Sodium-sensitive and -insensitive copper accumulation by isolated intestinal cells of rainbow trout Oncorhynchus mykiss
School of Biological Sciences, University of Plymouth, Drake Circus, Plymouth PL4 8AA, UK
* Author for correspondence (e-mail: rhandy{at}plymouth.ac.uk)
Accepted 9 November 2004
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Summary |
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Key words: rainbow trout, Oncorhynchus mykiss, dietary copper, sodium, amiloride, phenamil, low pH
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Introduction |
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In mammals and fish, Cu uptake from the gut lumen to the blood involves (i)
electrostatic adsorption of Cu to the surface of the mucosal membrane, (ii)
entry into the gut cells by facilitated diffusion, probably through ion
channels, (iii) transfer of Cu across the cell by metal chaperones, (iv)
export from the cell to the blood against the electrochemical gradient
(Linder, 1991;
Harrison and Dameron, 1999
;
Handy et al., 2000
,
2002
). The latter step
involves both exocytosis of vesicular Cu derived from the Golgi complex
(Harrison and Dameron, 1999
;
Huffman and O'Halloran, 2001
),
and Cu export from the cytoplasm on a serosally located CuCl symporter
(Handy et al., 2000
). Copper
uptake across the gut is also negatively regulated at the intestine, Cu uptake
efficiency declining with increasing luminal Cu concentration in isolated
perfused catfish intestines (Handy et al.,
2000
), and dietary Cu bioavailability declining with increasing
dietary dose in vivo (e.g. trout;
Clearwater et al., 2002
).
Intestinal Cu uptake can also be downregulated by aqueous Cu exposure in trout
(Kamunde et al., 2002
),
implying some degree of systemic control of Cu absorption across the
intestine.
However the precise pathway for Cu entry into enterocytes from the gut
lumen is uncertain. Current evidence from a variety of epithelia (frog skin,
Flonta et al., 1998; rat
intestine, Wapnir, 1991
; trout
gills, Grosell and Wood, 2002
;
fish intestine, Handy et al.,
2002
) suggest at least two candidate pathways. These include Cu
leak through epithelial Na+ channels (EnaCs; e.g. fish gills,
Grosell and Wood, 2002
) and Cu
uptake on a Cu-specific carrier, Ctr1 (Lee et al.,
2000
,
2002a
,b
).
Ctr1 is located in the plasma membrane of cells and appears to be ubiquitously
expressed in mammalian tissues (Lee et
al., 2000
; Klomp et al.,
2002
). Ctr1 has also been recently identified in zebrafish
(Mackenzie et al., 2004
).
In the intestine, Cu uptake on Ctr1 is more likely than through ENaCs for
several reasons. Firstly, the relatively high external Na+
concentration (Na+o, circa 100 mmol
l1) in the gut lumen compared to gills or frog skin in
freshwater suggest that Cu will be less able to compete for entry through
ENaCs (Handy et al., 2002).
Secondly, Cu2+ ions bind externally to the alpha subunit of ENaCs
rather than going through the channel pore, and Cu exposure does not stop the
Na+ current through ENaCs (frog skin;
Flonta et al., 1998
). Removal
of external Na+ or Cl also tends to lower
intestinal Cu uptake rates (rat, Wapnir
and Stiel, 1987
; catfish,
Handy et al., 2000
), and this
is not consistent with Cu ions competing with Na+ for entry through
ENaCs in the gut mucosa (Handy et al.,
2002
).
Alternatively, amiloride-dependent depression of Cu uptake by perfused
intestines is most easily explained by Cu uptake through ENaCs
(Wapnir, 1991;
Handy et al., 2002
). In
Na+o removal experiments, the indirect effects of low
cell Na+ on Cl balance could also alter Cu
status. For example, decreasing intracellular chloride during
Na+o removal experiments could slow Cu export to the
blood on CuCl symport (Handy et al.,
2000
,
2002
), prevent Cu binding to
vesicular Cu-ATPase (Davis-Kaplan et al.,
1998
), and reduce the probability of opening of ENaCs
(Kunzelmann et al., 2001
; for
a discussion, see Handy et al.,
2002
). These indirect effects would raise whole cell Cu content as
Na+o declines, giving the impression of apparent
Na+o-dependent Cu uptake. In vivo, there may
also be systemic Na+-dependent modulation of Cu uptake (systemic
Na+ effects on Cu uptake by trout gills;
Kamunde et al., 2003
;
Pyle et al., 2003
).
In this experiment we resolve some of these controversies for fish, and
explore the Na+o-dependence of Cu accumulation by
isolated intestinal cells from rainbow trout. Freshly isolated intestinal
cells have the advantage of retaining the biochemical and metabolic
characteristics of the gut mucosa for several hours
(Kimmich, 1990). Primary
cultures of intestinal cells are not used because they gradually become
quiescent as they reach confluence, and for this reason intestinal cell lines
derived from hybrids with cancer stem cells are preferred by most researchers
for studies on ion transport
(Dharmsathaphorn and Madara,
1990
). Furthermore, external Cu concentrations as low as 5 µmol
l1 may compromise the tight junctions in monolayers of
cultured intestinal cells (Caco-2 cells;
Ferruzza et al., 1999
). Such
cell lines are not readily available in rainbow trout, and so in the present
study we use freshly isolated intestinal cells. This approach also enables Cu
accumulation by the mucosal cells to be measured without contributions from
the underlying muscularis or enteric nervous system, and avoids systemic/organ
level Cu-dependent endocrine modulation of gut function
(Handy, 2003
). Putative loss
of polarity in cell suspensions is not so problematic for studies on Cu
accumulation by intestinal cells, because unlike major electrolytes, Cu
appears only to move from the luminal side across the mucosal membrane,
through the cell, and out of the cell across the serosal membrane in a variety
of experimental conditions (Arredondo et
al., 2000
; Zerounian et al.,
2003
; Bauerly et al.,
2004
). There is no evidence that intestinal cells can secrete
accumulated Cu (back flux) across the mucosal membrane
(Arredondo et al., 2000
), and
this is not surprising since the endosomal recycling of Ctr1 back to the
mucosal membrane does not colocalise with Golgi Cu stores
(Bauerly et al., 2004
). Thus
Cu-loaded Caco-2 cells retain 40% or more of their Cu for at least 18 h
(Zerounian et al., 2003
), and
the observed Cu efflux from the cells is best explained by vesicular
trafficking via metal chaperones to the serosal membrane
(Arredondo et al., 2000
;
Zerounian et al., 2003
;
Bauerly et al., 2004
).
