Altered plasma membrane ion permeability in mercury-induced cell injury: studies in hepatocytes of elasmobranch Raja erinacea.
The effects of HgCl2, CH3HgCl, p-chloromercuribenzene sulfonate (PCMBS), and CdCl2 on plasma membrane and cell metabolic functions of skate (Raja erinacea) hepatocytes in suspension culture were assessed by measuring (a) the rates of Na+-dependent and -independent L-[14C]alanine uptake, (b) Na+-dependent 86Rb+ uptake, a measure of Na-K-ATPase activity, (c) 86Rb+ efflux, a measure of K+ permeability, (d) the difference between the 3H2O and [14C]inulin distribution spaces, a measure of intracellular water volume, (e) cellular ATP concentrations, and (f) glutathione (GSH) and glutathione disulfide (GSSG) levels. The initial rates of L-alanine and 86Rb+ uptake were inhibited by each of these metals in the following order: HgCl2 greater than CH3HgCl greater than PCMBS greater than CdCl2. Inorganic mercury significantly inhibited the initial rates of Na+-dependent L-alanine and 86Rb uptakes at a concentration of 10 microM, whereas 100 microM produced nearly complete inhibition. These effects were dose-dependent, immediate (observed after less than 5 min of incubation with the metal), and persistent. Mercuric chloride also impaired volume regulatory mechanisms in skate hepatocytes: cells treated with 50 microM HgCl2 swelled slowly over a 60-min interval to volumes nearly double those of control cells. In addition, HgCl2 prevented the normal volume regulatory decrease observed after swelling the hepatocytes in hypotonic media. Mercuric chloride (5-50 microM) produced a rapid initial loss of a large fraction of intracellular 86Rb, followed by a slower rate of release of the remaining isotope. These effects were prevented if GSH was added with, but not following HgCl2. In contrast, dithiothreitol, a more permeable thiol, both prevented and even partially reversed the effects of mercury. Mercuric chloride (10 microM) had no effect on cellular ATP, GSH, or GSSG levels for up to 4 hr incubation. These findings indicate that 86Rb+ (K+) efflux is a sensitive indicator of mercury toxicity, and are consistent with the hypothesis that the plasma membrane is a primary target for mercury's effects. A change in membrane permeability to K+ would dissipate transmembrane electrochemical gradients, and may contribute to the apparent inhibition of transport processes energized by these gradients, such as Na+-alanine cotransport, and volume regulatory mechanisms.
Ballatori
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N
Mount Desert Island Biological Laboratory, Salsbury Cove, Maine 04672.
Shi
C
C
Boyer
J L
JL
D000409:Alanine / Q000493:pharmacokinetics; D000596:Amino Acids / Q000493:pharmacokinetics; D000818:Animals; D002104:Cadmium / Q000633:toxicity; D002463:Cell Membrane Permeability / Q000187:drug effects*; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism; D019803:Glutathione Disulfide; D008099:Liver / Q000187:drug effects*; D008297:Male; D008628:Mercury / Q000633:toxicity*; D011188:Potassium / Q000378:metabolism; D012413:Rubidium / Q000493:pharmacokinetics; D012860:Skates, Fish; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism
D000596:Amino Acids; D002104:Cadmium; D000254:Sodium-Potassium-Exchanging ATPase; D008628:Mercury; D005978:Glutathione; D012413:Rubidium; D000409:Alanine; D011188:Potassium; D019803:Glutathione Disulfide