Ion-motive ATPases and active, transbranchial NaCl uptake in the red freshwater crab, Dilocarcinus pagei (Decapoda, Trichodactylidae)
1 Department of Animal Physiology, University of Osnabrueck, 49076
Osnabrueck, Germany
2 Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras
de Ribeirão Preto, Universidade de São Paulo, Avenida
Bandeirantes 3900, Ribeirão Preto 14040-901, São Paulo,
Brasil
3 Mount Desert Island Biological Laboratory, Salsbury Cove, ME 04672,
USA
4 School of Biological Sciences, Washington State University, Pullman, WA
99164-4236, USA
* Author for correspondence (e-mail: weihrauch{at}biologie.uni-osnabrueck.de)
Accepted 5 October 2004
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Summary |
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Key words: osmoregulation, crab, Dilocarcinus pagei, gill epithelia, phylogenetic analysis, NaCl uptake, Na+/K+-ATPase, V-type H+-ATPase, transepithelial voltage, short-circuit current, ouabain, concanamycin
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Introduction |
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A current model for NaCl uptake in freshwater crustaceans has been proposed
based on data obtained using gills of the hyperosmoregulating, diadromous
crab, Eriocheir sinensis (for reviews see
Péqueux et al., 1988;
Péqueux, 1995
;
Onken and Riestenpatt, 1998
).
Sodium uptake in this crab is Cl-independent and proceeds
via apical Na+-channels and the basolateral
Na+/K+-ATPase
(Zeiske et al., 1992
).
Na+-independent, chloride uptake may take place via apical
Cl/HCO3 exchange and
basolateral Cl channels, driven by the apical V-ATPase
(Onken et al., 1991
). Carbonic
anhydrase (CA) may rapidly supply the intracellular substrates, H+
and HCO3, necessary for apical transporter
function.
Most studies of NaCl absorption in hyperosmoregulating crabs concern
diadromous or migratory species (for reviews, see
Péqueux, 1995;
Onken and Riestenpatt, 1998
);
information on true, continental, freshwater brachyurans is fairly scarce. In
a recent study (Onken and McNamara,
2002
) we established the South American freshwater crab,
Dilocarcinus pagei, endemic to the Amazon and Paraguay/Paraná
river basins, as a transport model for such hololimnetic crabs. Our data
suggest that active, electrogenic NaCl uptake across the posterior gills of
D. pagei occurs via mechanisms similar to those of other
freshwater animals. However, in contrast to the known epithelia responsible
for NaCl absorption from highly dilute media, Na+ and
Cl uptake in the posterior gills of D. pagei are
effected across epithelia spatially separated within the gill lamellae, which
exhibits a marked structural asymmetry. The epithelium of the proximal
lamellar surface consists of thick cells, and generates a positive,
Na+-dependent, ouabain-sensitive, short-circuit current; the distal
epithelium is composed of thin, pillar cell flanges, and generates a negative,
Cl-dependent, short circuit current, which is inhibited by
diphenylamine-2-carboxylate (DPC) and acetazolamide (AZ).
To better characterize the mechanisms of gill ion absorption in hololimnetic crabs, in the present investigation we analyze the microanatomy, expression of mRNA and the activity of ion-motive ATPases in the anterior and posterior gills of D. pagei. We also provide electrophysiological data that support a role for apical V-ATPases in active, transbranchial Cl uptake.
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Materials and methods |
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To obtain the gills, the crabs were quickly killed by destroying the dorsal
brain and the ventral ganglia using large scissors. The carapace was removed
and anterior gill no. 4 and posterior gill no. 7 were excised at their bases
with fine scissors, and removed with tweezers (see also
Onken and McNamara, 2002). The
gills were immediately prepared for light microscopic analysis and
physiological experiments, or stored in RNA Later buffer (Ambion; Austin,
Texas, USA) for subsequent RNA isolation.
