Electrophysiology of posterior, NaCl-absorbing gills of Chasmagnathus granulatus: rapid responses to osmotic variations
1 Dept Biodiversity and Experimental Biology, F.C.E.N. University of Buenos
Aires, Pab. II, Ciudad Universitaria, C1428EHA Buenos Aires,
Argentina
2 Dept Biological Sciences, F.F.C.L.R.P. University of Sao Paulo,
Brazil
* Author for correspondence (e-mail: 3guerres{at}bg.fcen.uba.ar)
Accepted 30 October 2002
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Summary |
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Key words: Chasmagnathus granulatus, crab, cyclic AMP, gills, hyperosmoregulation, perfused gills, Na+/K+-ATPase, short-circuit current, split gill lamellae, transepithelial conductance, transepithelial voltage
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Introduction |
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With respect to active NaCl secretion in hypoosmoregulating C.
granulatus, Luquet et al.
(2002) demonstrated its
presence in posterior gills and its dependence on a functioning
Na+/K+-ATPase. However, as in other hypoosmoregulating
crabs (Green et al., 1959
;
Baldwin and Kirschner,
1976a
,b
;
Evans et al., 1976
), the
entire mechanism is still unknown.
C. granulatus has developed bimodal ventilation and spends long
periods on land (Halperin et al.,
2000). These semi-terrestrial habits force C. granulatus
to deal with sudden salinity changes: the water available in supratidal areas
can vary from tide pools concentrated by evaporation to rain pools with
significantly diluted seawater. Besides, during land visits the water retained
within the gill chambers might evaporate, exposing the gills to high salinity
conditions (Schmidt and Santos,
1993
). As a consequence, the gills of this species must be able to
rapidly switch between absorption, secretion and no transport. One way of
changing between different transport states would be endocrine regulation, as
observed in a couple of crab species
(Sommer and Mantel, 1988
;
Kamemoto, 1991
;
Mo et al., 1998
;
Onken et al., 2000
;
Morris, 2001
). An alternative
would be hormone-independent transport regulation triggered by the osmolarity
of the internal medium, as has been demonstrated in the amphibian skin
(Ussing, 1965
) and other
vertebrate epithelia (for a review, see
Macknight, 1991
) but also in
the gills of Chinese crabs Eriocheir sinensis
(Onken, 1996
). A decreasing
internal osmolarity resulted in a rapid stimulation of NaCl absorption,
whereas an increasing internal osmolarity resulted in decreased transport
rates. Thus, the osmotic influence on transport rates stabilises the
hemolymph/blood osmolarity and was, therefore, called autoregulation (cf.
Onken, 1996
). In the gills of
E. sinensis, the osmotic variations were shown to modulate the apical
transporters involved in NaCl absorption (V-type H+-ATPase and
Na+ channels; Onken,
1996
).
The presence of an autoregulatory mechanism in C. granulatus has
been suggested in a previous study of the transepithelial voltage generated by
isolated and perfused gills of C. granulatus adapted to diluted
seawater (Luquet et al.,
2002). To verify and to further characterise this regulation of
NaCl absorption by osmotic changes, the present study investigated the
influences of short-term osmotic variations on the transepithelial voltage
generated by isolated and perfused gills, the short-circuit current across
split gill lamellae mounted in a modified Ussing chamber, and the activity of
the Na+/K+-ATPase.
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Materials and methods |
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Gill perfusion and measurement of the transepithelial potential
difference (Vte)
Crabs were sacrificed by destroying the ventral nervous ganglion with a
spike. After removing the dorsal carapace, gill pair no. 6 (representative for
posterior gills; Luquet et al.,
2002) was removed and used for the experiments. The afferent and
efferent vessels were connected by 0.4-mm diameter polyethylene tubing to a
peristaltic pump (afferent) and to a glass tube (efferent). Perfusion rate was
kept at 0.1 ml min-1. The tubing was held in position by an acrylic
clamp and the preparation put into a glass beaker with the appropriate saline
and constant aeration. Under these conditions, isolated perfused gill
preparations can remain viable for up to 15 h
(Siebers et al., 1985
).
For the measurements of transepithelial voltage, Ag/AgCl electrodes were connected by agar bridges to the external bath and to the glass tube collecting the perfusate. Vte was measured with a millivoltmeter (Metrix, Paris, France) and is given as the difference in electrical potential between the external and internal medium (reference electrode connected to the internal perfusate).
