We studied the intracellular pH of Vero cells parasitised by Trypanosoma cr
uzi, using different methods: fluorimetric measurement after labelling the
cells with the pH-sensitive intracellular fluorescent dye 2', 7',-bis-(2-ca
rboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester; flow cytomet
ry; and image analysis after staining the cells with neutral-red vital stai
n. The results show that the intracellular pH of the parasitised cells rose
in comparison with that of the uninfected control cells. A study of the po
pulation of parasitised cells made by flow cytometry allowed us to subdivid
e the cells from the infected cultures into two populations according to th
eir pH as obtained by fluorimetric measurements. Image analysis showed that
the cell cytoplasm was more alkaline in the vicinity of the sites containi
ng parasites. Treatment of the parasitised cells with amiloride, ouabain, o
r with 4,4'-diisothiocyano-2,2'-stilbene disulphate consistently lowered th
e pH Values of the parasitised cells, but not sufficiently to return to the
values of the non-parasitised control cells. When the control cells were s
ubject to similar treatments with the inhibitors, only amiloride acidified
the cytoplasm to any extent. The basification undergone by the parasitised
cells was independent of the transport systems and may be a consequence of
the release of NH4+ by the intracellular amastigotes. (C) 1998 Australian S
ociety for Parasitology. Published by Elsevier Science Ltd. All rights rese
rved.