Regulation of cell volume and ion concentrations in aHalobacterium

Summary Changes in cell volume and ion content of aHalobacterium species are described in terms of the NaCl concentration (0.5–3.5m) and pH (4–8) of the suspending medium. Cell volume, per unit content of protein of bacteria in stationary phase cultures, rose as the [NaCl] of the growth medium was increased. Logarithmic-phase bacteria shrank as the pH fell from 7 to 5.5. These changes are characteristic of bacteria with a moderate or rapid rate of O₂ consumption. Starving (i.e. nonmetabolizing) bacteria, on the other hand, did not change in size within the above ranges of [NaCl] and pH. At lower values, however, such bacteria swelled and eventually lysed. Effects of low pH on cell ions are compared in metabolizing and starving bacteria, and it is shown that changes in the state of the cell K are correlated with movements of cell Na. It appears that the cell K is used to maintain cell [Na] below the NaCl concentration of the medium. The results are explained in terms of a model involving interactions between polyelectrolytes, salts and water in the concentrated cytoplasm of these halophilic organisms.     

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.     

Bioenergetic properties of alkalophilic Bacillus sp. strain C-59 on an alkaline medium containing K2CO3.

Alkalophilic Bacillus sp. strain C-59 could grow well on an alkaline medium containing K2CO3, as well as Na2CO3, but did not grow on K+-depleted medium. Right-side-out membrane vesicles, energized in the absence of Na+, however, could not take up [14C]methylamine actively, while vesicles equilibrated with 10 mM NaCl actively took up [14C]methylamine. The uptake of [14C]serine was also stimulated by the addition of Na+, and the imposition of a sodium gradient caused transient uptake. These results indicated that an Na+/H+ antiporter was involved in pH homeostasis and generation of an electrochemical sodium gradient in strain C-59 even though a growth requirement for Na+ was not evident. The efflux of 22Na+ from 22Na+-loaded vesicles was more rapid at pH 9.5 than at pH 7 in the presence of an electron donor. On the other hand, vesicles at pH 7 showed more rapid efflux than at pH 9.5 when the antiporter was energized by a valinomycin-mediated K+ diffusion potential (inside negative).     

Further studies of the volume-regulatory response of Amphiuma red cells in hypertonic media. Evidence for amiloride-sensitive Na/H exchange

When Amphiuma red cells are shrunken in hypertonic media, they return toward their original volume by gaining Na through an amiloride-sensitive pathway. As cells recover their volume during this volume-regulatory increase (VRI) response, acid is extruded into the medium. Medium acidification is correlated with cell Na uptake. Both medium acidification and cell Na uptake are blocked by 10(-3) M amiloride or by replacing medium Na with K or choline. Perturbations that increase cell Na uptake (such as increasing medium osmolality) also increase medium acidification. As the medium becomes more acidic, the cells become more alkaline. These changes in cell and medium pH are increased if pH equilibration across the cell membrane is prevented by inhibiting the anion exchanger with SITS (4-acetamido-4'-isothiocyano-2,2'-stilbene disulfonic acid). The quantity of acid extruded by SITS-treated cells is the same as the quantity of Na gained, which strongly suggests 1:1 exchange of Na for H. Cell enlargement in SITS-treated cells results from the exchange of osmotically active Na ions for H ions that are not osmotically active when combined with cellular buffers. Previous evidence indicates that the normal VRI response involves an increase in the cellular content of Cl as well as Na. We show that SITS completely blocks net Cl uptake, which suggests that Cl enters via the anion exchanger. SITS also slows Na entry, presumably as a result of the above-mentioned increase in cell pH caused by SITS. We suggest that the initial event in the VRI response is net Na uptake via a Na/H exchanger, and that net Cl uptake results from secondary Cl/HCO3 exchange via the anion exchanger.     

