Constant Internal pH Research Articles

The relationship between intracellular pH, growth characteristics and calcium in the cyanobacterium Anabaena sp. strain PCC7120 exposed to low pH

summaryChanges in various cell functions were examined during a shift of the cyanobacterium Anabaena sp. strain PCC7120 to acidic external pH (pHex) in the presence and absence of added calcium. In the presence of 0–25 mM Ca (the standard Ca concentration of the growth medium), growth and O2 evolution were inhibited at pH values lower than 6. The cyanobacterium was unable to maintain a relatively constant internal pH (pHi) at pH 6 and below, which led to acidification of the cytoplasm. In the absence of Ca, the acidification found at acid pH values was even more pronounced. The elimination of Ca did not affect pHi at pHex of 6–5 and above. Increased acidification of the internal cell contents correlated well with a general impairment of growth in Ca‐deficient cells exposed to external acid pH values. On the other hand, Ca enrichment of cells grown under acidic conditions resulted in a significant improvement of several physiological processes. The protein pattern of cell extracts of Anabaena sp. strain PCC7120 was altered by acidity. The most significant modifications of the protein profiles induced at low pH were not evident in the presence of high concentrations of Ca. Increasing concentrations of Ca allowed Anabaena sp. strain PCC7120 to perform better at lower pH.

Mechanism of H(+)-coupled formate transport in rabbit renal microvillus membranes.

It has been proposed that a major fraction of Cl- absorption in the mammalian proximal tubule occurs by Cl-/formate exchange across the apical membrane with recycling of formate by nonionic diffusion. The purpose of this study was to characterize the mechanism of formate recycling in rabbit renal microvillus membrane vesicles. Formate uptake was stimulated by an inside-alkaline pH gradient. When external pH (pH alpha) was varied at constant internal pH (pHi), the initial rate of formate uptake was less than predicted for nonionic diffusion of formic acid at constant formic acid permeability. When pHi was varied at constant pHi, the initial rate of formate uptake exhibited cooperative and saturable kinetics with respect to pHi, in contrast to the pHi independence predicted for nonionic diffusion. pH gradient-stimulated [14C]formate uptake was stimulated by internal formate, indicating formate/formate exchange. pH gradient-stimulated formate influx was sensitive to inhibition by 1 mM 4,4'-diisothiocyanostilbene-2, 2'-disulfonic acid but not by furosemide or hydroxycinnamate. We conclude that pH gradient-stimulated formate uptake takes place by a carrier-mediated process of H(+)-formate cotransport, OH-/formate exchange, or facilitated formic acid diffusion, rather than solely by passive nonionic diffusion through the lipid bilayer.

PH sensitivity of the Na+:HCO3- cotransporter in basolateral membrane vesicles isolated from rabbit kidney cortex.

HCO3- exit across the basolateral membrane of the kidney proximal tubule cell is mediated via an electrogenic Na+:HCO3- cotransporter. We have studied the effect of pH on the activity of this cotransport system in basolateral membrane vesicles isolated from rabbit renal cortex. At constant internal pH 6.0, increasing the external pH and [HCO3-] increased the rate of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive 22Na+ influx into the vesicles. To determine the role of internal pH on the activity of the Na+:HCO3- cotransport system, the influx of 22Na+ via HCO3-dependent Na(+)-Na+ exchange was measured in the absence of an initial pH and [HCO3-] gradient (pH(i) = pH(o), 5% CO2). Increasing the pH from 6.8 to 7.2 increased whereas, increasing the pH from 7.4 to 8.0 decreased the rate of 22Na+ influx via this exchange. Increasing pH at constant [HCO3-] (pH(i) = pH(o) = 8.0, 1.5% CO2 versus pH(i) = pH(o) = 7.2, 10% CO2) reduced the influx of 22Na+ via HCO3-dependent Na(+)-Na+ exchange. Increasing pH at constant [CO3(2-)](pH(i) = pH(o) = 8.0, 1.5% CO2 versus pH(i) = pH(o) = 7.2, 60% CO2) was associated with reduced 22Na+ uptake. Decreasing the pH (pH(i) = pH(o) = 6.3, 60% CO2 versus pH(i) = pH(o) = 7.2, 5% CO2) was associated with a reduced rate of HCO3(-)-dependent Na(+)-Na+ exchange. We conclude that the Na+:HCO3- cotransporter displays a significant pH sensitivity profile with the cotransporter being more functional at pH 7.0-7.4 and less active at more acid or alkaline pH. In addition, the results suggest that the pH sensitivity arises at the inner surface of the basolateral membrane.

