Ion transport across posterior gills of hyperosmoregulating shore crabs (Carcinus maenas): amiloride blocks the cuticular Na+ conductance and induces current-noise
1 Departmento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Avenida Bandeirantes 3900, 14040-901 Ribeirão Preto, SP, Brasil and
2 Biologische Anstalt Helgoland, Zentrale Hamburg, Notkestrasse 31, 22607 Hamburg, Germany
*e-mail: onkenh{at}ffclrp.usp.br
Accepted 29 November 2001
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Summary |
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Key words: amiloride, shore crab, Carcinus maenas, conductance, gills, cuticle, ion flux, NaCl absorption, short-circuit current, current-noise analysis, Ussing chamber.
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Introduction |
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The electrogenic and coupled absorption of Na+ and Cl seems to proceed via apical Na+/K+/2Cl cotransport and basolateral Cl channels and Na+/K+-ATPases. Transcellular current flow occurs via apical K+ channels and basolateral Cl channels. The presence of additional transapical pathways for NaCl absorption, such as Na+/H+ and Cl/HCO3 antiports, could not be confirmed in this study. Active Cl absorption was found to be directly related to the negative Isc, suggesting the absence of any electroneutral NaCl absorption via parallel cation and anion antiports. The latter interpretation is also consistent with the finding that blockers of carbonic anhydrase inhibit neither Cl absorption across isolated and perfused gills (Böttcher et al., 1991) nor the negative Isc (Onken and Siebers, 1992
).
Amiloride has long been used as a probe for epithelial Na+ channels and Na+/H+ antiports in a variety of Na+-absorbing epithelial tissues (Benos, 1982; Garty and Benos, 1988
). In two studies, external amiloride was observed to affect the transport characteristics of the gills of shore crabs. Na+ fluxes, but not Cl fluxes, across isolated perfused gills were inhibited by external amiloride in a dose-dependent manner (KAmi=4070 µmol l1), and the inward negative transbranchial potential difference (PDte) became hyperpolarised (KAmi=50 µmol l1) (Lucu and Siebers, 1986
; Siebers et al., 1987
). It was proposed that these results reflected Na+ absorption via apical Na+/H+ antiport. It has also been suggested that a passive, conductive and amiloride-sensitive paracellular pathway is at least partly responsible for the observed effects of external amiloride (Siebers et al., 1987
, 1989
). In a study of split gill lamellae (Onken and Siebers, 1992
), the negative Isc was increased to more negative values by the addition of external amiloride (KAmi=10 µmol l1), and the transepithelial resistance (Rte) increased simultaneously. These results led to the proposal of an electrogenic Na+ uptake via apical Na+ channels or electrogenic 2Na+/1H+ antiports in addition to symporter-mediated NaCl absorption (see above). In fact, in gill membrane vesicles of the shore crab, an electrogenic, amiloride-sensitive (KAmi=280 µmol l1) 2Na+/1H+ antiporter was identified (Shetlar and Towle, 1989
). In addition to these effects at the cellular level, the isolated gill cuticle was demonstrated to be amiloride-sensitive: at a concentration of 1 mmol l1, the diuretic reduced the conductance of the cuticle of Carcinus maenas (Lignon and Péqueux, 1990
). Thus, the location of the amiloride-sensitive site in the gills of the shore crab remains unclear.
In the present investigation, we focus on the effects of amiloride on NaCl absorption across split gill lamellae and on the amiloride-sensitivity of the cuticle. Our results indicate that the amiloride-induced changes in PDte, Gte, Isc and Na+ fluxes are based on the inhibition of Na+ movements across the cuticle, rather than the interaction between the drug and ion-transport proteins in the apical membrane.
