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First published online January 5, 2005
Journal of Experimental Biology 208, 391-407 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01379

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

J. Burke and R. D. Handy^*

School of Biological Sciences, University of Plymouth, Drake Circus, Plymouth PL4 8AA, UK

View larger version (153K): [in a new window]   Fig. 1. Examples of isolated trout intestinal cells in normal physiological saline (no added Cuo) after 15 min (A) and 3 h (B), compared to cells exposed to saline containing 800 µmol l–1 Cuo for 15 min (C) and 3 h (D). Cells were photographed in phase contrast (not fixed or stained) using a Leica DMIRB microscope and a Nikon coolpix digital camera. Scale bar, 2 µm.

View larger version (14K): [in a new window]   Fig. 2. Preliminary experiment to define (A) the time course of Cu accumulation by freshly isolated intestinal cells, and (B) Cu retention by Cu-loaded cells placed in normal physiological saline. (A) The cells were incubated in external Cu (Cuo) concentrations of no added Cu (control, filled circles), 10 (open circles), 50 (filled triangles), 100 (open triangles), 200 (filled squares), 400 (open squares), or 800 µmol l–1 Cu (filled diamonds) for up to 30 min. Cells were then quickly washed in a 0.1 µmol l–1 EDTA washing solution and pelleted prior to determination of cell Cu content (expressed as nmol Cu mg–1 cell protein). Values are means ± S.E.M. (N=6 experiments). Rectangular hyperbola are fitted to the raw data using Sigma plot. All cells had reached a stable Cu content by 15 min of exposure time (all significantly higher than the control with no added Cu, Student's t-test, P<0.05, except for the 10 µmol l–1 Cuo treatment). (B) Cells were loaded with Cu by exposing them to 800 µmol l–1 Cu for 15 min, and the subsequent loss of accumulated Cu was followed for 1 h by placing cells back into normal physiological saline (no added Cu). Values are means ± S.E.M. (N=9 experiments) and expressed as nmol Cu mg–1 cell protein. Values in parentheses are the percent decrease in Cu content (means ± S.E.M.) of the cells relative to the start of the post-exposure period where the Cu content of the Cu-loaded cells is defined as 100%. *Statistically different from time 0 post-exposure by Kruskall–Wallis test (P=0.00005). Note, cells did not leak Cu for at least the first 15 min in normal saline (no statistical difference from time 0 post-exposure control).

View larger version (13K): [in a new window]   Fig. 4. The effect of external Cu (Cuo) on Cu accumulation by intestinal cells in the presence of normal (140 mmol l–1, triangles) and low (11 mmol l–1, squares) external [Na+] (Na+o). (A) Total Cu accumulation by intestinal cells during 15 min incubation in Cu-containing saline and (B) net Cu accumulation revealed after rapid Cu accumulation in/on cells at time 0 (Fig. 3) had been deducted from the total Cu accumulation over 15 min. Values are means ± S.E.M. of N=5–6 separate experiments using fresh cells from different individual fish. *Significantly different from the control with no added Cu within treatment; Kruskal–Wallis test (P<0.05). Significantly different from normal Na+o at the indicated Cuo; Mann–Whitney U-test (P<0.05). Accumulation is normalised mg–1 cell protein and h–1 to allow comparison between plots, and with Fig. 3.

View larger version (16K): [in a new window]   Fig. 3. Rapid or instantaneous Cu accumulation in/on intestinal cells incubated in external Cu (Cuo) concentrations between 0 (no added Cu control) and 800 µmol l–1. A Cu-free EDTA control is also included (no added Cu + 1 µmol l–1 EDTA, open symbol). Cells were exposed to solutions of various Cuo and then immediately (<1 min to prepare all tubes) washed in a 0.1 µmol l–1 EDTA washing solution and pelleted (1 min in a bench centrifuge). These rapid measurements represent a time 0 control for the main Cu exposures, which lasted 15 min (see Figs 4, 5, 6, 7, 8, 9, 10 for 15 min exposures), and were performed mainly as an additional check to confirm that spontaneous Cu adsorption/accumulation (Handy and Eddy, 2004) was only a small component of net Cu accumulation over a 15 min period. Fig. 3 shows time 0 rapid Cu accumulation in/on cells in the presence of normal (140 mmol l–1, triangles) and low (11 mmol l–1, squares) external [Na+] (Na+o). Values are means ± S.E.M. of N=5–6 separate experiments using fresh cells from different individual fish. *Significantly different from the control with no added Cu within treatment by ANOVA (P <0.05). Significantly different from normal Na+o at the indicated Cuo; Student's t-test (P<0.05). Accumulation is normalised mg–1 cell protein and h–1 to allow comparison between plots in Figs 3 and 4.

