bafilomycin-a1 and 2--7--bis(carboxyethyl)-5(6)-carboxyfluorescein

Giardia intestinalis is a pathogenic fermentative parasite, which inhabits the gastrointestinal tract of animals and humans. G. intestinalis trophozoites are exposed to acidic fluctuations in vivo and must also cope with acidic metabolic endproducts. In this study, a combination of independent techniques ((31)P NMR spectroscopy, distribution of the weak acid pH marker 5,5-dimethyl-2,4-oxazolidinedione (DMO) and the fluorescent pH indicator 2',7'-bis (carboxyethyl)-5,6-carboxyfluorescein (BCECF)) were used to show that G. intestinalis trophozoites exposed to an extracellular pH range of 6.0--7.5 maintain their cytosolic pH (pH(i)) within the range 6.7--7.1. Maintenance of the resting pH(i) was Na(+)-dependent but unaffected by amiloride (or analogs thereof). Recovery of pH(i) from an intracellular acidosis was also Na(+)-dependent, with the rate of recovery varying with the extracellular Na(+) concentration in a saturable manner (K(m) = 18 mm; V(max) = 10 mm H(+) min(-1)). The recovery of pH(i) from an acid load was inhibited by amiloride but unaffected by a number of its analogs. The postulated involvement of one or more Na(+)/H(+) exchanger(s) in the regulation of pH(i) in G. intestinalis is discussed.

Topics: Amiloride; Animals; Anti-Bacterial Agents; Cell Membrane; Cytosol; Dicyclohexylcarbodiimide; Dimethadione; Ethylmaleimide; Fluoresceins; Fluorescent Dyes; Giardia lamblia; Hydrogen-Ion Concentration; Kinetics; Macrolides; Magnetic Resonance Spectroscopy; Phosphorus; Sodium; Vanadates

The hypothesis that changes in cytosolic pH effect the release from intracellular compartments of stored calcium in Trypanosoma brucei was addressed by the use of procyclic and bloodstream trypomastigotes of T. brucei loaded with the fluorescent reagents 2',7'-bis-(2-carboxyethyl)-5(and 6)-carboxyfluorescein (BCECF) to measure intracellular pH (pHi), or fura 2 to measure intracellular free calcium ([Ca2+]i). Experiments were performed in EGTA-containing buffers, so increases in [Ca2+]i reflected release of stored calcium rather than Ca2+ entry. Nigericin reduced pHi and increased [Ca2+]i in loaded cells, whilst propionate reduced pHi, but did not affect [Ca2+]i, and NH4Cl increased both variables, so there appears to be no correlation between pHi and [Ca2+]i. Treatment of the cells with the calcium ionophore ionomycin under similar conditions (nominal absence of extracellular Ca2+) resulted in an increase of [Ca2+]i which was greatly increased by addition of either NH4Cl, nigericin or the vacuolar H(+)-ATPase inhibitor bafilomycin A1. Similar results were obtained when the order of additions was reversed or when digitonin-permeabilized cells were used with the Ca2+ indicator arsenazo III. The results suggest that more Ca2+ is stored in this acidic compartment in procyclic than in bloodstream forms. Taking into account the relative importance of the ionomycin-releasable and the ionomycin-plus-NH4Cl-releasable Ca2+ pools, it is apparent that a significant amount of the Ca2+ stored in T. brucei trypomastigotes is present in the acidic compartment thus identified.

Topics: Ammonium Chloride; Animals; Anti-Bacterial Agents; Calcium; Cytosol; Enzyme Inhibitors; Fluoresceins; Fluorescent Dyes; Fura-2; Hydrogen-Ion Concentration; Ionomycin; Ionophores; Kinetics; Macrolides; Nigericin; Propionates; Proton-Translocating ATPases; Trypanosoma brucei brucei; Vacuoles

The role of plasma membrane V-ATPase activity in the regulation of cytosolic pH (pHi) was determined for resident alveolar and peritoneal macrophages (m theta) from sheep. Cytosolic pH was measured using 2',7'-biscarboxyethyl-5,6-carboxyfluorescein (BCECF). The baseline pHi of both cell types was sensitive to the specific V-ATPase inhibitor bafilomycin A1. Bafilomycin A1 caused a significant (approximately 0.2 pH units) and rapid (within seconds) decline in baseline pHi. Further, bafilomycin A1 slowed the initial rate of pHi recovery (dpHi/dt) from intracellular acid loads. Amiloride had no effects on baseline pHi, but reduced dpHi/dt (acid-loaded pHi nadir < 6.8) by approximately 35%. Recovery of pHi was abolished by co-treatment of m theta with bafilomycin A1 and amiloride. These data indicate that plasma membrane V-ATPase activity is a major determinant of pHi regulation in resident alveolar and peritoneal m theta from sheep. Sheep m theta also appear to possess a Na+/H+ exchanger. However, Na+/H+ exchange either is inactive or can be effectively masked by V-ATPase-mediated H+ extrusion at physiological pHi values.

