling of the choline caused only an increase to 78%. When the initial condition was intracellular Na+ ([Na+]i) = [Na+]o = 100 mM, there was a 94% net increase in cellular ATP compared with only 18 and 11% for Li+ and K+ controls, respectively, indicating that Nai+ may be the only cation tested that the cells extruded to generate the electrochemical gradient required to drive ATP synthesis. The Na(+)-dependent ATP synthesis was inhibited completely by monensin (12 microM), but only transiently by the protonophore 3,5-di-tert-butyl-4-hydroxybenzaldehyde (100 microM), further evidence that the Na+ gradient and not a H+ gradient was driving the ATP synthesis. ATP synthesis in response to an artificially imposed H+ gradient (delta pH approximately 3) in the absence of an added cation, or in the presence of Li+, K+, or choline, yielded similar delta ATP/delta pH ratios of 0.98-1.22. In the presence of Na+, however, this ratio dropped to 0.23, indicating that Na+ inhibited H(+)-coupling to ATP synthesis and possibly that H+ and Na+ coupling to ATP synthesis share a common catalyst. The above evidence adds to previous findings that under normal growth conditions Na+ is probably the main coupling cation for ATP synthesis in Vitreoscilla.     

Temperature sensitivity of catecholamine secretion and ion fluxes in bovine adrenal chromaffin cells

The effects of temperature on ion fluxes and catecholamine secretion that are mediated by nicotinic acetylcholine receptors (nAChRs), voltage-sensitive calcium channels (VSCCs), and voltage-sensitive sodium channels (VSSCs) were investigated using bovine adrenal chromaffin cells. When the chromaffin cells were stimulated with DMPP, a nicotinic cholinergic agonist, or 50 mM K+, the intracellular calcium ([Ca2+]i) elevation reached a peak and decreased more slowly at lower temperatures. The DMPP-induced responses were more sensitive to temperature changes compared to high K+-induced ones. In the measurement of intracellular sodium concentrations ([Na+]i), it was found that nicotinic stimulation required a longer time to attain the maximal level of [Na+]i at lower temperatures. In addition, the VSSCs-mediated [Na+]i increase evoked by veratridine was also reduced as the temperature decreased. The measurement of [3H]norepinephrine (NE) secretion showed that the secretion within the first 3 min evoked by DMPP or high K+ was greatest at 37 degrees C. However, at 25 degrees C, the secretion evoked by DMPP, but not that by the 50 mM K+, was greater after 10 min of stimulation. This data suggest that temperature differentially affects the activity of nAChRs, VSCCs, and VSSCs, resulting in differential [Na+]i and [Ca2+]i elevation, and in the [3H]NE secretion by adrenal chromaffin cells.     

The molecular size required varies according to the reaction step round the sodium pump cycle

Progress along the path of the sodium pump cycle requires a stepwise recruitment of additional subunits for maximal activity. These results show that whereas a particle the size of the alpha beta protomer presents Na+,K+-ATPase activity at 10 microM ATP, an additional subunit, perhaps a second alpha-chain, is required to obtain the much greater Na+,K+-ATPase activity resulting from the occupation of low-affinity ATP sites at physiological ATP concentrations. A non-phosphorylating ATP analogue, however, will modestly stimulate the Na+,K+-ATPase activity acting at an alternative low-affinity site or step on the alpha beta protomer.     

The Na+,K+/H+ -antiporter Nha1 influences the plasma membrane potential of Saccharomyces cerevisiae

There are three different sodium transport systems (Ena1-4p, Nha1p, Nhx1p) in Saccharomyces cerevisiae. The effect of their absence on the tolerance to alkali-metal cations and on the membrane potential was studied. All three sodium transporters were found to participate in the maintenance of Na+, Li+, K+ and Cs+ homeostasis. Measurements of the distribution of a fluorescent potentiometric probe (diS-C3(3) assay) in cell suspensions showed that the lack of all three transporters depolarizes the plasma membrane. The overexpression of the Na+,K+/H+ antiporter Nha1 resulted in the hyperpolarization of the plasma membrane and consequently increased the sensitivity to Cs+, Tl+ and hygromycin B. This is the first evidence that the activity of a Na+,K+/H+ antiporter could play a role in the homeostatic regulation of the plasma membrane potential in yeast cells.     

