Contraction of cultured human uterine smooth muscle cells after stimulation with endothelin-1.

To our knowledge, the problem of how to maintain isolated smooth cells in a "contractile" phenotypic state without deviation after subculturing has yet to be resolved. The present study characterized the in vitro contractile response of human uterine smooth muscle cell to endothelin-1, which induces contractions in isolated uterine strips. Contractile effects were qualitatively investigated using silicone rubber substrata. Endothelin-1 was able to distort and reduce the wrinkles in the silicone surface. Contractions were also quantified by measuring the resulting change in the collagen lattice area. Endothelin-1 significantly increased the contractile response in a dose-dependent manner by selectively activating endothelin A receptors. When myometrial cells were cultured within collagen lattices, a microfilament-disrupting agent, cytochalasin B, abolished contractions, and no change was observed in smooth muscle alpha-actin immunostaining. Taken together, these observations show that the uterine smooth muscle cells are contractile and respond appropriately to a potent uterotonic agent. Based on these findings, a cultured uterine smooth muscle cell model, which could be used to elucidate the mechanisms controlling uterine activity, is proposed.

Biogenesis of Leishmania-harbouring parasitophorous vacuoles following phagocytosis of the metacyclic promastigote or amastigote stages of the parasites.

Protozoan parasites Leishmania alternate between a flagellated promastigote form and an amastigote form. In their mammalian hosts, Leishmania survive and multiply in macrophages. Both forms can be internalized by these host cells at different stages of the infectious process and eventually establish themselves within parasitophorous vacuoles exhibiting phagolysosomal properties. To determine whether the biogenesis of these organelles differs according to the parasitic stage used to initiate infection, we compared their formation kinetics after phagocytosis of either metacyclic promastigotes or amastigotes of L. amazonensis or of L. major by mouse bone-marrow-derived macrophages pre-exposed or not to IFN-gamma. After 10 minutes of contact, an accumulation of F-actin was observed around the promastigotes and amatigotes undergoing phagocytosis or those that had already been internalized. This accumulation was transient and rapidly disappeared at later times. At 30 minutes, most of the promastigotes were located in long, narrow organelles that were exactly the same shape as the parasites. The latter were elongated with their cell bodies near to the macrophage nucleus and their flagella towards the periphery. This suggests that promastigote phagocytosis mainly occurs in a polarized manner, with the cell body entering the macrophages first. Most, if not all, of the phagocytosed promastigotes were located in organelles that rapidly acquired phagolysosomal properties. At 30 minutes, lamp-1, macrosialin, cathepsins B and D were detected in 70-98% of these compartments and about 70% of them were surrounded by rab7p. These late endosome/lysosome 'markers' were recruited through fusion with late endocytic compartments. Indeed, when late endosomes/lysosomes were loaded with fluorescein dextran, 81-98% of the promastigote-harbouring compartments contained the endocytic tracer 30 minutes after infection. Electron microscopy of infected macrophages previously loaded with peroxidase confirmed that the phagosomes rapidly fused with late endocytic compartments. When the amastigote stage of L. amazonensis was used to initiate infection, the kinetics of acquisition of the different late endosome/lysosome 'markers' by the phagosomes were similar to those measured after infection with metacyclics. However, more rab7p(+)-phagosomes were observed at early time points (e.g. 90% were rab7p(+) at 30 minutes). The early endosome 'markers', EEA1 and the transferrin receptor, were hardly detected in parasite-containing compartments regardless of the parasitic stage used to infect macrophages and the time after infection. In conclusion, both metacyclic- and amastigote-containing phagosomes fuse with late endosomes/lysosomes within 30 minutes. However, with L. amazonensis, the time required for the formation of the huge parasitophorous vacuoles, which are characteristic of this species, was much shorter after infection with amastigotes than after infection with metacyclic promastigotes. This indicates that the initial fusions with late endosomes/lysosomes are followed by a stage-specific sequence of events.