Estimation of intramitochondrial pCa and pH by fura-2 and 2,7 biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) fluorescence

Isolated heart mitochondria hydrolyze the acetoxymethyl esters of the Ca2+-sensitive fluorescent probe fura-2 and the fluorescent pH indicator biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). The free acid forms of both probes are retained in the matrix and their fluorescence can be used to monitor the pCa and pH, respectively, of this compartment. When fura-2 loaded rat heart myocytes are lysed with digitonin, a portion of the dye is retained in the mitochondrial fraction and its fluorescence reports the uptake and release of Ca2+ by the mitochondria. It is concluded that fura-2 and BCECF may report mitochondrial as well as cytosol parameters when the probes are used in intact cells.     

Correcting for artifacts in complex aqueous solutions when using the pH-sensitive dye 2',7'-bis-(2-carboxyethyl)- 5-(and -6)carboxyfluorescein

The pH-sensitive fluorescent indicator dye 2', 7'-bis-(2-carboxyethyl)-5-(and -6)carboxyfluorescein (BCECF) is routinely used to measure intracellular pH within cells. Surprisingly, no studies have been performed to see if various solution parameters modulate the fluorescence intensity of BCECF even though viscosity artifacts have been reported for particular Ca2+ selective dyes. In this report we demonstrate that even minor increases in the concentration of a number of different agents significantly decrease the excitation fluorescence intensity at two wavelengths routinely used to determine solution pH. Solution viscosity was varied using a number of different agents including glycerol, sucrose, polyethylene glycol, polyvinylpyrrolidone, and methylcellulose. In general, there was a detectable and significant decrease in the maximum fluorescence excitation ratio as the viscosity was increased, although the effect was more dramatic with Newtonian solutions than with non-Newtonian solutions. This same general effect was seen at pH 6.5, 7.0, and 7.3, a range of pH levels where BCECF is found to be particularly sensitive. To correct for these artifactually low values we used different combinations of excitation wavelengths to determine which could be used to accurately measure pH while minimizing the artifact. Choosing excitation wavelengths so that excitation ratios were collected at 470 and 435 nm allowed a significant signal to quantitatively measure pH while the artifact was nearly abolished.     

Calcium and proton activities in rat cardiac mitochondria. Effect of matrix environment on behaviour of fluorescent probes.

The ionic composition of the mitochondrial matrix, under both physiological and pathophysiological conditions, remains controversial. Although fura-2 and 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), fluorescent probes for [Ca2+] and [H+] respectively, have successfully been loaded into mitochondria [Lukács & Kapus (1987) Biochem. J. 248, 609-613; Davis, Altschuld, Jung & Brierley (1987) Biochem. Biophys. Res. Commun. 49, 40-45], the adaptation of fluorescence-ratio spectroscopy to the study of the matrix ion content poses unique problems. In this report, we describe a method for successfully attaching viable rat cardiac mitochondria to glass coverslips, allowing continuous superfusion of isolated organelles during fluorescence microscopy. This technique obviated the need to correct for the accumulation of ion-sensitive and -insensitive fluorescent species of dye both within the matrix and outside of mitochondria in suspension in a cuvette, a particular problem with fura-2. By using this technique for superfusion of immobilized mitochondria, we found the pKa of BCECF for H+ at 25 degrees C shifted from 6.8 in buffer to 7.2 in rat cardiac mitochondria, with a marked hysteresis effect noted for intramitochondrial BCECF calibration curves. At higher pH, photobleaching of BCECF was enhanced. The dissociation constant (Kd) of fura-2 for Ca2+ was found to be 315 nM at 25 degrees C, pH 8.0, but only at [Ca2+] below 1 microM. At matrix [Ca2+] greater than 1 microM, the Kd shifted into the micromolar range, an effect that appeared to be pH-dependent. Importantly, the matrix [Ca2+] was determined to be between 10 and 100 nM at perfusion buffer [Ca2+] below 500 nM, but rose rapidly at the higher extramitochondrial [Ca2+] reported to occur in ischaemic cardiac myocytes. Importantly, mitochondrial transmembrane H+ and Ca2+ gradients both appeared to be maximal at perfusion buffer [H+] and [Ca2+] that approximate those of the cytosol of many resting cells.     