Our aims are fourfold: (i) to demonstrate concentration-dependent Cu
accumulation in isolated intestinal cells and the effect of
Na+o removal on Cu uptake; (ii) to critically evaluate
the effect of the ENaC blocking agent amiloride compared to other more
specific ENaC inhibitors, 6-chloro-3, 5-diamino-2 pyrazinecarboxamide (CDPC)
and phenamil (Garvin et al.,
1985; Flonta et al.,
1998
; Reid et al.,
2003
), on intestinal Cu uptake; (iii) to determine the indirect
effects of manipulations of anion transport on Cu accumulation and
Na+ content of intestinal cells; (iv) explore Cu accumulation in
the low pH conditions known to stimulate Ctr1
(Lee et al., 2002a
).
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Materials and methods |
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Intestinal cell isolation
Intestinal cells were freshly isolated by a method modified from Kimmich
(1990). Briefly, each rinsed
intestine was cut open longitudinally and divided into 1 cm2
pieces, then added to 50 ml of ice-cold isolation medium (in mmol
l1: NaCl, 125; NaHCO3, 10;
K2HPO4, 3; MgCl2, 1; CaCl2, 1;
dithiothreitol, 0.1; glucose, 10; Hepes, 10; Trizma base 10; pH 7.4). The
tissue was gently agitated using a Pasteur pipette to detach enterocytes from
the mucosa. The resulting cell suspension was then filtered through a 200
µm mesh to remove debris, and then divided between 4 x 13 ml tubes
and centrifuged (5 min at 200 g, Denley 401 refrigerated
centrifuge; Thermo-Denley, Basingstoke, UK). The supernatant was discarded and
the pellet of cells in each test tube was resuspended in 850 µl of
physiological saline (in mmol l1: NaCl, 125;
NaHCO3, 10; NaH2PO4, 1; KCl, 3;
MgSO4, 2; CaCl2, 1.8; glucose, 10; adjusted to pH 7.4
with 2 drops of 1 mmol l1 HCl or NaOH). The tubes were then
combined, typically providing a total of 3.4 ml of cell suspension per fish
intestine (mean ± S.E.M., 1.05±0.005
x107 cells ml1 and 94.1±1.08% cell
viability by Trypan Blue exclusion; N=54 separate cell isolations).
At least six separate cell isolations were used for each experiment. Cell
counts and cell viability were measured immediately before experiments, and
only cell isolations showing in excess of 80% viability were used.
Preliminary experiments
Several preliminary experiments were performed to explore cell viability
and to determine the optimum exposure times/Cu concentrations needed to
measure physiological Cu accumulation. In an initial trial the survival of a
batch of cells was assessed by Trypan Blue exclusion over 4 h in normal
physiological saline without Cu (no added Cu). In this trial, viability was
initially 82% and remained at 82% for the first 3 h, then decreased only
slightly to 78% by 4 h (4% decline over 4 h). Thus freshly isolated intestinal
cells remained intact for at least 4 h on the bench in normal physiological
saline. Experiments were then repeated in the presence of 800 µmol
l1 Cuo. Cells were intact and morphology also
remained normal for several hours, with or without added Cu
(Fig. 1). Other batches of
cells were then used to establish whether or not Cu accumulation could be
detected in cells exposed to Cuo for periods lasting several hours.
Cells (N=6 batches from separate isolations) were treated for 2 h
with either nominally Cu-free saline (normal saline + 1 µmol
l1 ethylenediaminetetraacetic acid, EDTA), control with no
added Cu (normal saline with no EDTA or added Cu), and normal saline
containing 10, 50, 100 or 200 µmol l1 Cu. The Cu content
of cells was easily detected, and mean values after 2 h were 0.01±0.002
and 0.96±0.09 nmol Cu mg1 cell protein in controls
with no added Cu and in saline containing 200 µmol l1
Cuo, respectively (mean ± S.E.M., N=6;
significantly different, student's t-test, P<0.05). We
therefore elected to explore Cu accumulation over a much shorter time scale
(030 min) and with Cuo up to 800 µmol
l1 (Fig. 2A).
Cells that were exposed to Cu for 30 min showed a dose-dependent increase in
Cu content, with the Cu content of cells reaching a plateau by 10 min at most
exposure concentrations, and Cu content remained steady for at least another
20 min (Fig. 2). The leakiness
of these cells was assessed by measuring lactate dehydrogenase (LDH) release
into the medium (for LDH assay, see
Campbell et al., 1999) and
cell Na+ and K+ content
(Table 1). Control cells (no
added Cu) showed stable Na+ and K+ contents over a 30
min period with no changes in membrane permeability measured by LDH leak
(Table 1). Similarly Cu-treated
cells did not leak LDH, the release rate of which remained at or below
detection limits for all external Cu concentrations (<0.1 µmol LDH
min1 ml1 medium, N=6). Cu-treated
cells also showed stable Na+ and K+ contents over 30 min
(Table 1). In another series of
experiments, cells were loaded with Cu by exposing them to 800 µmol
l1 Cuo for 15 min and then placed in normal
saline (no added Cu) to determine whether the accumulated Cu would leak out of
the cells. This was not observed (Fig.
2B) over a 15 min period. Cu-loaded cells retained 86±5.1%
(mean ± S.E.M., N=9) of the accumulated Cu after 15
min recovery in normal saline (not statistically different from the control,
Fig. 2B). Even after 1 h in
clean saline, cells still retained 50% of the accumulated Cu. Overall, these
preliminary experiments demonstrated that freshly isolated intestinal cells
could survive physiological concentrations of Cuo of up to 800
µmol l1 and did not leak LDH, Cu or electrolytes over
exposure periods of 15 min. We therefore elected to conduct the main
experiments using total external Cu concentrations of between 0800
µmol l1 (added as CuSO4.5H2O to the
physiological saline above), for exposures lasting 15 min.
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Cu exposure protocol
Copper exposures lasting 15 min were used in the main experiments, and the
protocol was derived from the preliminary experiments outlined above. Briefly,
125 µl of the washed cell suspension was added to 500 µl of the
appropriate Cu concentration (final concentrations of 0, 10, 50, 100, 200, 400
or 800 µmol l1 Cu, as CuSO4.5H2O)
in physiological saline (saline as above), and incubated in an Eppendorf tube
at room temperature (20°C) for 15 min (in triplicate). Copper
concentrations in all solutions were measured prior to experiments, and if
necessary solutions were prepared again to meet the exact concentrations
indicated above. Controls included incubation in physiological saline with no
added Cu (saline only control) and a Cu-free control (saline without Cu, + 1
µmol l1 EDTA). At the end of the exposure period the
cells were quickly pelleted (1 min at 13 000 rpm, Sanyo Microcentaur, Fischer
Scientific, Loughborough, UK), and the pellet was washed gently (3 times) with
100 µl of 0.1 µmol l1 EDTA. Finally the cells were
lysed with 0.5 ml of analytical grade 0.1 mol l1 nitric acid
prior to metal analysis (see below). The entire protocol was repeated on at
least six separate occasions for each experiment, using gut tissue from a new
fish each time.
