Mechanism of mercurial inhibition of sodium-coupled alanine uptake in liver plasma membrane vesicles from Raja erinacea.
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.
Sellinger
M
M
Mount Desert Island Biological Laboratory, Salsbury Cove, Maine 04672.
Ballatori
N
N
Boyer
J L
JL
D000409:Alanine / Q000493:pharmacokinetics; D026905:Amino Acid Transport Systems; D026921:Amino Acid Transport Systems, Neutral; D000818:Animals; D002352:Carrier Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D008099:Liver / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D008297:Male; D008627:Mercuric Chloride / Q000633:toxicity; D008628:Mercury / Q000633:toxicity*; D012860:Skates, Fish; D012964:Sodium / Q000378:metabolism; D012979:Sodium Radioisotopes; D013347:Subcellular Fractions / Q000378:metabolism; D027981:Symporters
D026905:Amino Acid Transport Systems; D026921:Amino Acid Transport Systems, Neutral; D002352:Carrier Proteins; D012979:Sodium Radioisotopes; D027981:Symporters; C052168:sodium-alanine cotransporter; D008627:Mercuric Chloride; D012964:Sodium; D008628:Mercury; D000409:Alanine