Effects of Hg2+ on cytosolic Ca2+ in isolated skate hepatocytes

Hg2+ is an environmental pollutant that adversely affects a range of cellular functions, including those that regulate free cytosolic Ca2+ (Ca(i)2+). To investigate the mechanism of Hg(2+)-induced Ca(i)2+ signaling, we examined the effects of Hg2+ on Ca(i)2+ in isolated skate hepatocytes, and developed a method to assess cytosolic Hg2+ (Hgi2+) in these cells as well. At lower concentrations (1-5 microM), Hg2+ induced little detectable change in Ca(i)2+. At higher concentrations (10 microM-1 mM), Hg2+ induced a dose-dependent, progressive increase in Ca(i)2+, which occurred even in Ca(2+)-free medium. Pretreatment of hepatocytes with the membrane-impermeant Hg2+ chelator glutathione (GSH) blocked the Hg(2+)-induced Ca(i)2+ increase, whereas addition of GSH after exposure to Hg2+ slowed but did not prevent further increases in Ca(i)2+. Pretreatment with the membrane-permeant Hg2+ chelator dithiothreitol (DTT) also blocked Hg(2+)-induced increases in Ca(i)2+. Unlike GSH, however, addition of DTT after Hg2+ significantly decreased Ca(i)2+, returning it to near-baseline levels. Thapsigargin induced a sustained increase in Ca(i)2+, but subsequent addition of Hg2+ resulted in a further, progressive Ca(i)2+ increase. We also describe the use of the fluorescent dye BTC-5N to measure Hgi2+, and with it found that Hgi2+ reaches nanomolar levels within minutes of extracellular application, but that these measurable levels of Hgi2+ do not precede elevations in Ca(i)2+. Hg2+ 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) Hg2+ increases Ca(i)2+ in skate hepatocytes; (ii) Hg2+ must enter the hepatocytes for this Ca(i)2+ increase to occur; (iii) this increase is mediated by release of Ca2+ from endogenous stores that are distinct from the thapsigargin-sensitive Ca2+ stores; and (iv) this increase occurs in association with measureable levels of Hg2+ in the cytosol. Adverse cellular effects of Hg2+ may be mediated by changes in Ca(i)2+ that result from intracellular accumulation of this toxic metal.