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.     

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.     

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.     

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.     

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.     

Ataxia telangiectasia cells exhibit the same radiosensitization response by incorporation of BrdUrd or IdUrd as do normal human cells

Normal and ataxia telangiectasia (AT) human cells were exposed to 10(-5) mole/liter bromodeoxyuridine (BrdUrd) or iododeoxyuridine (IdUrd). High-pressure liquid chromatography (HPLC) measurements showed that up to 26 and 23% of the thymidine in DNA was substituted by BrdUrd in normal and AT cells, respectively. The incorporation of BrdUrd or IdUrd into DNA resulted in radiosensitization in normal and AT cells. When exposed to equal concentrations of BrdUrd and IdUrd, the BrdUrd caused greater radiosensitization than IdUrd in both normal and AT cells.     

Possible importance of PLD repair in the modulation of BrdUrd and IdUrd-mediated radiosensitization in plateau-phase C3H10T1/2 mouse embryo cells

C3H10T1/2 mouse embryo cells exhibiting strong contact inhibition of growth at confluency were grown in the presence of 5-bromodeoxyuridine (BrdUrd) or 5-iododeoxyuridine (IdUrd) (0-1.2 microM) with daily refeeding and exposed to gamma-rays (6 Gy) either in the logarithmic or the plateau phase of growth. Sensitization to radiation was observed in both growth states with increasing concentration of BrdUrd or IdUrd but the degree of sensitization achieved was lower for plateau-phase cells. Because the degree of [H3]BrdUrd incorporation was found to be similar in exponentially growing and plateau-phase cells, it is hypothesized that the radiosensitization caused by pyrimidine analogues may be affected by the physiological state of the cells at the time of irradiation. Delayed plating of plateau-phase cells (6 h) caused an increase in survival, indicating repair of potentially lethal damage (PLD). A greater increase in cell survival was observed in cells that had been grown in the presence of BrdUrd and IdUrd and it was found to increase with increasing concentrations. This analogue-concentration dependent PLD repair activity resulted in an almost complete loss of the radiosensitizing effect in delayed plated plateau-phase cells up to a concentration of about 0.6 microM of BrdUrd and IdUrd. Both compounds, but especially BrdUrd, caused a relaxation in the mechanism of contact inhibition and led to higher cell densities in the plateau phase. The results suggest that repair and/or expression of PLD might be involved in the mechanism underlying BrdUrd and IdUrd-mediated radiosensitization and point out the potential importance of PLD repair in the modulation of the radiosensitizing effect of these compounds in their clinical application.     

Effects of acidic pH on the formation of vinblastine-induced paracrystals in Chinese hamster ovary cells

Summary— The pH-related change in morphology of vinblastine (VLB)-induced paracrystals formed in Chinese hamster ovary (CHO) cells was examined immunohistochemically in order to determine both the mechanism of tubulin crystallization and the influence of acidic pHs on cytoskeletal microtubules. Lowering the extracellular pH (pHe) rapidly reduced the intracellular pH (pHi) in CHO cells. Lowering the pHi to near the neutral range significantly accelerated the growth of VLB-induced paracrystals, compared to that of paracrystals formed at a physiological pHe. However, further cytoplasmic acidification caused by the addition of sodium azide into the culture medium induced the disappearance of typical paracrystals and the appearance of a highly organized meshwork of tubulin appearing as short, thick filaments at the light microscopic level. Treatments using different concentrations of VLB at different pHe's showed that low pHi's (6.7 and 6.3) suppressed paracrystal-formation at lower concentrations of VLB (5×10^?6 M and 10^?5 M). At higher concentrations of VLB (5×10^?5 M and 10^?4 M), only short filaments were formed at pHi 6. 3. Electron microscopy revealed that the filaments had a ladder-like structure probably consisting of a stacked series of fused rings. This indicates that paracrystals may be modified by extremely low pH. These results show that paracrystals are unstable in living cells and that their formation is regulated by environmental pH.     

Intracellular pH (pHi) of red cells stored in acid citrate dextrose medium. Effects of temperature and citrate anions.

The intracellular pH (pHi) of red cells stored in acid citrate dextrose (ACD) medium was estimated by the 5,5'-dimethyloxazoldine,-2,4-dione (DMO) method. The initial pHi at 4degrees was about 7.6 and was higher than the extracellular pH (pHe) at 4degrees. During storage, both pHi and pHe decreased, but the former was always higher than the latter and the former decreased more slowly than the latter. The high pHi of ACD blood was a results of the temperature at which the pHe and the pHi were measured (4degrees) and the presence of citrate anions in the medium, and could be explained by application of the Donnan-Gibbs equilibrium. ATP and 2,3-diphosphoglycerate (DPG) were well-maintained in heparinized blood when it was acidified and pHe and pHi at 4degrees were both about 7.4, which suggests that improvement of blood preservation may be attained by suitable adjustment of the pHi and pHe of the blood.     

