Mechanisms of copper accumulation in isolated mantle cells of the marine clam Mesodesma mactroides
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In vivo copper accumulation was determined in tissues (mantle, gills, digestive gland, and hemolymph) following exposure to Cu (5 µM) for up to 96 h. Mantle was the tissue that accumulated the most Cu, followed by gill, digestive gland, and hemolymph. Therefore, in vitro Cu accumulation was evaluated in isolated mantle cells exposed to 0.5, 1.0, 2.5, and 5.0 µM Cu for 1 and 3 h. After both exposure times, no change in cell viability was observed. However, a significant Cu accumulation was observed in cells exposed to 2.5 and 5.0 µM Cu. Cell exposure to 2.5 µM Cu for 1 h did not affect the ionic (Na(+), K(+), Ca(2+), and Cl(-)) content of isolated mantle cells, characterizing an "ideal" noneffect concentration for the study of the involvement of different ion-transporting proteins (Na(+), K(+), and Cl(-) channels; Na(+)/K(+) 2Cl(-) and Na(+)/Cl(-) cotransporters; Na(+)/Ca(2+), Cl(-)/HCO3-, and Na(+)/H(+) exchangers; Na(+)/K(+) -ATPase; V-ATPase; and carbonic anhydrase) in Cu accumulation. Isolated cells were pre-exposed (30 min) to specific blockers or inhibitors of the ion-transporting proteins and then exposed (1 h) to Cu (2.5 µM) in the presence of the drug. A significant increase of 29.1 and 24.3% in Cu accumulation was observed after cell incubation with acetozalamide (carbonic anhydrase inhibitor) and NPPB (Cl(-) channels blocker), respectively. On the other hand, a significant decrease (48.2%) in Cu accumulation was observed after incubation with furosemide (Na(+) /K(+)/2Cl(-) blocker). Taken together, these findings indicate the mantle as an important route of Cu entry in M. mactroides, pointing to the cotransporter Na(+)/K(+)/2Cl(-) as a major mechanism of Cu accumulation in mantle cells of the clam.