NMR studies on Na+ transport in Synechococcus PCC 6311.

The freshwater cyanobacterium Synechococcus PCC 6311 is able to adapt to grow after sudden exposure to salt (NaCl) stress. We have investigated the mechanism of Na+ transport in these cells during adaptation to high salinity. Na+ influx under dark aerobic conditions occurred independently of delta pH or delta psi across the cytoplasmic membrane, ATPase activity, and respiratory electron transport. These findings are consistent with the existence of Na+/monovalent anion cotransport or simultaneous Na+/H+ +anion/OH- exchange. Na+ influx was dependent on Cl-, Br-, NO3-, or NO2-. No Na+ uptake occurred after addition of NaI, NaHCO3, or Na2SO4. Na+ extrusion was absolutely dependent on delta pH and on an ATPase activity and/or on respiratory electron transport. This indicates that Na+ extrusion via Na+/H+ exchange is driven by primary H+ pumps in the cytoplasmic membrane. Cells grown for 4 days in 0.5 m NaCl medium, "salt-grown cells," differ from control cells by a lower maximum velocity of Na+ influx and by lower steady-state ratios of [Na+]in/[Na+]out. These results indicate that cells grown in high-salt medium increase their capacity to extrude Na+. During salt adaptation Na+ extrusion driven by respiratory electron transport increased from about 15 to 50%.     

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.     

Respiratory-driven Na+ electrical potential in the bacterium Vitreoscilla

Vitreoscilla is a Gram-negative bacterium with unique respiratory physiology in which Na+ was implicated as a coupling cation for the generation of a transmembrane electrical gradient (delta psi). Thus, cells respiring in the presence of 110 mM Na+ generated a delta psi of -142 mV compared to only -42 and -56 mV for Li+ and choline, respectively, and even the -42 and -56 mV were insensitive to the protonophore 3,5-di-tert-butyl-4-hydroxybenzaldehyde (DTHB). The kinetics of delta psi formation and collapse correlated well with the kinetics of Na+ fluxes but not with those of H+ fluxes. Cyanide inhibited respiration, Na+ extrusion, and delta psi formation 81% or more, indicating that delta psi formation and Na+ extrusion were coupled to respiration. Experiments were performed to distinguish among three possible transport systems for this coupling: (1) a Na(+)-transporting ATPase; (2) an electrogenic Na+/H+ antiport system; (3) a primary Na+ pump directly driven by the free energy of electron transport. DCCD and arsenate decreased cellular ATP up to 86% but had no effect on delta psi, evidence against a Na(+)-transporting ATPase. Low concentrations of DTHB had no effect on delta psi; high concentrations transiently collapsed delta psi, but led to a stimulation of Na+ extrusion, the opposite of that expected for a Na+/H+ antiport system. Potassium ion, which collapses delta psi, also stimulated Na+ extrusion. The experimental evidence is against Na+ extrusion by mechanisms 1 and 2 and supports the existence of a respiratory-driven primary Na+ pump for generating delta psi in Vitreoscilla.     

The Na+-motive terminal oxidase activity in an alkalo- and halo-tolerant Bacillus

An alkalo- and halo-tolerant aerobic microorganism has been isolated which, according to microbiological analysis data and the ribosomal 5S RNA sequence, is a Bacillus similar, but not identical, to B. licheniformis and B. subtilis. The microorganism, called Bacillus FTU, proved to be resistant to the protonophorous uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP). The fast growth of Bacillus FTU in the presence of CCCP was shown to require a high Na+ concentration in the medium. A procedure was developed to exhaust endogenous respiratory substrates in Bacillus FTU cells so that fast oxygen consumption by the cells was observed only when an exogenous respiratory substrate was added. The exhausted cells were found to oxidize ascorbate in the presence of N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) in a cyanide-sensitive fashion. The ascorbate oxidation was coupled to the uphill Na+ extrusion which was stimulated by CCCP and a penetrating weak base, diethylamine, as well as by valinomycin with or without diethylamine. Operation of the Bacillus FTU terminal oxidase resulted in the generation of a delta psi which, in the Na+ medium, was slightly decreased by CCCP and strongly decreased by CCCP + diethylamine. In the K+ medium, CCCP discharged delta psi even without diethylamine. Ascorbate oxidation was competent in ATP synthesis which was resistant to CCCP in the Na+ medium and sensitive to CCCP in the K+ medium as if Na+- and H+-coupled oxidative phosphorylations were operative in the Na+ and K+ media, respectively. Inside-out subcellular vesicles of Bacillus FTU were found to be competent in the Na+ uptake supported by oxidation of ascorbate + TMPD or diaminodurene. CCCP or valinomycin + K+ increased the Na+ uptake very strongly. The process was completely inhibited by cyanide or monensin, the former, but not the latter, being inhibitory for respiration. The data obtained indicate that in Bacillus FTU there is not only H+-motive but also Na+-motive terminal oxidase activity.     

