Mechanistic studies of lantibiotic-induced permeabilization of phospholipid vesicles.
AuthorsDriessen AJ, van den Hooven HW, Kuiper W, van de Kamp M, Sahl HG, Konings RN, Konings WN
JournalBiochemistry
PubMed ID7849020
'Nisin is a cationic polycyclic bacteriocin secreted by some lactic acid bacteria. Nisin has previously been shown to permeabilize liposomes. The interaction of nisin was analyzed with liposomes prepared of the zwitterionic phosphatidylcholine (PC) and the anionic phosphatidylglycerol (PG). Nisin induces the release of 6-carboxyfluorescein and other small anionic fluorescent ... More
Luminal pH regulated calcium release kinetics in sarcoplasmic reticulum vesicles.
AuthorsDonoso P, Beltrán M, Hidalgo C
JournalBiochemistry
PubMed ID8873610
'Calcium binding to triads isolated from rabbit skeletal muscle followed a single hyperbolic function in the pH range 5.5-8.0. Maximal binding was obtained at pH 8.0; decreasing the pH decreased the binding capacity and, at pH < or = 6.0, increased Kd 2-fold. These results indicate that lowering the pH ... More
Modulation of the hyperpolarization-activated cation current of rat thalamic relay neurones by intracellular pH.
AuthorsMunsch T, Pape HC
JournalJ Physiol
PubMed ID10457065
'1. Properties of the hyperpolarization-activated cation current (Ih) were investigated in thalamocortical neurones of an in vitro slice preparation of the rat ventrobasal thalamic complex (VB) before and during changes of pipette pH (pHp), intracellular pH (pHi) and bath pH (pHb) using the whole-cell patch-clamp technique and fluorescence ratio imaging ... More
Correcting for artifacts in complex aqueous solutions when using the pH-sensitive dye 2',7'-bis-(2-carboxyethyl)- 5-(and -6)carboxyfluorescein.
AuthorsCardullo RA, Dandala S
JournalAnal Biochem
PubMed ID10036141
'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 ... More
Reduced surface area in mitotic rounding of human Chang liver cells.
AuthorsSit KH, Bay BH, Wong KP
JournalAnat Rec
PubMed ID8420388
'The rounding up of mitotic human Chang liver cells in monolayer culture was studied quantitatively. It was surprising to find significant reduction in cell surface area considering that endocytosis has been demonstrated to be at a complete standstill in M phase. Uptake studies using impermeant BCECF (2'',7''-bis(2-carboxyethyl)-4(5)-carboxyfluorescein free acid) pH ... More
SNARF-1 as an intracellular pH indicator in laser microspectrofluorometry: a critical assessment.
AuthorsSeksek O, Henry-Toulmé N, Sureau F, Bolard J
JournalAnal Biochem
PubMed ID2042742
'The use of SNARF-1-AM (seminaphtorhodafluor-1-acetoxymethylester) to measure the internal pH of a single living cell by laser microspectrofluorometry has been analyzed with a lymphocyte murine B cell line A20. After incubation of the cells at 37 degrees C in the presence of 10 microM SNARF-1-AM, the internal concentration of SNARF-1 ... More
Measuring the adsorption of Fatty acids to phospholipid vesicles by multiple fluorescence probes.
AuthorsSimard JR, Kamp F, Hamilton JA,
JournalBiophys J
PubMed ID18296488
'Fatty acids (FA) are important nutrients that the body uses to regulate the storage and use of energy resources. The predominant mechanism by which long-chain fatty acids enter cells is still debated widely as it is unclear whether long-chain fatty acids require protein transporters to catalyze their transmembrane movement. We ... More
Characterization of the activation of Na+/H+ exchange in lymphocytes by phorbol esters: change in cytoplasmic pH dependence of the antiport.
AuthorsGrinstein S, Cohen S, Goetz JD, Rothstein A, Gelfand EW
JournalProc Natl Acad Sci U S A
PubMed ID2983345
'Phorbol 12-myristate 13-acetate and other phorbol esters induce an intracellular alkalinization in rat thymic lymphocytes. An extracellular acidification can be recorded concomitantly. This transmembrane H+ (equivalent) flux is dependent on external Na+ and is amiloride sensitive. Phorbol esters also stimulate an amiloride-sensitive uptake of 22Na+, suggesting activation of Na+o/H+i exchange. ... More
Na+-H+ and Cl(-)-OH-(HCO3-) exchange in gastric glands.
AuthorsParadiso AM, Negulescu PA, Machen TE
JournalAm J Physiol
PubMed ID3008575
'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 ... More
Anomalous driving force for renal brush border H+/OH-transport characterized by using 6-carboxyfluorescein.
AuthorsVerkman AS, Ives HE
JournalBiochemistry
PubMed ID3013286
'The pH, delta pH, and membrane potential dependences of H+/OH-permeability in renal brush border membrane vesicles (BBMV) were studied by using the entrapped pH indicator 6-carboxyfluorescein (6CF). Quantitative H+/OH-fluxes (JH) were obtained from a calibration of the fluorescence response of 6CF to intravesicular pH using vesicles prepared with varying intravesicular ... More
AuthorsFreudenrich CC, Murphy E, Levy LA, London RE, Lieberman M
JournalAm J Physiol
PubMed ID1566808
'To assess the role of pH in cellular Mg homeostasis, cytosolic pH (pHi) was manipulated by the NH4Cl prepulse technique; pHi, cytosolic Mg2+ (Mgi), and cytosolic Ca2+ (Cai) were measured fluorometrically in single cultured embryonic chicken heart cells loaded with 2'',7''-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), FURAPTRA, and fura-2, respectively. The basal values obtained ... More
Quick and accurate method to convert BCECF fluorescence to pHi: calibration in three different types of cell preparations.