The Membrane Potential of Nitella translucens

The effects of changing the external concentrations of Na, K,Ca, and Cl on the potentials of the cytoplasm and the vacuolewith respect to the bathing medium of the internodal cells ofNitella translucens have been investigated. The potential differencebetween the vacuole and the cytoplasm is practically unaffectedby the concentration changes. The observed changes of potentialdifference are therefore attributed to the boundary separatingthe cytoplasm from the medium; this boundary is possibly a plasmalemma–cellwall complex. The difference of potential between the cell walland the medium has also been measured and, in the presence ofCa, shown to be markedly sensitive only to the external Ca concentration.The results are divided into two sections: (a) for cells pretreatedin 5 mM NaCl, the subsequent experiments being carried out inCa-free media, and (b) for cells initially immersed in a standardartificial pond water containing the chlorides of Na, K, Ca.With the pretreated cells the external Na/K ratio was variedwith the total NaCl+KCl concentration kept constant at 1.1 mM.The results suggest that over a limited range of concentrationsthe cytoplasm-medium potential difference can be described byan equation similar in form to a Goldman equation but containingonly terms for Na and K, the average value of the permeabilityratio (= P_Na/P_K) being 0.27. In the presence of Ca the effectsof Na and K on the cytoplasm-medium potential difference aregreatly reduced, while the effect of Ca is relatively large.The results cannot be fitted to any form of Goldman equationcontaining terms for the major ions. The possibility of a contributionto the plasmalemma potential from electrogenic pumps is brieflydiscussed. Measurements of the Na and K content of the cytoplasmand the vacuole have been made for the pretreated cells. TheNa concentration in the cytoplasm is 37 mM and in the vacuole73 mM; the K concentration is 93 mM in the cytoplasm and 67mM in the vacuole. The Nernst potentials for both ions are comparedwith the cytoplasm-medium and cytoplasm-vacuole potential differences.This analysis shows that Na is actively transported from thecytoplasm into the medium as well as into the Vacuole; K ispumped into the cytoplasm from the medium but appears to beclose to electrochemical equilibrium across the tonoplast. ThisConfirms previously published work.     

Mechanism of the stimulatory effect of a potassium-rich medium on the phosphorylation of glucose in isolated rat hepatocytes.

We have investigated the mechanism by which the replacement of a Na(+)-rich medium by a K(+)-rich medium causes an increase in the apparent affinity of glucokinase (hexokinase IV or D) for glucose in isolated hepatocytes [Bontemps, F., Hue, L. & Hers, H. G. (1978) Biochem. J. 174, 603-611]. The stimulatory effect of a K(+)-rich medium on the rate of glucose phosphorylation, as assessed by the release of tritium from [2-3H]glucose, was only partially additive with the effect of fructose, suggesting that it was also due to a decrease in the inhibition exerted on glucokinase by its regulatory protein. Measurements of metabolites indicated that the effect of the K(+)-rich medium was neither due to the formation of fructose 1-phosphate, nor to changes in the concentrations of fructose 6-phosphate or Pi, two other effectors of the regulatory protein. Replacement of Na+ by K+ in the medium resulted in a time-dependent and dose-dependent increase in cell volume that paralleled the changes in the rate of detritiation observed at 5 mM glucose. The water and chloride contents, estimated using radiolabelled compounds, were threefold and tenfold higher, respectively, in K+ cells than in Na+ cells, and the intracellular Cl- concentration about threefold higher (94 versus 29 meq/l). The effects of the K(+)-rich medium on cell volume, Cl- concentration and rate of detritiation were greatly reduced by including 80 mM trehalose or sucrose in the medium at the start of the incubation. Addition of trehalose to cells incubated for 45-50 min in the K(+)-rich medium caused an immediate decrease in cell volume whereas the rate of detritiation and the Cl- concentration underwent a transient increase followed by a decrease. Replacement of KCl by KBr, potassium acetate or potassium trichloroacetate in the K(+)-rich medium resulted in different relationships between cell volume and the rate of detritiation, in agreement with the differential effect of these salts on the activity of purified glucokinase assayed in the presence of regulatory protein. From these results we conclude that the increase in the activity of glucokinase induced by a KCl-rich medium is at least partly due to an increase in the concentration of Cl-, which relieves the inhibition exerted by the regulatory protein on purified glucokinase.     