Na+ is translocated at NADH:quinone oxidoreductase segment in the respiratory chain of Vibrio alginolyticus

The coupling site of the Na+ pump to the respiratory chain of Vibrio alginolyticus was examined using membrane fractions prepared from the wild type, Na+ pump-deficient mutants, and spontaneous revertant. NADH oxidase of the wild type and revertant specifically required NA+ for maximum activity, whereas Na+ was not essential for the NADH oxidase of mutants. Similar to the Na+ pump in whole cells, the Na+-dependent NADH oxidase in membranes had a pH optimum in the alkaline region. A respiratory inhibitor, 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO), inhibited the Na+-dependent NADH oxidase but had little effect on the NA+-independent activity of mutant membranes. NADH:quinone oxidoreductase was found to be the Na+-dependent HQNO-sensitive site of the NADH oxidase. In the wild type cells, HQNO was also found to cause a strong inhibition of the Na+ pump with little effect on the overall H+ extrusion by respiration. The inhibition of the Na+ pump by HQNO was overcome by oxidized, but not reduced, N,N,N',N'-tetra-methyl-p-phenylenediamine (TMPD). In the presence of oxidised TMPD, the electron flow NADH to oxygen seemed to bypass the HQNO-sensitive site and energize the Na+ pump. From these results, it was concluded that the Na+ pump is coupled to the respiratory chain at the step of NADH:quinone oxidoreductase.     

Na+/H+ antiporters

Na+/H+ antiports or exchange reactions have been found widely, if not ubiquitously, in prokaryotic and eukaryotic membranes. In any given experimental system, the multiplicity of ion conductance pathways and the absence of specific inhibitors complicate efforts to establish that the antiport observed actually results from the activity of a specific secondary porter which catalyzes coupled exchanged of the two ions. Nevertheless, a large body of evidence suggests that at least some prokaryotes possess a delta psi-dependent, mutable Na+/H+ antiporter which catalyzes Na+ extrusion in exchange for H+; in other bacterial species, the antiporter my function electroneutrally, at least at some external pH values. The bacterial Na+/H+ antiporter constitutes a critical limb of Na+ circulation, functioning to maintain a delta mu Na+ for use by Na+-coupled bioenergetic processes. The prokaryotic antiporter is also involved in pH homeostasis in the alkaline pH range. Studies of mutant strains that are deficient in Na+/H+ antiporter activity also indicate the existence of a relationship, e.g., a common subunit or regulatory factor, between the Na+/H+ antiporter and Na+/solute symporters in several bacterial species. In eukaryotes, an electroneutral, amiloride-sensitive Na+/H+ antiport has been found in a wide variety of cell and tissue types. Generally, the normal direction of the antiport appears to be that of Na+ uptake and H+ extrusion. The activity is thus implicated as part of a complex system for Na+ circulation, e.g., in transepithelial transport, and might have some role in acidification in the renal proximal tubule. In many experimental systems, the Na+/H+ antiport appears to influence intracellular pH. In addition to a role in general pH homeostasis, such Na+-dependent changes in intracellular pH could be part of the early events in a variety of differentiating and proliferative systems. Reconstitution and structural studies, as well as detailed analysis of gene loci and products which affect the antiport activity, are in their very early stages. These studies will be important in further clarification of the precise structural nature and role(s) of the Na+/H+ antiporters. In neither prokaryotes nor eukaryotes systems is there yet incontrovertible evidence that a specific protein carrier, that catalyzes Na+/H+ antiport, is actually responsible for any of the multitude of effects attributed to such antiporters. The Na+-H+ exchange might turn out to be side reactions of other porters or the additive effects of several conductance pathways; or, as appears most likely in at least some bacteria and in renal tissue, the antiporter may be a discrete, complex carr     

Characterization of the Na+-dependent Mg2+ transport in sheep ruminal epithelial cells

