Norah M. Bradford

Evidence for the existence of an ornithine/citrulline antiporter in rat liver mitochondria

In the urea cycle in rat liver, the enzymes carbamoyl phosphate synthase and ornithine transcarbamylase are situated in the mitochondria while the other enzymes of the cycle are cytoplasmic. Thus during the operation of the urea cycle, it is necessary for ornithine to enter the mitochondria and for citrulline to leave. These two amino acids are presumably transported by specific carrier systems, but the systems involved have not been adequately characterised. Citrulline penetrates the mitochondrial membrane relatively slowly at low concentrations. Accordingly, it was originally suggested that citrulline may cross the mitochondrial membrane rapidly only via an ornithine/citrulline antiport system [l]. Gamble and Lehninger ]a] presented evidence which they interpreted to show the existence of an electrogenic ornithine uniporter and a separate citrulline uniporter. However, Bryla and Harris [33 later showed that the transport of ornithine was not energy-dependent and was not linked in an obligatory manner to citrulline transport. McGivan et al. ]4] showed that in the absence of citr&ine, omithine enters the mitochondrial matrix via an ornithine/H’ antiport system. It is possible that two ornithine transport systems exist: an ornithine/H’ antiporter [4] and an ornithinej citrulline exchange system, In this paper, a method for loading mitochondria with citrulline is described, and evidence for an ornith~e~citru~~ne antiporter is presented.

The transport of branched-chain amino acids into isolated rat liver cells

Isolated liver cells may constitute a useful system for the study of the penetration of metabolites across the liver cell plasma membrane. Hepatocytes isolated from rat liver have been used to study the transport of taurocholic acid [I] and of hexoses [2] . Recently, the uptake of the non-metabolisable amino acid analogues a-aminobutyrate and cycloleucine has been investigated in detail [3,4] . Although the liver is one of the major sites of amino acid metabolism in mammalian systems, relatively little is known about the quantitative characteristics of the transport of naturally-occurring amino acids across the liver cell plasma membrane. The kinetics of amino acid transport can studied in isolated liver cells using radioactive substrates, but a major problem in this approach is the prevention of the metabolism of the labelled compound after it has been transported into the cell. However, it is known that the activity of the branched-chain amino acid transaminase in rat liver is very low [5] . The transport of the branched chain amino acids can therefore be studied in liver cells over short time intervals when no significant breakdown of these compounds will occur. In this paper, the characteristics of the uptake of L-leucine, L-isoleucine and L-valine into isolated liver cells are described. 2. Experimental

The transport of alanine and glutamine into isolated rat intestinal epithelial cells.

(1) The transport of alanine and glutamine into isolated rat intestinal epithelial cells, prepared as described previously (Watford, M., Lund, P. and Krebs, H.A. (1979) Biochem. J. 178, 589-596), was studied. (2) Cells isolated by this method accumulated alanine 7-fold from an external concentration of 0.5 mM, and by this criterion appear more suitable for transport studies than do previous rat intestinal cell preparations. (3) In these cells, it was shown using several different approaches that the major part of the transport of alanine and glutamine is mediated by a common carrier which is Na+ dependent and is sensitive to inhibition by 2-methylaminoisobutyric acid. (4) These results are in contrast to the situation in isolated hepatocytes were glutamine is transported by a carrier system (System N) distinct from that that which mediates the transport of alanine. (5) It is suggested that a major metabolic function of this transport system in intestinal cells is the exchange of extracellular glutamine for intracellular alanine, which is a major product of glutamine metabolism in the gut.

Characteristics of the activation of glutaminase by ammonia in sonicated rat liver mitochondria.

The time course of the glutaminase reaction in sonicated liver mitochondria exhibited a variable lag period before the final steady rate of glutamate formation was obtained. Added NH4Cl reduced the lag period without altering the final rate. Measurements of time courses under various conditions of pH and protein concentration showed that a half-maximum rate occurred at a critical concentration of NH3 which was independent of pH. It was concluded that in the pH range 7.0-8.0 liver glutaminase is activated by NH3 rather than by NH4+.

The inhibition of 2-oxoglutarate entry into rat liver mitochondria by L-aspartate

The transport of 2-oxoglutarate across the inner membrane of rat liver mitochondria is mediated by a specific carrier system which is activated by phos phate and L-malate [l-4]. 2-n-Butyl malonate inhibits this carrier by competition with the malate activator [5] . In this communication, evidence is presented that the entry of oxoglutarate into mitochondria is specifically inhibited by L-aspartate. The inhibition is competitive with respect to oxoglutarate (Ki = 0.15 mM), and non-competitive with respect to L malate or malonate. Externally added aspartate does not appear to influence oxoglutarate efflux from mitochondria. It is suggested that in vivo the oxoglutarate carrier is effectively unidirectional.

The use of 36Cl− to measure cell plasma membrane potential in isolated hepatocytes — effects of cyclic AMP and bicarbonate ions

The plasma membrane potential of hepatocytes was calculated from the distribution of 36Cl-. The potential observed under several conditions was equivalent to that previously measured using microelectrodes in perfused liver. Dibutyryl cAMP increased the membrane potential. Replacement of bicarbonate ions by morpholinosulphonate decreased the potential and reduced the effect of cAMP. The effect of both bicarbonate and cAMP was abolished by ouabain. Both bicarbonate and cAMP stimulated the activity of the (Na+ + K+)-ATPase as measured by ouabain-inhibitable 86Rb+ uptake. It is suggested that the stimulation of alanine transport by these effectors is mediated by an increase in cell membrane potential via stimulation of the (Na+ + K+)-ATPase.