The role of low intracellular or extracellular pH in sensitization to hyperthermia

Cells are more sensitive to heat when they are heated in an acidic environment, and this study confirms (K. G. Hofer and N. F. Mivechi, J. Natl. Cancer Inst., 65, 621, 1980) that intracellular pH (pHi) and not extracellular pH (pHe) is responsible for the sensitization. The relationship between pHe, pHi, and heat survival of cells heated in vitro in various buffers at pHe 6.3-8.0 was investigated. Cells' adaptation to low environmental pH in terms of increases in pHi and heat survival also was investigated. Finally, we studied the relationships among pHe, pHi, and survival from heat for cells heated in sodium-free reconstructed medium. Intracellular pH was measured by the distribution of the weak acid, [2-14C]5,5-dimethyl-2,4-oxazolidinedione. Our results are summarized as follows: (1) CHO cells maintained the same relationship between pHe and pHi in four different media or buffers (McCoy's 5a medium buffered with CO2 and NaHCO3 or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) and 2-(N-morpholino)ethanesulfonic acid (Mes), Krebs-Ringer bicarbonate solution, and Krebs-Ringer phosphate solution) with pHi being 0.05 to 0.20 pH units higher than pHe as it varied from 7.0 to 6.4; furthermore, heat sensitization by acid was the same in medium buffered with NaHCO3 or Hepes and Mes. (2) The low pHe adapted cells multiplied with an increased doubling time of 20.7 +/- 0.7 h and appeared morphologically similar to the unadapted cells. However, the pHi of these cells was 0.15-0.30 pH units higher than that of the unadapted cells when pHe was varied between 7.0 and 6.3. (3) After being heated at 43.5 degrees C for 55 min or at 42.5 degrees C for 150 min at pHe 6.3-7.2, the pHi of the adapted cells increased by 0.2-0.1 pH units. However, heat caused no significant change in the unadapted cells. (4) Heat survival plotted versus pHe was 1000-fold higher for the adapted cells than for the unadapted cells at pHe of 6.3. However, heat survival plotted versus pHi was identical for the two cell types. (5) In sodium-free reconstructed McCoy's 5a medium, pHi was 0.25-0.1 pH units lower than that in the sodium-containing counterpart at pHe 6.3-7.2, and heat sensitization increased accordingly; however, heat survival plotted versus pHi was identical for the two types of media.     

Determination of the intravesicular pH of fragmented sarcoplasmic reticulum with 5,5-dimethyl-2,4-oxazolidinedione.

The intravesicular pH (pHi) of fragmented sarcoplasmic reticulum (SR) of the skeletal muscle was determined from the distribution of 5,5-dimethyl-2,4-oxazolidinedione (DMO), a weak organic acid, between the intra- and extravesicular spaces. The pHi's thus obtained were found to be slightly lower (0.02-0.17 pH unit) than the pH's of the external medium (pHe) at pH 6.5-8.5 in the presence of 105 mM KCl and 40 mM Tris-maleate buffer. The higher the pHe, the greater the pH gradient. When pHe was changed, pHi attained equilibrium within about 20 min, the time necessary for the separation of the SR by centrifugation. When 0.25 M sucrose and 5 mM Tris-maleate buffer were used instead of 105 mM KCl and 40 mM buffer, the pH gradient increased to 0.56. It was also demonstrated by direct measurements of pHe with a glass-electrode pH meter that K+ ions added to the external medium exchanged the intravesicular H+ ions. From these results it appears that the pH gradient across the SR membrane was at the Donnan equilibrium. In this state, the Donnan potentials corresponding to pH gradients of 0.17 and 0.56 were -9.3 and -30.6 mV, respectively.     

Differential effect of pH on the density and volume of rat reticulocytes and erythrocytes. Relevance to their fractionation by centrifugation.

