Control of proteolysis in perifused rat hepatocytes

Abstract

The mechanism by means of which amino acids inhibit intrahepatic protein degradation has been studied in perifused rat hepatocytes. Proteolysis was extremely sensitive to inhibition by low concentrations of amino acids. A mixture of 0.5 mM leucine and l-2 mM alanine, concentrations found in the portal vein of the rat after feeding, inhibited proteolysis to the same extent as a complete physiological mixture of amino acids. Inhibition by these two amino acids was accompanied by a rise in the intracellular concentrations of glutamate and aspartate, and was largely prevented by addition of glucagon, by addition of the transaminase inhibitor aminooxyacetate, or by omission of K+. Acceleration of proteolysis by K+ depletion was accompanied by a fall in intracellular glutamate caused by an increased rate of transport of this amino acid to the extracellular fluid. It is concluded that intracellular leucine, glutamate and aspartate are important elements in the control of hepatic protein degradation.

Control of hepatic proteolysis by amino acids

“…Although the antiproteolytic effect of alanine was significantly diminished in the presence of aminooxyacetate, an inhibitor of transaminase reactions, the alanine-induced cell swelling was lowered to a similar extent. This suggests that inhibition of alanine metabolism by aminooxyacetate exerts only a minor effect (if any) on the antiproteolytic potency of this amino acid; this is at variance with conclusions from other studies [6]. Interestingly, no significant effect of alanine (2 mmol .…”

“…1-I) or leucine (0.5 mmol . 1-') on proteolysis was reported in perifused hepatocytes [6], but a 45% inhibition of proteolysis was found when both amino acids were added together. Thus, the presence of leucine (0.1 mmol .…”

Cell Swelling and the sensitivity of autophagic proteolysis to inhibition by amino acids in isolated rat hepatocytes

“…The final concentration of amino acids in the basal mixture of amino acids (1 X AA) used in our studies was as described previously [ 131 and approximated the concentration in the portal vein of a starved rat, except that arginine was replaced by ornithine to avoid overestimation of ornithinecycle flux and that valine was omitted from the mixture, to allow accurate measurements of proteolytic flux [14].…”

“…Glycogen [21, 221, ATP [23] and lactate [24] were measured according to standard procedures. Amino acids and urea were determined as described previously [14] with a Pharmacia-LKB alpha-plus amino acid analyser.…”

“…The final pH of the incubation medium was 7.4. The osmolarity of the medium (305 mOsm under iso-osmotic conditions) was varied by changing the concentration of NaC1, the main component of the medium, from 120 mM (iso-osmotic) to lower (hypo-osmotic) or higher (hyper-osmotic) values, as indicated in the legends to the figures.The final concentration of amino acids in the basal mixture of amino acids (1 X AA) used in our studies was as described previously [ 131 and approximated the concentration in the portal vein of a starved rat, except that arginine was replaced by ornithine to avoid overestimation of ornithinecycle flux and that valine was omitted from the mixture, to allow accurate measurements of proteolytic flux [14].For the determinations of ATP and lactate, incubations were terminated in the cold by addition of HClO, (final con- …”