Altered plasma membrane ion permeability in mercury-induced cell injury: Studies in hepatocytes of elasmobranch Raja erinacea

Abstract

The effects of HgCl 2, CH 3HgCl, p-chloromercuribenzene sulfonate (PCMBS), and CdCl 2 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 3H 2O 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: HgCl 2 > CH 3HgCl > PCMBS > CdCl 2. Inorganic mercury significantly inhibited the initial rates of Na +-dependent l-alanine and 86Rb uptakes at a concentration of 10 μ m, whereas 100 μ m produced nearly complete inhibition. These efects were dose-dependent, immediate (observed after <5 min of incubation with the metal), and persistent. Mercuric chloride also impaired volume regulatory mechanisms in skate hepatocytes: cells treated with 50 μ m HgCl 2 swelled slowly over a 60-min interval to volumes nearly double those of control cells. In addition, HgCl 2 prevented the normal volume regulatory decrease observed after swelling the hepatocytes in hypotonic media. Mercuric chloride (5–50 μ m) 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 HgCl 2. In contrast, dithiothreitol, a more permeable thiol, both prevented and even partially reversed the effects of mercury. Mercuric chloride (10 μ m) 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.