2--7--bis-(2-carboxyethyl)-5(6)-carboxyfluorescein-acetoxymethyl-ester and 2--7--bis(carboxyethyl)-5(6)-carboxyfluorescein

Vacuolar and cytosolic pH are highly regulated in yeast cells and occupy a central role in overall pH homeostasis. We describe protocols for ratiometric measurement of pH in vivo using pH-sensitive fluorophores localized to the vacuole or cytosol. Vacuolar pH is measured using BCECF, which localizes to the vacuole in yeast when introduced into cells in its acetoxymethyl ester form. Cytosolic pH is measured with a pH-sensitive GFP expressed under control of a yeast promoter, yeast pHluorin. Methods for measurement of fluorescence ratios in yeast cell suspensions in a fluorimeter are described. Through these protocols, single time point measurements of pH under different conditions or in different yeast mutants have been compared and changes in pH over time have been monitored. These methods have also been adapted to a fluorescence plate reader format for high-throughput experiments. Advantages of ratiometric pH measurements over other approaches currently in use, potential experimental problems and solutions, and prospects for future use of these techniques are also described.

Topics: Cytosol; Fluoresceins; Fluorescent Dyes; Fluorometry; Hydrogen-Ion Concentration; Saccharomyces cerevisiae; Vacuoles

Fluorescence intensity of the pH-sensitive carboxyfluorescein derivative 2,7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) was monitored by high-throughput flow cytometry in living yeast cells. We measured fluorescence intensity of BCECF trapped in yeast vacuoles, acidic compartments equivalent to lysosomes where vacuolar proton-translocating ATPases (V-ATPases) are abundant. Because V-ATPases maintain a low pH in the vacuolar lumen, V-ATPase inhibition by concanamycin A alkalinized the vacuole and increased BCECF fluorescence. Likewise, V-ATPase-deficient mutant cells had greater fluorescence intensity than wild-type cells. Thus, we detected an increase of fluorescence intensity after short- and long-term inhibition of V-ATPase function. We used yeast cells loaded with BCECF to screen a small chemical library of structurally diverse compounds to identify V-ATPase inhibitors. One compound, disulfiram, enhanced BCECF fluorescence intensity (although to a degree beyond that anticipated for pH changes alone in the mutant cells). Once confirmed by dose-response assays (EC(50)=26 microM), we verified V-ATPase inhibition by disulfiram in secondary assays that measured ATP hydrolysis in vacuolar membranes. The inhibitory action of disulfiram against V-ATPase pumps revealed a novel effect previously unknown for this compound. Because V-ATPases are highly conserved, new inhibitors identified could be used as research and therapeutic tools in cancer, viral infections, and other diseases where V-ATPases are involved.

Topics: Drug Evaluation, Preclinical; Enzyme Inhibitors; Flow Cytometry; Fluoresceins; High-Throughput Screening Assays; Hydrogen-Ion Concentration; Macrolides; Saccharomyces cerevisiae; Spectrometry, Fluorescence; Vacuolar Proton-Translocating ATPases; Vacuoles; Yeasts

The present study quantitatively compared the drug efflux transport kinetics of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and its fluorescent metabolite 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) in various blood-brain barrier (BBB) models. BCECF-AM was exposed to freshly isolated bovine brain microvessels (BBM), primary cultured bovine brain microvessel endothelial cells (BBMEC), and MDCK-MDR1 cells for 30 min in the presence or absence of the P-glycoprotein (P-gp) inhibitor N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918). P-gp transport kinetics were determined indirectly by calculating the difference in BCECF accumulation when P-gp was functional and completely inhibited by GF120918 (3.2 microM). Multidrug resistance-associated protein (MRP) transport kinetics were determined by measuring the amount of BCECF transported out of the cell over time. For P-gp-related transport, Km values for BCECF-AM were approximately the same in all three models (around 2 microM), whereas the Vmax was 4-fold greater in the BBM than in the BBMEC or MDCKII-MDR1 cells. For MRP-related transport, Km values for BCECF varied widely among the three BBB models with a rank order of MDCKII-MDR1 < BBMEC < BBM. Like P-gp, the Vmax of BCECF for MRP-related transport was overwhelmingly higher in the BBM compared with the cultured cells. Because differences in the expression of P-gp, MRP5, and MRP6 were observed in the various BBB models using reverse transcription-polymerase chain reaction techniques, the disparity in transport kinetics between the BBB models may be linked to variations in the amount or type of drug efflux transporters expressed in each model. The present study introduces a method of quantitatively evaluating drug efflux transport kinetics in the BBB.