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Materials and methods |
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Preparations
When the crabs had been killed by destroying their ventral ganglion by pressing a needle through the ventral side of the body wall and lifting the carapace, the three posterior gills were removed. Single gill lamellae, consisting of the gill epithelium and the adherent apical cuticle, were isolated and split according to the method described by Schwarz and Graszynski (1989). Isolated cuticles were obtained by mechanically peeling off the epithelium (Lignon, 1987
). Separation of cuticle and epithelium could be easily controlled under the microscope since cuticle and epithelium differ in colour under the light of a halogen cold light. Split gill lamellae or isolated cuticles were mounted in an Ussing-type chamber modified after De Wolf and Van Driessche (1986
) with an epithelial area of 0.02 cm2 or 0.01 cm2. To minimise edge damage, silicone grease was used. The chamber compartments (50 µl) were continuously perfused with salines by gravity flow (approximately 2 ml min1) or by means of a peristaltic pump (0.5 ml min1, to measure fluxes of radioactive tracers).
Salines and chemicals
The haemolymph-like saline used was composed of (mmol l1): 248 NaCl, 5 KCl, 2 NaHCO3, 4 MgCl2, 5 CaCl2, 5 Hepes and 2 glucose. Immediately before use, the pH was adjusted to 7.7 with Tris. To prepare Cl-free saline, the respective gluconates were used. In Na+-free salines, NaCl was substituted with choline chloride, KHCO3 was used instead of NaHCO3 and KCl concentration was reduced to 3 mmol l1. Ouabain was purchased from Fluka. Amiloride was a gift from Merck, Sharp and Dohme (München, Germany).
Electrophysiological measurements
To measure the transepithelial potential difference (PDte), calomel electrodes were connected via agar bridges (3 % agar in 3 mol l1 KCl) with the chamber compartments (distance to the preparation <0.1 cm). The reference electrode was in the basolateral bath. Ag/AgCl electrodes served as current electrodes to short-circuit the preparation (measurement of short-circuit current, I*sc) using an automatic clamping device (VCC 600, Physiologic Instruments, San Diego, CA, USA). The area-specific resistance between the tips of the voltage electrodes (Rtot) was calculated from small imposed voltage pulses (PDte) and the resulting current deflections (
I). Rtot is the sum of the serial resistances of the solutions (Rs) and the tissue (Rte or Rcut). Because of the low values of Rtot, it was necessary to correct the Rtot and I*sc data to obtain values directly related to the preparations (Rte or Rcut, Isc or Icut). Rs was measured in the absence of a preparation separating the chamber compartments and was found to be 9
cm2 for NaCl saline (N=15) and Na+-free saline (N=8) and 13
cm2 for Cl-free saline (N=8). The corrected values of Rte and Rcut, respectively, result from subtracting Rs from Rtot, while the correction of I*sc followed Ohms law (see Riestenpatt et al., 1996
). In the results, only the corrected values of area-specific Isc, Icut, Gte (=1/Rte) and Gcut (=1/Rcut) are given.
For the current fluctuation (noise) analysis experiments, we used a specially constructed low-noise voltage-clamp apparatus, designed and modified after the original version of Van Driessche and Lindemann (1978). Current fluctuations were digitally recorded after passing the clamp current through a set of (anti-aliasing) high-pass and low-pass filters and after appropriate amplification at each step. Fast Fourier analysis of the current fluctuations yields the so-called power density spectrum, a double-logarithmic representation of the variance of Isc over the frequency (see Fig. 3 for examples). Lorentzian components in the current noise spectra were obtained by adding amiloride to the external perfusion saline. To evaluate the spectra, the two-state model was used, producing the Lorentzian parameters So (low-frequency plateau):
|
| (1) |
and fc (corner frequency):
![]() | (2) |
where INa(Ami) is the amiloride-sensitive Na+ current in the presence of submaximal concentration of amiloride, i is the single-channel current, Po is the channel open probability, k01 and k10 are the association and dissociation rate constants, respectively, of the channel/amiloride interaction and cAmi is the amiloride concentration. The channel open probability Po:
| (3) |
was determined using values of k obtained from the linear 2fc/cAmi plots or by using:
| (4) |
where INa(Ctrl) is the overall amiloride-blockable (100 µmol l1) current. The two procedures resulted in similar Po values. To calculate the single-channel current, equation 1 was solved for i. To determine the number of channels per cm2 (M), we used:
| (5) |
For further details, see Zeiske et al. (1992).
