Comparative analysis of trehalose production by Debaryomyces hansenii and Saccharomyces cerevisiae under saline stress

The comparative analysis of growth, intracellular content of Na⁺ and K⁺, and the production of trehalose in the halophilic Debaryomyces hansenii and Saccharomyces cerevisiae were determined under saline stress. The yeast species were studied based on their ability to grow in the absence or presence of 0.6 or 1.0 M NaCl and KCl. D. hansenii strains grew better and accumulated more Na⁺ than S. cerevisiae under saline stress (0.6 and 1.0 M of NaCl), compared to S. cerevisiae strains under similar conditions. By two methods, we found that D. hansenii showed a higher production of trehalose, compared to S. cerevisiae; S. cerevisiae active dry yeast contained more trehalose than a regular commercial strain (S. cerevisiae La Azteca) under all conditions, except when the cells were grown in the presence of 1.0 M NaCl. In our experiments, it was found that D. hansenii accumulates more glycerol than trehalose under saline stress (2.0 and 3.0 M salts). However, under moderate NaCl stress, the cells accumulated more trehalose than glycerol. We suggest that the elevated production of trehalose in D. hansenii plays a role as reserve carbohydrate, as reported for other microorganisms.     

Extracellular pH determines the rate of Ca2+ entry into Madin-Darby canine kidney-focus cells

We investigated the relationship between intracellular Ca²⁺ and pH homeostasis in Madin-Darby canine kidney-focus (MDCK-F) cells, a cell line exhibiting spontaneous oscillations of intracellular Ca²⁺ concentration (Ca _i ²⁺ ). Ca _i ²⁺ and intracellular pH (pH _i ) were measured with the fluorescent dyes Fura-2 and BCECF by means of video imaging techniques. Ca²⁺ influx from the extracellular space into the cell was determined with the Mn²⁺ quenching technique. Cells were superfused with HEPES-buffered solutions. Under control conditions (pH 7.2), spontaneous Ca _i ²⁺ oscillations were observed in virtually all cells investigated. Successive alkalinization and acidification of the cytoplasm induced by an ammonia ion prepulse had no apparent effect on Ca _i ²⁺ oscillations. On the contrary, changes of extracellular pH value strongly affected Ca _i ²⁺ oscillations. Extracellular alkalinization to pH 7.6 completely suppressed oscillations, whereas extracellular acidification to pH 6.8 decreased their frequency by 40%. Under the same conditions, the respective pH _i changes were less than 0. 1 pH units. However, experiments with the Mn²⁺ quenching technique revealed that extracellular alkalinization significantly reduced Ca²⁺ entry from the extracellular space. Large increases of Ca _i ²⁺ triggered by the blocker of the cytoplasmic Ca²⁺-ATPase, thapsigargin, had no effect on pH _i We conclude: intracellular Ca²⁺ homeostasis in MDCK-F cells is pH dependent. pH controls Ca²⁺ homeostasis mainly by effects on the level of Ca²⁺ entry across the plasma membrane. On the contrary, the intracellular pH value seems to be insensitive to rapid changes of Ca _i ²⁺ .The project was supported by the Deutsche Forschungsgemeinschaft, SFB-176 (A6) and by the Jubilämusstiftung of the University of Würzburg.The authors gratefully acknowledge the valuable discussions with Drs. M.J. Berridge, M. Carew, I. Davidson, G. Law and B. Somasundraman. We are grateful to Applied Imaging for financial and technical support and to the Medical Research Council for financial support.     

Responses of Listeria monocytogenes to Acid Stress and Glucose Availability Revealed by a Novel Combination of Fluorescence Microscopy and Microelectrode Ion-Selective Techniques

