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.     

Regulation of the mitochondrial Na+/Ca2+ antiport by matrix pH

The effect of matrix pH (pHi) on the activity of the mitochondrial Na+/Ca2+ antiport has been studied using the fluorescence of SNARF-1 to monitor pHi and Na(+)-dependent efflux of accumulated Ca2+ to follow antiport activity. Heart mitochondria respiring in a KCl medium maintain a large delta pH (interior alkaline) and show optimal Na+/Ca2+ antiport only when the pH of the medium (pH0) is acid. Addition of nigericin to these mitochondria decreases delta pH and increases the membrane potential (delta psi). Nigericin strongly activates Na+/Ca2+ antiport at values of pH0 near 7.4 but inhibits antiport activity at acid pH0. When pHi is evaluated in these protocols, a sharp optimum in Na+/Ca2+ antiport activity is seen near pHi 7.6 in the presence or absence of nigericin. Activity falls off rapidly at more alkaline values of pHi. The effects of nigericin on Na+/Ca2+ antiport are duplicated by 20 mM acetate and by 3 mM phosphate. In each case the optimum rate of Na+/Ca2+ antiport is obtained at pHi 7.5 to 7.6 and changes in antiport activity do not correlate with changes in components of the driving force of the reaction (i.e., delta psi, delta pH, or the steady-state Na+ gradient). It is concluded that the Na+/Ca2+ antiport of heart mitochondria is very sensitive to matrix [H+] and that changes in pHi may contribute to the regulation of matrix Ca2+ levels.     

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.     

The opposite effect of bivalent cations on cytochrome b5 reduction by NADH:cytochrome b5 reductase and NADPH:cytochrome c reductase.

The effects of bivalent cations on cytochrome b5 reduction by NADH:cytochrome b5 reductase and NADPH:cytochrome c reductase were studied with the proteinase-solubilized enzymes. Cytochrome b5 reduction by NADH:cytochrome b5 reductase was strongly inhibited by CaCl2 or MgCl2. When 1.2 microM-cytochrome b5 was used, the concentrations of CaCl2 and MgCl2 required for 50% inhibition (I50) were 8 and 18 mM respectively. The inhibition was competitive with respect to cytochrome b5. The extent of inhibition by CaCl2 or MgCl2 was much higher than that by KCl or other alkali halides. In contrast, cytochrome b5 reduction by NADPH:cytochrome c reductase was extremely activated by CaCl2 or MgCl2. In the presence of 5 mM-CaCl2, the activity was 24-fold higher than control when 4.4 microM-cytochrome b5 was used. The magnitude of activation by CaCl2 was 2-3-fold higher than that by MgCl2. The activation by these salts was much higher than that by KCl, indicating that bivalent cations play an important role in this activation. The mechanisms of inhibition and activation by bivalent cations of cytochrome b5 reduction by these two microsomal reductases are discussed.     

Preparation and polymerization of skeletal muscle ADP-actin

Skeletal muscle ADP-G-actin was prepared from ADP-F-actin, which had been freed of residual ATP by repeated sonication, by depolymerization in 5 mM Tris-HCl, 0.2 mM ADP, 0.2 mM dithiothreitol, 0.1 mM CaCl2, 0.1 mM MgCl2, and 0.01% NaN3, pH 8.0. The ADP had been freed of traces of ATP by DEAE-chromatography, and 5 microM diadenosine pentaphosphate was added to inhibit myokinase activity. The kinetics of the spontaneous polymerization of ADP-actin in 1 mM MgCl2 + 0.1 M KCl were compatible with the simple nucleation-elongation model previously used to explain the polymerization of ATP-actin. The critical concentrations of ADP-actin were 8.0 and 2.0 microM in 1 mM MgCl2 and 1 mM MgCl2 + 0.1 M KCl, respectively. These values are 20-30-fold higher than the corresponding values in ATP. Using cross-linked actin trimers to nucleate polymerization, the association rate constants were found to be 0.8 and 0.9 microM-1 S-1 in MgCl2 and MgCl2 + KCl, respectively, which are 0.4 and 0.2 times the values for ATP-actin. The dissociation rate constants, calculated from the critical concentrations and the association rate constants, were 6.4 and 1.8 S-1, respectively, which are 10 and 5 times the corresponding values for ATP-actin.     

