Transepithelial potential differences and Na+ flux in isolated perfused gills of the crab Chasmagnathus granulatus (Grapsidae) acclimated to hyper- and hypo-salinity
1 Department of Biological Sciences, FCEN University of Buenos Aires, Building II, Ciudad Universitaria, C1428EHA Buenos Aires, Argentina,
2 Alfred Wegener Institute for Polar and Marine Research, Columbusstrasse, D-27568 Bremerhaven, Germany,
3 CONICET-Argentina and
4 Department of Inorganic Chemistry, FCEN University of Buenos Aires, Argentina
*e-mail: luquet{at}bg.fcen.uba.ar
Accepted 1 October 2001
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Summary |
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The gills of crabs acclimated to low salinity, perfused and bathed with 10 saline solutions, produced the following TEPDs (hemolymph side with respect to bath side): 0.4±0.7, 10.2±1.6, 10.8±1.3 and 6.7±1.3 mV for gills 5, 6, 7 and 8, respectively. Gills 6, 7 and 8 did not differ significantly. Reducing the saline concentration of bath and perfusate from 30
to 20
or 10
increased significantly the TEPDs of these gills. TEPDs of gill 6 (representative of posterior gills) were reduced by 69±5 % and 60±5 % after perfusion with ouabain or BaCl2 (5 mmol l1 each), respectively. The same gill showed a net ouabain-sensitive Na+ influx of 1150±290 µequiv g1 h1.
Gill 6 of crabs acclimated to high salinity produced TEPDs of 1.5±0.1 and 1.3±0.09 mV after perfusion with 30 or 40
salines, respectively. Perfusion with ouabain or BaCl2 reduced TEPDs by 76±7 % and 86±4 %, respectively. A net ouabain-sensitive Na+ efflux of 2282±337 µequiv g1 h1 was recorded in gill 6 perfused with 38
saline.
Key words: isolated perfused gill, transepithelial potential difference, Na+K+-ATPase, ion flux, hypo-regulation, hyper-regulation, ouabain, crab, Chasmagnathus granulatus.
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Introduction |
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The gills of euryhaline crabs are histologically and functionally differentiated. While anterior gills are lined with thin epithelium and have a mainly respiratory function, the posterior ones play a key role in compensatory active uptake of ions (Siebers et al., 1982; Gilles and Péqueux, 1986
; Towle and Kays, 1986
; Compère et al., 1989
). The predominant cells in posterior gills are thick and possess large basolateral membrane interdigitations together with mitochondria (Copeland and Fitzjarrell, 1968
; Compère et al., 1989
; Luquet et al., 1997
). There is much evidence from electrophysiological and ion flux studies on the ion-uptake capacity of posterior gills of different crab species (Gilles and Péqueux, 1981
, 1985
; Péqueux et al., 1988
; Siebers et al., 1985
; Lucu and Siebers, 1986
; Burnett and Towle, 1990
) (for a review, see Péqueux, 1995
).
Two models for the mechanisms involved in such active ion transport through the gills, have been reviewed recently (Onken and Riestenpatt, 1998). (1) In the gills of species such as Carcinus maenas, which possess limited capacity to invade low-salinity environments (weak hyper-regulators), it is proposed that the ions enter the cell by crossing the apical membrane through Na+/2Cl/K+ symports coupled to K+ channels. (2) In strong hyper-regulators, such as Eriocheir sinensis, Onken and Riestenpatt suggest that Na+ crosses the apical membrane through epithelial Na+ channels while Cl is exchanged with HCO3, driven by an apical H+-V-ATPase. At the basolateral side, Na+K+-ATPase and K+ and Cl channels are thought to drive ions into the hemolymph in both weak and strong hyper-regulating species.