This study examines the routes by which Mg2+ leaves cultured ovine ruminal epithelial cells (REC). Mg2+-loaded (6 mM) REC were incubated in completely Mg2+-free solutions with varying Na+ concentrations, and the Mg2+ extrusion rate was calculated from the increase of the Mg2+ concentration in the incubation medium determined with the aid of the fluorescent probe mag-fura 2 (Na+ salt). In other experiments, REC were also studied for the intracellular free Mg2+ concentration ([Mg2+]i; using mag-fura 2), the intracellular Na+ concentration (using Na+-binding benzofuran isophthalate), the intracellular cAMP concentration ([cAMP]i; using an enzyme-linked immunoassay), and Na+/Mg2+ exchanger existence [using a monoclonal antibody (mAb) raised against the porcine red blood cell Na+/Mg2+ exchanger]. Mg2+-loaded REC show a Mg2+ efflux that was strictly dependent on extracellular Na+. The Mg2+ extrusion rate increased from 0.018+/-0.009 in a Na+-free medium to 0.73+/-0.3 mM.l cells-1.min-1 in a 145 mM Na+ medium and relates to extracellular Na+ concentration ([Na+]e) according to a typical saturation kinetic (Km value for [Na+]e=24 mM; maximal velocity=11 mM.l cells-1.min-1). Mg2+ efflux was reduced by imipramine (48%) and increased after application of dibutyryl-cAMP (55%) or PGE2 (17%). These effects are completely abolished in Na+-free media. Furthermore, an elevation of [cAMP]i led to an [Mg2+]i decrease that amounted to 375+/-105 microM. The anti-Na+/Mg2+ exchanger mAb inhibits Mg2+ extrusion; moreover, it detects a specific 70-kDa immunoreactive band in protein lysates of ovine REC. The data clearly demonstrate that a Na+/Mg2+ exchanger is existent in the cell membrane of REC. The transport protein is the main pathway (97%) for Mg2+ extrusion and can be assumed to play a considerable role in the process of Mg2+ absorption as well as the maintenance of the cellular Mg2+ homeodynamics.     

Amplification of the Na+-ATPase of Streptococcus faecalis at alkaline pH

The Na+-ATPase activity of Streptococcus faecalis was influenced by the medium pH. Activities of the protonophore-resistant Na+ extrusion and the KtrII (active K+ uptake by the Na+-ATPase) were maximal in the cells grown at pH 9.5, and were minimal in those grown at pH 6.0. In the cells grown at pH 7.5, they were moderately observed. The Na+-stimulated ATPase activity of the cells grown at pH 9.5 was about 4-fold higher than that of the cells grown at pH 6.0. Thus, amplification of the Na+-ATPase is remarkable at alkaline pH in this organism, possibly by an increase of the cytoplasmic Na+ level as a signal.     

Regulation of intracellular free Ca2+ concentration in the outer segments of bovine retinal rods by Na-Ca-K exchange measured with fluo-3. II. Thermodynamic competence of transmembrane Na+ and K+ gradients and inactivation of Na(+)-dependent Ca2+ extrusion.

Regulation of cytosolic free Ca2+ in the physiologically relevant submicromolar range was measured in isolated intact bovine rod outer segments (ROS) with the intracellular Ca(2+)-indicating dye fluo-3. Changes in free Ca2+ were compared with changes in total Ca2+ measured with 45Ca fluxes and a good qualitative correlation was observed. Ca2+ homeostasis in isolated bovine ROS was exclusively mediated via the Na-Ca-K exchanger. Free cytosolic Ca2+ concentration was lowered by an increase in the inward Na+ gradient, was raised by an increase in external K+, and was raised by depolarization of the plasma membrane. The simplest stoichiometry consistent with these qualitative observations is 4Na:(1Ca + 1K). The individual K:Ca, Na:Ca, and K:Na coupling ratios were deduced from quantitative changes in cytosolic free Ca2+ upon changes in the transmembrane Na+ and K+ gradients. The observed changes in free Ca2+ did not agree with changes in free Ca2+ calculated on the basis of the above fixed stoichiometry which may reflect the flexibility in the Ca:K coupling ratio observed before in flux experiments (Schnetkamp, P. P. M., Szerencsei, R. T., and Basu, D. K. (1991) J. Biol. Chem. 266, 198-206). The most dramatic discrepancy was observed for the Na:Ca coupling ratio: the expected very large changes in cytosolic free Ca2+ upon changes in the transmembrane Na+ gradient were not observed. Rapid Na(+)-induced Ca2+ extrusion was unable to lower cytosolic free Ca2+ below 100 nM, even under nonequilibrium conditions and despite the observation that Ca2+ influx via reverse Na-Ca-K exchange readily occurred at a free external Ca2+ concentration of 20 nM. We conclude that the Na(+)-dependent extrusion mode of the Na-Ca-K exchanger occurs in a brief (20-s) burst of high maximal velocity transport followed by a nearly complete inactivation of transport. The importance of our findings for Ca2+ homeostasis in functioning rod photoreceptors is discussed.     