Ionic and osmotic equilibria of human red blood cells treated with nystatin

Human red blood cells have been incubated in the presence of nystatin, which allows Na and K, as well as Cl and pH to equilibrate rapidly when cell volume is set with external impermeant sucrose. The intracellular mean ionic activity coefficients, relative to values in the extracellular solution, for KCl and NaCl are 1.01 +/- 0.02 and 0.99 +/- 0.02 (SD, n = 10), respectively, and are independent of external pH, pH o, and of [sucrose]o. With nystatin the dependence of red cell volume on [sucrose]o deviates from ideal osmotic behavior by as much as a factor of three. A virial equation for the osmotic coefficient, phi, of human hemoglobin, Hb, accounts for the cell volumes, and is the same as that which describes Adair's measurements of phi Hb for Hb isolated from sheep and ox bloods. In the presence of nystatin the slope of the acid-base titration curve of the cells is independent of cell volume, implying that the charge on impermeant cellular solutes is independent of Hb concentration at constant pH. By modifying the Jacobs-stewart equations (1947. J. Cell. Comp. Physiol. 30: 79--103) with the osmotic coefficients of Hb and of salts, a nonideal thermodynamic model has been devised which predicts equilibrium Donnan ratios and red cell volume from the composition of the extracellular solution and from certain parameters of the cells. In addition to accounting for the dependence of cell volume on osmotic pressure, the model also describes accurately the dependence of Donnan ratios and cell volumes on pHo either in the presence or absence of nystatin.     

Factors affecting the lytic susceptibility of some marine and terrestrial bacteria

Eighteen gram-negative marine bacteria and two terrestrial species, Escherichia coli and Pseudomonas aeruginosa, were examined for their sensitivity to lysis in distilled water after exposure to a salt solution containing a sea water concentration of Mg2+ (0.05 M) or to 0.5 M NaCl. A spectrum of lytic susceptibility was observed among the marine bacteria ranging from those organisms which lysed in distilled water after exposure to the Mg2+-containing solution, through organisms which could be sensitized to lysis by washing with the NaCl solution, to organisms which failed to lyse in distilled water even after having been washed with a solution of 0.5 M NaCl. Pseudomonas aeruginosa and E. coli fell within this spectrum, the former being capable of being induced to lyse in distilled water by washing with 0.5 M NaCl, while the latter failed to lyse in distilled water after this treatment. It was thus concluded that no overall distinction could be made between marine and terrestrial bacteria on the basis of the sensitivity of the two groups of organisms to lysis in freshwater. Quite large decreases in optical density and increases in the release of ultraviolet-absorbing material took place when cells preexposed to the Mg2+-containing solution or to 0.5 M NaCl were subsequently suspended in distilled water even though in some cases no loss of cell numbers could be detected. In most cases two to three times as much K+ as Na+ and 1/10 to 1/100 as much Mg2+ was required to prevent these changes. For three of the marine bacteria and P. aeruginosa grown in a terrestrial type medium little difference in the requirements for Na+ and K+ to prevent the optical density changes was noted. For P. aeruginosa grown in a marine type medium, cells required more K+ than Na+ to prevent these changes.     

Imidazole chloride and tris-chloride substitute for sodium chloride in inducing high-affinity AdoPP[NH]P binding to (Na+ + K+)-ATPase

Optimal binding of [2,8-3H]AdoPP[NH]P to (Na+ + K+)-ATPase requires 25 mM Na+ (Cl-), 50 mM imidazole+ (Cl-) or 50 mM Tris+ (Cl-). Chloride is essential as counterion. We conclude that imidazole+ and Tris+ are able to bind to the Na+ site, and recommend the use of dilute buffers for studying the partial reactions of (Na+ + K+)-ATPase. In NaCl or the substituting buffers the dissociation constant for the enzyme-AdoPP[NH]P complex at 0 degrees C and pH 7.25 is 0.4 microM, whereas in millimolar MgCl2 it is about 2 microM. These distinct levels in affinity with MgCl2 as compared to NaCl, together with the MgCl2-dependence of photolabelling of the enzyme with ATP analogues (Rempeters, G. and Schoner, W. (1981) Eur. J. Biochem. 121, 131-137), suggest significant changes within the substrate site of (Na+ + K+)-ATPase upon binding of Mg2+ (Cl-)2.     

