Activation by lithium ions of the inside sodium sites in (Na+ + K+)-ATPase.

1. Purified pig kidney ATPase was incubated in 30--160 mM Tris-HCl with various monovalent cations. 130 mM LiCl stimulated a ouabain-sensitive ATP hydrolysis (about 5% of the maximal (Na+ + K) activity), whereas 160 mM Tris-HCl did not stimulate hydrolysis. Similar results were obtained with human red blood cell broken membranes. 2. In the absence of Na+ and with 130 mM LiCl, the ATPase activity as a function of KCl concentration showed an initial slight inhibition (50 micrometer KCl) followed by an activation (maximal at 0.2 mM KCl) and a further inhibition, which was total at mM KCl. In the absence of LiCl, the rate of hydrolysis was not affected by any of the KCl concentrations investigated. 3. The lithium-activation curve for ATPase activity in the absence of both Na+ and K+ had sigmoid characteristics. It also showed a marked dependence on the total LiCl + Tris-HCl concentration, being inhibited at high concentrations. This inhibition was more noticeable at low LiCl concentrations. 4. In the absence of Na+, 130 mM Li+ showed promoted phosphorylation of ATPase from 1 to 3 mM ATP in the presence of Mg2+. In enzyme treated with N-ethylmaleimide, the levels of phosphorylation in Li+-containing solutions, amounted to 40% of those in Na+- and up to 7 times of those in K+-containing solutions. 5. The total (Na+ + K+)-ATPase activity was markedly inhibited at high buffer concentrations (Tris-HCl, Imidazole-HCl and tetramethylammonium-HEPES gave similar results) in cases when either the concentration of Na+ or K+ (or both) was below saturation. On the other hand, the maximal (Na+ + K+)-ATPase activity was not affected (or very slightly) by the buffer concentration. 6. Under standard conditions (Tris-HCl + NaCl = 160 mM) the Na+-activation curve of Na+-ATPase had a steep rise between 0 and 2.5 mM, a fall between 2.5 and 20 mM and a further increase between 20 and 130 mM. With 30 mM Tris-HCl, the curve rose more steeply, inhibition was noticeable at 2.5 mM Na+ and was completed at 5 mM Na+. With Tris-HCl + NaCl = 280 mM, the amount of activation decreased and inhibition at intermediate Na+ concentrations was not detected.     

Physiological basis for the high salt tolerance of Debaryomyces hansenii.

The effects of KCl, NaCl, and LiCl on the growth of Debaryomyces hansenii, usually considered a halotolerant yeast, and Saccharomyces cerevisiae were compared. KCl and NaCl had similar effects on D. hansenii, indicating that NaCl created only osmotic stress, while LiCl had a specific inhibitory effect, although relatively weaker than in S. cerevisiae. In media with low K+, Na+ was able to substitute for K+, restoring the specific growth rate and the final biomass of the culture. The intracellular concentration of Na+ reached values up to 800 mM, suggesting that metabolism is not affected by rather high concentrations of salt. The ability of D. hansenii to extrude Na+ and Li+ was similar to that described for S. cerevisiae, suggesting that this mechanism is not responsible for the increased halotolerance. Also, the kinetic parameters of Rb+ uptake in D. hansenii (Vmax, 4.2 nmol mg [dry weight]-1 min-1; K(m), 7.4 mM) indicate that the transport system was not more efficient than in S. cerevisiae. Sodium (50 mM) activated the transport of Rb+ by increasing the affinity for the substrate in D. hansenii, while the effect was opposite in S. cerevisiae. Lithium inhibited Rb+ uptake in D. hansenii. We propose that the metabolism of D. hansenii is less sensitive to intracellular Na+ than is that of S. cerevisiae, that Na+ substitutes for K+ when K+ is scarce, and that the transport of K+ is favored by the presence of Na+. In low K+ environments, D. hansenii behaved as a halophilic yeast.     

