Fluorescent pH Indicator Research Articles

Angiotensin II stimulates sodium-hydrogen exchange in adult rabbit ventricular myocytes

The aim was to characterise the effects of angiotensin II on Na+/H+ exchange in adult ventricular myocytes. Intracellular pH (pHi) was continuously measured with the fluorescent pH indicator, SNARF-1, in single resting myocytes obtained from adult rabbits by enzymatic dissociation. In some experiments cells were electrically paced to elicit contractions. All experiments were performed at 36 degrees C in HEPES buffered solution containing no added CO2 or HCO3- (pHo 7.4). Rapid application of angiotensin II caused pHi to rise. The initial rate of rise and initial net H+ efflux responded to angiotensin II in a concentration dependent manner, EC50 = 7.8. Buffering of cytosolic calcium with the calcium chelator BAPTA did not affect the initial net H+ efflux elicited by 1 microM angiotensin II. The increase in steady state pHi was blocked by inhibitors of Na+/H+ exchange, amiloride (1 mM) and EIPA (10 microM). Angiotensin II also increased the rate of pHi recovery from intracellular acidosis at pHi values above approximately 6.9. During inhibition of Na+/H+ exchange the application of angiotensin II decreased steady state pHi. This acidosis was blocked by preincubation in dextrose-free solution containing 20.0 mM 2-deoxy-D-glucose and 10 microM EIPA. The positive inotropic effect of angiotensin II was markedly suppressed by amiloride. Angiotensin II exerts a concentration dependent stimulatory effect on Na+/H+ exchange in adult rabbit ventricular myocytes. This effect does not appear to involve changes in cytosolic calcium. During inhibition of Na+/H+ exchange, angiotensin II causes pHi to fall, perhaps by stimulating metabolic acid production. The positive inotropic action of angiotensin II depends, in part, on stimulation of Na+/H+ exchange.

Role of sodium-hydrogen exchange in the proliferation of immortalised lymphoblasts from patients with essential hypertension and normotensive subjects

The aim was to investigate the effect of ethylisopropylamiloride (EIPA), an inhibitor of Na+/H+ exchange, and of extracellular pH on the proliferation of immortalised lymphoblasts derived from patients with essential hypertension and normotensive controls. Na+/H+ exchange activity was determined in cells loaded with the fluorescent pH indicator BCECF. Cell proliferation was determined by FACS analysis, by cell counting, and from the incorporation of [3H]-thymidine. EIPA inhibited the Na+/H+ exchanger with an average KI value of 14 nM in all cell lines. Cell growth and DNA synthesis were only inhibited at EIPA concentrations > 10 microM suggesting a non-specific effect independent of Na+/H+ exchanger blockade. When extracellular pH was varied from 7.1 to 7.7 by changing the HCO3- concentration at constant PCO2, cell proliferation was optimal at pH 7.4, but reduced at acidic and alkaline pH in cells from normotensive and hypertensive subjects. The increased proliferation of lymphoblasts from hypertensive subjects persisted over the whole pH range. Comparable results were obtained when pH was altered by varying the PCO2 at constant HCO3-. Preincubation of cells with pertussis toxin inhibited serum stimulated DNA synthesis by 14.5% and 23.5% (P = 0.02) in cell lines from normotensive and hypertensive subjects. The enhanced Na+/H+ exchanger activity in lymphoblasts from patients with essential hypertension is obviously not the major determinant of the enhanced proliferation of these cells. The increased sensitivity of the growth of "hypersensitive" cell lines to pertussis toxin suggests a cellular alteration which resides upstream of Na+/H+ exchange activity and proliferation control.

