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Net uptake of chloride across the posterior gills of the Chinese crab (Eriocheir sinensis)
Comparative Biochemistry and Physiology Part A 137 (2004) 51–55
Net uptake of chloride across the posterior gills of the Chinese crab (Eriocheir sinensis) Martina Rathmayera,*, Dietrich Siebersb a
University of Konstanz, Faculty of Biology, Fach M624, Konstanz D-78457, Germany Alfred-Wegener-Institute for Polar and Marine Research, Am Handelshafen 12, Bremerhaven D-27570, Germany
b
Received 4 December 2002; received in revised form 3 September 2003; accepted 4 September 2003
Abstract Two methods are commonly used for the determination of transbranchial net fluxes of Naq and Cly: direct analysis of changes in ion concentrations in the external medium using flame spectrophotometry or titration (net flux method), and measurement of unidirectional ion fluxes by means of radioactive tracers (tracer method). When we applied both methods in the same preparation, the isolated perfused posterior gill of freshwater-acclimated Eriocheir sinensis, to determine net fluxes of Cly, the results differed substantially. In artificial fresh water (AFW) containing NaCl, the net flux method yielded a net uptake, but the tracer method showed a net efflux of Cly. The net uptake of Cly was abolished in Naq-free AFW indicating that Cly uptake is coupled with the uptake of Naq . Applying the tracer method, net efflux of Cly remained almost unchanged in Naq-free AFW. This suggests the opposite mechanism, i.e. uncoupled uptake of Naq and Cly. The discrepancy in the results obviously depends on the method employed. Since the data obtained with the net flux method explain the osmoregulatory performance of crabs living in fresh water, we consider this method as appropriate for determining net transbranchial ion fluxes. 䊚 2003 Elsevier Inc. All rights reserved. Keywords: Crustacea; Coupling of ion uptake; Gill perfusion; Salinity adaptation; Ion transport; Net flux method; Tracer method; Short circuit current method; Osmoregulation
1. Introduction The Chinese mitten crab, Eriocheir sinensis, spends most of its life in fresh water. Crabs migrate into estuaries and coastal waters only for reproduction (Peters and Panning, 1933). To cope with the large ion gradients between hemolymph and environment, the crabs compensate loss of ions in fresh water through gills and urine by active uptake of Naq and Cly across the gills (for summary see ´ Pequeux, 1995). We have recently determined the *Corresponding author. Tel.: q49-7531-61012; fax: q497531-883894. E-mail address: [email protected] (M. Rathmayer).
ion balance and kinetics of net uptake of Naq and Cly by isolated perfused posterior gills under fresh water conditions (Rathmayer and Siebers, 2001). These data accord with environmental demands for animals living in fresh water. We also reported coupling of Naq and Cly net uptake. The data were obtained by direct determination of Naq and Cly concentrations in the medium bathing the gills using flame photometry and titration (net flux method). Our data differ from those obtained by measuring unidirectional fluxes by means of radi´ oactive tracers (tracer method: Pequeux and Gilles, 1981; Gocha et al., 1987; Riestenpatt et al., 1996b) or by measuring short circuit currents in split gill lamellae preparations mounted in a micro-Ussing
1095-6433/04/$ - see front matter 䊚 2003 Elsevier Inc. All rights reserved. doi:10.1016/S1095-6433(03)00270-8
52
M. Rathmayer, D. Siebers / Comparative Biochemistry and Physiology Part A 137 (2004) 51–55
chamber (Isc method: Schwarz and Graszynski, 1989; Riestenpatt et al., 1994, 1996b; Onken and Riestenpatt, 1998). The latter two methods indicated net efflux instead of uptake of ions in fresh water of low salinity, kinetics inappropriate for animals living in fresh water, and independent rather than coupled uptake of Naq and Cly. To resolve these discrepancies, we have applied both the tracer and the net flux method in parallel in the same preparations of perfused posterior gills of freshwater-adapted E. sinensis. We show that the two methods indeed yield different results with respect to net ion uptake and to coupling of this process. The data obtained with the net flux method match the performance of crabs adapted to fresh water. 