Split gill lamellae and measurement of short-circuit current
(Isc) and transepithelial conductance
(Gte)
Single gill lamellae were isolated and split under microscopic control
according to Schwarz and Graszynski
(1989). The split gill
lamellae were mounted in a modified Ussing chamber
(De Wolf and Van Driessche,
1986
). An epithelial area of 0.002 cm2 was exposed to
the chamber compartments (volume approximately 50 µl), bathing the internal
and external sides of the tissue. Continuous perfusion of both chamber
compartments with aerated saline was achieved by gravity flow at a constant
rate of approximately 2 ml min-1.
To measure Vte, Ag/AgCl electrodes were connected
via agar bridges (3% agar in 3 mol l-1 KCl) to both sides
of the preparation (distance from the tissue, <1 mm). The reference
electrode was in the internal bath. Silver wires coated with AgCl served as
electrodes to short-circuit the transepithelial voltage by an automatic
clamping device (VCC 600; Physiological Instruments, San Diego, USA). The
transepithelial conductance (Gte) was calculated from
imposed voltage pulses and the resulting current deflections
(I). As the resistance of the preparation was small, the
values of Gte and Isc were corrected
for the influence of the resistance of the salines (for details, see
Riestenpatt et al., 1996
). In
the Results, only the corrected values are shown.
Activity of the Na+/K+-ATPase
Posterior gill pair no. 6 was dissected and perfused as described above.
The two gills from the same crab were perfused at the same time with
Isosucrose saline, and Vte was monitored. Once
Vte became stable, one of the gills was perfused with
Hyposmotic saline (treated), while the other continued as before (control).
When the treated gill achieved a new stable Vte value,
both gills were disconnected from the peristaltic pump, cut at the clamp level
and immediately frozen at -40°C until the
Na+/K+-ATPase activity assay was performed. In previous
assays, freezing and thawing the gills did not produce any significant effect
on the Na+/K+-ATPase activity, as the obtained values
for frozenthawed and freshly excised tissues were in the same
range.
For the measurement of Na+/K+-ATPase activity, the
gills were placed in 20 volumes of cold buffer [12.5 mmol l-1 NaCl,
1 mmol l-1 Hepes, 0.5 mmol l-1 EDTA, 0.5 mmol
l-1 PMFS (phenylmethylsulfonyl fluoride), adjusted to pH 7.6 with
NaOH] and homogenized in a teflonglass homogeniser (20 strokes).
Homogenates were centrifuged at 11 000 g for 20 min at
4°C. Supernatants were discarded and pellets were resuspended in cold
buffer [in C. granulatus gills, the greatest
Na+/K+-ATPase activity is detected in this fraction
(Rodriguez Moreno et al.,
1998; G. Genovese, C. Luchetti and C. M. Luquet, unpublished
data)].
Na+/K+-ATPase activity was determined as described
previously by Lucu and Flik
(1999) by incubating 10 µl
of sample with 500 µl assay solution, containing 100 mmol l-1
NaCl; 5 mmol l-1 MgCl2; 0.1 mmol l-1 EDTA; 15
mmol l-1 imidazol; 3 mmol l-1 Na2ATP and 12.5
mmol l-1 KCl with or without 1 mmol l-1 ouabain (pH 7.5;
histidine-imidazol). Assay mixtures were then incubated for 30 min at
37°C. The reaction was stopped by the addition of 1 ml 8.6% cold
trichloroacetic acid. Liberated phosphate was quantified colorimetrically
according to the modified method of Bonting and Cavaggio
(1963
) by adding 1 ml of a
freshly made solution of 9.2% Fe2SO4.7H2O and
1.14% ammonium heptamolybdate in 3.63% H2SO4. After 30
min incubation at room temperature, the absorbance was recorded at 700 nm. The
difference between phosphate released with and without ouabain was attributed
to Na+/K+-ATPase activity. Protein concentration was
determined in triplicate using the method of Lowry et al.
(1951
). Specific
Na+/K+-ATPase activity was expressed in µmol
Pih-1 mg protein-1.