View larger version (26K): [in a new window]   Fig. 5. The effect of pre-incubating intestinal cells with epithelial Na+ channel (ENaC) inhibitors on rate of Cu accumulation at external [Cu] (Cuo) ranging from 0–800 µmol l–1. Cells were pre-incubated for 15 minwithout Cuo (no added Cu) but with either 100 µmol l–1 phenamil (open squares), 10 µmol l–1 CDPC (open diamonds), or 2 mmol l–1 amiloride (open triangles), compared to controls with no added drug (normal NaCl and no inhibitors, filled triangles) and cells incubated at 4°C without inhibitors (open circles). Drugs were then washed off and cells exposed to 0–800 µmol l–1 Cuo for 15 min. All experiments used normal Na+o (140 mmol l–1 NaCl) throughout. Rapid Cu accumulation in/on cells at time 0 was not deducted from the data. Values are means ± S.E.M. of N=5–6 separate experiments using fresh cells from different individual fish. Different letters (a, b, c or d) indicate a statistically significant difference between adjacent treatments within Cuo concentration (looking up at adjacent data points between plots at each Cuo, Kruskal–Wallis test, P<0.05). 1Significant effect of phenamil compared to amiloride within Cuo (Kruskal–Wallis test, P<0.05). For clarity, statistical differences between treatment effects (between plots) for 10, 50 and 100 µmol l–1 Cuo, respectively, are shown on the insert with the axis expanded for the lowest Cuo concentrations. The insert also shows the Cu-free (+1 µmol l–1 EDTA) control (EDTA on the axis label) compared to no added Cu (zero on the axis label). No statistical differences were observed between EDTA and controls with no added Cu (Student's t-tests between EDTA and no added Cu within drug treatment, P>0.05). Cu accumulation rates within drug treatments were all significantly different from the no added Cu control at Cuo=50 µmol l–1 or greater (labels not added for clarity; Kruskal–Wallis test, P<0.05). Cuo effect within ice-cold treatment was significantly different from no added Cu ice control at Cuo=800 µmol l–1 only (Kruskal–Wallis test, P=0.0035).

View larger version (19K): [in a new window]   Fig. 6. Copper accumulation by intestinal cells exposed to external [Cu] (Cuo) ranging from 0 (no added Cu) to 800 µmol l–1 for 15 min in normal saline (NaCl=140 mmol l–1 at pH 7.4, filled triangles), compared with the effects of either low external chloride (Cl–o=6.6 mmol l–1, Cl–o replaced by sodium gluconate, filled circles), 0.1 mmol l–1 DIDS in normal saline (open squares), or with low external pH (pH 5.5, open circle). Values are means ± S.E.M. of N=5–6 separate experiments using fresh cells from different individual fish. Rapid Cu accumulation in/on cells at time zero was not deducted from the data. Different letters (a, b, c or d) indicate a statistically significant difference between adjacent treatments within Cuo (looking up at adjacent data points between plots at each Cuo, Kruskal–Wallis test, P<0.05). For clarity, statistical differences between treatment effects at 10 µmol l–1 Cuo are not shown, but are identical to those at 50 µmol l–1 Cuo. Cu accumulation within treatment (Cu-dose effects within each experiment) were significantly different from the control with no added Cu at Cuo=50 µmol l–1 or greater for the low Cl–o and low pH experiments; and at 10 µmol l–1 Cuo or greater for the DIDS experiment (labels not added for clarity, Kruskal–Wallis test, P<0.05). The effect of DIDS reached a plateau at 200 µmol l–1 Cuo because Cu accumulation rates between 200–800 µmol l–1 Cuo were not statistically different from each other within DIDS treatment (from post-hoc box whisker plots following Kruskal–Wallis test). Similarly, the low pH effect reached a plateau at 400 µmol l–1 Cuo because Cu accumulation rates between 400–800 µmol l–1 Cuo were not statistically different from each other within the low pH treatment (from post-hoc box whisker plots following Kruskal–Wallis test). The above statistics for Cu-dose effect within treatment are not shown for clarity.