AuthorsJames-Kracke MR
JournalJ Cell Physiol
PubMed ID1295907
'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 ... More
Rapid flip-flop of oleic acid across the plasma membrane of adipocytes.
AuthorsKamp F, Guo W, Souto R, Pilch PF, Corkey BE, Hamilton JA
JournalJ Biol Chem
PubMed ID12499383
'Nonesterified long-chain fatty acids may enter cells by free diffusion or by membrane protein transporters. A requirement for proteins to transport fatty acids across the plasma membrane would imply low partitioning of fatty acids into the membrane lipids, and/or a slower rate of diffusion (flip-flop) through the lipid domains compared ... More
Photophysics of the fluorescent pH indicator BCECF.
AuthorsBoens N, Qin W, Basaric N, Orte A, Talavera EM, Alvarez-Pez JM,
JournalJ Phys Chem A
PubMed ID16869681
'The photophysical behavior of BCECF [2'',7''-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein]--currently the most widely used fluorescent pH indicator for near-neutral intracellular pH measurements--has been explored by using absorption and steady-state and time-resolved fluorescence measurements. The influence of ionic strength as well as total buffer concentration on the absorbance and steady-state fluorescence has been investigated. The ... More
Determinants of the translational mobility of a small solute in cell cytoplasm.
AuthorsKao HP, Abney JR, Verkman AS
JournalJ Cell Biol
PubMed ID8416987
'The purposes of this study were: (a) to measure the translational mobility of a small solute in cell cytoplasm; (b) to define quantitatively the factors that determine solute translation; and (c) to compare and contrast solute rotation and translation. A small fluorescent probe, 2,7-bis-(2-carboxyethyl)-5-(and 6-)-carboxyfluorescein (BCECF), was introduced into the ... More
Cellular pH and Na+-H+ exchange activity in lens epithelium of Bufo marinus toad.
AuthorsWolosin JM, Alvarez LJ, Candia OA
JournalAm J Physiol
PubMed ID2847535
'Incubation of lenses with the acetoxymethyl ester of 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF-AM) led to selective, prolonged entrapment of the deesterified, pH-sensitive, membrane-impermeable form of the dye (BCECF) in the lens epithelial cells allowing, thereby, for the continuous fluorometric monitoring of epithelial intracellular pH (pHi) in intact lenses. Under anaerobic conditions in physiological ... More
Maintenance of acidic lateral intercellular spaces by endogenous fixed buffers in MDCK cell epithelium.
AuthorsDzekunov SM, Spring KR
JournalJ Membr Biol
PubMed ID9784582
'The lateral intercellular spaces (LIS) of MDCK cell epithelia grown on permeable supports are about 0.4 pH units acidic to the bathing solutions, presumably because of buffering by the fixed negative charges on the surface of the lateral cell membranes. To test the hypothesis that fixed buffers are responsible for ... More
Continuous measurement of the cytoplasmic pH in Lactococcus lactis with a fluorescent pH indicator.
AuthorsMolenaar D, Abee T, Konings WN
JournalBiochim Biophys Acta
PubMed ID1958707
'The cytoplasmic pH of Lactococcus lactis was studied with the fluorescent pH indicator 2'',7''-bis-(2-carboxyethyl)-5 (and-6)-carboxyfluorescein (BCECF). A novel method was applied for loading bacterial cells with BCECF, which consists of briefly treating a dense cell suspension with acid in the presence of the probe. This results in a pH gradient, ... More
Multiple spectral parameter imaging.
AuthorsWaggoner A, DeBiasio R, Conrad P, Bright GR, Ernst L, Ryan K, Nederlof M, Taylor D
JournalMethods Cell Biol
PubMed ID2648118
Construction and evaluation of a frequency-domain epifluorescence microscope for lifetime and anisotropy decay measurements in subcellular domains.
AuthorsVerkman AS, Armijo M, Fushimi K
JournalBiophys Chem
PubMed ID1873470
'The measurement of time-resolved fluorescence parameters in living cells provides a powerful approach to study cell structure and dynamics. An epifluorescence microscope was constructed to resolve multi-component fluorescence lifetimes and complex anisotropy decay rapidly in labile biological samples. The excitation source consisted of focused, polarized laser light modulated by an ... More
The fluorescent indicator pyranine is suitable for measuring stromal and cameral pH in vivo.
'Three pH-sensitive fluorescent indicators (fluorescein, bis-carboxyethyl-carboxyfluorescein and pyranine) were studied to determine their usefulness as probes for the living cornea and anterior chamber. Of the three dyes, pyranine had properties which render it almost ideally suited for this purpose. Adequate concentrations are reached in the cornea and anterior chamber after ... More
Relationship of the effects of nigericin on the aggregation and cytoplasmic pH of bovine platelets in the presence of different cations.
AuthorsKitagawa S, Awai M, Kametani F
JournalBiochim Biophys Acta
PubMed ID2441757
'The effect of nigericin on aggregation of bovine platelets was investigated in media containing the chloride salts of various alkali metal cations of quaternary ammonium cations. In medium with K+, which has the highest permeability with the ionophore among the cations tested, nigericin slightly enhanced both ADP- and thrombin-induced aggregation. ... More
Cytosolic pH regulation in osteoblasts. Regulation of anion exchange by intracellular pH and Ca2+ ions.
