Arsenic inhibits SGK1 activation of CFTR Cl- channels in the gill of killifish, Fundulus heteroclitus.

Seawater acclimation in killifish, Fundulus heteroclitus, is mediated in part by a rapid (1h) translocation of CFTR Cl(-) channels from an intracellular pool to the plasma membrane in gill and increased CFTR-mediated Cl(-) secretion. This effect is mediated by serum and glucocorticoid-inducible kinase 1 (SGK1), which is stimulated by plasma hypertonicity rather than cortisol. Since arsenic exposure prevents acclimation to seawater by decreasing CFTR protein levels we tested the hypothesis that arsenic (as sodium arsenite) blocks acclimation to seawater by down regulating SGK1 expression. Freshwater adapted killifish were exposed to arsenic (48h) and transferred to seawater containing arsenic, and SGK and CFTR expression were measured. Arsenic reduced the seawater induced increase in SGK1 mRNA and protein abundance, and reduced both the total amount of CFTR and the amount of CFTR in the plasma membrane. The decrease in membrane CFTR reduced Cl(-) secretion. Arsenic also increased the amount of ubiquitinated CFTR and its degradation by the lysosome. Thus, we propose a model whereby arsenic reduces the ability of killifish to acclimate to seawater by blocking the seawater induced increase in SGK1, which results in increased ubiquitination and degradation of CFTR.

The role of SGK and CFTR in acute adaptation to seawater in Fundulus heteroclitus.

Killifish are euryhaline teleosts that adapt to increased salinity by up regulating CFTR mediated Cl(-) secretion in the gill and opercular membrane. Although many studies have examined the mechanisms responsible for long term (days) adaptation to increased salinity, little is known about the mechanisms responsible for acute (hours) adaptation. Thus, studies were conducted to test the hypotheses that the acute homeostatic regulation of NaCl balance in killifish involves a translocation of CFTR to the plasma membrane and that this effect is mediated by serum-and glucocorticoid-inducible kinase (SGK1). Cell surface biotinyation and Ussing chamber studies revealed that freshwater to seawater transfer rapidly (1 hour) increased CFTR Cl(-) secretion and the abundance of CFTR in the plasma membrane of opercular membranes. Q-RT-PCR and Western blot studies demonstrated that the increase in plasma membrane CFTR was preceded by an increase in SGK1 mRNA and protein levels. Seawater rapidly (1 hr) increases cortisol and plasma tonicity, potent stimuli of SGK1 expression, yet RU486, a glucocorticoid receptor antagonist, did not block the increase in SGK1 expression. Thus, in killifish SGK1 does not appear to be regulated by the glucocorticoid receptor. Since SGK1 has been shown to increase the plasma membrane abundance of CFTR in Xenopus oocytes, these observations suggest that acute adaptation (hours) to increased salinity in killifish involves translocation of CFTR from an intracellular pool to the plasma membrane, and that this effect may be mediated by SGK1.

The influence of exposure history on arsenic accumulation and toxicity in the killifish, Fundulus heteroclitus.

Exposure to arsenic is known to cause adverse effects in aquatic biota and wildlife and is of major concern to human health. Although numerous studies have investigated the toxicity of arsenic, little is known about the effects of acquired tolerance on arsenic accumulation and toxicity outside of cell culture models. Accordingly, studies were conducted on the estuarine fish, Fundulus heteroclitus, that were preexposed to nontoxic concentrations of arsenic (as sodium arsenite; 0.7 and 106 micromol As/L) for 96 h or naïve to elevated arsenic to determine the effects of acclimation on arsenic toxicity and accumulation. Tolerance to arsenic was rapidly (96 h) acquired in killifish that were preexposed. In toxicity tests with arsenic-acclimated killifish, preexposure to 106 micromol As/L resulted in a reduction in toxicity when compared to naïve animals. Toxicity in arsenic-acclimated fish also was distinguished by a delayed onset of mortality that manifested in dose-dependent fashion and was significant even for the lower acclimation concentration (0.7 micromol As/L). The increase tolerance acquired following preexposure to 106 micromol As/L for 96 h was associated with lower concentrations of arsenic in all monitored tissues (e.g., gill, liver, kidney) and the whole body when fish were exposed to 240 micromol As/L for an additional 96 h. In accordance with these observations, expression of the multidrug resistance- associated protein (MRP)-2 gene, which is responsible for transporting arsenic conjugated to glutathione out of cells, was increased in the liver of arsenic-acclimated fish.