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

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Abstract
Amino acid and K+ transport during development has been investigated in hepatocyte monolayer cultures with .alpha.-amino[1-14C]isobutyrate or 86Rb+ used as a tracer for K+. Parenchymal cells from neo- and postnatal rat livers were isolated by an improved nonperfusion technique and the resulting hepatocyte suspensions purified from nonhepatocytes 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-Km component (Km 0.91 mM) with a Vmax of 2.44 nmol/mg of protein per 4 min, 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 actvity of the electrogenic Na+/K+-pump during early stages of development. Kinetic evaluation of the enhanced rates of Rb+ uptake indicates multiple and cooperative binding sites of the enzyme involved in the Rb+ uptake, and the transport system is positively cooperative. Amino acid transport in neonatal rat hepatocytes is increased as a result of an existing low-Km 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.