Amino acid transport and rubidium-ion uptake in monolayer cultures of hepatocytes from neonatal rats

Abstract

Amino acid and K+ transport during development has been investigated in hepatocyte monolayer cultures with either α-amino[1-14C]isobutyrate or 86Rb+ 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 α-aminoisobutyrate was decreased and it was not affected by the age of the animals. Kinetic analysis of Na+-dependent α-aminoisobutyrate transport revealed the existence of a high-affinity low-Km component (Km0.91mm) with a Vmax. 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-Km component for the Na+-dependent α-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 α-aminoisobutyrate transport in the alanine-preferring system during postnatal age.