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.     

Clastogenicity of low pH to various cultured mammalian cells.

It has been reported that low pH itself can be clastogenic to Chinese hamster ovary cells or mouse lymphoma L5178Y cells. On the other hand, there was no indication that low pH is clastogenic to rat or human lymphocytes. Therefore, in order to evaluate the generality of clastogenicity of low pH conditions, chromosomal aberration tests were carried out on Chinese hamster cell line cells (CHO-K1, CHL, Don and V79 379A) and human cells (HeLa and peripheral lymphocytes used as whole-blood cultures). The cytotoxicity of low pH to each cell line was also evaluated by counting surviving cells. The treatment medium used was Eagle's MEM containing 15 mM MES or Bis-Tris as an organic buffer to maintain the acidity of the medium for the 6-h or 24-h treatment period, and pH adjustment was done with NaOH or HCl. Chromosomal aberrations were induced at pH 6.5 or below in CHO or CHL cells, and the maximum frequency was 24.7% at pH 6.0 or 34% at pH 6.3, respectively. About 5-10% of Don or HeLa cells had aberrations over the range of pH 6.6-6.0 or pH 6.6-6.3, respectively. In V79 379A cells or human lymphocytes, however, aberrant cells amounted to about 8% at near pH 6.0, where cell survival was low (less than 20%). About 90% of aberrations induced in each cell line examined were chromatid-type gaps and breaks. When CHO or CHL cells were treated with acidic medium for 6 h plus 18 h recovery in fresh medium, about 20% of cells had aberrations including chromatid exchanges at pH 5.5 or pH 5.7, respectively. These results indicate that clastogenicity of low pH is a general finding, although the extent of it varies with cell type, and that the clastogenicity is associated with varying extents of cytotoxicity. The mechanisms of clastogenesis at low pH are not known, but might involve inhibition of DNA or protein synthesis or DNA-repair enzymes.     

Inducible pH homeostasis and the acid tolerance response of Salmonella typhimurium.

The acid tolerance response (ATR) is an adaptive system triggered at external pH (pHo) values of 5.5 to 6.0 that will protect cells from more severe acid stress (J. Foster and H. Hall, J. Bacteriol. 172:771-778, 1990). Correlations between the internal pH (pHi) of adapted versus unadapted cells at pHo of 3.3 indicate that the ATR system produces an inducible pH-homeostatic function. This function serves to maintain the pHi above 5 to 5.5. Below this range, cells rapidly lose viability. Development of this pH homeostasis mechanism was sensitive to protein synthesis inhibitors and operated only to augment the pHi at pHo values below 4. In contrast, classical constitutive pH homeostasis was insensitive to protein synthesis inhibitors and was efficient only at pHo values above 4. Physiological studies indicated an important role for the Mg(2+)-dependent proton-translocating ATPase in affording ATR-associated survival during exposure to severe acid challenges. Along with being acid intolerant, cells deficient in this ATPase did not exhibit inducible pH homeostasis. We speculate that adaptive acid tolerance is important to Salmonella species in surviving acid encounters in both the environment and the infected host.     

Activation of vacuolar-type proton pumps by protein kinase C. Role in neutrophil pH regulation.

Activated neutrophils undergo a large burst of metabolic acid generation, yet maintain their cytosolic pH (pHi) within physiological limits. To analyze the underlying regulatory mechanisms, pHi was measured fluorimetrically in suspensions of human neutrophils. In acid loaded but otherwise unstimulated cells, pHi recovered rapidly via Na+/H+ exchange. Upon Na+ removal, recovery from an imposed acid load was negligible. Phorbol ester activation of acidified cells induced a rapid recovery of pHi partly due to a Zn(2+)-sensitive H(+)-conductive pathway. A third component of the regulatory response was apparent in Na(+)-free media containing Zn2+. Acid extrusion through this alternate pathway was voltage sensitive and capable of translocating H+ equivalents against their electrochemical gradient. This active H+ transport was inhibited by N-ethylmaleimide, by N,N'-dicyclohexylcarbodiimide and by nanomolar doses of bafilomycins A1 or B1, suggesting the involvement of vacuolar (V)-type H+ pumps. Cytosolic alkalinization was accompanied by extracellular acidification, indicative of translocation of H+ equivalents across the surface membrane and consistent with the sensitivity of the alkalinization to changes in plasma membrane potential. The activity of the V-type H+ pumps was virtually undetectable in resting cells, becoming apparent only after treatment with phorbol esters or other, chemically unrelated agonists of protein kinase C. These H+ pumps are likely to play a role in pHi homeostasis during the metabolic burst that accompanies neutrophil activation during infection and inflammation.     