Topics: Acridines; Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Blood-Brain Barrier; Brain; Cattle; Cell Line; Dogs; Endothelial Cells; Fluoresceins; Fluorescent Dyes; Kinetics; Microcirculation; Multidrug Resistance-Associated Proteins; RNA, Messenger; Tetrahydroisoquinolines; Transfection

2',7'-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) is a fluorescent probe used to examine multidrug resistance-associated protein (MRP) transporter activity in cells. BCECF is introduced into the cell as the nonfluorescent membrane permeable acetoxymethyl ester, BCECF-AM, where it is hydrolyzed to the membrane impermeable BCECF. The lipophilic nature of BCECF-AM suggests it may be a substrate for other drug efflux transporters such as P-glycoprotein (P-gp) and the breast cancer resistance protein (BCRP). To assess the drug efflux transporter interactions of BCECF-AM and BCECF, accumulation studies were examined in various drug efflux-expressing cells. Inhibition of P-gp, BCRP, and/or MRP produced distinct changes in the time-dependent accumulation of BCECF in the cells. Treatment with GF120918 produced an immediate and sustained effect throughout the entire time course examined. Fumitremorgin C only affected BCECF accumulation at the early time points, whereas the impact of indomethacin on BCECF accumulation was observed only at the latter time points. Permeability studies in bovine brain microvessel endothelial cells indicated an increased basolateral-to-apical transport of BCECF, which could be reduced in the presence of either indomethacin or GF120918. These results indicate that the intracellular accumulation and transcellular permeability of BCECF are sensitive to a variety of drug efflux interactions. These results likely reflect an interaction of the ester form with P-gp and BCRP during the initial accumulation process, and an interaction of the free acid form with MRP after hydrolysis in the cell.

Topics: Acridines; Animals; Anti-Inflammatory Agents, Non-Steroidal; Area Under Curve; ATP Binding Cassette Transporter, Subfamily B, Member 1; Carrier Proteins; Cattle; Cell Separation; Cells, Cultured; Endothelial Cells; Fluoresceins; Fluorescent Dyes; Indicators and Reagents; Indomethacin; Permeability; Spectrometry, Fluorescence; Tetrahydroisoquinolines

