Interpretation of the phenomenon of K+ transport activation in Escherichia coli during transition to anaerobiosis

Earlier it was demonstrated that the transition of E. coli K-12 cells to anaerobiosis is accompanied by the activation of K+ uptake. K+ that are additionally accumulated during the transition to anaerobiosis are released from the cells after the turning on of the respiratory chain. The K+ accumulation by the cells is potential-dependent both under aerobic and anaerobic conditions. A correlation was found between the degree of acidification of the cytoplasm and the rate of K+ uptake during the transition to anaerobiosis. It was assumed that under aerobic conditions the functioning of the electrogenic system of K+ uptake is concomitant with the operation of the K+ release system, K+/H+ antiporter, which is inactivated at the beginning of anaerobiosis, presumably as a result of cytoplasm acidification. This effect manifests itself as the activation of K+ uptake. The trigger function of the K+/H+ antiporter in E. coli cells was suggested to provide for the control of the intracellular pH as well as the switching from the aerobic to the anaerobic pathway of energy metabolism.     

The internal pH of the forespore compartment of Bacillus megaterium decreases by about 1 pH unit during sporulation.

Previous work has shown that the internal pH of dormant spores of Bacillus species is more than 1 pH U below that of growing cells but rises to that of growing cells in the first minutes of spore germination. In the present work the internal pH of the whole Bacillus megaterium sporangium was measured by the distribution of the weak base methylamine and was found to decrease by approximately 0.4 during sporulation. By using fluorescence ratio image analysis with a fluorescein derivative, 2',7'-bis(2-carboxyethyl)-5 (and -6)-carboxyfluorescein (BCECF), whose fluorescence is pH sensitive, the internal pH of the mother cell was found to remain constant during sporulation at a value of 8.1, similar to that in the vegetative cell. Whereas the internal pH of the forespore was initially approximately 8.1, this value fell to approximately 7.0 approximately 90 min before synthesis of dipicolinic acid and well before accumulation of the depot of 3-phosphoglyceric acid. The pH in the forespore compartment was brought to that of the mother cell by suspending sporulating cells in a pH 8 potassium phosphate buffer plus the ionophore nigericin to clamp the internal pH of the cells to that of the external medium. We suggest that at a minimum, acidification of the forespore may regulate the activity of phosphoglycerate mutase, which is the enzyme known to be regulated to allow 3-phosphoglyceric acid accumulation during sporulation.     

Kinetic studies of the variations of cytoplasmic pH, nucleotide triphosphates (31P-NMR) and lactate during normoxic and anoxic transitions in maize root tips

We have followed the dynamic evolution of intracellular pH and of the intracellular concentration of nucleotides (NDP, NTP), Pi and lactate in maize root tips during the course of normoxia and anoxia transition. The intracellular pH, determined from the 31P-NMR chemical shift of the cytoplasmic P1 peak, dropped from 7.5 to 6.9 during the first few minutes after anaerobiosis. It increased again, then settled to a steady-state value of 7.1-7.2, 25 min after the beginning of the anoxic treatment. Following oxygenation, the chemical shift of the cytoplasmic Pi peak drifted gradually to its initial value. The cytoplasmic pH followed an oscillatory time course which was almost identical to the time course of NTP. Intracellular lactate accumulated steadily during the first 30 min after anaerobiosis, then its intracellular concentration remained almost constant. Following oxygenation, the intracellular concentration of lactate decreased slowly. The cytoplasmic pH followed a time course which was not identical to the time course of lactate. Following hypoxia, the pH dropped to low values long before the intracellular lactate concentration reached a steady-state equilibrium. Conversely, subsequent to oxygenation, the pH returned to normal values long before lactate. These results do not agree with the statement that cytoplasmic acidification in hypoxic maize root tips is necessarily associated with lactic acid synthesis.     

