[Role of the parasitophorous vacuole of murine macrophages infected with Leishmania amazonensis in molecule acquisition].
BACKGROUND
Leishmania are intracellular parasites of macrophages, confined into compartments known as parasitophorous vacuoles. The permeability of this compartment depends on its interaction with the endocytic pathway and transport proteins present on its membrane.


OBJECTIVE
The membrane permeability of the parasitophorous vacuole was studied in J774.A1-macrophage like cells infected with Leishmania amazonensis, in situ and on isolated compartments.


METHODS
The parasitophorous vacuoles were isolated by density gradients. Fluorescent probe distribution and electrophysiological recordings were used to determine parasitophorous vacuole membrane permeability. Proton transport was evaluated indirectly by acridine orange staining. Probenecid sensitive ABC transporters were detected using the fluorescent probes lucifer yellow and calcein. For the first time ion currents were recorded on the membrane of isolated parasitophorous vacuoles using the patch clamp technique.


RESULTS
The parasitophorous vacuole stains red with acridine orange indicating an acidic compartment. It concentrates lucifer yellow by means of a probenecid sensitive transporter but excludes calcein. Isolated vacuoles stained red with acridine orange and concentrated lucifer yellow by means of a probenecid sensitive transporter. These vacuoles excluded calcein and showed an ion current in their membrane which is activated at potentials close to 60 mV with a mean conductance of 46 +/- 3 pS.


CONCLUSIONS
Isolated parasitophorous vacuoles with permeability properties preserving transport mechanisms similar to those found in situ can be purified. A poorly selective ion current on the parasitophorous vacuole membrane is reported for the first time.
Cortázar
Tania M
TM
Laboratorio de Biofísica, Centro Internacional de Física, Universidad Nacional de Colombia, Bogóta, D.C., Colombia.
Hernández
Joselín
J
Echeverry
María Clara
MC
Camacho
Marcela
M
D000818:Animals; D007425:Intracellular Membranes; D007894:Leishmania mexicana; D008264:Macrophages / Q000378:metabolism / Q000469:parasitology* / Q000648:ultrastructure*; D051379:Mice; D010539:Permeability; D014617:Vacuoles / Q000378:metabolism* / Q000469:parasitology*