Rats were injected with 59Fe-ferrous citrate and bled thereafter at different times (16 h to 49 d). This gave rise to red cell populations in which cells corresponding in age to the time elapsed between injection and bleeding were labeled. The anticoagulant used was either acid-citrate-dextrose (ACD) with a pH adjusted to 7.3 or ACD (pH 5.1). Final pH of the collected blood was about 7.2-7.4 in the former case and 6.4-6.7 in the latter. Red cells were then centrifuged (5) and approximately 7-10% of the packed cells from the top and 7-10% from the bottom of the cell column collected. When reticulocytes are the predominant labeled red cell population, as in blood obtained for about 24 h after isotope injection, a fractionation of these cells and mature erythrocytes is in evidence only when blood is collected at the higher pH. Thus, at pH 7.2-7.4 ratios of specific radioactivities of cells in top fraction/cells in an unfractionated sample are about 3, whereas at pH 6.4-6.7, the analogous ratios are 1 or less. These differences in specific activity ratios, as a function of pH at collection, virtually disappear after about 4 d following isotope injection. The lower pH is known to increase the volume and decrease the density of mature red blood cells. The marked effect of pH on cellular fractionation could be correlated with the smaller change in rat reticulocyte density and volume in acid medium. At pH 6.4-6.7, the densities of mature erythrocytes and reticulocytes are so close that their physical separation by centrifugation is not feasible.     

Comparison of DMO and flow cytometric methods for measuring intracellular pH and the effect of hyperthermia on the transmembrane pH gradient

Intracellular pH (pHi) was measured in both unheated and heated cells by the distribution of the weak acid, 5,5-dimethyl-2,4-oxazolidinedione-2-14C (14C-DMO), and by the fluorescence intensity ratio (I530/I630) of the pH sensitive fluorescent dye, 2',7'-bis(carboxyethyl)-5,6-carboxy-fluorescein (BCECF), analyzed by flow cytometry (FCM). BCECF-loaded Chinese hamster ovary (CHO) cells were analyzed by FCM after they had incubated in fresh medium at 37 degrees C for 90 min, during which time a decrease in fluorescence ratio stabilized. After stabilization, the pHi determined for CHO cells by the FCM method at pHe values of 6.0-8.1 agreed-within 0.1 pH units with that determined by the 14C-DMO method. There is a pH gradient across the plasma membrane that is not affected by heat. In CHO cells, the gradient, determined by DMO and FCM, is less or greater than pHe by 0.30 and 0.15 pH units at pHe 7.4 and 6.3, respectively, and in NG108-15 cells, the gradient determined by DMO increases to 0.50 pH units at pHe 6.3. Both cells maintained their pH gradients for at least 4 h after heating, although 99.9% of the cells were reproductively dead (survival of 10(-3)) after heating at 45.5 degrees C either at the normal pHe of 7.4 or at a low pHe of 6.4-6.7.     

The regulation of intracellular pH studied by 31P- and 1H-NMR spectroscopy in superfused guinea-pig cerebral cortex slices.

(1) The intracellular pH (pHi) of superfused slices of guinea-pig cerebral cortex was measured in 31P-NMR spectra using the chemical shifts of intracellular inorganic phosphate (Pi) and of 2-deoxyglucose 6-phosphate (DOG6P). The pHi was found to be 7.30 +/- 0.04 (SD, n = 15) in bicarbonate-buffered medium and 7.20 +/- 0.05 (n = 10, P < 0.001) in bicarbonate-free HEPES buffer of the same pH (7.4). (2) Decreases in pHe below 7.05 resulted in pHi falling to similar values, with a decrease in the energy state. There was no change in intracellular lactate as assessed by 1H-NMR. (3) The tissues showed an ability to buffer higher pH: increasing pHe to 8.0 had no effect on pHi, PCr or lactate. (4) In order to characterize possible mechanisms of pH regulation in the tissue, the recovery from acid insult was investigated under various conditions. Initially pHi was decreased to 6.44 +/- 0.15 (n = 15) by exposure to media containing 6 mM bicarbonate gassed with O2/CO2, 80:20 (pHe 6.4). When this medium was replaced by normal bicarbonate buffer (pH 7.4) there was full recovery of pHi to 7.31 +/- 0.05 (n = 15), whereas replacing the buffer with HEPES resulted in incomplete recovery of pHi to 6.88 +/- 0.15 (n = 15, P < 0.001). (5) In the presence of the carbonic anhydrase inhibitor, acetazolamide (1 mM), or the sodium/proton exchange inhibitor, amiloride (1 mM), there was an incomplete return of pHi to the control value (pHi 6.90 +/- 0.20, n = 5, P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxygen binding and acid base status of the blood from the freshwater turtle, Phrynops hilarii

