Slender and stumpy bloodstream forms of Trypanosoma brucei display a differential response to extracellular acidic and proteolytic stress.
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Natural infections of mammals with African trypanosomes, such as Trypanosoma brucei, are generally pleomorphic, the population consisting of different forms, termed slender and stumpy forms, that vary in number as the parasitaemia develops. We show that the differentiation of slender into stumpy forms is characterized by the acquisition by the parasite of the ability to regulate its internal pH, even in the face of a large, inwardly directed gradient of H+, as well as a tolerance towards external proteolytic stress. These adaptations effectively abbrogate cellular stress-activated signalling pathways involving adenylate cyclase and glycosylphosphoinositol-specific phospholipase-C mediated release of the surface coat. Although in metabolic terms stumpy forms of the parasite are considered to be preadapted to life in the arthropod vector, these data clearly demonstrate that these forms also possess additional cellular adaptations designed to deal with the immediate and potentially harmful changes in the extracellular environment that occur upon ingestion of a bloodmeal by the tsetse fly vector. (+info)
Hydrogen ion gradients across the mitochondrial, endosomal and plasma membranes in bloodstream forms of trypanosoma brucei solving the three-compartment problem.
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Conditions for the use of both [14C]methylamine and 5, 5-dimethyl[14C]oxa-azolidine-2,4-dione (DMO) to measure the H+ concentration of intracellular compartments of monomorphic long thin bloodstream forms of Trypanosoma brucei were established. Neither probe was actively transported or bound to internal components of the cell and both probes equilibrated passively with a t1/2 close to 8 min. DMO was excluded from cells, while methylamine was accumulated but not metabolized. Solution of the three-compartment problem revealed that, when cells were respiring aerobically on glucose at an external pH of 7.5, the cytoplasmic pH was in the range 6.99-7.03, the pH of the mitochondrial matrix was 7.71-7.73, and the algebraic average pH of the various endosomal compartments was 5.19-5.50. Similar values were found when cells were respiring aerobically on glycerol. However, bloodstream forms of T. brucei could not maintain a constant internal H+ concentration outside the external pH range 7.0-7.5, and no evidence for the presence of an H+/Na+ exchanger was found. Full motility and levels of pyruvate production were maintained as the external pH was raised as high as 9.5, suggesting that these cells tolerate significant internal alkalinisation. However, both motility and pyruvate production were severely inhibited under acidic conditions, and the cells deteriorated rapidly below an external pH of 6.5. Physiologically, the plasma membrane of T. brucei had low permeability to H+ and the internal pH was unaffected by changes in Deltapsip, which is dominated by the potassium diffusion potential. However, in the presence of FCCP, the internal pH fell rapidly about 0.5 pH unit and came into equilibrium with Deltapsip. Oligomycin abolished the mitochondrial pH gradient (DeltapHm) selectively, whereas chloroquine abolished only the endosomal pH gradient (DeltapHe). The pH gradient across the plasma membrane (DeltapHp) alone could be abolished by careful osmotic swelling of cells. The plasma membrane had an inwardly directed proton-motive force (DeltaPp) of -52 mV and an inwardly directed sodium-motive force (DeltaNp) of -149 mV, whereas the mitochondrial inner membrane had only an inwardly directed DeltaPm of -195 mV. The pH gradient across the endosomal membranes was not accompanied by an electrical gradient. Consequently, endosomal membranes had an algebraically average outwardly directed DeltaPl within the range + 89 to +110 mV, depending on the measurement method. (+info)
Na(+)-dependent pH regulation by the amitochondriate protozoan parasite Giardia intestinalis.
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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. (+info)
Altering intracellular pH disrupts development and cellular organization in preimplantation hamster embryos.
