bafilomycin-a1 and propionic-acid

Disruption of cellular acid-base status alters the host defence functions of alveolar macrophages (m phi). These pH effects might be mediated by pH-sensitive changes in the signalling pathways of the effector functions of m phi. The present study examined the effects of intracellular pH (pH(i)) on the free cytosolic calcium concentration ([Ca(2+)](i)), an important second messenger for cell functions. [Ca(2+)](i) and pH(i) of rabbit resident alveolar m phi were measured using fluorescent dyes. With extracellular pH (pH(o)) of 7.4, the steady-state pH(i) and [Ca(2+)](i) were approx. 7.14 and 123 nM respectively. Incubation at pH(o) 6.8 caused a sustained cytosolic acidosis, but did not affect [Ca(2+)](i). Likewise, [Ca(2+)](i) was unchanged when m phi at pH(o) 7.4 were acidified using bafilomycin A(1) or sodium propionate. In contrast, [Ca(2+)](i) was markedly sensitive to cytosolic alkalosis. Exposure to NH(4)Cl at pH(o) 7.4 caused transient increases in both pH(i) and [Ca(2+)](i). The Ca(2+) response was mediated by release of intracellular Ca(2+) from thapsigargin-sensitive stores and was potentiated by capacitative entry of extracellular Ca(2+). Incubation at high pH(o) values (>7.4) produced sustained increases in pH(i) and [Ca(2+)](i). The sustained elevation of [Ca(2+)](i) was consistent with pH-sensitive inhibition of plasma-membrane Ca(2+)-ATPase. The response to high pH(o) was unaffected by blockade of L-type or receptor-operated Ca(2+) channels with nifedipine or SKF-96365, and was independent of extracellular Na(+). The findings indicate that pH impacts cytosolic Ca(2+) homoeostasis at multiple levels. The data suggest that cellular acid-base status can influence Ca(2+)-dependent signalling events in resident alveolar m phi, especially during alkaline disruptions of pH(i).

Topics: Ammonium Chloride; Animals; Anti-Bacterial Agents; Calcium; Cells, Cultured; Cytosol; Fluorescent Dyes; Fura-2; Hydrogen-Ion Concentration; Macrolides; Macrophages, Alveolar; Propionates; Rabbits; Second Messenger Systems; Spectrometry, Fluorescence; Vacuolar Proton-Translocating ATPases

Changes in plasma volume in vivo cause rapid changes in extracellular pH by altering the plasma bicarbonate concentration at a constant Pco(2) (Garella S, Chang BS, and Kahn SI. Kidney Int 8: 279, 1975). Few studies have examined the possibility that changes in cell volume produce comparable changes in intracellular pH (pH(i)). In the present study, alveolar macrophages were exposed to hyperosmotic medium in the absence or presence of the open-system buffers CO(2)-HCO(3)(-), propionic acid-propionate, or NH(3)-NH(4)(+). In the absence of open-system buffers, exposure to twice-normal osmolarity (2T) produced a slow cellular alkalinization [change in pH(i) (DeltapH(i)) approximately 0.38; exponential time constant (tau) approximately 120 s]. In the presence of 5% CO(2), 2T caused a biphasic pH(i) response: a rapid increase (DeltapH(i) approximately 0.10, tau approximately 15 s) followed by a slower pH(i) increase. Identical rapid pH(i) increases were produced by 2T in the presence of propionic acid (20 mM). Conversely, 2T caused a rapid pH(i) decrease (DeltapH(i) approximately -0.21, tau approximately 10 s) in the presence of NH(3) (20 mM). Thus osmotic cell shrinkage caused rapid pH(i) changes of opposite direction in the presence of a weak acid buffer (contraction alkalosis with CO(2) or propionic acid) vs. a weak base buffer (contraction acidosis with NH(3)). Graded DeltapH(i) were produced by varying extracellular osmolarity in the presence of open-system buffers; osmolarity increases of as little as 5-10% produced significant DeltapH(i). The rapid pH(i) responses to 2T were insensitive to inhibitors of membrane H(+) transport (ethylisopropylamiloride and bafilomycin A(1)). The results are consistent with shrinkage-induced disequilibria in the total cellular buffer system (i.e., intrinsic buffers plus added weak acid-base buffer).

Topics: Amiloride; Animals; Anti-Bacterial Agents; Buffers; Cell Size; Cells, Cultured; Hydrogen-Ion Concentration; Hypertonic Solutions; Kinetics; Macrolides; Macrophages, Alveolar; Mannitol; Models, Biological; Propionates; Rabbits; Saline Solution, Hypertonic; Sucrose

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