Mechanisms of ion transport in Potamotrygon, a stenohaline freshwater elasmobranch native to the ion-poor blackwaters of the Rio Negro
1 Laboratory of Ecophysiology and Molecular Evolution, National Institute
for Amazon Research (INPA), Alameda Cosme Ferreira, 1756-Aleixo, 69083-000
Manaus, Amazonas, Brazil
2 Department of Biology, McMaster University, 1280 Main St. West, Hamilton,
Ontario, Canada L8S 4K1
3 Department of Biology, University of San Diego, 5998 Alcala Park, San
Diego, CA 92110, USA
4 School of Biological Sciences, Hatherly Laboratories, University of
Exeter, Exeter EX4 4PS, UK
5 Department of Cell Biology and Neuroscience, University of California,
Riverside, CA 92521, USA
* Author for correspondence at address 2 (e-mail: woodcm{at}mcmail.cis.mcmaster.ca)
Accepted 3 July 2002
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Summary |
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Key words: Potamotrygonidae, freshwater elasmobranch, ion transport kinetics, Na+ flux, Cl- flux, Ca2+ flux, ammonia excretion, low pH, blackwater
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Introduction |
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The exact route and timing of evolutionary invasion from the ocean remains
controversial, either from the Pacific Ocean during the late Cretaceous 90-100
million years ago (Brooks et al.,
1981; Brooks, 1995
)
or from the northern coast of South America (i.e. Caribbean Atlantic) during
the early Miocene 15-23 million years ago
(Lovejoy, 1996
;
Lovejoy et al., 1998
).
Regardless of which scenario proves correct, it is apparent that the
Potamotrygonidae have experienced as long a period for evolutionary adaptation
to freshwater as many teleosts endemic to the same areas
(Lovejoy et al., 1998
). It is
therefore possible that they have evolved ionoregulatory strategies similar to
those of freshwater teleosts and different from those of their euryhaline
cousins, stingrays of the family Dasyatidae. Indeed, it is known that the
Potamotrygonidae are ammoniotelic rather than ureotelic
(Gerst and Thorson, 1977
;
Barcellos et al., 1997
) and
regulate their blood salts, osmolality and urea at levels similar to those of
teleosts (Thorson et al.,
1967
; Griffith et al.,
1973
; Gerst and Thorson,
1977
; Mangum et al.,
1978
; Bittner and Lang,
1980
) and at levels lower than those of freshwater-adapted
Dasyatidae (Thorson, 1967
;
Shuttleworth, 1988
;
Piermarini and Evans, 1998
).
However, little is known about the details of the mechanisms involved.
Some species of the Potamotrygonidae are endemic to the most extreme
freshwater environment of the continent, the acidic, ion-poor blackwaters of
the Rio Negro and its tributaries, so called because of their high content of
dissolved organic carbon (DOC) comprising humic, fulvic and other organic
acids derived from the breakdown of jungle vegetation. Recently, Gonzalez and
colleagues have begun to characterize ionoregulatory mechanisms in various
teleost species endemic to these blackwaters
(Gonzalez, 1996; Gonzalez et
al., 1997
,
1998
,
2002
;
Gonzalez and Preest, 1999
;
Gonzalez and Wilson, 2001
).
Two basic strategies have emerged in the Rio Negro teleosts. In one strategy,
as seen in many members of the Family Characidae, the presence of a
high-affinity (i.e. low Km), high-capacity Na+
transport system, which is relatively insensitive to inhibition by low pH,
compensates for high rates of diffusive loss. In the other, as seen in some
members of the Family Cichlidae, the transporter is sensitive to inhibition by
low pH, and affinity is much lower (i.e. higher Km),
although capacity may still be high. However, the key to adaptation in these
species is that diffusive loss rates are much lower. In both strategies,
diffusive loss rates are relatively resistant to stimulation by low pH, but
show variable patterns of change in response to alterations in external
Na+ concentration.
With this background in mind, the objectives of the present study were
several-fold. First, using radiotracers to measure unidirectional flux rates,
we described the basic concentration-dependent kinetics of both Na+
and Cl- uptake and efflux in a Potamotrygon species
collected from and tested in its native blackwater. This point is important
because Gonzalez et al. (2002)
concluded that transport mechanisms of Rio Negro teleosts bred in captivity
and/or held in other water qualities for long periods overseas by aquarists
may differ from those of native fish in natural blackwater. Second, we
examined whether these kinetic relationships changed when the freshwater rays
were acclimated to ion-rich hard water. Third, we evaluated the responses of
Na+, Cl- and Ca2+ transport and ammonia
excretion to acute short-term exposure to low pH (pH 4.0) in natural
blackwater. The only previous work on ammonia excretion in Rio Negro teleosts
indicated that it generally increases at low pH and does not appear to be
linked to Na+ uptake (Wilson,
1996
; Wilson et al.,
1999
). Lastly, we used a range of pharmacological inhibitors to
characterize the nature of the Na+, Cl- and ammonia
transport systems in stingrays acclimated to and tested in blackwater. To
date, little research has been done on Cl- or Ca2+
transport or on the pharmacological characterisation of any of the
transporters in Rio Negro fish.