Lithium inhibits amplification or action of the maturation-promoting factor (MPF) in meiotic maturation of starfish oocytes

Microinjection of LiCl reversibly inhibits hormone-induced meiotic maturation of starfish oocytes. Microinjection of NaCl (even in ouabain-treated oocytes) or KCl, or external application of LiCl have no such effect. Blockade of meiotic maturation by Li+ occurs even when microinjection is performed after the hormone dependent period has ended, that is the period during which the hormone must be present in the medium in order that meiosis can take place. Li+ microinjection prevents oocytes from meiosis reinitiation following transfer of cytoplasm taken from maturing oocytes, which contain a maturation-promoting factor (MPF). Cytoplasm taken from Li+-injected and hormone-treated oocytes does not trigger meiosis reinitiation when transferred in control immature oocytes. Intracellular pH does not change following LiCl microinjection. Simultaneous microinjection of either K+, Na+, or EGTA does not prevent Li+-dependent inhibition in oocytes.     

Capacity of the outer membrane of a gram-negative marine bacterium in the presence of cations to prevent lysis by Triton X-100.

Cells of marine pseudomonad B-16 (ATCC 19855) washed with a solution containing 0.3 M NaCl, 50 mM MgCl2, and 10 mM KCl (complete salts) could be protected from lysis in a hypotonic environment if the suspending medium contained either 20 mM Mg2+, 40 mM Na+, or 300 mM K+. When the outer double-track layer (the outer membrane) of the cell envelope was removed to yield mureinoplasts, the Mg2+, Na+ or K+, requirements to prevent lysis were raised to 80, 210, and 400 mM, respectively. In the presence of 0.1% Triton X-100, 220, 320, and 360 mM Mg2+, Na+ or K+, respectively, prevented lysis of the normal cells. Mureinoplasts and protoplasts, however, lysed instantly in the presence of the detergent at all concentrations of Mg2+, Na+, or K+ tested up to 1.2 M. Thus, the structure of the outer membrane appears to be maintained by appropriate concentrations of Mg2+ or Na+ in a form preventing the penetration of Triton X-100 and thereby protecting the cytoplasmic membrane from dissolution by the detergent. K+ was effective in this capacity with cells washed with complete salts solution but not with cells washed with a solution of NaCl, suggesting that bound Mg2+ was required in the cell wall membrane for K+ to be effective in preventing lysis by the detergent. At high concentrations (1 M) K+ and Mg2+, but not Na+, appeared to destabilize the structure of the outer membrane in the presence of Triton X-100.     

Melibiose transport of Escherichia coli.

Transport of [3H]melibiose, prepared from [3H]raffinose, was investigated in Escherichia coli. Na+ stimulated the transport of melibiose via the melibiose system, whereas Li+ inhibited it. Kinetic parameters of melibiose transport were determined. The Kt values were 0.57 mM in the absence of Na+ or Li+, 0.27 mM in the presence of 10 mM NaCl, and 0.29 mM in the presence of 10 mM LiCl. The Vmax values were 40 and 46 nmol/min per mg of protein in the absence and in the presence of NaCl and 18 nmol/min per mg of protein in the presence of LiCl. Melibiose transport via the melibiose system was temperature sensitive in a wild-type strain of Escherichia coli and was not inhibited by lactose. On the other hand, melibiose uptake via the lactose system was not temperature sensitive, was inhibited by lactose, and was not affected by Na+ and Li+. Methyl-beta-D-thiogalactoside, a substrate for both systems, inhibited the transport of melibiose via both systems.     

Characterization of lithium-induced enzyme release from human polymorphonuclear leukocytes

Lysozyme release from purified human polymorphonuclear leukocytes was found to be uniquely enhanced by 2.5-20 mM LiCl. This effect was dose dependent and was not detected when the media was supplemented with NaCl, KCl, MgCl2, or CaCl2. The purified isotopes of Li+, 6Li, and 7Li were equally effective in enhancing lysozyme release from the cells at 10 and 20 mM, but 6Li was more effective than 7Li at 5 mM. The enhancement of enzyme release in the presence of Li+ was comparable to the enhancement observed in the presence of N-formylmethionylleucylphenylalanine (fMLP). Addition of LiCl plus fMLP did not result in lysozyme release in excess of each stimulant alone, except when the cells were incubated with 20 mM 6Li + 10(-5) M fMLP. In addition, enzyme release induced by these two agents could be further enhanced to the same degree by addition of cytochalasin D to the incubation mixtures. While similarities between enzyme release induced by LiCl and fMLP were detected, optimal stimulation of enzyme release by Li+ was much more sensitive to inhibition by pertussis toxin than was maximal fMLP stimulation. Therefore, the intracellular events altered by Li+ and the peptide may share some metabolic steps, but they differ in their sensitivity to alterations in cAMP metabolism.     