Continuous measurement of the cytoplasmic pH in Lactococcus lactis with a fluorescent pH indicator

The cytoplasmic pH of Lactococcus lactis was studied with the fluorescent pH indicator 2',7'-bis-(2-carboxyethyl)-5 (and-6)-carboxyfluorescein (BCECF). A novel method was applied for loading bacterial cells with BCECF, which consists of briefly treating a dense cell suspension with acid in the presence of the probe. This results in a pH gradient, which drives accumulation of the probe in the cytoplasm. After neutralization the probe was well retained in cells stored on ice. BCECF-loaded cells were metabolically active, and were able to generate a pH gradient upon energization. The probe leaks out slowly at elevated temperatures. Efflux is stimulated upon energization of the cells, and is most likely catalyzed by an active transport system. It is a first-order process, and the rate constant could be deduced from the decrease of the fluorescence signal in periods of constant intracellular pH. This allowed a correction of the fluorescence signal for efflux of the probe. After calibration the cytoplasmic pH could be calculated from efflux-corrected fluorescence traces.     

Regulation of cytoplasmic pH under extreme acid conditions in suspension cultured cells of Catharanthus roseus: a possible role of inorganic phosphate

Changes in cytoplasmic pH of suspension-cultured cells of Catharanthus roseus under extreme acid conditions were measured with the pH-dependent fluorescence dye; 2',7'-bis-(2-carboxyethyl)-5 (and-6) carboxyfluorescein (-acetoxymethylester) (BCECF). When cells were treated with 1 mM HCl (pH 3 solution), the cytoplasmic pH first decreased then returned to the original level. Treatment with 10 mM HCl (pH 2 solution) acidified the cytoplasm to a greater extent, and the acidification continued at a constant level throughout the measurement. Treatment with a pH 2 solution resulted in a gradual decrease of the malate content, indicating the operation of biochemical pH regulation mechanism. The pH 2 treatment also caused a sudden decrease of the intracellular level of Pi. The cellular content of total phosphorus did not change during the acidification. The Pi was converted to the organic phosphate form. The ATP level was not increased by the pH 2 treatment, but slightly decreased. The role of Pi, which might be functioning as a regulatory factor of cytoplasmic pH, a non-competitive inhibitor of the H+-pumps of both the plasma membrane and tonoplast is discussed.     

Effects of nutrient and non-nutrient stimuli on cytosolic pH in cultured insulinoma (HIT-T15) cells

Intracellular pH (pHi) was measured in the insulin-secreting HIT-T15 cell line using the pH-sensitive fluorescent dye, 2',7'-bis(carboxyethyl)-5'(6')-carboxyfluorescein (BCECF). It was observed that the addition of a weak acid (e.g., acetate or propionate) caused a rapid decrease in pHi, followed by a slower recovery to the resting pH value. Conversely the addition of N4Cl caused an increase in pHi followed by recovery. The addition of amiloride caused a fall in pHi; however, in this case no recovery to basal pH levels was observed. Subsequent addition of a weak acid caused a further fall in pHi with no recovery. The addition of glucose caused a transient acidification followed by alkalinization. When glucose was added to cells which had been pretreated with amiloride, the initial acidification was not followed by recovery or alkalinization. Addition of glyceraldehyde, alpha-ketoisocaproate, lactate or pyruvate to HIT cells also resulted in intracellular acidification followed by recovery. Similarly, depolarisation of HIT cells by treatment with high K+ or with Ba2+ was associated with a pronounced fall in pHi, followed by a gradual recovery. Insulin secretion from HIT cells was stimulated by glucose, glyceraldehyde, alpha-ketoisocaproate, lactate, pyruvate and KCl, whilst amiloride and weak acids exerted only modest effects in the absence of glucose, but amiloride in particular markedly potentiated glucose-induced insulin release. Thus, HIT cells appear to have an amiloride-sensitive mechanism for the extrusion of protons, probably Na+-H+ exchange. Whilst intracellular acidification appears to potentiate secretory responses to nutrient stimuli, it seems unlikely that the activation of HIT cells by these nutrients occurs as a result of intracellular acidification. The mechanisms by which various nutrient and non-nutrient stimuli might exert distinct effects on pHi are discussed.     