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Materials and methods |
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After capture, most (16) of the rays were transported to the Laboratory of Ecophysiology and Molecular Evolution [Instituto Nacional de Pesquisas da Amazonia (INPA)], Manaus, Brazil, where they were held for 10 days in a recirculating, filtered tank (3001) in which the INPA wellwater (which is very soft) had been supplemented with NaCl and limestone chips, a common procedure used to reduce the stress of captivity. This provided us with an opportunity to examine the kinetics of both Na+ and Cl- uptake and efflux (see below) in this species after acclimation to ion-rich hard water (composition in Table 1). After completion of tests at INPA, the fish were transported back to the Anavilhanas Archipelago on board the research vessel Amanai II. The research vessel was moored in an embayment close to the Scientific Base of the Anavilhanas Archipelago of the Instituto Brasiliero do Meio Ambiente e dos Recursos Naturais Renovaveis. During this time, the 3001 holding tank was continually flushed with fresh blackwater (which is very soft) from the Rio Negro (composition in Table 1), i.e. the original water quality from which the fish had been collected. The rays were held in this water for at least 5 days prior to further tests, and all other experiments were performed in this water quality on board the research vessel. During the 14-day period of research at Anavilhanas, an additional five rays were collected locally, from the wild and added to the pool of experimental animals. The fish were offered food (pellets, tubifex worms) but did not appear to eat in captivity. Holding and experimental temperatures were 28-31°C throughout.
|
Chemicals
The radioisotopes 22Na (as NaCl), 36Cl (as HCl) and
45Ca (as CaCl2) were manufactured by New England Nuclear
(Dupont) and supplied by REM (Sao Paulo). Dimethylsulphoxide (DMSO), amiloride
hydrochloride, 5-(N,N)-hexamethylene amiloride (HMA),
4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt
(DIDS), 4-acetomido-4'-isothiocyanatostilbene-2,2'-disulfonic acid
sodium salt (SITS), sodium thiocyanate, lithium heparin and tricaine methane
sulfonate (MS-222) were all obtained from Sigma. Diphenylamine-2-carboxylic
acid (DPC: N-phenylanthranilic acid) and phenamil methane sulphonate
were obtained from RBI Pharmaceuticals.
General methods
In all experiments, rays were placed in individual polyethylene containers
and allowed to settle for at least 12h prior to tests. The containers were
fitted with airstones and lids and flushed with water from the holding
reservoir or directly from the river at approximately 200 ml min-1.
Container volume was 2.61, except for the largest rays (450-770 g), for which
61 chambers were used. At the start of all flux experiments, the inflowing
water was stopped, and the volume was set to a known volume, generally 2.01
for fish up to 450 g, and 3.0 or 4.01 as appropriate for the largest rays.
In most experiments, unidirectional Na+ and Cl- flux
rates were determined simultaneously by adding both 22Na (a dual
gamma and beta emitter) and 36Cl (a beta emitter only; neutralized
with KOH) to the external water in a 1:2.5 ratio
(Wood, 1988). Differential
gamma and scintillation counting (see below) was employed to monitor the
disappearance of 22Na and 36Cl counts into the fish over
time. Initial tests using `cold displacement' (a rinse with 20 mmol
l-1 NaCl, approximately 1000x ambient concentrations, at the
end of an experiment) demonstrated that there was no significant adsorption of
either radioisotope to the walls of the container or the surface of the fish.
Unidirectional influx rates were low in these fish, so flux periods of 2 h (or
longer) were routinely employed. Water samples (generally 15 ml) were taken at
the beginning and end of each flux period for the measurement of
22Na and 36Cl radioactivity and total concentrations of
water Na+ (by flame photometry) and Cl- (by colorimetric
assay). Influx rates (JinX, by convention
positive) were calculated from the mean external specific activity and the
disappearance of counts from the external water (factored by time, volume and
fish mass), net flux rates (JnetX) were
calculated from the change in total Na+ or Cl-
concentration in the water (similarly factored) and unidirectional efflux
rates (JoutX, by convention negative) were
calculated by difference, as outlined in detail by Wood
(1988
). This approach makes no
assumptions about steady state and allows comparisons of different treatments
over time. Experiments were designed such that internal specific activity,
calculated from the measured accumulation of counts in the fish (i.e.
cumulative disappearance from the water) and an estimated internal
Na+ or Cl- pool of 40 mmol kg-1, was never
more than 5% of measured external specific activity at the end of an
experiment, so there was no need for `backflux correction'. In these same
experiments, net ammonia flux rates were determined simultaneously by the
appearance of total ammonia (measured colorimetrically) in the external water.
Net urea-N excretion was similarly measured in a few experiments.
Measurements of the kinetics of Na+ and Cl-
uptake and efflux
Kinetic relationships were determined by starting at the lowest water
Na+ and Cl- concentrations and then moving upwards in
approximately twofold steps until approximately 2000 µmol l-1
was reached. For stingrays acclimated to ion-rich hard water
(Table 1), this was achieved by
replacing the acclimation water immediately prior to the first test period
with INPA wellwater ([Na+]20 µmol l-1,
[Cl-]
20 µmol l-1, [Ca2+]
10 µmol
l-1) supplemented 24 h previously with sufficient CaCO3
to raise the measured [Ca2+] to 912 µmol l-1,
approximately equal to that in the acclimation water. For stingrays acclimated
to ion-poor Rio Negro blackwater (Table
1), the starting point was Rio Negro water. Radioisotopes (18.5
kBq of 22Na, 46.3 kBq of 36Cl or proportionately more
for the largest rays) were added, allowed to mix for 10 min, and then an
initial 15 ml water sample was taken, followed by a final 15 ml water sample
after 2h. Thereafter, sufficient quantities of NaCl, 22Na and
36Cl were added (as a single stock solution) to approximately
double the concentrations of all in the external bath. The process was
repeated until six flux determinations had been made, each at progressively
twofold higher external Na+ and Cl- concentrations and
radioactivity. This procedure ensured that external specific activity remained
approximately constant throughout the kinetics experiment. To achieve
comparable resolution at these progressively higher external concentrations,
the length of the flux period was gradually increased from 2 h to 2.5 h, 3.25
h, 4.5 h, 7 h and finally 10 h. As both Na+ and Cl-
uptake clearly exhibited saturation kinetics, the
JinNa and JinCl
data for each individual were fitted separately to a MichaelisMenten
model by EadieHofstee regression
(Michal, 1985
) to yield
individual estimates of Km (affinity constant) and
Jmax (maximum transport rate). Grand means ± S.E.M.
were then calculated for all fish in a treatment group.