The modifications of the final stages of the complement reaction by alkali metal cations.

We studied the effects of alkali metal cations on the terminal stages of complement lysis of human and sheep HK erythrocytes. Sensitized erythrocytes (EA) were reacted with limited amounts of complement for 1 hr at 37 degrees C in buffer containing 147 mM NaCl (Na buffer), which resulted in 10-40% lysis. The unlysed cells were washed with Na buffer at 0-2 degrees C and incubated for 1 hr at 37 degrees C in buffers containing 147 mM of the various alkali metal cations. Although additional lysis (25 to 65%) occurred with K, Rb, or Cs buffer, only minor degrees developed with Na or Li buffer, only minor degrees developed with Na or Li buffer. Intermediate levels occurred with 100 mM of the divalent alkali cations. Halogen ions and SCN-(147 MM), Ca++ (0.15mM), and Mg++ (0.5 mM) did not alter the effect of the alkali metal cations. Lysis occurring in K+, Rb+ or Cs+ proceeded without lag, was temperature dependent with an optimum of 43 degrees C, and had a pH optimum of 6.5. Lysis in K and Na buffers was unaffected by 10(-3) to 10(-5) M ouabain. Experiments with mixtures of cations indicated that Na+ had a mild inhibitory effect that could be totally overcome by K+, partially by Rb+, and not at all by Cs+. Li+ had a strong inhibitory effect, 6 X 10(-5) M causing 50% inhibition in buffers containing 147 mM K+, Rb+, or Cs+. By using intermediate complexes of EA and purified complement components we demonstrated that K+ enhances the lytic action of C8 on EAC1-7 as well as that of C9 on EAC1-8. It was known that Li+ facilitates lysis when acting on the entire complement reaction. We found that Li+ enhanced the lytic action of C8 on EAC1-7, with a kinetic that differed from that of the K+ effect. In addition, Li+ inhibited the enhancing effect of K+ upon lysis of EAC1-8 by C9. This occurred at concentration of Li+ similar to those which inhibited the additional lysis by K+, Rb+, and Cs+ of cells that were pretreated in Na buffer with the entire complement sequence. We propose that the major effects of alkali metal cations on complement lysis are due to their interaction with C8 and/or membrane constitutes.     

Cation coupling to melibiose transport in Salmonella typhimurium.

Melibiose transport in Salmonella typhimurium was investigated. Radioactive melibiose was prepared and the melibiose transport system was characterized. Na+ and Li+ stimulated transport of melibiose by lowering the Km value without affecting the Vmax value; Km values were 0.50 mM in the absence of Na+ or Li+ and 0.12 mM in the presence of 10 mM NaCl or 10 mM LiCl. The Vmax value was 140 nmol/min per mg of protein. Melibiose was a much more effective substrate than methyl-beta-thiogalactoside. An Na+-melibiose cotransport mechanism was suggested by three types of experiments. First, the influx of Na+ induced by melibiose influx was observed with melibiose-induced cells. Second, the efflux of H+ induced by melibiose influx was observed only in the presence of Na+ or Li+, demonstrating the absence of H+-melibiose cotransport. Third, either an artificially imposed Na+ gradient or membrane potential could drive melibiose uptake in cells. Formation of an Na+ gradient in S. typhimurium was shown to be coupled to H+ by three methods. First, uncoupler-sensitive extrusion of Na+ was energized by respiration or glycolysis. Second, efflux of H+ induced by Na+ influx was detected. Third, a change in the pH gradient was elicited by imposing an Na+ gradient in energized membrane vesicles. Thus, it is concluded that the mechanism for Na+ extrusion is an Na+/H+ antiport. The Na+/H+ antiporter is a transformer which converts an electrochemical H+ gradient to an Na+ gradient, which then drives melibiose transport. Li+ was inhibitory for the growth of cells when melibiose was the sole carbon source, even though Li+ stimulated melibiose transport. This suggests that high intracellular Li+ may be harmful.     

