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AMINO ACID TRANSPORT, Na+/K+‐ATPASE, AND TAT INDUCTION IN MONOLAYER CULTURES OF HEPATOCYTES FROM DEVELOPING RATS

Hepatocellular membrane is an important locus for the transport of substrates involved in the regulation of metabolic events that are related to proliferation. Cellular growth is strongly associated with an increase in the capacity to accumulate amino acids and in cation transport, which has been demonstrated in a variety of cell types, e.g., during liver regeneration.' The present study reports an enhanced amino acid and potassium transport, and the inducibility of tyrosine aminotransferase in primary monolayer culture from liver tissue during development. The isolation of parenchymal cells was preceded by a complete flushing of the organ free of erythrocytes and plasma. Livers from newborn, 5-, lo-, 20-, and 30day-old rats were retrogradely perfused for 3 min with Ca'+-free Swim's S-77 medium via the inferior vena cava cannulated near the right auricle. Isolation of hepatocytes was performed by an improved slicing technique,2 since pooling of livers from newborn, 5-and 10-day-old rats was necessary in order to obtain sufficient cells for at least 200 culture dishes. Isolated cells became attached to collagen-coated tissue culture plates in 4 hr while incubating in WOBA-M2 medium plus 5 &ml insulin.3 Transport of a-aminoisobutyric acid (I4C-AIB) and potassium (using &Rb+) and induction of tyrosine aminotransferase (TAT) were initiated at this time. The slicing technique, with which aseptic cell suspensions of small liver samples (e.g., of newborn) are very easy to obtain, produced optimal cell yields with high viability as judged by Trypan Blue exclusion. Purified suspensions of newborn and 5 day old liver cells contained more than 90% hepatocytes. Studies on the ultrastructure demonstrated distinct cell membranes, normal mitochondria, and many parallel strands of polyribosomes (courtesy of Dr. C. Sattler). In the presence of sodium (I40 mM), amino acid transport in 5-day-old liver cells was dramatically enhanced compared with 30-day-cells, but gradually declined with time of development. When sodium was replaced by choline-the sodium-independent component-the uptake of AIB was reduced and accumulation was not affected by the animal's age. The 4 min rates of the sodium-dependent AIB transport, calculated by subtracting the sodium-independent component at each AIB concentration, also showed an enhanced amino acid uptake 5 days after birth. This gradually declined with progressive development, reaching rates of AIB uptake after 30 days that are comparable to normal adult livers. Preliminary analysis of the sodiumdependent AIB transport based on 4 min rates revealed evidence for the emergence of a low K, component (approx. l.0)4 that contributed to the AIB transport during early stages of development. Potassium transport measured with '?ubidium as a tracer was also dramatically enhanced during the first 5 days after birth and thereafter declined with progressive development. The increased potassium transport suggests enhanced Na'/K'-ATPase activity that was effectively inhibited by ouabain ( 1 mM) and demonstrated the ouabain sensitivity of the potassium transport.