The effect of extracellular pH on radiosensitization by misonidazole and acidic or basic analogues

The effect of extracellular pH (pHe) on the radiosensitization of hypoxic Chinese hamster V79 cells in vitro by the 2-nitroimidazole, misonidazole, and analogues substituted with basic or acid functions has been studied. Misonidazole (1 mmol dm-3) gave an enhancement ratio (e.r.) of 1.6 which remained unchanged over the pHe range of 3.8-9.5. Control hypoxic survival curves in the absence of sensitizer also remained essentially unchanged over this pHe range. These results contrast with those seen for 0.1 mmol dm-3 Ro 03-8799 (1-(2-nitro-1-imidazolyl)-3-N-piperidino-2-propanol), a base with pKa = 8.9): the ER increased from 1.4 to 2.1 as pHe increased from 5.6 to 8.4. However, with the weaker bases, Ro 03-8800 and nimorazole (morpholino derivatives with pKa = 6.3 and 5.2 respectively) the e.r. remained constant over a wide pHe range. Nitroimidazoles substituted with acidic functions gave decreasing sensitization with increasing pHe. For azomycin (pKa = 7.2) at 1 mmol dm-3 the e.r. decreased from 1.9 at pHe 4 to 1.0 at pHe 9. The effect of the proton conductor carbonyl cyanide-3-chlorophenylhydrazone (CCCP, 10 mumol dm-3) on radiosensitization by Ro 03-8799 (0.1 mmol dm-3) and misonidazole (1.0 mmol dm-3) was also studied. At pHe 6.67 the e.r. for Ro 03-8799 was increased from 1.36 to 1.76 by the presence of CCCP, whereas at pHe 7.33 the e.r. was unchanged. In contrast the e.r. for misonidazole was unchanged at pHe 6.65 and 7.33. These results are consistent with pH differentials across the cell membrane creating intracellular:extracellular concentrations gradients for radiosensitizers with acidic or basic functions.     

The effect of single versus double-strand substitution on halogenated pyrimidine-induced radiosensitization and DNA strand breakage in human tumor cells

To better understand the mechanism underlying halogenated pyrimidine-mediated cytotoxicity and radiosensitization in human tumor cells, a study was undertaken to determine the influence of unifilar (one DNA strand) versus bifilar (both DNA strands) substitution of thymidine by the halogenated bases 5-iodo-2'-deoxyuridine (IdUrd) and 5-bromo-2'-deoxyuridine (BrdUrd) in HT29 human colon cancer cells. Unifilar labeling was obtained by incubating cells with IdUrd or BrdUrd for one doubling time. Cells were incubated for at least three doublings to approximate bifilar substitution. Only IdUrd caused significant cytotoxicity, which correlated with incorporation into DNA. Both BrdUrd and IdUrd were potent radiosensitizers. Radiosensitization was linearly correlated with incorporation of both bases regardless of the number of strands in which thymidine was substituted. In contrast, the relationship between radiosensitization and DNA double-strand breakage was critically dependent in the case of IdUrd, but not for BrdUrd, on whether substitution was unifilar or bifilar. These findings suggest that incorporation is the best predictor of radiation sensitivity, and that the induction of DNA double-strand breaks alone does not account for radiosensitization mediated by halogenated pyrimidines in these human tumor cells.     

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)     

Autophagy on acid

The microenvironment of solid tumors tends to be more acidic (6.5–7.0) than surrounding normal (7.2–7.4) tissue. Chaotic vasculature, oxygen limitation and major metabolic changes all contribute to the acidic microenvironment. We have previously proposed that low extracellular pH (pHe) plays a critical role in the development and progression of solid tumors. While extracellular acidosis is toxic to most normal cells, cancer cells can adapt and survive under this harsh condition. In this study, we focused on identifying survival strategies employed by cancer cells when challenged with an acidic pHe (6.6–6.7) either acutely or for many generations. While acutely acidic cells did not grow, those acclimated over many generations grew at the same rate as control cells. We observed that these cells induce autophagy in response to acidosis both acutely and chronically, and that this adaptation appears to be necessary for survival. Inhibition of autophagy in low pH cultured cells results in cell death. Histological analysis of tumor xenografts reveals a strong correlation of LC3 protein expression in regions projected to be acidic. Furthermore, in vivo buffering experiments using sodium bicarbonate, previously shown to raise extracellular tumor pH, decreases LC3 protein expression in tumor xenografts. These data imply that autophagy can be induced by extracellular acidosis and appears to be chronically employed as a survival adaptation to acidic microenvironments.     