2. Materials and methods Composition of the perfusion saline was (mmol ly1): NaCl 300, KCl 4.0, MgCl2 5.0, CaCl2 12.5, NaHCO3 6.0, Tris 10.0 titrated to pH 7.5 by addition of 1 mol ly1 maleic acid anhydride, and aerated with 1% CO2 in air resulting in a pH value of 7.3. The external medium was artificial fresh water (AFW) of the composition (mmol ly1): NaCl 1.8, K-gluconate 0.04, Ca-gluconate 1.25, MgSO4 0.31, Tris-maleate 1.0 of pH 7.6; on aeration the pH changed to 7.8–8.0. Animals, preparation, experimental set up, and probe sampling procedures have been described in detail by Rathmayer and Siebers (2001). Net Cly fluxes were determined by analysing the samples for Cly by titration. The tracer method followed the description of Riestenpatt et al. (1996a). At the beginning of an experiment, 40 ml of 36Cly (Amersham, 120 mCi in 0.1–0.3 mol ly1 HCl) were added to 40 ml of AFW in which the gill was suspended. This changed the pH from 8 to 7.7, which was still in the normal range for uptake (see Rathmayer and Siebers, 2001). After taking two 2 ml samples at an interval of 20 min for net flux determination, samples for the tracer flux measurements were collected in the following sequence: Three samples of 20 ml were taken from the bath to set the standard. Then, for the determination of Cly influx, the perfusion saline leaving the efferent gill vessel was collected in scintillation vessels in five samples, each covering a period of 5 min. After 30 min required completing these measurements, the sampling from the bath for the net flux determination was resumed
with two 2 ml probes at an interval of 20 min. Finally, to determine 36Cly efflux, the gill was washed and perfused with 25 ml saline containing 25 ml 36Cly. After the complete exchange of the perfusate (see below), the gill was suspended in 35 ml AFW (1.8 mmol ly1 NaCl). Six samples of 2 ml each were taken at 5 min intervals for counting, where the time of the first sample was designated t0. The same procedure was used for flux measurements in Naq-free AFW (1.8 mmol ly1 choline chloride). The dye Kiton pure blue (Chroma, Stuttgart, Germany) was added to the perfusion saline at a concentration of 50 mg ly1. This allowed detection of leaking gills through the appearance of blue colour in the bath. Leaking preparations were discarded. In experiments where the perfusion saline was substituted, the disappearance of colour in the perfusate indicated completeness of exchange. 3. Calculations The rates of net ion uptake and radio tracer fluxes were calculated as micrmolar gram gill weight (gw)y1 hy1. All data are presented as means "S.D. Statistical significance was tested with Student’s t-test. To convert the short circuit current ICl (mA cmy2 hy1) and the unidirectional flux of Cly (mmol cmy2 hy1) published by Riestenpatt et al. (1996b) and Onken and Riestenpatt (1998) into ion flux rates (mmol gy1 hy1), we used the relation 1 As37.2 mmol cmy2 hy1. The resulting value was multiplied by 319 cm2 posterior gill surface area per 100 g body weight (bw) which corresponds to 602 mg gw per 100 g bw (Rathmayer and Siebers, 2001). 4. Results The posterior gills of E. sinensis perform net uptake of Cly even in low salinity. In AFW of 1.8 mmol ly1 NaCl, the net uptake of Cly was 145"17 mmol gy1 hy1 (ns3, Table 1) when determined by the net flux method. The fact that ion uptake occurs in freshwater of such low salinity and the amount of uptake measured corresponds with results reported previously (Rathmayer and Siebers, 2001 and Table 1). When the tracer method was applied in the same preparation a net efflux of 133"110 mmol gy1 hy1 (ns6) was
No.
Method
External Cly (mmol ly1)
Internal Cly (mmol ly1)
Influx of Cly (mmol g gwy1 hy1)
Efflux of Cly (mmol g gwy1 hy1)
Net influx of Cly (mmol g gwy1 hy1)
Reference
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Net flux Net flux Net flux Net flux Tracer Tracer Tracer Tacer Net flux Net flux Tracer Tracer Isc Isc
1.0 NaCl 1.8 NaCl 1.8 NaCl 4–8 NaCla 1.0 NaCl 1.8 NaCl 10.0 NaCl 10.0 NaCl 1.8 choline-Cl 1.8 choline-Cl 1.8 choline-Cl 10 choline-Cl 10 choline-Cl 10 choline-Cl
300 300 300 300 280 300 280 300 300 300 300 300 300 300
– – – – 16"3 177"44 (10) 568"66 2544"424 – – 93"39 (9)
– – – – 138"39 300"149 (6) 469"2 1855"583 – – 249"148 (6)
– –
– –
65"1.7 (3) 170"0.9 (108) 145"17 (3) 240"69 (16) y122 (3) y133"110 (6) 99 (3) 689"742 (5) y6.5"17 (5) 3.8 b(1) y143"165 (6) 689 1850"493 (11) 1700
Rathmayer and Siebers, 2001 Rathmayer and Siebers, 2001 This paper Rathmayer and Siebers, 2001 Gocha et al., 1987 This paper Gocha et al., 1987 Riestenpatt et al., 1996b Rathmayer and Siebers, 2001 This paper This paper Riestenpatt et al., 1996b Riestenpatt et al., 1996b Onken and Riestenpatt, 1998
a b
NaCl NaCl NaCl NaCl NaCl NaCl NaCl NaCl NaCl NaCl NaCl NaCl NaCl NaCl
data for uptake were pooled from measurements in 4, 6 and 8 mmol ly1 NaCl. with regard to ns1, see discussion with reference to additional experiments. The number of experiments is shown in parentheses.