Microscopic studies
After dissection on ice, the gills were immediately perfused via
the afferent vessel over a 23 min period with 1 mlice-cold, primary
fixative containing (in mmol l1): paraformaldehyde 200,
glutaraldehyde 250, and Na+ 100, K+ 10, Ca2+
13, Mg2+ 2 (as chlorides), buffered in 100 mmol
l1 sodium cacodylate, at pH 7.5. Medial portions of selected
gills consisting of approximately five lamellae each were then fixed on ice in
fresh primary fixative for 1.5 h. After rinsing in buffered saline alone
(3x5 min), the gill lamellae were post-fixed in 1% osmium tetroxide in
buffered saline for 1 h,dehydrated in an ethanol/propylene oxide series and
embedded in Araldite 502 resin. Thick (0.5 µm) sections were prepared using
a Porter-Blum Sorvall MT-2B ultramicrotome and stained using a mixture of 1%
methylene and Toluidine Blue in 1% aqueous borax. Digital images of sections
at 200400x magnification were taken using a Zeiss Axiophot
microscope and Zeiss AxioVision 3 image acquisition software.
Molecular cloning of partial Na+/K+-ATPase and V-type H+-ATPase cDNA sequences
The total RNA from anterior gill no. 4, and from posterior gill no. 7,
respectively, was extracted under RNAse-free conditions using reagents
supplied by Promega Corporation. After DNAse I treatment
(Gibco-BRL/Invitrogen, Karlsruhe, Germany), mRNA reverse transcription was
performed using oligo(dT) primers and Superscript II reverse transcriptase
(Gibco-BRL/Invitrogen, Karlsruhe, Germany).
Degenerate primers, previously used successfully with other crab species
for the Na+/K+-ATPase (-subunit), primer pair NAK
10F/NAK 16R (Towle et al.,
2001
), and for the V-type H+-ATPase (B-subunit), primer
pair HATF2/HATR4 (Weihrauch et al.,
2001
) (see Table
1), were employed in a polymerase chain reaction (PCR) procedure
(REDTaq, Sigma, Taufkirchen, Germany) to initially amplify the partial cDNA
for both cation pumps from D. pagei gills.
|
PCR products were separated electrophoretically on 1% agarose gels,
extracted from the gel slices (Qiagen, Valencia, CA, USA), and sequenced
automatically using the dideoxynucleotide method
(Sanger et al., 1977) at the
Marine DNA Sequencing Center of Mount Desert Island Biological Laboratory,
Maine, USA employing the degenerate PCR primers in the sequencing reaction.
Fragment sequences were assembled using DNASTAR software and were analyzed for
open reading frames employing DNASIS. A search of GenBank using the BLAST
algorithm (Altschul et al.,
1997
) revealed close matches with sequences previously published
for the Na+/K+-ATPase (
-subunit) and the V-type
H+-ATPase (B-subunit). The sequences obtained were then employed to
design specific primers for D. pagei
(Table 1) using Primer Premier
software. Multiple alignments were performed with Multalin
(Corpet, 1988
) and GeneDoc
software
(http://www.psc.edu/biomed/genedoc/).
A phylogenetic tree was generated using Drawtree
(Felsenstein, 1993
).
Semi-quantitative RT-PCR
The relative abundance of the Na+/K+-ATPase
-subunit and the V-type H+-ATPase B-subunit in total RNA
extracts was estimated by semi-quantitative RT-PCR using identical amounts (2
µg) of total RNA in each reverse transcription reaction. The PCR procedure
was performed under conditions in which product formation was directly
dependent on cDNA template availability. To illustrate, as shown in
Fig. 1, different amounts
(0.25, 0.5 and 1 µl) of template cDNA were employed in a polymerase chain
reaction (primers DiloHATF1/DiloHATR1, see
Table 1) using a number of
cycles in which PCR product amplification was clearly in the logarithmic phase
(23 cycles). Band densities were estimated under UV light by digital analysis
using a Kodak Digital Science DC 120 Zoom Digital Camera and Kodak Digital
Science 1D 2.0.2 software (Kodak).
|
ATPase activity measurements
Gills were homogenized on ice using a 1 ml glass homogenizer in ice-cold
homogenization buffer, containing 10 mmol l1 Hepes, 5 mmol
l1 EDTA and 250 mmol l1 sucrose (pH 7.25).