Oxygenation studies with the whole blood of Phrynops hilarii show a P50 of 38 torr at extracellular pH (pHe) of 7.4 which corresponds to an intracellular pH (pHi) of 7.05 at 25 degrees C. The blood CO2 Bohr effect was -0.56 when related to pHi. pHi is related to pHe by the following equation: pHi = 0.75.pHe + 1.54 (r = 0.99); pHi = 0.72. pHe + 1.72 (r = 0.96) at 10 and 25 degrees C respectively. Blood pHe, for 25 degrees C, was 7.519 +/- 0.254 (n = 6). Blood gas partial pressures were: pCO2 = 25.8 +/- 3.8 torr (n = 6); pO2 = 61.7 +/- 21.2 torr (n = 6). The major red cell phosphates, in mmole/l erythrocytes, n = 6, were: ATP (3.66 +/- 0.86); GTP (0.53 +/- 0.28); 2.3-DPG (0.32 +/- 0.12) and inorganic phosphates (2.00 +/- 0.35). The plasma inorganic ion composition, n = 6, was, in mEq/l: K+ (3.04 +/- 0.40); Na+ (148.4 +/- 12.6); Ca2+ (4.75 +/- 1.32); Cl- (106.6 +/- 5.0). Additional blood parameters of interest (n = 6) were: lactate (2.07 +/- 1.72 mM in plasma); erythrocytes/mm3 (416 X 10(3) +/- 4.6 X 10(3)); leucocytes/mm3 (44636 +/- 2618); haematocrit (%) (14.5 +/- 3.6); haemoglobin, g/dl (3.2 +/- 0.5); plasma protein g/dl (4.4 +/- 0.4); osmolarity (293 +/- 10 mOsm/l). The non-bicarbonate buffer value was -22.6 mmol/kg H2O/pH. For a constant CO2 content, delta pHe/delta t = 0.0141 +/- 0.002 (n = 18) and delta pHi/delta t = 0.0157 +/- 0.003 (n = 18).     

The role of low intracellular or extracellular pH in sensitization to hyperthermic radiosensitization

Previous work showed that intracellular pH (pHi) and not extracellular pH (pHe) was the determinant in the low pH sensitization of hyperthermic killing. The present studies show that the same is true for heat-induced radiosensitization and loss of cellular DNA polymerase activities. Chinese hamster ovary cells after they had adapted to low pH (6.7) had an increase in pHi which rendered cells partially resistant to the low pH sensitization of heat-induced cell killing, radiosensitization, and loss of cellular DNA polymerase activities. These results were quantified by plotting versus pHe, both the thermal enhancement ratio (TER), defined as the ratio of the X-ray dose without heat to the X-ray dose with heat to give an isosurvival value of 0.01, and the thermal enhancement factor (TEF), defined as the ratio of the D0 of the radiation survival curve to the D0 of the radiation survival curve for heat plus radiation. Both the TER and TEF were higher for the unadapted cells than for the adapted cells, i.e., 1.3-1.4 fold higher at a pHe of 6.3. However, the TER or TEF plotted versus pHi was identical for the two cell types. Finally, heat-induced loss of cellular DNA polymerase activities correlated with pHi and not pHe. Therefore, we conclude that pHi and not pHe is responsible for the increase by acid in heat-induced radio-sensitization and loss of cellular DNA polymerase activities.     

Role of intramitochondrial pH in the energetics and regulation of mitochondrial oxidative phosphorylation.