Flow cytometric measurement of cytoplasmic pH: a critical evaluation of available fluorochromes

Three pH-sensitive fluorochromes-4-methyl-umbelliferone(4MU),2, 3-dicyano-hydroquinone (DCH), and 2',7'-bis(carboxyethyl)-5,6-carboxy fluorescein (BCECF)--were evaluated for their resolution, range, and stability of cellular fluorescence. Flow cytometric techniques for determining cytoplasmic pH (pHi) have been fully described for 4MU and DCH; BCECF has previously been used for fluorimetric estimation of pHi, and was adapted to flow cytometry. For each fluorochrome, the ratio of fluorescence intensity at two wavelengths gives a measure of pHi, which may be calibrated by obtaining the fluorescence ratios for cells suspended in buffers of varying pH in the presence of a proton ionophore. Reliable calibration proved difficult using 4MU, partly because of poor retention within cells. Both DCH and BCECF could be calibrated using a fluorescence ratio and had resolutions of 0.2 and 0.4 pH units, respectively. The fluorescence of DCH is so strongly pH dependent that there were practical difficulties in its use over a wide pH range; however, pHi measurements are possible between pH 6.0 and pH 7.5 using either DCH or BCECF. Substantial dye leakage was found for 4MU and, to a lesser extent, DCH, while BCECF was retained by cells for up to 2 hours. Despite its lower resolution BCECF had a usable range of more than 1.5 pH units and this coupled with its stable fluorescence and excitation at 488 nm rather than UV suggests a wide application.     

Dual probe fluorescence monitoring of intracellular free calcium during ischemia in mouse heart by using continuous compensation for pH dependence of the dissociation constant of Fura-2, and the interference of myoglobin

Mitochondrial damage is the main source of cellular injury upon ischemia-reperfusion, and calcium loading has been implicated in this phenomenon. The use of optical probes for calcium monitoring of the intact heart is hampered by internal filter effects of intracellular hemoproteins, endogenous fluorescence, and their sensitivity to pH. We describe here a method for measurement of intracellular free calcium in isolated myoglobin-deficient perfused mouse hearts under conditions of large intracellular pH fluctuations by simultaneous fluorescence monitoring of the calcium-probe Fura-2 and the pH probe BCECF through dual wavelength excitation of both probes. In myoglobin-containing mouse heart endogenous chromophores interfere with Fura-2 fluorometry. It is shown that a paradoxical decrease in Fura-2 fluorescence occurs during ischemia in isolated mouse hearts. Simultaneous recording of BCECF fluorescence (calibrated against pH measurement with phosphorus NMR) and data reduction based on continual recalculation of the apparent dissociation constant of the calcium-probe complex revealed that a marked increase in intracellular free calcium occurs, and that the Fura-2 fluorescence decrease was caused by an increase in dissociation constant due to intracellular acidification. Intracellular free calcium rose almost linearly during a 20-min period of ischemia and returned to basal values rapidly upon the commencement of perfusion.     

Dual probe fluorescence monitoring of intracellular free calcium during ischemia in mouse heart by using continuous compensation for pH dependence of the dissociation constant of Fura-2, and the interference of myoglobin

Mitochondrial damage is the main source of cellular injury upon ischemia–reperfusion, and calcium loading has been implicated in this phenomenon. The use of optical probes for calcium monitoring of the intact heart is hampered by internal filter effects of intracellular hemoproteins, endogenous fluorescence, and their sensitivity to pH.We describe here a method for measurement of intracellular free calcium in isolated myoglobin-deficient perfused mouse hearts under conditions of large intracellular pH fluctuations by simultaneous fluorescence monitoring of the calcium-probe Fura-2 and the pH probe BCECF through dual wavelength excitation of both probes. In myoglobin-containing mouse heart endogenous chromophores interfere with Fura-2 fluorometry.It is shown that a paradoxical decrease in Fura-2 fluorescence occurs during ischemia in isolated mouse hearts. Simultaneous recording of BCECF fluorescence (calibrated against pH measurement with phosphorus NMR) and data reduction based on continual recalculation of the apparent dissociation constant of the calcium-probe complex revealed that a marked increase in intracellular free calcium occurs, and that the Fura-2 fluorescence decrease was caused by an increase in dissociation constant due to intracellular acidification. Intracellular free calcium rose almost linearly during a 20-min period of ischemia and returned to basal values rapidly upon the commencement of perfusion.     