Effect of hydrogen peroxide on intracellular pH in the human atrial myocardium

The cardiac injury observed during myocardial ischemia and reperfusion has been shown to be a consequence of a complex mechanism in which the accumulation of hydrogen peroxide (H2O2) and other oxygen free radicals (OFRs), and intracellular pH (pHi) are believed to play a major role. However, the effect of H2O2 on pHi has not been well characterized in the human atrial myocardium. In the present study, we superfused hydrogen peroxide into the human atrial tissue in order to assess the effects of oxygen free radicals on the pHi, and, furthermore, to test the ability of certain potential cardioprotective agents, including scavengers of the *OH free radical (N-(mercaptopropionyl)-glycine; N-MPG) and the HOCl free radical (L-methionine), to protect against oxidative-induced pHi challenge. The human atrial tissues were obtained from patients undergoing corrective open-heart surgery. The ratiometric recordings of pHi were measured using the pH-sensitive, dual-excitation and dual-emission fluorescent dye BCECF (2', 7'-bis(carboxyethyl)-5, 6-carboxyfluorescein acetoxymethyl ester). By continuously monitoring pHi changes in human atrial myocardium, we have found, for the first time, that (a) H2O2 (30 microM-3 mM) induced a significant dose-dependent intracellular acidosis, (b) N-MPG caused a significant block on the intracellular acidosis induced by 3 mM H2O2, whereas L-methionine did not, and (c) Hoe 694, a specific Na+/H+ exchanger (NHE) inhibitor, caused a similar extents like that induced by 3 mM H2O2. Our data suggest that the effects of H2O2 are caused mainly through the generation of *OH, which is attributed to the intracellular acidosis seen in the human atrial trabecular muscle. The possible underlying mechanism for H2O2-induced acidosis is likely due to its inhibition on the activity of NHE and other acid extruders, as the pHi changes after H2O2 exposure could be detected even though the activity of NHE was completely blocked by 30 mM Hoe 694.     

Potassium-Induced Release of Endogenous Amino Acids in the Guinea Pig Cochlea

Guinea pig cochleae were perfused with high-potassium solutions to depolarize hair cells artificially and induce the release of afferent neurotransmitter. Sequential injections of artificial perilymph containing 5 mM KCl, then 50 mM KCl, and finally 5 mM KCl were made into the scala tympani. This injection sequence was conducted under either normal divalent-cation conditions (2.0 mM CaCl2, 1.0 mM MgCl2) or calcium-deficient conditions intended to antagonize evoked transmitter release (0.1 mM CaCl2, 20.0 mM MgCl2). The levels of 21 endogenous primary amines in effluent collected from the scala vestibuli were determined by gradient-elution, reverse-phase HPLC using o-phthaldialdehyde-thiol adducts with fluorescence detection. Analyses indicated effluent concentrations of glutamate, taurine, and a coeluting taurine-gamma-aminobutyrate (GABA) fraction (but not GABA alone) increased significantly after exposure to 50 mM KC1 and returned to baseline levels after reintroduction of 5 mM KC1 under normal divalent-cation conditions. Correspondent changes in the release of these constituents were significantly attenuated under calcium-deficient conditions. This was not the case for potassium-induced changes in the release of arginine, aspartate, and isoleucine. These data are consistent with the hypothesis that the receptoneuronal transmitter is glutamate and further suggest a calcium-dependent mechanism involving taurine.     

Intracellular pH in individual pituitary cells: measurement with a dual emission pH indicator

Intracellular pH (pHi) can now be measured at the single cell level using dual emission wavelength microspectrofluorimetry with the fluorescent pH indicator SNARF 1 and its membrane permeant acetoxymethyl ester (SNARF 1/AM). We measured pHi of individual pituitary cells under both basal and stimulated conditions. The emitted fluorescence of SNARF 1 probe was calibrated following experimental manipulations of pHi in two types of rat pituitary cells. The calibration curves obtained in the two cell types were identical. We observed a Gaussian distribution of individual pHi with a wide dispersion (6.95 to 8) in the two cell populations. TRH (10(-7) M) and ionomycin (5 microM) induced a transient acidification followed by a sustained alkalinization, whereas K+ (50 mM) depolarization only exerted a transient acidification. These results show that the dual emission pH indicator SNARF 1 can be used to reliably investigate changes in pHi in individual endocrine cells.     