Early work on salt and water regulation by intact fiddler crabs of the genus Uca (Green et al., 1959; Baldwin and Kirschner, 1976a
; Evans et al., 1976
) suggests that active ion excretion follows an extra-renal route. There is some physiological and histological evidence to suggest that the gills of hypo-regulating crabs are the organs involved in this function. Martínez et al. (1998
), working with isolated perfused gills of Ucides cordatus, reported that gill 6 is capable of active ion excretion, while gill 5 is specialized in ion uptake. Ultrastructural studies also suggested an ion excretion capacity of the gills of crabs acclimated to hypersaline media; Martelo and Zanders (1986
) and Luquet et al. (1997
) described a cell architecture characteristic of an ion-excreting epithelium in the gills of grapsids and ocypodids. Some features of this cell architecture, such as the apparently low-resistance cell junctions, resemble those of the extensively studied vertebrate salt-secreting organs such as the avian salt gland (Riddle and Ernst, 1979
), the teleost opercular epithelium (Ernst et al., 1980
) and the rectal gland of elasmobranchs (Ernst et al., 1981
).
Chasmagnathus granulatus Dana 1851 is a strong ion hyper- and hypo-regulating crab species that inhabits intertidal estuarine coasts of Brazil, Uruguay and Argentina (Boschi, 1964; Mougabure Cueto, 1998
). The posterior gills of this species are believed to be involved in both ion uptake and excretion, since their epithelium thickness is increased to the same extent after transfer from full seawater to either dilute or concentrated seawater (Genovese et al., 2000
).
The aim of this work was to establish, by electrophysiological and ion tracer flux experiments, the possible role of the different gill pairs of C. granulatus in ion-transport functions at low and high salinity. The participation of Na+K+-ATPase and K+ channels was also studied.
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Materials and methods |
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Gill perfusion
Crabs were killed by destroying the ventral nervous ganglion with a spike. After removing the dorsal carapace, the gills were gently excised and placed in a Petri dish with saline solution. The afferent and efferent vessels of gills 58 were connected by fine polyethylene tubing of 0.4 mm diameter to a peristaltic pump (afferent) and to a glass tube (efferent). Perfusion rate was kept at 0.1 ml min1. The tubing was held in position by an acrylic clamp and the preparation put into a glass beaker with the appropriate saline solution and constant aeration.
Gills 58 from crabs acclimated to low (12 ) and high (45
) salinity were perfused and bathed with identical solutions, except that the perfusate contained 2 mmol l1 glucose. Table 1 shows the composition of the saline solutions used in the different experiments. All solutions were adjusted with Tris-base to the physiological pH 7.75 for C. granulatus (Luquet and Ansaldo, 1997
). The effects of the following drugs were tested in gills perfused with 10
saline for low-salinity crabs and 38
saline for high-salinity crabs: ouabain (a specific Na+K+-ATPase inhibitor) (Skou, 1965
), 5 mmol l1 applied basolaterally, and BaCl2 and CsCl (K+ channel blockers) (Zeiske, 1990
; Draber and Hansen, 1994
), 510 mmol l1 applied at both apical and basolateral sides.
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Na+ flux
Na+ outward and inward movements were measured in the same gill by applying 22Na first in the perfusate and afterwards in the bathing solution. Identical ionic concentrations (20 or 38
, for low- and high-salinity crabs, respectively) were used in the bath and perfusate. For Na+ efflux, 22Na was included in the perfusate at a final concentration of 0.25 µCi ml1. After stabilization for 15 min, three samples (1 ml each) were collected from the perfusate and the bath at intervals of 15 min. Na+ efflux was calculated from the radioactivity that appeared in the bath. To avoid overestimated flux due to a possible leak, we also measured the radioactivity lost by the perfusate after passing through the gill. Gills showing large discrepancies between both methods of measurement were discarded. Results from measurements of radioactivity that appeared in the bath were chosen for the study.
After washing with non-radioactive solution, Na+ influx was measured by applying 22Na in the bath at a final concentration of 9 kBq (0.25 µCi) ml1. After stabilization for 15 min, radioactivity was measured in the collected perfusate at 10 min intervals during the subsequent 30 min.
Radioactivity was measured with a Canberra Series 35 plus gamma scintillation counter. Na+ efflux and influx were calculated according to the formula of Lucu and Siebers (1986):
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where J is the calculated unidirectional flux of Na+ in µequiv h1 g1; 22Na is the radioactivity (cts min1) collected during each interval; S is the number of samples collected during 1 h (6 and 4 for influx and efflux, respectively); SRA is the specific radioactivity (cts min1 µequiv1) and m is the fresh mass of the gill (g).