Measurements of unidirectional NaCl fluxes
Radioactive isotopes, 36Cl (ICN) and 22Na (NEN, Dupont), were used at a final activity of 1 mCi l1 (1 Ci=3x1010 Bq). Unidirectional influxes (Jab) or effluxes (Jb
a) were measured over a period of 60 min in a closed perfusion circuit (5 ml in each chamber compartment) allowing the accumulation of radioactivity in the superfusate. Net influxes of the respective ions were calculated as the differences between the means of Ja
b and Jb
a. The radioactivity of 36Cl contained in 2 ml samples was determined with a PRIAS liquid scintillation counter (Packard; model PLD) after addition of 4 ml of Insta Gel (Packard; no. 6013008). The radioactivity of 22Na was measured in 2 ml samples using the same procedure or was determined directly in 2 ml samples using a gamma spectrometer (Fischer, Hamburg). Flux data were calculated from the respective specific activities, the volume of the perfusion compartment (5 ml) and time (1 h) and expressed as µmol h1 cm2. Influxes and effluxes of 22Na and 36Cl were measured in separate experiments. It was impossible to measure influxes and effluxes in the same preparation since a complete wash-out of the radioactivity required an incubation of more than 3 h because of the high doses of radioactivity necessary.
Statistical analyses
All results represent means ± S.E.M. Differences between groups were tested with the paired Students t-test. Significance was assumed for P<0.05.
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Results |
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Current-noise analysis was used to characterise the effect of amiloride in five split gill lamellae. Under short-circuit conditions, no Lorentzian components were observed in the presence of 5 µmol l1 amiloride in the external bath. After increasing the driving force for inward movement of Na+ by applying outside-positive clamp voltages (1050 mV), positive currents were measured at unchanged conductances. Under these conditions, external amiloride caused a fast and reversible decrease in current and conductance, and Lorentzian components appeared in the power density spectra. Fig. 3A,B shows the dependence of the Lorentzian components in the power density spectra on the presence of external amiloride and on externally positive clamp voltages. At a clamp voltage of 50 mV, we analysed the dose-dependence of the effects of amiloride on macroscopic (current and conductance) and microscopic (corner frequency and low-frequency plateau) parameters. When applying different amiloride concentrations, simple MichaelisMenten kinetics was observed for the reductions in transepithelial current and conductance. The half-maximal decreases (KAmi) in current and conductance induced by amiloride were analysed from HanesWoolf plots to be at 3.6±0.1 µmol l1 (N=5; see Fig. 4) and 2.0±0.4 µmol l1 (N=5; not shown), respectively. As theoretically predicted for a first-order rate process between blocker and ion channel (Van Driessche and Zeiske, 1980; Lindemann and Van Driessche, 1977
), the plateau value (So) showed a bell-shaped response to increasing cAmi (maximum close to KAmi; see Fig. 5), whereas the corner frequency increased linearly with the amiloride concentration (see Fig. 6). From the slope of the line (k01=126.4±11.8 µmol1 s1 l) and from its intercept with the 2
fc axis (k10=70.9±2.1 s1), a KAmi of 1.8±0.2 µmol l1 (N=5) was calculated, which is close to the values obtained from the inhibition of macroscopic currents and conductances.
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The dose-dependence of the inhibition of Icut and Gcut by amiloride was studied in five experiments. Stepwise increases in concentration (0.5100 µmol l1) of amiloride in the external perfusion saline resulted in stepwise decreases in current (control value 28 530±4373 µA cm2; N=5) and conductance (control value 600±100 mS cm2). After transformation of the data into HanesWoolf plots, straight lines were obtained, indicating simple MichaelisMenten kinetics for the reduction in current (Fig. 4) and conductance (data not shown) elicited by amiloride. The average half-maximal effects of the drug on Icut (KAmi=0.7±0.1 µmol l1; N=5) and Gcut (KAmi=0.6±0.1 µmol1; N=5) were determined from the intercepts of the lines with the abcissa. In another set of experiments (N=5), the dose/response curves of external amiloride were measured at a clamp voltage of +10 mV. The mean KAmi values were at very similar amiloride concentrations (0.9±0.1 µmol l1 in both cases) to those in the experiments with a clamp voltage of 50 mV (see Fig. 4).