An Na(+)-independent short-chain fatty acid transporter contributes to intracellular pH regulation in murine colonocytes

Short-chain fatty acids (SCFAs) are the major anions in the colonic lumen. Experiments studied how intracellular pH (pHi) of isolated colonocytes was affected by exposure to SCFAs normally found in the colon. Isolated crypt fragments were loaded with SNARF-1 (a fluorescent dye with pH-sensitive excitation and emission spectra) and studied in a digital imaging microscope. Intracellular pH was measured in individual colonocytes as the ratio of fluorescence intensity in response to alternating excitation wavelengths (575/505 nm). After exposure to 65 mM acetate, propionate, n-butyrate, or iso-butyrate in isosmotic Na(+)- free media (substituted with tetramethylammonia), all colonocytes acidified rapidly and then > 90% demonstrated a pHi alkalinization (Na(+)-independent pHi recovery). Upon subsequent removal of the SCFA, pHi alkalinized beyond the starting pHi (a pHi overshoot). Using propionate as a test SCFA, experiments demonstrate that the acidification and pHi overshoot are explained by transmembrane influx and efflux of nonionized SCFA, respectively. The basis for the pHi overshoot is shown to be accumulation of propionate during pHi alkalinization. The Na(+)-independent pHi recovery (a) demonstrates saturable propionate activation kinetics; (b) demonstrates substrate specificity for unmodified aliphatic carbon chains; (c) occurs after exposure to SCFAs of widely different metabolic activity, (d) is electroneutral; and (e) is not inhibited by changes in the K+ gradient, Cl- gradient or addition of the anion transport inhibitors DIDS (1 mM), SITS (1 mM), alpha-cyano-4-hydroxycinnamate (4 mM), or probenicid (1 mM). Results suggest that most mouse colonocytes have a previously unreported SCFA transporter which mediates Na(+)-independent pHi recovery.     

Use of a D-optimal design with constrains to quantify the effects of the mixture of sodium,potassium, calcium and magnesium chloride salts on the growth parameters of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The combined effect of NaCl, KCl, CaCl(2), and MgCl(2) on the water activity (a (w)) and the growth parameters of Saccharomyces cerevisiae was studied by means of a D-optimal mixture design with constrains (total salt concentrations < or = 9.0%, w/v). The a (w) was linearly related to the concentrations of the diverse salts; its decrease, by similar concentrations of salts, followed the order NaCl > CaCl(2) > KCl > MgCl(2), regardless of the reference concentrations used (total absence of salts or 5% NaCl). The equations that expressed the maximum specific growth (mu (max)), lag phase duration (lambda), and maximum population reached (N (max)) showed that the values of these parameters depended on linear effects and two-way interactions of the studied chloride salts. The mu (max) decreased as NaCl and CaCl(2) increased (regardless of the presence or not of previous NaCl); however, in the presence of a 5% NaCl, a further addition of KCl and MgCl(2) markedly increased mu (max). The lambda was mainly affected by MgCl(2) and the interactions NaCl x CaCl(2) and CaCl(2) x MgCl(2). The further addition of NaCl and CaCl(2) to a 5% NaCl medium increased the lag phase while KCl and MgCl(2) had negligible or slightly negative effect, respectively. N (max) was mainly affected by MgCl(2) and its interactions with NaCl, KCl, and CaCl(2); MgCl(2) stimulated N (max) in the presence of 5% NaCl while KCl, NaCl, and CaCl(2) had a progressive decreasing effect. These results can be of interest for the fermentation and preservation of vegetable products, and foods in general, in which this yeast could be present.     