Selective Accumulation of the Fluorescent pH Indicator,BCECF, in Vacuoles of Maize Root-Hair Cells

The vacuolar-type H+ -ATPase localized on the tonoplast membrane is believed to regulate aciditywithin the vacuolar lumen. However, the ability to monitor vacuolar pH in situ can be difficult because of the use of destructive assays based on cell sap pH or accumulation of weak bases, or elicit ion of wound responses after impaling cells with a pH-sensing microelectrode. The goal of our present study was to identify a fluorescent pH probe that is localized in vacuoles of ( Zea mays L.) root cells. When excised corn root segments were exposed to the ester derivative of 2′,7′ -bis-2-(carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) for 30 to 60 min, the root-hair cells preferentially became fluorescent. The distribution of the fluorescence within cells followed closest to that of the vacuole when fluorescent and phase contrast images were compared. The ratio of the fluorescence intensities at 535 nm using excitation wavelengths of 440 and 490 nm varied directly with pH between 4.5 and 7.5. The ratio of fluorescent intensities of BCECF-Iabeled root-hair cells indicated that the majority of the dye was localized in a compartment with an average pH of 5.8. The above data indicated that the BCECF was localized in the vacuoles of root-hair cells from maize seedlings, and the changes in the fluorescence ratio of BCECF may provide a means of monitoring changes of vacuolar pH in situ .

Depolarization-induced alkalinization (DIA) in rat hippocampal astrocytes.

1. Depolarization of glial cells causes their intracellular pH (pHi) to increase. To more completely characterize this depolarization-induced alkalinization (DIA) in mammalian astrocytes, we studied DIA in cultured rat hippocampal astrocytes. Astrocytes were loaded with the fluorescent pH indicator 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF), and pHi was monitored with the use of an imaging system. Cells were studied approximately 24 h after removing them from serum-containing culture medium. In HCO-3-buffered solution containing 3 mM K+, mean baseline pHi was 7.14 +/- 0.14 (mean +/- SD). 2. Astrocyte pHi rapidly and reversibly alkalinized when bath [K+] was increased from 3 to 12 mM. In HCO-3-buffered solution, mean DIA amplitude was 0.16 +/- 0.01 pH units, and mean rate of pHi change was 0.076 +/- 0.03 pH units/min. In contrast, DIA elicited in nominally HCO-3-free, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered solution was much smaller (0.03 +/- 0.01 pH units, 0.04 +/- 0.01 pH units/min; P < 0.0001), indicating that DIA was, in large part, a HCO-3-dependent process. Subsequent experiments were carried out in HCO-3-buffered solution. 3. The relationship between DIA and variable changes in bath [K+] was examined. Increasing bath [K+] from 3 to 6 mM produced a DIA of 0.07 +/- 0.04 pH units, and lowering [K+] to 0.5 mM resulted in an acid shift of 0.08 +/- 0.05 pH units. The effects of these changes in [K+] on membrane potential were measured in separate experiments by whole cell patch-clamp recording. On the basis of these data, it was possible to construct a relationship between Vm and pHi; shifting membrane potential approximately 10 mV resulted in a pHi shift of approximately 0.07. 4. Application of 0.5 mM Ba2+ depolarized Vm and elicited DIA in astrocytes. This indicated that depolarization, in the absence of an increase in [K+], could cause DIA. Application of Ba2+ also blocked K(+)-induced DIA, presumably because blockade of K+ channels prevented any depolarization by K+. 5. Cells with more alkaline baseline pHis exhibited larger and more rapidly developing DIAs. The mechanism of this effect is not known. 6. The timing of serum removal affected astrocyte DIA. Cells studied approximately 24 h after serum removal always exhibited robust DIA (mean = 0.16 +/- 0.01 pH units).(ABSTRACT TRUNCATED AT 400 WORDS)

Functional reconstitution in lipid vesicles of influenza virus M2 protein expressed by baculovirus: evidence for proton transfer activity