Amiloride-sensitive sodium transport in lamprey red blood cells: evidence for two distinct transport pathways

To determine Na+/H+ exchange in lamprey erythrocyte membranes, the cells were acidified to pH(i) 6.0 using the K+/H+ ionophore nigericin. Incubation of acidified erythrocytes in a NaCl medium at pH 8.0 caused a considerable rise in 22Na+ influx and H+ efflux during the first 1 min of exposure. In addition, exposure of acidified red cells to NaCl medium was associated with rapid elevation of intracellular Na+ content. The acid-induced changes in Na+ influx and H+ efflux were almost completely inhibited by amiloride and dimethylamiloride. In native lamprey erythrocytes, amiloride-sensitive Na+ influx progressively increased as the osmolality of incubation medium was increased by addition of 100, 200, or 300 mmol/l sucrose. Unexpectedly, the hypertonic stress induced a small, yet statistically significant decrease in intracellular Na+ content in these cells. The reduction in the cellular Na+ content increased with hypertonicity of the medium. The acid- and shrinkage-induced Na+ influxes were inhibited by both amiloride and 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) in a dose-dependent manner. For both blockers, the half-maximal inhibitory values (IC50) were much greater for the shrinkage-induced (44 and 15 micromol/l for amiloride and EIPA, respectively) than for the acid-induced Na+ influx (5.1 and 3.3 micromol/l, respectively). The data obtained are the first demonstration of the presence of a Na+/H+ exchanger with high activity in acidified (pH(i) 6.0) lamprey red blood cells (on average, 512 +/- 56 mmol/l cells/h, n = 13). The amiloride-sensitive Na+ influxes produced by hypertonic cell shrinkage and acid load are likely to be mediated by distinct ion transporters in these cells.     

Regulation of internal solute concentrations of marine Vibrio alginolyticus in response to external NaCl concentration.

Slightly halophilic marine Vibrio alginolyticus grown in the range of NaCl from 0.2 to 1.5 M maintained the total internal solute concentration always higher than the external medium by about 0.25 osM. The concentrations of macromolecules such as DNA, RNA, and protein were little affected by the increase in medium NaCl. The internal K+ concentration was kept to about 400 mM in the range of medium NaCl from 0.4 to 0.8 M; it rose to 510 mM when the bacterium was grown in 1.5 M NaCl, indicating that K+ increased only slightly in response to the large increase in medium NaCl. Thus, in contrast to the case of nonhalophilic and extremely halophilic bacteria, K+ was unlikely to act as a major component to regulate the internal solute concentration of marine V. alginolyticus. The internal Na+ and Cl- concentrations were maintained always lower than those in the growth medium, but they increased in response to the increase in medium NaCl. The concentration of internal Na+ was close to that of K+ at the concentration of medium NaCl that supports the optimal growth of this organism. The total amino acid content of V. alginolyticus increased from 76 to 413 mM by the increase in medium NaCl from 0.2 to 1.5 M. The concentrations of glutamic acid and prolined were 254 and 72 mM, respectively, when grown in 1.5 M NaCl. These results indicated that Na+, Cl- and amino acids, especially glutamic acid and proline, contributed to the regulation of internal solute concentration of V. alginolyticus in response to the increased external NaCl.     