M. Rathmayer, D. Siebers / Comparative Biochemistry and Physiology Part A 137 (2004) 51–55
Table 1 Chloride fluxes across isolated gills of E. sinensis in low external concentrations of NaCl and choline chloride
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M. Rathmayer, D. Siebers / Comparative Biochemistry and Physiology Part A 137 (2004) 51–55
obtained instead of a net uptake derived from the difference between influxes and effluxes. The average of Cly influx was 177"44 (ns10) and of efflux 300"149 (ns6) mmol gy1 hy1 (Table 1). A discrepancy in the results obtained with the two methods was also apparent when Naq of the external medium was replaced by choline (Naqfree AFW). In 1.8 mmol ly1 choline chloride, the net flux method showed that the net uptake of Cly was almost abolished (3.8 mmol gy1 hy1). This figure corresponds to the values published earlier (Rathmayer and Siebers, 2001, and Table 1). The uptake was completely restored by reintroducing Naq (154 mmol gy1 hy1 before and 159 mmol gy1 hy1 after choline chloride). Analysis of the unidirectional fluxes by means of the tracer method showed reduction of influx to 93"39 (P-0.001, ns9) and of efflux to 249"148 (Ps0.6, ns6) mmol gy1 hy1. The net efflux remained almost unchanged at 143"165 (ns6) mmol gy1 hy1 (Table 1). The reduction of Cly uptake in the presence of 36Cly in 1.8 mmol ly1 choline chloride was observed with the net flux method only when 36Cly was added as HCl (see Section 2). When 36Cly was added as NaCl, which introduces 0.15 mmol ly1 Naq to the Naq-free solution, no reduction of Cly uptake occurred (121 mmol gy1 hy1 control and 120 mmol gy1 hy1 from ‘Naq-free’ AFW). This shows that the presence of a very small amount of Naq is sufficient to enable Cly uptake. With the tracer method the source of 36 Cly was without effect on the results. The potentiating effect of the presence of small concentrations of Naq is also seen by the addition of 3=10y4 mol ly1 Na-vanadate (Na3VO4), which was used to test for the possible presence of a PATPase. This increased the Naq concentration from 1.8 to 2.6 mmol ly1, but the uptake of both Naq and Cly was enhanced on average by 95"60% (P-0.01, ns9). The enhancement of net uptake of both ions by an increase of the concentration of only one ion was also observed by addition of 6 mmol ly1 Na-gluconate, or of 6 mmol ly1 choline chloride to the external saline of 1.8 mmol ly1 NaCl. In the first instance, the net uptake of Naq increased by 311%, Cly by 202%. In the second case, net uptake of Naq increased by 100% and Cly by 178%.
5. Discussion When posterior gills of E. sinensis suspended in fresh water (FW) of less than 3 mmol ly1 NaCl were perfused with hemolymph-like saline, net efflux of Cly was found when the tracer method was employed (Gocha et al., 1987). However, using the net flux method, we found a net uptake. Even in salinity as low as 0.18 mmol ly1 NaCl, a small net uptake and no efflux were determined (Rathmayer and Siebers, 2001). In the present investigation we show that in experiments using the two methods simultaneously in the same gill preparation, the tracer method indeed yielded a net efflux similar to that reported by Gocha et al. (1987). However, using the net flux method, an uptake comparable to that obtained in a previous study (Rathmayer and Siebers, 2001) was measured. To decide which result reflects the in vivo performance of the gills, it helps to consider the environmental constraints a crab experiences when living in fresh water. The compensation of loss of ions via the gills and the urine can only be achieved by net uptake mechanisms. In uptake measurements with the net flux method, net uptake saturates at a low concentration of 4 mmol ly1 and the concentration for half maximum transport rate Kt is 1.5 mmol ly1 NaCl (Rathmayer and Siebers, 2001). Similar low values have been obtained by Shaw (1961) in his studies of intact Chinese crabs. Low Kt and saturation values must be anticipated for animals living in fresh water. The two parameters are high when determined ´ with the tracer method (Pequeux and Gilles, 1981; Gocha et al., 1987), and even higher with the short circuit current (Isc) method (Riestenpatt et al., 1994). The discrepancy in results obtained by the different methods is even larger when the net uptake across gills is determined in salines where the counter ion is missing or in the presence of specific inhibitors for one ion (Rathmayer and Siebers, 2001). When the tracer method and the Isc method were applied simultaneously on the same preparation in the Ussing chamber to determine ion fluxes, both methods showed uptake from salines where either Cly (10 mmol ly1 Na-gluconate) or Naq (10 mmol ly1 choline chloride) was missing (Riestenpatt et al., 1996b; see also Table 1). In contrast, the data of the present study with the net flux method show that uptake of Cly is reversibly abolished when the external medium is Naq-free.