The influenza virus M2 protein was expressed from a recombinant baculovirus in Spodoptera frugiperda Sf9 cells, purified and reconstituted into artificial membrane vesicles. The specific inhibitor amantadine overcame the toxic activity of the protein and boosted the rate of M2 synthesis by a factor of 10, allowing yields of about 1 mg of purified M2 protein per g of Sf9 cells. M2 protein expressed in this system was phosphorylated and palmitoylated and displayed properties similar to the authentic virus protein. Purified wild-type M2 protein and an amantadine-resistant mutant M2 (M2 delta) with a deletion in the trans-membrane domain (amino acids 28 to 31) were incorporated into lipid vesicles, which were loaded with the fluorescent pH indicator pyranine. On imposition of an ionic gradient, M2 caused a decrease in intravesicular pH, which was susceptible to inhibition by 0.1 to 1 microM-rimantadine or N-ethyl-rimantadine. M2 delta behaved similarly but exhibited the expected drug resistance. These experiments indicate that isolated M2 functions as an ion channel and demonstrates in vitro M2-mediated proton translocation.

Polarized Na+/H+ exchange function is pliable in response to transepithelial gradients of propionate.

Short-chain fatty acids are produced at high concentration in the colonic lumen and stimulate electroneutral Na+ absorption by activating apical Na+/H+ exchange in colonocytes. We used an epithelial cell line derived from a human colon carcinoma (HT29-18-C1) to study activation of apical and basolateral Na+/H+ exchange by a short-chain fatty acid, propionate. Confluent cell monolayers on membrane filters were loaded with 2',7'-bis(2-carboxyethyl)-5 (and 6)-carboxyfluorescein (a fluorescent pH indicator) and intracellular pH was monitored with a digital fluorescence imaging microscope. Cells acidified by transient exposure to NH4Cl demonstrated both apical and basolateral Na+/H+ exchange. In this condition, apical Na+/H+ exchange was 50% of the total Na+/H+ exchange activity. Similar results were obtained when cells were bilaterally perfused with apical and basolateral propionate in an isosmotic medium (130 mM propionate at each membrane surface). However, apical Na+/H+ exchange was a significantly larger fraction (76%) of the total Na+/H+ exchange activity when cells were acidified by exposure to apical propionate alone. Conversely, in cells acidified by basolateral propionate alone, apical Na+/H+ exchange was 21% of the total Na+/H+ exchange activity. The change in relative activity was observed in individual cells which expressed both apical and basolateral Na+/H+ exchange and occurred rapidly (within 7 min). In the presence of transepithelial propionate gradients, all Na(+)-dependent alkalinization was sensitive to 3 microM 5-(N-ethyl-N-isopropyl)amiloride, a potent Na+/H+ exchange inhibitor. These results suggest that transepithelial gradients of short-chain fatty acids, which occur in vivo, can cause preferential activation of apical Na+/H+ exchange.

An increase in intracellular pH during neural induction in Xenopus.

In this paper, we show that an intracellular alkalinization of the dorsal ectoderm cells is among the earliest responses to neural induction in Xenopus. Planar explants of the dorsal marginal zone were prepared from embryos that had been microinjected during cleavage stages with the fluorescent pH indicator bis-carboxyethyl-carboxyfluorescein-dextran (BCECF-dextran), and intracellular pH (pHi) was monitored continuously by emission ratio microfluorimetry. During stage 10.5, the dorsal ectoderm cells undergo a sustained intracellular alkalinization of approximately 0.1 pH units in response to neural induction; in the absence of the inductive signal, the pH of the dorsal ectoderm cells decreases slightly. Ectoderm cells within planar explants of the ventral marginal zone show little change in pH during a similar period. This increase in intracellular pH is inhibited by 4, 4'-dihydrodiisothiocyanatostilbene-2, 2'-disulfonate (H2DIDS) or a low Na+/high Cl- medium, treatments that presumably affect anion transport. Under these conditions, expression of the anterior neural-specific homeobox gene engrailed is not detected, while the notochord-specific epitope recognized by the Tor-70 antibody is expressed in the presence of H2DIDS. This characteristic alkalinization is not evoked by pharmacological agents that reportedly alter ectodermal developmental pathways in Xenopus embryos, such as NH4Cl, phorbol esters, or cAMP-dependent protein kinase agonists. Our results suggest that an ionic regulatory event may participate in the regulation of gene expression in response to neural induction.