At least in normal-pressure glaucoma a vascular genesis with hypoperfusion and regulation impairment is discussed. This may lead to malnutrition of retinal ganglion cells and apoptosis. The retinal microvasculature has a small functional reserve. In addition, the retinal microvessels lack the autonomic nerves that are normally found in other tissues. Thus, no systemic influences reach the retinal capillaries apart from circulating hormones or transmitters. Blockers of carbonic anhydrase (CA) may modulate regional blood flow by mediating changes in extra- and intracellular pH. However, it is still unclear (1) whether blockers of CA really change the pH near the retinal capillaries and (2) how changes in the local pH affect the capillary tone in situ. Therefore, we tested dorzolamide and acetazolamide in our model of the freshly enucleated rat retina.. Adult Sprague-Dawley rats (of both sexes, 250-350 g) were killed and retinae were prepared. The retinae were gently separated from the retinal pigmented epithelium and were observed in a chamber for electronic light microscopy or were fixed for immunohistochemistry. Electronic light microscopy of the retinal cells was performed with a Zeiss Axiovert microscope equipped with differential interference contrast (DIC) optics. Changes in capillary diameter were measured using an Openlab acquisition system and analyzed statistically using ANOVA. In addition to light microscopy the intracellular pH was analyzed in the whole mounts by ratio imaging of the pH using the special dye BCECF-AM (2,7 -bis-(2-carboxyethyl)-5-(and -6)-carboxyfluoresceinacetoxymethyl ester) and the extracellular pH using BCECF (2,7 -bis-(2-carboxyethyl)-5-(and -6)-carboxyfluorescein).. Pericytes of most segments of retinal capillaries are immunoreactive for alpha-smooth muscle actin (SMA). The SMA immunostaining is strong around the nucleus; the endothelial tube is visible by virtue of the slight immunoreactivity of the surrounding pericyte processes. Acetazolamide and dorzolamide showed statistically significant vasoactive effects in retinal capillaries. Vasodilation increased by up to 105% of that in control capillaries after 5, 10 and 15 min. CA inhibitors were found to be able to induce intracellular alkalization in retinal cells. After addition of dorzolamide or acetazolamide the extracellular pH decreased from 7.4 to 7.2 concomitant with diameter changes.. The tube-like pattern of SMA immunoreactivity demonstrates the presence of contractile elements within the pericyte processes of the rat retina. Thus, pericytes may act as a regulation element within the retinal microcirculation. Our results further suggest that CA inhibitors are able to decrease pH in the extracellular space; however, the pH within the cells increases. The increase in capillary diameter is concomitant with these pH changes. Thus, we may conclude that CA inhibitors can relax pericytes and might improve the retinal blood supply.

Topics: Acetazolamide; Actins; Animals; Capillaries; Carbonic Anhydrase Inhibitors; Extracellular Space; Female; Fluoresceins; Fluorescent Dyes; Hydrogen-Ion Concentration; Male; Muscle, Smooth, Vascular; Organ Culture Techniques; Pericytes; Rats; Rats, Sprague-Dawley; Retina; Retinal Vessels; Sulfonamides; Thiophenes; Vasodilation

The fluorescent dye 2',7'-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF) has been widely used as an indicator of cytosolic pH. Here we report that BCECF localizes to hydrogenosomes (hydrogen-generating organelles found in several phylogenetically separate groups of anaerobic protists) in Trichomonas vaginalis, where it was observable by fluorescence microscopy. Its cellular location was confirmed by treatment of BCECF-loaded cells with diaminobenzidine and hydrogen peroxide together with UV illumination. This produced an osmiophilic precipitate in the matrix of hydrogenosomes, observable by electron microscopy. Use of a short (7.5 min) loading period, loading on ice, use of concentrations of BCECF (acetoxymethyl ester) down to 10 nM, and inclusion of the anion channel blockers probenicid or sulfinpyrazone, or the K+/H+ ionophore nigericin in the loading buffer all failed to prevent hydrogenosomal accumulation of BCECF. This uptake was best observed when intact cells were loaded with the ester form of BCECF, but could also be seen using free BCECF following either incubation with ruptured cells or electroporation of intact cells. Hydrogenosomal BCECF loading was also obtained with washed cell lysates, without cytoplasm or metabolic substrates. We tested a range of other fluorogenic dyes designed for cytosolic labeling, and found that the calcium indicator fura-2 (acetoxymethyl ester) and the cell viability marker fluorescein diacetate also labeled hydrogenosomes. The results illustrate a novel use for BCECF as a fluorescent marker for hydrogenosomes (the first such marker), but present a warning against the indiscriminate use of fluorogenic ester dyes to measure properties of the cytosol in hydrogenosome-containing organisms - the dyes may also be indicating the properties of the hydrogenosome.

