Copper uptake across rainbow trout gills : mechanisms of apical entry
McMaster University, Department of Biology, 1280 Main Street West,
Hamilton, Ontario, Canada L8S 4K1
*
Present address: Zoophysiological Laboratory, August Krogh Institute,
University of Copenhagen, Universitetsparken 13, DK-2100 Copenhagen Ø,
Denmark
Accepted 25 January 2002
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Summary |
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Key words: copper, homeostasis, sodium uptake, copper/sodium interactions, gill, rainbow trout, Oncorhynchus mykiss
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Introduction |
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At the organ level, fish resemble higher vertebrates with respect to copper
homeostasis. As in mammals, the liver is the major homeostatic organ, and
biliary copper excretion is elevated in situations of elevated copper uptake
(Grosell et al.,
1998a,b
,
2001b
;
Kamunde et al., 2001
).
Circulating levels of copper in the plasma are under tight regulation (Grosell
et al., 1997
,
2001b
), and copper derived
from recent uptake from the water is associated predominantly with a 70 kDa
protein (presumably albumin) (Grosell,
1996
), as in mammals (Cousins,
1985
; Frieden,
1980
; Weiner and Cousins,
1983
). Renal loss of copper from fish and mammals is low
(Grosell et al., 1998b
) and
does not appear to be stimulated under conditions of copper excess.
While part of the copper requirements in fish is clearly met by dietary
intake, the role of the gills in copper homeostasis has been overlooked until
recently. However, it has long been known from toxicological studies that
elevated copper concentrations in water can lead to increased copper levels,
particularly in the gills and the liver
(Buckley et al., 1982; Grosell
et al., 1996
,
1997
,
1998a
,b
;
Laurén and McDonald,
1987a
,b
;
McCarter and Roch, 1984
),
suggesting that the gills can serve as a route of copper uptake. In the light
of the high volume of water passing through the gills (18 l kg-1
h-1) (Wood and Jackson,
1980
), even the low ambient copper concentrations normally present
in fresh water (8-80 nmol l-1=0.5-5.0 µg l-1)
(Spry et al., 1981
) offer a
potential source for normal copper assimilation by the gills. A recent study
revealed that significant copper uptake occurs across the gills under normal
conditions and that as much as 60 % of the whole-body copper requirement in
fast-growing juvenile rainbow trout fed a copper-deficient diet can be
obtained directly from the water across the gills
(Kamunde et al., 2002
).
Recent findings of regulated copper transport across the branchial
epithelium suggest the involvement of more specific copper transport proteins.
For example, in rainbow trout Oncorhynchus mykiss, copper uptake
across the gills was reduced by exposure to elevated ambient copper
concentrations (Grosell et al.,
1997). Furthermore, Kamunde et al.
(2001
,
2002
) reported reduced
branchial copper uptake in rainbow trout fed a copper-rich diet and increased
branchial copper uptake in trout fed a copper-deficient diet. In addition,
copper deficiency in juvenile rainbow trout could be induced only when copper
levels in both the diet and the water were reduced. Fish held in water with
normal copper levels and fed a copper-deficient diet maintained normal growth
through the increased branchial copper uptake rate
(Kamunde et al., 2002
).
Branchial copper uptake thus appears to be modulated depending on the copper
status of the fish, strongly suggesting the involvement of specific, regulated
transport pathways.
The main objective of the present study was to characterize the branchial
copper uptake pathways in rainbow trout. Our initial hypothesis was that
copper shares transport pathways with sodium since copper, at higher
concentrations, impairs branchial sodium uptake, a vital part of freshwater
osmoregulation (Laurén and McDonald,
1985,
1987a
,b
).
Furthermore, recent evidence indicates that silver, which is known to serve as
an analogue of copper in certain specific transport pathways (e.g.