Increase of Na+ gradient-dependent L-glutamate and L-aspartate transport in high K+ dog erythrocytes associated with high activity of (Na+, K+)-ATPase

As reported previously, some dogs possess red cells characterized by low Na+, high K+ concentrations, and high activity of (Na+, K+)-ATPase, although normal dog red cells contain low K+, high Na+, and lack (Na+, K+)-ATPase. Furthermore, these red cells show increased activities of L-glutamate and L-aspartate transport, resulting in high accumulations of such amino acids in their cells. The present study demonstrated: (i) Na+ gradient-dependent L-glutamate and L-aspartate transport in the high K+ and low K+ red cells were dominated by a saturable component obeying Michaelis-Menten kinetics. Although no difference of the Km values was observed between the high K+ and low K+ cells, the Vmax values for both amino acids' transport in the high K+ cells were about three times those of low ones. (ii) L- and D-aspartate, but not D-glutamate, competitively inhibited L-glutamate transport in both types of the cells. (iii) Ouabain decreased the uptake of the amino acids in the high K+ dog red cells, whereas it was not effective on those in the low K+ cells. (iv) The ATP-treated high K+ cells [(K+]i not equal to [K+]o, [Na+]i greater than [Na+]o) showed a marked decrease of both amino acids' uptake rate, which was almost the same as that of the low K+ cells. (v) Valinomycin stimulated the amino acids' transport in both of the high K+ and the ATP-treated low K+ cells [( K+]i greater than [K+]o, [Na+]o), suggesting that the transport system of L-glutamate and L-aspartate in both types of the cells might be electrogenic. These results indicate that the increased transport activity in the high K+ dog red cells was a secondary consequence of the Na+ concentration gradient created by (Na+, K+)-ATPase.     

Futile Na+ cycling at the root plasma membrane in rice (Oryza sativa L.): kinetics, energetics, and relationship to salinity tolerance

Globally, over one-third of irrigated land is affected by salinity, including much of the land under lowland rice cultivation in the tropics, seriously compromising yields of this most important of crop species. However, there remains an insufficient understanding of the cellular basis of salt tolerance in rice. Here, three methods of 24Na+ tracer analysis were used to investigate primary Na+ transport at the root plasma membrane in a salt-tolerant rice cultivar (Pokkali) and a salt-sensitive cultivar (IR29). Futile cycling of Na+ at the plasma membrane of intact roots occurred at both low and elevated levels of steady-state Na+ supply ([Na+]ext=1 mM and 25 mM) in both cultivars. At 25 mM [Na+]ext, a toxic condition for IR29, unidirectional influx and efflux of Na+ in this cultivar, but not in Pokkali, became very high [>100 micromol g (root FW)(-1) h(-1)], demonstrating an inability to restrict sodium fluxes. Current models of sodium transport energetics across the plasma membrane in root cells predict that, if the sodium efflux were mediated by Na+/H+ antiport, this toxic scenario would impose a substantial respiratory cost in IR29. This cost is calculated here, and compared with root respiration, which, however, comprised only approximately 50% of what would be required to sustain efflux by the antiporter. This suggests that either the conventional 'leak-pump' model of Na+ transport or the energetic model of proton-linked Na+ transport may require some revision. In addition, the lack of suppression of Na+ influx by both K+ and Ca2+, and by the application of the channel inhibitors Cs+, TEA+, and Ba2+, questions the participation of potassium channels and non-selective cation channels in the observed Na+ fluxes.     

Biphasic effect of protein kinase C on rat renal cortical Na+, K+-ATPase.

We examined the dependence of rat renal Na+, K+-ATPase activity on protein kinase C (PKC) stimulation. Infusion of either phorbol 12, 13-dibutyrate (PDBu) or phorbol 12-myristate 13-acetate (PMA) into rat abdominal aorta resulted in dose-dependent changes of renal cortical Na+, K+-ATPase activity. Low doses of these esters (3 x 10(-11) mol/kg/min) increased activity of Na+, K+-ATPase whereas high doses (3 x 10(-9) mol/kg/min) decreased it. The changes in Na+, K+-ATPase activity induced by PDBu and PMA were prevented by staurosporine, a PKC inhibitor. 4Alpha phorbol didecanoate (4alpha PDD), phorbol ester which does not activate PKC had no effect on cortical Na+, K+-ATPase. PDBu and PMA did not change Na+, K+-ATPase activity in the renal medulla. The stimulatory effect of PDBu (3 x 10(-11) mol/kg/min) was neither mimicked by amphotericin B, a sodium ionophore nor blocked by amiloride, an inhibitor of Na+/H+-exchanger. The inhibitory effect of 3 x 10(-9) mol/kg/min PDBu was not mimicked by amiloride indicating that the observed effects of PKC stimulation are not secondary to alterations in intracellular sodium concentration. The inhibitory effect of PDBu was prevented by infusion of ethoxyresorufin, an inhibitor of cytochrome P450-dependent arachidonate metabolism. These results suggest that the inhibitory effect of PKC on renal cortical Na+, K+-ATPase is mediated by cytochrome P450-dependent arachidonate metabolites.