Relationship between intracellular pH and energy metabolism in dog brain as measured by 31P-NMR

The relationships between pHi (intracellular pH) and phosphate compounds were evaluated by nuclear magnetic resonance (NMR) in normo-, hypo-, and hypercapnia, obtained by changing fractional inspired concentration of CO2 in dogs anesthetized with 0.75% isoflurane and 66% N2O. Phosphocreatine (PCr) fell by 2.02 mM and Pi (inorganic phosphate) rose by 1.92 mM due to pHi shift from 7.10 to 6.83 during hypercapnia. The stoichiometric coefficient was 1.05 (r2 = 0.78) on log PCr/Cr against pHi, showing minimum change of ADP/ATP and equilibrium of creatine kinase in the pH range of 6.7 to 7.25. [ADP] varied from 21.6 +/- 4.1 microM in control (pHi = 7.10) to 26.8 +/- 6.3 microM in hypercapnia (pHi = 6.83) and 24.0 +/- 6.8 microM in hypocapnia (pHi = 7.17). ATP/ADP X Pi decreased from 66.4 +/- 17.1 mM-1 during normocapnia to 25.8 +/- 6.3 mM-1 in hypercapnia. The ADP values are near the in vitro Km; thus ADP is the main controller. The velocity of oxidative metabolism (V) in relation to its maximum (Vmax) as calculated by a steady-state Michaelis-Menten formulation is approximately 50% in normocapnia. In acidosis (pH 6.7) and alkalosis (pH 7.25), V/Vmax is 10% higher than the normocapnic brain. This increase of V/Vmax is required to maintain cellular homeostasis of energy metabolism in the face of either inhibition at extremes of pH or higher ATPase activity.     

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.     

Resolution of the paradox of red cell shape changes in low and high pH

The molecular basis of cell shape regulation in acidic pH was investigated in human erythrocytes. Intact erythrocytes maintain normal shape in the cell pH range 6.3-7.9, but invaginate at lower pH values. However, consistent with predicted pH-dependent changes in the erythrocyte membrane skeleton, isolated erythrocyte membranes evaginate in acidic pH. Moreover, intact cells evaginate at pH greater than 7.9, but isolated membranes invaginate in this condition. Labeling with the hydrophobic, photoactivatable probe 5-[125I]iodonaphthyl-1-azide demonstrated pH-dependent hydrophobic insertion of an amphitropic protein into membranes of intact cells but not into isolated membranes. Based on molecular weight and on reconstitution experiments using stripped inside-out vesicles, the most likely candidate for the variably labeled protein is glyceraldehyde-3-phosphate dehydrogenase. Resealing of isolated membranes reconstituted both the shape changes and the hydrophobic labeling profile seen in intact cells. This observation appears to resolve the paradox of the contradictory pH dependence of shape changes of intact cells and isolated membranes. In intact erythrocytes, the demonstrated protein-membrane interaction would oppose pH-dependent shape effects of the spectrin membrane skeleton, stabilizing cell shape in moderately abnormal pH. Stabilization of erythrocyte shape in moderately acidic pH may prevent inappropriate red cell destruction in the spleen.     

Na+/H+ exchange activation mediates the lipopolysaccharide-induced proliferation of human B lymphocytes and is impaired in malignant B-chronic lymphocytic leukemia lymphocytes

