Ellen F. Barber

Characteristics and hormonal regulation of amino acid transport system A in isolated rat hepatocytes

Publisher Summary Nutrient transport across biological membranes is gaining increased recognition as an important site for metabolic regulation. In many instances, the transport of a nutrient across the plasma membrane represents the first step in its catabolism. The characterization of amino acid transport in eukaryotic cells has received a great deal of attention in the recent years. As a result, more information has been obtained for amino acid transport in rat hepatocytes than for any other cell type with the possible exception of the Ehrlich ascites tumor cell. Although nine separate systems have been shown to mediate amino-acid transport in isolated hepatocytes, System A has been studied in detail than any of the others. Despite the large number of descriptive investigations that have focused on hepatic System A, there are several areas of characterization that remain to be explored. Some of these should wait until isolation of the proteins responsible for System A activity makes them feasible, but several interesting studies can be undertaken at the cellular level as well.

An enzyme-linked immunoadsorbent assay for rat ceruloplasmin

A noncompetitive, colorimetric enzyme-linked immunoadsorbent assay (ELISA)† was developed for measuring rat ceruloplasmin in serum and in medium from culture hepatocytes. The assay utilized polystyrene immobilized antibody which bound ceruloplasmin which then bound biotinylated antibody. The biotinylated antibody-antigen complex was detected with strepavidin-alkaline phosphatase conjugate. Standard curves for rat ceruloplasmin were constructed in the range between 10 and 50 ng/mL. Increases of 10 ng produced an increase inA403 of more than 0.2. With this immunoassay, serum ceruloplasmin levels were found to average 35 mg Cp/dL in control rats and 87 mg/dL in rats with experimental inflammation. Liver parenchymal cells secreted 1.6μg Cp/5×105 cells/24h. This ELISA assay for ceruloplasmin will facilitate studies on the regulation of ceruloplasmin synthesis and secretion in both intact rats and isolated hepatocytes.

Amino acid-dependent inactivation of glucagon-induced system A transport activity in cultured rat hepatocytes

Hepatocytes isolated from glucagon-treated rats contain stimulated System A activity. If these cells are placed in primary culture, the enhanced transport decays rapidly provided the culture medium contains substrate amino acids. This amino acid-dependent inactivation can be composed of trans-inhibition (protein synthesis-independent), repression (protein synthesis-dependent), or both depending on the particular substrate tested. Repression was most prominently observed with a group of small neutral amino acids that are commonly found in proteins. A strong trans-inhibition response was induced by a variety of amino acid analogs. Amino acids showing no reactivity with System A produced neither trans-inhibition nor repression. Repression of System A activity in culture was blocked by inhibitors of both RNA and protein synthesis. In contrast to inhibitors of RNA biosynthesis such as actinomycin and alpha-amanitin, inhibitors of poly(A) polymerase (cordycepin and adenine-9-beta-D-arabinopyranoside) did not prevent the inactivation of the transport activity. These results demonstrate that both the stimulation of activity and the turnover of the hepatic System A activity are controlled at the transcriptional level.

Regulation of Ceruloplasmin Synthesis by Retinoic Acid and Interleukin-1

Research conducted over a period of fifty years, both in a clinical setting with human subjects, as well as in experiments with animals, has shown that plasma ceruloplasmin levels increase during periods of stress, inflammatory disease and chronic infection. However, there is little known about the factors which influence ceruloplasmin synthesis. The physiological role for the increase in circulating levels of this acute phase protein is equally obscure. The plethora of hormonal changes that precede and/or accompany these conditions suggest a variety of signals contribute to the phenomenon.