Cytoplasmic pH dynamics in maize pulvinal cells induced by gravity vector changes

In maize (Zea mays) and other grasses, changes in orientation of stems are perceived by pulvinal tissue, which responds to the stimulus by differential growth resulting in upward bending of the stem. The amyloplast-containing bundle sheath cells are the sites of gravity perception, although the initial steps of gravity perception and transmission remain unclear. In columella cells of Arabidopsis roots, we previously found that cytoplasmic pH (pH(c)) is a mediator in early gravitropic signaling (A.C. Scott, N.S. Allen [1999] Plant Physiol 121: 1291-1298). The question arises whether pH(c) has a more general role in signaling gravity vector changes. Using confocal ratiometric imaging and the fluorescent pH indicator carboxy seminaphtorhodafluor acetoxymethyl ester acetate, we measured pH(c) in the cells composing the maize pulvinus. When stem slices were gravistimulated and imaged on a horizontally mounted confocal microscope, pH(c) changes were only apparent within the bundle sheath cells, and not in the parenchyma cells. After turning, cytoplasmic acidification was observed at the sides of the cells, whereas the cytoplasm at the base of the cells where plastids slowly accumulated became more basic. These changes were most apparent in cells exhibiting net amyloplast sedimentation. Parenchyma cells and isolated bundle sheath cells did not show any gravity-induced pH(c) changes although all cell types responded to external stimuli in the predicted way: Propionic acid and auxin treatments induced acidification, whereas raising the external pH caused alkalinization. The results suggest that pH(c) has an important role in the early signaling pathways of maize stem gravitropism.     

pH regulation in apoplastic and cytoplasmic cell compartments of leaves

 The regulation of pH in the apoplast, cytosol and chloroplasts of intact leaves was studied by means of fluorescent pH indicators and as a response of photosynthesis to acid stress. The apoplastic pH increased under anaerobiosis. Aeration reversed this effect. Apoplastic responses to CO₂, HCl or NH₃ differed considerably. Whereas HCl and ammonia caused rapid acidification or alkalinization, the return to initial pH values was slow after cessation of fumigation. Addition of CO₂ either did not produce the acidification expected on the basis of known apoplastic buffering or even caused some alkalinization. Removal of CO₂ shifted the apoplastic pH into the alkaline range before the pH returned to initial steady-state levels. In the presence of vanadate, the alkaline shift was absent and the apoplastic pH returned slowly to the initial level when CO₂ was removed from the atmosphere. In contrast to the response of the apoplast, anaerobiosis acidified the cytosol or, in some species, had little effect on its pH. Acidification was rapidly reversed upon re-admission of oxygen. The CO₂-dependent pH changes were very fast in the cytosol. Considerable alkalinization was observed after removal of CO₂ under aerobic, but not under anaerobic conditions. Rates of the re-entry of protons into the cytosol during recovery from CO₂ stress increased in the presence of oxygen with the length of previous exposure to high CO₂. Effective pH regulation in the chloroplasts was indicated by the recovery of photosynthesis after the transient inhibition of photosynthetic electron flow when CO₂ was increased from 0.038% to 16% in air. As photosynthesis became inhibited under high CO₂, reduction of the electron transport chain increased transiently. The time required for recovery of photosynthesis from inhibition during persistent CO₂ stress was similar to the time required for establishing steady-state pH values in the cytosol under acid stress. The high capacity of leaf cells for the rapid re-attainment of pH homeostasis in the apoplast and the cytoplasm under acid or alkaline stress suggested the rapid activation or deactivation of membrane-localised proton-transporting enzymes and corresponding ion channel regulation for co-transport of anions or counter-transport of cations together with proton fluxes. Acidification of the cytoplasm appeared to activate energy-dependent proton export primarily into the vacuoles whereas apoplastic alkalinization resulted in the pumping of protons into the apoplast. Proton export rates from the cytosol into the apoplast after anaerobiosis were about 100 nmol (m² leaf area)⁻¹ s⁻¹ or less. Proton export under acid stress into the vacuole was about 1200 nmol m⁻² s⁻¹. The kinetics of pH responses to the addition or withdrawal of CO₂ indicated the presence of carbonic anhydrase in the cytosol, but not in the apoplast. Received: 19 July 1999 / Accepted: 29 December 1999     

P NMR Observations on the Effect of the External pH on the Intracellular pH Values in Plant Cell Suspension Cultures