Measurement of Ca2+ flux rates
We found that the methodology used for Na+ and Cl-
flux determinations would not work for measuring unidirectional
Ca2+ fluxes, because Ca2+ influx rates were lower and
there was a significant adsorption of 45Ca to the body surface of
the stingrays (in all experiments) and to the walls of the container (in some
experiments). Therefore, a longer-term measurement with correction for the
latter was developed. The method assumed steady-state conditions and therefore
allowed only a single measurement of JinCa
under a single treatment condition. However, the method also provided an
estimate of the amount of Ca2+ bound to the surface of the fish. At
the start of the experiment, 148 kBq of 45Ca was added to the 2.01
of water in each container, allowed to mix for 10 min, and 15 ml water samples
(for measurement of 45Ca radioactivity and total Ca2+
concentration) were then taken at 0 h, 3 h, 6 h and 9 h. The slope of the
regression line of the natural logarithm of total water radioactivity against
time yielded the rate constant (k) for turnover, and the product of
k and the mean external total Ca2+ pool, factored by mass,
yielded an uncorrected estimate of JinCa (see
Kirschner, 1970).
After the 9 h sample, the ray was gently transferred to new container
containing 1.01 of fresh Rio Negro water. A water sample was taken, and then
10 ml of 1 mol l-1 Ca(NO3)2 was added to
raise the ambient Ca2+ concentration to 10 mmol l-1
(approximately 1000x ambient concentration) for `cold displacement'. A
second water sample was taken, and the increase in 45Ca
radioactivity, factored by volume, mean specific activity of the original
water and mass, was taken as the amount bound to the surface of the fish
(B). Similarly, the water remaining in the original container
(approximately 2.01) was spiked with 20 ml of 1 mol l-1
Ca(NO3)2, and any increase in 45Ca
radioactivity that occurred, factored by volume and mean specific activity of
the original water, was taken as the amount bound (C) to the
container's surface. Corrected JinCa was then
calculated as:
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Responses to low pH
The responses to low pH in Rio Negro water were assessed by measuring
unidirectional and net Na+ and Cl- flux rates and net
ammonia flux rates during a 2 h control period, followed by a 2 h low-pH
period and a 2 h recovery period, using each fish as its own control. Water
samples (15 ml) were taken at the beginning and end of each period. At 10 min
prior to the start of the control period, 37 kBq of 22Na and 92.5
kBq of 36Cl were added to the external water (or proportionately
more if the volume was greater than 2.01). A 20-min interval between each
period was used to lower the pH to approximately 4.0 with 0.5 mol
l-1 HNO3 and then raise it back to approximately 6.1
with 0.5 mol l-1 KOH for recovery. In practice, because pH tended
to rise during the low-pH period as a result of ammonia production by the
fish, pH was monitored in each container every 30 min and was held between 3.8
and 4.2 by addition of more HNO3. During the control and recovery
periods, pH was held between 5.9 and 6.3.
To assess the effects of low pH on unidirectional Ca2+ fluxes, two separate experiments were performed using the Ca2+ methodology outlined above. In the first 9 h experiment (control), pH was held at approximately 6.1. In the second experiment, water pH was lowered to approximately 4.0 approximately 30 min before the start of the experiment and held at this level for the ensuing 9 h by monitoring at 30 min intervals.
Responses to pharmacological treatments
We tested the effects of various pharmacological agents on the
unidirectional and net flux rates of Na+ and/or Cl- and
the net flux rates of ammonia in Rio Negro water. The basic protocol was a 2 h
control period followed by three successive 2 h experimental periods in the
presence of the drug, using each fish as its own control. Water samples (15
ml) were taken at the beginning and end of each period. At 10 min prior to the
start of the control period, 37 kBq of 22Na and 92.5 kBq of
36Cl were added to the external water (or proportionately more if
the volume was greater than 2.01). In tests with amiloride (10-4
mol l-1), DIDS (2x10-5 mol l-1),
phenamil (4x10-5 mol l-1) and HMA
(4x10-5 mol l-1), both Na+ and
Cl- exchanges were evaluated. In tests with DIDS (10-4
mol l-1), SITS (10-4 mol l-1), DPC
(10-4 mol l-1) and thiocyanate (10-4 mol
l-1), only Cl- exchanges were evaluated. In all cases,
drugs were initially dissolved in DMSO, yielding a final concentration of 0.1%
DMSO in the experimental water. The same concentration of DMSO was therefore
used during the control periods.
Blood sampling
At the end of the 14-day period on the Amanai II, stingrays
acclimated to Rio Negro water were lightly anaesthetized in MS-222.
Approximately 1 ml of blood was withdrawn non-terminally from either the
caudal arch or the heart using a 23-gauge needle and syringe heparinized with
50 µl of 5000 i.u. ml-1 lithium heparin. Haematocrit and plasma
protein (by refractometry) were measured immediately after centrifugation of
blood in microcapillary tubes at 500 g for 10 min. The remainder of
the plasma was separated by centrifugation at 10 000 g, portioned,
and frozen in liquid nitrogen for later analysis of Na+,
Cl-, glucose, urea and total ammonia concentrations and
osmolality.
Analytical methods
22Na radioactivity was determined by manually counting 2 ml
water samples in a Picker Cliniscaler gamma counter fitted with a 10 cm NaI
crystal with a well. 22Na plus 36Cl radioactivity was
determined by mixing 3.0 ml water samples with 7.5 ml of Ecolite fluor and
manually counting on a Triathler portable scintillation counter.
36Cl radioactivity was obtained by subtraction after correcting for
differences in efficiency of 22Na counting between the two
instruments, determined by counting the same standards on both
(Wood, 1988). 45Ca
radioactivity was determined directly by scintillation counting. Water and
plasma total Na+ and water total Ca2+ concentrations
were determined by flame photometry (CELM flame photometer) or atomic
absorption spectrophotometry (Perkin Elmer 1100B), water DOC by a Rosemount
total carbon analyzer, and water pH using an Orion (model 266) portable meter
and electrode. Water total Cl- concentrations were determined by
the colorimetric assay of Zall et al.