The bioenergetics of ammonia and hydroxylamine oxidation in Nitrosomonas europaea at acid and alkaline pH

Autotrophic ammonia oxidizers depend on alkaline or neutral conditions for optimal activity. Below pH 7 growth and metabolic activity decrease dramatically. Actively oxidizing cells of Nitrosomonas europaea do not maintain a constant internal pH when the external pH is varied from 5 to 8. Studies of the kinetics and pH-dependency of ammonia and hydroxylamine oxidation by N. europaea revealed that hydroxylamine oxidation is moderately pH-sensitive, while ammonia oxidation decreases strongly with decreasing pH. Oxidation of these oxogenous substrates results in the generation of higher proton motive force which is mainly composed of a ΔΨ. Hydroxylamine, but not ammonia, is oxidized at pH 5, which leads to the generation of a high proton motive force which drives energy-dependent processes such as ATP-synthesis and secondary transport of amino acids. Endogenoussubstrates can be oxidized between pH 5 to 8 and this results in the generation of a considerable proton motive force which is mainly composed of a ΔΨ. Inhibition of ammonia-mono-oxygenase or cytochrome aa3 does not influence the magnitude of this gradient or the oxygen consumption rate, indicating that endogenous respiration and ammonia oxidation are two distinct systems for energytransduction. The results indicate that the first step in ammonia oxidation is acid sensitive while the subsequent steps can take place and generate a proton motive force at acid pH.

Activation of yeast plasma membrane ATPase by acid pH during growth

When yeast grows on media in which a final external pH lower than 4 is attained there is a 2–3-fold increase in plasma membrane ATPase activity. This acid-mediated activation produces a 2-fold increase in the ATPase affinity for ATP but does not modify its optimum pH. The acid-mediated activation is dependent on the stage of growth, only late logarithmic or stationary cells being capable of being activated by incubation in an acidic buffer. Only cells able to activate the ATPase maintain a constant internal pH when incubated in acidic buffers. It is concluded that acid-mediated activation of the plasma membrane ATPase is a mechanism of maintaining constant internal pH during growth of yeast on acid media.

Active transport in phototrophic bacteria

Phototrophic bacteria utilize light-driven, cyclic electron flow to pump protons out of their cytoplasm, creating an electrochemical proton gradient, ΔμH+, outside acid and positive. These bacteria exchange external protons for internal cations (Na(+), K(+) and Ca(+2)), allowing the cells to maintain a nearly constant internal pH while maintaining the electrical component of ΔμH+. Na(+)/H(+) exchange also establishes an electrochemical Na(+) gradient. Phototrophic bacteria are able to utilize these electrochemical gradients as energy sources for the uptake of a wide variety of metabolites (e.g., sugars, organic acids and amino acids) via metabolite/cation symports.

Effect of External pH on the Internal pH of Chlorella saccharophila

The overall internal pH of the acid-tolerant green alga, Chlorella saccharophila, was determined in the light and in the dark by the distribution of 5,5-dimethyl-2-[(14)C]oxazolidine-2,4-dione ([(14)C]DMO) or [(14)C]benzoic acid ([(14)C]BA) between the cells and the surrounding medium. [(14)C]DMO was used at external pH of 5.0 to 7.5 while [(14)C]BA was used in the range pH 3.0 to pH 5.5. Neither compound was metabolized by the algal cells and intracellular binding was minimal. The internal pH of the algae obtained with the two compounds at external pH values of 5.0 and 5.5 were in good agreement. The internal pH of C. saccharophila remained relatively constant at pH 7.3 over the external pH range of pH 5.0 to 7.5. Below pH 5.0, however, there was a gradual decrease in the internal pH to 6.4 at an external pH of 3.0. The maintenance of a constant internal pH requires energy and the downward drift of internal pH with a drop in external pH may be a mechanism to conserve energy and allow growth at acid pH.

Characterization of the Na+/H+ antiporter of alkalophilic bacilli in vivo: delta psi-dependent 22Na+ efflux from whole cells.