Quick and accurate method to convert BCECF fluorescence to pHi: calibration in three different types of cell preparations.

A rapid, easy, and accurate method for converting the fluorescence of BCECF to pH, as an alternative to the nigericin method, is described. The ratio of the fluorescence intensities for BCECF can be converted to pH between 4 and 9 by a formula similar to the one used to calculate [Ca2+]i from the fluorescence of fura2. The formula is inverted because H+ binding to BCECF causes a decrease in fluorescence, whereas Ca2+ binding to fura2 causes an increase in fluorescence. The ratio of the fluorescence intensities is a sigmoidal function of the [H+] between pH 4 and 9 with an essentially linear mid region from pH 6 to 8. This calibration procedure in cells is similar to the popular method for fura2 where ionomycin, Ca2+, and an alkaline EGTA solution are added in succession to change the intracellular pCa from 4 to 9. For BCECF in cells, a protonophore, FCCP or CCCP, is added and the cells are titrated with acid to an intracellular pH of 4 and then back to pH 9 with base by observing the gradual change in fluorescence as it asymptotically reaches its limiting minimum and maximum values. This method does not require changing the medium to one with high KCl to depolarize the membrane potential nor does the proton concentration need to be equilibrated across the plasma membrane. The technique can be used to calibrate BCECF in sheets of cells, as well as suspensions of cells over a wide range of pH sensitivities.     

2′,7′-Bis-(2-carboxyethyl)-5(6)-carboxyfluorescein as a dual-emission fluorescent indicator of intracellular pH suitable for argon laser confocal microscopy

The widely used fluorescent probe 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) serves as a pH-sensitive indicator in classical microscopy. Characteristics of BCECF were studied and a way of employing the probe in a confocal laser scanning microscope equipped with an argon laser at 488 nm was developed, based on the fact that the emission fluorescence spectra are pH-dependent with spectral maximum shift from 518 to 529 nm. Optical filters for the dual-emission ratio method were set to 506 and 529 nm. pH values measured inside a single cell of Saccharomyces cerevisiae were similar to those obtained with other pH estimation methods.     

Ratiometric measurement of intracellular pH in cultured human keratinocytes using carboxy-SNARF-1 and flow cytometry

In this study we describe a method to measure intracellular pH in cultured human keratinocytes using flow cytometry. Keratinocytes pose a technical problem because the population is heterogeneous with respect to size and metabolic activity (nonspecific esterase activity), resulting in variability in dye uptake. In order to compensate for this, dyes were selected that change colour with pH. The ratio of fluorescence intensities at two wavelengths was recorded and used as a measure of intracellular pH by reference to the pH in the presence of the proton ionophore nigericin. However, methods published till now do not routinely combine the ratiometric technique and excitation with an argon ion laser set at 488 nm. Therefore we have tested the recently developed pH-sensitive dye carboxyseminaphthorhodafluor-1 (SNARF-1) as a possible candidate for flow cytometric pH measurements and compared it with 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) and 2,3-dicyanohydroquinone (DCH) with respect to emission spectra, resolution, range, and stability of cellular fluorescence. SNARF-1 had a practical and stable excitation wavelength of 488 nm rather than UV, it offered the possibility of ratiometric measurements on the basis of a real emission shift, and had superior resolution for the pH range 7-8. With SNARF-1 we found that keratinocytes cultured under low serum conditions (0.2%) contain a higher proportion of cells with relatively low intracellular pH compared to high serum cultures (6%). Furthermore, pH changes were followed by changes in relative DNA content. These findings suggest that intracellular pH can be an early functional proliferation marker for human keratinocytes.     