Regulation of respiration by Na+ and K+ in the halotolerant bacterium, Ba1

In the obligate aerobe, moderate halophile bacterium, Ba1, the ion composition of the medium was found to have a profound influence on the response of the respiratory system to changes in the external pH. In the pH range 6.5 to 8.5 the respiratory activity either increased or decreased progressively, depending whether K+ or Na+ ions were omitted from the medium. A nearly constant rate of respiration was observed in the entire pH range when both K+ and Na+ were present simultaneously. The stimulatory effect of Na+ was expressed especially in the alkaline pH range, where it induced acidification of the intracellular milieu. It was manifest in whole cells as well as in inverted membrane vesicles, and was not affected by either uncoupler or inhibitor of H+-ATPase. In contrast, the respiratory stimulation induced by K+ was most prominent in the acidic pH range and was accompanied by alkalinization of the internal pH. The effect of K+ was observed only in intact cells. Agents which interfered with energy transfer suppressed the effect of K+. With ethanol as the electron donor, Na+ was found to decrease the extent of reduction of the cellular NAD+ in the aerobic steady state, and to cause increased reduction of the cytochromes. K+ had no appreciable effect on the extent of reduction of any component in the respiratory chain. The implications of the above findings are discussed in relation to the mechanism(s) involved in the cation-mediated regulation of respiration and intracellular pH.     

Generation of a transmembrane gradient of Na+ in Methanosarcina barkeri

A transmembrane Na+ gradient was generated by Methanosarcina barkeri during methanogenesis. The intracellular Na+ concentration amounted to approximately one fifth of the extracellular one. A secondary Na+/H+ antiport system was shown to be responsible for Na+ extrusion. This system could be inhibited by amiloride. In the presence of amiloride the delta pH across the cytoplasmic membrane increased and a transmembrane Na+ gradient could neither be generated nor maintained. The possible role of Na+ in the oxidation of methanol to the level of formaldehyde is discussed.     

Amiloride-sensitive Na+/H+ exchange in human neutrophils: mechanism of activation by chemotactic factors

The cytoplasmic pH (pHi) of human blood neutrophils was measured using trapped carboxyfluorescein derivatives. Cells were acid-loaded using propionate or by pretreatment with NH4+. Acid-loaded cells were found to regain near-normal pHi by means of a Na+-dependent process. A concomitant Na+ uptake was recorded as a change in cell volume. Both events were amiloride-sensitive, indicating involvement of a Na+/H+ antiport. Activation of Na+/H+ exchange was also observed with chemotactic factors. Studies of the pHi-dependence of the H+ extrusion rate indicate that chemotactic factors increase the [H+i] sensitivity of the antiport.     