The dependence of ATP synthesis coupled to electron transfer from 3-hydroxy-butyrate (3-OH-B) to cytochrome c on the intramitochondrial pH (pHi) was investigated. Suspensions of isolated rat liver mitochondria were incubated at constant extramitochondrial pH (pHe) with ATP, ADP, Pi, 3-OH-B, and acetoacetate (acac) (the last two were varied to maintain [3-OH-B]/[acac] constant), with or without sodium propionate to change the intramitochondrial pH. Measurements were made of the steady-state water volume of the mitochondrial matrix, transmembrane pH difference, level of cytochrome c reduction, concentration of metabolites and rate of oxygen consumption. For each experiment, conditions were used for which transmembrane pH was near maximal and minimal values and the measured extramitochondrial [ATP], [ADP], and [Pi] were used to calculate log[ATP]/[ADP][Pi]. When [3-OH-B]/[acac] and [cyt c2+]/[cyt c3+] were constant, and pHi was decreased from approx. 7.7 to 7.2, log [ATP]/[ADP][Pi] at high pHi was significantly (P less than 0.02) greater than at low pHi. The mean slope (delta log [ATP]/[ADP][Pi] divided by the change in pHi) was 1.08 +/- 0.15 (mean +/- S.E.). This agrees with the slope of 1.0 predicted if the energy available for ATP synthesis is dependent upon the pH at which 3-hydroxybutyrate dehydrogenase operates, that is, on the pH of the matrix space. The steady-state respiratory rate and reduction of cytochrome c were measured at different pHi and pHe values. Plots of respiratory rate vs.% cytochrome c reduction at different intra- and extramitochondrial pH values indicated that the respiratory rate is dependent upon pHi and not on pHe. This implies that the matrix space is the source of protons involved in the reduction of oxygen to water in coupled mitochondria.     

Differential effect of pH on the density and volume of rat reticulocytes and erythrocytes

Rats were injected with⁵⁹Fe-ferrous citrate and bled thereafter at different times (16 h to 49 d). This gave rise to red cell populations in which cells corresponding in age to the time elapsed between injection and bleeding were labeled. The anticoagulant used was either acid-citrate-dextrose (ACD) with a pH adjusted to 7.3 or ACD (pH 5.1). Final pH of the collected blood was about 7.2–7.4 in the former case and 6.4–6.7 in the latter. Red cells were then centrifuged (5) and approximately 7–10% of the packed cells from the top and 7–10% from the bottom of the cell column collected. When reticulocytes are the predominant labeled red cell population, as in blood obtained for about 24 h after isotope injection, a fractionation of these cells and mature erythrocytes is in evidence only when blood is collected at the higher pH. Thus, at pH 7.2–7.4 ratios of specific radioactivities of cells in top fraction/cells in an unfractionated sample are about 3, whereas at pH 6.4–6.7, the analogous ratios are 1 or less. These differences in specific activity ratios, as a function of pH at collection, virtually disappear after about 4 d following isotope injection. The lower pH is known to increase the volume and decrease the density of mature red blood cells. The marked effect of pH on cellular fractionation could be correlated with the smaller change in rat reticulocyte density and volume in acid medium. At pH 6.4–6.7, the densities of mature erythrocytes and reticulocytes are so close that their physical separation by centrifugation is not feasible.     

Use of flow cytometry and SNARF to calibrate and measure intracellular pH in NS0 cells.

BACKGROUND: Two calibration methods have been proposed for determining the relation between the fluorescence ratio of a pH-sensitive fluorescent indicator and intracellular pH (pHi). The first method uses nigericin to clamp pHi to external pH (pHe) and the second is the null point method. We compared these different calibration methods, solution conditions, and temperatures by using flow cytometry and the fluorescent dye 1,5- (and-6)-carboxy seminaphtorhodafluor-1-acetoxymethyl ester with an NS0 cell line. METHODS: The nigericin method was performed in glucose solutions supplemented with KCl and 2-(N-morpholino)ethane sulphonic acid plus tris(hydroxymethyl)aminomethane (solution 1A), a mixture of K2HPO4/KH2PO4 in glucose-solution supplemented solutions (solution 2A), or bicarbonate buffered growth medium supplemented with K2HPO4/KH2PO4 (solution 2B); this allowed a range of pHe values to be used. The effect of temperature (22 degrees C or 37 degrees C) on the nigericin calibration curve was also investigated. The null point method was performed by using a series of solutions with a mixture of weak acid and base with a known pHi response. RESULTS: Using solution 1A as the calibration solution resulted in acidic values of pHi for cells cultured in medium as compared with the values achieved with solution 2A. Using solution 2B did not affect the calibration curve. For the temperatures considered in this study, there was no affect on the calibration curve, but temperature did affect the pHi value of cells in phosphate buffered saline. The pseudo-null point method used with flow cytometry resulted in a calibration curve that was significantly different (P<0.05) from that achieved using the nigericin method. CONCLUSIONS: Our data indicates that the choice of calibration solution can affect the reported pHi value; therefore, careful choice of solution is important.     