AuthorsGreen J, Yamaguchi DT, Kleeman CR, Muallem S
JournalJ Gen Physiol
PubMed ID2299328
'Measurements of cytosolic pH (pHi) 36Cl fluxes and free cytosolic Ca2+ concentration ([Ca2+]i) were performed in the clonal osteosarcoma cell line UMR-106 to characterize the kinetic properties of Cl-/HCO3- (OH-) exchange and its regulation by pHi and [Ca2+]i. Suspending cells in Cl(-)-free medium resulted in rapid cytosolic alkalinization from pHi ... More
Connexin43 gap junctions in normal, regenerating, and cultured mouse bone marrow and in human leukemias: their possible involvement in blood formation.
AuthorsKrenacs T, Rosendaal M
JournalAm J Pathol
PubMed ID9546360
'Communicating channels called gap junctions are thought to play a ubiquitous part in cell growth and development. Based on earlier work, we have recently found functional evidence of their presence in human and mouse bone marrow. In this study we studied the cell-type association of the gap junction channel-forming protein, ... More
Effects of alpha-adrenergic agonists on intracellular and intramitochondrial pH in rat proximal nephrons.
AuthorsGesek FA, Schoolwerth AC
JournalAm J Physiol
PubMed ID2572175
'Rat proximal tubular segments were used to examine alpha-adrenoceptor alterations in Na+-H+ exchange by monitoring intracellular pH (pHi) and mitochondrial matrix pH (pHm). To obtain pHi, tubules were incubated with the cell-permeant fluorescent probe, 2',7'-bis(2-carboxyethyl)-5(6) carboxyfluorescein acetoxymethyl ester in a HCO3--free Na+ buffer. The intracellular distribution of the weak acid ... More
Leukotriene D4-induced increases in the cytoplasmic pH of human myelocytic leukocytes.
AuthorsBaud L, Healy A, Cragoe EJ, Goetzl EJ, Koo CH
JournalJ Cell Physiol
PubMed ID2842349
'The effects of leukotriene D4 on the intracellular pH of human myelocytes, derived from cultured HL-60 cells by dimethylsulfoxide-induced differentiation, were quantified with the fluorescent indicator 2'',7''-bis-(2-carboxy-ethyl)-5,6-carboxyfluorescein. Leukotriene D4, but not C4 or E4, increased intracellular pH optimally by 3 min with a half-maximal effect at 1-2 nM. The increases ... More
Guanosine 5'-thiotriphosphate may stimulate phosphoinositide messenger production in sea urchin eggs by a different route than the fertilizing sperm.
AuthorsCrossley I, Whalley T, Whitaker M
JournalCell Regul
PubMed ID1650582
'We show that microinjecting guanosine-5''-thiotriphosphate (GTP gamma S) into unfertilized sea urchin eggs generates an intracellular free calcium concentration [( Ca]i) transient apparently identical in magnitude and duration to the calcium transient that activates the egg at fertilization. The GTP gamma S-induced transient is blocked by prior microinjection of the ... More
Two-photon fluorescence lifetime imaging of the skin stratum corneum pH gradient.
AuthorsHanson KM, Behne MJ, Barry NP, Mauro TM, Gratton E, Clegg RM
JournalBiophys J
PubMed ID12202391
'Two-photon fluorescence lifetime imaging is used to identify microdomains (1-25 microm) of two distinct pH values within the uppermost layer of the epidermis (stratum corneum). The fluorophore used is 2'',7''-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF), whose lifetime tau (pH 4.5, tau = 2.75 ns; pH 8.5, tau = 3.90 ns) is pH dependent over ... More
Ion transport, membrane potential, and cytoplasmic pH in lymphocytes: changes during activation.
AuthorsGrinstein S, Dixon SJ
JournalPhysiol Rev
PubMed ID2648419
Fluorescence ratio imaging microscopy.
AuthorsBright GR, Fisher GW, Rogowska J, Taylor DL
JournalMethods Cell Biol
PubMed ID2648109
Optical methods to measure membrane transport processes.
AuthorsVerkman AS
JournalJ Membr Biol
PubMed ID8606370
Fluorescent indicators for intracellular pH.
AuthorsHan J, Burgess K,
JournalChem Rev
PubMed ID19831417
This review is about intracellular pH sensors, includingsmall fluorescent organic molecules, nanoparticles, andfluorescent proteins, e.g., GFP. It focuses on their preparations, photophysical properties, and advantages/disadvantagesfor intracellular pH measurements. The discussion is limitedto fluorescent indicators that have been applied to measureintracellular pH values since 1980. ... More
Activation of Na+/H+ exchange in human platelets stimulated by thrombin and a phorbol ester.
AuthorsSiffert W, Siffert G, Scheid P
JournalBiochem J
PubMed ID3566711
We have investigated changes in cytoplasmic pH (pHi) in activated human platelets, using the fluorescent probe 2,7-biscarboxyethyl-5(6)-carboxyfluorescein. Stimulation of platelets by thrombin or 12-O-tetradecanoylphorbol 13-acetate increased pHi by about 0.11 pH unit above the resting value. This increase in pHi depended on the presence of external Na+ and was inhibited ... More
Patch-clamp, ion-sensing, and glutamate-sensing techniques to study glutamate transport in isolated retinal glial cells.