缺氧时大鼠红细胞变形性损伤的机制研究

本实验通过测定平原和模拟高原减压缺氧３０天大鼠红细胞滤过指数（ＩＦ）、红细胞内［ｐＨ］ｉ、［Ｋ＋］ｉ／［Ｎａ＋］ｉ比值、［Ｃａ２＋］ｉ、［Ｍｇ２＋］ｉ、平均红细胞体积（ＭＣＶ）及平均红细胞血红蛋白浓度（ＭＣＨＣ）,从而探讨缺氧条件下大鼠红细胞变形性损害的机制。结果发现：１．缺氧组大鼠红细胞［Ｃａ２＋］ｉ明显升高,且与ＩＦ呈显著正相关,但［Ｍｇ２＋］ｉ无明显差异;２．缺氧组［Ｋ＋］ｉ／［Ｎａ＋］ｉ值较平原组明显降低,且与ＩＦ呈显著负相关;３．缺氧组ＭＣＨＣ与平原组无明显差异,但ＭＣＶ显著升高;４．缺氧组红细胞内［ｐＨ］ｉ较平原组明显升高。提示：缺氧时红细胞［Ｃａ２＋］ｉ升高,［Ｋ＋］ｉ／［Ｎａ＋］ｉ值降低,ＭＣＶ增大以及红细胞［ｐＨ］ｉ值的改变在其变形性损伤中起重要作用。     

The Response of Duck Erythrocytes to Hypertonic Media : Further evidence for a volume-controlling mechanism

The addition of a hypertonic bathing medium to duck erythrocytes results in an initial instantaneous phase of osmotic shrinkage and, when the [K]_o of the hypertonic solution is larger than "normal," in a second, more prolonged phase, the volume regulatory phase. During the latter, which also requires extracellular Na, the cells swell until they approach their initial isotonic volume. The increase in cell volume during the volume regulatory phase is accomplished by a gain in the cell content of K, Cl, and H₂O. There is also a smaller increase in the Na content of the cell. Potassium is accumulated against an electrochemical gradient and is therefore actively transported into the cell. This accumulation is associated with an increase, although dissimilar, in both K influx and efflux. Changes in cell size during the volume regulatory phase are not altered by 10^-4 M ouabain, although this concentration of ouabain does change the cellular cation content. The response is independent of any effect of norepinephrine. The changes in cell size during the volume regulatory phase are discussed as the product of a volume controlling mechanism identical in principle to the one reported in the previous paper which controls cell volume in hypotonic media. Similarly, this mechanism can regulate cell size, when the Na-K exchange, ouabain-inhibitable pump mechanism is blocked.     

The protective action of betaine on the deleterious effects of NaCl on preimplantation mouse embryos in vitro

The development of outbred mouse (CF1) zygotes in vitro has been studied using medium SOM in which the concentrations of NaCl (85, 105, 125 mM), glutamine (0, 1, 2 mM), and betaine (0, 1, 2 mM) were varied. The effects of the compounds were studied using a 3³ factorial experimental arrangement. The inhibitory effect of relatively high concentrations of NaCl and the protective effect of glutamine were confirmed. Betaine, an organic osmolyte, can also protect against the deleterious effects of relatively high concentrations of NaCl. The intracellular contents of potassium and sodium have also been measured in single zygotes using X-ray electron probe spectrometry. When medium SOM contains 85 mM or 125 mM NaCl, the intracellular content of Na rises and the content of K decreases. These changes are partially reduced in the presence of 125 mM NaCl if betaine is also in the medium. Betaine has no effect on the intracellular content of K and Na if the concentration of NaCl is 85 mM. These results suggest that organic osmolytes may be required in embryo culture media to prevent excessive changes in the intracellular ionic concentration. © 1993 Wiley-Liss, Inc.     

Ionic regulation of the unicellular green algadunaliella tertiolecta

Summary Different techniques were investigated in order to determine the Na, K and Cl concentrations ofDunaliella tertiolecta cells adapted to a large range of salinity (20 to 1640 mM NaCl). The K cell concentrations were 6 to 13 times higher than the K concentration of the external medium (11 mM). The The Na and Cl cell concentrations, on the other hand, were lower than in the external medium at all salinities tested. Considerable differences in the absolute values of Na and Cl were, however, found according to the technique employed. These results are interpreted in terms of compartmentalization of the cells (at least two compartments). It is postulated that the larger compartment regulates its ion concentrations, maintaining low Na and Cl and high K concentrations, whereas the second compartment equilibrates with the external medium. The cation permeability of the membrane limiting the regulating compartment is altered by the antibiotics nystatin and monensin. Incubation of cells in K-free medium leads to a decrease of K and to an increase of the cell Na, this effect being reversed by addition of KCl to the medium. A good correlation is found between gain of K and loss of Na, suggesting a stoichiometric exchange of these two ions. The magnitude of this apparent Na/K exchange increases as the salinity increases. The external K concentration necessary to mediate half-saturation of the Na/K exchange is a function of the NaCl concentration of the adaptation medium. This Na/K exchange is partially light-dependant and inhibited by cold, cyanide and DCCD. It is suggested that this mechanism helps in the regulation of the ionic composition ofDunaliella cells.     