Buffer power and intracellular pH of frog sartorius muscle.

Intracellular pH (pHi) and buffer power of frog muscle were measured using pH-sensitive microelectrodes under conditions used previously in energy balance experiments because pH strongly influences the molar enthalpy change for phosphocreatine splitting, the major net reaction during brief contractions. The extracellular pH (pHe) of HEPES buffered Ringer's solution influenced pHi, but change in pHi developed slowly. Addition or removal of CO2 or NH3 from the extracellular solution caused a rapid change in pHi. The mean buffer power measured with CO2 was 38.4 mmol.l-1.pH unit-1 (+/- SEM 2.1, n = 49) and with NH3 was 36.2 (+/- SEM 5.5, n = 4) at 20-22 degrees C. At 5 degrees C, in experiments with CO2 the mean buffer power was 40.3 (+/- SEM 2.6, n = 3). For pHi values above approximately 7.0, the observed buffer power was greater than that expected from the values in the literature for the histidine content of intracellular proteins, carnosine and inorganic phosphate in the sarcoplasm. The measured pHi values were similar to those assumed in energy balance calculations, but the high measured buffer power suggests that other buffering reactions occur in addition to those included in energy balance calculations.     

Effect of hyperthermia on intracellular pH in Chinese hamster ovary cells

Chinese hamster ovary cells were heated at 45.5 or 43.0 degrees C at acidic pH (6.7) or normal physiological pH (7.4) to have a survival of 10(-3). The weak acid, 5,5-dimethyl-2,4-oxazolidinedione-2-14C), was used to measure the intracellular pH (pHi) both during and following hyperthermia. Tritiated water and a Particle Data machine were used to measure cellular volume as well. With 99.9% of the cell population destined to die clonogenically, the physiologically alive cells, as determined by the exclusion of trypan blue dye, maintained their pH differential between pHe and pHi as well as unheated cells. Furthermore, the cell's ability to regulate its pHi in response to changes in pHe was not affected by the same hyperthermic treatment. However, cellular volume decreased by 15-30% by 5 h after the onset of heat treatment. We conclude that heat does not perturb the cellular regulation of intracellular H+ concentration. Therefore, there is no thermal damage to the pHi-regulatory mechanism that could be responsible for either heat-induced reproductive cell death or low pH sensitization of heat killing.     

Rapid method for measuring intracellular pH in vivo.

Intracellular pH (pHi) was simultaneously measured in 6 normal tissues and a malignant tumour of rats by a rapid triple isotope technique, based on the in vivo distribution of 5,5-dimethyl-2,4-oxazolidinedione-2-14C (DMO), tritiated water and sodium chloride-36. Results compared favourably with pH measured directly in the same rat by capillary glass electrode, and with values of other workers for pHi in rat tissues. Mean pHi of normal tissues was close to pH 7, and in each organ there was a linear relationship between pHi and extracellular pH (pHe) over the normal range of pHe encountered (pH 6.9-7.6). Organ pHi altered in response to administration of NH4Cl or NaHCO3 to the host.     

Viability, ATP and 2,3-DPG content of resuspended erythrocytes during several-week storage in cold

Leukocyte-and thrombocyte-poor packed red cells obtained from ACD or. ACD-AG blood were resuspended to a hematocrit of about 55% and stored at 4 degrees C. The resuspension solution consisted of xylitol, inorganic phosphate, bicarbonate, adenine (A) and guanosine (G) solved in water. In one case glucose, citrate and sucrose were also added, in another one, sorbitol. The 2,3-DPG and the ATP level remained for a longer period in the sorbitol-xylitol-medium than in the glucose-xylitol-medium. The ATP content in the red cell suspension was higher than in packed cells. Higher ATP values were obtained in red blood cells from whole blood with adenine and guanosine. The survival rate of resuspended red blood cells in glucose-AG-citrate-sucrose medium was about 80--85% after 3 weeks of storage and 77% after 6 weeks with a higher range.     