M. Rathmayer, D. Siebers / Comparative Biochemistry and Physiology Part A 137 (2004) 51–55
This finding demonstrates coupled uptake of Naq and Cly and accords with previous results where we have demonstrated that no net uptake occurs when the counter ion is missing (ns8) (Rathmayer and Siebers, 2001). The simultaneous measurement with the tracer method in the present study showed only reduced influx and efflux of Cly, but practically no change of net efflux. That measurement of net uptake from Naq-free external medium with the two methods leads to different results is in agreement with results in intact crayfish (Shaw, 1960). Our finding that the presence of a small amount of external Naq is sufficient to enable substantial Cly uptake sheds new light on earlier reports on FW-adapted crabs (Krogh, 1938). Since animals in KCl saline, i.e. in the absence of external Naq, still showed uptake of Cly, it was concluded that this uptake does not depend on Naq. However, it is conceivable—even when urine flow is prevented—that small amounts of Naq were lost by the animal and were responsible for the observed Cly uptake. In addition, the finding that net uptake of both ions is enhanced when the concentration of only one ion is increased shows mutual dependence of the net uptake of Naq and Cly, and supports the conclusion of coupled net uptake of these ions. We, therefore, conclude that both the tracer and the Isc method are unsuitable for determining the exact net uptake of Naq and Cly from FW by the gills of E. sinensis. However, these methods offer advantages for the study of other aspects: With the tracer method, influx and efflux can be studied separately, and this method can be applied at higher external ion concentrations. But other effects, for example exchange diffusion (Shaw, 1960; Ussing, 1947, 1978), or erroneous ion effluxes of radioactive tracer because of the large osmotic and ion concentration gradients, which can blur the results, must be considered. For studying the mechanisms of uptake, the Isc method is a most appropriate tool. To explain the large discrepancy in the results on kinetics and coupling of NaCl transport obtained by the Isc method and the net uptake method, an additional mechanism to those suggested by Onken and Riestenpatt (1998) for the regulation of net uptake of NaCl must be inferred.
55
Acknowledgments We thank Mary A. Cahill for help with the English. The experiments were performed according to the current laws of animal protection in Germany. References ´ Gocha, N., Pequeux, S., Wanson, S., Gilles, R., 1987. Cly fluxes across isolated, perfused gills of the Chinese crab Eriocheir sinensis acclimated to fresh water. Comp. Biochem. Physiol. A 88, 581–584. Krogh, A., 1938. The active absorption of ions in some fresh water animals. Z. Vergl. Physiol. 25, 335–350. Onken, H., Riestenpatt, S., 1998. NaCl absorption across split gill lamellae of hyper-regulating crabs: transport mechanisms and their regulation. Comp. Biochem. Physiol. A 119, 883–893. ´ Pequeux, A., 1995. Osmotic regulation in crustaceans. J. Crustacean Biol. 15, 1–60. ´ Pequeux, A., Gilles, R., 1981. Naq fluxes across isolated perfused gills of the Chinese crab Eriocheir sinensis. J. Exp. Biol. 92, 173–186. Peters, N., Panning, A., 1933. Die chinesische wollhandkrabbe in Deutschland. Zool. Anz. 104, 1–180. Rathmayer, M., Siebers, D., 2001. Ionic balance in the freshwater-adapted Chinese crab, Eriocheir sinensis. J. Comp. Physiol. B 171, 271–281. Riestenpatt, S., Onken, H., Siebers, D., 1996a. Active absorption of sodium and chloride across the gill epithelium of the shore crab Carcinus maenas: voltage-clamp and ion flux studies. J. Exp. Biol. 199, 1545–1554. Riestenpatt, S., Siebers, D., Onken, H., 1996b. NaCl-absorption across the gill epithelium of the Chinese crab, Eriocheir sinensis: tracer flux studies and electrophysiological investigations on split gill lamellae. Verh. Dtsch. Zool. Ges. 89, 175. Riestenpatt, S., Zeiske, W., Onken, H., 1994. Cyclic AMP stimulation of electrogenic uptake of Naq and Cly across the gill epithelium of the Chinese crab Eriocheir sinensis. J. Exp. Biol. 188, 159–174. Schwarz, H.J., Graszynski, K., 1989. Ion transport in crab gills: a new method using isolated half platelets of Eriocheir gills in an Ussing type chamber. Comp. Biochem. Physiol. A 92, 601–604. Shaw, J., 1960. The absorption of chloride ions by the crayfish, Astacus pallipes Lereboullet. J. Exp. Biol. 37, 557–572. Shaw, J., 1961. Sodium balance in Eriocheir sinensis. The adaptation of the Crustacea to fresh water. J. Exp. Biol. 38, 153–162. Ussing, H.H., 1947. Interpretation of the exchange of radiosodium in isolated muscle. Nature 160, 262. Ussing, H.H., 1978. Interpretation of tracer fluxes. In: Giebisch, G., Andersen, O.S. (Eds.), Membrane Transport in Biology, Concepts and Models, Vol. 1. Springer Verlag, Berlin, Heidelberg, New York, pp. 115–140.