Acidosis-Induced Enhanced Activity of the Na-K Exchange Pump in the In Vivo Choroid Plexus: An Ontogenetic Analysis of Possible Role in Cerebrospinal Fluid pH Homeostasis

Abstract: Changes in cellular [K] and [Na] in the choroidal epithelium (as a reflection of Na-K pump activity) were analyzed in Sprague-Dawley rats subjected to acute systemic acidosis. In the lateral and 4th ventricle choroid plexus (CP) of adult rats in which metabolic acidosis was induced for 1 h, cell [K] and [Na] increased and decreased by 35 and 15 m m /kg water, respectively, indicating marked stimulation of the Na-K exchange pump in the CSF-facing membrane; in contrast, this striking response of the CP to acidosis could not be elicited in immature animals (1 week old). Since the effects of respiratory acidosis on CP cell [K] and [Na] were similar to those of metabolic acidosis, the reduction in plasma pH (rather than in [HCO₃]) is likely the mechanism underlying the enhanced turnover of Na and K across the CP in adults. The concentration of Na and K in the cerebral cortex, medulla, and CSF was generally not altered during acute acid-base distortions in both mature and immature animals. The striking difference in the response of CNS tissue protected by the blood-CSF barrier (i.e., CP) and the blood-brain barrier (BBB) to systemic acidosis emphasizes a unique role, presumably homeostatic, for the plexus. Since propranolol substantially attenuated the acidosis-induced changes in choroidal cell [K] and [Na], it is possible that there is β-receptor modulation of the Na,K-ATPase (Na-K pump) in the CP. We postulate that the generally observed enhanced electropositivity in the CSF in systemic acidosis is brought about, at least in part, by facilitation of Na-K pumping in the CP, although induced changes in membrane permeability may also be a factor.     

Expression of the Na+/H+ antiporter gene (g1-nhaC) of alkaliphilic Bacillus sp. G1 in Escherichia coli

A Na(+)/H(+) antiporter gene was isolated from alkaliphilic Bacillus sp. G1. The full-length sequence of the Na(+)/H(+) antiporter gene was obtained using a genome walking method, and designated as g1-nhaC. An ORF preceded by a promoter-like sequence and a Shine-Dalgarno sequence, and followed by a terminator-like sequence was identified. The deduced amino acid sequence consists of 535 amino acids, and a calculated molecular mass of 57 776 Da. g1-nhaC was subsequently cloned into pET22b(+) and expressed in Escherichia coli BL21 (DE3). Recombinant E. coli harboring the g1-nhaC gene was able to grow in modified L medium at various concentrations of NaCl (0.2-2.0 M) at different pH values. The recombinant bacteria grew well in the medium with concentrations of NaCl as high as 1.75 M at pH 8.0-9.0. Minimal growth was observed at 2.0 M NaCl, pH 8.0-9.0. At pH 10, the recombinant bacteria grew well in a medium with a low concentration of NaCl (0.2 M). These results suggested that the g1-NhaC antiporter from Bacillus sp. G1 plays a role in Na(+) extrusion at lower pH values and in pH homeostasis at pH 10 under Na(+)-limiting conditions.     