Inhibition of Na(+)-independent H+ pump by Na(+)-induced changes in cell Ca2+

Apical membrane H+ extrusion in the renal outer medullary collecting duct, inner stripe, is mediated by a Na(+)-independent H+ pump. To examine the regulation of this transporter, cell pH and cell Ca2+ were measured microfluorometrically in in vitro perfused tubules using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein and fura-2, respectively. Apical membrane H+ pump activity, assayed as cell pH recovery from a series of acid loads (NH3/NH+4 prepulse) in the total absence of ambient Na+, initially occurred at a slow rate (0.06 +/- 0.02 pH units/min), which was not sufficient to account for physiologic rates of H+ extrusion. Over 15-20 min after the initial acid load, the rate of Na(+)-independent cell pH recovery increased to 0.63 +/- 0.09 pH units/min, associated with a steady-state cell pH greater than the initial pre-acid load cell pH. This pattern suggested an initial suppression followed by a delayed activation of the apical membrane H+ pump. Replacement of peritubular Na+ with choline or N-methyl-D-glucosamine resulted in an initial spike increase in cell Ca2+ followed by a sustained increase in cell Ca2+. The initial rate of Na(+)-independent cell pH recovery could be increased by elimination of the Na+ removal-induced sustained cell Ca2+ elevation by: (a) performing studies in the presence of 135 mM peritubular Na+ (1 mM peritubular amiloride used to inhibit basolateral membrane Na+/H+ antiport); (b) clamping cell Ca2+ low with dimethyl-BAPTA, an intracellular Ca2+ chelating agent; or (c) removal of extracellular Ca2+. Cell acidification induced a spike increase in cell Ca2+. The late acceleration of Na(+)-independent cell pH recovery was independent of Na+ removal and of the method used to acidify the cell, but was eliminated by prevention of the cell Ca2+ spike and markedly delayed by the microfilament-disrupting agent, cytochalasin B. This study demonstrates that peritubular Na+ removal results in a sustained elevation in cell Ca2+, which inhibits the apical membrane H+ pump. In addition, rapid cell acidification associated with a spike increase in cell Ca2+ leads to a delayed activation of the H+ pump. Thus, cell Ca2+ per se, or a Ca(2+)-activated pathway, can modulate H+ pump activity.     

A novel mechanism of cation/substrate cotransport: Na+/H+/adenosine cotransport in Vibrio parahaemolyticus

Adenosine is actively transported with Na+ in Vibrio parahaemolyticus (Sakai, Y., Tsuda, M., Tsuchiya, T. (1987) Biochim, Biophys. Acta 893, 43-48). The proton conductor carbonylcyanide m-chlorophenylhydrazone, CCCP, strongly inhibited active transport of adenosine at pH 8.5 as well as at pH 7.0. This seemed peculiar because the driving force, an electrochemical potential of Na+, is established by the Na(+)-extruding respiratory chain at pH 8.5 in this organism, although it is established by the function of the Na+/H+ antiporter at pH 7.0. This suggested that H+ might be involved in the adenosine transport. We detected H+ uptake induced by adenosine influx in V. parahaemolyticus cells in the presence of Na+, but not in its absence, suggesting the occurrence of Na+/H+/adenosine cotransport. We isolated formycin A-resistant mutants which showed defective adenosine transport. The mutation resulted in simultaneous losses of Na+ uptake and H+ uptake induced by adenosine. In revertants from these mutants the Na+ uptake and H+ uptake were restored simultaneously. The frequencies of reversion were in the order of 10(-7), indicating that the mutations were single mutations; namely that Na+/adenosine cotransport and H+/adenosine cotransport took place via the same carrier. Thus, we conclude that adenosine is transported by the novel mechanism of Na+/H+/adenosine cotransport in V. parahaemolyticus.     

Na+, K+ and Cl- transport in isolated small intestinal cells from guinea pig. Evidences for the existence of a second Na+ pump

Isolated small intestinal epithelial cells, after incubation at 4 degrees C for 30 min, reach ion concentrations (36 mM K+, 113 mM Na+ and 110 mM Cl-) very similar to those of the incubation medium. Upon rewarming to 37 degrees C, cells are able to extrude Na+, Cl- and water and to gain K+. Na+ extrusion is performed by two active mechanisms. The first mechanism, transporting Na+ by exchanging it for K+, is inhibited by ouabain and is insensitive to ethacrynic acid. It is the classical Na+ pump. The second mechanism transports Na+ with Cl- and water, is insensitive to ouabain but is inhibited by ethacrynic acid. Both mechanisms are inhibited by dinitrophenol and anoxia. The second Na+ extruding mechanism could be the Na+/K+/2Cl- cotransport system. However, this possibility can be ruled out because the force driving cotransport would work inwards, and because Na+ extrusion with water loss continues after substitution of Cl- by NO3-. We propose that enterocytes have a second Na+ pump, similar to that proposed in proximal tubular cells.     