(1956
), and water total
ammonia concentrations by the salicylate hypochlorite method of Verdouw et al.
(1978
). In both assays,
standards were made up with the same concentrations of DMSO and the
appropriate drug, as in the experimental medium. In addition, we ran blanks to
correct for the absorbance due to the natural color of blackwater, and
confirmed that the slopes of the assays were not altered by this color. Water
and plasma urea concentrations were determined by the diacetyl monoxime method
of Rahmatullah and Boyde
(1980
). Plasma Cl-
concentration was determined by coulometric titration (Radiometer CMT10),
plasma osmolality by vapour pressure osmometry (Wescor 5100C), plasma protein
concentration by refractometry (American Optical TS meter;
Alexander and Ingram, 1980
),
plasma total ammonia concentration by the L-glutamate dehydrogenase method
(Mondzac et al., 1965
; Sigma
kit 171-UV) and plasma glucose concentration by the hexokinase method
(Bergmeyer, 1985
; Sigma kit
17).
Statistical analyses
All data are reported as means ± 1 S.E.M. (N).
Relationships were assessed by analysis of variance (ANOVA), and individual
means were compared using Student's paired or unpaired two-tailed
t-tests, as appropriate, with the Bonferroni correction for multiple
comparisons (Nemenyi et al.,
1977). A significance level of P
0.05 was used
throughout.
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Results |
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Kinetics of Na+ and Cl- uptake and efflux
Both JinNa and
JinCl exhibited clear saturation kinetics as
external Na+ and Cl- concentrations were increased
(Fig. 1). There were no
differences in the kinetic variables (Km,
Jmax) for these relationships between animals acclimated
to their native ion-poor blackwater of the Rio Negro or to ion-rich hard water
(Table 3), so single lines have
been fitted to the data of Fig.
1. The relationships for Cl- were significantly
different from those for Na+, with 1.7- to twofold higher
Jmax values but virtually identical Km
values (Table 3). As an
additional check that the relationships for Rio Negro water had not changed as
a result of the animals' holding and manipulation in the laboratory, uptake
kinetics of Na+ and Cl- were also measured in four
stingrays freshly collected from the Rio Negro. These yielded values of
Km=563±86 µmoll-1 and
Jmax=400±73 µmol kg-1 h-1
for Na+ and Km=569±98
µmoll-1 and Jmax=487±160 µmol
kg-1 h-1 for Cl-, not significantly different
from the corresponding values (Table
1) for fish acclimated to Rio Negro water in the laboratory. The
slightly lower Jmax values in the freshly collected
animals probably reflected their 2.5-fold greater mean body mass, and we
therefore elected not to pool these data in the kinetic relationships.
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To evaluate whether there was any effect of acclimation water chemistry on ammonia excretion or any direct coupling of ammonia excretion to JinNa, the net ammonia flux was measured at the lowest and highest external Na+ concentrations used in establishing the kinetic relationships (Table 4). There were no differences in ammonia excretion associated with acclimation water chemistry, and no differences in ammonia excretion between the lowest and highest water Na+ levels, despite approximately sevenfold differences in JinNa at the two concentrations. The four freshly collected Rio Negro fish exhibited the same patterns (data not shown).
|
JoutNa did not vary significantly with external Na+ concentration in either acclimation condition and was generally lower than JinNa, so that JnetNa was positive over most of the range tested (Fig. 2). In the lower range of external Na+ levels, JoutNa was lower in the stingrays acclimated to Rio Negro water, with a significant difference at around 400 µmoll-1 (Fig. 2). While the absolute differences appear small, the net effect was to shift the balance point (marked by arrows in Fig. 2), where JinNa=JoutNa (and hence JnetNa is zero), from 220 µmoll-1 in ion-rich hard water to 75 µmoll-1 in ion-poor Rio Negro water.
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|
All other experiments were performed with stingrays held in Rio Negro water. Under the conditions of our experiments, blackwater Na+ and Cl- concentrations were generally around 20-40 µmol l-1 (Table 1; i.e. slightly below the balance points), so the animals were in slight negative balance for both Na+ and Cl- during control treatments (Figs 4,5,6,7). JinNa and JinCl were low, approximately 30-80 µmol kg-1 h-1 (generally higher for JinCl than for JinNa), while efflux rates were approximately twice these values. DMSO (0.1%) had no effect on any of the flux rates measured.
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Responses to low pH
Acute exposure to pH 4.0 induced substantial reductions (approximately 80%)
in both JinNa and
JinCl (Fig.
4A). However, there was no increase in net ion loss rates because
efflux rates were slightly reduced (Fig.
4A). At the same time, net ammonia excretion was increased by
approximately 70% (Fig. 4B).
When the water pH was returned to 6.1 after 2 h of exposure to low pH, the
recovery of JinNa was only partial, and
JoutNa was now significantly lower (i.e.
Na+ turnover was reduced). In contrast, Cl- fluxes
returned to normal levels, and ammonia flux was no longer significantly
elevated.
At pH 6.1, JinCa was only approximately 19 µmol kg-1 h-1 (Table 5) under control conditions, lower than JinNa or JinCl. JoutCa was approximately 3.5 times higher than JinCa, so JnetCa was negative. Exposure to pH 4.0 completely abolished JinCa, while JoutCa and JnetCa did not change significantly. Interestingly, the amount of Ca2+ bound to the surface of the fish (6-7 µmol kg-1) was not significantly altered by exposure to low pH (Table 5).
|
Responses to pharmacological treatments
Exposure to the cationic transport inhibitor amiloride (10-4 mol
l-1) caused a 70% inhibition of
JinNa, an effect that was stable throughout all
three 2 h periods of the experimental treatment
(Fig. 5A). There was no
significant effect on JoutNa or
JnetNa, or on the unidirectional or net flux
rates of Cl-. The non-significant tendency for a large stimulation
of JinCl and
JoutCl probably reflected the fact that the
drug was presented as the hydrochloride salt, which therefore raised the water
Cl- concentration by 100 µmol l-1 to around 130
µmol l-1. In this range, both
JinCl (Fig.