For V-ATPase activity measurements, the homogenization buffer also contained
4.5 mmol l1 mercaptoethanol and 0.05% (w/v)
C12E10.
ATPase activities were estimated according to the method of Onishi et al.
(1976) and quantified as
released inorganic phosphate. SDS activation and measurement of
Na+/K+-ATPase activity were performed according to the
method of Kosiol et al.
(1988
), using 1020
µg protein in each assay. Na+/K+-ATPase activity was
defined as the difference in SDS-activated activities obtained with or without
5 mmol l1 ouabain (a Na+/K+ATPase
inhibitor). Mitochondrial F1F0-ATPase activity was
estimated similarly; however, inhibition by 1 mmol l1 sodium
azide was used to define F1F0-ATPase activity.
To measure V-ATPase activity, a reaction buffer containing 50 mmol
l1 MopsTris, 1 mmol l1 sodium
vanadate (inhibition of P-type ATPases), 1 mmol l1 sodium
azide (inhibition of F1F0-ATPase), 0.01% (w/v)
C12E10, 3 mmol l1 TrisATP and 3
mmol l1 MgCl2 (pH 8.0) was preincubated for 10
min at 30°C. The reaction was initiated by adding the respective gill
homogenate (50100 µg protein), and after 30 min, the amount of
phosphate released was measured as above
(Kosiol et al., 1988).
V-ATPase activity was defined as the difference between the activities
obtained with or without 1 µmol l1 concanamycin (a
V-ATPase inhibitor; Dröse and
Altendorf, 1997
).
Protein content was measured according to Bradford's method
(Bradford, 1976), modified
following Sedmark and Grossberg
(1977
), with bovine serum
albumin as the standard.
Electrophysiological measurements
Gill lamellae were isolated from a medial portion of the posterior gills
and mechanically split using two pairs of ultrafine tweezers (cf.
Onken et al., 1991). Distal
split lamellae were mounted using a dissecting microscope in a modified Ussing
chamber, such that an epithelial surface area of 0.01 cm2 was
exposed to the chamber compartments (approximately 50 µl volume) bathing
the respective external and internal surfaces of the split lamella. Both
chamber compartments were continuously perfused with aerated hemolymph-like
saline (see Onken and McNamara,
2002
) by gravity flow at a rate of approximately 2 ml
min1. For voltage measurements, calomel electrodes were
connected via agar bridges (3% agar in 3 mol l1 KCl) to both
sides of the preparation, the distance from the bridge tip to the tissue being
less than 1 mm. The reference electrode was placed in the internal,
hemolymph-side bath. Silver wires coated with AgCl served as electrodes to
apply current for short-circuiting the transepithelial voltage (i.e.
measurement of the short-circuit current, Isc) through an
automatic clamping device (model VCC 600; Physiologic Instruments, San Diego,
CA, USA). The conductance of the preparations (Gte) was
calculated from imposed voltage pulses (
V) and the resulting
current deflections (
I). The data were recorded continuously
on a chart recorder (type 3229 I/85; Linseis, Germany).
Statistical analyses
All data are given as the mean ± S.E.M. Differences
between mean values were compared using Student's t-test, employing a
significance level of P=0.05.
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Results |
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mRNA expression of Na+/K+-ATPase and V-type H+-ATPase
To identify expression of the Na+/K+-ATPase
-subunit and V-type H+-ATPase B-subunit in gill epithelia of
the freshwater crab, D. pagei, we employed degenerate oligonucleotide
primer pairs (NAK F10/NAK 16R for the
-subunit, and HATF2/HATR4 for the
B-subunit, see Table 1), known
to specifically amplify the desired target sequences in other crab species
(Weihrauch et al., 2001
;
Towle et al., 2001
). PCR
amplification, employing the primer pair NAK F10/NAK 16R, resulted in a 700 bp
product, which was gel-purified and sequenced. A BLAST search of GenBank
clearly identified the amplified sequence as a
Na+/K+-ATPase
-subunit fragment (GenBank
accession no. AF409119). Comparison of the deduced amino acid sequence
revealed a high homology with the Na+/K+-ATPase
-subunits of other invertebrate and vertebrate species
(Fig. 3). Employing the primer
combination HATF2/HATR4 resulted in a 392 bp product (GenBank accession no.