Solutions and chemicals
Table 1 shows the
composition of the salines used in the different experiments with perfused
gills and with split gill lamellae. The osmolarity and ionic composition of
Isosmotic saline is similar to the hemolymph of C. granulatus adapted
to 30 salinity, where the crabs hardly maintain an osmotic gradient
across their body surface (cf. Mougabure
Cueto, 1998
; Charmantier et
al., 2002
). Hyposmotic saline is composed according to the
hemolymph of crabs adapted to a hyposmotic ambient medium of approximately
2
salinity (cf. Mougabure Cueto,
1998
; Charmantier et al.,
2002
). The osmolarity of the salines Clred and
Nared is adjusted to the hemolymph of crabs adapted to 30
salinity. However, the concentrations of sodium or chloride are reduced to the
level of Hyposmotic saline by partly substituting NaCl with choline chloride
or NaNO3, respectively. In the Hyposucrose salines
(Hyposucrose 1-4), the osmolarity of Hyposmotic saline is stepwise
increased by addition of sucrose until reaching the osmolarity of Isosmotic
saline (Isosucrose). All solutions were adjusted with Tris base to
the physiological pH of C. granulatus (7.75;
Luquet and Ansaldo, 1997
).
|
Theophylline was obtained from Serva, New York, USA. Forskolin and ouabain were purchased from Sigma, St Louis, USA. All other salts and reagents were purchased from Merck, Buenos Aires, Argentina.
Statistics
All data are given as means ± S.E.M. Differences between groups were
tested using Student's t-test, paired Student's t-test or
repeated-measures analysis of variance (RM-ANOVA) when appropriate.
Differences were considered significant at P<0.05.
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Results |
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With Isosmotic saline as perfusate and bath, the transbranchial voltage
stabilised at 2.14±0.32 mV and 1.36±0.13 mV in the
Clred and Nared experiments, respectively (external side
positive; N=8 for each experiment); i.e. at very similar values to
those observed in a previous study under similar conditions with C.
granulatus adapted to 12 salinity
(Luquet et al., 2002
). No
significant changes were observed after reducing the concentrations of
chloride (2.16±0.41 mV; N=8) or sodium (1.34±0.13 mV;
N=8) at constant osmolarity. However, when changing to Hyposmotic
saline, Vte rapidly increased to significantly higher
values (11.89±1.48 mV and 5.36±0.44 mV, respectively;
N=8 in each case). These results are summarised in
Fig. 1.
|
In another series of experiments, we changed between Isosmotic saline, Hyposmotic saline and salines of intermediate osmolarities prepared by adding sucrose to the basolateral Hyposmotic saline (salines Hyposucrose 1-4 and Isosucrose). A representative time-course of one of these experiments is shown in Fig. 2, demonstrating the fast and reversible effects of osmotic changes. With Isosmotic saline and with Isosucrose saline, the transepithelial voltage is low and not significantly different (1.14±0.30 mV and 1.88±0.25 mV, respectively; N=5). However, reduction of the saline's osmolarity significantly increased Vte (to 9.14±1.29 mV; N=5). In Fig. 3, the response of Vte to gradually reduced osmolarities is summarised, normalising the Vte increase with Hyposmotic saline to 100% response.
|
|
Osmotic variations and their effects on Isc
and Gte across split gill lamellae
The transepithelial voltage is not a measure of transport rates and depends
significantly on the resistance of the paracellular pathway. Thus, it may be
that the above-demonstrated Vte changes are due to
variations in the paracellular resistance at different osmolarities. To verify
that osmotic changes influence the transport rates and not only the
paracellular resistance, we conducted a series of experiments (N=3)
with split gill lamellae mounted in a modified Ussing chamber, measuring the
short-circuit current (Isc; which depends only on
transcellular parameters) and the transepithelial conductance
(Gte).
