digitonin and 2--7--bis(carboxyethyl)-5(6)-carboxyfluorescein

A non-invasive microspectrofluorimetric technique was used to investigate experimentally induced changes in cell water volume in single N1E-115 murine neuroblastoma cells, using calcein, a derivative of fluorescein, as a marker of the intracellular water compartment. The osmotic behavior of N1E-115 cells exposed to media of various osmolalities was studied. Exposure to hyperosmotic (up to +28%) or hyposmotic (up to -17%) solutions produced reversible decreases and increases in cell water volume, respectively, which agreed with near-osmometric behavior. Increases in [Ca2+]i produced by exposing the cells to the ionophore ionomycin (1 microM) in isosmotic medium, resulted in a gradual decrease in cell water volume. Cells shrank to 40 +/- 7% (n = 7) below their initial water volume at an initial rate of -1.2 +/- 0.2%/min. It is concluded that N1E-115 cells are endowed with Ca2+-sensitive mechanisms for volume control, which can produce cell shrinkage when activated under isosmotic conditions. Because the technique used for measuring cell water volume changes is new, we describe it in detail. It is based on the principle that relative cell water volume in single cells can be measured by introducing an impermeant probe into cells and measuring its changes in concentration. If the intracellular content of the probe is constant, changes in its concentration reflect changes in cell water volume. Calcein was used as the probe because its fluorescence intensity is directly proportional to its concentration and independent of changes in the concentration of native intracellular ions within the physiological range. Because calcein is two to three times more fluorescent that other fluorophores such as 2,7,-bis-[2-carboxyethyl]-5-[and 6]-carboxyfluorescein or Fura-2, and it is used at its peak excitation and emission wavelengths, it has a better signal to noise ratio and baseline stability than the other dyes. Calcein can also be esterified allowing for cell loading and because of the possibility of reducing the intensity of the excitation light, measurements can be performed producing minimal photodynamic damage. The technique allows for measurements of cell water volume changes of < 5% and it can be applied to single cells which can be grown or affixed to a rigid substratum, e.g., a coverslip.

Topics: Animals; Calcium; Digitonin; Fluoresceins; Fluorescent Dyes; Indicators and Reagents; Intracellular Membranes; Mice; Models, Neurological; Neuroblastoma; Osmolar Concentration; Osmosis; Tumor Cells, Cultured; Type C Phospholipases; Water

Intracellular pH (pHi) regulation has been investigated in cells isolated from the proximal ceca of the chicken. pHi was measured with the pH-sensitive dye, 2',7'-bis(carboxyethyl)-5 (6)-carboxyfluorescein in nominally HCO(3-)-free solutions. Under resting conditions the pHi was 7.08. Removal of extracellular Na+ decreased pHi by approx. 0.24 pH units and the subsequent addition of Na+ increased pHi towards the control value. This Na(+)-dependent pHi recovery was inhibited by 5-(N-ethyl-N-isopropyl)amiloride (EIPA). Following an intracellular acidification, by abrupt withdrawal of NH4Cl, pHi alkalinized in the nominally absence of Na+. Rotenone, N-ethylmaleimide, N,N'-dicyclohexylcarbodiimide, 4-chloro-7-nitrobenz-2-oxa-1,3-diazole, iodoacetic acid and SCH 28080 inhibited the Na(+)-independent pHi recovery rate by 82, 82, 67, 74, 77 and 50% respectively. Bafilomycin A1 was without effect. Na(+)-independent cell alkalization was stimulated by external K+. In the presence of N-ethylmaleimide addition of Na+ induced a rapid pHi recovery. The initial rate of this recovery exhibited first-order dependence on Na+ concentration and it was inhibited by EIPA. The initial rate of Na(+)-dependent cell alkalization increased with a Hill coefficient greater than one when pHi was reduced from 7.2 to 6.2. The 'set point' for the exchanger is approx. 7.5. These studies demonstrate that in cecal epithelial cells exist at least two mechanisms for proton secretion: a Na(+)-H+ exchanger and a Na(+)-independent proton transport system.

Topics: 4-Chloro-7-nitrobenzofurazan; Animals; Anti-Ulcer Agents; Cecum; Cells, Cultured; Chickens; Dicyclohexylcarbodiimide; Digitonin; Epithelium; Ethylmaleimide; Fluoresceins; Fluorescent Dyes; Hydrogen-Ion Concentration; Imidazoles; Iodoacetates; Iodoacetic Acid; Kinetics; Nigericin; Rotenone; Sodium; Spectrometry, Fluorescence; Time Factors; Valinomycin

Sea urchin eggs were loaded with the pH-sensitive fluorescent probe BCECF. Homogenization of these eggs, followed by centrifugation, resulted in 25% of the total homogenate fluorescence remaining with the pellet. Fluorescence microscopy revealed brightly fluorescing punctate organelles whose fluorescence was not quenched by acidification. The excitation spectrum of intracellular BCECF was markedly red-shifted compared to probe calibrated in buffer. Model excitation spectra based on a two compartment model mimicked the intracellular BCECF spectrum, therefore, supporting the possibility that organelles in sea urchin eggs accumulate large amounts of BCECF in a relatively pH-insensitive form.