Fluorescence ratio imaging microscopy and microelectrode ion flux estimation techniques were combined to study mechanisms of pH homeostasis in Listeria monocytogenes subjected to acid stress at different levels of glucose availability. This novel combination provided a unique opportunity to measure changes in H⁺ at either side of the bacterial membrane in real time and therefore to evaluate the rate of H⁺ flux across the bacterial plasma membrane and its contribution to bacterial pH homeostasis. Responses were assessed at external pHs (pH_o) between 3.0 and 6.0 for three levels of glucose (0, 1, and 10 mM) in the medium. Both the intracellular pH (pH_i) and net H⁺ fluxes were affected by the glucose concentration in the medium, with the highest absolute values corresponding to the highest glucose concentration. In the presence of glucose, the pH_i remained above 7.0 within a pH_o range of 4 to 6 and decreased below pH_o 4. Above pH_o 4, H⁺ extrusion increased correspondingly, with the maximum value at pH_o 5.5, and below pH_o 4, a net H⁺ influx was observed. Without glucose in the medium, the pH_i decreased, and a net H⁺ influx was observed below pH_o 5.5. A high correlation (R = 0.75 to 0.92) between the pH_i and net H⁺ flux changes is reported, indicating that the two processes are complementary. The results obtained support other reports indicating that membrane transport processes are the main contributors to the process of pH_i homeostasis in L. monocytogenes subjected to acid stress.     

Lowering extracellular sodium or pH raises intracellular calcium in gastric cells

Summary The dependence of cytoplasmic free [Ca] (Ca _i ) on [Na] and pH was assessed in individual parietal cells of intact rabbit gastric glands by microfluorimetry of fura-2. Lowering extracellular [Na] (Na _o ) to 20mm or below caused a biphasic Ca _i increase which consisted of both release of intracellular Ca stores and Ca entry across the plasma membrane. The Ca increase was not blocked by antagonists of Ca-mobilizing receptors (atropine or cimetidine) and was independent of the replacement cation. Experiments in Ca-free media and in Na-depleted cells indicated that neither phase was due to reversal of Na/Ca exchange. The steep dependence of the Ca _i increase on Na _o suggested that the response was not due to lowering intracellular [Na] (Na _i ). The effects of low Na _o on Ca _i were also completely independent of changes in intracellular pH (pH _i ). Ca _i was remarkably stable during changes of pH _i of up to 2 pH units, indicating that H and Ca do not share a cytoplasmic buffer system. Such large pH excursions required determination of the pH dependence of fura-2. Because fura-2 was found to decrease its affinity for Ca as pH decreased below 6.7, corrections were applied to experiments in which large pH _i changes were observed. In contrast to the relative insensitivity of Ca _i to changes in pH _i , decreasing extracellular pH (pH _o ) to 6.0 or below was found to stimulate release of intracellular Ca stores. Increased Ca entry was not observed in this case. The ability of decreases in Na _o and pH _o to stimulate release of intracellular Ca stores suggest interactions between Na and H with extracellular receptors.     

pH Homeostasis and Citric Acid Utilization: Differences Between Leuconostoc mesenteroides and Lactococcus lactis

This study presents the effects of citric acid and extracellular pH (pH_e) on the intracellular pH (pH_i) of wild-type and citrate negative variants (cit⁻) Leuconostoc mesenteroides subsp. mesenteroides (Ln. mesenteroides M) and Lactococcus lactis subsp. lactis bv. diacetylactis (L. lactis LD). A recent method using a pH-sensitive fluorescent indicator carboxyfluorescein succinimidyl ester (cFSE) was adapted to measure the pH_i of these two lactic acid bacteria in resting cells. Energized cells with 10 mM lactose of Ln. mesenteroides M and L. lactis LD modified their pH gradient (ΔpH) in the same manner; when the pH_e was decreased from 7 to 4, the pH_i decreased from 7 to about 5. The adjunction of 10 mM citric acid had no effect on the pH_i of wild-type and cit(−) variant of L. lactis LD, nor on the pH_i of Ln. mesenteroides cit(−) variant. Nevertheless, in Ln. mesenteroides M wild-type, citric acid utilization increased the pH_i, which was maintained at about 6.5–7.0 when the pH_e was decreased from 7 to 4. It could be concluded that citric acid allows the maintenance of pH homeostasis in Leuconostoc mesenteroides. Received: 7 March 1997 / Accepted: 14 April 1997     

Dynamic Changes of Intracellular pH in Individual Lactic Acid Bacterium Cells in Response to a Rapid Drop in Extracellular pH