Solioz and Odermatt, 1995
), is
taken up via the apical sodium pathway in rainbow trout
(Bury and Wood, 1999
). This
sodium pathway is via an apical, H+-ATPase-coupled,
Na+ channel (Fenwick et al.,
1999
) and a basolateral Na+/K+-ATPase
extruding sodium from the gill epithelial cells to the blood plasma (for a
review, see Wood, 2001
). We
tested this hypothesis by cation competition studies and pharmacological
manipulation of the apical sodium entry step. A detailed analysis of copper
and sodium uptake kinetics and their interactions at environmentally realistic
copper levels was performed to test whether multiple copper uptake pathways
exist. Because of the essential nature of copper, relatively high background
levels occur in most organs, making an isotopic approach necessary for
detailed studies of copper uptake at environmentally realistic copper
concentrations. In the present study, we employed the radioactive
64Cu isotope to trace copper uptake rates of the order of pmol
g-1 h-1 accurately.
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Materials and methods |
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Time course of copper accumulation
To establish the appropriate incubation time and sampling protocol for
copper influx studies in softwater-acclimated juvenile rainbow trout (mean
mass 4.44 g; range 2.12-10.40 g), copper accumulation as a function ambient
copper concentration was measured during 0.5, 1, 2, 4 and 8 h of
64Cu incubation at a range of copper concentrations (29±3,
30±2, 44±2, 88±2 and 145±5 nmol l-1,
means ± S.E.M., N=6 in all cases). The 64Cu was
obtained by radiation of CuNO3 (solid form) in the Nuclear Research
Reactor at McMaster University. After radiation, 64Cu was dissolved
in 0.5 % HNO3. For all the above concentrations, groups of eight
fish were placed in Pyrex beakers (1 l) and allowed to settle, after which
each of the beakers was spiked with 64Cu (specific activity at the
time of addition; 3.26 MBq µmol l-1) to yield each of the above
copper concentrations. The addition of small amounts of the highly
concentrated 64Cu stock solution had negligible effects on water
pH. After the isotope incubation period (see above), fish were netted out of
the exposure containers and placed in a rinsing solution containing 200
µmol l-1 non-radioactive CuSO4 and 0.1 g
l-1 MS-222, an anaesthetic that completely anaesthetized the fish.
This cold displacement rinse was performed to remove any non-specifically
surface-bound isotope from the gills and skin of the fish. After 1 min, the
fish were removed and the gills were obtained by dissection. The gills and the
rest of the body were then blotted dry, placed in pre-weighed plastic vials
and assayed for gamma radioactivity. On the basis of the results of this
experiment, all the following experiments employed 2 h incubation periods, and
copper uptake was measured by assaying the appearance of 64Cu in
the whole fish.
Cation competition studies
To test interactions between the major freshwater cations and copper
uptake, copper uptake rates were measured in separate experiments in the
presence of 0.05, 1, 2.5, 10 and 20 mmol l-1 Na+,
K+ and Ca2+ (all as chloride salts). Juvenile rainbow
trout (mean mass 0.52 g; range 0.19-1.46 g) were placed in 15 individual 100
ml Nalgene containers (eight fish per chamber) containing 50 ml of standard
soft water and one of the above concentrations of either Na+,
K+ or Ca2+. After a 1 h pre-incubation period,
64Cu was added to each container to reach a final concentration of
200 nmol l-1. After a 2 h isotope incubation period, fish were
netted out of the exposure containers and placed in a rinsing solution
containing 200 µmol l-1 non-radioactive CuSO4 and 0.1
g l-1 MS-222, after which each fish was blotted dry, placed in a
pre-weighed plastic vial and assayed for gamma radioactivity.
Pharmacological studies
To test the involvement of an H+-ATPase in branchial copper
uptake, juvenile rainbow trout (mean mass 0.18 g; range 0.09-0.29 g) were
placed in four 50 ml Nalgene containers (eight fish per chamber) each
containing 20 ml of aerated soft water. Bafilomycin A1 (2 µmol
l-1; Biomol, PA, USA), a specific H+-ATPase inhibitor,
dissolved in dimethylsulphoxide (DMSO) (final concentration 0.2 %) was added
to two of the four containers. The other two chambers contained only 0.2 %
DMSO and acted as controls. Very small fish were employed because of the cost
of the drug.
