Osmoregulation in the parasitic protozoan Tritrichomonas foetus

Abstract

Tritrichomonas foetus was shown to undergo a regulatory volume increase (RVI) when it was subjected to hyperosmotic challenge, but there was no regulatory volume decrease after hypoosmotic challenge, as determined by using both light-scattering methods and measurement of intracellular water space to monitor cell volume. An investigation of T. foetus intracellular amino acids revealed a pool size (65 mM) that was similar to that of Trichomonas vaginalis but was considerably smaller than those of Giardia intestinalis and Crithidia luciliae. Changes in amino acid concentrations in response to hyperosmotic challenge were found to account for only 18% of the T. foetus RVI. The T. foetus intracellular sodium and potassium concentrations were determined to be 35 and 119 mM, respectively. The intracellular K(+) concentration was found to increase considerably during exposure to hyperosmotic stress, and, assuming that there was a monovalent accompanying anion, this increase was estimated to account for 87% of the RVI. By using light scattering it was determined that the T. foetus RVI was enhanced by elevated external K(+) concentrations and was inhibited when K(+) and/or Cl(-) was absent from the medium. The results suggested that the well-documented Na(+)-K(+)-2Cl(-) cotransport system was responsible for the K(+) influx activated during the RVI. However, inhibitors of Na(+)-K(+)-2Cl(-) cotransport in other systems, such as quinine, ouabain, furosemide, and bumetanide, had no effect on the RVI or K(+) influx in T. foetus.

FIG. 1.

Time course of relative cell volume changes in response to hypoosmotic challenge. T. foetus suspensions in PBS at 25°C were subjected to a range of hypotonic conditions by dilution with appropriate amounts of distilled H₂O, and the OD₅₅₀ was recorded. The data were converted into relative cell volumes by using a previously described mathematical approach (21). Symbols: ▪, 120 mosmol kg⁻¹; ○, 150 mosmol kg⁻¹; ♦, 180 mosmol kg⁻¹; ▵, 210 mosmol kg⁻¹; •, 240 mosmol kg⁻¹; □, 270 mosmol kg⁻¹; ▴, 300 mosmol kg⁻¹.

FIG. 2.

Relationship between volume change and osmotic shift. The cellular water space of T. foetus (Vf) was measured 5 min after hypoosmotic challenge (•) and 2 min after hyperosmotic challenge (▴) by using inulin [¹⁴C]carboxylic acid and ³H₂O in PBS at 25°C. The ratio of the initial volume (Vi) to the final volume (Vi/Vf) is plotted versus osmolality. The values are means ± standard errors from five separate experiments. Where error bars are not shown, the standard error is smaller than the symbol.

FIG. 3.

Time course of relative cell volume changes in response to hyperosmotic challenge. T. foetus suspensions in culture medium at 25°C were subjected to hypertonic conditions by addition of appropriate volumes of 2.5 M NaCl, and the OD₅₅₀ was recorded. The data were converted into relative cell volumes by using a previously described mathematical approach (21). Symbols: ▴, 300 mosmol kg⁻¹; □, 375 mosmol kg⁻¹; ♦, 450 mosmol kg⁻¹; ○, 525 mosmol kg⁻¹.

FIG. 4.

Time course of cellular water space changes in response to hyperosmotic challenge (495 mosmol kg⁻¹), as determined with d-[1-¹⁴C]mannitol and ³H₂O in culture medium at 25°C. The values are means ± standard errors from five separate experiments.

FIG. 5.

Response to multiple changes in medium osmolality. T. foetus cells suspended in isotonic PBS at 25°C were exposed at time zero to hypoosmotic stress (120 mosmol kg⁻¹) (▪) or were maintained isotonically (300 mosmol kg⁻¹) (▴ and ○). At time A the hypoosmotic cells (▪) were returned to isotonic conditions by appropriate addition of 2.5 M NaCl, while control cell preparations (▴ and ○) were diluted with an equivalent volume of isotonic PBS. At time B, the test cells and one lot of control cells (○) were subjected to hyperosmotic stress (450 mosmol kg⁻¹) by addition of 2.5 M NaCl, while the other control cell preparation (▴) was diluted with an equivalent volume of isotonic PBS.

FIG. 6.

Time course of intracellular potassium changes in response to hyperosmotic challenge (495 mosmol kg⁻¹), as determined by flame photometry at 25°C. The values are means ± standard errors from at least three separate experiments. Where error bars are not shown, the standard error is smaller than the symbol.

FIG. 7.

Effect of external potassium on RVI. T. foetus cells suspended in K⁺-free buffer (▴), PBS (□), and all-K⁺ buffer (•) were subjected to hyperosmotic stress (495 mosmol kg⁻¹) by addition of 2.5 M choline chloride, and the OD₅₅₀ was recorded.