Recent Developments in Organically Doped Sol-Gel Sensors: A Microns-Scale Probe; Successful Trapping of Purified Polyclonal Antibodies; Solutions to the Dopant-Leaching Problem

ABSTRACTWe describe recent advances made in our laboratories in the general field of organically and bio-organically doped sol-gel sensors. The developments described are: (a) The first miniaturization of a sol-gel sensor down to the microns scale, with potential applications to near-field optical microscopy, using a fluorescent pH-indicator. (b) The first successful sol-gel encapsulation of purified polyclonal antibodies, and in particular an anti-nitroaromatics immunoglobulin, with which selective sensing of nitroaromatics, an important class of environmental pollutants, was demonstrated, (c) The leaching problem, occasionally encountered in doping procedures, is solved by two methodologies: First, TMOS polymerization at high acidity and low water content was found to result in non-leachable yet reactive matrices, as demonstrated with O2 sensing by excited state pyrene and with H+ sensing by excited state pyranine; and second, doping with molecules capable of forming a covalent bond within the encapsulating cage results in the permanent anchoring of the dopant. Thus, Methyl-Red, a pH indicator, was derivatized with a silylating residue, and a polymerizing TMOS was doped with it forming a pH-shifted indicator. With both methodologies, leachability was practically zero.

Continuous fluorometric measurement of intracellular pH and Ca2+ in perfused salivary gland and pancreas

Intracellular pH (pHi) has been measured in intact, perfused rat mandibular salivary glands loaded with the fluorescent pH indicator BCECF [2',7'-bis(2-carboxyethyl)-5(6)- carboxyfluorescein]. Glands mounted in the cuvette of a conventional bench-top spectro-fluorometer were perfused for 5 min with the acetoxymethyl ester of BCECF and fluorescence was measured ratiometrically at 6-s intervals. The mean value of pHi in glands perfused with a HCO3(-)-free, N-2-hydroxyethylpiperazine-N'-2- ethanesulphonic acid (HEPES)-buffered solution at 37 degrees C was 7.36 +/- 0.01 (n = 52) which is comparable with values obtained by 31P nuclear magnetic resonance (NMR) spectroscopy. NMR data confirmed that the BCECF loading period was accompanied by a transient acidification of the cells, but there was no significant change in the content of the major phosphorus metabolites. Changes in pHi in response to NH4Cl pulses and acetylcholine stimulation were comparable with results reported previously for isolated acini. Additional, preliminary experiments show that the method can also be used to monitor intracellular Ca2+ (using fura-2) in perfused salivary glands, and can be adapted for studies of the isolated, perfused pancreas.

Effects of repetitive stimulation, veratridine and ouabain on cytoplasmic pH in frog nerve fibres: role of internal Na +

Changes of cytoplasmic pH (pH i) in frog nerve fibres during repetitive stimulation have been measured using the fluorescent pH indicator dye fluorescein diacetate (FDA). Under control conditions repetitive (10–50 Hz) stimulation caused only a very small decrease in pH i (by 0.015–0.06 pH units). Modification of Na + channels by veratridine (VER, 10, μM) greatly increased this stimulus-evoked (SE) internal acidification. Blockade of the Na +-K + pump by ouabain (0.5 mM) enhanced the effects VER and prevented pH i recovery after the termination of repetitive stimulation. A similar inhibition of post-stimulatory recovery of pH i was observed after replacement of external Na + with Li +, which is not accepted by the Na +-K + pump instead of Na +. These data suggest that SE intracellular acidification in nerves results from or is closely associated with an increase in [Na +] i. Treatments that promote Na + influx and accumulation of Na + inside the fibre enhance reduction of pH i. Li + can be substituted for Na + in this process.

Hypertensive sodium-proton exchanger phenotype persists in immortalized lymphoblasts from essential hypertensive patients. A cell culture model for human hypertension.