Net uptake of chloride across the posterior gills of the Chinese crab (Eriocheir sinensis) Martina Rathmayera,*, Dietrich Siebersb a
University of Konstanz, Faculty of Biology, Fach M624, Konstanz D-78457, Germany Alfred-Wegener-Institute for Polar and Marine Research, Am Handelshafen 12, Bremerhaven D-27570, Germany
b
Received 4 December 2002; received in revised form 3 September 2003; accepted 4 September 2003
Abstract Two methods are commonly used for the determination of transbranchial net fluxes of Naq and Cly: direct analysis of changes in ion concentrations in the external medium using flame spectrophotometry or titration (net flux method), and measurement of unidirectional ion fluxes by means of radioactive tracers (tracer method). When we applied both methods in the same preparation, the isolated perfused posterior gill of freshwater-acclimated Eriocheir sinensis, to determine net fluxes of Cly, the results differed substantially. In artificial fresh water (AFW) containing NaCl, the net flux method yielded a net uptake, but the tracer method showed a net efflux of Cly. The net uptake of Cly was abolished in Naq-free AFW indicating that Cly uptake is coupled with the uptake of Naq . Applying the tracer method, net efflux of Cly remained almost unchanged in Naq-free AFW. This suggests the opposite mechanism, i.e. uncoupled uptake of Naq and Cly. The discrepancy in the results obviously depends on the method employed. Since the data obtained with the net flux method explain the osmoregulatory performance of crabs living in fresh water, we consider this method as appropriate for determining net transbranchial ion fluxes. 䊚 2003 Elsevier Inc. All rights reserved. Keywords: Crustacea; Coupling of ion uptake; Gill perfusion; Salinity adaptation; Ion transport; Net flux method; Tracer method; Short circuit current method; Osmoregulation
1. Introduction The Chinese mitten crab, Eriocheir sinensis, spends most of its life in fresh water. Crabs migrate into estuaries and coastal waters only for reproduction (Peters and Panning, 1933). To cope with the large ion gradients between hemolymph and environment, the crabs compensate loss of ions in fresh water through gills and urine by active uptake of Naq and Cly across the gills (for summary see ´ Pequeux, 1995). We have recently determined the *Corresponding author. Tel.: q49-7531-61012; fax: q497531-883894. E-mail address: [email protected] (M. Rathmayer).
ion balance and kinetics of net uptake of Naq and Cly by isolated perfused posterior gills under fresh water conditions (Rathmayer and Siebers, 2001). These data accord with environmental demands for animals living in fresh water. We also reported coupling of Naq and Cly net uptake. The data were obtained by direct determination of Naq and Cly concentrations in the medium bathing the gills using flame photometry and titration (net flux method). Our data differ from those obtained by measuring unidirectional fluxes by means of radi´ oactive tracers (tracer method: Pequeux and Gilles, 1981; Gocha et al., 1987; Riestenpatt et al., 1996b) or by measuring short circuit currents in split gill lamellae preparations mounted in a micro-Ussing
1095-6433/04/$ - see front matter 䊚 2003 Elsevier Inc. All rights reserved. doi:10.1016/S1095-6433(03)00270-8
52
M. Rathmayer, D. Siebers / Comparative Biochemistry and Physiology Part A 137 (2004) 51–55
chamber (Isc method: Schwarz and Graszynski, 1989; Riestenpatt et al., 1994, 1996b; Onken and Riestenpatt, 1998). The latter two methods indicated net efflux instead of uptake of ions in fresh water of low salinity, kinetics inappropriate for animals living in fresh water, and independent rather than coupled uptake of Naq and Cly. To resolve these discrepancies, we have applied both the tracer and the net flux method in parallel in the same preparations of perfused posterior gills of freshwater-adapted E. sinensis. We show that the two methods indeed yield different results with respect to net ion uptake and to coupling of this process. The data obtained with the net flux method match the performance of crabs adapted to fresh water. 