AuthorsBillups B, Szatkowski M, Rossi D, Attwell D
JournalMethods Enzymol
PubMed ID9779478
We have described how a combination of electrical, ion-sensing, and glutamate-sensing techniques has advanced our understanding of glutamate uptake into isolated salamander retinal glial cells. The next steps in understanding glutamate transport will inevitably depend strongly on molecular biological methods, as described elsewhere in this book, but will also require ... More
Continuous monitoring of transport by fluorescence on cells and vesicles.
AuthorsEidelman O, Cabantchik ZI
JournalBiochim Biophys Acta
PubMed ID2686755
Fluorescence techniques are gaining wider applicability in the field of membrane transport due to their high temporal resolution, modest demand for biological material and the kinetic information which is made available by fluorescence tracings. The development of novel fluorescent substrates for particular transport systems and of novel fluorescent indicators for ... More
pH-regulated anion antiport in nucleated mammalian cells.
AuthorsOlsnes S, Tønnessen TI, Sandvig K
JournalJ Cell Biol
PubMed ID3949885
The uptake of 36Cl- into cells was measured after preincubation in medium containing nigericin and KCl to allow control of the intracellular pH. When the pH was increased from pH 7.0 to pH 7.3 there was a 10-fold increase in the rate of 36Cl- uptake. The increase was half maximal ... More
Renal intercalated cells are rather energized by a proton than a sodium pump.
AuthorsChambrey R, Kurth I, Peti-Peterdi J, Houillier P, Purkerson JM, Leviel F, Hentschke M, Zdebik AA, Schwartz GJ, Hübner CA, Eladari D,
JournalProc Natl Acad Sci U S A
PubMed ID23610411
The Na(+) concentration of the intracellular milieu is very low compared with the extracellular medium. Transport of Na(+) along this gradient is used to fuel secondary transport of many solutes, and thus plays a major role for most cell functions including the control of cell volume and resting membrane potential. ... More
Increased glycolytic ATP synthesis is associated with tafenoquine resistance in Leishmania major.
AuthorsManzano JI, Carvalho L, Pérez-Victoria JM, Castanys S, Gamarro F,
JournalAntimicrob Agents Chemother
PubMed ID21199921
Tafenoquine (TFQ), an 8-aminoquinoline used to treat and prevent Plasmodium infections, could represent an alternative therapy for leishmaniasis. Indeed, TFQ has shown significant leishmanicidal activity both in vitro and in vivo, where it targets Leishmania mitochondria and activates a final apoptosis-like process. In order not to jeopardize the life span ... More
Advantages and limitations of commonly used methods to assay the molecular permeability of gap junctional intercellular communication.
AuthorsAbbaci M, Barberi-Heyob M, Blondel W, Guillemin F, Didelon J,
JournalBiotechniques
PubMed ID18611167
The role of gap junctional intercellular communication (GJIC) in regulation of normal growth and differentiation is becoming increasingly recognized as a major cellular function. GJIC consists of intercellular exchange of low molecular weight molecules, and is the only means for direct contact between cytoplasms of adjacent animal cells. Disturbances of ... More
A continuous fluorimetric method to monitor the enzymatic hydrolysis of medicinal esters.
AuthorsSalvi A, Mayer JM, Carrupt PA, Testa B
JournalJ Pharm Biomed Anal
PubMed ID8933416
This paper describes a new spectrofluorimetric assay for continuously monitoring the enzymatic hydrolysis of medicinal esters. The procedure is based on the stoichiometric quantity of protons generated by the hydrolysis of the substrate, which produces changes in the fluorescence of a pH-sensitive dye. The pH indicator, 2', 7'-bis(car-boxyethyl)-5(6)-carboxyfluorescein, was selected ... More
A synthetic peptide of the pseudosubstrate domain of protein kinase C blocks cytoplasmic alkalinization during activation of the sea urchin egg.
AuthorsShen SS, Buck WR
JournalDev Biol
PubMed ID2373253
Multiple second messenger pathways have been proposed for transduction of the sperm-egg fusion event during fertilization of sea urchin eggs. Cytoplasmic alkalinization due to increased Na(+)-H+ antiport has been causally linked to many of the metabolic events during fertilization. Two possible second messenger pathways coupling sperm-egg fusion and antiporter activity ... More
Evaluation of algorithms for ratio imaging in fluorescence microscopy.
AuthorsRogowska J, Bright GR, Preston K, Taylor DL
JournalCytometry
PubMed ID2766881
Ratio imaging in fluorescence microscopy is used in measuring parameters such as pH, pCa, cytoplasmic porosity, and the relative concentration of fluorescent analogs within single cells. The fastest method for ratio imaging is to use lookup tables on special-purpose image processors. Since lookup tables store integers in integer addresses, using ... More
Barley aleurone cells contain two types of vacuoles. Characterization Of lytic organelles by use of fluorescent probes
AuthorsSwanson SJ, Bethke PC, Jones RL
JournalPlant Cell
PubMed ID9596630
Light microscopy was used to study the structure and function of vacuoles in living protoplasts of barley (Hordeum vulgare cv Himalaya) aleurone. Light microscopy showed that aleurone protoplasts contain two distinct types of vacuole: the protein storage vacuole and a lysosome-like organelle, which we have called the secondary vacuole. Fluorescence ... More
Inositol phosphates turnover, cytosolic Ca++ and pH: putative signals for the control of cell growth.
