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Sellinger M, Ballatori N, Boyer JL: Mechanism of mercurial inhibition of sodium-coupled alanine uptake in liver plasma membrane vesicles from Raja erinacea. Toxicol Appl Pharmacol. 1991 Feb;107(2):369-76.
In mammalian hepatocytes the L-alanine carrier contains a sulfhydryl group that is essential for its activity and is inhibited by mercurials. In hepatocytes of the evolutionarily primitive little skate (Raja erinacea), HgCl2 inhibits Na (+)-dependent alanine uptake and Na+/K (+)-ATPase and increase K+ permeability. To distinguish between direct effects of HgCl2 on the Na (+)-alanine cotransporter and indirect effects on membrane permeability, [3H] alanine transport was studied in plasma membrane vesicles. [3H] Alanine uptake was stimulated by an "out-to-in" Na+ but not K+ gradient and was saturable confirming the presence of Na (+)-alanine cotransport in liver plasma membranes from this species. Preincubation of the vesicles with HgCl2 for 5 min reduced initial rates of Na (+)-dependent but not Na (+)-independent alanine uptake in a dose-dependent manner (10-200 microM). In the presence of equal concentrations of NaCl or KCl inside and outside of the vesicles, 75 microM HgCl2 directly inhibited sodium-dependent alanine-[3H] alanine exchange, demonstrating that HgCl2 directly affected the alanine cotransporter. Inhibition of Na (+)-dependent alanine uptake by 30 microM HgCl2 was reversed by dithiothreitol (1 mM). HgCl2 (10-30 microM) also increased initial rates of 22Na uptake (at 5 sec), whereas 22Na uptake rates were decreased at HgCl2 concentrations greater than 50 microM. Higher concentrations of HgCl2 (100-200 microM) produced nonspecific effects on vesicle integrity. These studies indicate that HgCl2 inhibits Na (+)-dependent alanine uptake in skate hepatocytes by three different concentration-dependent mechanisms: direct interaction with the transporters, dissipation of the driving force (Na+ gradient), and loss of membrane integrity. Inactivation of the Na (+)-coupled alanine carrier by mercury in hepatocytes of this evolutionarily primitive vertebrate, as in mammals, suggests that the sulfhydryl groups on this transport protein are highly conserved. |
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