2. Materials and methods Composition of the perfusion saline was (mmol ly1): NaCl 300, KCl 4.0, MgCl2 5.0, CaCl2 12.5, NaHCO3 6.0, Tris 10.0 titrated to pH 7.5 by addition of 1 mol ly1 maleic acid anhydride, and aerated with 1% CO2 in air resulting in a pH value of 7.3. The external medium was artificial fresh water (AFW) of the composition (mmol ly1): NaCl 1.8, K-gluconate 0.04, Ca-gluconate 1.25, MgSO4 0.31, Tris-maleate 1.0 of pH 7.6; on aeration the pH changed to 7.8–8.0. Animals, preparation, experimental set up, and probe sampling procedures have been described in detail by Rathmayer and Siebers (2001). Net Cly fluxes were determined by analysing the samples for Cly by titration. The tracer method followed the description of Riestenpatt et al. (1996a). At the beginning of an experiment, 40 ml of 36Cly (Amersham, 120 mCi in 0.1–0.3 mol ly1 HCl) were added to 40 ml of AFW in which the gill was suspended. This changed the pH from 8 to 7.7, which was still in the normal range for uptake (see Rathmayer and Siebers, 2001). After taking two 2 ml samples at an interval of 20 min for net flux determination, samples for the tracer flux measurements were collected in the following sequence: Three samples of 20 ml were taken from the bath to set the standard. Then, for the determination of Cly influx, the perfusion saline leaving the efferent gill vessel was collected in scintillation vessels in five samples, each covering a period of 5 min. After 30 min required completing these measurements, the sampling from the bath for the net flux determination was resumed
with two 2 ml probes at an interval of 20 min. Finally, to determine 36Cly efflux, the gill was washed and perfused with 25 ml saline containing 25 ml 36Cly. After the complete exchange of the perfusate (see below), the gill was suspended in 35 ml AFW (1.8 mmol ly1 NaCl). Six samples of 2 ml each were taken at 5 min intervals for counting, where the time of the first sample was designated t0. The same procedure was used for flux measurements in Naq-free AFW (1.8 mmol ly1 choline chloride). The dye Kiton pure blue (Chroma, Stuttgart, Germany) was added to the perfusion saline at a concentration of 50 mg ly1. This allowed detection of leaking gills through the appearance of blue colour in the bath. Leaking preparations were discarded. In experiments where the perfusion saline was substituted, the disappearance of colour in the perfusate indicated completeness of exchange. 3. Calculations The rates of net ion uptake and radio tracer fluxes were calculated as micrmolar gram gill weight (gw)y1 hy1. All data are presented as means "S.D. Statistical significance was tested with Student’s t-test. To convert the short circuit current ICl (mA cmy2 hy1) and the unidirectional flux of Cly (mmol cmy2 hy1) published by Riestenpatt et al. (1996b) and Onken and Riestenpatt (1998) into ion flux rates (mmol gy1 hy1), we used the relation 1 As37.2 mmol cmy2 hy1. The resulting value was multiplied by 319 cm2 posterior gill surface area per 100 g body weight (bw) which corresponds to 602 mg gw per 100 g bw (Rathmayer and Siebers, 2001). 4. Results The posterior gills of E. sinensis perform net uptake of Cly even in low salinity. In AFW of 1.8 mmol ly1 NaCl, the net uptake of Cly was 145"17 mmol gy1 hy1 (ns3, Table 1) when determined by the net flux method. The fact that ion uptake occurs in freshwater of such low salinity and the amount of uptake measured corresponds with results reported previously (Rathmayer and Siebers, 2001 and Table 1). When the tracer method was applied in the same preparation a net efflux of 133"110 mmol gy1 hy1 (ns6) was
No.
Method
External Cly (mmol ly1)
Internal Cly (mmol ly1)
Influx of Cly (mmol g gwy1 hy1)
Efflux of Cly (mmol g gwy1 hy1)
Net influx of Cly (mmol g gwy1 hy1)
Reference
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Net flux Net flux Net flux Net flux Tracer Tracer Tracer Tacer Net flux Net flux Tracer Tracer Isc Isc
1.0 NaCl 1.8 NaCl 1.8 NaCl 4–8 NaCla 1.0 NaCl 1.8 NaCl 10.0 NaCl 10.0 NaCl 1.8 choline-Cl 1.8 choline-Cl 1.8 choline-Cl 10 choline-Cl 10 choline-Cl 10 choline-Cl
300 300 300 300 280 300 280 300 300 300 300 300 300 300
– – – – 16"3 177"44 (10) 568"66 2544"424 – – 93"39 (9)
– – – – 138"39 300"149 (6) 469"2 1855"583 – – 249"148 (6)
– –
– –
65"1.7 (3) 170"0.9 (108) 145"17 (3) 240"69 (16) y122 (3) y133"110 (6) 99 (3) 689"742 (5) y6.5"17 (5) 3.8 b(1) y143"165 (6) 689 1850"493 (11) 1700
Rathmayer and Siebers, 2001 Rathmayer and Siebers, 2001 This paper Rathmayer and Siebers, 2001 Gocha et al., 1987 This paper Gocha et al., 1987 Riestenpatt et al., 1996b Rathmayer and Siebers, 2001 This paper This paper Riestenpatt et al., 1996b Riestenpatt et al., 1996b Onken and Riestenpatt, 1998
a b
NaCl NaCl NaCl NaCl NaCl NaCl NaCl NaCl NaCl NaCl NaCl NaCl NaCl NaCl
data for uptake were pooled from measurements in 4, 6 and 8 mmol ly1 NaCl. with regard to ns1, see discussion with reference to additional experiments. The number of experiments is shown in parentheses.