³¹P nuclear magnetic resonance (NMR) spectroscopy was used to monitor the response of oil palm (Elaeis guineensis) and carrot (Daucus carota) cell suspensions to changes in the external pH. An airlift system was used to oxygenate the cells during the NMR measurements and a protocol was developed to enable a constant external pH to be maintained in the suspension when required. Phosphonoacetic acid was used as an external pH marker and the intracellular pH values were measured from the chemical shifts of the cytoplasmic and vacuolar orthophosphate resonances. In contrast to earlier studies the cytoplasmic pH was independent of the external pH over the range 5.5 to 8.0 and it was only below pH 5.5 that the cytoplasmic pH varied, falling at a rate of 0.12 pH unit per external unit. Loss of pH control was observed in response to sudden increases in external pH with the response of the cells depending on the conditions imposed. A notable feature of the recovery from these treatments was the transient acidification of the cytoplasm that occurred in a fraction of the cells and overshoot phenomena of this kind provided direct evidence for the time dependence of the regulatory mechanisms.     

Effect of starvation on cytoplasmic pH, proton motive force, and viability of an acidophilic bacterium, Thiobacillus acidophilus.

The question of whether Thiobacillus acidophilus maintains its cytoplasmic pH at values close to neutrality by active or passive means was explored by subjecting the organism to long-term starvation (up to 22 days). Starving cells maintained a delta pH of 2 to 3 U throughout starvation, although cellular poly-beta-hydroxybutyric acid and ATP, the proton motive force, and culture viability were low or not detectable after 200 h. Cells exposed to azide or azide plus N,N'-dicyclohexylcarbodiimide immediately exhibited characteristics of cells starved for more than 200 h. Thus, a large delta pH in T. acidophilus was maintained in the absence of ATP, ATPase activity, respiration, significant levels of proton motive force, and cell viability and was therefore not dependent on chemiosmotic ionic pumping. The transition from a metabolically active to an inactive state was accompanied by a large increase in the positive membrane potential, which nearly completely compensated for the delta pH in the inactive cells. The longevity of the acidophile during starvation was comparable to that reported previously for neutrophiles, and the loss of viability occurred not because of the acidification of the cytoplasm but apparently because of energy depletion.     

Intracellular pH homeostasis in the filamentous fungus Aspergillus niger.

Intracellular pH homeostasis in the filamentous fungus Aspergillus niger was measured in real time by 31P NMR during perfusion in the NMR tube of fungal biomass immobilized in Ca2+-alginate beads. The fungus maintained constant cytoplasmic pH (pH(cyt)) and vacuolar pH (pH(vac)) values of 7.6 and 6.2, respectively, when the extracellular pH (pH(ex)) was varied between 1.5 and 7.0 in the presence of citrate. Intracellular metabolism did not collapse until a Delta pH over the cytoplasmic membrane of 6.6-6.7 was reached (pH(ex) 0.7-0.8). Maintenance of these large pH differences was possible without increased respiration compared to pH(ex) 5.8. Perfusion in the presence of various hexoses and pentoses (pH(ex) 5.8) revealed that the magnitude of Delta pH values over the cytoplasmic and vacuolar membrane could be linked to the carbon catabolite repressing properties of the carbon source. Also, larger Delta pH values coincided with a higher degree of respiration and increased accumulation of polyphosphate. Addition of protonophore (carbonyl cyanide m-chlorophenylhydrazone, CCCP) to the perfusion buffer led to decreased ATP levels, increased respiration and a partial (1 microm CCCP), transient (2 microm CCCP) or permanent (10 microm CCCP) collapse of the vacuolar membrane Delta pH. Nonlethal levels of the metabolic inhibitor azide (N3-, 0.1 mm) caused a transient decrease in pH(cyt) that was closely paralleled by a transient vacuolar acidification. Vacuolar H+ influx in response to cytoplasmic acidification, also observed during extreme medium acidification, indicates a role in pH homeostasis for this organelle. Finally, 31P NMR spectra of citric acid producing A. niger mycelium showed that despite a combination of low pH(ex) (1.8) and a high acid-secreting capacity, pH(cyt) and pH(vac) values were still well maintained (pH 7.5 and 6.4, respectively).     