Binding of fluorescent lanthanides to rat liver mitochondrial membranes and calcium ion-binding proteins.

(1) Tb3+ binding to mitochondrial membranes can be monitored by enhanced ion fluorescence at 545 nm with excitation at 285 nm. At low protein concentrations (less than 30 mug/ml) no inner filter effects are observed. (2) This binding is localized at the external surface of the inner membrane and is unaffected by inhibitors of respiration or oxidative phosphorylation. (3) A soluble Ca2+ binding protein isolated according to Lehninger, A.L. ((1971) Biochem. Biophys. Res. Commun. 42, 312-317) also binds Tb3+ with enhanced ion fluorescence upon excitation at 285 nm. The excitation spectrum of the isolated protein and of the intact mitochondria are indicative of an aromatic amino acid at the cation binding site. (4) Further characterization of the Tb3+-protein interaction revealed that there is more than one binding site per protein molecule and that these sites are clustered (less than 20 A). Neuraminidase treatment or organic solvent extraction of the protein did not affect fluorescent Tb3+ binding. (5) pH dependency studies of Tb3+ binding to the isolated protein or intact mitochondria demonstrated the importance of an ionizable group of pK greater than 6. At pH less than 7.5 the amount of Tb3+ bound to the isolated protein decreased with increase in pH as monitored by Tb3+ fluorescence. With intact mitochondria the opposite occurred with a large increase in Tb3+ fluorescence at higher pH. This increase was not observed when the mitochondria were preincubated with antimycin A and rotenone.     

Activation of Na+/H+ exchange and Ca2+ mobilization start simultaneously in thrombin-stimulated platelets. Evidence that platelet shape change disturbs early rises of BCECF fluorescence which causes an underestimation of actual cytosolic alkalinization.

Although an increase in cytosolic pH (pHi) caused by Na+/H+ exchange enhances Ca2+ mobilization in platelets stimulated by low concentrations of thrombin [Siffert & Akkerman (1987) Nature (London) 325, 456-458], studies using fluorescent indicators for pHi (BCECF) and [Ca2+]i (fura2) suggest that Ca2+ is mobilized while the cytosolic pH decreases. Several lines of evidence indicate that the initial fall in BCECF fluorescence is not due to cytosolic acidification but is caused by a platelet shape change. (1) Pulse stimulation of platelets by successive addition of hirudin (4 unit/ml) and thrombin (0.2 unit/ml) induced a shape change of 43 +/- 8% and a fall in BCECF fluorescence, which both remained unchanged when Na+/H+ exchange was inhibited by ethylisopropylamiloride (EIPA, 100 microM). (2) Increasing the thrombin concentration to 0.4 unit/ml doubled the shape change and the fall in BCECF fluorescence, but again EIPA had no effect on these responses. (3) Treating platelets with 2 microM-ADP induced shape change and a decline in BCECF fluorescence that was unaffected by EIPA. (4) A second addition of thrombin to platelets that had already undergone shape change induced an immediate increase in BCECF fluorescence without a prior decrease. (5) Activation of protein kinase C by 1,2-dioctanoyl-sn-glycerol (DiC8) neither induced shape change nor a decline in BCECF fluorescence; in contrast BCECF fluorescence rapidly increased indicating an immediate cytosolic alkalinization. Concurrent analysis of [Ca2+]i under conditions in which shape change did not interfere with BCECF fluorescence showed that cytosolic alkalinization and Ca2+ mobilization started almost simultaneously. These observations suggest that cytosolic alkalinization is not preceded by a fall in pHi and can support Ca2+ mobilization induced by weak agonists.     