After a 1 h pre-incubation period, 64Cu was added to one bafilomycin-treated container and to the corresponding control container to reach a final concentration of 200 nmol l-1. The two remaining containers received 74 kBq l-1 22Na (Amersham, specific activity 11.21 MBq g-1 Na+). After a 2 h isotope incubation period, fish were netted out of the exposure containers and placed in a rinsing solution containing 200 µmol l-1 non-radioactive CuSO4 or 100 mmol l-1 non-radioactive NaCl for the 64Cu and 22Na experiments, respectively. Both rinsing solutions contained 0.1 g l-1 MS-222. Each fish was blotted dry, placed in a pre-weighed plastic vial and assayed for gamma radioactivity.
To test the involvement of the apical Na+ channel in branchial
copper uptake, a similar experiment was conducted using phenamil (RBI,
Sigma-Aldrich, Canada), an amiloride analogue that is an irreversible
Na+ channel inhibitor (Garvin
et al., 1985; Kleyman and
Cragoe, 1988
). Studies with amiloride analogues are complicated by
the fact that these compounds bind metals avidly (cf.
Bury and Wood, 1999
). However,
the irreversible nature of phenamil, unlike many other analogues, means that,
after a pre-incubation period, the compound can be removed from the bathing
solution and still be pharmacologically active. This approach avoids the
problem of the drug complexing copper in solution and inadvertently affecting
copper influx by this non-specific mechanism.
Juvenile rainbow trout (mean mass 0.80 g; range 0.26-1.49 g) were placed in four 100 ml Nalgene containers (eight fish per chamber) each containing 50 ml of aerated soft water. To two of these four containers, 100 µmol l-1 phenamil (RBI, Sigma-Aldrich, Canada), dissolved in DMSO (final concentration 0.1 %), was added. Again, the remaining two tanks contained only 0.1 % DMSO and acted as controls. Exploiting the fact that phenamil acts irreversibly on Na+ channels, fish were removed from the pre-incubation containers, quickly rinsed and placed in identical containers holding pure aerated soft water. Subsequently, 64Cu and 22Na incubations were performed. Performing the 64Cu exposure after removal of phenamil eliminated any potential problem with phenamil-64Cu complex formation possibly rendering copper unavailable for uptake.
Interactions between copper and sodium uptake: duallabelling
experiments
To investigate the interactions between copper uptake and the ambient
sodium concentration further, a detailed analysis of simultaneous copper and
sodium uptake was performed. For these experiments, the short half-life of the
64Cu isotope (12.7 h) was a clear advantage because it allowed for
a dual radio-labelling experimental approach (see below). Juvenile rainbow
trout (mean mass 4.23 g; range 1.66-9.91 g) were exposed to combinations of
copper concentrations (background, no added 64Cu; 18±1 and
29±3, 30±2, 44±2, 88±2 and 145±5 nmol
l-1) and sodium concentrations (10±1, 51±3,
96±2, 142±2, 217±2, 583±14 and 1059±12
µmol l-1; means ± S.E.M., N=6-7); the 42
combinations each contained eight fish per experimental group. For each of the
above sodium concentrations, six Pyrex beakers (11) containing aerated soft
water (prepared with a background sodium concentration of only 10 µmol
l-1) supplemented with the appropriate sodium concentration were
prepared. Of the six beakers with identical sodium concentrations, each was
spiked with 22Na (37 kBq) and a different concentration of
64Cu (specific activity at the time of addition 3.26 MBq
µmol-1) to yield each of the six target copper concentrations.
After a 2 h isotope incubation period, fish were netted out of the exposure
containers and placed in a rinsing solution containing 200 µmol
l-1 non-radioactive CuSO4, 1 mol l-1 NaCl and
0.1 g l-1 MS-222, after which each fish was blotted dry, placed in
a pre-weighed plastic vial and assayed for gamma radioactivity.
Analytical techniques and calculations
For all isotope flux experiments, an initial water sample (5 ml) was
obtained 10 min after isotope addition, and a final water sample (5 ml) was
obtained just before termination of the flux experiment. The water samples
were acidified with 1 % HNO3 (trace metal analysis grade; BHD
Chemicals). From the metal uptake kinetic studies, an additional set of water
samples was passed through a 45 µm filter (Acrodisc polyethersulphone
inline filters; Gelman) prior to acidification to reveal the amount of metal
truly in solution. Water copper concentrations were measured by graphite
atomizer, atomic absorption spectroscopy (Varian, SpectrAA220 with a SpectrAA
GTA110), while concentrations of other cations were measured by atomic
absorption spectroscopy (Varian Spectra AA220).