Intracellular pH and Catecholamine Secretion from Bovine Adrenal Chromaffin Cells

To study the role of intracellular pH (pHi) in catecholamine secretion and the regulation of pHi in bovine chromaffin cells, the pH-sensitive fluorescent indicator [2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein] was used to monitor the on-line changes in pHi. The pHi of chromaffin cells at resting state is approximately 7.2. The pHi was manipulated first by incubation of the cells with NH4+, and then the solution was replaced with a NH4(+)-free solution to induce acidification of the cytoplasm. The pHi returned toward the basal pH value after acidification within 5-10 min in the presence of Na+ or Li+, but the pHi stayed acidic when Na(+)-free buffers were used or in the presence of amiloride and its analogues. These results suggest that the pH recovery process after an acid load is due to the Na+/H+ exchange activity in the plasma membrane of the chromaffin cells. The catecholamine secretion evoked by carbachol and Na+ removal was enhanced after the cytoplasm had been made more acidic. It appears that acidic pH favors the occurrence of exocytosis.     

Changes in lysosome shape and distribution correlated with changes in cytoplasmic pH

Lysosomes labeled by uptake of extracellular horseradish peroxidase display remarkable changes in shape and cellular distribution when cytoplasmic pH is experimentally altered. Normally, lysosomes in macrophages and fibroblasts cluster around the cell center. However, when the cytoplasmic pH is lowered to approximately pH 6.5 by applying acetate or by various other means, lysosomes promptly move outward and accumulate in tight clusters at the very edge of the cell, particularly in regions that are actively ruffling before acidification but become quiescent. This movement follows the distribution of microtubules in these cells, and does not occur if microtubules are depolymerized with nocodazole before acidification. Subsequent removal of acetate or the other stimuli to acidification results in prompt resumption of ruffling activity and return of lysosomes into a tight cluster at the cell center. This is correlated with a rebound alkalinization of the cytoplasm. Correspondingly, direct application of weak bases also causes hyperruffling and unusually complete withdrawal of lysosomes to the cell center. Thus, lysosomes appear to be acted upon by microtubule-based motors of both the anterograde (kinesin) type as well as the retrograde (dynein) type, or else they possess bidirectional motors that are reversed by changes in cytoplasmic pH. During the outward movements induced by acidification, lysosomes also appear to be smaller and more predominantly vesicular than normal, while during inward movements they appear to be more confluent and elongated than normal, often becoming even more tubular than in phorbol-treated macrophages (Phaire-Washington, L., S. C. Silverstein, and E. Wang. 1980. J. Cell Biol. 86:641-655). These size and shape changes suggest that cytoplasmic pH also affects the fusion/fission properties of lysosomes. Combined with pH effects on their movement, the net result during recovery from acidification is a stretching of lysosomes into tubular forms along microtubules.     

Early death at medium acidification and survival after low pH adaptation inCryptococcus neoformans

WhenCryptococcus neoformans was grown in yeast nitrogen base (YNB) supplemented with 0.5% glucose, the medium was acidified to below pH 3 during the exponential growth phase, which caused early growth-phase death in susceptible strains. Even in resistant strains, 30–70% cells died if incubated for 2 d in YNB supplemented with 1.5% glucose, whereas the remaining cells survived long. Two types of fatal alterations have been observed in dead cells. In the first type, release of cytoplasm occurred through weakened parts of the cell wall; structures attached to cell walls of dead cells were shown to be rich in proteins by FITC staining, indicating their cytoplasmic origin. In the second type, cells shrank distinctly with no sign of wall rupture. The shrinkage may be due to dysfunction of the plasma membrane at low pH. The mechanism of cell survival in medium below pH 3 was also examined. Aniline blue alone, or calcofluor together with methylene blue, allowed cell wall glucan or chitin and dead cell cytoplasm to be stained simultaneously. In the later stages of incubation, cells showing bright staining for cell wall glucan and chitin emerged. These changes in cell wall synthesis could be considered as an adaptation mechanism to acidification of the medium, because such cells survived longer than cells showing no change in the cell wall staining pattern.     