Na+ and K+ binding by glycerinated muscle fibers at equal concentrations of the chlorides of these cations in the medium

The Na+ and K+ equilibrium distribution between the medium and glycerinated muscle fibres of the frog has been investigated under equal concentrations of NaCl and KCl in solutions. Concentrations of NaCl and KCl varied from 0.5-1.5 mkM till 50 mM. Ion strength (0.11) was constant owing to the imidazol--HCl buffer. The binding of Na+ and K+ by model fibres occurred in accordance with the Langmur equation. Two kinds of cation-binding sites were found. The one with a low limiting ion sorption (A infinity approximately 1.3 mmol/kg dry weight of fibres) and high affinities (-delta F0 approximately 4.3 kcal/mol) was saturated at 0.5 mM concentrations (Na+ = K+) in the medium, and the other--with A infinity exceeding the previous one by an order and low -delta F0 (2.5 kcal/mol) was discovered at Na+, K+-1-10 mM. At ion concentrations equal to 0.5-1 mM the Langmur-binding is disturbed. At Na+-K+ less than or equal to 1 mM Na+ bound:K+ bound approximately to 1:1. At higher concentrations of cations Na+ bound:K+ bound approximately equal to 3:2. It is concluded that at least part of the sites in model fibres is capable of interacting only with Na+, but not with K+. It is supposed that at equal concentrations of Na+ and K+ in the medium the cations are bound by Na+, K+-ATPase of glycerinated muscle fibres.     

Sodium chloride stimulated respiration of Anacystis nidulans.

With certain salts a stimulation of respiration of the blue-green alga Anacystis nidulans was found in the dark. The stimulation was observed only at high concentrations (10(-2)M--10(-1)M). NaCl or LiCl are the most effective salts and on addition the increase of the respiration is about 2.5fold. Li is assumed to function as a substitute for Na. Potassium salts, except KCl, are ineffective. The order for the effectiveness is: NaCl greater than NaNO3, Na2SO4 greater than KCl greater than KNO3, K2SO4 (=zero). Accordingly, the cation Na+, and to a less degree the anion Cl- are responsible for the stimulatory effect. K, which is ineffective, is passively accumulated by Anacystis according to the membrane potential. Na is actively extruded. At 0.1 M external NaCl, the passive influx of Na is high, but even then it is balanced by an active efflux. This increases the energy consumption of the cells and leads to a stimulated respiration. With DCCD (N,N'-dicyclohexylcarbodiimide) or NEM (N-ethylmaleimide), the Na efflux is inhibited, simultaneously the stimulation of respiration is abolished and the passive influx of Na becomes detectable. At 0.1 M NaCl, the passive influx of Na measured in presence of DCCD is 5 x 10(-6) moles Na/min and ml packed cells. In absence of DCCD on addition of 0.1 M NaCl the extra oxygen consumption is 2 x 10(-6) moles O2/min and ml cells. This may prove that the stimulation of respiration is mainly caused by the active Na extrusion.     

Mode of interaction of the single a subunit with the multimeric c subunits during the translocation of the coupling ions by F1F0 ATPases.

