System a transport activity in normal rat hepatocytes and transformed liver cells: Substrate protection from inactivation by sulfhydryl‐modifying reagents

Abstract

The transport of amino acids by normal rat hepatocytes and several hepatoma cell lines has been examined for inactivation by various protein-modifying reagents, including the sulfhydryl-preferring reagents N-ethylmaleimide (NEM) and p-chloromercuribenzene sulfonate (PCMBS). Uptake of 2-aminoisobutyric acid (AIB), a specific probe for hepatic System A-mediated transport, was equally sensitive to inhibition by the organic mercurial PCMBS in each of the cell types tested. In contrast, the sensitivity of System A to inactivation by NEM was substantially different among the five cell types. Normal hepatocytes showed the greatest sensitivity, while the hepatoma cells varied in their responsiveness from moderate to no inhibition. PCMBS inactivated greater than 85% of the System A activity in rat H4 hepatoma cells within 10 min (t1/2 = 3 min). The inhibition by PCMBS was rapidly reversed by treatment of the cells with dithiothreitol. Amino acids showing a high affinity for System A protected the transport system from inactivation, whereas non-substrates produced little or no protection. Amino acid-dependent protection was stereospecific and system-specific. L-norleucine competitively inhibited AIB uptake (Ki = 1.9 +/- 0.1 mM) in H4 cells and also protected System A from PCMBS-dependent inactivation (half-maximal protection occurred at an amino acid concentration of 0.6 +/- 0.1 mM). N-bromosuccinimide was completely ineffective as an inhibitor of System A activity in hepatocytes, whereas treatment of H4 rat hepatoma cells with this reagent resulted in greater than 95% inhibition.

ATA2-mediated amino acid uptake following partial hepatectomy is regulated by redistribution to the plasma membrane

“…Inherent differences in System A transport between normal liver and transformed hepatic cell lines have been previously suggested by differential responses to sulfhydryl-specific reagents [6,7]. NEM decreased System A-mediated transport in normal hepatocytes, but did not affect System A activity in hepatoma cells [6,7].…”

Up‐regulation of system A activity in the regenerating rat liver

“…Altered sensitivity to NEM was consistent with carriers structurally different in hepatoma cells. Most hepatoma cell lines (HTC, H4, FAO, HepG2) show very low or nil sensitivity to this agent, but liver cells are highly sensitive [14]. Preincubation with NEM resulted in a concentration-dependent inhibition of Na'-dependent L-alanine transport, at a substrate concentration where system A contribution was predominant.…”

“…Results are shown in Table I. 2.4. Effect of N-ethylmaleimide on Na'-dependent t-alanine uptake Table I The effect of NEM on Nab-dependent L-alanine uptake was studied as previously described [14]. Liver plasma membrane vesicles were preincubated with increasing concentrations of NEM (0.625-5 mM) for 30 min at 25°C.…”

“…Hepatoma cell lines show system A transporters with altered sensitivity to sulfhydryl-modifying reagents [14,29], thus, we wondered whether the activity that develops during liver cell proliferation after partial hepatectomy could have any similarity with the one present in hepatoma cells. Sensitivity of system A to agents like NEM results in irreversible inactivation, which can be protected by the presence of system A substrate [14,29,30], thus supporting the idea that -SH groups are essential for substrate recognition and translocation. Altered sensitivity to NEM was consistent with carriers structurally different in hepatoma cells.…”

“…Liver system A activity is induced in different physiological conditions associated with liver hypertrophia and/or hyperplasia, like pregnancy, development or genetic obesity fl I-13]. Hepatoma cell lines also express system A activity, although the sensitivity of the carrier to some suhhydryfmodifying reagents is markedly different to the one found in cultured hepatoqtes [14]* Interestingly, the activity of an uncharacterized high-afhnity component of transport that was attributable to system A is also induced in rat hepatocytes isolated from regenerating livers, and it has even been suggested that induction of system A may be a permissive factor in liver proliferation after partial hepatectomy [15]. The biochemical basis of this likely up-regulation is not well understood.…”