Each experiment also included a `time zero' control, where batches of cells
were added to Cuo solutions (<1 min to prepare all tubes),
immediately washed in the EDTA solution above, then pelleted. These rapid
measurements were used mainly as an additional check within each experiment to
confirm that net Cu accumulation was a progressive accumulation during the 15
min incubations rather than spontaneous Cu adsorption/surface binding in the
first minute of incubation (Handy and
Eddy, 2004). The time zero controls showed a rapid Cu accumulation
component, which was maximally (when Cuo=800 µmol
l1) around 1216% of the total apparent accumulation
(see Results, and Table 2) and
at Cuo <800 µmol l1 this component was
<1%. Thus over most of the Cuo range used in the experiment
(0400 µmol l1), the instantaneous component was
small and was therefore not deducted from the Cu accumulation data after the
15 min incubations.
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Copper uptake in normal and low Na+o
This series of experiments was conducted to determine dose-dependent net Cu
uptake by freshly isolated cells in the presence of normal NaCl
(Na+o=140 mmol l1;
Handy et al., 2002) using the
protocol and saline described above. The experiment was then repeated with Cu
incubations on ice at 4°C (normal NaCl, 140 mmol l1
Na+o) to determine whether net Cu uptake was
energy/temperature-dependent. Then the Na+o-dependence
of Cu accumulation was explored (at laboratory temperature, 19°C) using a
low-Na+o version of the physiological saline above for
the incubations, where the NaCl was replaced by 125 mmol l1
choline chloride (Na+o=11 mmol
l1).
Effect of epithelial Na+ channel blocking agents on Cu accumulation
This series of experiments explored the effects of epithelial
Na+ channel (ENaC) blocking agents on net Cu uptake when
Na+o was normal. Drug concentrations were selected to
produce complete blockade of ENaC, and included: (i) 2 mmol
l1 amiloride (blocks Cu uptake in perfused catfish
intestine; Handy et al.,
2002), (ii) 10 µmol l1 6-chloro-3,
5-diamino-2 pyrazinecarboxamide (CDPC; Acros Chemical Co., Morris Plains, NJ,
USA), which is an amiloride analogue that does not chelate Cu
(Flonta et al., 1998
) and
alters Cu uptake in perfused catfish intestine (R. D. Handy, unpublished
observations), or (iii) 100 µmol l1 phenamil (Sigma
Aldrich, Poole, UK), which partly inhibits Cu uptake
(Grosell and Wood, 2002
) and
Na+ influx (Reid et al.,
2003
) by trout gills. In order to eliminate theoretical Cu
chelation by the drugs, 150 µl of cell suspension was incubated with 200
µl of the appropriate ENaC inhibitor in normal physiological saline for 15
min prior to the addition of Cu (at the final drug concentrations indicated
above; drugs dissolved in water, except for phenamil, which was dissolved in
DMSO, final concentration <1% DMSO). This also ensured that inhibitors had
free access to bind to the epithelium. Cells were then briefly washed in
drug-free saline (as above) and resuspended in 500 µl of physiological
saline (normal NaCl throughout) containing the appropriate Cu concentration
for 15 min (for the remainder of the experiment and replication as above).
This series of experiments also included controls with no added Cu + drug, and
no drug + DMSO, in addition to those described above.
Effect of manipulating anion transport and external pH on Cu accumulation
Copper uptake is sensitive to removal of external chloride
(Clo) from the gut lumen and the serosal
additions of the anion transport inhibitor 4, 4-diisothiocyanato-stilbene-2,
2'-disulfonic acid (DIDS) in perfused catfish intestine
(Handy et al., 2000). We
therefore conducted similar experiments with isolated trout cells. Cells were
exposed to a range of Cuo concentrations as above, but this time in
low Clo conditions (NaCl replaced by sodium
gluconate, Clo=6.6 mmol l1), or
in the presence of 0.1 mmol l1 DIDS throughout the 15 min
incubation with Cu (normal NaCl).
Finally, Lee et al. (2002a)
demonstrated that Cu flux through the Cu-specific pathway, Ctr1, was
stimulated at pH 5.5 compared to neutral pH. We therefore repeated our first
series of Cu-uptake experiments (in normal NaCl) at pH 5.5 (physiological
saline acidified with a few drops of 2 mmol l1
HNO3).
Trace metal analysis
In all experiments, cell lysates were analysed by inductively coupled
plasma atomic emission spectrophotometry (ICP-AES, Varian Liberty 200, Walton
on Thames, UK) for total Cu and Na+, and in some experiments for
K+, according to Handy et al.
(2002). Cell metal content was
normalised per mg of cell protein, following protein assays (in triplicate) on
each cell suspension using a modified Lowry method
(Handy and Depledge,
1999
).
Statistics and calculations
In some preliminary experiments on the time course of Cu accumulation, data
are presented as an absolute metal content and normalised for cell protein
content (e.g. nmol Cu mg1 cell protein). Similarly
electrolyte contents of cells are expressed as µmol mg1
cell protein. In other experiments where data are expressed as a rate of Cu
accumulation, the cell Cu content was divided by incubation time to give net
accumulation rates in nmol Cu mg1 cell protein
h1. Rapid Cu accumulation in `time zero' controls was also
expressed as nmol Cu mg1 cell protein h1
to allow comparison with data on net accumulation rates over the 15 min
period. The rapid component was not deducted from any of the data, except in
Fig. 4B where deduction of the
fast component reveals a saturable Cu component (see Results). Statistics were
performed using Statgraphics 4.0 Plus software. The variance of the data was
checked using Bartlett's test. Cu-dose effects within treatment, or the
effects of experimental manipulations within Cu concentrations, were compared
using one-way ANOVA followed by the least-squares difference (LSD)
multiple-range test. Where data were non-parametric, the KruskalWallis
test was applied instead. The location of statistical differences from the
KruskalWallis test were identified from the post-hoc box
whisker plots generated by Statgraphics. Where visual differences in overlap
in box whisker plots were difficult to discern, the MannWhitney
U-test was applied to the pairs of data points concerned to confirm
the statistical difference. In some experiments where only two treatments were
used, data were compared between treatments at fixed Cu concentration using
either the Student's t-test or the MannWhitney
U-test. All statistical analysis used a rejection level of
P<0.05 and included Bonferroni correction where appropriate.
Curve-fitting was used to describe some of the experimental data, where
appropriate, and equations describing the best fits to the raw data were
derived using Sigma Plot version 8.0.