AuthorsVicentini LM, Villereal ML
JournalLife Sci
PubMed ID3014243
The signals that induce a cell to divide are usually external and in the form of a binding of growth factors. We focussed our attention in defining the sequence of events which occurs after the binding of the mitogens to their surface receptors. One of the early membrane events stimulated ... More
Intracellular pH measurement with fluorescent dye in canine basilar arteries.
AuthorsYu J, Zheng JJ, Ong BY, Bose R
JournalBlood Vessels
PubMed ID1782402
The intracellular pH (pHi) of basilar artery rings was determined with 2',7'-bis-(carboxyethyl)-5,(6)-carboxyfluorescein (BCECF) by measuring the ratio of emitted (540 nm) fluorescence intensities (FI) at excitation wavelengths of 500 and 440 nm. There was a dye loss from the rings in 90 min (39.3 +/- 3.6%, p less than 0.001). ... More
Phorbol ester-induced changes of cytoplasmic pH in neutrophils: role of exocytosis in Na+-H+ exchange.
AuthorsGrinstein S, Elder B, Furuya W
JournalAm J Physiol
PubMed ID2579574
Cytoplasmic pH homeostasis was studied in intact and granule-free porcine neutrophils following activation with 12-O-tetradecanoyl-phorbol-13-acetate (TPA). In intact cells, TPA activated at least two separate processes: a Na+-independent and amiloride-insensitive acidification, and a compensatory acid extrusion. The latter is Na+ dependent and blocked by amiloride and is likely to represent ... More
Internal calcium release and activation of sea urchin eggs by cGMP are independent of the phosphoinositide signaling pathway.
AuthorsWhalley T, McDougall A, Crossley I, Swann K, Whitaker M
JournalMol Biol Cell
PubMed ID1320962
We show that microinjecting cyclic GMP (cGMP) into unfertilized sea urchin eggs activates them by stimulating a rise in the intracellular free calcium ion concentration ([Ca2+]i). The increase in [Ca2+]i is similar in both magnitude and duration to the transient that activates the egg at fertilization. It is due to ... More
Na+/H+ exchange and cytoplasmic pH in the action of growth factors in human fibroblasts.
AuthorsMoolenaar WH, Tsien RY, van der Saag PT, de Laat SW
JournalNature
PubMed ID6410286
The mechanisms by which growth factors stimulate metabolism and cell proliferation are largely unknown. Recent evidence suggests that mitogens rapidly activate a Na+/H+ exchange mechanism in the plasma membrane of their target cells, implicating cytoplasmic pH (pH1) as a potential 'messenger'. Indeed, growth stimulation of quiescent fibroblasts leads to intracellular ... More
Intracellular pH on adrenergic-stimulated cAMP and cGMP production in rat pinealocytes.
AuthorsHo AK, Girard M, Young I, Chik CL
JournalAm J Physiol
PubMed ID1656768
In rat pinealocytes, activation of alpha 1-adrenergic receptors leads to increases in intracellular pH (pHi). In this study, the role of pHi on adrenergic regulation of cyclic nucleotide accumulation was investigated using ammonium chloride, which increased pHi, and sodium propionate, which reduced pHi. Ammonium chloride significantly enhanced the norepinephrine-stimulated adenosine ... More
Transmembrane gradients of free Na+ in isolated heart mitochondria estimated using a fluorescent probe.
AuthorsJung DW, Apel LM, Brierley GP
JournalAm J Physiol
PubMed ID1566810
The concentration of free Na+ in the matrix of isolated pig heart mitochondria has been monitored using the fluorescent probe sodium-binding benzofuran isophthalate (SBFI) developed by Minta and Tsien (J. Biol. Chem. 264: 19449-19457, 1989). SBFI was sequestered in the matrix by hydrolysis of the permeant acetoxymethyl ester. The sequestered ... More
Acidic pH of the lateral intercellular spaces of MDCK cells cultured on permeable supports.
AuthorsChatton JY, Spring KR
JournalJ Membr Biol
PubMed ID7932651
The pH of the lateral intercellular space (LIS) of Madin-Darby canine kidney (MDCK) cell monolayers grown on permeable supports was investigated by microspectrofluorimetry using BCECF (2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein). The permeability of the support was selectively reduced by growing Zn-Al-silicate crystals inside its pores. The diffusion of BCECF across the filter was sufficiently ... More
Positive charges of the initial C-terminus domain of Cx32 inhibit gap junction gating sensitivity to CO2.
AuthorsWang XG, Peracchia C
JournalBiophys J
PubMed ID9251796
Gap junction channels close with CO2 exposure. To determine whether the carboxy-terminus (CT) of connexin32 (Cx32) participates in gating, the CO2 sensitivity of channels made of Cx32 or Cx32 mutants was studied by double voltage clamp. In Xanopus laevis oocytes expressing Cx32, junctional conductance (Gj) dropped to 85% and 47% ... More
Ca2+ depletion from granules inhibits exocytosis. A study with insulin-secreting cells.
AuthorsScheenen WJ, Wollheim CB, Pozzan T, Fasolato C
JournalJ Biol Chem
PubMed ID9668080
The secretory compartment is characterized by low luminal pH and high Ca2+ content. Previous studies in several cell types have shown that the size of the acidic Ca2+ pool, of which secretory granules represent a major portion, could be estimated by applying first a Ca2+ ionophore followed by agents that ... More
A fast kinetic method for assessing mitochondrial membrane potential in isolated hepatocytes with rhodamine 123 and flow cytometry.
