| The ability of Trypanosoma cruzi to regulate its intracellular volume in the face of hyposmotic stress was investigated. When the extracellular osmolarity was reduced from 300 to 150 mOsm, cells initially swelled but recovered normal cell volume within 5--10 minutes. Hyposmotic stress induced an efflux of neutral and anionic amino acids, accounting for 40--50% of the observed regulatory volume decrease. A small efflux of potassium accounting for 7% of the regulatory volume decrease was also observed. Sodium, chloride, methylamines, inositol, phosphate, and pyrophosphate were not released. Cell swelling was associated with an influx of calcium across the plasma membrane through channels partially sensitive to voltage-gated calcium channel blockers. However, intracellular calcium levels had no functional significance for volume recovery and only a small effect on amino acid efflux. These results ruled out direct contribution of acidocalcisomal osmolyte to volume recovery by extrusion from the cell as well as functional significance for the acidocalcisomal calcium pool during the response.; During cell swelling, acidocalcisomal alkalinization and concomitant cytosolic acidification was observed. This was paralleled by a rise in intracellular ammonium concentrations, and the cytosolic acidification could be inhibited by disrupting the acidocalcisomal proton gradient. These results are consistent with ammonium uptake into the acidocalcisome. Since alkalinization is known to activate acidocalcisomal polyphosphate hydrolysis, ammonium production may serve as a trigger for activating the osmotic potential of the acidocalcisome. Morphometric analysis showed a 50% increase in acidocalcisomal volume during cell swelling, which is consistent with osmolyte accumulation. RNAi-inducible mutants of acidocalcisomal H+-pyrophosphatase and vacuolar H+-ATPase in Trypanosoma brucei procyclic forms had reduced rates of volume recovery.; Electron microscopic observations revealed a contractile vacuole coupled to acidocalcisomes in all three life cycle stages of the parasite. An aquaporin homologue localized to the acidocalcisomes and contractile vacuole. In epimastigotes expressing a GFP-aquaporin fusion protein, acidocalcisomes trafficked toward the contractile vacuole during hyposmotic stress in a cAMP- and microtubule-dependent fashion. This phenomenon was functionally significant, since aquaporin and microtubule inhibitors reduced the rate of volume recovery. Isolated contractile vacuoles were enriched in alkaline phosphatase, aquaporin, and some acidocalcisomal markers. |