An enhancement of sodium-proton exchange activity is a frequently observed ion transport abnormality in essential hypertension. The cellular basis for this has not yet been elucidated. Due to the lack of a specific cell culture system it has been impossible to distinguish between intrinsic cellular abnormalities and influences exerted by the hypertensive neurohumoral milieu. Using Epstein-Barr virus we have immortalized lymphocytes from controls and from patients with essential hypertension that exhibited enhanced sodium-proton exchanger activity. Sodium-proton exchanger activity was determined in cells loaded with the fluorescent cytosolic pH indicator 2'7'-biscarboxyethyl-5,6-carboxyfluorescein acetoxymethylester (BCECF) after pretreatment with 250 nM of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate for 10 min. Cell lines from hypertensive patients displayed higher Vmax values of sodium-proton exchange than those from normotensive controls (129.6 +/- 30.0 vs. 77.1 +/- 13.2 mmol H+/min.; P < 0.001). Hill coefficients for H+ were distinctly lower in hypertension compared to normotension (1.12 +/- 0.12 vs. 1.50 +/- 0.14; P < 0.0001). The enhanced antiporter activity in cell lines from hypertensive patients was not accompanied by a corresponding increase in steady-state NHE-1 mRNA transcript levels, which argues against overexpression of antiporter protein in hypertension. The cells from hypertensive patients with high sodium-proton exchange activity proliferated distinctly faster than those from normotensive controls. These human cell lines represent a novel model to study the mutual interaction between sodium-proton exchange and cell proliferation, and may provide insights into the alterations in ion transport observed in a group of patients with essential hypertension.

Fluorescent cellular indicators are extruded by the multidrug resistance protein.

In this report we show that NIH-3T3 mouse fibroblasts stably expressing the human multidrug transporter (MDR1 or P-glycoprotein), in contrast to the control NIH-3T3 cells, actively extrude the hydrophobic acetoxymethyl ester (AM) derivatives used for cellular loading of various fluorescent calcium and pH indicators. This dye extrusion is blocked by competing substrates and inhibitors of the multidrug transporters, e.g. by verapamil, vincristine, sodium orthovanadate, oligomycin, and a monoclonal anti-MDR1 antibody. The hydrophilic free acid forms of the indicators are not exported by MDR1. We also demonstrate that in isolated cell membranes the MDR1-ATPase, similar to that by known substrates of the transporter, is stimulated by the AM derivatives of fluorescent dyes whereas the free acid forms of the dyes are without effect. Since (i) the AM derivatives of the fluorescent indicators rapidly permeate the cell membrane and are readily cleaved by high activity and large capacity cytoplasmic esterases and (ii) the free acid forms are not substrates for export by MDR1, the observations above suggest that dye extrusion by MDR1 may occur without a cytoplasmic appearance of the AM compounds. These data also call attention to the possible interaction of widely used hydrophobic fluorescent indicators with MDR1 and offer an efficient detection of MDR1-expressing tumor cells as well as a screening method for examining drug interactions with the multidrug transporter.

Intracellular pH‐based controlled cultivation of yeast cells: II. Cultivation methodology

Intracellular pH (pH(i)) was measured on-line in a bioreactor using a fluorescent pH(i) indicator, 9-aminoacridine, and controlled fed-batch cultivations of yeast cells based on pH(i) (FB-pH(i)) were performed. In FB-pH(i) cultivations, automated glucose additions were made to the culture in response to culture pH(i). The average ethanol (an-aerobic product) yield was significantly lower [0.12 g g(-1) glucose in fed-batch pH(i) cultivations with 100 ppm glucose additions (FB-pH(i)-100 cultivation) vs. 0.48 g g(-1) glucose in batch] and cell yield was higher (0.54 g g(-1) glucose in FB-pH(i)-100 cultivation vs. 0.3 g g(-1) glucose in batch) compared to batch cultivation. An expression has been derived to calculate changes in pH(i) from measured fluorescence values when the cell concentration increases during growth. Cultivations based on pH(i), performed with different magnitudes of glucose addition (100, 50, and 10 ppm additions), showed that lower magnitudes of glucose addition resulted in lower ethanol yields while cell yield remained unaffected. The ratio of specific oxygen uptake rate to specific glucose uptake rate (OUR/GUR) increased with decreased in magnitude of glucose additions in FB-pH(i) cultivations, suggesting that the culture aerobic state was higher when the magnitude of glucose addition was lower. The average cell productivity in FB-pH(i) cultivations was 29% higher than in batch cultivation. Cells were also cultivated at high OUR conditions, and the results are compared with other cultivations.

