monensin and trimethylamine

About 50% of Helicobacter pylori isolates produce a vacuolating toxin in vitro, which may be an important determinant of virulence. Because ammonium salts potentiate H. pylori toxin activity, the effect of other weak bases upon toxin activity was determined. Vacuolation of HeLa cells was quantitated using a neutral red uptake assay. As expected, ammonium chloride, trimethylamine, triethanolamine, and nicotine each induced vacuolation of HeLa cells when tested independently. In addition, each of these weak bases potentiated H. pylori vacuolating toxin activity, whereas sodium chloride or sodium hydroxide did not. Sequential incubation of cells with toxin followed by nicotine resulted in potentiation of vacuolation, whereas sequential incubation in the reverse order did not lead to potentiation. Monensin inhibited the formation of vacuoles by either H. pylori vacuolating toxin or nicotine. The potentiation of H. pylori toxin activity by ammonia and nicotine may contribute to gastroduodenal mucosal injury associated with this infection.

Topics: Ammonium Chloride; Bacterial Proteins; Bacterial Toxins; Drug Synergism; HeLa Cells; Helicobacter pylori; Humans; Kinetics; Methylamines; Monensin; Nicotine; Vacuoles; Virulence

Propionic acid induces a calcium mobilization in human neutrophils which is prevented by pretreatment with phorbol ester or pertussis toxin. The effect is reminiscent of that of chemotactic factors and leukotriene B4 and was attributed to cytoplasmic acidification (Naccache, P.H. et al. (1988) J. Cell. Physiol. 136, 118-124). We show there that other weak acids also induced cytoplasmic alkalinization and calcium mobilization. However, addition of trimethylamine together with propionic acid prevented the cytoplasmic acidification without modifying the calcium mobilization. Propionic acid increased the production of inositol phosphates but this effect was largely prevented by the joint addition of trimethylamine. The ionophores nigericin and monensin can both be forced to produce either cytoplasmic acidification or alkalinization by manipulating the extracellular concentrations of Na+, K+ or H+. Both ionophores produced calcium mobilization in all the cases, irrespective of the direction of the cytoplasmic pH shift. The ionophores were documented to collapse existing pH gradients among the cytoplasm and intracellular compartments. We conclude that the calcium-mobilizing effect of propionic acid and other weak acids is not due to the acidification of the cytoplasm. Our results are consistent, however, with calcium mobilization induced by weak acids and ionophores arising from acidification of an alkaline intracellular compartment.

Topics: Calcium; Cell Compartmentation; Cytoplasm; Humans; Hydrogen-Ion Concentration; Inositol Phosphates; Mathematics; Methylamines; Monensin; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Nigericin; Potassium; Propionates; Sodium

The intracellular pH (pHi) changes resulting from chemotactic factor-induced activation of Na+/H+ exchange in isolated human neutrophils were characterized. Intracellular pH was measured from the equilibrium distribution of [14C]-5,5-dimethyloxazolidine-2,4-dione and from the fluorescence of 6-carboxyfluorescein. Exposure of cells to 0.1 microM N-formyl-methionyl-leucyl-phenylalanine (FMLP) in 140 mM Na+ medium at extracellular pH (pHo) 7.40 led to a rise in pHi along an exponential time course (rate coefficient approximately 0.55 min-1). By 10 min, a new steady-state pHi was reached (7.75-7.80) that was 0.55-0.60 units higher than the resting pHi of control cells (7.20-7.25). The initial rate of H+ efflux from the cells (approximately 15 meq/liter X min), calculated from the intrinsic intracellular buffering power of approximately 50 mM/pH, was comparable to the rate of net Na+ influx (approximately 17 meq/liter X min), an observation consistent with a 1:1 stoichiometry for Na+/H+ exchange. This counter-transport could be inhibited by amiloride (apparent Ki approximately 75 microM). When either the external ([Na+]o) or internal Na ([Na+]i) concentrations, pHo, or pHi were varied independently, the new steady-state [Na+]i and pHi values in FMLP-stimulated cells were those corresponding to a chemical equilibrium distribution of Na+ and H+ across the cell membrane. By analogy to other activated cells, these results indicate that an alkalinization of pHi in human neutrophils is mediated by a chemotactic factor-induced exchange of internal H+ for external Na+.

Topics: Amiloride; Carrier Proteins; Fluoresceins; Humans; Hydrogen-Ion Concentration; Indicators and Reagents; Intracellular Fluid; Kinetics; Methylamines; Monensin; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Sodium; Sodium-Hydrogen Exchangers