M. Rathmayer, D. Siebers / Comparative Biochemistry and Physiology Part A 137 (2004) 51–55
Table 1 Chloride fluxes across isolated gills of E. sinensis in low external concentrations of NaCl and choline chloride
53
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obtained instead of a net uptake derived from the difference between influxes and effluxes. The average of Cly influx was 177"44 (ns10) and of efflux 300"149 (ns6) mmol gy1 hy1 (Table 1). A discrepancy in the results obtained with the two methods was also apparent when Naq of the external medium was replaced by choline (Naqfree AFW). In 1.8 mmol ly1 choline chloride, the net flux method showed that the net uptake of Cly was almost abolished (3.8 mmol gy1 hy1). This figure corresponds to the values published earlier (Rathmayer and Siebers, 2001, and Table 1). The uptake was completely restored by reintroducing Naq (154 mmol gy1 hy1 before and 159 mmol gy1 hy1 after choline chloride). Analysis of the unidirectional fluxes by means of the tracer method showed reduction of influx to 93"39 (P-0.001, ns9) and of efflux to 249"148 (Ps0.6, ns6) mmol gy1 hy1. The net efflux remained almost unchanged at 143"165 (ns6) mmol gy1 hy1 (Table 1). The reduction of Cly uptake in the presence of 36Cly in 1.8 mmol ly1 choline chloride was observed with the net flux method only when 36Cly was added as HCl (see Section 2). When 36Cly was added as NaCl, which introduces 0.15 mmol ly1 Naq to the Naq-free solution, no reduction of Cly uptake occurred (121 mmol gy1 hy1 control and 120 mmol gy1 hy1 from ‘Naq-free’ AFW). This shows that the presence of a very small amount of Naq is sufficient to enable Cly uptake. With the tracer method the source of 36 Cly was without effect on the results. The potentiating effect of the presence of small concentrations of Naq is also seen by the addition of 3=10y4 mol ly1 Na-vanadate (Na3VO4), which was used to test for the possible presence of a PATPase. This increased the Naq concentration from 1.8 to 2.6 mmol ly1, but the uptake of both Naq and Cly was enhanced on average by 95"60% (P-0.01, ns9). The enhancement of net uptake of both ions by an increase of the concentration of only one ion was also observed by addition of 6 mmol ly1 Na-gluconate, or of 6 mmol ly1 choline chloride to the external saline of 1.8 mmol ly1 NaCl. In the first instance, the net uptake of Naq increased by 311%, Cly by 202%. In the second case, net uptake of Naq increased by 100% and Cly by 178%.
5. Discussion When posterior gills of E. sinensis suspended in fresh water (FW) of less than 3 mmol ly1 NaCl were perfused with hemolymph-like saline, net efflux of Cly was found when the tracer method was employed (Gocha et al., 1987). However, using the net flux method, we found a net uptake. Even in salinity as low as 0.18 mmol ly1 NaCl, a small net uptake and no efflux were determined (Rathmayer and Siebers, 2001). In the present investigation we show that in experiments using the two methods simultaneously in the same gill preparation, the tracer method indeed yielded a net efflux similar to that reported by Gocha et al. (1987). However, using the net flux method, an uptake comparable to that obtained in a previous study (Rathmayer and Siebers, 2001) was measured. To decide which result reflects the in vivo performance of the gills, it helps to consider the environmental constraints a crab experiences when living in fresh water. The compensation of loss of ions via the gills and the urine can only be achieved by net uptake mechanisms. In uptake measurements with the net flux method, net uptake saturates at a low concentration of 4 mmol ly1 and the concentration for half maximum transport rate Kt is 1.5 mmol ly1 NaCl (Rathmayer and Siebers, 2001). Similar low values have been obtained by Shaw (1961) in his studies of intact Chinese crabs. Low Kt and saturation values must be anticipated for animals living in fresh water. The two parameters are high when determined ´ with the tracer method (Pequeux and Gilles, 1981; Gocha et al., 1987), and even higher with the short circuit current (Isc) method (Riestenpatt et al., 1994). The discrepancy in results obtained by the different methods is even larger when the net uptake across gills is determined in salines where the counter ion is missing or in the presence of specific inhibitors for one ion (Rathmayer and Siebers, 2001). When the tracer method and the Isc method were applied simultaneously on the same preparation in the Ussing chamber to determine ion fluxes, both methods showed uptake from salines where either Cly (10 mmol ly1 Na-gluconate) or Naq (10 mmol ly1 choline chloride) was missing (Riestenpatt et al., 1996b; see also Table 1). In contrast, the data of the present study with the net flux method show that uptake of Cly is reversibly abolished when the external medium is Naq-free.
