Sodium-sensitive and -insensitive copper accumulation by isolated intestinal cells of rainbow troutOncorhynchus mykiss

Abstract

The pathway for copper (Cu) uptake across the mucosal membrane into intestinal cells has not been elucidated in fish. Copper accumulation in freshly isolated intestinal cells from rainbow trout Oncorhynchus mykiss was measured after exposure to 0-800 micromol l(-1) CuSO(4) for 15 min. With external Cu concentration (Cu(o)) of 800 micromol l(-1), the rate of Cu accumulation by cells was 1.88+/-0.52 nmol Cu mg(-1) cell protein h(-1) compared to 0.05+/-0.01 nmol Cu mg(-1) cell protein h(-1) with no added Cu(o) (means +/- s.e.m., N=6). Deduction of a rapid Cu accumulation measured on/in cells at time zero (about 12% of the total Cu uptake when Cu(o) was 800 micromol l(-1)) revealed a saturable uptake curve, which reached a plateau at 400 micromol l(-1) Cu(o) (K(m)=216 micromol l(-1) Cu(o); V(max)=1.09 nmol Cu mg(-1) cell protein h(-1); 140 mmol l(-1) NaCl throughout). Incubation of cells at 4 degrees C did not prevent Cu accumulation. Lowering external [Na(+)] to 11 mmol l(-1) (low Na(+)(o)) generally did not alter the rate of Cu accumulation into the cells over a 15 min period. Under low Na(+)(o) conditions Cu accumulation was exponential (non-saturable). Na(+)-insensitive Cu accumulation dominated (59% of total Cu accumulation) when Cu(o) was 400 micromol l(-1) or less. At high Cu(o) (800 micromol l(-1)), removal of Na(+) caused a 45% increase in Cu accumulation. Pre-incubation of cells with blocking agents of epithelial Na(+) channel (ENaC) for 15 min (normal [NaCl] throughout) caused Cu accumulation rates to increase by 40-fold (100 micromol l(-1) phenamil), 21-fold (10 micromol l(-1) CDPC) or 12-fold (2 mmol l(-1) amiloride) when Cu(o) was 800 micromol l(-1) compared to those in drug-free controls. Lowering the external chloride concentration [Cl(-)](o) from 131.6 to 6.6 mmol l(-1) (replaced by sodium gluconate) caused the rate of Cu accumulation to increase 11-fold when Cu(o) was 800 micromol l(-1). Application of 0.1 mmol l(-1) DIDS (normal Cl(-)(o)) caused a similar effect. Lowering external pH from 7.4 to pH 5.5 produced a 17-fold, saturable, increase in Cu accumulation rate, which was not explained by increased instantaneous Cu accumulation on/in cells at low pH. We conclude that Cu accumulation by intestinal cells is mainly Na(+)-insensitive and more characteristic of a pH- and K(+)-sensitive Ctr1-like pathway than Cu uptake through ENaCs.