Topics: Animals; Biomarkers; Electroporation; Fluoresceins; Fluorescent Dyes; Hydrogen; Hydrogen-Ion Concentration; Microscopy, Electron; Microscopy, Fluorescence; Nigericin; Organelles; Pyruvic Acid; Sulfinpyrazone; Trichomonas vaginalis

2' ,7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein tetraacetoxymethyl ester (BCECF-AM), a fluorescence reagent for the measurement of intracellular pH with a molecular weight of 809 Da, was used to test the hypothesis that the blood-brain barrier (BBB) does not restrict the influx of substrate with a molecular weight greater than 600 Da. Using cultured bovine brain capillary endothelial cells (BCEC), the influx rate of BCECF-AM was found to be 151 +/- 2 microl/min/mg protein and was extrapolated to give 446 +/- 7 microl/min/g brain as a BBB permeability surface area product (PS). No significant saturation was observed for the initial in vitro uptake of BCECF-AM into BCEC at concentrations 0.1, 1.0 and 5.0 microM. The apparent activation energy of the initial uptake of BCECF-AM was found to be 5.09 kcal/mol. These results suggest that BCECF-AM is transported into the BBB by passive diffusion. The in vivo BBB PS value was also found to be 295 +/- 48 microl/min/g brain and 132 +/- 24 microl/min/g brain by the in situ brain perfusion and the carotid artery injection methods, respectively. No significant efflux of BCECF-AM from the brain was observed over a 120 sec washout period, suggesting that BCECF-AM is immediately hydrolyzed to BCECF, a hydrophilic analogue, in the brain after crossing the BBB. The octanol/water partition coefficient of BCECF-AM was found to be 5.66 +/- 0.27. The BBB PS value of BCECF-AM was predicted to be 105 microl/min/g brain, based on the relationship between the BBB PS value and the value of partition coefficient divided by the square root of the molecular weight. These results demonstrate that BCECF-AM transport across the BBB is not restricted despite its large molecular size.

Topics: 1-Butanol; 3-O-Methylglucose; Animals; Biological Transport; Blood-Brain Barrier; Butanols; Calorimetry; Capillaries; Carotid Arteries; Cattle; Cells, Cultured; Cerebrovascular Circulation; Diffusion; Endothelium, Vascular; Fluoresceins; Injections, Intra-Arterial; Male; Mannitol; Rats; Rats, Wistar

Monocytes may represent an important defense mechanism in disseminated cryptococcosis. We have developed a flow cytometric method to study the interaction of Cryptococcus neoformans with monocytes. For phagocytosis, C. neoformans was labelled with fluorescein isothiocynate (FITC). Monocytes were identified on the flow cytometer by labelling with anti-CD14-R-phycoerythrin. Discrimination between attached cells (association) and internalized cells (uptake) was made by quenching FITC-labelled C. neoformans with trypan blue. Only internalized cells kept their FITC fluorescence after quenching. For comparison under the microscope, specific staining of the cell wall of C. neoformans with Uvitex was used. Internalized C. neoformans cells were not stained, as Uvitex was occluded from phagocytes. To assay killing, C. neoformans was labelled with 0.2 mM 2'-7(1)-bis(2-carboxyethyl)-5-carboxyfluorescein acetoxymethylester. After phagocytosis of labelled cells by monocytes, blood cells were lysed with 25 mM deoxycholate. Viable yeast cells retained the fluorescence, but nonviable cells lost it. Quantitative counts of viable cells on Sabouraud dextrose agar were performed for comparison. The change in the relative fluorescence of green within the monocyte region was used to quantitate association, uptake, and killing of C. neoformans by monocytes on the flow cytometer. The flow cytometry methods showed that 18% +/- 2%, 35% +/- 14%, 50% +/- 11%, 51% +/- 6% of monocytes had become associated with C. neoformans after 0, 30, 60, and 120 min, respectively. After 2 h of phagocytosis time, 30% of C. neoformans-associated monocytes had taken up the cells, and killing rates of 23% +/- 17%, 22% +/- 9%, and 40% +/- 13% were obtained with effector-to-target cell ratios of 1:1, 10:1, and 50:1, respectively. Results with the flow cytometry methods compared favorably with those by the conventional methods used, but the flow cytometry methods are simpler, rapid, more reproducible, and objective.