Fermentation of citrate by Lactobacillus plantarum in the presence of a yeast under acid conditions

Summary At pH 3.6, Lactobacillus plantarum is unable to grow on citrate or to ferment it in the absence of another carbon source such as glucose. In a defined medium containing glucose and citrate, with a higher concentration of the former than the latter, as in many fermented alcoholic beverages, L. plantarum will first ferment the sugar. The production of lactate from glucose degradation increases the acidity of the medium and inhibits the fermentation of citrate. In co-culture with Saccharomyces cerevisiae, part of the glucose is fermented by the yeast, partly avoiding the pH drop and the inhibition of citrate fermentation by L. plantarum. Fermentation was still possible at pH values around 3.0. Offprint requests to: C. Kennes     

Regulation of intracellular pH values in higher plant cells. Carbon-13 and phosphorus-31 nuclear magnetic resonance studies.

The regulation of the cytoplasmic and vacuolar pH values (pHc and pHv) in sycamore (Acer pseudoplatanus L.) cells was analyzed using 31P and 13C nuclear magnetic resonance spectroscopy. Suspension-cultured cells were compressed in the NMR tube and perfused with the help of an original arrangement enabling a tight control of the pH (external pH, pHe) of the carefully oxygenated circulating nutrient medium. Intracellular pH values were measured from the chemical shifts of: CH2-linked carboxyl groups of citric acid below pH 5.7; orthophosphate between pH 5.7 and 8.0; 13C-enriched bicarbonate over pH 8.0. pHc and pHv were independent of pHe over the range 4.5-7.5. In contrast intracellular pH values decreased rapidly below pHe 4.5 and increased progressively at pHe over 7.5. There was an acceleration in the rate of O2 consumption accompanied with a decrease in cytoplasmic ATP concentration as pHe decreased. When the rate of O2 consumption was approaching the uncoupled O2 uptake rate, a loss of pHc control was observed. It is concluded that as pHe decreased, the plasma membrane ATPase consumed more and more ATP to reject the invading H+ ions in order to maintain pHc at a constant value. Below pHe 4.5 the efficiency of the H+ pump to react to back leakage of H+ ions became insufficient, leading to an acidification of pHc and to an alkalinization of pHe. On the other hand, over pHe 7.5 a passive influx of OH- ions was observed, and pHc increased proportionally to the increase of pHe. Simultaneously appreciable amounts of organic acids (malate and citrate) were synthesized by cells during the course of the alkalinization of the cytoplasmic compartment. The synthesis of organic acids which partially counteract the alkalinization of the cytoplasmic compartment may result from a marked activation of the cytoplasmic phosphoenolpyruvate carboxylase induced by an increase in cytoplasmic bicarbonate concentration. The fluctuations of pHv followed a similar course to that of pHc. It is concluded that the vacuole, which represents a potentially large H+ ions reservoir, can counteract H+ (or OH-) ion invasion observed at acidic (or alkaline) pHe contributing to the homeostasis of pHc.     

Effects of extracellular potassium on acid release and motility initiation in Toxoplasma gondii

The internal pH (pHi) of Toxoplasma gondii was estimated by measuring the accumulation of the weak base 9-aminoacridine in buffers with various ionic compositions. The pHi of the metabolizing parasite increased when the extracellular K+ was elevated in alkaline medium or when the external pH (pHe) was substantially increased in medium employing high external K+ (90 mM). The parasite in mouse peritoneal fluid, or in potassium sulfate buffer (pH 8.2), where the pHi was demonstrated to be increased to 7.9, became motile when acidic buffer was substituted for the original suspension medium. This acid-induced independent movement subsided within 5 min but was repeatedly induced if the pHe was serially lowered to 6.0. Basic buffers, on the other hand, abolished motility when applied to the moving parasites. Nigericin, which is known to collapse pH gradients across the membrane, also abolished motility.     

Low pH does not affect the dose response for 5'-amino-5'-deoxythymidine modulation of IdUrd DNA incorporation and radiosensitization in a human bladder cancer cell line.