We describe the dynamics of changes in the intracellular pH (pH_i) values of a number of lactic acid bacteria in response to a rapid drop in the extracellular pH (pH_ex). Strains of Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, and Lactococcus lactis were investigated. Listeria innocua, a gram-positive, non-lactic acid bacterium, was included for comparison. The method which we used was based on fluorescence ratio imaging of single cells, and it was therefore possible to describe variations in pH_i within a population. The bacteria were immobilized on a membrane filter, placed in a closed perfusion chamber, and analyzed during a rapid decrease in the pH_ex from 7.0 to 5.0. Under these conditions, the pH_i of L. innocua remained neutral (between 7 and 8). In contrast, the pH_i values of all of the strains of lactic acid bacteria investigated decreased to approximately 5.5 as the pH_ex was decreased. No pronounced differences were observed between cells of the same strain harvested from the exponential and stationary phases. Small differences between species were observed with regard to the initial pH_i at pH_ex 7.0, while different kinetics of pH_i regulation were observed in different species and also in different strains of S. thermophilus.     

The acid tolerance response of Bacillus cereus ATCC14579 is dependent on culture pH, growth rate and intracellular pH

The food pathogen Bacillus cereus is likely to encounter acidic environments (i) in food when organic acids are added for preservation purposes, and (ii) during the stomachal transit of aliments. In order to characterise the acid stress response of B. cereus ATCC14579, cells were grown in chemostat at different pH values (pH_o from 9.0 to 5.5) and different growth rates (μ from 0.1 to 0.8 h⁻¹), and were submitted to acid shock at pH 4.0. Cells grown at low pH_o were adapted to acid media and induced a significant acid tolerance response (ATR). The ATR induced was modulated by both pH_o and μ, and the μ effect was more marked at pH_o 5.5. Intracellular pH (pH_i) was affected by both pH_o and μ. At a pH_o above 6, the pH_i decreased with the decrease of pH_o and the increase of μ. At pH_o 5.5, pH_i was higher compared to pH_o 6.0, suggesting that mechanisms of pH_i homeostasis were induced. The acid survival of B. cereus required protein neo-synthesis and the capacity of cells to maintain their pH_i and ΔpH (pH_i - pH_o). Haemolysin BL and non-haemolytic enterotoxin production were both influenced by pH_o and μ.     

An increase in the intracellular pH of fertilized eggs of Xenopus laevis is associated with inhibition of protein and DNA syntheses and followed by an arrest of embryonic development

In many systems, events participating in cell division are controlled by intracellular pH (pH_i). In Xenopus eggs, fertilization is accompanied by an increase in pH_i which occurs concomitantly with an increase in protein synthesis and a reinitiation of DNA synthesis, leading the embryo to cell division. In this paper, we have shown that increasing pH_i of fertilized eggs from 7.8 to 8.2 by using weak bases produced an arrest in embryonic development. Such a change in pH_i was accompanied by a severe inhibition of both protein and DNA syntheses. In order to discriminate between a direct effect of pH_i and a pH-independent effect of weak bases on these biosyntheses, the situation was studied in vitro. For this purpose, cytoplasmic extracts were used in which weak base addition did not produce any change in pH. Under these conditions, protein synthesis was not inhibited, suggesting that pH is probably one of the events implicated in the regulation of protein synthesis. On the other hand, DNA synthesis was inhibited by weak bases in vitro, without any change in pH intervening.     

Preferential intracellular pH regulation represents a general pattern of pH homeostasis during acid–base disturbances in the armoured catfish,Pterygoplichthys pardalis

Preferential intracellular pH (pH_i) regulation, where pH_i is tightly regulated in the face of a blood acidosis, has been observed in a few species of fish, but only during elevated blood PCO₂. To determine whether preferential pH_i regulation may represent a general pattern for acid–base regulation during other pH disturbances we challenged the armoured catfish, Pterygoplichthys pardalis, with anoxia and exhaustive exercise, to induce a metabolic acidosis, and bicarbonate injections to induce a metabolic alkalosis. Fish were terminally sampled 2–3 h following the respective treatments and extracellular blood pH, pH_i of red blood cells (RBC), brain, heart, liver and white muscle, and plasma lactate and total CO₂ were measured. All treatments resulted in significant changes in extracellular pH and RBC pH_i that likely cover a large portion of the pH tolerance limits of this species (pH 7.15–7.86). In all tissues other than RBC, pH_i remained tightly regulated and did not differ significantly from control values, with the exception of a decrease in white muscle pH_i after anoxia and an increase in liver pH_i following a metabolic alkalosis. Thus preferential pH_i regulation appears to be a general pattern for acid–base homeostasis in the armoured catfish and may be a common response in Amazonian fishes.     