Chemicals
NaCl, KCl, MgCl2, BaCl2, KCN and glucose were obtained from Merck Argentina; NaHCO3 was obtained from Mallinckrodt USA; CaCl2 and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) were purchased from J. T. Baker, USA; ouabain was purchased from Sigma USA and 22Na was obtained from Amersham Pharmacia Biotech.
Statistics
Data were analyzed by one- or two-way repeated measures analysis of variance (ANOVA) or paired t-test when appropriate (Sokal and Rohlf, 1981). All values are means ± S.E.M.
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Results |
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Crabs acclimated to low salinity
Transepithelial potential difference
Posterior gills of crabs acclimated to low salinity, perfused and bathed with identical salines, showed hemolymph-side negative TEPDs, which increased significantly in absolute value as the concentration of saline solutions decreased; no significant TEPD was recorded in gill 5 (representative of anterior gills) (Fig. 1). Two-way repeated measures ANOVA comparing gills 6, 7 and 8 indicated that saline concentration was the only significant source of variation (P<0.001, N=6 for each gill). TEPDs for gill 6 ranged between 2.4±0.5 mV (N=6) in 30 saline and 10.2±1.6 mV (N=6) in 10
saline. Since the three posterior gills responded similarly to the different saline concentrations, gill 6 was chosen as representative for further experiments because it was bigger than gills 7 and 8 and therefore easier to handle.
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In a second series of experiments, gill 6 was used as representative of posterior gills and the concentration of the bath and perfusate was increased stepwise from 30 to 40
, in an attempt to minimize the effects of a possible osmotic shock. A slight tendency to increased TEPD at higher saline concentrations was observed, with the highest values recorded at 3638
(N=9). Nevertheless, these differences were not statistically significant (Fig. 4).
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Discussion |
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Acclimation to low salinity
Posterior gills of C. granulatus acclimated to low salinity, perfused and bathed with identical solutions, produce hemolymph-side negative TEPDs similar to those reported for hyper-regulating crabs (Lucu and Siebers, 1986; Siebers et al., 1985
) and also for the hyper-hyporegulating crabs Uca tangeri and Pachygrapsus marmoratus (Drews and Graszynski, 1987
; Krippeit Drews et al., 1989
; Pierrot et al., 1995a
,b
). These TEPDs are sensitive to the dilution of the perfusion and bathing media, being the lowest absolute values measured at 30
, which is near the physiological hemolymphatic concentrations of Na+ and Cl in this species (Mougabure Cueto, 1998
). This change in potential difference seems to reflect an autoregulatory mechanism of gill tissue, which involves enhanced ion transport activity and/or increased paracellular resistance in response to hypo-osmotic stress. Onken (1996
) and Onken and Riestenpatt (1998
) reported that changes in short-circuit currents measured in split gill lamellae of Eriocheir sinensis respond to osmotic variations at the basolateral side. Our results indicate that these changes in TEPD take place in C. granulatus within a few minutes and are totally reversed when the gill is perfused again with iso-ionic saline. This rapid response should be important for a species that is often observed emerging from brackish or seawater and entering into rain pools for feeding on supratidal plants.
Radioactive tracer flux suggests that posterior gills of this species actively take up Na+ at low salinity. Both influx and efflux rates as well as net influx are somewhat high compared with data reported for other species in similar experimental conditions (Lucu and Siebers, 1986; Pierrot et al., 1995a
). This high rate of Na+ uptake is possibly a response to a high rate of ion loss by the animal. Gill and whole animal ionic permeability should be studied in order to test this hypothesis.
As a first approach to understanding the mechanisms involved in gill ion uptake, it can be concluded that Na+K+-ATPase located at the basolateral membrane is the major driving force, since both TEPD and Na+ influx are inhibited by ouabain in similar proportions. The reason why ouabain does not cause total inhibition could be incomplete access of the drug to the enzyme molecules, due to the complex basolateral membrane interdigitations of gill ionocytes, as suggested by Burnett and Towle (1990) to explain similar results obtained with Callinectes sapidus.