Continuous recording of intramitochondrial pH with fluorescent pH indicators: novel probes and limitations of the method.

In addition to 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) used so far to monitor intramitochondrial pH, two other fluorescent pH indicators, 4',5'-dimethyl-5(6)-carboxyfluorescein (DMCF) and carboxyseminaphthofluorescein (carboxy-SNAFL-1), were applied for this purpose. These probes are taken up by isolated rat liver mitochondria in form of diacetate esters, hydrolyzed within mitochondria to free acids, and respond to changes of intramitochondrial pH by changing their fluorescence emission intensity. With all three probes energization of mitochondria by electron donors or acceptors was accompanied by fluorescence changes characteristic for alkalization, whereas deenergization by respiratory inhibitors or protonophores produced changes typical for acidification. Contrary to this, transition from State 4 to State 3, known to shift intramitochondrial pH towards acidification (equivalent to a decrease of delta pH), was accompanied by paradoxical responses of the fluorescent pH probes used: the fluorescence of DMCF increased as if the matrix compartment became more alkaline, the fluorescence of BCECF, measured in single excitation/emission wavelength mode, did not change, and the fluorescence of carboxy-SNAFL-1 could be interpreted as either alkalization or acidification, depending on the excitation/emission wavelength pair used. It was shown that depletion of intramitochondrial Mg2+ and Ca2+ using divalent metal ionophore A23187 decreased fluorescence intensity with all three probes examined, whereas subsequent addition of Mg2+ or Ca2+ increased the fluorescence. It is therefore proposed that the atypical response of intramitochondrial pH indicators upon State 4- State 3 transition is due to changes of intramitochondrial free Mg2+, as related to different complexing abilities of ATP and ADP towards magnesium.

Assay of Proton-Coupled Glycolate and D-Glycerate Transport into Chloroplast Inner Envelope Membrane Vesicles by Stopped-Flow Fluorescence

The transport of glycolate and D-glycerate across the inner envelope membrane of intact chloroplasts is rapid and mediated by a translocator with proton/substrate symport activity. The true initial rate of glycolate or D-glycerate transport could not be measured by conventional methods. To resolve the initial rates of glycolate and D-glycerate transport, a stopped-flow fluorescence assay was developed that allows the indirect observation of transport from about 4 ms after mixing. Inner envelope vesicles from pea (Pisum sativum) or spinach (Spinacia oleracea) chloroplasts were loaded with the fluorescent pH indicator pyranine (8-hydroxypyrene-1,3,6-trisulfonic acid) by a freeze-thaw sonication protocol. A rapid quenching of pyranine fluorescence was detected after mixing the vesicles with either glycolate or D-glycerate. This quenching was the result of acidification of the interior of the vesicles. D-Glycerate- or glycolate-induced acidification displayed saturation kinetics and was inhibited by pretreatment of the vesicles with N-ethylmaleimide. D-Glycerate was more effective than L-glycerate in causing the pH decrease. Also, L-mandelate inhibited D-glycerate-induced acidification much more strongly than D-mandelate. The glycolate/D-glycerate-induced pH decrease is consistent with glycolate/D-glycerate translocator activity. The assay was placed on a quantitative basis by converting fluorescence changes to pH and measuring the internal buffering capacity of the vesicles. The rates of transport across the inner envelope membrane were estimated to be as fast, if not faster, than those of transport in intact chloroplasts.