1) and JoutCl
(Fig. 3) are very sensitive to
small changes in external water Cl- concentration, and the
increases were approximately equal to those expected from the kinetic
relationships.
Exposure to the anionic transport inhibitor DIDS (2x10-5 mol l-1) had no effect on JinCl, JoutCl or JnetCl (Fig. 5B). Unidirectional and net flux rates of Na+ were also unaffected.
To pursue the amiloride effect, two analogues (HMA, phenamil) of reputedly greater specificity were tested at a lower concentration (4x10-5 mol l-1). Responses to the two drugs were very similar, although HMA appeared to be slightly more potent. Exposure to HMA (4x10-5 mol l-1) induced a 90% inhibition of JinNa, which persisted throughout all three 2 h periods of the treatment (Fig. 6A). Phenamil (4x10-5 mol l-1) caused a 70% inhibition of JinNa, which was significant during the first two experimental periods only (Fig. 6B). During the first and third periods of drug exposure, JinNa was significantly lower in the presence of HMA than in the presence of phenamil. Both drugs caused significant increases in JoutNa and more negative values of JnetNa throughout the exposures, whereas there were no significant effects on unidirectional or net flux rates of Cl-, apart from a decrease in JinCl at 2-4 h in the presence of HMA.
To pursue the surprising lack of response of Cl- transport to DIDS, both this drug and a related stilbene derivative (SITS) were tested at a higher concentration (10-4 mol l-1). In addition, two other anionic transport blockers (DPC, thiocyanate) were evaluated at this same concentration. To increase the sensitivity for Cl- uptake measurement, only 36Cl was used in these experiments, so that 36Cl counts could be obtained by direct scintillation counting, avoiding the need for differential gamma and scintillation detection to eliminate 22Na counts. Furthermore, DIDS, SITS and thiocyanate were all presented as Na+ salts, thereby raising water Na+ concentration significantly, which would have complicated interpretation of Na+ flux rates.
Even at 10-4 mol l-1, both DIDS (Fig. 7A) and SITS (Fig. 7B) were without significant effect on JinCl, JoutCl or JnetCl. However, both DPC (Fig. 7C) and thiocyanate (Fig. 7D) strongly inhibited JinCl, reducing it to almost zero by the third 2 h period. JoutCl was also strongly reduced during exposure to DPC and thiocyanate (significant in the second and third periods), so that Cl- turnover was greatly reduced, and therefore JnetCl became only slightly more negative.
Exposure to amiloride (at 10-4 mol l-1) and its
analogues (HMA and phenamil at 4x10-5 mol l-1)
exerted similar effects on net ammonia excretion, inhibiting it by
approximately 50% during the first 2 h of treatment, with recovery in
subsequent periods. This response was significant for amiloride
(Fig. 8A) and HMA
(Fig. 8B), but just below the
level of significance (P0.10) for phenamil
(Fig. 8C). None of the anionic
transport blockers tested had any effect on net ammonia excretion (data not
shown).
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Discussion |
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At least superficially, the freshwater stingrays appear to be
physiologically similar to some of the freshwater teleosts that are also
endemic to the dilute blackwaters of the Amazon basin. These teleosts may or
may not have shared the same route of invasion
(Brooks, 1995;
Lovejoy et al., 1998
).
Gonzalez et al. (2002
)
recently summarized kinetic variables and acid sensitivity for Na+
transport in a number of teleost species collected from the Rio Negro. The
data divided into two strategies for adaptation to acidic, ion-poor water: (i)
high-capacity, high-affinity (i.e. low Km) Na+
transport systems relatively resistant to inhibition by low pH, but linked
with high diffusive loss rates; and (ii) low-affinity (i.e. high
Km) transport systems relatively sensitive to inhibition
by low pH, but complemented by low diffusive loss rates. The freshwater rays
represent an extreme version of the second strategy, with
Km values for Na+ (and Cl-) of
approximately 400-500 µmol l-1
(Fig. 1;
Table 3), at least double that
of any of the teleosts surveyed, Jmax values at the lower
end of the range for the teleosts surveyed, normal influx and efflux rates of
less than 200 µmol kg-1 h-1 and an 80% inhibition of
influx but no increase in efflux or net loss rate upon challenge with pH 4.0
(Fig. 4). As pointed out by
Gonzalez et al. (2002
), this
strategy, typical of Amazonian cichlids, is designed to limit net ion loss and
thereby minimize physiological disturbance so as to `wait out' exposures to
especially acidic and dilute challenges.
Most of the teleosts surveyed by Gonzalez et al.
(2002) exhibited
JinNa=JoutNa
balance points below 43 µmol l-1, whereas the Na+ and
Cl- balance points in the stingrays were higher, 65-75 µmol
l-1 (Figs 2,
3), approximately twice the
normal levels in the blackwater in which they were living
(Table 1). Thus, the rays were
in negative balance in their native water, but they were not feeding in our
studies. We suspect the explanation is that these animals normally obtain
significant supplementary ions from the diet, as documented for some teleosts
(e.g. Smith et al., 1989
). The
importance of food-derived salts in successful adaptation to low environmental
pH has recently been highlighted for salmonids
(D'Cruz and Wood, 1998
). A
second explanation may be that the stingrays normally live at the bottom of
the water column, just above the benthos; ion concentrations in the water
column may be slightly higher here through remobilization from the
sediments.
A surprising result was the absence of any shift in the Na+ or
Cl- influx kinetic curves between acclimation to ion-rich hard
water and ion-poor Rio Negro water (Fig.
1; Table 3). The
`normal' pattern, documented in many freshwater teleosts and crustaceans (e.g.