Topics: Animals; Calibration; Cell Compartmentation; Cytoplasm; Digitonin; Fertilization; Fluoresceins; Fluorescent Dyes; Fluoroscopy; Hydrogen-Ion Concentration; Nigericin; Oocytes; Organelles; Sea Urchins

Three methods were used to measure intracellular pH (pHi) of Leishmania donovani promastigotes: (a) measurement of the fluorescence of the pH indicator 2',7'-bis-(carboxyethyl)-5,6-carboxyfluorescein; (b) pH null point assays; and (c) determination of the distribution across the promastigote plasma membrane of the fluorescent amine acridine orange and of the weak acid 5,5-dimethyl-2,4-oxazolidinedione. The three methods gave similar results and showed that promastigotes of L. donovani maintain pHi at a narrow range of 6.4-6.7, throughout an extracellular pH (pHo) range of 5.5-7.4. L-Proline transport in L. donovani promastigotes, which is known to be coupled to proton translocation, was used to estimate the proton electrochemical gradient across parasite plasma membrane. While proline uptake is optimal at pHo 7.5, an outward-directed concentration gradient is obtained at steady state throughout a pHo range of 5-8. The calculated electrochemical gradient of proline across the parasite plasma membrane at steady state is 90-100 mV within a pHo range of 5-8, suggesting an almost constant proton electrochemical gradient at this pHo range. Taken together, the results show that the parasites regulate both pHi and the size of the chemiosmotic energy required to drive active transport of nutrients.

Topics: Acridine Orange; Animals; Biological Transport, Active; Cytoplasm; Digitonin; Fluoresceins; Fluorescent Dyes; Hydrogen-Ion Concentration; Leishmania donovani; Nigericin; Proline; Protons

Inner medullary collecting duct cells were isolated from rat papillae and grown to confluence on cover slips. H+ secretion was estimated by intracellular pH (pHi) changes measured with the fluorescent probe 2,7-biscarboxyethyl-5(6)-carboxyfluorescein. In buffered NaCl, pHi was 7.14 +/- 0.04 (n = 78). After acidification about 40% of monolayers exhibited Na+-independent alkalinization. In 5 mM glucose, cell alkalinization occurred at a rate of 47 +/- 4 nM H+/min. However, cell alkalinization did not occur in the presence of 2-deoxy-D-glucose (5-15 mM), iodoacetate (5 mM), or KCN (5 mM). All monolayers tested exhibited amiloride-inhibitable Na+-dependent cell alkalinization that appeared to be a first-order kinetic process; Km [Na+] was approximately 52 mM and Vmax was approximately 250 nM [H+]/min. At a constant extracellular [Na+] (110 mM), Na+-dependent H+ efflux was a first-order function of pHi; Km for intracellular [H+] = 321 nM and Vmax = 182 nM H+/min. The data are consistent with the presence of a primary active H+ pump and a secondary active Na+ exchanger. The metabolic energy for the active H+ pump can be provided by glycolysis and oxidative phosphorylation.

Topics: Alkalies; Animals; Biomechanical Phenomena; Cells, Cultured; Digitonin; Energy Metabolism; Fluoresceins; Hydrogen; Hydrogen-Ion Concentration; Ions; Kidney Medulla; Kidney Tubules; Kidney Tubules, Collecting; Rats; Sodium

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.

Topics: Animals; Benzofurans; Calcium; Digitonin; Fluoresceins; Fluorescent Dyes; Fura-2; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Mitochondria, Heart; Rats; Spectrometry, Fluorescence

The pH-sensitive, fluorescent, cytoplasmic-trapped dye 2,7-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) has been used to measure intracellular (pHi) and pH electrode to measure extracellular pH (pHo) in suspensions of gastric glands isolated from rabbit stomachs. The fluorescence of BCECF-loaded glands was calibrated in terms of pHi by equilibrating pHo and pHi using ionophores or digitonin and titrating pHo to different values. An APPENDIX is included that covers details of dye calibration and interpretation of fluorescence signals. Glands incubated in NaCl Ringer solution had pHi 7.11. Na+-free Ringer solution caused pHi to decrease reversibly to 6.80. Na+-dependent alkalinization of pHi followed a similar time course to the acidification of pHo. These changes were blocked by 1 mM amiloride. When gland cells were acidified (using two different techniques) realkalinization was completely Na+ dependent but was independent of the presence of Cl-; also, neither high extracellular K+ concentration ([K+]o) nor high [K+]o plus 10(-5) M valinomycin affected the rates of Na+-dependent alkalinization. A neutral Na+-H+ exchanger was implicated. Glands also exhibited Cl(-)-dependent changes of pHi that were blocked by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (2 X 10(-4) M). A Cl(-)-OH-(HCO3-) exchanger was indicated. Other studies showed that intracellular buffering capacity was approximately 45 mM (pH-1) and that the apparent proton conductance of gland cell membranes was small.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amiloride; Ammonium Chloride; Animals; Carrier Proteins; Chloride-Bicarbonate Antiporters; Chlorides; Digitonin; Fluoresceins; Gastric Mucosa; Hydrogen-Ion Concentration; Male; Monensin; Nigericin; Ouabain; Potassium; Rabbits; Rotenone; Sodium; Sodium-Hydrogen Exchangers; Trialkyltin Compounds; Valinomycin