Cell to cell communication and pH in the frog lens

Fiber cells of the lens are electrically and diffusionally interconnected through extensive gap junctions. These junctions allow fluxes of small solutes to move between inner cells and peripheral cells, where the majority of transmembrane transport takes place. We describe here a method utilizing two intracellular microelectrodes to measure the cell to cell resistance between fiber cells at any given distance into the intact lens. We also use ion-sensitive microelectrodes to record intracellular pH at various depths in the intact lens. We find that gap junctions connecting inner fiber cells differ in pH sensitivity as well as normal coupling resistance from those connecting peripheral cells. The transition occurs in a zone between 500 and 650 microns into the lens. Fiber cells peripheral to this zone have a specific coupling resistance of 1.1 omega cm2, whereas those inside have a specific coupling resistance of 2.7 omega cm2. However, when the cytoplasm of fiber cells is acidified by bubbling with CO2, peripheral cells uncouple and the cell to cell resistance goes up more than 40-fold, whereas junctions inside this zone are essentially unaffected by changes in intracellular pH. In a normal frog lens, the intracellular pH in fiber cells near the lens surface is 7.02, a value significantly alkaline to electrochemical equilibrium. Our data suggest that Na/H exchange and perhaps other Na gradient-dependent mechanisms in the peripheral cells maintain this transmembrane gradient. Deep in the lens, the fiber cell cytoplasm is significantly more acidic (pHi 6.81) due to influx of hydrogen across the inner fiber cell membranes and production of H+ by the inner fiber cells. Because of the normally acid cytoplasm of interior fiber cells, their loss of gap junctional sensitivity to pH may be essential to lens survival.     

Measurement of intracellular (compartmental) pH by 31P NMR in Aspergillus niger

31P nuclear magnetic resonance (31P NMR) was used to monitor cytoplasmic and vacuolar pH values in the filamentous fungus Aspergillus niger. To obtain a homogeneous cell sample and to be able to perform long term in vivo NMR measurements A. niger mycelium was kept in a setup that allows perfusion of the cell plug within the NMR tube. Mycelial samples, however, became rapidly clogged during perfusion leading to (partial) anaerobiosis of the plug with subsequent acidification of the cytoplasm. As a result, only short-term NMR measurements (5-10 min) were possible using free mycelium. To increase and to prolong perfusion, A. niger was immobilized in Ca(2+)-alginate beads. Deteriorated spectra recorded under hypoxia could be completely restored in the presence of oxygen. With this system perfusion in the presence of citrate could be maintained for at least 18 h at much higher rates (15 ml min-1 compared with 4 ml min-1 for free mycelium). During this period 31P NMR spectra were highly invariable, indicating approximate steady-state intracellular conditions during long term measurements. Perfusion in the presence of glucose resulted in complete depletion of the vacuolar inorganic phosphate pool within 45 min and yielded a higher pH gradient over the tonoplast than when citrate was used (delta pH = 1.6 and 1.4, respectively).     

The response of GS-NS0 myeloma cells to pH shifts and pH perturbations

The perceived sensitivity of animal cells to hydrodynamic shear has limited agitation and aeration at large-scale. This makes it difficult to ensure adequate mixing of the vessel contents and may lead to inhomogeneities in operational parameters such as temperature, dissolved oxygen concentration, and especially pH. The effect of pH shifts and pH perturbations on the cellular responses, in batch culture, of a GS-NS0 mouse myeloma cell line, expressing a recombinant antibody, was investigated. In addition, the effect of extreme pH on the structure of the purified antibody product was studied using isoelectric focusing. The fermentation pH value was shifted abruptly from pH 7.3 to pH values ranging from 6.5 to 9.0. Culture pH was maintained at this new value for the remainder of the fermentation. All pH shifts of above 0.2 units caused a transient increase in apoptosis. However, cultures shifted to pH values between 7.0 and 8.0 continued to grow and the apoptotic fraction returned to initial levels. Cultures shifted to pH values above pH 8.0 and below pH 7.0 did not recover resulting in culture death. For example, a shift to pH 8.5 caused accumulation of cells in the G(2)/M phase of the cell cycle followed by apoptotic death. After the pH shift, maximum specific growth rate was observed over the range pH 7.3 to 7.5 and maximum viable cell number was seen at pH 7.3. Maximum volumetric antibody production, resulting from increased culture longevity, was seen at pH 7.0. It was also observed that glucose consumption increased with increasing pH. In a separate set of experiments cells were subjected to a single pH perturbation ranging in duration from 0 to 600 minutes. Exposure of cells to a pH value greater than 8.5 for more than 10 minutes caused a decrease in the proportion of viable cells and induced a lag in cell growth. At very low pH (6.5) similar effects were seen, but only for extended perturbations (600 min). However, after recovery from the pH perturbation, growth, product secretion and metabolism all returned to original levels. Incubation of the antibody, at the range of pH values investigated, indicated no alterations in the structure of the antibody as determined by the isoelectric focusing pattern.     