AuthorsJuan G, Cavazzoni M, Sáez GT, O'Connor JE
JournalCytometry
PubMed ID8026223
Rhodamine 123 (Rh123) is widely used as a flow cytometric probe for mitochondrial membrane potential (MMP) in metabolic, pharmacologic, and toxicological studies. However, the use of relatively high concentrations of Rh123 (up to 10 micrograms/ml) and prolonged incubation times (up to 1 h), including washing steps, may be inconvenient for ... More
Inositol trisphosphate and ryanodine receptors share a common functional Ca2+ pool in cerebellar Purkinje neurons.
AuthorsKhodakhah K, Armstrong CM
JournalBiophys J
PubMed ID9414245
Changes in the intracellular free calcium concentration ([Ca2+]i) control many important processes in excitable and nonexcitable cells. In cerebellar Purkinje neurons, increases in [Ca2+]i modulate excitability by turning on calcium-activated potassium and chloride conductances, and modifying the synaptic efficacy of inhibitory and excitatory inputs to the cell. Calcium release from ... More
pH regulation in the intracellular malaria parasite, Plasmodium falciparum. H(+) extrusion via a v-type h(+)-atpase.
AuthorsSaliba KJ, Kirk K
JournalJ Biol Chem
PubMed ID10559194
The mechanism by which the intra-erythrocytic form of the human malaria parasite, Plasmodium falciparum, extrudes H(+) ions and thereby regulates its cytosolic pH (pH(i)), was investigated using saponin-permeabilized parasitized erythrocytes. The parasite was able both to maintain its resting pH(i) and to recover from an imposed intracellular acidification in the ... More
Cytoplasmic pH and free Mg2+ in lymphocytes.
AuthorsRink TJ, Tsien RY, Pozzan T
JournalJ Cell Biol
PubMed ID6815204
Measurements have been made of cytoplasmic pH, (pHi) and free Mg2+ concentration, ( [Mg2+]i), in pig and mouse lymphocytes. pHi was measured in four ways: by a digitonin null-point technique; by direct measurement of the pH of freeze-thawed cell pellets; from the 31P nuclear magnetic resonance (NMR) spectrum of intracellular ... More
Acute inhibition of brain-specific Na(+)/H(+) exchanger isoform 5 by protein kinases A and C and cell shrinkage.
AuthorsAttaphitaya S, Nehrke K, Melvin JE
JournalAm J Physiol Cell Physiol
PubMed ID11546650
Little is known of the functional properties of the mammalian, brain-specific Na(+)/H(+) exchanger isoform 5 (NHE5). Rat NHE5 was stably expressed in NHE-deficient PS120 cells, and its activity was characterized using the fluorescent pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. NHE5 was insensitive to ethylisopropyl amiloride. The transport kinetics displayed a simple Michaelis-Menten relationship ... More
Simultaneous measurements of intracellular pH in the leech giant glial cell using 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein and ion-sensitive microelectrodes.
AuthorsNett W, Deitmer JW
JournalBiophys J
PubMed ID8804622
We have employed two independent techniques to measure the intracellular pH (pHi) in giant glial cells of the leech Hirudo medicinalis, using the fluorescent dye 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF) and double-barreled neutral-carrier, pH-sensitive microelectrodes, which also record the membrane potential. We have compared two procedures for calibrating the ratio of the BCECF ... More
Phorbol ester and diacylglycerol mimic growth factors in raising cytoplasmic pH.
AuthorsMoolenaar WH, Tertoolen LG, de Laat SW
JournalNature
PubMed ID6095096
There is now good evidence that cytoplasmic pH (pHi) may have an important role in the metabolic activation of quiescent cells. In particular, growth stimulation of mammalian fibroblasts leads to a rapid increase in pHi (refs 3-6), due to activation of a Na+/H+ exchanger in the plasma membrane, and this ... More
Dependence of Na+/H+ antiport activation in cultured rat aortic smooth muscle on calmodulin, calcium, and ATP. Evidence for the involvement of calmodulin-dependent kinases.
AuthorsLittle PJ, Weissberg PL, Cragoe EJ, Bobik A
JournalJ Biol Chem
PubMed ID2846549
The role of Ca2+/calmodulin-dependent processes in the activation of the Na+/H+ antiport of primary cultures of rat aortic smooth muscle was studied using 22Na+ uptake and measurement of intracellular pH (pHi) with the fluorescent pH dye 2',7'-bis-(2-carboxyethyl)-5(and 6)-carboxyfluorescein. Antiport activation following exposure to serum and by the induction of an ... More
Changes in intracellular calcium concentration and pH of target cells during the cytotoxic process: a quantitative study at the single cell level.
AuthorsRadosevic K, de Grooth BG, Greve J
JournalCytometry
PubMed ID7587715
This study reports on the changes in intracellular calcium concentration ([Ca2+]in) and intracellular pH ([pH]in) that occur in K562 target cells during interaction with human Natural Killer (NK) cells. The data were obtained using a quantitative fluorescence microscope and fluorescent ratio probes specific for [Ca2+]in (Fura-2-AM) and [pH]in (BCECF-AM). Results ... More
Acidosis of rat dorsal vagal neurons in situ during spontaneous and evoked activity.
AuthorsTrapp S, Lückermann M, Brooks PA, Ballanyi K
JournalJ Physiol
PubMed ID8930837
1. Rat brainstem slices were taken for simultaneous measurements of intracellular pH (pHi) and membrane currents or potentials in dorsal vagal neurons, dialysed with the pH-sensitive dye BCECF. 2. Intrinsic intracellular buffering power was 18 mM per pH unit, as determined by exposure to trimethylamine in CO2/HCO3(-)-free, Hepes-buffered saline. 3. ... More
Studies of the mechanism of activation of HIT-T15 cells by lactate.
