Nathanson M H, Mariwalla K, Ballatori N, Boyer J L
Center for Membrane Toxicity Studies, Mount Desert Island Biological Laboratory, Salsbury Cove, Maine, USA.
Cell Calcium. 1995 Nov;18(5):429-39. doi: 10.1016/0143-4160(95)90058-6.
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.
汞离子（Hg2+）是一种环境污染物，会对一系列细胞功能产生不利影响，包括那些调节游离胞质钙离子（Ca(i)2+）的功能。为了研究Hg2+诱导Ca(i)2+信号传导的机制，我们检测了Hg2+对分离的鳐鱼肝细胞中Ca(i)2+的影响，并开发了一种方法来评估这些细胞中的胞质汞离子（Hgi2+）。在较低浓度（1 - 5微摩尔）下，Hg2+诱导的Ca(i)2+变化几乎无法检测到。在较高浓度（10微摩尔 - 1毫摩尔）下，Hg2+诱导Ca(i)2+呈剂量依赖性、逐渐增加，即使在无钙培养基中也是如此。用不能透过细胞膜的Hg2+螯合剂谷胱甘肽（GSH）预处理肝细胞可阻断Hg2+诱导的Ca(i)2+升高，而在暴露于Hg2+后添加GSH虽减缓但并未阻止Ca(i)2+的进一步升高。用可透过细胞膜的Hg2+螯合剂二硫苏糖醇（DTT）预处理也可阻断Hg2+诱导的Ca(i)2+升高。然而，与GSH不同的是，在Hg2+处理后添加DTT可显著降低Ca(i)2+，使其恢复到接近基线水平。毒胡萝卜素诱导Ca(i)2+持续升高，但随后添加Hg2+会导致Ca(i)2+进一步逐渐升高。我们还描述了使用荧光染料BTC - 5N来测量Hgi2+，并由此发现，在细胞外施加Hg2+后几分钟内，Hgi2+就达到了纳摩尔水平，但这些可测量的Hgi2+水平在Ca(i)2+升高之前并未出现。在这段时间内（< 5分钟），通过碘化丙啶通透性和光学显微镜观察确定，Hg2+并未对肝细胞造成不可逆损伤。总之，这些发现表明：（i）Hg2+使鳐鱼肝细胞中的Ca(i)2+升高；（ii）Hg2+必须进入肝细胞才能使Ca(i)2+升高；（iii）这种升高是由内源性储存库释放Ca2+介导的，这些储存库与毒胡萝卜素敏感的Ca2+储存库不同；（iv）这种升高与胞质中可测量的Hg2+水平相关。Hg2+的不良细胞效应可能是由这种有毒金属在细胞内积累导致的Ca(i)2+变化介导的。