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Results |
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Copper accumulation with low Na+o
Dose-dependent Cu accumulation by the cells also occurred when
Na+o was lowered from 140 to 11 mmol
l1 (Fig. 4A,
rapid Cu accumulation not deducted). Cu accumulation in low
Na+o conditions remained linear over the Cuo
range used (linear fit, y=0.0039x, r2=0.96),
unlike the situation with normal Na+o. Cu accumulation
by the cells was twofold higher in low Na+o compared to
normal Na+o conditions (3.4±0.7 compared to
1.88±0.52 nmol Cu mg1 cell protein
h1, respectively, at 800 µmol l1
Cuo; significantly different, MannWhitney U-test,
P=0.045, Fig. 4A; mean
± S.E.M., N=6). However, the rapid Cu accumulation
component at time zero also increased twofold when Na+o
was removed (Fig. 3,
Table 2), and reached
0.52±0.05 and 0.24±0.14 nmol Cu mg1 cell
protein h1 (mean ± S.E.M., N=6)
in low and normal Na+o, respectively (significantly
different from each other, student's t-test, P<0.05).
When the rapid component (Fig.
3) was deducted from the total Cu accumulation over 15 min
(Fig. 4A), the low
Na+o Cu accumulation response showed an exponential rise
with Cuo (exponential fit to means,
y=0.0664+1.0045x, r2=0.99;
Fig. 4B), unlike the saturable
profile in the presence of Na+o when the rapid Cu
accumulation was deducted for net accumulation over 15 min
(Fig. 4B).
Effect of epithelial Na+ channel blocking agents on Cu accumulation
Pre-incubation of intestinal cells with epithelial Na+ channel
(ENaC) inhibitors (normal Na+o throughout) generally
caused increased rates of Cu accumulation by the cells compared to drug-free
controls at the appropriate Cuo
(Fig. 5). The greatest effect
of ENaC inhibitors were observed at the highest Cuo (800 µmol
l1), with cell Cu accumulation rates over a 15 min period of
75.3±13.4, 39.4±16.5 and 21.8±5.1 nmol Cu
mg1 cell protein h1 (mean ±
S.E.M., N=56) for phenamil, CDPC and amiloride,
respectively, compared to the drug-free control above (1.88 nmol Cu
mg1 cell protein h1, significantly
different from control; see Fig.
5 for details of statistics). In
Fig. 5, rapid Cu accumulation
at time zero was not deducted from the net Cu accumulation over 15 min.
However, although changes in rapid Cu accumulation occurred at time zero,
these were not large enough (maximally only 6 nmol mg1 cell
protein h1 when Cuo=800 µmol
l1; Table 2)
to explain the observed increases in net Cu accumulation rate over 15 min (20
nmol mg1 cell protein h1 or more in the
presence of ENaC inhibitors; Fig.
5 and Table 2).
Thus the relative proportions of rapid Cu accumulation to total accumulation
over 15 min remained similar to that observed in normal or low NaCl
(Table 2) and to drug-free
controls (not shown).
Interestingly, in the absence of added Cuo, intestinal cells contained more Cu after incubation with ENaC inhibitors compared to drug-free controls (also with no added Cuo). Rates of Cu accumulation were 0.04±0.01, 1.07±0.03, 0.66±0.12, 1.04±0.12 nmol mg1 cell protein h1 (mean ± S.E.M., N=6) for control with no added drug, phenamil, CDPC and amiloride, respectively, in no added Cuo conditions (drug effect significantly different from control, KruskalWallis test, P<0.05), implying that ENaC inhibitors influence background Cu accumulation even when Cuo is only at trace nanomolar levels. LDH permeability remained below detection limits for experiments with ENaC inhibitors, including DMSO solvent controls for phenamil (not shown).
Copper accumulation at 4°C
Incubation of cells in an iced water bath caused a small increase in Cu
accumulation compared to room temperature controls. The rate of copper
accumulation over 15 min inice-cold conditions was 1.5 nmol Cu
mg1 cell protein h1 at Cuo=400
µmol l1 or less, compared to 1.2± 0.18 nmol Cu
mg1 cell protein h1 or less in room
temperature controls at the same Cuo (mean ±
S.E.M., N=6; not statistically significant, see
Fig. 5). At 800 µmol
l1 Cuo, cells in ice showed a sudden rise in Cu
accumulation from the background level of <1.5 nmol Cu
mg1 cell protein h1 to 9.5±4.7 nmol
Cu mg1 cell protein h1 (significantly
different; KruskalWallis test, P<0.05,
Fig. 5), indicating a threshold
for Cu entry in the cold. However, overall the effects of ice treatment were
small compared to the effects of ENaC inhibitors
(Fig. 5).
Effect of manipulating anion transport and external pH on Cu accumulation
Lowering external [chloride] (Clo) from 131.6
to 6.6 mmol l1 by replacing NaCl with sodium gluconate
caused Cu accumulation rates to increase progressively in intestinal cells
with increasing Cuo (Fig.
6). At the highest Cuo, the rate of Cu accumulation in
low Clo was 11-fold higher (significantly
different, KruskalWallis test, P=5
x106) than in normal NaCl conditions (21.6±2.4
in low Clo compared to 1.88 nmol Cu
mg1 cell protein h1 in normal NaCl, means
± S.E.M., N=6; see above). Similar observations
were made on application of 0.1 mmol l1 DIDS instead of
Clo removal, except the DIDS effect reached a
plateau at Cuo=200 µmol l1 or more. However,
lowering external pH from 7.4 to pH 5.5 produced the greatest increases in Cu
accumulation rate compared to Clo-removal or
addition of DIDS (Fig. 6). At
low pH, the Cu accumulation rate was 17-fold above the control when
Cuo=800 µmol l1 (reaching 32.6±3.7 nmol
Cu mg1 cell protein h1 at low pH, mean
± S.E.M., N=5). Cu accumulation rate at low pH also
reached a plateau above 400 µmol l1 Cuo. The
accumulation responses above cannot be explained by increased rapid Cu
accumulation by the cells at time zero (not deducted from
Fig. 6), which at the highest
Cuo were only 12%, 15% and 27% of the total accumulation for low
Clo, DIDS and low pH treatments, respectively
(Table 2).
Effect of Cuo on cell Na+ content with normal or low Na+o
Additions of Cuo had no statistically significant
Cu-dose-dependent effect on cell Na+ content when
Na+o was normal or low
(Fig. 7A), nor did a decrease
of Na+o from 140 to 11 mmol l1,
regardless of Cuo treatment, alter cell Na+ content
(Fig. 7A). However, a
Cuo-dependent depletion of cell Na+ was revealed when
cells were chilled to 4°C in the presence of normal NaCl
(Fig. 7B). In the cold, cells
showed about a fivefold increase in cell Na+ content compared to
cells at room temperature (statistically significant at all Cuo
tested; student's t-test, P<0.05). Cell Na+
content also declined at Cuo of 400 µmol l1
and or more compared to no added Cu controls at 4°C, indicating a net
Na+ loss from cells at high Cuo in the cold.