AF409118). After gel purification and sequencing, a BLAST search of GenBank
revealed extremely high homologies between the derived amino acid sequence for
the putative H+-ATPase B-subunit from D. pagei and
H+-ATPase B-subunits from other invertebrate and vertebrate species
(Fig. 4). A phylogenetic
analysis of the B-subunit cDNA sequences from D. pagei, Carcinus
maenas (GenBank accession no. AF189779), Callinectes sapidus
(GenBank accession no. AF189780), Eriocheir sinensis (GenBank
accession no. AF189782), Chasmagnathus granulata (GenBank accession
no. AF189783) and Cancer irroratus (GenBank accession no. AF189781)
revealed the expected phylogenetic relationships among these species: C.
maenas and C. sapidus in the Portunidae (0.0706 percentage
divergence), E. sinensis and C. granulata in the Varunidae
(Martin and Davis, 2001
)
(0.0161 percentage divergence), D. pagei in the Trichodactylidae, and
C. irroratus in the Cancridae
(Fig. 5). Both D.
pagei and C. irroratus maintain large phylogenetic distances
from the portunid species (0.10.12 and 0.110.13 percentage
divergence, respectively) and the varunid species (1.11.2 and 1.4
percentage divergence, respectively).
|
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To evaluate the importance and distribution of the Na+/K+-ATPase and the V-ATPase within the gills of the true freshwater crab, D. pagei, mRNA expression analysis and enzyme activity measurements were performed.
Semi-quantitative RT-PCR
Semi-quantitative RT-PCR clearly revealed that the mRNA for both ion pumps,
the Na+/K+-ATPase (-subunit) and the V-ATPase
(B-subunit) are more highly expressed in the posterior than anterior gills.
Compared to the PCR signal for posterior gills, amplification of both ATPases
in the anterior gills required two additional PCR logarithm-phase cycles to
produce equivalent or lower signals (Fig.
6).
|
ATPase activities
The protein content and activities of the
Na+/K+-ATPase, the V-type H+-ATPase and the
F1F0-ATPase were measured in the anterior and posterior
gills of D. pagei (Table
2). Both protein content and Na+/K+-ATPase
activity were significantly higher in the posterior compared to anterior
gills. F1F0-ATPase and V-type H+-ATPase
activities did not differ significantly between anterior and posterior gill
homogenates.
|
Electrophysiological measurements
Onken and McNamara (2002)
have shown that ouabain inhibits the positive, Na+-dependent,
short-circuit current (Isc) across the thick, proximal,
split lamellae of D. pagei posterior gills when mounted in an
Ussing-type chamber. Here, we examined the effects of ouabain and concanamycin
on the negative, Cldependent Isc, and on
preparation conductance (Gte) in the thin, distal, split
lamellae. While internal ouabain (2 mmol l1) had no
significant effect on Isc, internal concanamycin (10
µmol l1) significantly reduced Isc by
approximately 50%, from 43±3 to 22±1 µA
cm2 (mean ± S.E.M.; N=3;
P<0.05) without affecting transepithelial conductance
(Gte). External concanamycin produced only a minor effect
on Isc. A representative experiment is shown in
Fig. 7.
|
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Discussion |
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Consequent to an initial study by Onken and McNamara
(2002), the present
investigation provides a detailed structural and functional analysis of the
gill epithelium in a true freshwater crab. In general, the known
specializations of the anterior and posterior gills of diadromous crabs are
also present in the hololimnetic crab, D. pagei. However, the gills
of D. pagei also exhibit striking modifications to this pattern that
may constitute a general feature of the trichodactylid crabs.
