trans

CALCIUM 243
forms of Trypanosoma brucei reveal that, as in
the mammalian host, the mitochondrion can
limit the free diffusion of Ca 2 . Selective accumulation
of Ca 2 within the mitochondrion
has been observed following influx across the
plasma membrane or following release from
the acidocalcisome. Only small amounts of the
remaining Ca 2 are free to contribute to the
signal process.
Ca 2 homeostatic pathways
A combination of Ca 2 transport into
organelles, polyanionic sites along plasma
membranes, and high capacity Ca 2 -binding
proteins help keep the level of [Ca 2 ] i low
(Figure 11.1A). The same homeostatic processes
that maintain a low level of [Ca 2 ] i can
also release Ca 2 during the signal process
(Figure 11.1B). In parasitic protozoa, the role
of Ca 2 homeostasis has been investigated
following disruption with ionophores, or conversely,
following stabilization of [Ca 2 ] i with
intracellular Ca 2 chelators. Cell processes as
diverse as secretion in Entamoeba and cell
invasion of T. cruzi, P. falciparum and T. gondii
are affected by these treatments. In mammalian
cells, it is difficult to find an organelle
that is not capable of Ca 2 transport. Along
with active transport across the plasma membrane,
Ca 2 sequestration occurs in mitochondria,
ER, Golgi apparatus, lysosomes, secretory
vesicles and within the nuclear envelope.
Organelles are necessary to maintain Ca 2
homeostasis and to sequester Ca 2 when the
signal is terminated. Parasitic protozoa vary in
organelle content from the relatively simple
Giardia and Entamoeba to the more elaborate
kinetoplastids. Parasites also encounter environments
that vary in Ca 2 content, such as
blood, the gut lumen of insects or mammals,
or the parasitophorous vacuole of intracellular
parasites. It is not yet known whether reliance
upon extracellular versus stored Ca 2 varies
with these different situations.
Mitochondrial Ca 2 transport
A variety of Ca 2 transporting organelles have
been detected in different protozoan parasites.
A permeabilized cell system has proven
to be generally useful in this regard. Cell membranes
are disrupted with cholesterol agents
such as digitonin, saponin or filipin. After permeabilization,
specific organelles can be targeted
for study with selective energy sources
or inhibitors. The azo dye arsenazo III changes
its absorption spectrum upon binding Ca 2 and
consequently can be used to monitor Ca 2
sequestration or release. Mitochondrial Ca 2
transport requires an oxidizable substrate or
ATP hydrolysis, is sensitive to electron transport
inhibitors and is not sensitive to vanadate.
Ca 2 transport into energized mitochondria of
apicomplexans and kinetoplastids has been
reported. Generally, mitochondria serve as a
high capacity and low affinity Ca 2 reservoir.
In permeabilized cells, the kinetoplastid mitochondrion
can buffer Ca 2 in the medium to
a fixed value around 700 nM. The remaining
Ca 2 buffering capacity of the cell depends
upon vanadate-sensitive ATPase pumps and
is sufficient to lower [Ca 2 ] in the medium to
a value around 50–100 nM. This value corresponds
to the resting level of Ca 2 in cytoplasm.
Within the mitochondrion of procyclic trypanosomes,
the resting level of Ca 2 is around
400 nM (measured with targeted aequorin).
When [Ca 2 ] i is perturbed with agents that
induce influx across the plasma membrane
or release from acidocalcisomes, a rapid and
transient accumulation of Ca 2 within the mitochondrion
is observed. As a consequence, the
cytosolic Ca 2 signal is attenuated, while the
intramitochondrial free Ca 2 concentration
reaches around 8–10 M. The rapid uptake of
BIOCHEMISTRY AND CELL BIOLOGY: PROTOZOA