Effect of epithelial Na+ channel blocking agents on cell Na+ and K+ content
Pre-incubation of intestinal cells with ENaC inhibitors followed by
Cuo exposure caused statistically significant depletions of cell
Na+ content compared to drug-free controls in the presence of
Cuo (Fig. 8A), with
amiloride and CDPC reducing cell Na+ content by ninefold or more.
This response was slightly different in the absence of added Cuo
(with or without EDTA), so that only amiloride produced a statistically
significant reduction in cell Na+ content
(Fig. 8A). However,
Cuo modulated the effectiveness of ENaC inhibitors at reducing cell
Na+ content, with all drugs being more effective with increasing
Cuo (e.g. with amiloride, cell Na+ showed an exponential
decrease with increasing Cuo). The biggest reductions in cell
Na+ content were observed at 800 µmol l1
Cuo, and of the ENaC blocking agents used, the effect of amiloride
was enhanced the most by the presence of Cu compared to drug-containing
controls without Cu (Fig.
9A).
Pre-incubation of intestinal cells with ENaC inhibitors followed by Cuo exposure caused statistically significant increases in cell K+ content compared to controls in normal saline (drug-free) at the same Cuo (Fig. 8B). No effect of ENaC inhibitors on cell K+ content was observed in the absence of Cu (Fig. 8B, KruskalWallis test, P>0.05), and there was no Cuo-dependent modulation on cell K+ content within drug treatments (Fig. 9B).
Effect of manipulating anion transport and external pH on cell Na+ and K+ content during Cuo exposure
Exposure to Cuo had no dose-dependent effect on either cell
K+ or Na+ content in low
Clo conditions
(Fig. 10A,
KruskalWallis for Na+, P=0.058; for K+,
P=0.59), except for an increase in cell [Na+] at 50
µmol l1 Cuo and a decrease in cell
[Na+] at 800 µmol l1, compared to the control
with no added Cu (Fig. 10A).
Removal of Clo caused cell Na+ content
to generally decline compared to controls in normal NaCl, regardless of Cu
exposure, but the differences were not statistically significant
(KruskalWallis test, P>0.05, compare
Fig. 10A with normal
Na+ in Fig. 7A).
Treatment with 0.1 mmol l1 DIDS caused a clear Cuo-dose-dependent decline in both cell [Na+] and [K+] (Fig. 10B; KruskalWallis test for Na+, P=1.8 x109; for K+, P=3.5 x106), with electrolyte levels decreasing at Cuo=10 µmol l1 or more. Incubation of cells with DIDS in the absence of Cuo also increased cell Na+ and K+ content compared to either the low Clo or normal NaCl treatments without Cuo (KruskalWallis test, P<0.05), suggesting effects of DIDS on cell electrolytes regardless of Cuo exposure.
Reducing external pH from 7.4 to 5.5 caused no Cuo-dependent effect on cell K+ content (KruskalWallis test, P=0.166), but did cause a Cuo-dependent fall in cell [Na+] (Fig. 10C; KruskalWallis test, P=0.0038). The latter effect occurred at Cuo=200 µmol l1 or more compared to the control with no added Cu. Acidification of the medium also tended to increase cell Na+ compared to normal pH in the absence of added Cuo (compare controls in Fig. 10C with Fig. 7A), but these were not statistically different (KruskalWallis test, P>0.05).
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Discussion |
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Copper accumulation with normal Na+o
Copper accumulation by isolated intestinal cells in normal physiological
saline was Cuo-dose dependent, with saturable and non-saturable
components (Fig. 4), and we
argue that this is indicative of at least two Cu accumulation processes
including a slower carrier-mediated Cu accumulation (saturable), and a faster
(non-saturable) diffusive process. Saturable Cu accumulation has been
demonstrated in cultured human intestinal cells (Caco-2 cells;
Arredondo et al., 2000), mouse
embryo cells (Lee et al.,
2002b
), jejunum segments of rat intestine
(Linder, 1991
), across
perfused catfish intestine (Handy et al.,
2000
); and is suggested in trout gut in vivo
(Clearwater et al., 2000
). The
Cu accumulation response here showed a curvilinear rise
(Fig. 4A), and when the rapid
Cu accumulation component (Fig.
3) was deducted, a saturable accumulation profile was revealed
(Fig. 4B). This is very similar
to rat intestine, where deduction of a linear component (assumed to be
diffusion by Linder, 1991
),
revealed a saturable Cu-uptake curve
(Linder, 1991
). The saturable
component in this study (Fig.
4B) probably involves a carrier-mediated process (as in mammals;
Linder, 1991
;
Arredondo et al., 2000
) and
cannot be explained by net Cu accumulation being a passive equilibrium between
diffuse influx and diffusive efflux. Electrochemical theory predicts that
passive diffusive efflux of Cu is impossible in our experimental conditions
(e.g. if intracellular free [Cu2+] is 10 nmol l1
or less, when Cuo=10 µmol l1, the equilibrium
potential = +90 V or more, membrane potential 70 mV). This is further
supported by the fact that exposure of intestines to micromolar concentrations
of Cu has no effect on transepithelial potential
(Handy et al., 2000
), and
trout intestinal mucosa shows no decrease in newly acquired Cu content 4 h
after exposure (Clearwater et al.,
2000
). Arredondo et al.
(2000
) argues the rapid
component is fast Cu uptake into the cells (see below) and the slow saturable
Cu accumulation component involves Cu storage by active uptake from the
cytoplasm into the Golgi network and/or cytosolic Cu buffering. The
observations on trout in the present work are also consistent with this
idea.
There may also be some species differences in Cu accumulation rates between
fish and mammalian intestinal cells. Cu accumulation rates measured in the
present work were around 0.053.4 nmol mg1 cell
protein h1 (Fig.
4A), which is higher than those measured in adherent Caco-2 cells
(2090 pmol mg1 cell protein h1,
Arredondo et al., 2000) or
isolated segments of rat intestine (72 pmol mg1 cell protein
h1, Linder
1991
), but are similar to rates measured in isolated fish
intestine (assuming 100 mg protein g1 gut tissue, 827
nmol mg1 cell protein h1;
Handy et al., 2000
). The
apparent Km was also higher in the present study (216
µmol l1 Cuo;
Fig. 4B) compared to Caco-2
cells (0.3 µmol l1;
Arredondo et al., 2000
) or rat
intestine (5 µmol l1;
Linder 1991
). These
differences may partly reflect species differences in Cu homeostasis, where
trout regulate plasma [Cu] to levels about tenfold higher than rats (compare
Clearwater et al., 2000
;
Linder, 1991
).
Is the rapid Cu accumulation component fast uptake into the cells or surface adsorption of Cu?