Endocytosis and intracellular fate of liposomes using pyranine as a probe

Lipid vesicles (liposomes) containing pH-sensitive fluorophores were used as probes for the study of liposome entry and intracellular fate. Pyranine [8-hydroxy-1,3,6-pyrenetrisulfonate (HPTS)] was entrapped in the liposome aqueous core during preparation to provide a means of detecting internalization into living cells. HPTS is highly water soluble and shows a strong pH-dependent shift in its fluorescence excitation spectrum. Fluorescence emission (FEM) is slightly pH dependent with excitation (lambda EX) at 350-415 nm but highly pH dependent with lambda EX at 450 nm. Liposomes bearing a net negative charge bound rapidly to CV-1 cells and underwent endocytosis. One hour after liposome addition, high FEM with lambda EX at 413 nm and low FEM with lambda EX at 450 nm suggest that most cell-associated liposomes had been internalized and resided at a mean pH of approximately 6.6. Collapse of cellular H+ gradients with NH4Cl or monensin treatment rapidly and reversibly increased FEM with lambda EX at 450 nm. Direct examination by fluorescence microscopy corroborates the fluorometric data on internalization; over time, FEM remained high with lambda EX at 350-405 nm but decreased with lambda EX at 450-490 nm, showing that all lipid vesicles were internalized within 40 min at 37 degrees C. Acidification of intracellular liposomes increased over 3 h, reaching a minimum value of approximately pH 5.5. HPTS persisted within acidic cellular vesicles for 2-3 days, and cytoplasmic dye was observed infrequently, suggesting that liposome fusion with cellular membranes seldom occurs. Material delivered to the endocytic pathway via lipid vesicles labeled an assortment of intracellular organelles of varying motility and morphology, including dynamic tubular structures whose lumen is acidic.     

Regulation of mitochondrial K+/H+ antiport activity by hydrogen ions

The effect of matrix pH (pHi) on the activity of the mitochondrial K+/H+ antiport has been studied using the fluorescence of 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) to monitor pHi and passive swelling in K+ acetate to follow antiport activity. Heart mitochondria suspended in hypotonic K+ acetate in the absence of respiration show an initial delta pH of -0.4 (interior acid) that decays slowly. Addition of A23187 to deplete matrix Mg2+ results in a further acid shift in pHi followed by equilibration of delta pH. This equilibration appears to depend on K+/H+ antiport and is slow at acid pHi but very rapid when the matrix is alkaline. Swelling of Mg(2+)-depleted mitochondria in K+ acetate is multiphasic with a slow initial rate, a period of maximum swelling, and a final period in which the rate declines. At constant external pH (pH0), the initial rate of swelling is faster with increasing pHi and the time to the onset of the maximum swelling rate decreases. The maximum swelling rate is initiated at pHi 7.4 when pH0 is 7.8 and at pHi 7.1 when pH0 is 7.4. The maximum rate of swelling increases linearly with increasing pH0 in the range from 7.0 to 8.2. This rate also shows a linear relationship to the value of pHi at the time the maximum rate is attained. Dixon plots of the reciprocal of the maximum swelling rate vs [H+]0 suggest that external [H+] is a noncompetitive inhibitor of K+ entry on the antiport. It is concluded that K+/H+ antiport in Mg(2+)-depleted heart mitochondria can be regulated by matrix [H+] (see Beavis, A. D., and Garlid, K. D. (1990) J. Biol. Chem. 265, 2538-2545), but that this antiport is also sensitive to external [H+] or to delta pH when it acts in the direction of K+ uptake.     

Glucose-induced oscillations of cytoplasmic Ca2+ in the pancreatic beta-cell

The cytoplasmic calcium concentration (Ca2+i) was measured in individual mouse pancreatic beta-cells loaded with fura-2 by recording the 340/380 nm fluorescence excitation ratio. An increase of the glucose concentration from 3 to 20 mM, caused initial lowering of Ca2+i followed by a rise with a peak preceding constant elevation at an intermediary level. However, at 11 mM glucose there were large Ca2+i oscillations with a frequency of 1 cycle per 2-6 min. The results indicate that both first and second phase secretion depend on elevated Ca2+i, and that many electrically coupled cells collectively determine the pace of rhythmic depolarization.     

