Mechanisms of sodium extrusion in isolated mitochondria-rich cells of the freshwater mussel Lasmigona costata after copper exposure

Gill tissues in aquatic animals are composed of various cell types, each with specific functions such as respiration, excretion, acid-base balance and ionic regulation. In the bivalves, the gill is the main route of contaminant uptake and metals can be absorbed through specific transporters in cell membranes. Among the gill cells, mitochondria-rich cells (MRCs) are considered to be the primary sites responsible for iron transport. In order to determine the mechanisms of sodium extrusion in MRCs after copper exposure, gill cells from Lasmigona costata were isolated and layered on a discontinuous Percoll gradient. The pavement cells (PCVs) and MRCs populations were confirmed by a specific mitochondrial dye (DASPEI) and Na, K-ATPase activity. It is the first time at literature that isolated fill cells from freshwater mussels are identified in two different populations and used in toxicological experiments. MRCs were then exposed to a range of copper concentrations environmentally relevant (5, 10 and 20 ug/L) for 3 hours. Copper accumulation increased in all copper exposures. In contrast, the sodium content did not change in low exposures and decreased significantly when cells were exposed to high copper concentrations. To further explore this result, both sodium and copper were measured after specific pharmacological blockade of Na, K-ATPase and carbonic anhydrase enzymes. Carbonic anhydrase inhibition suggest that sodium and copper uptake mechanisms are not dependent on a supply of protons and intracellular increase of both ions might be associated to blocked of Na extrusion via Na/HCO3-. The inhibition of Na, K-ATPase by ouabain also resulted in an increase in sodium and copper content in the MRCs. This enzyme is responsible for sodium extrusion from the cell, and the results suggest that it is also a route for copper extrusion.