In various mammalian cell types the stimulation of the plasma membrane amiloride-sensitive Na+/H+ exchange and the resulting increase of intracellular pH (pHi) play a key role in the initiation of cell proliferation. In the present work we have investigated whether Na+/H+ exchange is involved in normal human B cell proliferation and whether it is also operating in malignant B-chronic lymphocytic leukemia (B-CLL) lymphocytes. Our results show that: 1) normal human B cells contain an operating Na+/H+ exchanger, as inferred by their ability to recover pHi after acid-loading in a HCO3- -free medium and by evidences that LPS and phorbol ester PMA elicit a pHi rise inhibitable by either 5-(N-ethyl-N-isopropyl)amiloride (EIPA) or a Na+-free medium; 2) LPS-induced proliferation of normal human B cells is strongly inhibited when the amiloride analog EIPA (5 microM) is present in the culture medium (after 72 h the proportion of B cells incorporation bromodeoxyuridine falls from 13.9 +/- 3.9% to 2.8 +/- 1.1%); 3) EIPA does not affect BdR incorporation when B cells proliferation is induced by the co-mitogenic activity of IL-4 and low m.w. B cell growth factor (BCGF); 4) B-CLL cells, which proliferate in response to IL-4/BCGF but not to LPS, fail to increase pHi above their pHi resting levels when challenged with LPS or PMA and pHi recovery after acid-loading is highly impaired. These results lead to conclude that Na+/H+ exchange operation is necessary for LPS-(but not for IL-4/BCGF)-induced proliferation of human normal B lymphocytes and that Na+/H+ exchange activation is impaired in malignant B-CLL lymphocytes.     

Flow cytometric kinetic assay of the activity of Na+/H+ antiporter in mammalian cells.

BACKGROUND: The Na(+)/H(+) exchanger (NHE) of mammalian cells is an integral membrane protein that extrudes H(+) ion in exchange for extracellular Na(+) and plays a crucial role in the regulation of intracellular pH (pHi). Thus, when pHi is lowered, NHE extrudes protons at a rate depending of pHi that can be expressed as pH units/s. METHODS: To abolish the activity of other cellular pH-restoring systems, cells were incubated in bicarbonate-free Dulbecco's modified Eagle's medium buffered with HEPES. Flow cytometry was used to determine pHi with 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester or 5-(and-6)-carboxy SNARF-1 acetoxymethyl ester acetate, and the appropriate fluorescence ratios were measured. The calibration of fluorescence ratios versus pHi was established by using ionophore nigericin. The activity of NHE was calculated by a kinetic flow cytometric assay as the slope at time 0 of the best-fit curve of pHi recovery versus time after intracellular acidification with a pulse of exogenous sodium propionate. RESULTS: The kinetic method allowed determination of the pHi-dependent activity of NHE in cell lines and primary cell cultures. NHE activity values were demonstrated to be up to 0.016 pH units/s within the pHi range of 7.3 to 6.3. The inhibition of NHE activity by the specific inhibitor ethyl isopropyl amiloride was easily detected by this method. CONCLUSIONS: The assay conditions can be used to relate variations in pHi with the activity of NHE and provide a standardized method to compare between different cells, inhibitors, models of ischemia by acidification, and other relevant experimental or clinical situations.     

Escherichia coli glutamate- and arginine-dependent acid resistance systems increase internal pH and reverse transmembrane potential

Due to the acidic nature of the stomach, enteric organisms must withstand extreme acid stress for colonization and pathogenesis. Escherichia coli contains several acid resistance systems that protect cells to pH 2. One acid resistance system, acid resistance system 2 (AR2), requires extracellular glutamate, while another (AR3) requires extracellular arginine. Little is known about how these systems protect cells from acid stress. AR2 and AR3 are thought to consume intracellular protons through amino acid decarboxylation. Antiport mechanisms then exchange decarboxylation products for new amino acid substrates. This form of proton consumption could maintain an internal pH (pHi) conducive to cell survival. The model was tested by estimating the pHi and transmembrane potential (DeltaPsi) of cells acid stressed at pH 2.5. During acid challenge, glutamate- and arginine-dependent systems elevated pHi from 3.6 to 4.2 and 4.7, respectively. However, when pHi was manipulated to 4.0 in the presence or absence of glutamate, only cultures challenged in the presence of glutamate survived, indicating that a physiological parameter aside from pHi was also important. Measurements of DeltaPsi indicated that amino acid-dependent acid resistance systems help convert membrane potential from an inside negative to inside positive charge, an established acidophile strategy used to survive extreme acidic environments. Thus, reversing DeltaPsi may be a more important acid resistance strategy than maintaining a specific pHi value.     