Sodium coupled bicarbonate influx regulates intracellular and apical pH in cultured rat caput epididymal epithelium

BACKGROUND: The epithelium lining the epididymis provides an optimal acidic fluid microenvironment in the epididymal tract that enable spermatozoa to complete the maturation process. The present study aims to investigate the functional role of Na(+)/HCO(3)(-) cotransporter in the pH regulation in rat epididymis. METHOD/PRINCIPAL FINDINGS: Immunofluorescence staining of pan cytokeratin in the primary culture of rat caput epididymal epithelium showed that the system was a suitable model for investigating the function of epididymal epithelium. Intracellular and apical pH were measured using the fluorescent pH sensitive probe carboxy-seminaphthorhodafluor-4F acetoxymethyl ester (SNARF-4F) and sparklet pH electrode respectively to explore the functional role of rat epididymal epithelium. In the HEPES buffered Krebs-Henseleit (KH) solution, the intracellular pH (pHi) recovery from NH(4)Cl induced acidification in the cultured caput epididymal epithelium was completely inhibited by amiloride, the inhibitor of Na(+)/H(+) exchanger (NHE). Immediately changing of the KH solution from HEPES buffered to HCO(3)(-) buffered would cause another pHi recovery. The pHi recovery in HCO(3)(-) buffered KH solution was inhibited by 4, 4diisothiocyanatostilbene-2,2-disulfonic acid (DIDS), the inhibitor of HCO(3)(-) transporter or by removal of extracellular Na(+). The extracellular pH measurement showed that the apical pH would increase when adding DIDS to the apical side of epididymal epithelial monolayer, however adding DIDS to the basolateral side had no effect on apical pH. CONCLUSIONS: The present study shows that sodium coupled bicarbonate influx regulates intracellular and apical pH in cultured caput epididymal epithelium.     

Nucleases from Brevibacterium ammoniagenes differing in their effects on chromatin]

A method of isolation of three different, partially purified deoxyribonucleases from the cells of Brevibacterium ammoniagenes is descrirbed. The enzyme preparations were activated by various bivalent metal ions: 50mM MgCl2 (I), 5 mM CaCl2+5 mM MgCl2 (II), 10 mM CaCl2 (III), and had different pH optima -- 8.8 (I), 7.2 (II) and 8.2 (III). In the isolated nuclei of rat brain the first and third fractions split chromatin at the internucleosomal sites with a formation of nucleosomes -- structural subunits of chromatin. The second fraction exhibited no structural specificity for chromatin. A possible use of the enzymes for the analysis of chromatin structure is discussed.     

Inhibitory effect of Ca2+ on formation of Mg2(+)-mediated two-dimensional hexagonal lattice structure by an R-form lipopolysaccharide from Klebsiella pneumoniae

When the R-form lipopolysaccharide (LPS) from Klebsiella pneumoniae strain LEN-111 (O3-:K1-), from which cationic material had been removed by electrodialysis, was suspended in 50 mM Tris buffer at pH 8.5 containing 0.1 mM or higher concentrations of MgCl2, it formed an ordered two-dimensional hexagonal lattice structure and its center-to-center distance (lattice constant) depended upon the concentration of MgCl2 and reached the shortest value (14 nm) at 10 mM. In contrast, in the presence of 0.1 to 10 mM CaCl2 in place of MgCl2, the electrodialyzed LPS did not form such an ordered hexagonal lattice structure but formed an irregular network structure with a center-to-center distance of 19 to 20 nm. We investigated interaction of Mg2+ and Ca2+ in formation of the hexagonal lattice structure by the electrodialyzed LPS suspended in 50 mM Tris buffer at pH 8.5. When 0.1 mM or higher concentrations of CaCl2 were mixed with 1 mM MgCl2 or when 1 mM or higher concentrations of CaCl2 was mixed with 10 mM MgCl2, the electrodialyzed LPS did not form the hexagonal lattice structure of the magnesium salt type but formed the irregular network structure of the calcium salt type. In the coexistence of equimolar or higher concentrations of CaCl2 together with 1 or 10 mM MgCl2, the binding of Mg to the electrodialyzed LPS was significantly inhibited and, conversely, the binding of Ca was enhanced as compared with when MgCl2 or CaCl2 was present alone. However, the coexistence of 10 times less molar concentrations of CaCl2 did not significantly inhibit the binding of Mg to the electrodialyzed LPS. Therefore, the inhibition of formation of the Mg2(+)-mediated hexagonal lattice structure of the electrodialyzed LPS by equimolar or higher concentrations of CaCl2 accompanied the inhibition of binding of Mg but that by 10 times less molar concentrations of CaCl2 did not accompany it.     