The geochemical speciation model MINEQL revealed that more than 97 % of the free copper in solution in the experimental water at all concentrations was present as Cu2+.
Radioactivity from 64Cu and 22Na in the water, gills and fish samples was determined using a Canberra Packard Minaxi auto gamma 5000 series gamma-counter. The 64Cu isotope was counted immediately after the experiments, because of its short half-life, with a window of 433-2000 ke V, and decay correction was performed automatically by an onboard program.
For the dual-labelling experiments (64Cu and 22Na), the sum of the two isotopes was counted immediately using the appropriate 22Na window, after which the 64Cu isotope was allowed to decay. After a minimum of 12 half-lives, equivalent to 99.95 % loss of the 64Cu isotope, the samples were recounted to reveal the activity of only the 22Na in each sample. By subtracting the final activity measured (22Na counts only) from the activity measured initially (64Cu+22Na), the 64Cu radioactivity at the time the samples were taken could be calculated. All 64Cu activities obtained in this manner were decay-corrected mathematically, to a common reference time, prior to uptake calculations (see below).
The average of the measured specific activity (cts min-1 nmol-1) of the appropriate isotope in the initial and final water sample served as the basis for the uptake rate calculations. In practice, these values usually differed by no more than 10 %. In these calculations, the activity recorded in each gill or fish, the mass of the gill or fish and the isotope incubation time elapsed were related to the average specific activity to yield uptake rates (nmol g-1 h-1) of copper and sodium.
Statistical analyses
Non-linear regression analyses of both copper and sodium uptake kinetics
were performed with Sigmaplot for Windows version 4.00. Statistically
significant differences between control groups and treated groups from the
pharmacological studies were evaluated by a two-tailed Student's
t-test. General cation competition studies and affinity constants for
sodium uptake at different copper concentrations were evaluated by a
two-tailed Student's t-test with a Bonferroni multisample comparison
correction. A significance level of P<0.05 was employed
throughout.
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Results |
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Cation competition
Only sodium suppressed copper uptake across the gills of juvenile rainbow
trout in a statistically significant manner
(Fig. 2). Increasing the
ambient sodium concentration from 50 µmol l-1 to 1 mmol
l-1 reduced branchial copper uptake (at a concentration of 200 nmol
l-1) by more than 50%. Increasing the sodium concentration further
did not result in additional inhibition of copper uptake. Increasing potassium
(Fig. 2B) and calcium
(Fig. 2C) concentrations over
the same range had no significant effect on branchial copper uptake, although
there was a trend for inhibition by high potassium levels.
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Pharmacology of copper and sodium uptake
Both bafilomycin A1 (2µmol l-1) and phenamil (100 µmol
l-1) treatments were successful in inhibiting branchial sodium
uptake by 80% and 70%, respectively (Fig.
3). In addition, bafilomycin A1 and phenamil significantly reduced
branchial copper uptake by 68% and 39%, respectively
(Fig. 3). The different
absolute rates of copper and sodium uptake in the bafilomycin A1
versus the phenamil experiments reflects the very different sizes of
the fish used in the different trials. Smaller fish generally have higher
sodium uptake rates than larger fish. This is seen also in the present study,
where the controls in the bafilomycin A1 experiment with a mean mass of 0.18 g
exhibited sodium uptake rates of 950 nmol g-1 h-1
(Fig. 3A), which is
substantially higher than that of the controls in the phenamil experiment with
a mean mass of 0.80 g, which exhibited sodium uptake rates of 450 nmol
g-1 h-1 (Fig.
3B).
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Interactions between copper and sodium uptake
Increasing the ambient sodium concentration (from 10 to 1059 µmol
l-1) reduced branchial copper uptake considerably
(Fig. 4A-C). Copper uptake
rates at all copper concentrations exhibited a negative hyperbolic
relationship with ambient sodium concentration (illustrated for 145 nmol
l-1 copper in the inset of Fig.
