Phalloidin, a bicyclic heptapeptide isolated from the mushroom Amanita phalloides, was reported as a membrane specific toxin inducing membrane protrusions in the isolated hepatocyte. The molecular basis of this injury most likely lies in the
interaction
of phalloidin with the protein filaments of the plasma membrane.
Trypsin was reported as one of the agents which prevent membrane protrusions of hepatocyte from phalloidin. However, the exact mechanism of trypein to the phalloidin action is still obscure.
This study was designed to investigate the ultrastructures of isolated hepatocyte treated by phalloidin and trypsin, as a part of study to elucidate the mechanism of trypsin antagonism to phalloidin.
In our experiments, multiple protrusions on the surface of isolated rat hepatocytes were obseryed, whereas trypsinized hepatocytes showed mild degree membrane protrusions along with hepatocyte aggregation after 20 min incubation with 10 μg/ml of
phalloidin. The total number of aggregated cells were increased with gradual increase in trypsin concentration. These trypsin effects were disappeared spontaneously after twice washing of trypsinized heptocytes with Hank's balanced salt solution
(HBSS)
or treatment with soy bean trypsin inhibitor. Without trypsin, both Tween 80 treated hepatocytes and hepatocyte ghosts were not aggregated but showed membrane protrusions with phalloidin. However, electron microscopy revealed mild membrane
deformity on
trypsin and phal-loidin treated cells that was determined as normal under light microscopy.
In conclusion, these results suggest that certain membrane structures in isolated rat liver cell, to which trypsin is reversely reactive, are required to recognze or to uptake phalloidin. These trypsin altered membrane structures lead to
aggregation of
hepatocytes upon reaction to phalloidin. And more studies should be performed to evaluate the mechanism of trypsin especially in regard to the hepatocyte aggregation.
|