Development of thermotolerance and changes in intracellular pH in CHO cells heated at 45.0 degrees C at pH 6.6

Chinese hamster ovary (CHO) cells were given short heat pulses (5 to 20 min) at 45.0 degrees C and incubated at 37 degrees C for up to 20 h under either pH 7.3 or 6.6 conditions. Thermotolerance developed under both pH conditions, but at a slower rate in the pH 6.6 medium. Intracellular pH (pHi) was measured with the dye, 1,4-diacetoxy-2,3-dicyanobenzene, combined with flow cytometry. Time-dependent changes in the intracellular pH occurred under either pH condition. CHO cells incubated under normal pH conditions had a transient increase in the pHi. This pHi elevation was followed by a rapid intracellular acidification of approximately 0.15 to 0.25 pH units. The timing of both the increases and decreases in the pHi was dependent on the magnitude of the initial heat dose. With heat doses less than or equal to 10 min, the pHi returned to normal unheated levels after the acidification phase. Although cells incubated under low pH (6.6) conditions showed similar pHi alterations, differences in the kinetics were measured. The intracellular pH increased immediately after heating. In addition, when intracellular acidification occurred, the rate of acidification was significantly reduced. With heat doses longer than 5 min under the low pH conditions, the pHi did not return to normal unheated levels.     

Effect of amines on the pH in the lysosomes of 3T3 cells

The measurements of intralysosomal pH under the action of the number of amines earlier reported to block the process of the initiation of cell proliferation (Nikolsky et al., 1984) were made on Swiss 3T3 cells. The intralysosomal pH (pH1) value was estimated by parameters of fluorescence of fluorescein-labeled dextran in single intact cells. The pHl value was equal to 4.7 +/- 0.2 for both actively growing and quiescent cells. The pH gradient between lysosomes and the cytoplasm was completely destroyed by monensin and partially by carbonylcyamide-m-chlorophenylhydrasone. Methylamine and chloroquine rapidly enhanced the pHl, value to 6.4-7.0. Dansylcadaverine, 5-methoxytryptamine and dimethylurea did not affect pHl value. Intracellular accumulation of dansylcadaverine was shown to be due to the existence of acidic compartments into the cell and highly decreased in the presence of monensin. A conclusion is made that the inhibition of mitogenic signal by amines cannot be unequivocally accounted for by increasing the pH in organelles involved in the intracellular processing of growth factors.     

Selective perturbation of early endosome and/or trans-Golgi network pH but not lysosome pH by dose-dependent expression of influenza M2 protein

Many sorting stations along the biosynthetic and endocytic pathways are acidified, suggesting a role for pH regulation in protein traffic. However, the function of acidification in individual compartments has been difficult to examine because global pH perturbants affect all acidified organelles in the cell and also have numerous side effects. To circumvent this problem, we have developed a method to selectively perturb the pH of a subset of acidified compartments. We infected HeLa cells with a recombinant adenovirus encoding influenza virus M2 protein (an acid-activated ion channel that dissipates proton gradients across membranes) and measured the effects on various steps in protein transport. At low multiplicity of infection (m.o.i.), delivery of influenza hemagglutinin from the trans-Golgi network to the cell surface was blocked, but there was almost no effect on the rate of recycling of internalized transferrin. At higher m.o.i., transferrin recycling was inhibited, suggesting increased accumulation of M2 in endosomes. Interestingly, even at the higher m.o.i., M2 expression had no effect on lysosome morphology or on EGF degradation, suggesting that lysosomal pH was not compromised by M2 expression. However, delivery of newly synthesized cathepsin D to lysosomes was slowed in cells expressing active M2, suggesting that acidification of the TGN and endosomes is important for efficient delivery of lysosomal hydrolases. Fluorescence labeling using a pH-sensitive dye confirmed the reversible effect of M2 on the pH of a subset of acidified compartments in the cell. The ability to dissect the role of acidification in individual steps of a complex pathway should be useful for numerous other studies on protein processing and transport.