4B, r2=0.90, P<0.05). The sodium
concentration required for a 50% reduction in the rate of branchial copper
uptake (IC50) was 103.8±9.9 µmol l-1 (mean
± S.E.M., N=7) The copper uptake remaining at high sodium
concentrations was determined as the constant b in the hyperbolic
relationship, y=ax/(b+x), where y
is the copper uptake rate and x is the ambient sodium concentration,
and is referred to as Jmin in the following. Plotting the
Jmin kinetic constant from these hyperbolic relationships
as a function of ambient copper concentrations revealed the
`sodium-insensitive' component to branchial copper uptake. This
sodium-insensitive copper uptake exhibits a high-affinity, low-capacity
saturation kinetic component (Jmax=3.5 pmol g-1
h-1; Km=9.6 nmol l-1;
r2=0.92, P<0.05) at lower copper
concentrations; at copper concentrations above 44 nmol l-1, there
is a linear relationship between copper uptake rates and ambient copper
concentration (r2=0.91, P<0.05)
(Fig. 4B).
|
Subtracting the sodium-insensitive copper uptake (Fig. 4B) from the total copper uptake (Fig. 4A) revealed the sodium-sensitive copper uptake component (Fig. 4C). At the lowest sodium concentration (10 µmol l-1), this sodium-sensitive component exhibits saturation kinetics, with a comparable high affinity to that of the sodium-insensitive uptake, but a sixfold higher capacity at copper concentrations below 88 nmol l-1 (Jmax=21.2 pmol g-1 h-1; Km=7.1 nmol l-1; r2=0.89, P<0.05). At the higher sodium concentrations, copper uptake via this sodium-sensitive pathway is essentially totally inhibited at concentrations below 88-145 nmol l-1 copper.
Sodium uptake in control fish exhibited saturation kinetics characterized by a maximum transport capacity (Jmax) of 683 nmol g-1 h-1 and an affinity (Km) of 69 µmol l-1 and was clearly affected by copper exposure (Fig. 5). Ambient copper concentrations at and above 29 nmol l-1 tended to increase apparent sodium transport capacity (Jmax), a trend that was significant at 30, 44, 88 and 145 nmol l-1 copper, and to reduce sodium affinity (i.e. increase Km) significantly at 30 and 88 nmol l-1 copper.
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Discussion |
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Both the sodium-sensitive and the sodium-insensitive components of
branchial copper uptake exhibit saturation kinetics at low, environmentally
realistic concentrations (see below), but also a linear component at
concentrations above 88 and 44 nmol l-1 copper, respectively. These
linear components could reflect non-specific uptake or binding to
sodium-sensitive and sodium-insensitive sites directly on the gill surface.
Notably, they extend into the concentration range that can be acutely toxic to
rainbow trout (for a review, see Wood,
2001). Copper binding to rainbow trout gills exceeding an apparent
saturable component has previously been reported for rainbow trout
(Taylor et al., 2000
).
Time course of copper uptake
The tendency for whole-body 64Cu levels to stabilize after 2-4 h
observed in the present experiment and the decrease in gill copper
accumulation over time, especially at higher ambient copper concentrations,
reinforces the view that branchial copper uptake is subject to homeostatic
regulation.
Stabilization of the gill 64Cu concentration over time must
reflect the establishment of an equilibrium between uptake and elimination
from the gill tissue. At the highest copper concentration, a biphasic
accumulation pattern was observed, with an initial increase followed by a
marked drop in branchial 64Cu levels even during continuous
exposure. This is in close agreement with previous reports of an initial peak
in branchial copper levels in rainbow trout during continuous exposure
(Grosell et al., 1997). One
possible interpretation is that the basolateral transport mechanism is
limiting and that, as copper builds up in the cytosol of the gill transport
cells, this accumulation may initiate a stimulation of extrusion mechanisms
such as the copper-specific P-type ATPases (see below). Alternatively, there
may be a reduction in the apical entry of copper. Regardless of its origin,
the mechanism would appear to serve as a protective response against elevated
copper levels in the gill tissue because it occurs only at higher copper
concentrations.