在血清饥饿条件下CHP2调节NHE活性减少细胞死亡

钠氢离子交换蛋白（NHE）是维持细胞内pH值等内环境稳定的重要蛋白;钙调磷酸酶B同源蛋白（CHP）是NHE的一个活性调节亚单位。研究CHP2对NHE1的调节作用时发现,在血清饥饿的条件下,PS120细胞依赖于CHP2的表达来调节外源性NHE1的活性,使细胞维持必要的钠氢交换生理活性和较高水平的细胞内pH值（pHi 7．4）,明显减少细胞因自身的胞浆酸性化而死亡,延长细胞存活时间（70％以上的细胞存活时间超过7天）。实验结果提示,通过研究减少CHP2表达或抑制其活性,可望找到加速细胞死亡的新方法。     

Influence of Physico-Chemical Factors on the Oligomerization and Biological Activity of Bacteriocin AS-48

Bacteriocin AS-48 forms a mixture of monomers and oligomers in aqueous solutions. Such oligomers can be clearly differentiated by SDS-PAGE after formaldehyde crosslinking, and we have verified that these associates are stable to acid treatment after fixation. In addition, they show antimicrobial activity and are recognized by anti-AS-48 antibodies. AS-48 oligomers can be dissociated by the detergents SDS and Triton X-100. The degree of oligomerization of AS-48 depends on the pH of the solution and the protein concentration. At pH below 5, AS-48 is in the monomeric state at protein concentrations below 0.55.mg ml⁻¹, but it also forms dimers above this protein concentration. This bacteriocin forms oligomers at pH values above 5, in agreement with the observation that it is also more hydrophobic at neutral pH. AS-48 is stable to mild heat treatments irrespectively of pH. At 120°C it is more heat resistant under acidic conditions, but it inactivates at neutral pH. Activity of AS-48 against E. faecalis is highest at neutral pH, but it is highest at pH 4 for E. coli. The influence of pH on bacteriocin activity could be owing to changes in the conformation/oligomerization of the bacteriocin peptide as well as to changes in the surface charge of the target bacteria. Received: 3 July 2000\t/\tAccepted: 11 August 2000     

The regulation of intracellular pH in monkey kidney epithelial cells (BSC-1). Roles of Na+/H+ antiport, Na+-HCO3(-)-(NaCO3-) symport, and Cl-/HCO3- exchange

Using the pH-sensitive absorbance of 5 (and 6)-carboxy-4',5'- dimethylfluorescein, we investigated the regulation of cytoplasmic pH (pHi) in monkey kidney epithelial cells (BSC-1). In the absence of HCO3-, pHi is 7.15 +/- 0.1, which is not significantly different from pHi in 28 mM HCO3-, 5% CO2 (7.21 +/- 0.07). After an acid load, the cells regulate pHi in the absence of HCO3- by a Na+ (or Li+)-dependent, amiloride-inhibitable mechanism (indicative of Na+/H+ antiport). In 28 mM HCO3-, while still dependent on Na+, this regulation is only blocked in part by 1 mM amiloride. A partial block is also observed with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) (1 mM). With cells pretreated with DIDS, 1 mM amiloride nearly totally inhibits this regulation. Cl- had no effect on pHi regulation in the acidic range. In HCO3(-)-free saline, Na+ removal leads to an amiloride-insensitive acidification, which is dependent on Ca2+. In 28 mM HCO3-, Na+ (and Ca2+) removal led to a pronounced reversible and DIDS-sensitive acidification. When HCO3- was lowered from 46 to 10 mM at constant pCO2 (5%), pHi dropped by a DIDS-sensitive mechanism. Identical changes in pHo (7.6 to 6.9) in the nominal absence of HCO3- led to smaller changes of pHi. In the presence but not in the absence of HCO3-, removal of Cl- led to a DIDS-sensitive alkalinization. This was also observed in the nominal absence of Na+, which leads to a sustained acidification. It is concluded that in nominally bicarbonate-free saline, the amiloride-sensitive Na+/H+ antiport is the predominant mechanism of pHi regulation at acidic pHi, while being relatively inactive at physiological values of pHi. In bicarbonate saline, two other mechanisms effect pHi regulation: a DIDS-sensitive Na+-HCO3- symport, which contributes to cytoplasmic alkalinization, and a DIDS-sensitive Cl-/HCO3- exchange, which is apparently independent of Na+.     