Chaperone Stress 70 Protein (STCH) Binds and Regulates Two Acid/Base Transporters NBCe1-B and NHE1

Regulation of intracellular pH is critical for the maintenance of cell homeostasis in response to stress. We used yeast two-hybrid screening to identify novel interacting partners of the pH-regulating transporter NBCe1-B. We identified Hsp70-like stress 70 protein chaperone (STCH) as interacting with NBCe1-B at the N-terminal (amino acids 96–440) region. Co-injection of STCH and NBCe1-B cRNA into Xenopus oocytes significantly increased surface expression of NBCe1-B and enhanced bicarbonate conductance compared with NBCe1-B cRNA alone. STCH siRNA decreased the rate of Na⁺-dependent pH_i recovery from NH₄⁺ pulse-induced acidification in an HSG (human submandibular gland ductal) cell line. We observed that in addition to NBCe1-B, Na⁺/H⁺ exchanger (NHE)-dependent pH_i recovery was also impaired by STCH siRNA and further confirmed the interaction of STCH with NHE1 but not plasma membrane Ca²⁺ ATPase. Both NBCe1-B and NHE1 interactions were dependent on a specific 45-amino acid region of STCH. In conclusion, we identify a novel role of STCH in the regulation of pH_i through site-specific interactions with NBCe1-B and NHE1 and subsequent modulation of membrane transporter expression. We propose STCH may play a role in pH_i regulation at times of cellular stress by enhancing the recovery from intracellular acidification.     

Interactions of intracellular pH and intracellular calcium in primary cultures of rabbit corneal epithelial cells

Summary Homeostasis of intracellular calcium ([Ca⁺⁺]_i) and pH (pH_i) is important in the cell's ability to respond to growth factors, to initiate differentiation and proliferation, and to maintain normal metabolic pathways. Because of the importance of these ions to cellular functions, we investigated the effects of changes of [Ca⁺⁺]_i and pH_i on each other in primary cultures of rabbit corneal epithelial cells. Digitized fluorescence imaging was used to measure [Ca⁺⁺]_i with fura-2 and pH_i with 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Resting pH_i in these cells was 7.37±0.05 (n=20 cells) and resting [Ca⁺⁺]_i was 129±10 nM (n=35 cells) using a nominally bicarbonate-free Krebs Ringer HEPES buffer (KRHB), pH 7.4. On exposure to 20 mM NH₄Cl, which rapidly alkalinized cells by 0.45 pH units, an increase in [Ca⁺⁺]_i to 215±14 nM occurred. Pretreatment of the cells with 100 μM verapamil or exposure to 1 mM ethylene bis-(oxyethylenenitrilo)-tetraacetic acid (EGTA) without extracellular calcium before addition of 20 mM NH₄Cl did not abolish the calcium increase, suggesting that the source of the calcium transient was from intracellular calcium stores. On removal of NH₄Cl or addition of 20 mM sodium lactate, there were minimal changes in calcium even though pH_i decreased. Treatment of CE cells with the calcium ionophores, ionomycin and 4-bromo A23187, increased [Ca⁺⁺]_i, but produced a biphasic change in pH_i. Initially, there was an acidification of the cytosol, and then an alkalinization of 0.10 to 0.11 pH units above initial values. When [Ca⁺⁺]_i was decreased by treating the cells with 5 mM EGTA and 20 μM ionomycin, pH_i decreased by 0.35±0.02 units. We conclude that an increase in pH_i leads to an increase in [Ca⁺⁺]_i in rabbit corneal epithelial cells; however, a decrease in pH_i leads to minor changes in [Ca⁺⁺]_i. The ability of CE cells to maintain proper calcium homeostasis when pH_i is decreased may represent an adaptive mechanism to maintain physiological calcium levels during periods of acidification, which occur during prolonged eye closure.     