The open-circuit voltage measured with split gill lamellae was in the same range as observed with isolated and perfused gills. Fig. 4 shows a representative time-course of an Isc measurement where we changed between Isosmotic saline, Isosucrose saline and Hyposmotic saline. The negative Isc was hardly affected when NaCl was reduced at constant osmolarity (changing from Isosmotic saline to Isosucrose saline). However, Gte was decreased after reduction of the NaCl concentration. When the osmolarity was reduced, the negative Isc approximately doubled and Gte also increased again. The results of these experiments are shown in Table 2.
|
|
Osmotic variations and their effect on
Na+/K+-ATPase activity
In order to investigate the possible involvement of a functioning
Na+/K+-ATPase in the response to osmotic variations, we
measured the specific activity of this enzyme in homogenates obtained from
gills perfused with isosmotic (Isosucrose) or Hyposmotic saline. As
shown in Fig. 5, the specific
activity of the Na+/K+-ATPase in gills perfused and
bathed with Hyposmotic saline (41.84±14.54 µmol Pi
h-1 mg-1) was significantly higher than in the gills
perfused with Isosucrose saline (18.73±6.35 µmol
Pi h-1 mg-1).
|
Osmotic variations and intracellular cyclic AMP
In the final series of experiments, we studied a possible interaction
between the effects of osmotic variations and the intracellular messenger
cyclic AMP (cAMP). First, we measured Vte of isolated and
perfused gills and analysed the effect of theophylline, a blocker of cAMP
degradation by phosphodiesterases (Johnsen
and Nielsen, 1978), before and after stimulating adenylate cyclase
with forskolin (Seamon et al.,
1981
). As can be seen in Fig.
6A, a small stimulation of the outside positive
Vte with Isosmotic saline can be observed after addition
of theophylline (2.5 mmol l-1) to the perfusate. Addition of
forskolin (0.01 mmol l-1) results in a much more pronounced
Vte stimulation, and addition of theophylline after the
forskolin treatment sustains the voltage elevated by forskolin. Thus,
theophylline apparently keeps cAMP levels high. In a second experiment
(Fig. 6B), we replaced the
forskolin treatment with Vte stimulation by Hyposmotic
saline. After the stimulation with Hyposmotic saline plus theophylline, the
Vte level in the presence of Isosmotic saline plus
theophylline was significantly higher than before treatment with Hyposmotic
saline. These results (summarised in Fig.
7) suggest that Hyposmotic saline increased the cellular cAMP
level.
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Discussion |
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When NaCl concentration and osmolarity of the salines were reduced, the
outside positive transepithelial voltage (Vte) increased
in the same manner as observed before
(Luquet et al., 2002). The
presented results with respect to reduction of the Na+ or
Cl- concentration (Fig.
1) clearly show that the observed effects on
Vte are due to the reduction in osmolarity and do not
depend on the change in the concentrations of sodium or chloride. The
possibility that the Vte increase in the presence of
Hyposmotic saline may just reflect changes in paracellular resistance and not
in transport rates can be excluded after measuring the short-circuit current
(Isc) across spilt gill lamellae
(Fig. 4). The negative
Isc, which is independent of the paracellular pathway and
reflects active transcellular charge transport, increased when Hyposmotic
saline was used but was unaffected by a reduction in the NaCl concentration at
constant osmolarity (Fig. 4;
Table 2). We always used
solutions of identical ionic composition on both sides of the epithelium to
avoid superposition of the signal reflecting active transport with
transepithelial diffusive ion movements. However, when we studied the
influence of gradually increased osmolarities (Figs
2,
3), sucrose was added only to
the basolateral perfusion saline. The observed effects were almost identical
to the results with bilateral osmolarity changes, indicating that this
response is mainly caused by the hemolymph-side osmolarity change. The effects
of reduced osmolarity on isolated gills and split gill lamellae are fast,
reversible (Figs 2,
4) and gradually dependent on
the magnitude of the osmotic variation
(Fig. 3). Thus, the underlying
mechanism perfectly accomplishes the demands for a hormone-independent
regulation of hemolymph NaCl concentration and osmolarity by adjusting the
rates of active NaCl absorption.
The results of the experiments with split gill lamellae allow a first
insight into the mechanisms underlying the effects of osmotic variations. The
Gte reduction at constant Isc after
reducing NaCl at constant osmolarity (see
Table 2) probably reflects a
decrease in paracellular conductance due to the reduction of the ionic
strength of the solutions. On the other hand, the Gte
increase at increased Isc after also reducing the
osmolarity (but at constant ionic strength) indicates a change in the
transcellular conductance. In many epithelia, including crab gills (Onken et
al., 1991,
1995
), the apical membrane is,
by far, the barrier of highest resistance along the transcellular pathway, and
significant conductance changes are most likely due to modulations in this
membrane. Thus, it seems likely that the Gte increase in
the presence of Hyposmotic saline is related to the modulation of an apical,
electrogenic transporter. If we assume that active NaCl absorption across the
gills of C. granulatus also follows the same mechanism as proposed
for C. maenas (cf. Riestenpatt et
al., 1996
; see Introduction), it seems conclusive that the osmotic
stimulation of NaCl absorption is at least partly based on the increase of an
apical K+ conductance. However, a detailed analysis of the mode of
active NaCl absorption in C. granulatus is still missing and the
above hypothesis needs to be readdressed after successful characterization of
the transport mechanism.