Measurement of myosin adenosinetriphosphatase and myosin content in cultured heart cells

An assay specific for myosin ATPase in whole-cell extracts of cultured heart cells has been developed. Myosin ATPase is measured by the production of Pi from ATP in the presence of high ionic strength (0.5 M KCl) at pH 9.1. Enzyme activity is maximal with 10 mM CaCl2 and completely inhibited with 5 mM MgCl2. Spontaneously beating myocytes grown in the presence of 10% newborn calf serum and 0.1 mM 5-bromo-2'-deoxyuridine show a significant rise in myosin ATPase between Days 1 and 4 in culture. The measurement of myosin ATPase allows for the quantitation of cellular myosin content, and can be used to assess changes in myosin content that occur during growth, development, and cellular repair.     

Difference in kinetic properties of phosphorylated intermediates formed in the forward and backward reactions of solubilized Ca2+-ATPase of sarcoplasmic reticulum

Solubilized Ca2+-ATPase (SSR) was prepared by solubilizing fragmented sarcoplasmic reticulum (FSR) with a nonionic detergent (C12E8) then displacing the detergent with Tween 80, using a DEAE-cellulose column. The kinetic properties of the phosphorylated intermediate (EP) formed by the reaction of SSR with ATP were compared with those of EP formed by the reaction with Pi. The time course of decay of E32P formed with 4 microM AT32P in the presence of 19 mM CaCl2 and 10 mM MgCl2 (forward reaction) was measured by adding 0.4 mM unlabeled ATP and 10 mM Pi at pH 6.0 and 30 degrees C. The rate of E32P decay was accelerated by 0.4 mM ADP. On the other hand, when the time course of decay of E32P formed with 10 mM 32Pi in the presence of 5 mM EGTA and 10 mM MgCl2 (backward reaction) was measured by adding 0.4 mM unlabeled ATP and 15 mM CaCl2, the rate of E32P decay was unaffected by 0.4 mM ADP. AT32P was produced on adding ADP to E32P formed with AT32P in the presence of 10 mM CaCl2 and 10 mM MgCl2, while no AT32P was produced on adding ADP to E32P formed with 32Pi in the presence of 5 mM EGTA and 10 mM MgCl2, even when 15 mM CaCl2 was added simultaneously with ADP.     

Intracellular calibration of a pH-sensitive dye in isolated, perfused salamander proximal tubules

We evaluated the dye 4',5'-dimethyl-5-(and -6-) carboxyfluorescein (Me2CF) for determining the intracellular pH(pHi) of isolated, perfused proximal tubules of the salamander. The intracellular absorbance spectrum, corrected for the intrinsic absorbance of the tubule, was obtained once per second. The dye was incorporated into tubule cells by exposing them to the membrane-permeable precursor 4',5'-dimethyl-5- (and -6-) carboxyfluorescein diacetate. The introduction of the dye had no significant effect on either pHi or cell voltage transients. Compared with dye contained in a cuvette, intracellular dye had a peak absorbance that was red-shifted by approximately 5 nm, and an apparent pK that was increased by approximately 0.3. These differences precluded an accurate calculation of pHi by the comparison of intracellular spectra with in vitro calibration spectra. However, when Me2CF was calibrated intracellularly, using the K-H exchanger nigericin to equalize external pH and pHi, the dye-derived, steady state pHi was within approximately 0.1 of the value obtained with pH-sensitive microelectrodes. Furthermore, when pHi was simultaneously measured with dye and microelectrodes during rapid pHi transients, the pHi time courses measured by the two methods were very similar. We conclude that the intracellular absorbance spectrum of Me2CF can be used to measure steady state pHi and rapid pHi transients reliably, provided the dye is calibrated intracellularly.     

