Effects of Hg 2+ on cytosolic Ca 2+ in isolated skate hepatocytes

Abstract

Hg 2+ is an environmental pollutant that adversely affects a range of cellular functions, including those that regulate free cytosolic Cal 2+ (Ca 2+). To investigate the mechanism of Hg 2+-induced Ca 2+ signaling, we examined the effects of Hg 2+ on Cal 2+ in isolated skate hepatocytes, and developed a method to assess cytosolic Hg 2+ (Hg 2+) in these cells as well. At lower concentrations (1–5 μM), Hg 2+ induced little detectable change in Ca 2+. At higher concentrations (10 μM-1 mM), Hg 2+ induced a dose-dependent, progressive increase in Ca 2+ which occurred even in Ca 2+-free medium. Pretreatment of hepatocytes with the membrane-impermeant Hg 2+ chelator glutathione (GSH) blocked the Hg 2+-induced Cal 2+ increase, whereas addition of GSH after exposure to Hg 2+ slowed but did not prevent further increases in Cai 2+. Pretreatment with the membrane-permeant H g2+ chelator dithiothreitol (DTT) also blocked Hg 2+-induced increases in Ca 2+. Unlike GSH, however, addition of DTT after Hg 2+ significantly decreased Ca 2+, returning it to near-baseline levels. Thapsigargin induced a sustained increase in Ca 2+, but subsequent addition of Hg 2+ resulted in a further, progressive Ca 2+ increase. We also describe the use of the fluorescent dye BTC-5N to measure Hg 2+, and with it found that Hg 2+ reaches nanomolar levels within minutes of extracellular application, but that these measurable levels of Hg; 2+ do not precede elevations in Ca 2+. Hg 2+ did not irreversibly damage the hepatocytes over this time period (< 5 min), as determined both by propidium iodide permeability and light microscopic appearance. Together, these findings suggest: (i) Hg 2+ increases Cai 2+ in skate hepatocytes; (ii) Hg 2+ must enter the hepatocytes for this Cal` increase to occur; (iii) this increase is mediated by release of Cat+ from endogenous stores that are distinct from the thapsigargin-sensitive Ca 2+ stores; and (iv) this increase occurs in association with measureable levels of Hg 2+ in the cytosol. Adverse cellular effects of Hg 2+ may be mediated by changes in Ca 2+ that result from intracellular accumulation of this toxic metal.