Sodium-sensitive copper uptake
Metal transport through the apical Na+ channel of rainbow trout
gills has been reported previously. Bury and Wood
(1999) demonstrated that silver
uptake across rainbow trout gills was sensitive to both phenamil and
bafilomycin A1 at the concentrations used here. Furthermore, silver uptake was
reduced in the presence of 500 µmol l-1 sodium, but was not
influenced by 200 µmol l-1 sodium. This indicates that the
IC50 of sodium against silver uptake is higher than the 104 µmol
l-1 sodium reported for sodium against copper uptake in the present
study (Fig. 4). In turn, this
suggests that the affinity of the Na+ channel for silver is higher
than for copper. In parallel, it is known that the affinity of the whole gill
surface for silver is more than two orders of magnitude higher than for copper
(Wood, 2001
). It appears from
the copper uptake kinetics of the sodium-insensitive pathway that it comprises
a high-affinity, low-capacity transport system. The sodium-sensitive copper
uptake has a similar high affinity but a much higher capacity. Depending on
the ambient copper concentration, the copper uptake rates via this
mechanism are 2-5 times greater than via the sodium-insensitive
copper uptake pathway (Fig. 4B
versus Fig. 4C).
Clearly, in low-sodium fresh water, typical of ion-poor softwater areas, this
will be the dominant mechanism of branchial copper uptake. Particularly
noteworthy for both mechanisms are their extremely high affinities (i.e. low
Km values of 7.1-9.6 nmol l-1), which means
that they will be functional at environmental copper concentrations in
non-contaminated environments (8-80 nmol l-1)
(Spry et al., 1981
). This
again points to an important role for the gill in normal copper
homeostasis.
Additional support for a link between sodium and copper transport is
provided by the recent work of Pyle and co-workers (G. G. Pyle, C. Kamunde, C.
M. Wood and D. G. McDonald, unpublished observations), who found that rainbow
trout fed a high-sodium diet for 1 week exhibited reduced unidirectional
uptake of both sodium and copper at the gills. This suggests that the
sodium-sensitive pathway may be modulated by internal sodium status, in
addition to external water sodium levels. A link between sodium and copper
transport has been largely overlooked in higher vertebrates, although it has
previously been suggested in rat intestine on the basis of
amiloride-sensitivity and sodium-sensitive tissue copper retention
(Wapnier, 1991). These
observations are in agreement with the present study suggesting a role for the
apical Na+ channel in copper uptake not only across fish gills but
possibly also across mammalian intestinal epithelia.
Effects of copper on branchial sodium transport
Sodium clearly inhibited copper uptake, and the reverse was also true
(Fig. 5). This is in parallel
to findings of an immediate partial inhibition of sodium uptake in rainbow
trout during exposure to toxic levels of silver
(Morgan et al., 1997) followed
by a greater inhibition at 8 h. It is also in agreement with findings of a
progressively developing inhibition of sodium uptake over 24 h in rainbow
trout exposed to elevated copper levels
(Laurén and McDonald,
1985
). The effect of both silver and copper on sodium uptake is
associated with inhibition of the basolateral
Na+/K+-ATPase (for a review, see
Wood, 2001
). Detailed
measurements of the parallel time course of inhibition of sodium uptake and
maximal Na+/K+-ATPase activity during copper or silver
exposure in rainbow trout gills have not, to our knowledge, been reported.
Clearly, these would be useful in determining whether all the inhibition can
be explained by a slowly developing blockade of the
Na+/K+-ATPase or whether there is an additional more
rapid effect on apical or intracellular mechanisms.
In the present study, which employed only 2 h of exposure, the inhibition
observed might reflect actions of copper on these latter components, rather
than on basolateral Na+/K+-ATPase activity. In support
of this suggestion are the different effects of copper on sodium transport
kinetics observed in the present study and in the study by Laurén and
McDonald (1987a). Both studies
found a reduced apparent affinity of branchial sodium transport (increased
Km); however, where the present study documents a somewhat
increased maximal transport capacity (Jmax) after only 2 h
of exposure, the study by Laurén and McDonald
(1987b
) reported a decreased
Jmax after 24 h, when Na+/K+-ATPase
activity was inhibited. This difference may mean that different components of
the branchial sodium transport pathway are affected by copper, depending on
the duration of the exposure. The increased Jmax observed
in the present study is in agreement with findings of stimulated
transepithelial short-circuit current and conductance in copper-exposed
isolated ventral frog skin, as reported by Flonta et al.