Intracellular pH mapping with SNARF-1 and confocal microscopy. I: A quantitative technique for living tissue and isolated cells

A fluorescent pH indicator in conjunction with confocal microscopy, was used to map intracellular pH in a variety of cells and tissues with high spatial resolution. The new pH-sensitive fluorescent probe SNARF-1 was excited with the 488 nm band of the argon ion laser of a Bio-Rad MRC-500 confocal microscope. Ratio images were created with pixel-by-pixel division, with the intensity of these images representing a function of pH, that is independent of dye concentration, photobleaching or path length. Cell cultures of rat aortic smooth muscle were loaded with 20 μм SNARF-1/AM for 20 min at 37°C. Intracellular pH levels were calibrated in situ by treatment of each cell with nigericin (20 μм) in solutions of known pH. The cytosolic pH of the majority of cells was uniform, however, pH gradients were evident between the cytosol and nuclear regions, indicating the ability of this technique to map intracellular and intraorganelle pH. Rat C6 glioblastoma spheroids were cultured then loaded with SNARF-1/AM at 10°C for 90 min. The pH values were calibrated in vitro, using SNARF-1 acid in buffered solutions of known pH. Ratio images of the bisected spheroids showed a marked gradient in pH from the outer cells compared with central necrotic cells. The degree of involvement of acidification in muscle fatigue was investigated by simultaneously determining force generation and intracellular pH in individual fibres of an intact rat muscle. The investigation was performed during a stimulation protocol which induced significant fatigue in the force response of the muscle. The fatigue protocol induced little change in cytosolic pH in the fibres. We show that the use of SNARF-1, in conjunction with confocal microscopy is a powerful technique for accurately mapping pH within single cells, multicellular tissues and intact organs, as well as for accurately recording dynamic changes in pH.

A novel method for measuring intracellular pH: Effect of neutron irradiation on pH i of transformed cells

A new flow cytometric method was developed for measuring the intracellular pH (pH i) of mammalian cells using a fluorescent pH indicator dye 2′,7-bis-(2-carboxyethyl)-5(and -6)-carboxyfluorescein (BCECF). Emission intensities (or their ratios) measured from BCECF-loaded cells can be converted into absolute pH i values using appropriate calibration curves. By comparison of several possible measuring and data evaluation procedures a double-ratio method was suggested as the most advantageous protocol to yield reliable intracellular pH data. This method allows pH i to be determined on a cell-by-cell basis corrected for cell volume and change in geometry of input—output optics of the flow cytometer. Our method applies a standard calibration curve and does not necessitate its reconstruction for each new set of measurements. Cells of the OKT-4 and OKT-8 hybridoma lines were exposed to neutron irradiation of different doses. Irradiated cells underwent a biphasic alkalinization; an instantaneous effect detected within 1.5 h was found to be intensified over 24 h. For the interpretation of data we suggest that the increase in cytoplasmic pH following neutron treatment is evoked by two mechanisms.

Phenylephrine and ATP enhance an amiloride insensitive bicarbonate-dependent alkalinizing mechanism in rat single cardiomyocytes

To expel the excess protons generated during a cellular acidification and to fully recover basal intracellular pH (pHi), cardiac cells rely on the amiloride-sensitive Na/H antiport. We report that rat single ventricular cardiomyocytes, loaded with the fluorescent pH indicator Snarf-1 and treated with inhibitors of the Na/H antiport, amiloride or its analogues, partially restored their pHi through a bicarbonate-dependent mechanism following an acidosis (imposed by the ammonia-pulse technique). In the presence of ethylisopropylamiloride (10 microM) or amiloride (1 mM) and 25 mM bicarbonate in the extracellular solution, the average time that cells needed to recover half of their pHi, following the removal of 20 mM NH4Cl, was 3.4 min, while the rate of proton efflux was calculated to be 2.0 mM/min. The stilbene derivative, 4-4'-di-isothiocyanostilbene-2,2'-disulphonate (DIDS 200 microM), a known blocker of anion transporters, inhibited this recovery. Both phenylephrine (100 microM, 3 microM propranolol present), an alpha 1-adrenoceptor agonist, and ATP (10 microM), a purinergic agonist, significantly enhanced the rate of proton efflux that was due to this HCO3-dependent alkalinizing mechanism. Phenylephrine and ATP also shortened by three-fold the time that a myocyte needed to recover half of its initial pHi. This bicarbonate-dependent alkalinizing mechanism could provide an additional means by which cardiac cells recover their pHi from acidosis, especially under conditions in which the Na/H antiport is inhibited. Furthermore, catecholamines and ATP, which are released under various pathophysiological conditions often associated with intracellular acidosis, could play an important role in the modulation of pHi under these conditions.