Some of the `time zero' control experiments in this study
(Fig. 3, Table 2) revealed a rapid
component of Cu accumulation that occurred in less than 1 min. This component
was probably very rapid Cu uptake into the cells, rather than just surface
binding (adsorption) of Cu on to the outside of the cells
(Handy et al., 2002). The
characteristics of the rapid component include sensitivity to ENaC inhibitors,
DIDS and ice-cold chilling (Table
2), and are best explained by rapid changes in the rate of Cu
transport rather than adsorption chemistry. Cells were also washed several
times in 0.1 µmol l1 EDTA, and this would chelate/wash
off some of the surface-bound Cu2+. Furthermore, the Cu content of
cells in Cu-free conditions (no added Cu + 1 µmol l1
EDTA) were the same as in conditions of no added Cu (saline without EDTA) in
all experiments (Figs 4,
5,
6), suggesting that
surface-bound Cu was a small component relative to the total background Cu
content of the cells (albeit after washing the cells in EDTA). Although we
cannot completely exclude some instantaneous surface binding of
Cu2+ to the cells in our experiments, it is probably fortuitous
that the rapid Cu accumulation component in normal saline (12% of the total,
Table 2) is similar to that for
apparent Cu binding to trout gills at the tissue Cu levels found here
(MacRae et al., 1999
).
Does mucosal Cu entry control Cu accumulation by intestinal cells?
In our experimental conditions, passive diffusion of Cu from the cells into
the external medium is thermodynamically impossible, and intestinal cells are
not known to secrete Cu across the mucosal membrane into the gut lumen by
active transport (Linder,
1991; Arredondo et al.,
2000
). Therefore, Cu accumulation by intestinal cells must be
controlled by passive Cu influx, and active Cu efflux across the serosal
membrane (Arredondo et al.,
2000
). In cell suspensions there is a thermodynamic possibility of
passive Cu influx across the serosal membrane into the cells. However this is
unlikely, because the putative ion channels involved in passive Cu entry into
the cells (e.g. ENaC, Ctr1, see below) are mainly localised on the mucosal
membrane (Staub et al., 1992
;
Bauerly et al., 2004
). Thus
there is no obvious pathway for passive Cu leak into intestinal cells across
the serosal membrane. Indeed, the absence of such a pathway partly contributes
to Cu overload diseases because excess Cu cannot be excreted via the
intestine to the gut lumen (e.g. Wilson's disease;
Menkes, 1999
). Copper
accumulation in our cells is therefore the sum of Cu influx across the mucosal
membrane and active efflux across the serosal membrane.
We believe that Cu accumulation by isolated trout cells is mainly
controlled by influx across the mucosal membrane into the cell, as in perfused
catfish intestine (Handy et al.,
2000), and in mammalian gut cells
(Arredondo et al., 2000
). If
active Cu efflux controlled cell Cu accumulation, then lowering metabolism by
chilling should slow active efflux and cause a large increase in cell Cu
content. This was not observed (Fig.
5). Chilling cells in an iced water bath
(Fig. 5) had no effect on cell
Cu accumulation, except for a small increase in Cu accumulation at the highest
Cuo compared to cells at room temperature. Furthermore Cu-loaded
cells that are placed in normal saline should show rapid Cu depletion if
efflux controls cell Cu content, but this was not observed over the 15 min
period in the present work (Fig.
2B), or in mammalian intestinal cells
(Zerounian et al., 2003
).
Zerounian and Linder (2002
)
also argue that Cu is tightly bound to intracellular chaperones and cannot be
simply dialysed from ligands to facilitate passive efflux. Instead, we suggest
that passive Cu entry into the cell down the electrochemical gradient is more
important in controlling cell Cu content. Arredondo et al.
(2000
) made similar
observations in Caco-2 cells using 64Cu, where 70% or more of the
cell Cu content was controlled by flux across the mucosal membrane in cells
equilibrated with 1 mmol l1 Cu or less. Copper accumulation
by mouse embryo cells also continued in ice-cold conditions (passive influx
conditions), as measured by net influx of 64Cu
(Lee et al., 2002b
).
Is Na+o-sensitive Cu accumulation through epithelial Na+ channels?
The main candidate pathways for Cu entry into intestinal cells across the
mucosal (apical) membrane are incidental Cu entry through ENaCs or Cu influx
through the Cu-specific pathway encoded by Ctr1 (for a review, see
Handy et al., 2002). The
available evidence suggests that diffusive Cu entry through ENaCs is unlikely
at the Na+o concentrations found in the intestine (+100
mmol l1 NaCl, Handy et
al., 2002
) or frog skin (short circuit conditions;
Flonta et al., 1998
), although
Cu entry into epithelial cells through ENaCs is more likely at much lower
Na+o (around 1 mmol l1, e.g.
freshwater fish gills, Grosell and Wood,
2002
; frogs in freshwater,
Handy et al., 2002
). In the
present study our data support the notion of Cu accumulation by a pathway(s)
that is characteristic of Ctr1, with some Cu entry through ENaCs only when
Cuo is high (800 µmol l1) in combination with
low Na+o.
Cu uptake in competition with Na+ through ENaCs requires
increased Cu accumulation by the cell on removal of
Na+o, and at least slowed Cu accumulation in the
presence of ENaC inhibitors. This is not the case in isolated intestinal cells
(Figs 4 and
5). Lowering
Na+o to 11 mmol l1 generally did not
increase Cu accumulation rates (Fig.
4A), and even after deduction of the rapid Cu-component
(Fig. 3), a non-saturable
Cuo-dependent and mainly Na+o-insensitive
curvilinear (diffusive) component remained. The latter
Na+o-insensitive component to accumulation was not
significantly different from Cu accumulation rates in normal NaCl over most of
the Cuo range (Fig.
4B). Removal of Na+o also slows Cu uptake in
intact intestine (fish, Handy et al.,
2002; rats, Wapnir and Stiel,
1987
), an observation that also excludes Cu uptake through ENaCs.
However, reducing Na+o does stimulate Cu accumulation
when Cuo is also high (800 µmol l1
Cuo; Fig. 4A),
implying some Cu entry through EnaCs, but only when Cuo is above
normal (nutritional range equates to 200400 µmol
l1 Cuo,
Watanabe et al., 1997
;
Clearwater et al., 2002
) and
in combination with abnormally low Na+o
(non-physiological).
The rapid Cu accumulation component was
Na+o-dependent (Fig.
3), and this is consistent with the similar charge densities and
ionic mobilities of hydrated Cu2+ and Na+ ions that
ensure close competition of these ions for binding on cell membranes
(Handy et al., 2002). The
possibility that the instantaneous component also includes some rapid Cu
uptake through ENaCs is unlikely, since Flonta et al. (1988) showed that Cu
binds externally on the channel rather than going through it. However, we
cannot exclude rapid Cu uptake through Cu specific channels (Ctr1, see
below).