The basolateral Na+/K+-ATPase was shown to energize
NaCl absorption across C. granulatus posterior gills
(Luquet et al., 2002). In the
present study, the activity of this ATPase was approximately doubled when
Hyposmotic saline was used instead of Isosmotic saline (see
Fig. 5). This finding clearly
demonstrates that, apart from a possible modulation of an apical conductive
pathway (see above), the basolateral Na+/K+-ATPase is
modulated by the osmotic variation and participates in the stimulation of NaCl
absorption in the presence of hyposmotic salines. Fast activation of the
Na+/K+-ATPase in hyposmotic media has been observed with
myocytes (Venosa, 1991
;
Whalley et al., 1993
) and also
with epithelial cells (Coutry et al.,
1994
). A modulation of the in situ activity of the ATPase
due to changes in transapical NaCl absorption and respective alterations in
cellular sodium would not be detectable in activity measurements as conducted
in the present study. Thus, it seems that osmotic variations modulate the
activity of the ATPase by phosphorylation/dephosphorylation processes (cf.
Cheng et al., 1999
) or by rapid
insertion of ATPase-containing vesicles into the basolateral membrane (cf.
Venosa, 1991
;
Carranza et al., 1998
). The
activation of the Na+/K+-ATPase during hyposmotic
conditions represents a major difference compared with the autoregulatory
response of the gills of E. sinensis, in which only apical
transporters (V-type H+-ATPase and Na+ channels) are
involved (Onken, 1996
). Some
results obtained at our laboratory suggest that dopamine, an extensively
studied bioamine likely to be involved in the adaptation to low salinity (for
a review, see Morris, 2001
),
also modulates the electrical properties and
Na+/K+-ATPase activity of the gills of C.
granulatus (J. Halperin, M. Tresguerres, G. Genovese, A. Pozzi and C. M.
Luquet, unpublished data).
Apart from the cell volume, a multitude of cellular parameters has been
shown to change following exposure of cells to anisosmotic media (see
Lang et al., 1998). One of
these factors is an increase in cellular cAMP by activation of adenylate
cyclase in hyposmotic media (Watson,
1989
,
1990
). As shown in
Fig. 6A, an artificial increase
in intracellular cAMP concentration in C. granulatus posterior gills
by the addition of theophylline and/or forskolin in isosmotic salines results
in an increase in Vte. So far, these findings are
consistent with an increase in the activity of the
Na+/K+-ATPase after increasing cellular cAMP (J.
Halperin, M. Tresguerres, G. Genovese, A. Pozzi and C. M. Luquet, unpublished
data). When theophylline was present in the Isosmotic saline after stimulation
with Hyposmotic saline, Vte was maintained at a higher
level than under the same conditions before the hyposmotic stimulation (see
Figs 6B,
7), suggesting that the change
to hyposmotic salines was accompanied by a rise in cellular cAMP. The same
second messenger has already been shown to mediate the stimulatory effects of
dopamine and serotonin in E. sinensis
(Trausch et al., 1989
;
Mo et al., 1998
; see
Morris, 2001
for a review).
However, since theophylline preserved only part of the hyposmotic activation,
other second messengers might also be involved in the autoregulatory response
observed in C. granulatus gills.
In summary, we propose that reduction of the hemolymph-side osmolarity results in an increase of cellular cAMP that, in turn, stimulates active NaCl absorption across the posterior gills of C. granulatus by activating the basolateral Na+/K+-ATPase. A conductive pathway, probably at the apical membrane, is also stimulated by signalling events that are still to be elucidated. Under physiological conditions, this autoregulation could be an important mechanism to rapidly stabilise the hemolymph osmolarity and NaCl concentration when the animals move between ambient media of different salinities.
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Acknowledgments |
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