AJP - Cell Physiology, Vol 258, Issue 4 C700-C712, Copyright © 1990 by American Physiological Society
Alterations of bile acid and bumetanide uptake during culturing of rat hepatocytes
W. Follmann, E. Petzinger and R. K. Kinne
Justus-Liebig Universitat Giessen, Institut fur Pharmakologie und Toxikologie, Federal Republic of Germany.
Uptake by the multispecific bile acid transport system of [3H]taurocholate,
[14C]cholate, and [3H]-bumetanide into primary cultures of rat hepatocytes
was compared with their uptake into freshly isolated rat hepatocytes. The
uptake maximum velocity (Vmax) of all compounds declined in primary
culture, whereas the Michaelis constant (Km) values remained stable. Loss
of uptake was not due to the reduction of driving forces as evaluated from
the level of ATP and the activity of Na(+)-K(+)-ATPase. No
alpha-fetoprotein was detectable in culture supernatants. Neither growth
factors (glycylhistidyl-lysine, epidermal growth factor), peroxisome and
cell proliferators (nafenopin, dimethyl sulfoxide), nor bile acids
prevented the loss of transport in hepatocyte culture. However, addition of
dibutyryl adenosine 3'5'-cyclic monophosphate protracted the transport
activity significantly. When cultured rat hepatocytes with reduced
transport were detached by trypsin, cells rounded up and showed the same
uptake capacity for bumetanide, cholate, and taurocholate as seen in
freshly isolated hepatocytes. "Cryptic" transport activity in the lower
basolateral membrane facing the support was found using an incubation
chamber for cultured hepatocytes, which allowed us to distinguish
simultaneously between uptake via the upper and lower basolateral membrane
of the cultured cells.