Calcium ionophores increase intracellular pH in chicken granulosa cells.

Several hormone agonists exert their physiological actions by triggering an inositol phospholipid-Ca2+ signalling cascade and cytosolic alkalinization. Although calcium ionophores have been used extensively to probe the role of Ca2+ in the regulation of steroidogenesis in granulosa cells, the precise relationship between changes in intracellular Ca2+ (Ca2+i) and pH (pHi) is unclear. In the present study we have used a fluorescent pH indicator, 2'7'-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein, to examine the influence of two Ca2+ ionophores, ionomycin and 4-Bromo-A23187 (4-Br-A23187), on pHi in chicken granulosa cells. Chicken granulosa cells from the largest preovulatory follicle were incubated with Ca2+ ionophores (0-2 microM) and/or inhibitors of Na+/H+ antiport (amiloride, dimethylamiloride and ethylisopropyl amiloride; 0.5, 5 and 50 microM respectively) in the presence of Na+ (or choline+; 0-144 mM) and/or Ca2+ (0-10 mM). Ionomycin or 4-Br-A23187 elicited a rapid and sustained cytosolic alkalinization. The magnitude of increase in pHi was dependent on the concentration of the Ca2+ ionophore and the presence of extracellular Ca2+ but independent of extracellular Na+. Pretreatment of the cells with amiloride or its analogues failed to affect the increase in pHi induced by the Ca2+ ionophores significantly. These findings demonstrate that, in addition to their widely reported effects on Ca2+i redistribution in granulosa cells, 4-Br-A23187 and ionomycin cause Ca(2+)-dependent cytosolic alkalinization. This action of the Ca2+ ionophores is independent of the Na+/H+ antiport. Caution must be exercised in using Ca2+ ionophores as probes to define the role of Ca2+ in the regulation of granulosa cell function.

Cytoplasmic Acidification Induced by Inorganic Phosphate Uptake in Suspension Cultured Catharanthus roseus Cells

Cytoplasmic acidification during inorganic phosphate (Pi) absorption by Catharanthus roseus cells were studied by means of a fluorescent pH indicator, 2',7'-bis-(2-carboxyethyl)-5 carboxyfluorescein (acetomethylester) (BCECF), and (31)P-nuclear magnetic resonance spectroscopy. Cytoplasmic acidification measured by decrease in the fluorescence intensity started immediately after Pi application. Within a minute or so, a stable state was attained and no further acidification occurred, whereas Pi absorption was still proceeding. As soon as Pi in the medium was exhausted, cytoplasmic pH started to recover. Coincidentally, the medium pH started to recover toward the original acidic pH. The Pi-induced changes in the cytoplasmic pH were confirmed by (31)P-nuclear magnetic resonance study. Maximum acidification of the cytoplasm induced by 1.7 millimolar Pi was 0.2 pH units. Vacuolar pH was also affected by Pi. In some experiments, but not all, pH decreased reversibly by 0.2 to 0.3 pH units during Pi absorption. Results suggest that the cytoplasmic pH is regulated by proton pumps in the plasma membrane and in the tonoplast. In addition, other mechanisms that could consume extra protons in the cytoplasm are suggested.