We have recently isolated a mutant (aK220R, aV264E, aI278N) of the Na+-translocating Escherichia coli/Propionigenium modestum ATPase hybrid with a Na+-inhibited growth phenotype on succinate. ATP hydrolysis by the reconstituted mutant ATPase was inhibited by external (N side) NaCl but not by internal (P side) NaCl. In contrast, LiCl activated the ATPase from the N side and inhibited it from the P side. A similar pattern of activation and inhibition was observed with NaCl and the ATPase from the parent strain PEF42. We conclude from these results that the binding sites for the coupling ions on the c subunits are freely accessible from the N side. Upon occupation of these sites, the ATPase becomes more active, provided that the ions can be further translocated to the P side through a channel of the a subunit. If by mutation of the a subunit this channel becomes impermeable for Na+, N side Na+ ions specifically inhibit the ATPase activity. These conclusions were corroborated by the observation that proton transport into proteoliposomes containing the mutant ATPase was abolished by N side but not by P side Na+ ions. In contrast, LiCl affected proton translocation from either side, similar to the sidedness effect of Na+ ions on H+ transport by the parent hybrid ATPase. If the ATPase carrying the mutated a subunit was incubated with 22NaCl and ATP, 1 mol 22Na+/mol enzyme was occluded. With the parent hybrid ATPase, 22Na+ occlusion was not observed. The occluded 22Na+ could be removed from its tight binding site by 20 mM LiCl, while incubation with 20 mM NaCl was without effect. Li+ but not Na+ is therefore apparently able to pass through the mutated a subunit and make the entrapped Na+ ions accessible again to the aqueous environment. These results suggest an ion translocation mechanism through F0 that in the ATP hydrolysis mode involves binding of the coupling ions from the cytoplasm to the multiple c subunits, ATP-driven rotation to bring a Na+, Li+, or H+-loaded c subunit into a contact site with the a subunit and release of the coupling ions through the a subunit channel to the periplasmic surface of the membrane.     

Activation and stabilization of diaphragm-associated acetylcholinesterase by monovalent (Na+, Li+) cations

Acetylcholinesterase (AChE) activity was determined at varied pH values between 6 and 11 in rat homogenated diaphragm and in eel E. electricus soluble AChE, in the presence or absence of 115 mM NaCl or LiCl. It was observed that by using homogenated diaphragm Li+ stimulated AChE at physiological pH (7-7.4). In control (no cations) a pH "optimum" of 8.6-9 was found, while in presence of NaCl or LiCl "optima" of 9.5 and 10.2 were observed respectively. At optimum pH, AChE activity was about 2 times higher with NaCl, while with LiCl 5 times higher than the control. Preincubation of the enzyme or the homogenate in cations presence at pH 5.5 or pH 12.8 had no effect on the activity, when it was measured at pH "optima". However, without cations only 76% of the activity in optimum pH after preincubation at pH 5.5 was found. These results suggest that: (a) Li+ may neutralize negative charges of AChE more successfully than Na+, resulting in better enzyme activation and stabilization; (b) a possible enzyme desensitization induced by pH changes can be avoided by increasing Na+ concentrations and especially Li+.     

The ATP dependence of a ouabain-sensitive sodium efflux activated by external sodium, potassium and lithium in human red cells.

The stimulation of ouabain-sensitive Na+ efflux by external Na+, K+ and Li+ was studied in control and ATP-depleted human red cells. In the presence of 5 mM Na+, with control and depleted cells, Li+ stimulated with a lower apparent affinity than K+, and gave a smaller maximal activation than K+. The ability of Na+, K+ and Li+ to activate Na+ efflux was a function of the ATP content of the cells. Relative to K+ both Na+ and Li+ became more effective activators when the ATP was reduced to about one tenth of the control values. At this low ATP concentration Na+ was absolutely more effective than K+.     