In squid axons intracellular Mg2+ is essential for ATP-dependent Na+ efflux in the absence and presence of strophanthidin

The effect on Na+ efflux of removal of intracellular Mg2+ was studied in squid giant axons dialyzed without internal Ca2+. In the absence of Mg2i+, ATP was unable to stimulate any efflux of Na+ above the baseline of about 1 pmol . cm-2 . s-1. This behavior was observed in otherwise normal axons and in axons poisoned with 50 microM strophanthidin in the sea water. Reinstatement of 4 mM MgCl2 in excess to ATP in the dialysis solution brought about the usual response of Na+ efflux to ATP, external K+ and strophanthidin. The present experiments show that, regardless of the mechanism for the ATP-dependent Na+ efflux in strophanthidin-poisoned axons, this type of flux shares with the active Na+ extrusion the need for the simultaneous presence of intracellular ATP and Mg2+.     

Bacterial Na+ energetics

Novel observations related to the Na+-linked energy transduction in bacterial membranes are considered. It is concluded that besides the well-known systems based on the circulation of protons, there are those based on the circulation of Na+. In some cases, H+ and Na+ cycles co-exist in one and the same membrane. Representatives of the 'sodium world', i.e. cells possessing primary Na+ pumps (delta mu Na generators and consumers) are found in many genera of bacteria. Among the delta mu Na generators, one should mention Na+-NADH-quinone reductase and Na+-terminal oxidase of the respiratory chain, Na+-decarboxylases and Na+-ATPases. For delta mu Na consumers, there are Na+-ATP-synthases, Na+-metabolite symporters and Na+ motors. Sometimes, one and the same enzyme can transport H+ or, alternatively, Na+. For instance, an Na+-ATP-synthase of the F0F1 type translocates H+ when Na+ is absent. Employment of the Na+ cycle, apart from or instead of the H+ cycle, increases the resistance of bacteria to alkaline or protonophore-containing media and, apparently, to some other unfavourable conditions.     

Impairment of Na+/H+ exchange underlies inhibitory effects of Na+-free media on leukocyte function

Inhibition of activation has been reported when neutrophils are suspended in Na+-free media. We considered the possibility that impairment of cellular pH (pHi) regulation due to elimination of Na+/H+ exchange underlies this effect. In the absence of Na+, the phorbol ester-induced respiratory burst was partially inhibited and a concomitant cytoplasmic acidification recorded. Using nigericin/K+ to clamp pHi we demonstrated that the acidification accounts for the inhibition of O2 uptake. Moreover, in Na+-free media, relieving the acidification by means of ionophores restored maximal O2 consumption. It was concluded that Na+ is not directly involved in signal transduction during stimulation. Instead, omission of Na+ affects neutrophils activation indirectly, by impairing pHi regulation.     

Volume regulation in Mycoplasma gallisepticum: evidence that Na+ is extruded via a primary Na+ pump.

The primary extrusion of Na+ from Mycoplasma gallisepticum cells was demonstrated by showing that when Na+-loaded cells were incubated with both glucose (10 mM) and the uncoupler SF6847 (0.4 microM), rapid acidification of the cell interior occurred, resulting in the quenching of acridine orange fluorescence. No acidification was obtained with Na+-depleted cells or with cells loaded with either KCl, RbCl, LiCl, or CsCl. Acidification was inhibited by dicyclohexylcarbodiimide (50 microM) and diethylstilbesterol (50 microM), but not by vanadate (100 microM). By collapsing delta chi with tetraphenylphosphonium (200 microM) or KCl (25 mM), the fluorescence was dequenched. The results are consistent with a delta chi-driven uncoupler-dependent proton gradient generated by an electrogenic ion pump specific for Na+. The ATPase activity of M. gallisepticum membranes was found to be Mg2+ dependent over the entire pH range tested (5.5 to 9.5). Na+ (greater than 10 mM) caused a threefold increase in the ATPase activity at pH 8.5, but had only a small effect at pH 5.5. In an Na+-free medium, the enzyme exhibited a pH optimum of 7.0 to 7.5, with a specific activity of 30 +/- 5 mumol of phosphate released per h per mg of membrane protein. In the presence of Na+, the optimum pH was between 8.5 and 9.0, with a specific activity of 52 +/- 6 mumol. The Na+-stimulated ATPase activity at pH 8.5 was much more stable to prolonged storage than the Na+-independent activity. Further evidence that two distinct ATPases exist was obtained by showing that M. gallisepticum membranes possess a 52-kilodalton (kDa) protein that reacts with antibodies raised against the beta-subunit of Escherichia coli ATPase as well as a 68-kDa protein that reacts with the anti-yeast plasma membrane ATPases antibodies. It is postulated that the Na+ -stimulated ATPases functions as the electrogenic Na+ pump.