Disappearance of phalloidin-transporting ability in rat hepatocytes in culture and retardation of this process by demethylphalloin or antamanide.
Uptake of phalloidin was studied in cultured rat hepatocytes. In the initial phase of culturing (0-3 hr) the toxin was taken up at a V(max) of 43.5 ng/mg protein/hr and a K(M) of 2.48 μm. During culturing the rate of phalloidin transport decreased rapidly in a logarithmic way. After 12 hr of culturing the transport of the phallotoxin was 50%, and after 45 hr was only 6% of the initial value. The decrease was reflected by a lower V(max), while the K(M) value remained unchanged. This suggests a decrease in the number of transporting units rather than an alteration in their structure. By contrast, when the cultured cells were continuously exposed to 1 μm-toxin for several hours, the rate of transport was found to be unchanged. This indicates that in the presence of substrate the process leading to the decrease in rate of phalloidin transport was retarded. Antamanide, which is a strong competitive inhibitor of phalloidin transport in freshly isolated rat hepatocytes, also inhibited phalloidin uptake into cultured hepatocytes. However, pre-incubation of cultured hepatocytes with antamanide prior to phalloidin exposure resulted in the preservation of phalloidin-transporting ability. This indicates that a competitive inhibitor of phalloidin transport may also be able to retard the decrease in the rate of transport when present during the culturing period. These results suggest that during culturing rat hepatocytes lose their phalloidin-transporting ability, probably by a decrease in the number of transporting units, and that this process can be prevented or retarded by exposure to phalloidin or antamanide. This phenomenon is interpreted as a kind of substrate protection of the transporting unit.
Münter
K
K
Abt. Physiologie, Max-Planck-Institut für Medizinische Forschung, Jahnstr. 29, 6900 Heidelberg, FRG.
Mayer
D
D
Faulstich
H
H