Cytosolic and vacuolar Ca2+ concentrations in yeast cells measured with the Ca2+-sensitive fluorescence dye indo-1

Cells of Saccharomyces cerevisiae were loaded with indo-1, by incubation in a medium of pH 4.5, which contained penta-potassium indo-1. Cells were then washed and resuspended in a buffer of pH 4.0. The emission fluorescence spectra were recorded between 390 and 500 nm (excitation at 355 nm) and the autofluorescent spectra of the matched controls were subtracted. A 19-fold cellular accumulation of indo-1 was achieved. By permeabilization of plasma membranes, leaving the vacuolar membrane intact, it was proved that indo-1 was accumulated in the cytosol. It was also shown that intracellular indo-1 did not leak out of the cells and was not modified by cellular metabolism. Using the emission fluorescence ratio at 410/480 nm, the concentration of a free cytosolic Ca2+ was found to be 346 nM. Vacuolar Ca2+ concentration, calculated from indo-1 fluorescence after lysis of vacuolar and cellular membranes, was found to be 1.3 mM.     

Changes in FAD and NADH Fluorescence in Neurosecretory Terminals Are Triggered by Calcium Entry and by ADP Production

We measured changes in the intrinsic fluorescence (IF) of the neurosecretory terminals of the mouse neurohypophysis during brief (1-2 s) trains of stimuli. With fluorescence excitation at either 350 +/- 20 or 450 +/- 50 nm, and with emission measured, respectively, at 450 +/- 50 or > or = 520 nm, DeltaF/F(o) was approximately 5-8 % for a 2 s train of 30 action potentials. The IF changes lagged the onset of stimulation by approximately 100 ms and were eliminated by 1 microM tetrodotoxin (TTX). The signals were partially inhibited by 500 microM Cd(2+), by substitution of Mg(2+) for Ca(2+), by Ca(2+)-free Ringer's with 0.5 mM EGTA, and by 50 microM ouabain. The IF signals were also sensitive to the mitochondrial metabolic inhibitors CCCP (0.3 microM), FCCP (0.3 microM), and NaN(3) (0.3 mM), and their amplitude reflected the partial pressure of oxygen (pO(2)) in the bath. Resting fluorescence at both 350 nm and 450 nm exhibited significant bleaching. Flavin adenine dinucleotide (FAD) is fluorescent, while its reduced form FADH(2) is relatively non-fluorescent; conversely, NADH is fluorescent, while its oxidized form NAD is non-fluorescent. Thus, our experiments suggest that the stimulus-coupled rise in [Ca(2+)](i) triggers an increase in FAD and NAD as FADH(2) and NADH are oxidized, but that elevation of [Ca(2+)](i), alone cannot account for the totality of changes in intrinsic fluorescence.     

Measuring cytosolic free calcium concentration in endothelial cells with indo-1: the pitfall of using the ratio of two fluorescence intensities recorded at different wavelengths

Indo-1 is a new fluorescent indicator of the intracellular free calcium concentration Cai++. Indo-1 may be used in a similar manner as its predecessor quin2 but offers the principal advantage that the Ca++ saturated form of the Ca++ chelator has a emission maximum different in wavelength from that of free indo-1 (400 nm versus 483 nm). Therefore, the ratio of the fluorescence intensity F emitted at 400 nm to that of the fluorescence intensity G emitted at 483 nm (or 500 nm) should be a measure of Cai++ independent of the total amount of intracellular dye. However, when indo-1 is loaded into endothelial cells (grown in culture on quartz coverslips) by incubation with the acetoxymethylester of indo-1 (indo-1/AM), the ester in not completely hydrolysed to indo-1 intracellularly. Fluorescence emitted by uncleaved indo-1/AM at wavelengths 483-500 nm interferes with the fluorescence of indo-1. Ester fluorescence is influenced not only by ester concentration but by the fluorescence emitted at 400 nm by Ca++ bound indo-1 as well. Therefore, the ratio F/G cannot reliably evaluate increases in Cai++ in endothelial cells although F/G would indicate a basal Cai++ constant with time. By contrast, the fluorescence F is a sensitive parameter of the intracellular concentration of Ca++ bound indo-1, in particular when the excitation wavelength is set to 332 nm. F was used to measure resting Cai++ in endothelial cells (132 +/- 22 nM; n = 22) and to demonstrate dose-dependent and reversible increases in Cai++ in response to stimulation with bradykinin.     