The Na+/H+ antiporter of Bacillus alcalophilus was studied by measuring 22Na+ efflux from starved, cyanide-inhibited cells which were energized by means of a valinomycin-induced potassium diffusion potential, positive out (delta psi). In the absence of a delta psi, 22Na+ efflux at pH 9.0 was slow and appreciably inhibited by N-ethylmaleimide. Upon imposition of a delta psi, a very rapid rate of 22Na+ efflux occurred. This rapid rate of 22Na+ efflux was competitively inhibited by Li+ and varied directly with the magnitude of the delta psi. Kinetic experiments with B. alcalophilus and alkalophilic Bacillus firmus RAB indicated that the delta psi caused a pronounced increase in the Vmax for 22Na+ efflux. The Km values for Na+ were unaffected by the delta psi. Upon imposition of a delta psi at pH 7.0, a retardation of the slow 22Na+ efflux rate at pH 7.0 was caused by the delta psi. This showed that inactivity of the Na+/H+ antiporter at pH 7.0 was not secondary to a low delta psi generated by respiration at this pH. Indeed, 22Na+ efflux activity appeared to be inhibited by a relatively high internal proton concentration. By contrast, at a constant internal pH, there was little variation in the activity at external pH values from 7.0 to 9.0; at an external pH of 10.0, the rate of 22Na+ efflux declined. This decline at typical pH values for growth may be due to an insufficiency of protons when a diffusion potential rather than respiration is the driving force. Non-alkalophilic mutant strains of B. alcalophilus and B. firmus RAB exhibited a slow rate of 22Na+ efflux which was not enhanced by a delta psi at either pH 7.0 or 9.0.

Regulation of intracellular pH by human peripheral blood lymphocytes as measured by 19F NMR.

We have measured the intracellular pH of human peripheral blood lymphocytes by means of high-resolution 19F NMR spectroscopy using D,L-2-amino-3,3-difluoro-2-methylpropanoic acid (F2MeAla) as a probe. Lymphocytes readily took up the methyl ester of F2MeAla, and endogenous esterase hydrolyzed the ester to the free amino acid inside the cell. This alpha-methyl amino acid is not metabolized by the cell, and its 19F NMR spectrum exhibits large pH-dependent shifts as the alpha-amino group is protonated. The size of the 19F shifts, the high sensitivity of 19F NMR, and the favorable pKa of the alpha-amino group of F2MeAla (pKa = 7.3) allowed us to measure intracellular pH of lymphocytes at 25-30 degrees C with approximately 5-min acquisition times. Measurements at various external pH values demonstrated that human peripheral blood lymphocytes regulate their internal pH, a process requiring expenditure of metabolic energy. In the pH range between 6.8 and 7.4, lymphocytes maintain a constant internal pH of 7.17 +/- 0.06 pH unit. Outside this range, intracellular pH changes with extracellular pH. The accuracy of this 19F pH probe has been confirmed by independent measurements of intracellular pH using equilibrium distributions of 5,5-dimethyloxazolidine-2,4-dione.

Effect of membrane potential and internal pH on active sodium-potassium transport and on ATP content in high-potassium sheep erythrocytes

Ouabain-sensitive Na + and K + fluxes and ATP content were determined in high potassium sheep erythrocytes at different values of membrane potential and internal pH. Membrane potential was adjusted by suspending erythrocytes in media containing different concentrations of MgCl 2 and sucrose. Concomitantly either the external pH was changed sufficiently to maintain a constant internal pH or the external pH was kept constant with a resultant change of internal pH. The erythrocytes were preincubated before the flux experiment started in a medium which produced increased ATP content in order to avoid substrate limitation of the pump. p] It was found that an increased cellular pH reduced the rates of active transport of Na + and K + without significantly altering the ratio of pumped Na + K + . This reduction was not due to limitation in the supply of ATP although ATP content decreased when internal pH increased. Changes of membrane potential in the range between −10 and +60 mV at constant internal pH did not affect the rates of active transport of Na + or K +.

An anion-exchange method for the determination of the ionic charge and the dissociation constant of acid species

An anion-exchange method for determining the average anionic charge of acid species which involve a dissociation equilibrium in solution and for determining their dissociation constants is proposed. This method is based on measurements of the distribution ratio of the acid at a constant internal pH of the anion-exchanger phase. An experiment with ethylenediamine-tetraacetic acid the dissociation behaviour of which is well known indicated that the present method is reliable in studying the solution species. This method was applied to fairly strong acids which can be studied only with extreme difficulty by ordinary potentiometric pH titrations. The first dissociation constants of some oxoacids of phosphorus were successfully determined at 21 ± 1°C: p k 1 = 1.0 ( I = 0.075) for hypophosphoric acid: p k 1 = 0.6 ( I = 0.16), 0.9 (0.05) and 1.7 (0.015) for pyrophosphoric acid, respectively.