Regulation mechanisms of intracellular pH of Xenopus laevis oocyte.

Intracellular pH values (pHi) of Xenopus oocytes were optically measured using a fluorescent dye, 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The oocytes were loaded with dye by incubation with a membrane-permeable form (BCECF-AM). Mean pHi of the oocytes in pH 7.6 solution was 7.69. Increasing ambient pCO2 rapidly decreased pHi and estimated buffering power was 23.8 mM/pH unit. Changing ambient HCO3- from 5 to 30 mM did not alter pHi. After incubation in a Na(+)-free solution, Na+ addition to the bath rapidly increased pHi and this response was blocked by amiloride (ED50 2 microM). The addition of NH4Cl to the bath caused an initial transient increase in PHi followed by a secondary decrease. The secondary decrease was greatly inhibited by a histidine specific reagent, diethylpyrocarbonate. It was also slightly inhibited by ouabain, Ba2+ and furosemide, but not by amiloride. These data suggest that (1), fluorescence technique is applicable to PHi measurements of Xenopus oocytes; (2), Xenopus oocytes have an amiloride sensitive Na+/H(+)-exchange, and permeabilities to CO2, NH3, and NH+4. These observation may be useful in studying the relationship between pHi and oocytes development, and the expression of acid/base transporters in Xenopus oocytes.     

Subcellular compartmentation of different lipophilic fluorescein derivatives in maize root epidermal cells

Summary Fluorescence microscopy offers some distinct advantages over other techniques for studying ion transport processes in situ with plant cells. However, the use of this technology in plant cells has been limited by our lack of understanding the mechanisms that influence the subcellular distribution of dyes after loading with the lipophilic precursors. In this study, the subcellular distribution of 5-(and 6-)carboxydichlorofluorescein (CDCF), carboxy-SNAFL-1, and carboxy-SNARF-1 was compared to that of 2,7-bis-(2-carboxyethyl)-5-(and 6-)carboxyfluorescein (BCECF) after incubation of maize roots with their respective lipophilic precursors. Previously, we reported that incubation of roots with BCECF-acetomethyl ester (BCECF-AM) led to vacuolar accumulation of this dye. Similar results were found when roots were incubated with CDCF-diacetate. In contrast, carboxy-SNAFL-1 appeared to be confined to the cytoplasm based on the distribution of fluorescence and the excitation spectra of the dye in situ. On the other hand, incubation of roots with carboxy-SNARF-1-acetoxymethyl acetate yielded fluorescence throughout the cell. When the cytoplasm of epidermal cells was loaded with the BCECF acid by incubation at pH 4 in the absence of external Ca, the dye was retained in the cytoplasm at least 3 h after the loading period. This result indicated that vacuolar accumulation of BCECF during loading of BCECF-AM was not due to transport of BCECF from cytoplasm to vacuole. The esterase activities responsible for the production of either carboxy-SNAFL-1 or BCECF from their respective lipophilic precursor by extracts of roots were compared. The characterization of esterase activities was consistent with the subcellular distribution of these dyes in root cells. The results of these experiments suggest that in maize root epidermal cells the subcellular distribution of these fluorescein dyes may be determined by the characteristics of the esterase activities responsible for hydrolysis of the lipophilic precursor.Abbreviations BCECF (BCECF-AM) 2,7-bis-(2-carboxyethyl)-5-(and 6-)carboxyfluorescein (its acetoxymethyl ester) - BTB bis-trispropane - CDCF (CDCF-DA) 5-(and 6-)carboxy-2,7-dichlorofluorescein (its diacetate derivative) - DAPI 4,6-diamidino-2 phenylindole dihydrochloride - DMSO dimethylsulfoxide - HEPES N-[2-hydroxyethyl] piperazine-N-[2-ethanesulfonic acid] - MES 2-[N-morpholino]ethane-sulfonic acid - SNAFL-1 (SNAFL-1-DA) carboxyl SNAFL-1 (its diacetate) - SNARF-1 (SNARF-1-AM) carboxyl SNARF-1 (its acetoxymethyl acetate)