AuthorsLynch AM, Trebilcock R, Tomlinson S, Best L
JournalBiochim Biophys Acta
PubMed ID1995073
L-Lactate, D-lactate, propionate and acetate (all 20 mM) caused a marked intracellular acidification in HIT-T15 cells loaded with 2'7'-bis(carboxyethyl)-5'(6')-carboxyfluorescein (BCECF), followed by recovery to more alkaline values. The effects of L- and D-lactate, but not propionate or acetate, were inhibited by 5 mM alpha-fluorocinnamate. Both L- and D-lactate caused a ... More
4-aminopyridine acts as a weak base and a Ca2+ mobilizing agent in triggering oocyte meiosis reinitiation and activation in the Japanese clam Ruditapes philippinarum.
AuthorsGobet I, Lippai M, Tomkowiak M, Durocher Y, Leclerc C, Moreau M, Guerrier P
JournalInt J Dev Biol
PubMed ID7577439
Ovarian oocytes of the prosobranch mollusc Patella vulgata and the pelecypod Ruditapes philippinarum are arrested during prophase of the first maturation division. Release from this blockade, which is revealed by germinal vesicle breakdown, drives these oocytes to a second arrest in metaphase I, at which time the oocytes become fertilizable. ... More
Effect of acidosis on intracellular pH and calcium concentration in the newborn and adult rabbit myocardium.
AuthorsNakanishi T, Seguchi M, Tsuchiya T, Yasukouchi S, Takao A
JournalCirc Res
PubMed ID2364486
This study investigated developmental changes in the effect of acidosis on intracellular pH (pHi) and [Ca]i in the isolated heart and isolated myocyte preparations. The whole heart or myocytes of newborn (5-7 days old) and adult rabbits were loaded with the fluorescent pH indicator 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) or calcium indicator fura-2. ... More
Regulation of intracellular pH in resting and in stimulated parietal cells.
AuthorsParadiso AM, Townsley MC, Wenzl E, Machen TE
JournalAm J Physiol
PubMed ID2476937
Microspectrofluorimetry of the pH-sensitive, fluorescent dye 2',7'-biscarboxyethyl-5 (6)-carboxyfluorescein (BCECF) was used to measure intracellular pH (pHi) in single parietal cells (PC) of intact rabbit gastric glands during resting and stimulated conditions. In 61% of PC, histamine plus isobutylmethylxanthine (IBMX) (both 100 microM) caused a small increase in pHi, ranging from ... More
Plasma membrane Na+/H+ exchanger isoforms (NHE-1, -2, and -3) are differentially responsive to second messenger agonists of the protein kinase A and C pathways.
Na+/H+ exchanger (NHE) activity is regulated by several types of receptors directly coupled to distinct classes (i.e. Gs, Gi, Gq, and G12) of heterotrimeric (alpha beta gamma) GTP-binding proteins (G proteins), which, upon activation, modulate production of various second messengers (e.g. cAMP, cGMP, diacylglycerol, inositol trisphosphate, and Ca2+). Recently, four ... More
Ca2+ mobilization can occur independent of acceleration of Na+/H+ exchange in thrombin-stimulated human platelets.
AuthorsZavoico GB, Cragoe EJ
JournalJ Biol Chem
PubMed ID2838474
Intracellular free Ca2+ [( Ca2+]i) and pH (pHi) were measured simultaneously by dual wavelength excitation in thrombin-stimulated human platelets double-labeled with the fluorescent probes fura2 and 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein to determine the relationship between changes in [Ca2+]i and pHi, respectively. At 37 degrees C, thrombin (0.5 or 0.1 units/ml) increased [Ca2+]i with ... More
The application of pH-sensitive fluorescent dyes in lactic acid bacteria reveals distinct extrusion systems for unmodified and conjugated dyes.
AuthorsGlaasker E, Konings WN, Poolman B
JournalMol Membr Biol
PubMed ID8905646
Intracellular pH in bacteria can be measured efficiently between internal pH values of 6.5 and 8.5 with the fluorescent pH indicator 2',7'-bis-(2-carboxyethyl)-5[and-6]-carboxyfluorescein (BCECF). A new fluorescent pH probe with a lower pKa(app) than BCECF was synthesized from fluorescein isothiocyanate and glutamate. The new probe, N-(fluorescein thio-ureanyl)-glutamate (FTUG), was much less ... More
Electrophoretic repatterning of charged cytoplasmic molecules within tissues coupled by gap junctions by externally applied electric fields.
AuthorsCooper MS, Miller JP, Fraser SE
JournalDev Biol
PubMed ID2917693
Ionic currents and cytoplasmic voltage gradients have been observed in a variety of polarizing cells and developing tissues. In certain cases, it has been determined that these endogenous electric fields can segregate intracellular charged molecules by electrophoresis; in other cases, the endogenous fields are suspected to have such an influence. ... More
Na+-H+ exchange in gastric glands as measured with a cytoplasmic-trapped, fluorescent pH indicator.