Shaw, 1959;
Maetz, 1974
;
McDonald and Rogano, 1986
;
Perry and Laurent, 1989
;
Potts, 1994
), is for a higher
affinity (lower Km) and/or greater
Jmax upon acclimation to ion-poor water. The explanation
may be that modification of ion uptake systems originally designed for
function in seawater (see below) has been pushed to a maximum during the
evolutionary invasion of freshwater, and there is no capacity in these
elasmobranchs for further modification to differentiate between normal
freshwater and very dilute freshwater. In this regard, examination of teleost
families with a similar distribution to the Potamotrygonidae, and which today
have both marine and blackwater representatives (e.g. Scianidae, Engraulidae,
Clupeidae, Belonidae, Soleidae; Gery,
1969
; Chao, 1978
;
Fink and Fink, 1979
), may be
instructive.
On the other hand, upon acclimation to ion-poor blackwater, there were
subtle but significant reductions in both
JoutNa and
JoutCl, which substantially shifted the balance
points to much lower external Na+ and Cl- concentrations
(Figs 2,
3). Efflux rates may consist of
two components in these fish (see below), but it appears likely that an
attenuation of the simple diffusive component via epithelial
tightening was responsible for the overall reductions. This is a classic
response to softwater acclimation (e.g.
McDonald and Rogano, 1986).
Possibly, DOC may also play a role. Since dissolved organic matter is now
known to bind to fish gills, especially at low pH
(Campbell et al., 1997
),
Gonzalez et al. (1998
,
2002
) have speculated that the
high DOC content of ion-poor blackwaters
(Table 1) may also be important
in limiting gill permeability.
Mechanisms of Ca2+ transport and binding
To our knowledge, there are no previous measurements of unidirectional
Ca2+ fluxes (Table
5) in any elasmobranch, marine or freshwater, or in any Amazonian
blackwater teleosts. In other freshwater teleosts, in which transport rates
are similar, Ca2+ uptake is active and is mediated by apical
Ca2+ channels and basolateral high-affinity Ca2+-ATPase
and/or Na+/Ca2+ exchange mechanisms in gill ionocytes
(Flik et al., 1995;
Jurss and Bastrop, 1995
;
Perry, 1997
). As for
Na+ and Cl- balance, these stingrays were in negative
Ca2+ balance when acclimated to their native blackwater
(Table 5), again suggesting an
important role for dietary uptake or ionic microclimates in benthic waters in
normal homeostasis. In two northern hemisphere teleost species, Hobe et al.
(1984
) reported 65-75%
reductions of JinCa upon acute exposure to pH
4.0. In stingrays, JinCa was abolished at pH
4.0 (Table 5), similar to the
severe reduction of JinNa and
JinCl during acid exposure
(Fig. 4). Interestingly, for
all three ions, there was no stimulation of the efflux components. Gonzalez
and coworkers (Gonzalez and Dunson,
1989
; Gonzalez et al.,
1997
,
1998
,
2002
;
Wood et al., 1998
;
Gonzalez and Preest, 1999
;
Wilson et al., 1999
;
Gonzalez and Wilson, 2001
)
have developed the idea that some blackwater teleosts are able to minimize or
prevent low-pH-induced increases in diffusive permeability by having very high
affinity Ca2+ binding sites which stabilize external cell
junctions. The present measurements showing that surface-bound Ca2+
in stingrays are unaffected by exposure to pH 4.0
(Table 5) provide direct
support for this hypothesis.
Mechanisms of Na+ and Cl- transport
In the following discussion, we assume that ion exchanges occur mainly at
the gills, as in other fish. However, given the large surface area and soft
texture of the well-perfused skin (which becomes pink with blood flow during
hypoxia), we cannot eliminate a significant role for the skin in both ion
uptake and efflux, and of course for the renal system in ion efflux (see
Mangum et al., 1978). Another
important caveat is that the present experiments were performed on whole
animals, in which side effects of the pharmacological treatments or water
chemistry manipulations may potentially confound interpretation. These might
include changes in transepithelial potential
(Kirschner, 1997
) or
alterations in the potency of drugs by trapping in mucus or by interactions
with dissolved organic matter in the natural blackwater
(Campbell et al., 1997
).
The presence of traditional MichaelisMenten saturation kinetics
(Fig. 1) for both
JinNa and JinCl
(Fig. 1) against large
water-to-blood concentration gradients
(Table 1) suggests that both
occur by carrier-mediated active transport in these freshwater elasmobranchs.
They were of different magnitude and responded entirely differently to various
pharmacological treatments (Figs
5,
6), suggesting that they are
independent, as in most freshwater teleosts (Kirschner,
1970,
1997
;
Goss et al., 1992
;
Potts, 1994
;
Jurss and Bastrop, 1995
;
Perry, 1997
;
Evans et al., 1998
).
JoutCl appeared to exhibit a strong kinetic
linkage with JinCl
(Fig. 3), a conclusion
reinforced by the inhibitory effects of several drugs on both components
(Fig. 7C,D). This pattern is
reminiscent of the `exchange diffusion' phenomenon seen in many freshwater
teleosts and crustaceans (e.g. Shaw,
1960
; Kerstetter and
Kirschner, 1972
; de Renzis,
1975
; Wood et al.,
1984
; Goss and Wood,
1990
), in which the same transport mechanism may perform both
Cl-/Cl- self-exchange and vectorial transport (e.g.
Cl-/base exchange). The phenomenon can be equally well explained by
an exchange protein or a selective channel mechanism linked to an electrogenic
pump (Potts, 1994
). Exchange
diffusion has also been seen for Na+ transport where
Na+/Na+ and Na+/acid exchange may co-exist
(e.g. Shaw, 1959
;
Wood and Randall, 1973
;
Goss and Wood, 1990
), but this
was not apparent in the stingrays. JoutNa and
JinNa appeared to be kinetically independent
(Fig. 2), and did not co-vary
when JinNa was pharmacologically inhibited
(Figs 5A,
6A,B). Interestingly, Gonzalez
et al. (2002
) reported kinetic
linkage between JoutNa and
JinNa in approximately half the Rio Negro
teleost species they surveyed (Cl- fluxes were not measured), but
were unable to relate the presence or absence of this exchange diffusion
phenomenon to either of the strategies outlined above.