Analyses of the current fluctuations (N=3) in the presence of amiloride revealed similar results to those obtained with split gill lamellae. Lorentzian components appeared in the power density spectra only when the clamp voltage was increased to 1050 mV (outside-positive). The dependence of the Lorentzian component in the power density spectra on the presence of amiloride and the clamp voltage is shown in Fig. 3C,D. At a clamp voltage of 50 mV, changes in the Lorentzian variables So and 2fc with varying external cAmi (1.2510 µmol l1) were observed. As expected for a first-order rate process between blocker and ion channel (Van Driessche and Zeiske, 1980
; Lindemann and Van Driessche, 1977
), the plateau value (So) showed a bell-shaped response to increasing cAmi (maximum close to KAmi; Fig. 5), whereas the corner frequency increased linearly with the amiloride concentration (Fig. 6). From the slope of the line (k01=110.8±6.3 µmol1 s1 l) and from its intercept with the 2
fc axis (k10=57.8±1.1 s1), a KAmi of 1.9±0.1 µmol l1 (N=3) was calculated. Although true ion channels are unlikely to be present in the non-cellular cuticle, calculations of single-channel currents (i) and channel densities (M) were conducted. An increase in i and a decrease in M were observed with increasing external amiloride concentration (Fig. 7).
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Discussion |
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The electrophysiological parameters and the flux data under control conditions measured in the present study with split gill lamellae of posterior gills of hyperosmoregulating shore crabs are very similar to the values published in previous studies of the same preparation (Onken and Siebers, 1992; Riestenpatt et al., 1996
). The polarity and magnitude of the short-circuit current (Isc), the magnitude of the transepithelial conductance (Gte) and the magnitudes of the measured unidirectional and calculated net fluxes of Na+ and Cl are in the same range as observed recently. The same applies to the experiments with isolated cuticles. Given that the cuticle is cation-selective (Lignon, 1987
) (see below), the cuticular conductances measured in the present study are consistent with the molar area-specific Na+ conductance of 2.05±0.53 mS cm2/mmol l1 determined in an earlier study of the isolated gill cuticles of Carcinus maenas (Lignon, 1987
).
In the present study, external addition of amiloride did not significantly affect the Isc, but decreased the conductance of the split gill lamellae. With respect to the increased Isc/Gte ratio, these results are consistent with findings on isolated and perfused gills, in which amiloride caused a hyperpolarization of the outside-positive transbranchial potential difference (Lucu and Siebers, 1986; Siebers et al., 1987
). When studying split gill lamellae of Carcinus maenas, Onken and Siebers (1992
) also observed a decrease in the conductance of split gill lamellae after application of external amiloride. However, in these experiments, the negative Isc increased to more negative values. On the basis of this observation, the authors proposed an active, electrogenic and transcellular Na+ absorption via Na+ channels or electrogenic Na+/H+ antiports independent of the coupled NaCl absorption via apical symporters.
Amiloride-induced increases in Isc were observed in approximately one-third of the preparations in the present study. However, in the remaining two-thirds, Isc did not change or even decreased after the addition of amiloride. The unidirectional fluxes and the net influx of Na+ across split gill lamellae of shore crabs were markedly reduced by amiloride, whereas Cl fluxes remained unchanged (Fig. 1). Similar results have been obtained with isolated and perfused gills (Lucu and Siebers, 1986; Siebers et al., 1987
). These findings may suggest that external amiloride induces very complex effects at the epithelial level: blockade of active and electrogenic Na+ absorption via apical Na+ channels or electrogenic antiports and a change in Cl absorption from a Na+-coupled to a Na+-independent mode.