(1998
). A similar response was
reported for zinc uptake across the gills of rainbow trout as ambient calcium
concentration was increased (Spry and
Wood, 1989
); these two metals are also thought to share a common
apical uptake mechanism (for a review, see
Wood, 2001
).
The present study shows that copper may be transported by the apical
Na+ channel, and inhibitory interactions between sodium and copper
could thus occur at this channel. Another possible site of inhibition of
sodium transport is the branchial carbonic anhydrase. This enzyme provides the
substrate for the apical proton pump (from carbon dioxide and water), and
inhibition of carbonic anhydrase could thus reduce the activity of the proton
pump simply by depletion of substrate. Carbonic anhydrase readily binds copper
(Ditusa et al., 2001), and
copper-induced inhibition of branchial carbonic anhydrase has been reported in
the estuarine crab Chasmagnathus granulata
(Vitale et al., 1999
). It is
not known whether this also occurs in the freshwater fish gill, although
silver is effective in this regard (Morgan
et al., 1997
).
Sodium-insensitive copper transport
The saturable component of sodium-insensitive copper uptake suggests the
involvement of a specific carrier in addition to the apical Na+
channel. The recent identification of a group of high-affinity copper uptake
carriers, Ctr-type copper transporters, offer an appealing potential mechanism
for sodium-insensitive copper uptake across trout gills. This type of copper
transporter has been documented in phylogenetically distinct species such as
yeast (Dancis et al., 1994;
Kamphenkel et al., 1995
;
Knight et al., 1996
), mouse
(Lee et al., 2000
) and human
(Zhou and Gitschier, 1997
) and
is essential for normal development (Lee
et al., 2001
). Thus, it may well be present in teleost fish.
The mouse Ctr1 copper transporter exhibits an affinity constant of 1000-2000 nmol l-1 copper (J. Lee and D. Thiele, personal communication). The affinity constant of the sodium-insensitive copper uptake across trout gills in the present study was only 9.6 nmol l-1 (similar to that of the sodium-sensitive pathway) and is therefore very relevant to normal environmental copper levels in natural fresh water. However, this value is at least two orders of magnitude lower than that found for the murine Ctr1 copper transporter. Assuming that sodium-insensitive copper uptake across trout gills is mediated by a Ctr1-type transporter, this difference in apparent affinity could be explained by the different incubation media employed in the mouse Ctr1 transport study and the present study. Copper uptake kinetic measurements for the mouse Ctr1 transporter were performed in cell culture media that offer a large number of complexing agents, possibly rendering much of the copper unavailable for copper-specific transporters. In contrast, the present study was performed in ion-poor fresh water at slightly acidic pH, at which more than 90% of the copper occurs in ionic form. The difference in apparent affinity between the present study and the Ctr transporter study could simply reflect different chemical forms of copper present in the different media.
While the sodium-sensitive copper uptake pathway clearly dominates at low
ambient sodium concentrations, the sodium-insensitive copper uptake pathway
dominates at sodium concentrations above 200 µmol l-1. It
therefore probably plays the dominant role in most natural fresh waters, all
except those endemic to very ion-poor watersheds. Both the sodium-sensitive
and the sodium-insensitive copper uptakes exhibited saturation kinetics within
the range of concentrations employed in the present study. This observation is
in agreement with a previous report of copper uptake kinetics in juvenile
rainbow trout, employing similarly low copper concentrations, in which
saturation occurred at copper concentrations below 95 nmol l-1
(Kamunde et al., 2002). The
concentrations employed in the present study range from low environmentally
realistic concentrations (Spry et al.,
1981
) to levels that will probably cause toxic effects in ion-poor
water (for a review, see Wood,
2001
). At higher copper concentrations, which cause severe acute
toxicity, saturation kinetics for branchial copper uptake and binding by
rainbow trout has been observed (Campbell
et al., 1999
; Laurén and McDonald,
1987a
,b
;
Taylor et al., 2000
). The
interpretation of the saturation transport kinetics at concentrations of
copper that may cause acute toxicity is, however, complicated by potential
lamellar damage and gill surface mucification
(Wilson and Taylor, 1993
),
which may create an artificial condition of `apparent saturation'.