Apical and basolateral Na+/H+ exchange in the rabbit outer medullary thin descending limb of henle: Role in intracellular pH regulation

Summary The present study was designed to investigate the apical and basolateral transport processes responsible for intracellular pH regulation in the thin descending limb of Henle. Rabbit thin descending limbs of long-loop nephrons were perfused in vitro and intracellular pH (pH _i ) was measured using BCECF. Steady-state pH _i in HEPES buffered solutions (pH 7.4) was 7.18±0.03. Following the removal of luminal Na⁺, pH _i decreased at a rate of 1.96±0.37 pH/min. In the presence of luminal amiloride (1mm), the rate of decrease of pH _i was significantly less, 0.73±0.18 pH/min. Steady-state pH _i decreased 0.18 pH units following the addition of amiloride (1mm) to the lumen (Na⁺ 140mm lumen and bath). When Na⁺ was removed from the basolateral side of the tubule, pH _i decreased at a rate of 0.49±0.05 pH/min. The rate of decrease of pH _i was significantly less in the presence of 1mm basolateral amiloride, 0.29±0.04 pH/min. Addition of 1mm amiloride to the basolateral side (Na⁺ 140mm lumen and bath) caused steady-state pH _i to decrease significantly by 0.06 pH units. When pH _i was acutely decreased to 5.87±0.02 following NH₄Cl removal (lumen, bath), pH _i failed to recover in the absence of Na⁺ (lumen, bath). Addition of 140mm Na⁺ to the lumen caused pH _i to recover at a rate of 2.17±0.59 pH/min. The rate of pH _i recovery was inhibited 93% by 1mm luminal amiloride. When 140mm Na⁺ was added to the basolateral side, pH _i recovered only partially at 0.38±0.07 pH/min. Addition of 1mm basolateral amiloride inhibited the recovery of pH _i , by 97%. The results demonstrate that the rabbit thin descending limb of long-loop nephrons possesses apical and basolateral Na⁺/N⁺ antiporters. In the steady state, the rate of Na⁺-dependent H⁺ flux across the apical antiporter exceeds the rate of Na⁺-dependent H⁺ flux via the basolateral antiporter. Recovery of pH _i following acute intracellular acidification is Na⁺ dependent and mediated primarily by the luminal antiporter.     

In Vivo Microdialysis of 2-Deoxyglucose 6-Phosphate into Brain

Abstract : A unique method for simultaneously measuring interstitial (pH_e) as well as intracellular (pH_i) pH in the brains of lightly anesthetized rats is described. A 4-mm microdialysis probe was inserted acutely into the right frontal lobe in the center of the area sampled by a surface coil tuned for the collection of ³¹P-NMR spectra. 2-Deoxyglucose 6-phosphate (2-DG-6-P) was microdialyzed into the rat until a single NMR peak was detected in the phosphomonoester region of the ³¹P spectrum. pH_e and pH_i values were calculated from the chemical shift of 2-DG-6-P and inorganic phosphate, respectively, relative to the phosphocreatine peak. The average in vivo pH_e was 7.24 ± 0.01, whereas the average pH_i was 7.05 ± 0.01 (n = 7). The average pH_e value and the average CSF bicarbonate value (23.5 ± 0.1 mEq/L) were used to calculate an interstitial Pco₂ of 55 mm Hg. Rats were then subjected to a 15-min period of either hypercapnia, by addition of CO₂ (2.5, 5, or 10%) to the ventilator gases, or hypocapnia (Pco₂ < 30 mm Hg), by increasing the ventilation rate and volume. pH_e responded inversely to arterial Pco₂ and was well described (r² = 0.91) by the Henderson-Hassel-balch equation, assuming a pK_a for the bicarbonate buffer system of 6.1 and a solubility coefficient for CO₂ of 0.031. This confirms the view that the bicarbonate buffer system is dominant in the interstitial space. pH_i responded inversely and linearly to arterial Pco₂. The intracellular effect was muted as compared with pH_e (slope = -0.0025, r² = 0.60). pH_e and pH_i values were also monitored during the first 12 min of ischemia produced by cardiac arrest. pH_e decreases more rapidly than pH_i during the first 5 min of ischemia. After 12 min of ischemia, pH_e and pH_i values were not significantly different (6.44 ± 0.02 and 6.44 ± 0.03, respectively). The limitations, advantages, and future uses of the combined microdialysis/³¹P-NMR method for measurement of pH_e and pH_i are discussed.     