Na+-independent Cl absorption via apical Cl/HCO3 antiports and basolateral Cl channels driven by an apical H+ pump has been observed to generate a negative Isc across split gill lamellae of the Chinese crab Eriocheir sinensis adapted to fresh water (Onken and Putzenlechner, 1996; Onken and Riestenpatt, 1998
). This negative, Cl-dependent Isc across the gill lamellae of the Chinese crab was independent of a functioning Na+/K+-ATPase. In the present study, however, the negative Isc across shore crab gill lamellae was almost completely blocked by ouabain, even in the presence of external amiloride (see Fig. 2). Thus, a change in Na+-coupled Cl absorption to Na+-independent Cl absorption had not been induced by amiloride.
A more detailed analysis of the effects of amiloride on split gill lamellae and on isolated cuticles (present study) also indicated the absence of active and electrogenic Na+ absorption via Na+ channels or electrogenic 2Na+/1H+ antiports in the apical membrane. Both split lamella preparations and isolated cuticles showed very similar responses to external amiloride. At a clamp voltage of +50 mV, and thus in the presence of an increased driving force for inward Na+ movement, the addition of the diuretic resulted in fast and reversible reductions in currents and conductances across split gill lamellae and isolated cuticles. Even the values of KAmi were similar for split gill lamellae and isolated cuticle (see Results and Fig. 4). These findings indicate that the effect of amiloride is due to an interaction between the drug and the external side of the cuticle and not with transporters in the apical membrane or with the paracellular junctions. Thus, our findings clearly support the data obtained on isolated cuticle in a previous study using high amiloride concentrations (Lignon and Péqueux, 1990).
The similarities between the effects of amiloride on split gill lamellae and isolated cuticle were also observed with respect to the parameters obtained by amiloride-induced current-noise analysis. Noise analysis was used in the present study with the expectation that amiloride-induced Lorentzian components in the power density spectra would be visible only if the diuretic were to interfere with epithelial Na+ channels. In fact, under short-circuit conditions, no Lorentzian components could be detected in the presence of amiloride. However, when the driving force for inward movement of Na+ was increased by clamping to an outside-positive voltage, amiloride-induced Lorentzian components were clearly expressed in split gill lamellae and isolated cuticles (Fig. 3). Moreover, the amiloride-dependent shifts in the low-frequency plateau (So; bell-shaped) and the corner frequency (fc; linear) agreed perfectly with the theoretical two-state model of pseudo-first-order channel blockade (see Figs 5, 6) (cf. Lindemann and Van Driessche, 1977; Van Driessche and Zeiske, 1980
). The clearly higher values of So for isolated cuticle (see Fig. 5) are probably due to the larger currents in these preparations in which the epithelium does not act as a series resistance. Of course, amiloride-sensitive, epithelial Na+ channels cannot be expected to be present in a non-cellular layer such as the cuticle. Consequently, it is hardly surprising that the noise analysis data obtained in the present study also show clear differences from results obtained with Na+ channels in epithelial tissues. The association (k01) and dissociation (k10) rate constants for the interaction between amiloride and its binding site, which can be determined from plots of 2
fc versus cAmi, seem to be considerably higher in shore crab cuticle than observed for Na+ channels in epithelial tissues, including the gill epithelium of Chinese crabs (Helman and Kizer, 1990
; Zeiske et al., 1992
). Moreover, when calculating the single-channel current (i) and the channel density (M), the changes observed with increasing blocker concentration (Fig. 7) are not consistent with the theoretical model, which assumes constancy of i and M. In this light, there appears to be little point in interpreting the measured single-channel currents and channel densities with respect to the gill cuticle. Nevertheless, to our knowledge, it is a new and important observation that a biological, but non-membraneous, barrier shows such a high degree of similarity with epithelial Na+ channels.