Calcium inhibition of the ATPase and phosphatase activities of (Na+ + K+)-ATPase

In experiments performed at 37 degrees C, Ca2+ reversibly inhibits the Na+-and (Na+ + K+)-ATPase activities and the K+-dependent phosphatase activity of (Na+ + K+)-ATPase. With 3 mM ATP, the Na+-ATPase was less sensitive to CaCl2 than the (Na+ + K+)-ATPase activity. With 0.02 mM ATP, the Na+-ATPase and the (Na+ + K+)-ATPase activities were similarly inhibited by CaCl2. The K0.5 for Ca2+ as (Na+ + K+)-ATPase inhibitor depended on the total MgCl2 and ATP concentrations. This Ca2+ inhibition could be a consequence of Ca2+-Mg2+ competition, Ca . ATP-Mg . ATP competition or a combination of both mechanisms. In the presence of Na+ and Mg2+, Ca2+ inhibited the K+-dependent dephosphorylation of the phosphoenzyme formed from ATP, had no effect on the dephosphorylation in the absence of K+ and inhibited the rephosphorylation of the enzyme. In addition, the steady-state levels of phosphoenzyme were reduced in the presence both of NaCl and of NaCl plus KCl. With 3 mM ATP, Ca2+ alone sustained no more than 2% of the (Na+ + K+)-ATPase activity and about 23% of the Na+-ATPase activity observed with Mg2+ and no Ca2+. With 0.003 mM ATP, Ca2+ was able to maintain about 40% of the (Na+ + K+)-ATPase activity and 27% of the Na+-ATPase activity seen in the presence of Mg2+ alone. However, the E2(K)-E1K conformational change did not seem to be affected. Ca2+ inhibition of the K+-dependent rho-nitrophenylphosphatase activity of the (Na+ + K+)-ATPase followed competition kinetics between Ca2+ and Mg2+. In the presence of 10 mM NaCl and 0.75 mM KCl, the fractional inhibition of the K+-dependent rho-nitrophenylphosphatase activity as a function of Ca2+ concentration was the same with and without ATP, suggesting that Ca2+ indeed plays the important role in this process. In the absence of Mg2+, Ca2+ was unable to sustain any detectable ouabain-sensitive phosphatase activity, either with rho-nitrophenylphosphate or with acetyl phosphate as substrate.     

Gill (Na+ + K+)- and Na+-stimulated Mg2+-dependent ATPase activities in the gilthead bream (Sparus auratus L.)

1. Gilthead gill 10(-3) M ouabain-inhibited (Na+ + K+)-ATPase and 10(-2) M ouabain-insensitive Na+-ATPase require the optimal conditions of pH 7.0, 160 mM Na+, 20 mM K+, 5 mM MgATP and pH 4.8-5.2, 75 mM Na+, 2.5 mM Mg2+, 1.0 mM ATP, respectively. 2. The main distinctive features between the two activities are confirmed to be optimal pH, the ouabain-sensitivity and the monovalent cation requirement, Na+ plus another cationic species (K+, Rb+, Cs+, NH4+) in the (Na+ + K+)-ATPase and only one species (Na+, K+, Li+, Rb+, Cs+, NH4+ or choline+) in the Na+-ATPase. 3. The aspecific Na+-ATPase activation by monovalent cations, as well as by nucleotide triphosphates, opposed to the (Na+ + K+)-ATPase specificity for ATP and Na+, relates gilthead gill ATPases to lower organism ATPases and differentiates them from mammalian ones. 4. The discrimination between the two activities by the sensitivity to ethacrynic acid, vanadate, furosemide and Ca2+ only partially agrees with the literature. 5. Present findings are viewed on the basis of the ATPase's presumptive physiological role(s) and mutual relationship.     

Insulin affects the sodium affinity of the rat adipocyte (Na+,K+)-ATPase

The K0.5 for intracellular sodium of the two forms of (Na+,K+)-ATPase which exist in rat adipocytes (Lytton, J., Lin, J. C., and Guidotti, G. (1985) J. Biol. Chem. 260, 1177-1184) has been determined by incubating the cells in the absence of potassium in buffers of varying sodium concentration; these conditions shut off the Na+ pump and allow sodium to equilibrate into the cell. The activity of Na+,K+)-ATPase was then monitored with 86Rb+/K+ pumping which was initiated by adding isotope and KCl to 5 mM, followed by a 3-min uptake period. Atomic absorption and 22Na+ tracer equilibration were used to determine the actual intracellular [Na+] under the different conditions. The K0.5 values thus obtained were 17 mM for alpha and 52 mM for alpha(+). Insulin treatment of rat adipocytes had no effect on the intracellular [Na+] nor on the Vmax of 86Rb+/K+ pumping, but did produce a shift in the sodium ion K0.5 values to 14 mM for alpha (p less than 0.025 versus control) and 33 mM for alpha(+) (p less than 0.005 versus control). This change in affinity can explain the selective stimulation of alpha(+) by insulin under normal incubation conditions. Measurement of the K0.5 for sodium ion of (Na+,K+)-ATPase in membranes isolated from adipocytes revealed only a single component of activation with a low K0.5 of 3.5 or 12 mM in the presence of 10 or 100 mM KCl, respectively. Insulin treatment of the isolated membranes or of the cells prior to membrane separation had no effect on these values.     