Growth inhibition of hybridoma cells by ammonium ion: correlation with effects on intracellular pH

The effect of NH₄Cl addition on intracellular pH (pH _i ) was determined by flow cytometric measurements of the fluorescence of a pH-sensitive dye. The effects of NH₄Cl on growth were determined for batch growth of cells in flasks in an incubator. The addition of NH₄Cl caused a cytoplasmic acidification. A new lower steady-state value of pH _i was attained within 20–40 min of NH₄Cl addition. A correlation was found between the effects of NH₄Cl on growth and on pH _i : whereas 3 mM NH₄Cl had little effect on growth and on pH _i , 10 mM NH₄Cl caused a substantial growth inhibition and a pH _i decrease of 0.2–0.3 units. The effects of NH₄Cl on growth and on pH _i were found to be independent of the external pH value (pH _e over the range 6.8 to 7.6, except that 10 mM NH₄Cl was more toxic at pH _e 7.6. The addition of NH₄Cl caused an increase in the average cell volume at pH _e 7.6, but had no effect on the average cell volume at pH _e 's 6.8 and 7.2. For comparison, the effects of pH _e alone on growth and on pH _i were determined. There was little difference in cell growth at pH _e 's 6.8, 7.2 and 7.6. At pH _e 6.6, there was a substantial growth inhibition. Some measurements of the effects of pH _e on pH _i were made, although the steady-state value of pH _i as a function of pH _e was not determined due to limitations in the pH _i -measuring technique. These measurements showed that pH _i remained constant from pH _e 7.6 to 6.8, but fell by 0.2 units at pH _e 6.6, in agreement with the growth results.     

Enhancement of succinic acid production by osmotic-tolerant mutant strain of Actinobacillus succinogenes

Fermentation and succinic acid production by Actinobacillus succinogenes YZ0819 was inhibited by high NaCl. To enhance the resistance of this strain to osmotic stress, an NaCl-tolerant mutant strain of A. succinogenes (CH050) was screened and selected through a continuous culture using survival in 0.7 M NaCl as the selection criterion. Using Na₂CO₃ as the pH regulator and glucose as the carbon source in batch fermentation, the isolated osmo-resistant stain, A. succinogenes CH050, produced up to 66 g/l succinic acid with a yield of 73.37% (w/w). The concentration of succinic acid and mass yield were increased by 37.5 and 4.37%, respectively, compared to the parent strain. The dry cell weight reached 10.1 g/l, which is 37% higher than that of the parent strain. The high tolerance of A. succinogenes CH050 to osmotic stress increased improved the succinic acid production from batch fermentation.     

Regulation of principal cell pH by Na/H exchange in rabbit cortical collecting tubule

Summary Changes in intracellular pH (pH _i ) were measured using the pH indicator, BCECF, in principal cells from split opened cortical collecting tubules (CCTs) derived from rabbits maintained on a normal diet. This monolayer preparation has the advantage of allowing us to visualize the morphological differences in the two major cell types in this nephron segment under transmitted light. The visual identification of the cell types was verified using emission measurements taken from single principal and intercalated cells in the opened tubule which had been exposed to fluorescein isothiocyanate (FITC)-labeled peanut lectin. We confirmed the existence of an amiloride-sensitive Na/H exchange process activated during intracellular acidosis in principal cells. In addition, the exchanger was active under basal conditions and over a wide range of pH _i . Because the exchanger was active under basal conditions we tested the hypothesis that changes in intracellular Na (Na _i ) would alter pH _i in a predictable way. Maneuvers designed to alter Na _i were without significant effects within a 10-min time frame. Specifically, addition of 100 m ouabain to increase Na _i or exposure of the tubules to 10^–5 m amiloride to decrease luminal Na entry and reduce Na _i did not have an effect on pH _i . In some experiments we did observe however, after a 30-min exposure to ouabain, a small decrease in pH _i . These results suggest that Na/H exchange is a major regulator of pH _i in principal cells. However, regulation of Na transport by changes in pH _i in principal cells of rabbit CCT via the activity of a Na/H exchanger do not seem to contribute to the feedback control of Na transport.This work was supported by U.S. Public Health Service grants DK27847 to L.G. Palmer and DK11489 to E.E. Windhager.     