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In early cleavage stage hamster embryos, the inability to regulate intracellular pH (pHi) properly is associated with reduced developmental competence in vitro. The disruption of mitochondrial organization is also correlated with reduced development in vitro. To determine the relationship between pHi and the disruption of cytoplasmic organization, we examined the effects of altering pHi on hamster embryo development, mitochondrial distribution, and cytoskeletal organization. The weak base trimethylamine was used to increase pHi and was found to reduce embryo development and disrupt the perinuclear organization of mitochondria. The weak acid 5,5-dimethyl-2,4-oxazolinedione was used to decrease pH(i) and was also found to reduce development and disrupt the perinuclear organization of mitochondria. With either treatment, the microfilament organization was perturbed, but the microtubule cytoskeleton was not. However, the temporal progression of the disruption of mitochondrial distribution was more rapid in alkalinized embryos than acidified embryos, as revealed by two-photon imaging of living embryos. Additionally, the disruption of the microfilament network by the two treatments was not identical. The cytoplasmic disruptions observed were not due to acute toxicity of the compounds because embryos recovered developmentally when the treatment compounds were removed. These observations link ionic homeostasis, structural integrity and developmental competence in preimplantation hamster embryos. (+info)
Intracellular pH of single crustacean muscle fibres by the DMO and electrode methods during acid and alkaline conditions.
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1. The intracellular pH of intact single muscle fibres of the giant barnacle was measured directly with a glass micro-electrode following prolonged (2-5 hr) equilibration in one of three solutions: normal Ringer, CO2 Ringer and NH4+ Ringer. 2. The intracellular pH of identically-prepared fibres from the same specimen was measured indirectly from the distribution of DMO following prolonged equilibration in the same solutions. 3. The DMO-pH compared favourably with the electrode-pHi provided DMO-pHi was calculated from the values of the indicator compounds, [14C]DMO and [3H]inulin, obtained by extrapolating the slow uptake phase to time zero. 4. Following prolonged equilibration, the transmembrane H+ ion distribution was found to vary with the membrane potential but not in accordance with a simple Gibbs-Donnan equilibrium. 5. A model which recognizes the existence of two independent net fluxes for H+ across the membrane in developed to explain the results. One of the fluxes represents passive diffusion and the other represents the so called H+-pump. The model predicts the H+-pump rate increases by two orders of magnitude when pHi is reduced from 7-2 to 6-7. (+info)
Determination of the intravesicular pH of fragmented sarcoplasmic reticulum with 5,5-dimethyl-2,4-oxazolidinedione.
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The intravesicular pH (pHi) of fragmented sarcoplasmic reticulum (SR) of the skeletal muscle was determined from the distribution of 5,5-dimethyl-2,4-oxazolidinedione (DMO), a weak organic acid, between the intra- and extravesicular spaces. The pHi's thus obtained were found to be slightly lower (0.02-0.17 pH unit) than the pH's of the external medium (pHe) at pH 6.5-8.5 in the presence of 105 mM KCl and 40 mM Tris-maleate buffer. The higher the pHe, the greater the pH gradient. When pHe was changed, pHi attained equilibrium within about 20 min, the time necessary for the separation of the SR by centrifugation. When 0.25 M sucrose and 5 mM Tris-maleate buffer were used instead of 105 mM KCl and 40 mM buffer, the pH gradient increased to 0.56. It was also demonstrated by direct measurements of pHe with a glass-electrode pH meter that K+ ions added to the external medium exchanged the intravesicular H+ ions. From these results it appears that the pH gradient across the SR membrane was at the Donnan equilibrium. In this state, the Donnan potentials corresponding to pH gradients of 0.17 and 0.56 were -9.3 and -30.6 mV, respectively. (+info)
The effects of diltiazem on hepatic drug metabolizing enzymes in man using antipyrine, trimethadione and debrisoquine as model substrates.