Bradykinin Release Avoids High Molecular Weight Kininogen Endocytosis

Human H-kininogen (120 kDa) plays a role in many pathophysiological processes and interacts with the cell surface through protein receptors and proteoglycans, which mediate H-kininogen endocytosis. In the present work we demonstrate that H-kininogen containing bradykinin domain is internalized and different endogenous kininogenases are present in CHO-K1 cells. We used CHO-K1 (wild type) and CHO-745 (mutant deficient in proteoglycans biosynthesis) cell lines. H-kininogen endocytosis was studied using confocal microscopy, and its hydrolysis by cell lysate fraction was determined by immunoblotting. Bradykinin release was also measured by radioimmunoassay. H-kininogen interaction with the cell surface of CHO-745 cells resulted in bradykinin release by serine proteases. In CHO-K1 cells, which produce heparan and chondroitin sulfate proteoglycans, internalization of H-kininogen through its bradykinin domain can occur on lipid raft domains/caveolae. Nevertheless bradykinin-free H-kininogen was not internalized by CHO-K1 cells. The H-kininogen present in acidic endosomal vesicles in CHO-K1 was approximately 10-fold higher than the levels in CHO-745. CHO-K1 lysate fractions were assayed at pH 5.5 and intact H-kininogen was totally hydrolyzed into a 62 kDa fragment. By contrast, at an assay pH 7.4, the remained fragments were 115 kDa, 83 kDa, 62 kDa and 48 kDa in size. The antipain-Sepharose chromatography separated endogenous kininogenases from CHO-K1 lysate fraction. No difference was detected in the assays at pH 5.5 or 7.4, but the proteins in the fraction bound to the resin released bradykinin from H-kininogen. However, the proteins in the unbound fraction cleaved intact H-kininogen at other sites but did not release bradykinin. H-kininogen can interact with extravascular cells, and is internalized dependent on its bradykinin domain and cell surface proteoglycans. After internalization, H-kininogen is proteolytically processed by intracellular kininogenases. The present data also demonstrates that serine or cysteine proteases in lipid raft domains/caveolae on the CHO cell can hydrolyze H-kininogen, thus releasing kinins.     

A Chinese hamster ovary cell mutant defective in the non-endocytic uptake of fluorescent analogs of phosphatidylserine: isolation using a cytosol acidification protocol

Transmembrane movement of phosphatidylserine (PS) and various PS analogs at the plasma membrane is thought to occur by an ATP-dependent, protein-mediated process. To isolate mutant CHO cells defective in this activity, we first obtained conditions which inhibited the endocytic, but not the non-endocytic pathway of lipid internalization since PS may enter cells by a combination of these two pathways. We found that acidic treatment of cells, which blocks clathrin-dependent endocytosis, enhanced the energy-dependent uptake of 1-palmitoyl-2-(6-[(7-nitrobenz- 2-oxa-1,3-diazol-4-yl)amino]caproyl -sn- glycero-3-phosphoserine (C6- NBD-PS) in CHO cells from donor vesicles (liposomes) by about twofold. Control experiments demonstrated that the enhanced uptake of C6-NBD-PS at acidic pH was not due to: (a) an increase in the capacity of the plasma membrane to incorporate C6-NBD-PS from the donor vesicles; (b) a decrease in the rate of loss of C6-NBD-PS from the cells; or (c) fusion or engulfment of the donor vesicles. When cytosolic acidification (to pH 6.3) was imposed without acidification of the extracellular medium, C6-NBD-PS uptake by intact cells was increased by about 50% compared to control values determined in the absence of acidification. These results suggested that a protein and energy dependent system(s) for transbilayer movement of the fluorescent PS was stimulated by cytosolic acidification. A screening method for mutant cells defective in the non- endocytic uptake of fluorescent PS analogs with replica cell colonies at acidic pH was then devised. After selection of mutagenized CHO-K1 cells by in situ screening, we obtained a mutant cell line in which uptake of fluorescent PS analogs was reduced to about 25% of the wild type level at either pH 6.0 or 7.4. Control experiments demonstrated that the reduced uptake of fluorescent PS analogs in the mutant cells was unrelated to multidrug resistance, and that endocytosis of another plasma membrane lipid marker occurred normally in the mutant cells. These results suggested that a non-endocytic pathway responsible for uptake of fluorescent PS analogs was specifically affected in the mutant cells.     