Cell pH and luminal acidification inNecturus proximal tubule

Summary Cellular potential and pH measurements (pH _i ) were carried out in the perfused kidney ofNecturus on proximal tubules with standard and recessed-tip glass microelectrodes under control conditions and after stimulation of tubular bicarbonate reabsorption. Luminal pH and net bicarbonate reabsorption were measured in parallel experiments with recessed-tip glass or antimony electrodes, both during stationary microperfusions as well as under conditions of isosmotic fluid transport. A mean cell pH of 7.15 was obtained in control conditions. When the luminal bicarbonate concentration was raised to 25 and 50mm, pH _i rose to 7.44 and 7.56, respectively. These changes in pH _i were fully reversible. Under all conditions intracellular H⁺ was below electrochemical equilibrium. Thus the maintenance of intracellular pH requires active H⁺ extrusion across one or both of the cell membranes. The observed rise in pH _i and the peritubular depolarization after stimulation of bicarbonate reabsorption are consistent with enhanced luminal hydrogen ion secretion and augmentation of peritubular bicarbonate exit via an anion-conductive transport pathway.     

Enhanced acid tolerance in <Emphasis Type="Italic">Lactobacillus casei</Emphasis> by adaptive evolution and compared stress response during acid stress

This study aimed to improve the acid tolerance of Lactobacillus casei Zhang and compare the stress response of the parental strain and the acid-resistant mutant during acidic conditions. Adaptive evolution was conducted for 70 days to generate acid-tolerant L. casei. The evolved mutant lb-2 exhibited more than a 60% increase in biomass as well as a 13.6 and 65.6% increase in concentrations of lactate and acetate, respectively, when cultured at pH 4.3 for 64 h. Lactic acid tolerances of the parental strain and the evolved mutant were determined. As a result, the evolved mutant showed a 318-fold higher survival rate than that of the parental strain. Physiological analysis showed that the evolved mutant exhibited higher intracellular pH (pH_i), NH₄ ⁺ concentration and lower inner membrane permeability than that of the parental strain during acid stress. Moreover, higher amounts of intracellular arginine and aspartate were also detected in lb-2 under acid stress. Validation of the relationship between the acid tolerance and the intracellular arginine and aspartate accumulation was conducted by experiments that showed the survival of L. casei at pH 3.3 was improved 1.36-, 2.10-, or 3.42-fold by the addition of 50 mM aspartate, arginine or both of them, respectively. Taken together, results presented here not only supply an effective way to select acid-resistant strains for the food industry, but also contribute to reveal the mechanisms of acid tolerance and provide new strategies to enhance the industrial utility and health-promoting properties of this species.     

Fused cells of frog proximal tubule: II. Voltage-dependent intracellular pH

Summary Experiments were performed in intact proximal tubules of the doubly perfused kidney and in fused proximal tubule cells ofRaha esculenta to evaluate the dependence of intracellular pH (pH_i) on cell membrane potential applying pH-sensitive and conventional microelectrodes. In proximal tubules an increase of the K^– concentration in the peritubular perfusate from 3 to 15 mmol/liter decreased the peritubular cell membrane potential from –55±2 to –38±1 mV paralleled by an increase of pH _i , from 7.54±0.02 to 7.66±0.02. The stilbene derivative DIDS hyperpolarized the cell membrane potential from –57 ± 2 to –71 ±4 mV and led to a significant increase of the K^–-induced cell membrane depolarization, but prevented the K^–-induced intracellular alkalinization. Fused proximal tubule cells were impaled by three microelectrodes simultaneously and cell voltage was clamped stepwise while pH _i changes were monitored. Cell membrane hyperpolarization acidified the cell cytoplasm in a linear relationship. This voltage-induced intracellular acidification was reduced to about one-third when HCO₃ ions were omitted from the extracellular medium. We conclude that in proximal tubule cells pH _i depends on cell voltage due to the rheogenicity of the HCO ₃ ^– transport system.