Gill microanatomy
Onken and McNamara (2002;
see also Fig. 2) described a
significant structural asymmetry in the posterior gill lamellae of D.
pagei, apparently unique among crabs studied so far. While the proximal
side of the lamella is lined by a thick epithelium, similar to that found
symmetrically distributed on both sides of the ion transporting cell patches
in the posterior lamellae of diadromous crabs (see
Taylor and Taylor, 1992
), the
distal side has a fine epithelium consisting exclusively of thin pillar cell
flanges. Thus, unlike the diadromous crabs, the distal side of the posterior
gill lamellae in D. pagei may contribute substantially to respiratory
gas exchange. Since a dense osmiophilic area, which delimits the thick
epithelium, was found only in the posterior gills, analysis of the structural
asymmetry lead to the premise that hololimnetic crab gills might exhibit
adaptations different from those of diadromous crab gills. Thus, the present
investigation directly compares the microanatomy of the anterior and posterior
gill lamellae. The anterior gills of D. pagei are symmetrically and
uniformly lined by a very attenuated epithelium (see
Fig. 2A), similar to the
situation in diadromous crabs. Comparison of this anterior lamellar epithelium
with the thin, distal epithelium of the posterior gill lamellae shows that the
latter is several times thicker, but, exhibiting more pronounced apical
membrane foldings and vesicles, reflecting a larger surface area. Thus, the
present morphological findings suggest that the anterior gills of D.
pagei share the same specialization for respiratory gas exchange as the
diadromous species. On a more microanatomical, architectural level, the
extraordinary difference between D. pagei and diadromous species is
restricted exclusively to the posterior gills. The notably different
thicknesses of the proximal and distal epithelia of the posterior gill
lamellae derive from two special features. First, the posterior lamellae of
diadromous crabs have pillar cells on both sides of the lamella, but in D.
pagei this cell type is restricted to the distal side. Second, the pillar
cells of D. pagei exhibit very broad, laterally attenuated, apical
flanges, a feature not seen in similar cells from diadromous species.
Interestingly, however, a similar structural arrangement is encountered in the
gills of freshwater, palaemonid shrimps
(Freire and McNamara, 1995
;
McNamara and Lima, 1997
), also
very efficient hyperosmoregulators (Freire
et al., 2003
).
Ion-motive ATPases
Transepithelial ion transport derives from the activity of members of three
ion-motive ATPase families. While mitochondrial F-ATPases generate ATP,
functioning as ATP synthetases under physiological conditions, P-ATPases and
V-ATPases use ATP to generate and maintain transmembrane ionic gradients
subsequently used as an energy source by passive transporters. P-ATPases and
V-type H+-pumps thus constitute the motors of transmembrane and
transepithelial transport.
The analysis of mRNA expression of the Na+/K+-ATPase
-subunit and the V-ATPase B-subunit clearly indicates that both cation
pumps are expressed in the gill tissue of the freshwater crab, D.
pagei. As shown in Figs 3
and 4, both the
-subunit
of the Na+/K+-ATPase and the B-subunit of the V-ATPase
are highly conserved across the animal kingdom. The deduced, partial amino
acid sequence of the Na+/K+-ATPase
-subunit from
D. pagei (202 amino acids) was 9599% homologous with the
-subunit sequences of other crab species published in GenBank. An even
higher homology (99100% identities) with other crab species was found
on comparing the partial amino acid sequences of the B-subunit (V-ATPase, 124
amino acids). The cDNA sequence of this highly conserved region of the
B-subunit was therefore used for a phylogenetic analysis among crab species.
The result, shown in Fig. 5,
confirms the previous classification and grouping based on morphological
criteria
mRNA expression and enzyme activity measurements of the
Na+/K+-ATPase in D. pagei showed significantly
higher levels in the posterior compared to anterior gills (see
Fig. 6),consistent with
molecular biological and physiological studies on other crab species
(Siebers et al., 1982;
Onken and Putzenlechner, 1995
;
Towle and Weihrauch, 2001
;
Lucu and Towle, 2003
), which
have designated the posterior gill epithelium, with its high abundance and
Na+/K+-ATPase activity, as the principal site of
osmoregulatory ion transport. Greater expression of V-ATPase mRNA was also
detected in posterior compared to anterior gills; however, V-ATPase activities
did not differ significantly between anterior and posterior gills. This
contrasts with findings from freshwater-adapted Chinese crabs, Eriocheir
sinensis, where enzyme activity
(Onken and Putzenlechner,
1995
) and mRNA expression
(Weihrauch et al., 2001
) are
significantly higher in posterior compared to anterior gills.