Does Cu accumulation fit the characteristics of Ctr1?
Our data show that Cu accumulation by trout cells over most of the
Cuo range used here has several of the key characteristics of Ctr1;
including Nao+-independence and stimulation by low pH.
This supports the notion of Cu uptake through Ctr1 rather than ENaC when
Cuo is 400 µmol l1 or less. In mammals, Cu
flux through the pathway encoded by Ctr1 is not
Na+o-dependent (Lee
et al., 2001; Lee et al.,
2002a
). Ctr1 is not affected by the removal of
Na+o or elevation of intracellular Na+
via ouabain addition (human Ctr1 in transfected Hek293 cells,
Lee et al., 2002a
). In the
present study, Cu accumulation is also independent of
Na+o over most of the Cuo range used (except
at 800 µmol l1 Cuo,
Fig. 4B). The low
Na+o used here (11 mmol l1, not
complete Na+o-removal) did not alter cell Na+
content (Fig. 7A). Even the ice
treatment, which caused large increases in cell Na+ content
(Fig. 7B), did not alter Cu
accumulation rates, except for a small increase in Cu accumulation rate at 800
µmol l1 Cuo
(Fig. 5).
Cu flux through Ctr1 is increased by the depolarising effects of high
K+o, and is strongly increased by acidification of the
external medium (Lee et al.,
2002a). Trout intestinal cells show both of these properties. The
incidental elevation of cell K+ associated with ENaC inhibitors in
the presence of Cuo (Fig.
8B) would aid depolarisation and Cu entry through Ctr1.
Importantly, acidification of the external medium to pH 5.5 enhanced Cu
accumulation up to 17-fold over that at normal pH
(Fig. 6). This effect occurred
without significant changes in cell Na+ or K+ below
Cuo=200 µmol l1
(Fig. 10C), and is
characteristic of Ctr1 (Lee et al.,
2002a
).
At Cuo >200 µmol l1, there is a big
increase in pH-dependent Cu accumulation
(Fig. 6) that coincides with a
reduction in cell Na+ content
(Fig. 10C). A fall in cell
Na+ content in conditions of low pH is not evidence against Ctr1,
but reveals some combined effects of low pH and Cu exposure on Na+
transporters. The decrease in cell Na+ content at low external pH
in the presence of high Cuo can be explained by: (i) reduced
competitive binding of external Na+ to the cell in the presence of
external Cu2+ with H+
(Handy and Eddy, 1991;
Handy et al., 2002
), and (ii)
reversal of the Na+/H+ exchanger during external
acidification (Ellis and Macleod,
1985
), which is stimulated by Cuo
(Kramhøft et al.,
1988
). Cu-dependent inhibition of
Na+K+-ATPase (Li et
al., 1996
) would normally raise intracellular [Na+],
but may not apply to uncoupled Na+K+-ATPase in acid
conditions (Skou, 1983
),
suggesting the Na+/H+ exchanger is more critical for
controlling cell Na+ content in conditions of high Cuo
and low external pH.
Alternative hypotheses to pHo-dependent Cu accumulation
via Ctr1, such as Cu uptake on other pH-sensitive divalent ion
transporters, or increased Cu entry into the cell as an artefact of Cu
speciation at low pH, are excluded. At the low pH used here, free
Cu2+ in the medium will only increase by 35%
(Sylva, 1976), and this is
insufficient to explain the 17-fold increase in Cu accumulation observed
(Fig. 6). Divalent metal ion
transporter 1 (DMT1) is stimulated by low external pH and will transport Cu in
oocytes expressing DMT1 (Gunshin et al.,
1997
). However, iron-deficient trout do not suffer Cu-overload and
show less than a twofold increase in intestinal Cu content during 8 weeks of
iron deficiency (Carriquiriborde et al.,
2004
). This suggests that Cu leak through DMT1 is not a major
factor in Cu accumulation by trout intestinal cells. Lee et al.
(2002b
) present a similar
argument for mouse embryo cells, and DMT1-deleted mutant mice do not show Cu
deficiency (Conrad et al.,
2000
).
Why do ENaC inhibitors increase Cu accumulation in isolated cells?
Our data (Fig. 4A) suggest
that Cu accumulation is mainly Na+o-insensitive at the
millimolar Na+o range used here, and is consistent with
previous reports on other epithelia at millimolar Na+o
(Handy et al., 2002). This
also suggests that ENaC inhibitors are unlikely to slow Cu accumulation, and
this notion is consistent with observations here on isolated cells, and
previous reports on gill and intestine. For example in trout gills, when
Na+o is below 200 µmol l1, Cu
uptake is inhibited by apical additions of 100 µmol l1
phenamil (Grosell and Wood,
2002
), an ENaC inhibitor
(Garvin et al., 1985
;
Reid et al., 2003
), or 2
µmol l1 bafilomycin, and can be explained by Cu entry
through a Na+ channel coupled to H+-ATPase
(Grosell and Wood, 2002
).
However, Grosell and Wood
(2002
) also identified a
Na+-insensitive Cu uptake pathway (possibly Ctr1), which dominates
when Na+o is >200 µmol l1. In
gut tissue where Na+o is much higher than in the gill,
we also see a Na+o-insensitive component (59% of Cu
accumulation rate when Cuo=400 µmol l1;
Fig. 4B), which is similar to
that in catfish intestine (59%; Handy et
al., 2002
). The Na+o-insensitive component
also dominates Cu uptake in rat intestine (85%;
Wapnir, 1991
). It would
therefore seem unlikely that ENaC inhibitors would decrease Cu uptake very
much in intestine (<20% in rat intestine,
Wapnir, 1991
; <50% in
catfish intestine; Handy et al.,
2002
).
The known pharmacology of ENaC inhibitors in the presence of Cuo
also excludes Cu entry through ENaCs, whilst still allowing cell
Na+ depletion due to drug-dependent blockade of Na+
uptake. Our data are consistent with this pharmacology, but also suggest that
ENaC inhibitors may create the conditions (depolarisation, altered pH gradient
across the cell membrane) that stimulate Cu uptake via a Ctr1-like
pathway. In the present study, there was a large increase in Cu accumulation
rate following incubation with ENaC inhibitors
(Fig. 5), which was not
consistent with blockade of Cu entry in to the cell through ENaCs by ENaC
inhibitors. Flonta et al.
(1998) demonstrated atypical
pharmacology of amiloride in the presence of Cuo, which resulted in
20% closure of ENaC. If the channel is closed, then it cannot be the pathway
for Cu entry into the cell in the presence of inhibitors. The ENaC inhibitors
used here were active because Cuo-dependent reductions in cell
Na+ content were observed (Fig.