Intracellular pH of sea urchin eggs measured by the dimethyloxazolidinedione (DMO) method

Intracellular pH (pH1) of sea urchin eggs and embryos was determined using DMO (5,5-dimethyl-2,4-oxazolidinedione). By this method, the pH1 of Lytechinus pictus eggs increased after fertilization from 6.86 to 7.27, and this higher pHi was maintained thereafter, as has been previously observed with pH microelectrodes. The same general result was obtained with the eggs of Strongylocentrotus purpuratus, in contrast to previous estimates of the pH of egg homogenates from this species, which had indicated a rise and then fall of pHi after fertilization. pHi did not significantly change during early cell divisions. Studies of treatments that alter pHi confirmed that ammonia alkalizes and acetate acidifies the cells. The regulation of pHi by embryos in the acidic seawater is impaired if sodium is absent, whereas unfertilized eggs can regulate pHi in acidic, sodium-free seawater.     

Intracellular pH modulates taste receptor cell volume and the phasic part of the chorda tympani response to acids

The relationship between cell volume and the neural response to acidic stimuli was investigated by simultaneous measurements of intracellular pH (pHi) and cell volume in polarized fungiform taste receptor cells (TRCs) using 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) in vitro and by rat chorda tympani (CT) nerve recordings in vivo. CT responses to HCl and CO2 were recorded in the presence of 1 M mannitol and specific probes for filamentous (F) actin (phalloidin) and monomeric (G) actin (cytochalasin B) under lingual voltage clamp. Acidic stimuli reversibly decrease TRC pHi and cell volume. In isolated TRCs F-actin and G-actin were labeled with rhodamine phalloidin and bovine pancreatic deoxyribonuclease-1 conjugated with Alexa Fluor 488, respectively. A decrease in pHi shifted the equilibrium from F-actin to G-actin. Treatment with phalloidin or cytochalasin B attenuated the magnitude of the pHi-induced decrease in TRC volume. The phasic part of the CT response to HCl or CO2 was significantly decreased by preshrinking TRCs with hypertonic mannitol and lingual application of 1.2 mM phalloidin or 20 microM cytochalasin B with no effect on the tonic part of the CT response. In TRCs first treated with cytochalasin B, the decrease in the magnitude of the phasic response to acidic stimuli was reversed by phalloidin treatment. The pHi-induced decrease in TRC volume induced a flufenamic acid-sensitive nonselective basolateral cation conductance. Channel activity was enhanced at positive lingual clamp voltages. Lingual application of flufenamic acid decreased the magnitude of the phasic part of the CT response to HCl and CO2. Flufenamic acid and hypertonic mannitol were additive in inhibiting the phasic response. We conclude that a decrease in pHi induces TRC shrinkage through its effect on the actin cytoskeleton and activates a flufenamic acid-sensitive basolateral cation conductance that is involved in eliciting the phasic part of the CT response to acidic stimuli.     

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.     

Dicarboxy-dichlorofluorescein: a new fluorescent probe for measuring acidic intracellular pH

Derivatives of fluorescein sensitive to pH are extensively utilized for the determination of intracellular pH (pHi). Available dyes have pKa values of approximately 7.0, and are not well suited for measuring acidic pHi. We examined the fluorescein derivative, 5 (and 6)-carboxy-2',7'-dichlorofluorescein (CDCF) for its potential in the microspectrofluorometric measurement of pHi during acidic conditions. CDCF showed intense fluorescence and pH sensitivity near its "effective" pKa value of 4.2, using a 495/440 nm dual excitation wave-length ratio method. Protein interactions caused fluorescence ratio deviations which were most pronounced at the extremes of pH, whereas calcium and magnesium concentrations had little effect on the fluorescent ratio intensity. Intracellular calibration performed using nigericin in the presence of high potassium eliminated the need to correct for protein interactions, and the ratio method minimized any variations due to dye concentration differences or instrument fluctuation. Intracellular retention of the dye was high, and 95% of the initial signal remained after 1 h. Fluorescence bleaching was 14.5% after 1 h of continuous excitation and cell survival was not affected by dye loading. We conclude that CDCF is an excellent intracellular pH indicator in the pH range of 4-5.