AuthorsParadiso AM, Tsien RY, Machen TE
JournalProc Natl Acad Sci U S A
PubMed ID6095295
We have used the pH-sensitive, fluorescent, cytoplasmic-trapped dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) to identify Na+-H+ exchange in gastric glands isolated from rabbit stomachs by high-pressure perfusion and collagenase digestion. The fluorescence of BCECF-loaded glands was calibrated in terms of cytosolic pH (pHc) by permeabilizing the cell membranes and titrating the extracellular solution ... More
Ca2+ and pH determine the interaction of chromaffin cell scinderin with phosphatidylserine and phosphatidylinositol 4,5,-biphosphate and its cellular distribution during nicotinic-receptor stimulation and protein kinase C activation.
AuthorsRodríguez Del Castillo A, Vitale ML, Trifaró JM
JournalJ Cell Biol
PubMed ID1331119
Nicotinic stimulation and high K(+)-depolarization of chromaffin cells cause disassembly of cortical filamentous actin networks and redistribution of scinderin, a Ca(2+)-dependent actin filament-severing protein. These events which are Ca(2+)-dependent precede exocytosis. Activation of scinderin by Ca2+ may cause disassembly of actin filaments leaving cortical areas of low cytoplasmic viscosity which ... More
Multiphasic action of glucose and alpha-ketoisocaproic acid on the cytosolic pH of pancreatic beta-cells. Evidence for an acidification pathway linked to the stimulation of Ca2+ influx.
AuthorsSalgado A, Silva AM, Santos RM, Rosário LM
JournalJ Biol Chem
PubMed ID8621508
Glucose stimulation raises the pHi of pancreatic beta-cells, but the underlying mechanisms are not well understood. We have now investigated the acute effects of metabolizable (glucose and the mitochondrial substrate alpha-ketoisocaproic acid, KIC) and nonmetabolizable (high K+ and the K-ATP channel blocker tolbutamide) insulin secretagogues on the pHi of pancreatic ... More
The intracellular calcium increase at fertilization in Urechis caupo oocytes: activation without waves.
AuthorsStephano JL, Gould MC
JournalDev Biol
PubMed ID9356171
The intracellular Ca2+ (Cai) increase at fertilization of the marine worm Urechis caupo (Echiura) was studied with conventional and confocal epifluorescence microscopy in oocytes microinjected with calcium green dextran or dually labeled with the calcium-insensitive dye tetramethylrhodamine dextran. Calcium green fluorescence was also measured with a photomultiplier system while the ... More
Characteristics of fluoroprobes for measuring intracellular pH.
AuthorsGraber ML, DiLillo DC, Friedman BL, Pastoriza-Munoz E
JournalAnal Biochem
PubMed ID3740410
We evaluated four different fluoroprobes to determine their capabilities and limitations in measuring intracellular pH by the fluorescent indicator technique. In vitro, carboxyfluorescein, dimethylcarboxyfluorescein, biscarboxyethyl carboxyfluorescein, and 4-methylumbelliferone (4MU) all showed comparably intense fluorescence and excellent pH sensitivity near their respective pKa values. Major differences were found between 4MU and ... More
pH-dependent fluorescence of a heterologously expressed Aequorea green fluorescent protein mutant: in situ spectral characteristics and applicability to intracellular pH estimation.
AuthorsRobey RB, Ruiz O, Santos AV, Ma J, Kear F, Wang LJ, Li CJ, Bernardo AA, Arruda JA
JournalBiochemistry
PubMed ID9665694
The green fluorescent protein of Aequorea victoria (GFP) is a natural peptide chromophore without substrate or cofactor requirements for fluorescence. In vitro, a recombinant F64L/S65T GFP mutant (GFPmut1) exhibited pH sensitive fluorescence within the physiologic range. When heterologously expressed in BS-C-1 cells or rabbit proximal tubule cells, uniform cytosolic and ... More
Upregulation of the hyperpolarization-activated cation current in rat thalamic relay neurones by acetazolamide.
AuthorsMunsch T, Pape HC
JournalJ Physiol
PubMed ID10457066
1. The effect of inhibition of brain carbonic anhydrase (CA) on the hyperpolarization-activated cation current (Ih) of thalamocortical (TC) neurones of the rat ventrobasal thalamic complex (VB) was investigated in an in vitro slice preparation using the whole-cell patch-clamp technique and fluorescence ratio imaging of the pH indicator 2',7'-bis(carboxyethyl)-5(and -6)-carboxyfluorescein ... More
The role of metabolism, cytoplasmic Ca2+, and pH-regulating exchangers in glucose-induced rise of cytoplasmic pH in normal mouse pancreatic islets.
AuthorsShepherd RM, Henquin JC
JournalJ Biol Chem
PubMed ID7713887
Intact mouse islets were loaded with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein to study the effects of glucose on cytoplasmic pH (pHi) in pancreatic B-cells. In HCO3- buffer, glucose produced a steady-state increase in pHi that required metabolism of the sugar and was concentration-dependent between 0 and 10 mM (Km approximately 5 mM) before plateauing ... More
Parathyroid hormone decreases HCO3 reabsorption in the rat proximal tubule by stimulating phosphatidylinositol metabolism and inhibiting base exit.
AuthorsPastoriza-Munoz E, Harrington RM, Graber ML
JournalJ Clin Invest
PubMed ID1314850
The mechanism of inhibition of HCO3 transport by parathyroid hormone (PTH) in the proximal tubule is not clearly defined. Previous studies in vitro have suggested that this effect is mediated via cAMP generation, which acts to inhibit Na/H exchange, resulting in cell acidification. To examine this question in vivo, intracellular ... More
The Toxoplasma Vacuolar H+-ATPase Regulates Intracellular pH and Impacts the Maturation of Essential Secretory Proteins.