Topics: Blood Bactericidal Activity; Cell Survival; Cryptococcus neoformans; Flow Cytometry; Fluorescein-5-isothiocyanate; Fluoresceins; Fluorescent Dyes; Humans; Hydrolysis; Leukocytes, Mononuclear; Phagocytosis; Trypan Blue; Yeasts

We examined cytoplasmic pH regulation in Schizosaccharomyces pombe and Saccharomyces cerevisiae using pH-sensitive fluorescent dyes. Of several different fluorescent compounds tested, carboxy-seminaphthorhodafluor-1 (C.SNARF-1) was the most effective. Leakage of C.SNARF-1 from S. pombe was much slower than leakage from C. cerevisiae. Using the pH-dependent fluorescence of C.SNARF-1 we showed that at an external pH of 7, mean resting internal pH was 7.0 for S. pombe and 6.6 for S. cerevisiae. We found that internal pH in S. pombe was maintained over a much narrower range in response to changes in external pH, especially at acidic pH. The addition of external glucose caused an intracellular alkalinization in both species, although the effect was much greater in S. cerevisiae than in S. pombe. The plasma membrane H(+)-ATPase inhibitor diethylstilbestrol reduced both the rate and extent of alkalinisation, with an IC50 of approximately 35 microM in both species. Amiloride also inhibited internal alkalinisation with IC50's of 745 microM for S. cerevisiae and 490 microM for S. pombe.

Topics: Benzopyrans; Diffusion; Fluoresceins; Fluorescent Dyes; Hydrogen-Ion Concentration; Intracellular Fluid; Naphthols; Rhodamines; Saccharomyces cerevisiae; Schizosaccharomyces

Fluorescein esters are employed in assays of cell viability, membrane permeability and esterase activity. The ester most widely used, fluorescein diacetate (FDA), has the disadvantage of rapid cellular efflux of its hydrolysis product fluorescein. This is particularly problematic for flow cytoenzymology (FCE), where fluorescence is measured in individual cells allowing identification of subpopulations differing in esterase activity and/or membrane characteristics. We present a comparison of FDA with two potentially improved substrate probes for FCE, carboxyfluorescein diacetate (CFDA) and bis(carboxyethyl)-carboxyfluorescein-tetra acetoxy methyl ester (BCECF-AM). Substrates were characterized in terms of reaction and product efflux kinetics in EMT6 mouse mammary tumour cells, together with inhibition kinetics for the carbamoylating agent BCNU. Intact viable cells were analysed by FCE and spectrofluorimetry, and the latter was also used for cell sonicates and purified esterase. CFDA and BCECF-AM enter cells and are hydrolysed more slowly than FDA. CFDA and FDA hydrolyses obey Michaelis-Menten kinetics with Km values of around 19 and 2 microM, respectively, whereas BCECF-AM hydrolysis deviates from this classical behaviour. BCNU (5 X 10(-4) M) inhibits FDA and BCECF-AM hydrolyses by approximately 50%, compared to 30% for CFDA. CFDA may be partly hydrolysed by membrane-bound esterases. Efflux half-lives were 16 min, 94 min and greater than 2 h for products of FDA, CFDA and BCECF-AM, respectively. We conclude that BCECF-AM is the optimal substrate probe for FCE. This study emphasizes the need to optimize various parameters when selecting a substrate for flow cytoenzymological assay or when loading other reporter fluorochromes into cells via lipophilic esters.

Topics: Animals; Carmustine; Cell Membrane Permeability; Cell Survival; Esterases; Flow Cytometry; Fluoresceins; Humans; Mice; Spectrometry, Fluorescence