Amino acid transport and rubidium-ion uptake in monolayer cultures of hepatocytes from neonatal rats.
Amino acid and K(+) transport during development has been investigated in hepatocyte monolayer cultures with either alpha-amino[1-(14)C]isobutyrate or (86)Rb(+) used as a tracer for K(+). Parenchymal cells from neo- and post-natal rat livers have been isolated by an improved non-perfusion technique [Bellemann, Gebhardt & Mecke (1977)Anal.Biochem.81, 408-415], and the resulting hepatocyte suspensions purified from non-hepatocytes before inoculation. In the presence of Na(+) (Na(+)-dependent component), the rates of amino acid uptake in neonatal hepatocytes were markedly enhanced compared with cells from 30-day-old rats. When Na(+) was replaced by choline (Na(+)-independent component) the accumulation of alpha-aminoisobutyrate was decreased and it was not affected by the age of the animals. Kinetic analysis of Na(+)-dependent alpha-aminoisobutyrate transport revealed the existence of a high-affinity low-K(m) component (K(m)0.91mm) with a V(max.) of 2.44nmol/mg of protein per 4min, which later declined gradually with progressive development. Rates of Rb(+) transport were concomitantly enhanced in neonatal hepatocytes and thereafter declined with postnatal age. The increased Rb(+) influx was effectively inhibited by ouabain and reflected elevated activity of the electrogenic Na(+)/K(+)-pump during early stages of development. Kinetic evaluation of the enhanced rates of Rb(+) uptake indicates multiple and co-operative binding sites of the enzyme involved in the Rb(+) uptake, and the transport system is positively co-operative (the Hill coefficient h is >1.0). In short, amino acid transport in neonatal rat hepatocytes is increased as a result of an existing low-K(m) component for the Na(+)-dependent alpha-aminoisobutyrate uptake, which endows the hepatocytes with a high capability for concentrating amino acids at low ambient values. The concomitant enhancement of K(+) transport reflects changes in the electrochemical gradient for Na(+) across the hepatocellular membrane and, along with this, presumably alterations in the membrane potential; the latter might be the driving force for the enhanced alpha-aminoisobutyrate transport in the alanine-preferring system during postnatal age.
D000621:Aminoisobutyric Acids / Q000378:metabolism*; D000818:Animals; D000831:Animals, Newborn / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D002469:Cell Separation; D002478:Cells, Cultured; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D051381:Rats; D011919:Rats, Inbred Strains; D012413:Rubidium / Q000378:metabolism*; D012964:Sodium / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors
D000621:Aminoisobutyric Acids; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium