Active NaCl absorption across posterior gills of hyperosmoregulating Chasmagnathus granulatus
1 Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras
de Ribeirão Preto, Universidade de São Paulo, Avenida
Bandeirantes 3900, Ribeirão Preto 14040-901, SP, Brasil
2 Departamento de Biodiversidad y Biología Experimental, Facultad de
Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pab. II, Ciudad
Universitaria, C1428EHA Buenos Aires, Argentina
* Author for correspondence (e-mail: onkenh{at}ffclrp.usp.br)
Accepted 10 January 2003
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Summary |
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Key words: Chasmagnathus granulatus, Crustacea, crab gill, flux measurement, ion transport, osmoregulation, short-circuit current, transepithelial conductance, ion substitution, inhibitor, Ussing chamber
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Introduction |
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After refining the study of ion transport across crab gills using split
gill lamellae mounted in modified Ussing chambers
(Schwarz and Graszynski,
1989), the basic epithelial properties and transport mechanisms
could be analysed more unambiguously. Posterior gills of crabs from freshwater
display a tight epithelium and a mechanism of NaCl absorption similar to that
of other freshwater animals such as fish and amphibia
(Goss et al., 1992
; Larsen,
1988
,
1991
). For Chinese crabs
Eriocheir sinensis adapted to freshwater, Na+ absorption
was shown to proceed via apical Na+ channels and the
basolateral Na+/K+-ATPase, generating a positive
short-circuit current in the absence of external Cl-
(Zeiske et al., 1992
). The
negative short-circuit current in the presence of external Cl- was
analysed to reflect Na+-independent Cl- absorption
via apical Cl-/HCO3- antiport and
basolateral Cl- channels, driven by an apical V-type H+
pump (Onken et al., 1991
;
Onken and Putzenlechner,
1995
). With in vivo-like, low external NaCl, the positive
and negative currents short-circuit each other and enable transcellular NaCl
absorption (Onken, 1999
). The
posterior gills of the hololimnetic Dilocarcinus pagei yielded
similar results to Chinese crab posterior gills
(Onken and McNamara, 2002
). On
the other hand, posterior gills of the shore crab Carcinus maenas,
which is a strong hyperosmoregulator but unable to survive in freshwater, were
found to be much more leaky (Onken and
Siebers, 1992
). Under short-circuit conditions a tight 1:2
coupling between Na+ and Cl- absorption was observed
(Riestenpatt et al., 1996
). In
these animals NaCl absorption is exclusively driven by the
Na+/K+-ATPase, and the negative short-circuit current
seems to reflect NaCl absorption, as in the thick ascending limb of Henle's
loop of the mammalian nephron (Greger and
Kunzelmann, 1990
) via apical
Na+/K+/2Cl- symport with an apical
K+ recycling via K+ channels
(Riestenpatt et al.,
1996
).
Chasmagnathus granulatus is a strong hyper- and
hypo-osmoregulating crab that inhabits intertidal, estuarine coasts of Brazil,
Uruguay and Argentina (Mougabure Cueto,
1998; Charmantier et al.,
2002
). The thickness of the posterior gill epithelium increases
when the animals are adapted to either low or high salinities, suggesting the
involvement of the gills in salt absorption of hyperosmoregulating crabs and
in salt secretion in hypo-osmoregulating animals
(Genovese et al., 2000
). In a
recent study with perfused posterior gills, the respective active
transbranchial absorption and secretion of Na+ have been directly
demonstrated (Luquet et al.,
2002
); however, a satisfying analysis of the transport mechanisms
is still lacking.
In the present study, the posterior gills of the euryhaline crab C. granulatus adapted to low salinity were investigated in order to obtain more detailed information about the transport mechanism responsible for active, transbranchial NaCl absorption in this species. The results indicate that at least the major part of NaCl is absorbed by an electrogenic mechanism that most likely resembles NaCl absorption across the gills of the shore crab C. maenas.
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Materials and methods |
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Preparations
After killing the animals by rapidly destroying the ventral and dorsal
ganglia, the carapace was lifted and the posterior gills removed.
For the Ussing chamber experiments, single posterior gill lamellae were
isolated and split according to Schwarz and Graszynski
(1989). The split gill
lamellae thus obtained were mounted in an Ussing chamber modified after De
Wolf and Van Driessche (1986
).
Preparation and mounting were conducted under microscopic control. Silicon
grease (Bayer, Germany) was used to minimise edge damage. An epithelial area
of 0.002 cm2 was exposed to the chamber compartments, bathing the
internal and external sides of the tissue. Continuous perfusion of both
chamber compartments with aerated saline was achieved by gravity flow
(approximately 2 ml min-1).
For the gill perfusion experiments, the afferent and efferent vessels of a posterior gill were connected via fine polyethylene tubings to a peristaltic pump (afferent) and to a glass tube (efferent). The tubings were fixed in position with a small Lucite clamp covered with smooth neoprene to avoid gill damage and to isolate the gill interior from the bathing medium. The gill was bathed in a beaker containing approximately 50 ml of aerated saline and was perfused at a rate of 0.1 ml min-1, the perfusate being collected in a second beaker.
Electrophysiology
In the Ussing chamber experiments the transepithelial voltage
(Vte) was measured using Ag/AgCl electrodes connected to
both sides of the preparation (distance <1 mm) by agar bridges (3% agar in
3 mol l-1 KCl). The reference electrode was in the internal bath.
Silver wires coated with AgCl served as electrodes to apply current for
short-circuiting Vte (measurement of the short-circuit
current, Isc*) from an automatic clamping device (VCC 600,
Physiologic Instruments, USA). The area-specific resistance between the tips
of the voltage electrodes (Rtot) was calculated from
imposed voltage pulses (Vte) and the resulting
current deflections (
I). Rtot is the sum
of the serial resistances of the solutions (Rs) and the
tissue (Rte). Because of the low values of
Rtot, it was necessary to correct the
Rtot and Isc* data to obtain values
directly related to the preparations (Rte,
Isc). Rs was measured in the absence
of a preparation separating the chamber compartments and was found to be 5.5
cm2 for NaCl saline, 7
cm2 for
Na+ saline, and 8
cm2 for Cl- saline.
The corrected values for Rte are obtained by substracting
Rs from Rtot, while the correction of
Isc* followed Ohm's law (see
Riestenpatt et al., 1996
). In
the Results, only the corrected values of Isc and
Gte (1/Rte) are given. In the figures
displaying time courses, the original Isc* is shown, and
the current deflections reflect the uncorrected conductance between the tips
of the voltage electrodes.
During flux measurements with isolated and perfused whole gills, the transbranchial voltage was controlled with a millivoltmeter (Metrix, France) by connecting Ag/AgCl electrodes via agar bridges to the external bath and to the glass tube collecting the perfusate (internal side).
Na+ influx measurements
Na+ influxes across isolated and perfused posterior gills were
measured by applying 22Na [final activity 9.25x103
Bq (0.25 µCi) ml-1] to the external bath. After 15 min of
stabilization, the radioactivity appearing in the perfusate was measured at 10
min intervals using a gamma scintillation counter (Canberra series 35 plus,
Canberra, USA). Sodium influx was calculated according to Lucu and Siebers
(1986) and related to the
fresh mass of the gill used.
Salines and chemicals
The basic NaCl saline contained (in mmol l-1): 468 NaCl, 9.5
KCl, 7.5 MgCl2, 12.5 CaCl2, 5 Hepes, 2.5
NaHCO3 and 5 glucose. In Na+-free saline NaCl was
replaced by choline chloride and NaHCO3 by KHCO3. KCl
was reduced to 7 mmol l-1. In Cl--free saline the
chlorides were replaced by the nitrates of the respective cations. Immediately
before use, the pH of all salines was adjusted to 7.75.
All chemicals and reagents not mentioned separately below were purchased from Merck (Argentina). NaHCO3 was obtained from Mallinckrodt (USA). CaCl2 and Hepes were from J. T. Baker (USA). Ouabain and acetazolamide were purchased from Sigma (Germany). Diphenylamine-2-carboxylate was obtained from Fluka (Germany) and 22Na (as chloride) was from Amersham Pharmacia Biotech. (USA).
Statistics
All values are means ± S.E.M. Differences between groups were tested
using the paired Student's t-test. Significance was assumed at
P<0.05.
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Results |
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To analyse the ionic nature of the short-circuit current, ion substitution experiments were performed. On replacing Cl- on both sides of the preparation by nitrate, the negative Isc significantly decreased by approximately 75% from -124±16 to -32±5 µA cm-2 (N=5). At the same time the conductance of the preparation also decreased from 45±3 to 22±2 mS cm-2 (N=5). After readministering Cl- on both sides of the tissue, Isc was found to be completely reversible (-149±29 µA cm-2; N=5). In most cases the new control current was even more negative than before Cl- substitution. The conductance of the split gill lamellae increased to 63±3 mS cm-2 (N=5) after readministering Cl-. A decrease in negative Isc and Gte similar to that obtained after substitution of Cl- was also observed when Na+ was replaced by choline. In this case the negative Isc decreased from -132±34 to -23±5 µA cm-2 (N=5) and the conductance of the split gill lamellae decreased from 61±8 to 45±8 mS cm-2 (N=5). After returning to NaCl salines, current and conductance returned to the control values seen before substitution of Na+. A representative example of the above-described ion substitution experiments is shown in Fig. 1.
|
Thus far, the polarity of Vte and
Isc, and the current reductions after substitution of
Na+ or Cl- indicate that the negative
Isc reflects Na+-coupled Cl-
absorption. To analyse the transport mechanism in more detail, several
inhibitors of transport proteins were used. Applying 5 mmol l-1
ouabain, a specific inhibitor of the Na+/K+-ATPase
(Skou, 1965), to the internal
perfusion medium, resulted in a significant decrease of the negative
Isc by approximately 80% from -110±16 to
-22±8 µA cm-2 (N=6). The effect of ouabain on
Isc was slowly reversible. The conductance of split gill
lamellae was not affected by internal ouabain. A representative time course of
an experiment with ouabain is shown in Fig.
2. Internal addition of 2 mmol l-1
diphenylamine-2-carboxylate (DPC), an inhibitor of Cl- channels
(Di Stefano et al., 1985
),
significantly reduced the negative Isc (from -92±11
to -25±6 µA cm-2; N=5) at constant
Gte. The effect of DPC on Isc was slowly but
completely reversible. The primary solvent for DPC, dimethylsulfoxide, was
always applied alone and at the same concentration before DPC. The minor
current decreases (see Fig. 3)
were taken into consideration when the DPC effects were quantified. Internal
BaCl2 (10 mmol l-1), a blocker of K+ channels
(Van Driessche and Zeiske,
1985
), decreased the negative Isc from
-135±13 to -31±7 µA cm-2 (N=4) at
unchanged Gte. To avoid influences of Cl- diffusion
currents, BaCl2 was first applied to the external bath (where it
had no effect on current or conductance), and afterwards added to the internal
perfusion medium. A representative experiment showing the influences of
BaCl2 and DPC is shown in Fig.
3. Internal addition of acetazolamide (0.1 mmol l-1),
an inhibitor of carbonic anhydrases
(Maren, 1967
), had no effect
on Isc or Gte (N=4; not shown).
|
|
Neither BaCl2 (10 mmol l-1; N=4, see above
and Fig. 3) nor furosemide
(N=4; not shown), a blocker of
Na+/K+/2Cl- symporters
(Greger and Kunzelmann, 1990),
showed any effects on current and conductance across C. granulatus
split gill lamellae when applied to the external bathing medium. External
Cs+ ions (50 mmol l-1 CsCl), another inhibitor of
K+ channels (cf. Van Driessche
and Zeiske, 1985
), however, reduced the negative
Isc from -135±18 to -29±10 µA
cm-2 (N=3) when applied to the external bath. The
conductance of the tissue remained unchanged in the presence of CsCl. To
reveal a possible contribution of inward-diffusing Cs+ ions to the
reduction of the negative Isc, the above values were also
determined after adding the same amount of CsCl to the internal bath. This,
however, resulted only in minimal recoveries of the negative current, showing
that Cs+ diffusion is very low or almost equal to Cl-
diffusion. The effect of external Cs+ on the current was
reversible. An example of the experiments with CsCl is shown in
Fig. 4.
|
Although external furosemide had no effect on Isc, the
results so far collected suggest that the negative current reflects
electrogenic, Na+-coupled Cl- absorption, as in the
gills of Carcinus maenas
(Riestenpatt et al., 1996). To
evaluate a possible contribution of an electroneutral uptake via
apical Na+/H+- and
Cl-/HCO3--antiports to overall NaCl
absorption, we measured the influence of acetazolamide (0.1 mmol
l-1) on unidirectional Na+ influx across isolated and
perfused posterior gills of C. granulatus. In five experiments the
drug significantly reduced the mean Na+ influx from 1211±179
to 979±188 µmol h-1 g-1 (N=5; see
Fig. 5) without affecting the
transbranchial voltage.
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Discussion |
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Nevertheless, despite the above mentioned disadvantages the results of the
present study clearly show that measurements with split gill lamellae of
C. granulatus can be successfully performed and result in useful
information about the characteristics of the epithelium. The voltage across
split gill lamellae was in exactly the same range as that in a recent study of
isolated and perfused gills (Luquet et
al., 2002), clearly showing that the splitting hardly damages the
epithelium, and that edge damage is not a serious problem.
Active NaCl absorption in C. granulatus posterior
gills
In diluted media, C. granulatus maintains an outward-directed
osmotic gradient (Mougabure Cueto,
1998; Charmantier et al.,
2002
). When the animals were adapted from seawater to brackish
water, the thickness of the posterior gill epithelium increased
(Genovese et al., 2000
),
indicating its involvement in osmoregulatory active NaCl absorption, as
observed in a variety of Crustacea
(Péqueux et al., 1988
).
Recently, a ouabain-sensitive net influx of Na+ was determined with
isolated and perfused posterior gills
(Luquet et al., 2002
). The
magnitudes of the transepithelial voltage (Vte),
short-circuit current (Isc) and conductance
(Gte) measured in the present study with NaCl salines on both sides
of split lamellae of C. granulatus characterise the tissue as a leaky
epithelium with a high conductance paracellular pathway. The negative
Isc was dependent on the presence of Na+ and
Cl- (see Fig. 1),
indicating that the current reflects active, electrogenic and
Na+-dependent Cl- absorption.
The cuticle
In Crustacea, the gill epithelium is covered on the apical side by cuticle.
On the one hand, the presence of this chitinous layer is of advantage for
measurements with split gill lamellae, because it gives the tissue a
mechanical stability essential for preparation and mounting. On the other
hand, the cuticle acts as serial resistance in front of the epithelium and as
a barrier for drugs. In the crab species studied so far, the cuticular
resistance was found to be very small when compared to the resistance of the
whole preparation. In C. maenas, for example, the resistance of split
gill lamellae was approximately 25 cm2 and the cuticular
resistance was below 2
cm2
(Riestenpatt et al., 1996
;
Onken and Riestenpatt, 2002
).
Thus, its influence as serial resistance seems of minor importance. With
respect to the cuticular permeability to inhibitors of transport proteins it
is of interest that in C. maenas, neither furosemide nor amiloride
seem to pass the cuticle (Riestenpatt et
al., 1996
; Onken and
Riestenpatt, 2002
). With respect to furosemide, the same may be
the case for the gill cuticle of C. granulatus (see below). In the
present investigation, the gill cuticle of C. granulatus has not been
studied separately, and a detailed investigation of this layer should be
performed in the future.
The apical membrane
Ba2+ and Cs+ ions are well-known blockers of
K+ channels (Van Driessche and
Zeiske, 1985). With C. maenas, split gill lamellae,
external Cs+ inhibited the negative Isc more
effectively than Ba2+
(Riestenpatt et al., 1996
),
although Cs+ is usually a less potent inhibitor than
Ba2+ (Van Driessche and Zeiske,
1985
). This result may reflect a different permeability of the
gill cuticle to the two cations, resulting in a better accessibility of the
apical membrane by Cs+. Similarly, with C. granulatus
split gill lamellae, external Ba2+ levels had no effect on the
negative Isc (see Fig.
3), whereas high concentrations of external Cs+
inhibited the current (see Fig.
4). The conductance of the preparation was not affected by
external Cs+. However, a possible effect on the transcellular
conductance might have been hidden by a conductance increase of the
paracellular pathway due to the augmentation of the solution ionic strength
after addition of CsCl. Besides their presence in tight NaCl absorbing
epithelia, where they serve for K+ secretion
(Van Driessche and Zeiske,
1985
), apical K+ channels are often present in
epithelia with K+-dependent cotransporters in the apical membrane,
as in the vertebrate gastric epthelium (K+/H+-ATPase;
Wolosin and Forte, 1984
) or
the thick ascending limb (TAL) of Henle's loop in the mammalian nephron
(Na+/K+/2Cl- symport;
Greger, 1985
). In the TAL, the
function of these K+ channels is not only to supply the
K+-dependent cotransporters with their substrate. They also
contribute to the cell's negativity, supporting the movement of Cl-
ions from the cell to the internal medium across the basolateral membrane, and
they are the basis for the electrogenicity of the overall absorption process,
allowing cation absorption along the paracellular pathway under open-circuit
conditions.
As seen with split gill lamellae of the shore crab C. maenas,
furosemide had no effect on Isc or Gte across
split gill lamellae of C. granulatus. In the shore crab, however, a
dependence of Cl- absorption on potassium was demonstrated, and
therefore it was concluded that NaCl absorption proceeds via apical
Na+/K+/2Cl- symporters
(Riestenpatt et al., 1996) in
this species. So the presence of apical K+ channels in an
epithelium generating coupled NaCl absorption and a negative
Isc suggests the involvement of this symporter (see above,
cf. Greger and Kunzelmann,
1990
). Thus in C. granulatus, as in C. maenas,
we propose that apical K+ channels and
Na+/K+/2Cl- symporters are the basis of
electrogenic, coupled NaCl absorption (see
Fig. 6).
|
As seen in the shore crab C. maenas
(Onken and Siebers, 1992),
acetazolamide, a blocker of the carbonic anhydrase
(Maren, 1967
), had no effect
on Isc or Gte across split gill lamellae of
C. granulatus. However, approximately 20% of the Na+
influx across isolated and perfused gills was inhibited by this drug (see
Fig. 5) without affecting the
transbranchial voltage. This finding suggests that at least a part of NaCl
absorption depends on the rapid supply of H+ and
HCO3-. We therefore propose that electrogenic NaCl
absorption as described above is accompanied by electroneutral NaCl absorption
via apical Na+/H+- and
Cl-/HCO3--antiports (see
Fig. 6), as has been described
for the cortical TAL of the mouse and rat nephron
(Greger, 1985
). However, this
proposal requires further investigation.
The basolateral membrane
The inhibition of Isc by internal ouabain (see
Fig. 2) is consistent with
recent Na+ flux measurements obtained with isolated and perfused
gills (Luquet et al., 2002)
and shows that Na+-dependent Cl- absorption is driven by
the basolateral Na+/K+-ATPase. The reduction in
Isc seen with the Cl- channel blocker DPC
(Di Stefano et al., 1985
; see
Fig. 3) indicates that
Cl- ions, which are most likely to be absorbed by a secondary
active mechanism across the apical membrane (see above), exit from the cells
via basolateral Cl- channels. The effect of internal
BaCl2 (see Fig. 3),
similarly already observed with isolated and perfused gills
(Luquet et al., 2002
),
indicates the presence of K+ channels in the basolateral membrane.
Their involvement in NaCl absorption can be considered as the basis for a
negative cellular potential, participating in the driving force for the inward
movement of Cl- ions through channels in the basolateral membrane.
Na+/K+-ATPase, Cl- channels and K+
channels (see above) are the usual `equipment' present in basolateral
membranes of many NaCl absorbing epithelia
(Greger and Kunzelmann, 1990
),
and have also been found in the other crab gill epithelia studied so far
(Onken et al., 1991
;
Riestenpatt et al., 1996
;
Onken and McNamara, 2002
).
Comparative aspects
The results obtained in the present study clearly indicate that the basic
epithelial characteristics and the mode of active NaCl absorption are very
similar in hyperosmoregulating C. maenas and C. granulatus.
Both animals are strong hyperosmoregulators which, however, are not able to
survive in freshwater. The epithelium of the posterior gills of both animals
displays a high conductance and a directly coupled NaCl absorption, similar to
the TAL. It might well be that these characteristics are general features of
animals from brackish waters, contrasting with the patterns of the gills of
freshwater crabs and the absorptive epithelia of freshwater vertebrates (low
conductance and electrical coupling of NaCl absorption via different
pathways; see Introduction). It is obvious, however, that this hypothesis
requires further studies on more animals from brackish waters.
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Acknowledgments |
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