Effects of Several Cations on the Neuronal Uptake of Dopamine and the Specific Binding of [3H]GBR 12783: Attempts to Characterize the Na+ Dependence of the Neuronal Transport of Dopamine

We have studied the effects of several cations on (1) the neuronal uptake of [3H]dopamine ([3H]DA) and (2) the specific binding of 1-[2-(diphenylmethoxy)ethyl]-4-(3-phenyl-2-[1-3H]propenyl)piperazi ne ([3H]GBR 12783) to a site associated with the neuronal carrier of DA, in preparations obtained from rat striatum. When studied under the same experimental conditions, both the uptake of [3H]DA and the binding of [3H]GBR 12783 were similarly impaired by the gradual replacement of NaCl by sucrose. In both processes, no convenient substitute for Na+ was found. Furthermore, potential substitutes of Na+ acted as inhibitors of the uptake with a rank order of potency as follows: K+ = Li+ > or = Cs+ > or = Rb+ > choline+ > Tris+ > sucrose, which was somewhat different from that observed in binding studies, i.e., Cs+ > Rb+ > choline+ > or = K+ > Li+ > Tris+ > sucrose. In the presence of either 36 mM or 136 mM Na+, [3H]DA uptake was optimal with 2 mM Mg2+, 1 mM K+, or 1 mM Ca2+. In contrast, higher concentrations of divalent cations competitively blocked the uptake process. K+ concentrations > 50 mM impaired the specific binding, whereas in the millimolar range of concentrations, K+ noncompetitively inhibited the uptake. Decreasing the Na+ concentration increased the inhibitory effect of K+, Ca2+, and Mg2+ on the specific uptake. An increase in NaCl concentration from 0 to 120 mM elicited a significant decline in the affinity of some substrates for the [3H]GBR 12783 binding site. An uptake study performed using optimal experimental conditions defined in the present study revealed that decreasing Na+ concentration reduces the affinity of DA for the neuronal transport. We propose a hypothetical model for the neuronal transport of DA in which both Na+ and K+ membrane gradients are involved.     

The ATP4- receptor-operated ion channel of human lymphocytes: inhibition of ion fluxes by amiloride analogs and by extracellular sodium ions.

Extracellular ATP is known to increase the membrane permeability of a variety of cells. Addition of ATP to human leukemic lymphocytes loaded with the Ca2+ indicator, fura-2, induced a rise in cytosolic Ca2+ concentration which was attenuated or absent in NaCl media compared with KCl, choline Cl, or NMG Cl media. In contrast, anti-immunoglobulin antibody gave similar Ca2+ transients in NaCl and KCl media. A half-maximal inhibition of peak ATP-induced Ca2+ response was observed at 10-16 mM extracellular Na+. Basal 45Ca2+ influx into lymphocytes was stimulated 9.6-fold by ATP added to cells in KCl media, but the effect of ATP was greatly reduced for cells in NaCl media. Hexamethylene amiloride blocked 74% of the ATP-stimulated Ca45 uptake of cells in KCl media. Flow cytometry measurements of fluo-3-loaded cells confirmed that the ATP-induced rise in cytosolic Ca2+ was inhibited either by extracellular Na+ or by addition of hexamethylene amiloride. Extracellular ATP stimulated 86Rb efflux from lymphocytes 10-fold and this increment was inhibited by the amiloride analogs in a rank order of potency 5-(N-methyl-N-isobutyl)amiloride greater than 5-(N,N-hexamethylene)amiloride greater than 5-(N-ethyl-N-isopropyl)amiloride greater than amiloride. ATP-induced 86Rb efflux showed a sigmoid dependence on the concentration of ATP and Hill analysis gave K1/2 of 90 and 130 microM and n values of 2.5 and 2.5 for KCl and NaCl media, respectively. However, the maximal ATP-induced 86Rb efflux was 3-fold greater in KCl than in NaCl media. Raising extracellular Na+ from 10 to 100 mM increased ATP-induced Na+ influx from a mean of 2.0 to 3.7 nEq/10(7) cells/min, suggesting either saturability or self-inhibition by Na+ of its own influx. These data suggest that ATP opens a receptor-operated ion channel which allows increased Ca2+ and Na+ influx and Rb+ efflux and these fluxes are inhibited by extracellular Na+ ions as well as by the amiloride analogs.     