View larger version (26K): [in a new window]   Fig. 7. (A) The effect of external Cu (Cuo) on the Na+ content of intestinal cells in the presence of normal (140 mmol l–1, open bars) and low (11 mmol l–1, black bars) external Na+ (Na+o) after a 15 min incubation in Cu-containing saline at room temperature. (B) Na+ content of intestinal cells incubated at the same Cuo range but at 4°C and with normal Na+o throughout. Values are means ± S.E.M. of N=5–6 separate experiments using fresh cells from different individual fish. *Significantly different from control with no added Cu within treatment by Kruskal–Wallis test (P <0.05). No differences were observed in cell Na+ content between low and normal Na+o within each Cuo at room temperature (Student's t-tests, P>0.05). Note the larger y-axis scale in B, all values at 4°C were significantly different from the respective room temperature control within Cuo treatment (comparison of normal NaCl in A with B, Student's t-tests, P>0.05; data not labelled for clarity). There were no differences between Cuo-free (EDTA, no added Cu but + 1 µmol l–1 EDTA) and controls with no added Cu within treatments (Student's t-test, P>0.05).

View larger version (22K): [in a new window]   Fig. 8. The effect of pre-incubation of intestinal cells with epithelial Na+ channel inhibitors on (A) cell Na+ content or (B) cell K+ content in Cu-free conditions (+ 1 µmol l–1 EDTA), with no added Cu, or after a 15 min incubation in 800 µmol l–1 Cuo in the presence of either 100 µmol l–1 phenamil (black bars), 2 mmol l–1 amiloride (light grey bars), or 10 µmol l–1 CDPC (cross-hatched bars), compared to no added drug controls (white bars). All experiments were at normal Na+o and room temperature throughout. Values are means ± S.E.M. of N=5–6 separate experiments using fresh cells from different individual fish. *Significant difference from control without drug within Cuo treatment (Kruskal–Wallis test, P<0.05). Significant difference from the control with no added Cu within drug treatment (Mann–Whitney U-test, P<0.05).

View larger version (21K): [in a new window]   Fig. 9. Relative reduction in (A) cell Na+ and (B) cell K+ content in 800 µmol l–1 Cuo, normalised, against the Na+ content of cells treated with the appropriate drug with no added Cu. 100 µmol l–1 phenamil (black bars), 2 mmol l–1 amiloride (grey bars), or 10 µmol l–1 CDPC (hatched bars), compared to drug containing controls without Cu (white bars, normalised to 100%). Values are means ± S.E.M. of N=5–6 separate experiments using fresh cells from different individual fish. Significant modulatory effect of Cuo on the ability of the drug to reduce cell electrolyte content (Kruskal–Wallis test, P=<0.05). Amiloride is relatively more effective than other ENaC inhibitors at reducing cell Na+ content in the presence of Cuo. No Cuo-dependent modulation of drug effects on cell K+ occurred.

View larger version (25K): [in a new window]   Fig. 10. Na+ (white bars) and K+ content (grey bars) content of intestinal cells exposed external [Cu] (Cuo) ranging from 0 (no added Cu) to 800 µmol l–1 for 15 min (A) in low external chloride (Cl–o=6.6 mmol l–1, Cl–o replaced by sodium gluconate), (B) with 0.1 mmol l–1 DIDS in normal saline, and (C) at low external pH (pH 5.5). Values are means ± S.E.M. of N=5–6 separate experiments using fresh cells from different individual fish. *Statistically significant difference from control with no added Cu within treatment and electrolyte (Kruskal–Wallis test, P<0.05).

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