Endocytosis of liposomes by macrophages: binding, acidification and leakage of liposomes monitored by a new fluorescence assay

The interaction of liposomes with macrophage cells was monitored by a new fluorescence method (Hong, K., Straubinger, R.M. and Papahadjopoulos, D., J. Cell Biol. 103 (1986) 56a) that allows for the simultaneous monitoring of binding, endocytosis, acidification and leakage. Profound differences in uptake, cell surface-induced leakage and leakage subsequent to endocytosis were measured in liposomes of varying composition. Pyranine (1-hydroxypyrene-3,6,8-trisulfonic acid, HPTS), a highly fluorescent, water-soluble, pH sensitive dye, was encapsulated at high concentration into the lumen of large unilamellar vesicles. HPTS exhibits two major fluorescence excitation maxima (403 and 450 nm) which have a complementary pH dependence in the range 5-9: the peak at 403 nm is maximal at low pH values while the peak at 450 nm is maximal at high pH values. The intra- and extracellular distribution of liposomes and their approximate pH was observed by fluorescence microscopy using appropriate excitation and barrier filters. The uptake of liposomal contents by cells and their subsequent exposure to acidified endosomes or secondary lysosomes was monitored by spectrofluorometry via alterations in the fluorescence excitation maxima. The concentration of dye associated with cells was determined by measuring fluorescence at a pH independent point (413 nm). The average pH of cell-associated dye was determined by normalizing peak fluorescence intensities (403 nm and 450 nm) to fluorescence at 413 nm and comparing these ratios to a standard curve. HPTS-containing liposomes bound to and were acidified by a cultured murine macrophage cell line (J774) with a t1/2 of 15-20 min. The acidification of liposomes exhibited biphasic kinetics and 50-80% of the liposomes reached an average pH lower than 6 within 2 h. A liposomal lipid marker exhibited a rate of uptake similar to HPTS, however the lipid component selectively accumulated in the cell; after an initial rapid release of liposome contents, 2.5-fold more lipid marker than liposomal contents remained associated with the cells after 5 h. Coating haptenated liposomes with antibody protected liposomes from the initial release. The leakage of liposomal contents was monitored by co-encapsulating HPTS and p-xylene-bis-pyridinium bromide, a fluorescence quencher, into liposomes. The time course of dilution of liposome contents, detected as an increase in HPTS fluorescence, was coincident with the acidification of HPTS. The rate and extent of uptake of neutral and negatively charged liposomes was similar; however, liposomes opsonized with antibody were incorporated at a higher rate (2.9-fold) and to a greater extent (3.4-fold).(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Dual‐color‐emitting green fluorescent protein from the sea cactus Cavernularia obesa and its use as a pH indicator for fluorescence microscopy

We isolated and characterized a green fluorescent protein (GFP) from the sea cactus Cavernularia obesa. This GFP exists as a dimer and has absorption maxima at 388 and 498 nm. Excitation at 388 nm leads to blue fluorescence (456 nm maximum) at pH 5 and below, and green fluorescence (507 nm maximum) at pH 7 and above, and the GFP is remarkably stable at pH 4. Excitation at 498 nm leads to green fluorescence (507 nm maximum) from pH 5 to pH 9. We introduced five amino acid substitutions so that this GFP formed monomers rather than dimers and then used this monomeric form to visualize intracellular pH change during the phagocytosis of living cells by use of fluorescence microscopy. The intracellular pH change is visualized by use of a simple long‐pass emission filter with single‐wavelength excitation, which is technically easier to use than dual‐emission fluorescent proteins that require dual‐wavelength excitation. Copyright © 2013 John Wiley & Sons, Ltd.