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Six healthy male subjects were given single oral doses of antipyrine (7 mg kg-1), trimethadione (4 mg kg-1) and debrisoquine (10 mg) before and during diltiazem treatment (30 mg three times daily orally for 8 days). Antipyrine clearance decreased from 33.7 +/- 9.1 to 22.5 +/- 4.9 ml min-1 (P less than 0.05, mean +/- s.e. mean) after diltiazem treatment without any significant change in apparent volume of distribution (0.59 +/- 0.06 to 0.60 +/- 0.04 1 kg-1), resulting in an increase in antipyrine elimination half-life from 13.4 +/- 4.8 to 19.7 +/- 3.2 h (P less than 0.05). The formation clearance of antipyrine to 4-hydroxyantipyrine was decreased significantly from 10.8 +/- 2.7 to 6.6 +/- 2.7 ml min-1 (P less than 0.05), while that to 3-hydroxymethylantipyrine and norantipyrine was not altered by diltiazem. The metabolic ratio of debrisoquine (urinary excretion of debrisoquine/4-hydroxydebrisoquine) was increased significantly from 0.70 +/- 0.05 to 1.95 +/- 0.20 (P less than 0.05), while that of trimethadione (serum concentration of dimethadione/trimethadione) was not changed significantly (0.48 +/- 0.08 vs 0.41 +/- 0.06) after diltiazem treatment. Diltiazem selectively inhibits cytochrome P-450 isoenzymes. (+info)
Intracellular pH and distribution of weak acids across cell membranes. A study of D- and L-lactate and of DMO in rat diaphragm.
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1. The steady-state distribution ratios of D- and L-lactate between fibre water and external fluid were measured in 'intact' rat hemidiaphragm preparations exposed for 2-5 hr to a variety of solutions of normal ionic strength and osmolarity. The studies were designed to minimize the effects, on these distributions, of conversion of lactate and of generation of lactic acid by the muscle. 2. At D-lactate concentrations between 2.3 and 118 mM, at normal pH and PCO2, the D-lactate distribution ratio, obtained from the distribution of [2-(3)H]D-lactate was independent of concentration; it averaged 0.349. As the concentration of D-lactate was reduced below 2.3 mM, its distribution ratio progressively fell to less than 0.1. 3. Radiochromatograms of extracts of incubated muscle showed that the tritium label was not attached to substances other than lactate. 4. At L-lactate concentrations of 59 and 108 mM, at normal pH and PCO2, the average L-lactate distribution ratios, obtained by enzymatic analysis, were respectively 0.395 and 0392. 5. At 19-89 mM D-lactate, depolarizing the muscle fibres by high K(49-127 mM), at normal pH, PCO2, and [K]0[Cl]0 product, only slightly affected the D-lactate distribution ratio which averaged 0.405. 6. The D-lactate distribution ratio and intracellular pH (pHi), obtained with the DMO method (5,5-dimethyl-2,4-oxazolidinedione), were measured in thirty sets of studies after exposure of the muscle to solutions buffered to pH values ranging between 5.99 and 8.13, and containing 18.5-118 mM D-lactate and 6-129 mM-K. 7. The relation between the distribution ratios of D-lactate ([TL]i/[TL]O) and of H ions ([Ho/[H]i) in these studies could be expressed by [TL]i/[T]O = 0.646 [H]o/[H]i+0.056. 8. It was concluded that it is predominantly the undissociated lactic acid molecules, rather than the much more numerous lactate ions, which permeate the fibre membrane; and that the steady-state lactate distribution ratio is determined by the transmembrane pH gradient, and not by membrane potential. 9. The expression of the steady-state lactate distribution ratio as function of relative membrane permeabilities of lactic acid molecule and lactate ion, membrane voltage, and internal and external H ion concentrations indicates that a finite permeability to the ion, three or four orders of magnitude less than that to the molecule, is compatible with the experimental data. When both ion and molecule of any weak acid are permeable, they act as a carrier system for the movement of protons down their electrochemical gradient. 10. Near-maintenance of pHi in the face of high fibre D-lactate (19-44 mM) and DMO (8-42 mM) indicates stimulation of proton extrusion by acid loans. 11. This extrusion is insensitive to ouabain, as judged from the lack of effect of the drug of pHi with acid loading. (+info)