We report that coincubation of 647V cells for one cell cycle with low concentrations (30 microM) of 5'-amino-5'-deoxythymidine increased IdUrd DNA incorporation and radiosensitivity at low extracellular pH (pHe 6.8) in a fashion similar to treatment at normal pHe. IdUrd DNA incorporation is inhibited by high (300 microM) 5'-AdThd concentrations at both normal and low pHe (7.4 and 6.8), resulting in no significant radiosensitization. These results at low pHe were not anticipated based on previously published studies of 5'-AdThd modulation of thymidine kinase (TK) activity and nucleoside cellular uptake. Our results suggest that regulation of intracellular pH (pHi) during the course of one cell cycle negates the 5'-AdThd dose-dependent modulation of TK activity demonstrated previously. Flow cytometric measurement of pHi in 647V cells showed that normal pHi (pH 7.4) was maintained in 647V cells over a 12- to 24-h exposure to low pHe (pH 6.8). Thus the concomitant use of IdUrd and high concentrations of 5'-AdThd (> 30 microM) is unlikely to result in selective in vivo radiosensitization of human tumors under conditions which are intermittently or chronically acidic. However, low concentrations of 5'-AdThd may prove to be an effective in vivo modulator of IdUrd radiosensitization of human tumors under both normal and acidic conditions.     

pH-dependent effects of the ionophore nigericin on response of mammalian cells to radiation and heat treatment

The extracellular pH (pHe) in many solid tumors is often lower than the pH of normal tissues. The K+/H+ ionophore nigericin is toxic to CHO cells when pHe is below but not above 6.5, and thus it has potential for selective killing of tumor cells in an acidic environment. This study examines the pH-dependent effects of nigericin on the response of CHO cells to radiation and heat treatment. Cells held for 4 h in Hank's balanced salt solution, after 9 Gy irradiation, exhibit potentially lethal damage recovery (PLDR) which is maximal at pHe 6.7-6.8. Addition of nigericin, postirradiation, not only inhibits PLDR when pHe is below 6.8, but interacts synergistically with radiation to reduce survival below that of cells plated immediately after irradiation when pHe is 6.4 or lower. Nigericin enhances heat killing of CHO cells perferentially under acidic conditions, and where neither heat nor drug treatment alone is significantly toxic. Survival of cells held for 30 min at 42.1 degrees C in the presence of 1.0 microgram/ml nigericin is 0.6, 0.08, 0.003, and 0.00003 at pHe 7.4, 6.8, 6.6, and 6.4, respectively, relative to survival of 1.0 in untreated cultures. The biochemical effects of nigericin at pHe 7.4 vs pHe 6.4 have been investigated. Nigericin inhibits respiration, stimulates glucose consumption, and causes dramatic changes in intracellular concentrations of Na+ and K+ at pHe 7.4 as well as 6.4. The drug reduces intracellular levels of ATP, GTP, and ADP but has more pronounced effects under acidic incubation conditions. Others have shown that nigericin equilibrates pHe and intracellular pH (pHi) only when pHe is 6.5 or lower. Our observations and those of others have led us to conclude that lowering of pHi by nigericin is either the direct or indirect cause of enhancement of radiation and heat killing of cells in an acidic environment.     

Role of monocarboxylic acid transport in intracellular pH regulation of isolated proximal cells

Isolated proximal cells were prepared from rabbit kidney cortex by mechanical dissociation. The intracytoplasmic pH (pHi) was measured in HCO3(-)-free media (external pH (pHe), 7.3) using the fluorescent dye 2,7-biscarboxyethyl-5,6-carboxyfluorescein (BCECF). Cells were acid-loaded by the nigericin technique. Addition of 70 mM Na+ to the cells caused a rapid pHi recovery, which was blocked by 0.5 mM amiloride. When the cells were exposed to 5 mM sodium butyrate in the presence of 1 mM amiloride, the H+ efflux was significantly increased and followed Michaelis-Menten kinetics. Increasing pHe from 6.4 to 7.6 at a constant pHi of 6.4 enhanced the butyrate activation of the H+ efflux. Increasing pHi from 6.5 to 7.2 at a constant pHe of 7.2 reduced the butyrate effect. 22Na uptake experiments in the presence of 1 mM amiloride showed that 1.5 mM butyrate increased the Na+ flux in the proximal cells (pHi 7.10). The efficiency of monocarboxylic anions in promoting a pHi recovery increased with the length of their straight chain (acetate less than propionate less than butyrate less than valerate). The data show that when the Na+/H+ antiporter is blocked, the proximal cells can regulate their pHi by a Na+-coupled absorption of butyrate followed by non-ionic diffusion of butyric acid out of the cell and probably also by OH- influx by means of the OH-/anion exchanger.