According to theory (Shaw,
1959; Kirschner,
1970
; Wood and Randall,
1973
; de Renzis,
1975
; Goss and Wood,
1990
), the exchange diffusion components of efflux, when present,
are always superimposed on the simple diffusive components, and the latter are
predominant at low external Na+ and Cl- concentrations.
It was probably a reduction in this simple diffusive permeability, rather than
a change in the exchange diffusion component, which explained the lower values
of JoutNa and
JoutCl, and the resulting reductions in
Jin=Jout balance points upon
acclimation to Rio Negro blackwater (Figs
2,
3).
Amiloride (10-4 mol l-1) inhibited
JinNa by 70% in these freshwater stingrays
(Fig. 5A). In this regard, the
stingray is similar to most freshwater teleosts, in which a 60-95% blockade of
Na+ uptake induced by this concentration of amiloride has been
widely reported (e.g. Kirschner et al.,
1973; Perry and Randall,
1981
; Perry et al.,
1981
; Wright and Wood,
1985
; Wilson et al.,
1994
; Nelson et al.,
1997
; Clarke and Potts,
1998
; Patrick and Wood,
1999
). Interestingly, in two native Rio Negro characids that
demonstrated the high-capacity, high-affinity, low-pH-resistant-strategy of
Na+ transport (i.e. opposite to that of the rays), amiloride
(10-4 mol l-1) had little effect (13-26% inhibition of
JinNa;
Gonzalez et al., 1997
;
Gonzalez and Preest, 1999
).
However, amiloride at this concentration is a relatively non-selective drug,
blocking both Na+ channels and Na+/H+
exchangers (Benos, 1982
). In an
attempt to separate these two possibilities in the stingrays, we employed two
drugs of reputedly greater specificity (HMA, phenamil) at lower concentration
(4x10-5 mol l-1). In higher vertebrate systems
(cell lines), HMA is reported to be over 500 times more potent against
Na+/H+ exchange and only 1/30th as potent
against Na+ channels as amiloride
(Kleyman and Cragoe, 1988
). In
contrast, phenamil is reported to be 17 times more potent against
Na+ channels, and only 1% as potent against
Na+/H+ exchange as amiloride
(Kleyman and Cragoe, 1988
).
While HMA was somewhat more effective than phenamil (90% versus 70%
inhibition, with greater persistence), our results were not clear-cut because
both drugs strongly reduced JinNa
(Fig. 6A,B). Amiloride analogue
specificities defined in mammalian cell lines may not necessarily apply in
intact lower vertebrates. No definitive conclusion as to the importance of
Na+/H+ exchange versus Na+
channel/H+-ATPase systems in mediating Na+ uptake can be
drawn.
Stilbene drugs such as DIDS and SITS are effective blockers of
JinCl in some freshwater teleosts
(Perry and Randall, 1981;
Perry et al., 1981
) although
they are ineffective in others (Marshall
et al., 1997
). In the present study, these agents were completely
ineffective against Cl- transport in freshwater stingrays, even at
concentrations (10-4 mol l-1) above those
(2x10-5 mol l-1) usually considered to be specific
for blockade of Cl-/HCO3- exchange (Figs
5B,
7A,B). We took precautions to
shield these agents from light and to make them up immediately before use, and
we also ran one test in water without DOC to ensure that the lack of effect
was not due to immobilization of DIDS by dissolved organic molecules in
blackwater. Nevertheless, from this result, we cannot eliminate a role for
anion exchange in Cl- uptake, because many members of the anion
exchange family are insensitive to stilbenes.
Thiocyanate is considered to be a more general blocker (competitive and
non-competitive) of many anion exchange systems and has been shown to inhibit
Cl- transport in several freshwater teleosts
(Epstein et al., 1973;
Kerstetter and Kirschner,
1974
; de Renzis,
1975
; Perry et al.,
1984
), even those in which stilbenes are ineffective
(Marshall et al., 1997
). In
the stingrays, at a concentration (10-4 moll-1) only
slightly higher than that of Cl- in the water, thiocyanate almost
abolished JinCl and also strongly reduced
JoutCl (Fig.
7D). These actions are very similar to those reported in the
goldfish, which were interpreted as a blockade of both
Cl-/HCO3- exchange and
Cl-/Cl- exchange diffusion in the very thorough study of
de Renzis (1975
). Since it is
known that thiocyanate is a `sticky' anion that will compete for and block
Cl- channels, while DIDS and SITS are generally ineffective against
Cl- channels, at least when applied externally
(McCarty, 2000
), this raised
the possibility that a Cl- channel might be involved.
DPC (10-4 moll-1) was employed because it is widely
effective in blocking Cl- channels
(Gogelein, 1988;
Distefano et al., 1985
;
Chang and Loretz, 1993
;
McCarty, 2000
), including
those recently characterized in isolated gill cells of freshwater teleost fish
(O'Donnell et al., 2001
),
although it has never been tested on intact freshwater teleosts. In the
stingrays, DPC (10-4 moll-1) essentially duplicated the
action of thiocyanate, abolishing JinCl and
greatly reducing JoutCl, indicating blockade of
both vectorial transport and Cl-/Cl- exchange diffusion.