Both substitution of Na+ and addition of amiloride resulted in similar decreases in transcuticular current and conductance (Table 1), suggesting that the diuretic inhibited a Na+ conductance. In the absence of Na+, amiloride had only a minor effect, which may be due to the inhibition of a small permeability of the cuticle for choline (which served as substitute for Na+) or to an effect on cuticular anion permeability. The permeability characteristics of the crustacean cuticle have been attributed to the lipoproteic, uncalcified, chitin-free epicuticle, which lacks the waterproofing wax layer of the insect cuticle (Lignon, 1987; Lignon and Péqueux, 1990
). It has been proposed that the selective permeability of the epicuticle is due to specific pores discriminating between anions and cations and between particles of different size (Lignon and Péqueux, 1990
). On the basis of this model, it is possible that amiloride inhibits the cation conductance of the shore crab gill cuticle in general. Nevertheless, further experiments with other cation species are needed to verify this hypothesis. The relatively large reductions in the transcuticular current and conductance after substitution of Cl with gluconate are puzzling and contrast with the results of a previous study of the shore crab gill cuticle (Lignon, 1987
). The sum of the conductance decreases induced by wash-out of Na+ and Cl (555±71+371±75=926±103 mS cm2) is far larger than the conductance in the presence of NaCl (583±71 mS cm2), suggesting that the presence of Cl has a positive influence on the permeability of the cuticle for Na+.
How could Na+-coupled Cl absorption continue after blockade of the cuticular Na+ conductance by amiloride? First, it is important to realise that amiloride did not completely abolish the influx and efflux of Na+ (Fig. 1). Even at the maximal dose of the diuretic, 2030 % of the Na+ fluxes were maintained. Moreover, the paracellular pathway of the shore crab gill epithelium seems to be cation-selective, with a high conductance (26±1 mS cm2) (Riestenpatt et al., 1996). Thus, Na+ actively absorbed from the subcuticular space might be replaced by recycling along this pathway. Such paracellular Na+ recycling may also explain the increases in Isc observed in individual preparations (Onken and Siebers, 1992
) (see Results). Of course, under otherwise unchanged conditions, a decrease in cuticular conductance should result in a decrease in Isc. However, this current-decreasing effect might be compensated, or even over-compensated, by the current-increasing effect of paracellular Na+ recycling.
Apart from direct measurements of changes in cuticular conductance, the most significant fingerprints of an amiloride-induced inhibition of the cuticular cation conductance of crustacean gills seem to be the hyperpolarization of the outside-positive PDte and the reduction in transbranchial influxes and effluxes of Na+ (see Fig. 1). The posterior gills of Uca tangeri and Carcinus maenas adapted to low salinities appear to be very similar with respect to the changes in PDte due to ion substitutions and transport inhibitors (cf. Drews and Graszynski, 1987; Krippeit-Drews et al., 1989
). External amiloride also induced a hyperpolarization of the outside-positive PDte in the posterior gills of Uca tangeri. As in shore crab gills, amiloride reduced both the influxes and the effluxes of Na+ across the gills of Callinectes sapidus (Cameron, 1979
). Thus, as in Carcinus maenas, and also in Uca tangeri and Callinectes sapidus, the effects of amiloride might be due to an interaction with the cuticle and not with the external surface of the gill epithelium. In contrast, however, in whole Procambarus spp. (Kirschner et al., 1973
) and Astacus leptodactylus (Ehrenfeld, 1974
) and in posterior gills of Eriocheir sinensis (Riestenpatt, 1995
), only the Na+ influxes were affected by amiloride, suggesting that, in these animals, the diuretic acted at the level of the apical membrane and not on the cuticle. In the posterior gills of Chinese crabs, amiloride has been reported to increase the cuticular conductance (Péqueux and Lignon, 1989
), and the presence of apical Na+ channels has been convincingly demonstrated using amiloride-induced current-noise analysis (Zeiske et al., 1992
). However, the cuticular conductance of the anterior gills of Chinese crabs has been reported to be reduced by the diuretic (Péqueux and Lignon, 1989
). It seems that the observed effects of amiloride on the cuticle of Carcinus maenas cannot be generalised for all Crustacea.
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Acknowledgments |
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