The interaction of pH and cyclic adenosine 3',5'-monophosphate on activation of motility in Triton X-100 extracted bull sperm

The motility of demembranated bull sperm was found to be governed by the concentrations of cyclic adenosine 3', 5'-monophosphate (cAMP) and Ca2+ at low pH (6.6-7.1), and was less sensitive to these variables at higher pH (7.4-7.8). Although motility was generally found to increase with increasing pH in the range from 6.6 to 7.8, the addition of exogenous cAMP markedly and selectively improved the motility at the lower end of the range (pH 6.6-7.1). In the presence of 10 microM cAMP, low Ca2+ (8.0 X 10(-8) M), and a high concentration of Mg-adenosine 5'-triphosphate (ATP, 8 mM), demembranated sperm at pH 6.8 and 7.1 exhibited swimming similar to that of live ejaculated sperm. At a free Ca2+ concentration of 4.4 X 10(-5) M, the motility was rapidly inhibited at pH 6.8-7.1, whereas at pH 7.4-7.8, the activity was not greatly affected. Since calcium is known to antagonize the cAMP pathway by activating Ca2+-dependent phosphodiesterase and Ca2+-dependent phosphatase, this further supports the idea that cAMP-dependent activation is crucial for motility at low pH. Our results demonstrate that the flagellar axoneme can function normally at relatively acidic pH, and produce vigorous swimming at high levels of ATP. The ATP content of live sperm was measured and found to be high enough (approximately 8 mM) to support the vigorous motility seen at pH 6.6-7.1 in the models.(ABSTRACT TRUNCATED AT 250 WORDS)     

Autophagy on acid

The microenvironment of solid tumors tends to be more acidic (6.5–7.0) than surrounding normal (7.2–7.4) tissue. Chaotic vasculature, oxygen limitation and major metabolic changes all contribute to the acidic microenvironment. We have previously proposed that low extracellular pH (pHe) plays a critical role in the development and progression of solid tumors. While extracellular acidosis is toxic to most normal cells, cancer cells can adapt and survive under this harsh condition. In this study, we focused on identifying survival strategies employed by cancer cells when challenged with an acidic pHe (6.6–6.7) either acutely or for many generations. While acutely acidic cells did not grow, those acclimated over many generations grew at the same rate as control cells. We observed that these cells induce autophagy in response to acidosis both acutely and chronically, and that this adaptation appears to be necessary for survival. Inhibition of autophagy in low pH cultured cells results in cell death. Histological analysis of tumor xenografts reveals a strong correlation of LC3 protein expression in regions projected to be acidic. Furthermore, in vivo buffering experiments using sodium bicarbonate, previously shown to raise extracellular tumor pH, decreases LC3 protein expression in tumor xenografts. These data imply that autophagy can be induced by extracellular acidosis and appears to be chronically employed as a survival adaptation to acidic microenvironments.     

Steady-state magnesium ion-adenosine triphosphatase activities of myosin and its subfragment 1 derivative

The Mg2+-ATPase activity of myosin and its subfragment 1 (ATP phosphohydrolase, EC 3.6.1.3) always followed normal Michaelis-Menten kinetics for ATP concentrations less than 10 microM. The average Km values at pH 7.4 and 25 degrees C are 0.33 +/- 0.04 microM for myosin and 0.43 +/- 0.11 microM for subfragment 1. At low salt concentration myosin yields a second hyperbolic increase in Mg2+-ATPase activity as the ATP rises from 10.2 microM to 153 microM: V doubles with a Km of 11 +/- 5 microM. This second low-salt-dependent increase in Mg2+-ATPase activity occurred between pH 6.8 and pH 8.7. It was not affected by the presence of 0.10 M EGTA to remove Ca2+ contamination. Solubilization of the catalytic sites by assaying myosin for ATPase activity in the presence of 0.60 M NaCl or by conversion of myosin to subfragment 1 eliminated the secondary hyperbolic increase. Subfragment 1 has a significantly different pH-activity curve from that of myosin. Subfragment 1 has an activity peak at pH 6.0, a rising activity as the pH goes from 8.7 to 9.8, and a deep activity valley between pH 6.8 and pH 8.4. Myosin has a very shallow trough of activity at pH 6.8 to 8.4, and in 1.0 mM ATP its activity drops as the pH decreases from 6.8 to 6.0. NaCl is a noncompetitive inhibitor of the Mg2+-ATPase activity of myosin and subfragment 1. Myosin has a greater affinity for NaCl (Ki = 0.101 +/- 0.004 M) than does subfragment 1 (Ki = 0.194 +/- 0.009 M).     

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.     

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.     

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.