Possible site of regulation of branchial copper uptake
Branchial copper uptake across rainbow trout gills is regulated depending
on the copper status of the fish. This regulated copper uptake is likely to be
mediated by one or more specific copper carriers rather than by the apical
Na+ channel primarily involved in maintaining sodium homeostasis.
Sodium-insensitive copper uptake, possibly through an apical Ctr-type
transporter, is one potential regulated copper uptake pathway in rainbow trout
gills. As noted above, the time-dependent pattern of branchial copper
accumulation and the increasing relative branchial contribution to whole-body
copper accumulation with increasing ambient copper concentration during
isotope incubation (Fig. 1B,
inset) strongly suggest that the basolateral membrane is the rate-limiting
site in branchial copper uptake in rainbow trout. This makes the basolateral
membrane another likely candidate for a site of regulation of branchial copper
uptake. The extrusion of copper across the basolateral membrane in the
copper-assimilating intestinal epithelium in mammals is mediated by a
copper-specific P-type ATPase, the Menke's (MNK) Cu-ATPase
(Camakaris et al., 1995).
Regulation of this step of intestinal copper uptake occurs via
copper-sensitive MNK protein trafficking between the trans-Golgi
network and the basolateral membrane
(Petris et al., 1996
).
A similar mechanism of copper transport across the basolateral membrane
could be involved in regulated copper uptake by the rainbow trout gill, an
organ clearly involved in copper assimilation. Copper uptake across the gills
of rainbow trout is sensitive to vanadate, a P-type ATPase inhibitor, which
could indicate the involvement of a Cu2+-ATPase
(Campbell et al., 1999).
Furthermore, a putative Cu2+-ATPase from teleost fish gills has
been identified on the basis of 80% amino acid homology with mammalian MNK
proteins (Grosell et al.,
2001a
), supporting the possible involvement of a
Cu2+-ATPase in branchial copper assimilation. In further support of
this hypothesis are reports of ATP-dependent, vanadate-sensitive silver
transport across basolateral membrane vesicles from trout gill cells
(Bury et al., 1999
). These
observations support the presence of a Cu2+-ATPase in the
basolateral membrane because silver has been shown to act as a substrate for
bacterial Cu2+-ATPases and also because vanadate is a specific
inhibitor of P-type ATPases (Solioz and
Odermatt, 1995
).
Copper uptake through Na+ channels may make an important
contribution to copper homeostasis not only in fish but also in mammals, in
which some evidence for an interaction between sodium and copper uptake has
been demonstrated (Wapnier,
1991). Copper uptake across the gills offers an exciting area for
further studies of the mechanisms of regulation of copper homeostasis. Studies
of the potential involvement of a Ctr-type transporter and
Cu2+-ATPases in branchial copper uptake may provide information
about evolutionary aspects of copper homeostasis. While copper transport by
Ctr-type transporters in yeast is regulated at the transcriptional level on
the basis of copper levels (Labbé
et al., 1997
), mammalian Ctr1 mRNA is not sensitive to cellular
copper availability (Lee et al.,
2000
). Similarly, transport by Cu2+-ATPase is regulated
by trafficking between the trans-Golgi network and the plasma
membrane in mammals rather than by changes in gene expression. The
Cu2+-ATPase resides in the trans-Golgi network under
conditions of normal copper concentration, but it relocates to the plasma
membrane in the presence of excess copper
(Camakaris et al., 1999
). In
contrast, Cu2+-ATPase mRNA levels in bacteria are sensitive to
copper levels (Odermatt and Solioz,
1995
). The possible involvement of these copper transporters in
the regulation of branchial copper transport in fish offers a fruitful area
for further understanding of the mechanisms of regulated copper transport not
only in fish but also in higher vertebrates.
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References |
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