Effect of volume changes on ouabain-insensitive net outward cation movements in human red cells

Summary The effect of cell volume changes in human red cells on ouabain-insensitive net outward cation movements through 1) the Na–K and Li–K cotransport, 2) the Li–Na counter-transport system and 3) the furosemide-insensitive Na, K and Li pathway was studied. Cell volume was altered by changing a) the internal cation content (isosmotic method) or b) the external osmolarity of the medium (osmotic method). Na–K and Li–K cotransport were measured as the furosemide-sensitive Na or Li and K efflux into (Na, Li and K)-free (Mg-sucrose replacement) medium from cells loaded to contain approximately equal concentrations of Na and K, or a constant K/Li concentration ratio of 91, respectively. Li–Na countertransport was assayed as the Na-stimulated Li efflux from Li-loaded cells and net furosemide-insensitive outfluxes in (Na, Li and K)-free media containing 1mm furosemide. Swelling of cells by the isosmotic, but not by the osmotic method reduced furosemide-sensitive Na and Li but not K efflux by 80 and 86%, respectively. Changes in cell volume by both methods had no effect on Li–Na countertransport. The effects of cell volume changes were measured on the rate constants of ouabain- and furosemide-insensitive cation fluxes and were found to be complex. Isosmotic shrinkage more than doubled the rate constants of Na and Li efflux but did not affect that of K efflux. Osmotic shrinkage increased the K efflux rate constant by 50% only in cells loaded for countertransport. Isosmotic cell swelling specifically increased the K⁺ efflux rate constants both in cells loaded for cotransport and countertransport assays while no effect was observed in cells swollen by the osmotic method. Thus, the three transport pathways responded differently to changes in cell volume, and, furthermore, responses were different depending on the method of changing cell water content.     

Effect of extreme salt concentrations on the physiology and biochemistry of Halobacteroides acetoethylicus.

Halobacteroides acetoethylicus grew in media with 6 to 20% NaCl and displayed optimal growth at 10% NaCl. When grown in medium with an [NaCl] of 1.7 M, the internal cytoplasmic [Na+] and [Cl-] were 0.92 and 1.2 M, respectively, while K+ and Mg2+ concentrations in cells were 0.24 and 0.02 M, respectively. Intracellular [Na+] was fourfold higher than intracellular [K+]. Since Na+ and Cl- ions were not excluded from the cell, the influence of high salt concentrations on key enzyme activities was investigated in crude cell extracts. Activities greater than 60% of the maximal activity of the following key catabolic enzymes occurred at the following [NaCl] ranges: glyceraldehyde-3-phosphate dehydrogenase, 1 to 2 M; alcohol dehydrogenase (NAD linked), 2 to 4 M; pyruvate dehydrogenase, 0.5 to 1 M; and hydrogenase (methyl viologen linked), 0.5 to 3 M. These studies support the hypothesis that obligately halophilic, anaerobic eubacteria adapt to extreme salt concentrations differently than do halophilic, aerobic eubacteria, because they do not produce osmoregulants or exclude Cl-. This study also demonstrated that these halophilic, anaerobic eubacteria have a physiological similarity to archaebacterial halophiles, since Na+ and Cl- are present in high concentrations and are required for enzymatic activity.     

Influence of the growth at high osmolality on the lipid composition, water permeability and osmotic response of Lactobacillus bulgaricus

Changes in water permeability and membrane packing were measured in cells of Lactobacillus bulgaricus and in vesicles prepared with lipids extracted from them. The osmotic response of whole cells and vesicles is compared with the one of bacteria grown in a high osmolal medium. Both bacteria and vesicles, behave as osmometers. This means that the volume decrease is promoted by the outflow of water, driven by the NaCl concentration difference, arguing that neither Na+ nor Cl- permeates the cell or the lipid membrane in these conditions. Therefore, the volume changes can be correlated with the rate of water permeation across the cell or the vesicle membranes. The permeation of water was analyzed as a function of the lipid species by measuring the volume changes and the saturation ratio of the lipids. To put into relevance the membrane processes, the permeation properties of lipid vesicles prepared with lipids extracted from bacteria grown in normal and high osmolality conditions were also analyzed. The permeation response was correlated with the physical properties of the membrane of whole cells and vesicles, by means of fluorescence anisotropy of diphenyl hexatriene (DPH). The modifications in membrane properties are related with the changes in the membrane composition triggered by the growth in a high osmolal medium. The changes appear related to an increase in the sugar content of the whole pool of lipids and in the saturated fatty acid residues.