In the present study on D. pagei, we employed the same assay used
by Onken and Putzenlechner
(1995) to measure ATPase
activities in E. sinensis. A comparison of the data shows that the
posterior gills of E. sinensis exhibits markedly higher F-, V- and
Na+/K+-ATPase activities than those of D.
pagei. This difference may reflect a smaller passive salt loss in D.
pagei, and thus, lower, compensatory active NaCl absorption. In
particular, low passive salt losses have been reported in hololimnetic crabs
(Shaw, 1959
;
Harris, 1975
;
Greenaway, 1981
;
Morris and Van Aardt, 1998
).
While the activities of all three ATPases are markedly reduced in the anterior
gills of E. sinensis, in D. pagei the anterior and posterior
gills exhibit similar V- and F-ATPase activities, which suggests that the
anterior gills of D. pagei may generate ATP for an as-yet-unknown
V-ATPase-driven process. One possibility is branchial ammonium excretion. In
the euryhaline crab Carcinus maenas, where V-ATPases seem not to be
involved in gill osmoregulatory NaCl uptake, V-ATPase mRNA levels are higher
in the anterior gills (Weihrauch et al.,
2001
), and higher active ammonia transport rates have been found
in the anterior compared to posterior gills
(Weihrauch et al., 1998
).
Furthermore, V-ATPases are directly involved in transbranchial ammonia
transport in C. maenas (Weihrauch
et al., 2002
). Whether V-ATPase activity in the anterior gills of
D. pagei is also related to increased branchial ammonia excretion
remains to be evaluated in further studies.
Transbranchial NaCl absorption
Onken and McNamara (2002)
demonstrated that the opposing sides of the posterior gill lamellae in D.
pagei generated short-circuit currents (Isc) of
opposite polarity. With perfusion of identical salines on both sides, the
thick, proximal epithelium generated a positive Isc
dependent on Na+ ions, and inhibited by hemolymph-side ouabain. The
thin, distal epithelium generated a negative Isc,
dependent on Cl ions, and inhibited by hemolymph-side,
Cl channel and carbonic anhydrase blockers. These authors
proposed that an apparently independent absorption of Na+ and
Cl is reflected in such currents and takes place across the
different sides of the lamellae: Na+/K+-ATPase-driven
Na+ absorption across the proximal epithelium, and V-ATPase-driven
Cl absorption across the distal epithelium. In the present
study, we used concanamycin to evaluate the role of V-ATPase in generating the
negative, Cl-dependent Isc. While
addition of the Na+/K+ATPase inhibitor ouabain
(Skou and Hilberg, 1969
) to
the hemolymph-side had no effect on the negative Isc, the
V-ATPase inhibitor concanamycin (Huss et
al., 2002
) significantly reduced this current
(Fig. 7), a result that
indicates that the negative current is indeed independent of a functioning
Na+/K+-ATPase, and that Cl transport
is apparently driven by the V-ATPase. In the NaCl-absorbing epithelia of
freshwater animals, V-ATPases are usually localized in the apical membrane
where they drive Na+ and/or Cl absorption
(Kirschner, 2004
). However,
the external application of concanamycin to the distal side of the posterior
gill lamella in D. pagei had only a minor effect on the negative
Isc (Fig.
7). A basolateral localization of the V-ATPase could not account
for this negative Isc and its inhibition by concanamycin.
Thus, we propose that internally perfused concanamycin exerts its effect only
after diffusing to the apical membrane, and that externally applied
concanamycin either does not, or barely reaches its site of action, owing to
the thick cuticle covering the apical membrane. Clearly, further studies are
necessary to establish the location of the V-ATPase in this epithelium.
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Acknowledgments |
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