8). Furthermore, the additional amiloride effect on cell
Na+ content in the presence of Cuo
(Fig. 9) was consistent with
the voltagecurrent recording made by Flonta et al.
(1998
) on the frog skin ENaC,
which showed that the Na+ current was inhibited more by amiloride
in the presence of Cuo.
In this study, the effect of ENaC inhibitors
(Fig. 5) are more easily
explained by indirect changes of intracellular pH and/or membrane potential
that would stimulate Ctr1-like Cu accumulation
(Lee et al., 2002a).
Amiloride, in addition to blocking ENaCs, also inhibits the
Na+/H+ exchanger (Ki=340
µmol l1 amiloride in colonic vesicles;
Dudeja et al., 1994
). This
would partly explain the fall in cell Na+ content in amiloride
treatment during Cuo exposure
(Fig. 8), but would also cause
intracellular acidification (the acidifying effects of Cu and amiloride are
additive; Kramhøft et al.,
1988
). A fall of intracellular pH would stimulate Cu flux through
Ctr1 (Lee et al., 2002a
),
resulting in the observed rise in cell Cu accumulation rate
(Fig. 5).
In trout gills, phenamil probably blocks ENaCs that are coupled to apical
H+-ATPase (Bury and Wood,
1999; Reid et al.,
2003
). However, in the intestine this is unlikely as
phenamil-sensitive ENaCs may not be coupled to H+-ATPase (no
blockade of apparent Na+/H+ activity,
Dudeja et al., 1994
;
Abdulnour-Nakhoul et al.,
1999
). Alternatively, phenamil inhibits inward rectifying
K+ channels (Guia et al.,
1996
) and depolarises the mucosal membrane of intestinal cells
(Sellin et al., 1993
). These
effects would cause Cu influx through a Ctr1-like pathway
(Lee et al., 2002a
) and cell
Cu accumulation (Fig. 5),
without depleting cell Na+ (Fig.
8A). Closure of K+ channels would also contribute to
the observed rise in cell K+ content in the presence of ENaC
inhibitors combined with high Cuo
(Fig. 8B).
CDPC is electroneutral at physiological pH and has a different binding site
on ENaCs to amiloride, and therefore unlike amiloride, does not compete
directly with Cu for binding (Fig.
9; Flonta et al.,
1998) to the extracellular loop of the alpha subunit of ENaC
(Li et al., 1995
). CDPC
therefore behaves more like phenamil in the presence of Cuo and
does not produce Cu-dependent depletion of cell Na+
(Fig. 9). CDPC reduces short
circuit current (Isc) in Ussing preparations (frog skin,
Flonta et al., 1998
; A6 cells,
Atia et al., 2002
) and makes
the transepithelial potential of catfish intestine slightly more positive (R.
D. Handy and H. Baines, unpublished observations), so might also depolarise a
Ctr1-like pathway.
How does Low Clo or DIDS treatment stimulate Cu accumulation?
In this study, reductions in Clo or additions
of 0.1 mmol l1 DIDS caused increases in Cu accumulation
rates (Fig. 6). These effects
can be partly explained by inhibition of Cu efflux across the serosal
membrane, and also by the likely acidifying effects of DIDS in the presence of
Cuo that would stimulate Ctr1-like activity (Cu influx) on the
mucosal membrane. The combination of reduced efflux and increased influx would
thus contribute to increased intracellular Cu accumulation. In fish intestine,
Cu export from intestinal cells to the blood across the serosal membrane
involves a CuCl symport and vesicular Cu trafficking
of Cu-loaded vesicles (Handy et al.,
2000). Both these processes are sensitive to Cl.
Lowering Clo would deplete intracellular
Cl and therefore inhibit Cu export on the
CuCl symporter
(Handy et al., 2000
). Failure
of Cu-loading into vesicles by Cu-ATPase at the Golgi complex is also possible
in low Clo conditions (Cl is an
allosteric effector of vesicular Cu loading in yeast;
Davis-Kaplan et al., 1998
). In
our experiments, the reduction of Clo in the
presence of Cuo did not deplete cell Na+ over most of
the Cuo range used (Fig.
10A), and so the effect of low Clo on
Cu accumulation rate is not an artefact of changes in Na+ balance.
DIDS is also known to inhibit anion-dependent divalent metal transport (Mg,
Bijvelds et al., 1996
; Cd,
Endo et al., 1998
), and
serosal application of DIDS causes tissue Cu accumulation in the perfused
catfish intestine (Handy et al.,
2000
). In the present study DIDS also caused Cu accumulation
(Fig. 6), and this observation
can be partly explained by blockade of the CuCl
symport.
DIDS also blocks a variety of other anion-transporters including
Cl channels (Bicho et
al., 1999), Cl/HCO3
exchanger (Ando, 1990
) and
Na+/HCO3 cotransporter, but not the
Na+K+2Cl cotransporter family
(Ramirez et al., 2000
). In
particular, the effect of DIDS on the
Na+/HCO3 cotransporter is likely to
cause intracellular acidification, and indirectly promote Ctr1-like activity.
DIDS blocks the electrogenic Na+/HCO3
cotransporter, which is ubiquitously expressed in vertebrate intestine
(Romero et al., 1997
), and
homologues are expressed in trout epithelia
(Perry et al., 2003
). In
rodents, the electrogenic Na+/HCO3
cotransporter is basolaterally located and protects the gut cell from acid
load by importing 1Na+ and 2HCO3
(Praetorius et al., 2001
;
Virkki et al., 2002
). Blockade
of basolateral Na+/HCO3 would cause
cell Na+ content to fall (Fig.
10), and Cuo-dependent acidification of the cell,
resulting in the observed DIDS-dependent Cu accumulation
(Fig. 6). DIDS block of apical
Cl/HCO3 to cause recovery of
intracellular pH can be excluded in our experiments because the anion
exchanger is only active during base secretion
(Grosell and Jensen, 1999
).
The depolarising effects of low Clo or DIDS on
the cell membrane (Bicho et al.,
1999
) might also directly contribute to stimulation of Ctr1-like
activity, and therefore Cu accumulation. However, this is a less likely
mechanism in the presence of both Cuo and DIDS because cell
[K+] did not rise in these conditions
(Fig. 10B).
In conclusion, isolated intestinal cells from rainbow trout show saturable Cu accumulation that is mainly controlled by passive Cu entry to the cell, as in mouse embryo and Caco-2 cells. The Cu accumulation is mostly (two thirds) Na+-insensitive and is not easily explained by Cu uptake through ENaC. Cu accumulation is more characteristic of a pH and K+-sensitive Ctr1-like pathway, where the known indirect effects of ENaC inhibitors and external acidification are also consistent with Ctr1 activation. Increased Na+-sensitive Cu accumulation can occur at high (non-nutritional) Cuo levels.
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Acknowledgments |
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