While this would suggest the involvement of a Cl- channel in both
processes, the result must be treated with caution because it is unclear where
this channel is located. DPC is lipophilic, so even though it was applied in
the external medium, it may well have penetrated to the basolateral membrane
of gill ionocytes, thereby blocking basolateral Cl- channels, which
in turn would disrupt the whole transcellular Cl- transport pathway
at the `exit' step rather than the `entry' step. A further caveat is that DPC
at this concentration has also been reported to block
Cl-/HCO3- exchange in at least one system
(Reuss et al., 1987
). Whatever
the mechanism of Cl- uptake, given a negative intracellular
potential, any reasonable estimate of intracellular Cl-
concentration in the low millimolar range and an extracellular concentration
in the low micromolar range, it would have to be `energized' at the apical
membrane, perhaps by a local elevation of intracellular
HCO3- concentration coupled to intracellular carbonic
anhydrase and/or H+-ATPase or
Cl-/HCO3--ATPase systems.
Nitrogen metabolism and ammonia excretion
Urea-N excretion was low (approximately 60 µmol kg-1
h-1) and comparable to rates previously reported in other
freshwater Potamotrygonidae, while ammonia-N excretion was high (500-600
µmol kg-1 h-1) but in the mid-range of previously
reported values (200-1000 µmol kg-1 h-1), confirming
the ammoniotelic nature of these fish
(Goldstein and Forster, 1971;
Gerst and Thorson, 1977
;
Barcellos et al., 1997
). The
high rate of ammonia-N production may have resulted from metabolism of
endogenous protein in these non-feeding animals. Since net ammonia flux was
approximately eight- to tenfold higher than normal
JinNa in animals held in Rio Negro blackwater,
any contribution from Na+/NH4+ exchange was
presumably small, and this was in accord with the complete insensitivity of
ammonia excretion to approximately sevenfold variations in
JinNa in the kinetic experiments
(Table 4). Furthermore, the 70%
increase in ammonia excretion at pH 4.0, when
JinNa was severely reduced, was consistent with
a dominant role for simple NH3 diffusion along
PNH3 gradients
(Wilson, 1996
;
Wilkie, 1997
). It was
therefore surprising that amiloride and its analogues (HMA, phenamil) all
caused a transitory 50% depression in ammonia excretion
(Fig. 8). Indeed, this was
larger than the marginal decreases in ammonia efflux (0-30%) normally seen in
freshwater teleosts in response to amiloride blockade of
JinNa
(Kirschner et al., 1973
;
Wright and Wood, 1985
;
Wilson et al., 1994
;
Nelson et al., 1997
;
Clarke and Potts, 1998
;
Patrick and Wood, 1999
). While
originally seen as evidence for Na+/NH4+
exchange, the latter observations have more recently been interpreted as an
inhibition of `diffusion trapping' of NH3 in gill boundary layer
water associated with blockade of H+ extrusion via
Na+/H+ and/or Na+
channel/H+-ATPase systems
(Wilson, 1996
;
Wilkie, 1997
). Perhaps this
diffusion trapping is particularly important in the poorly buffered
blackwater, especially if H+ and NH3 excretion sites are
`downstream' from alkalizing sites (e.g. HCO3-
excretion) on the gills. A related explanation is that blockade of
H+ excretion mechanisms by amiloride and its analogs may also
reduce internal pH, thereby trapping more ammonia internally as
NH4+ and transiently reducing the internal
PNH3 and, therefore, the
PNH3 gradient for outward NH3
diffusion.
Evolutionary perspective and future directions
Based on the limited knowledge of branchial Na+ and
Cl- transport in marine elasmobranchs (for a review, see
Shuttleworth, 1988), it is
possible to put the present results in an evolutionary perspective. In marine
elasmobranchs, the rectal gland normally performs the bulk salt excretion, and
the gills usually achieve a very small net uptake of Na+ and
Cl-. The gills exhibit unidirectional ion flux rates that are only
a small fraction of those in marine teleosts and are quantitatively more
similar to those of freshwater teleosts. Unidirectional Cl- fluxes
are generally greater than unidirectional Na+ fluxes
(Bentley et al., 1976
).
Although the fine details of both fluxes are unknown, the system appears to be
designed for pH regulation, normally achieving a small net uptake of
Na+ in exchange for `acid', as in freshwater teleosts (Payan and
Maetz, 1974; Evans et al.,
1979
). These characteristics are all similar to those we have
identified in these freshwater stingrays and suggest that gill transport
mechanisms already present in marine elasmobranchs may have been modified for
operation in dilute external solution during the evolutionary invasion of
freshwater by the Potamotrygonidae.
In the euryhaline cousins of the Potamotrygonidae, the Dasyatidae,
Piermarini and Evans (2000,
2001
) have recently identified
two types of ionocyte in the gills on the basis of immunocytochemistry, one of
which stains for Na+/K+-ATPase and the other for
H+-ATPase. After acclimation to freshwater, the expression of both
enzymes increased, as measured by western blotting, and the
H+ATPase appeared to become localized in the basolateral membrane.
Based solely on these data, for no flux measurements or pharmacological
characterizations were carried out, Piermarini and Evans
(2001
) have proposed a model
(see their Fig. 7) whereby
basolateral Na+/K+-ATPase energizes an apical
Na+/H+ exchanger in one cell type, while basolateral
H+-ATPase energizes an apical
Cl-/HCO3- exchanger in the other. This scheme
is certainly unusual compared to most current models for the gills of
freshwater teleosts (Goss et al.,
1992
; Potts, 1994
;
Jurss and Bastrop, 1995
;
Perry, 1997
;
Kirschner, 1997
;
Evans et al., 1998
), but is
coherent with our present data on potamotrygonid stingrays. Nevertheless, it
must be emphasized that the present pharmacological results cannot
conclusively differentiate between Na+/H+ exchange and a
Na+ channel/H+-ATPase system on the apical membrane, or
between a Cl- channel and
Cl-/HCO3- exchange on this membrane. Clearly,
the next step is to apply all techniques simultaneously (flux measurements,
pharmacology, immunocytochemistry and molecular characterization of
transporters) to these two different families of freshwater stingrays, one
euryhaline, the other stenohaline.
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