As our results imply, barium-sensitive K+ channels located in the basolateral membrane are also involved in generating the observed transepithelial potentials. The lack of effect of apical BaCl2 and CsCl (preliminary data) suggests the absence of barium-sensitive K+ channels in this membrane. It has been reported that these channels are necessary for electrogenic uptake of Na+ and Cl across the apical membrane through Na+/2Cl/K+ cotransporters in Uca tangeri and Carcinus maenas (Drews and Graszynski, 1987; Riestenpatt et al., 1996
).
Onken and Riestenpatt (1998) have proposed that in strong hyper-regulators, such as Eriocheir sinensis, Na+ and Cl cross the apical membrane through Na+ channels and Cl/HCO3 antiports, driven by an H+-V-ATPase. Although we have no direct evidence for apical transporters, expression of both Na+/H+ exchangers and H+-V-ATPase has been detected in gill 6 of C. granulatus in preliminary molecular biology experiments (D. Weihrauch and C. M. Luquet, unpublished observations). In addition, Genovese et al. (2000
) reported increased activity of cytosolic carbonic anhydrase in posterior gills of C. granulatus after acclimation to low salinity. This enzyme is believed to produce H+ and HCO3 as counterions for Na+ and Cl exchangers, respectively, and is also important for the function of an H+-V-ATPase (Henry, 1988
; Henry and Swenson, 2000
).
Acclimation to high salinity
The involvement of crab gills in hypo-regulatory ion excretion is still a matter of controversy. In the past two decades histological evidence has accumulated. There are reports of ultrastructural changes and increased posterior gill epithelium thickness after acclimation to hypersaline media (Martelo and Zanders, 1986; Luquet et al., 1997
; Rosa et al., 1999
; Genovese et al., 2000
). Martínez et al. (1998
) perfused gills 5 and 6 of Ucides cordatus acclimated in isosmotic medium, reporting net Na+ uptake by gill 5, even when the external saline was more concentrated than the perfusate. In contrast, they found net Na+ excretion by gill 6 at all concentrations tested. This experiment, however, is not comparable with the present results, since it was performed with asymmetrical perfusion. In addition, the authors acclimated the crabs to isosmotic medium. A clear difference does seem to exist between both species, however; whereas in U. cordatus different gills are specialized for transporting ions in opposite directions, the TEPDs measured on the three posterior gills of C. granulatus suggested that they have similar ion transport capacities. Thus gills 6, 7 and 8 of C. granulatus seem to be equally involved in both transport directions, after chronic acclimation to either low or high salinity.
At least two basolateral membrane proteins involved in ion uptake are also involved in ion excretion. These are Na+K+-ATPase, since both TEPD and Na+ flux are reduced by ouabain, and K+ channels, which are inhibited by basolateral application of BaCl2. By contrast, our results provide no evidence for any apical ion-transport proteins.
Current models for salt excretion in vertebrates (gills, and opercular epithelia of teleost fish, rectal glands of elasmobranchs and avian salt glands) consider Na+ pumping into the paracellular space and a transcellular flux of Cl, which in turn generates a positive transepithelial potential difference that drives Na+ efflux via low-resistance tight junctions (Ernst et al., 1980, 1981
; Lowy et al., 1987
). Our results on crabs acclimated to high salinity indicate that Na+K+-ATPase is the main driving force for ion extrusion. In addition, previous electron microscopic work shows shorter septate junctions in gills of Uca uruguayensis and C. granulatus acclimated to high salinity (Luquet et al., 1997
; Rosa et al., 1999
) compared with gills of the same species acclimated to low salinity. Thus, paracellular flux of ions through these junctions also seems possible. However, the vertebrate model predicts a positive transepithelial potential difference for driving paracellular Na+ efflux. This is not the case for the gills of C. granulatus, which produce a little negative potential difference. Therefore the routes followed by Na+ and Cl at the apical side seem to differ from known models for salt excretion and deserve further investigation.
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Acknowledgments |
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