Topics: Animals; Anti-Bacterial Agents; Cell Membrane; Fluoresceins; Hydrogen-Ion Concentration; Macrolides; Macrophages, Peritoneal; Proton-Translocating ATPases; Pulmonary Alveoli; Sheep; Vacuoles

We studied the influence of interstitial buffer capacity and CO2-HCO3- on oxyntic cell intracellular pH (pHi) in intact frog gastric mucosa. Oxyntic cells in stripped gastric mucosa of Rana esculenta were loaded with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, and pHi was assessed fluorometrically. In the presence of a constant serosal and luminal pH, oxyntic cell pHi was dependent on the serosal but not the luminal concentration of cell-impermeable buffer ions such as N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), tris(hydroxymethyl)aminomethane, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, and 3-(N-morpholino)propanesulfonic acid. The stepwise increase in oxyntic cell pHi from 6.74 +/- 0.05 with 1 mM HEPES to 7.23 +/- 0.08 was almost completely inhibited by removal of serosal Na+ and by amiloride and dimethyl amiloride, suggesting that it was largely due to Na+/H+ exchange. Increasing the serosal concentration of a CO2-HCO3- buffer from 1% CO2-4 mM HCO3- to 10% CO2-40 mM HCO3- increased oxyntic cell pHi from 7.03 +/- 0.06 to 7.39 +/- 0.07. This CO2-HCO(3-)-dependent pHi increase was also Na+ and amiloride sensitive, but high HCO3- concentrations increased pHi even in the absence of Na+, K+, or Cl-, and in the presence of omeprazole, bafilomycin A1, or acetazolamide. We suggest that in intact frog gastric mucosa, cellular proton extrusion acidifies the interstitial pH immediately adjacent to the basolateral membrane of the oxyntic cells in the absence of a high interstitial buffer capacity.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Anti-Bacterial Agents; Bicarbonates; Buffers; Carbon Dioxide; Fluoresceins; Gastric Mucosa; Hydrogen-Ion Concentration; Kinetics; Macrolides; Parietal Cells, Gastric; Rana esculenta; Sodium; Sodium-Hydrogen Exchangers

H+ secretory mechanisms and intrinsic intracellular buffering capacity were studied in crypt cells from rabbit distal colon. To this end crypts of Lieberkühn were isolated by microdissection, and intracellular pH (pHi) was measured using digital imaging fluorescence microscopy and the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)- 5(6)-carboxyfluorescein. In the absence of HCO(3-)-CO2 and presence of Na+, resting pHi was 7.51 +/- 0.04 (n = 237/23, cells/crypts). However, 6 min after superfusion with a solution containing zero Na+, 1 x 10(5) M Sch-28080 and 5 x 10(-8) M bafilomycin A1, pHi in cells at the bottom of the crypts was significantly reduced, whereas pHi in cells at the top of the crypts remained unchanged. The intrinsic buffering capacity of cells from the middle to the top portion of crypts was significantly higher in the pHi range 7.2-7.6 than of cells at the bottom of the crypt. H+ secretion after an NH(4+)-NH3 pulse amounted to 245 +/- 53 microM/s (n = 73/7) at pHi 7.1 and was largely Na+ dependent and ethylisopropylamiloride sensitive. The Na(+)-independent recovery of pHi after an acid load was insensitive to Sch-28080 and bafilomycin A1. In conclusion, pHi in colonic crypt cells is regulated through Na+/H+ exchange activity in the absence of HCO3-. In addition, intracellular buffering capacity varied with the position along the crypt axis, whereas Na+/H+ exchange activity and pHi did not.

Topics: Amiloride; Ammonia; Animals; Anti-Bacterial Agents; Buffers; Colon; Female; Fluoresceins; Fluorescent Dyes; Hydrogen-Ion Concentration; Imidazoles; Intracellular Membranes; Macrolides; Male; Quaternary Ammonium Compounds; Rabbits; Sodium