Ionic dependence of the extracellular ATP-induced permeabilization of transformed mouse fibroblasts: role of plasma membrane activities that regulate cell volume

Extracellular ATP rendered the plasma membrane of transformed mouse fibroblasts permeable to normally impermeant molecules. This permeability change was prevented by increasing the ionic strength of the isotonic medium with NaCl. Conversely, the cells exhibited increased sensitivity to ATP when the NaCl concentration was decreased below isotonicity, when the KCl concentration was increased above 5 mM while maintaining isotonicity, and when the pH of the medium was raised above 7.0. These conditions as well as the addition of ATP itself caused cell swelling. However, the effect of ATP was independent of cell volume and dependent upon the ionic strength and not the osmolarity of the medium since 1) addition of sucrose to isotonic medium did not prevent permeabilization although media made hypertonic with either sucrose or NaCl caused a decrease in cell volume; and 2) addition of sucrose or NaCl to hypotonic media caused a decrease in cell volume, but only NaCl addition decreased the response to ATP. Conditions that have been shown to inhibit plasma membrane proteins that play a reciprocal role in cell volume regulation had reciprocal effects on the permeabilization process, even though the effect of ATP was independent of cell volume. For example, inhibition of the Na+,K+-ATPase by ouabain increased sensitivity of cells to ATP while conditions which inhibit Na+,K+,Cl- -cotransporter activity, such as treatment of the cells with the diuretics furosemide or bumetanide or replacement of sodium chloride in the medium with sodium nitrate or thiocyanate, inhibited permeabilization. The furosemide concentration that inhibited permeabilization was greater than the concentration that inhibited Na+,K+,Cl- -cotransporter-mediated 86Rb+ (K+) uptake, suggesting that the effect of furosemide on the permeabilization process may not be specific for the Na+,K+,Cl- -cotransporter.     

Physiological roles of three Na+/H+ antiporters in the halophilic bacterium Vibrio parahaemolyticus

Vibrio parahaemolyticus mutants lacking three Na+/H+ antiporters (NhaA, NhaB, NhaD) were constructed. The DeltanhaA strains showed significantly higher sensitivity to LiCl regarding their growth compared to the parental strain. The DeltanhaA and DeltanhaB strains exhibited higher sensitivities to LiCl. The mutant XACabd lacking all of the three antiporters could not grow in the presence of 500 mM LiCl at pH 7.0, or 50 mM at pH 8.5. The XACabd mutant was also sensitive to 1.0 M NaCl at pH 8.5. These results suggest that Na+/H+ antiporters, especially NhaA, are responsible for resistance to LiCl and to high concentrations of NaCl. Reduced Na+/H+ and Li+/H+ antiport activities were observed with everted membrane vesicles of DeltanhaB strains. However, Li+/H+ antiport activities of DeltanhaB strains were two times higher than those of DeltanhaA strains when cells were cultured at pH 8.5. It seems that expression of nhaA and nhaB is dependent on medium pH to some extent. In addition, HQNO (2-heptyl-4-hydroxyquinoline N-oxide), which is a potent inhibitor of the respiratory Na+ pump, inhibited growth of XACabd, but not of the wild type strain. Moreover, survival rate of XACabd under hypoosmotic stress was lower than that of wild type strain. It is likely that the Na+/H+ antiporters are involved in osmoregulation under hypoosmotic stress. Based on these findings, we propose that the Na+/H+ antiporters cooperate with the respiratory Na+ pump in ionic homeostasis in V. parahaemolyticus.