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This finding demonstrates coupled uptake of Naq and Cly and accords with previous results where we have demonstrated that no net uptake occurs when the counter ion is missing (ns8) (Rathmayer and Siebers, 2001). The simultaneous measurement with the tracer method in the present study showed only reduced influx and efflux of Cly, but practically no change of net efflux. That measurement of net uptake from Naq-free external medium with the two methods leads to different results is in agreement with results in intact crayfish (Shaw, 1960). Our finding that the presence of a small amount of external Naq is sufficient to enable substantial Cly uptake sheds new light on earlier reports on FW-adapted crabs (Krogh, 1938). Since animals in KCl saline, i.e. in the absence of external Naq, still showed uptake of Cly, it was concluded that this uptake does not depend on Naq. However, it is conceivable—even when urine flow is prevented—that small amounts of Naq were lost by the animal and were responsible for the observed Cly uptake. In addition, the finding that net uptake of both ions is enhanced when the concentration of only one ion is increased shows mutual dependence of the net uptake of Naq and Cly, and supports the conclusion of coupled net uptake of these ions. We, therefore, conclude that both the tracer and the Isc method are unsuitable for determining the exact net uptake of Naq and Cly from FW by the gills of E. sinensis. However, these methods offer advantages for the study of other aspects: With the tracer method, influx and efflux can be studied separately, and this method can be applied at higher external ion concentrations. But other effects, for example exchange diffusion (Shaw, 1960; Ussing, 1947, 1978), or erroneous ion effluxes of radioactive tracer because of the large osmotic and ion concentration gradients, which can blur the results, must be considered. For studying the mechanisms of uptake, the Isc method is a most appropriate tool. To explain the large discrepancy in the results on kinetics and coupling of NaCl transport obtained by the Isc method and the net uptake method, an additional mechanism to those suggested by Onken and Riestenpatt (1998) for the regulation of net uptake of NaCl must be inferred.
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Acknowledgments We thank Mary A. Cahill for help with the English. The experiments were performed according to the current laws of animal protection in Germany. References ´ Gocha, N., Pequeux, S., Wanson, S., Gilles, R., 1987. Cly fluxes across isolated, perfused gills of the Chinese crab Eriocheir sinensis acclimated to fresh water. Comp. Biochem. Physiol. A 88, 581–584. Krogh, A., 1938. The active absorption of ions in some fresh water animals. Z. Vergl. Physiol. 25, 335–350. Onken, H., Riestenpatt, S., 1998. NaCl absorption across split gill lamellae of hyper-regulating crabs: transport mechanisms and their regulation. Comp. Biochem. Physiol. A 119, 883–893. ´ Pequeux, A., 1995. Osmotic regulation in crustaceans. J. Crustacean Biol. 15, 1–60. ´ Pequeux, A., Gilles, R., 1981. Naq fluxes across isolated perfused gills of the Chinese crab Eriocheir sinensis. J. Exp. Biol. 92, 173–186. Peters, N., Panning, A., 1933. Die chinesische wollhandkrabbe in Deutschland. Zool. Anz. 104, 1–180. Rathmayer, M., Siebers, D., 2001. Ionic balance in the freshwater-adapted Chinese crab, Eriocheir sinensis. J. Comp. Physiol. B 171, 271–281. Riestenpatt, S., Onken, H., Siebers, D., 1996a. Active absorption of sodium and chloride across the gill epithelium of the shore crab Carcinus maenas: voltage-clamp and ion flux studies. J. Exp. Biol. 199, 1545–1554. Riestenpatt, S., Siebers, D., Onken, H., 1996b. NaCl-absorption across the gill epithelium of the Chinese crab, Eriocheir sinensis: tracer flux studies and electrophysiological investigations on split gill lamellae. Verh. Dtsch. Zool. Ges. 89, 175. Riestenpatt, S., Zeiske, W., Onken, H., 1994. Cyclic AMP stimulation of electrogenic uptake of Naq and Cly across the gill epithelium of the Chinese crab Eriocheir sinensis. J. Exp. Biol. 188, 159–174. Schwarz, H.J., Graszynski, K., 1989. Ion transport in crab gills: a new method using isolated half platelets of Eriocheir gills in an Ussing type chamber. Comp. Biochem. Physiol. A 92, 601–604. Shaw, J., 1960. The absorption of chloride ions by the crayfish, Astacus pallipes Lereboullet. J. Exp. Biol. 37, 557–572. Shaw, J., 1961. Sodium balance in Eriocheir sinensis. The adaptation of the Crustacea to fresh water. J. Exp. Biol. 38, 153–162. Ussing, H.H., 1947. Interpretation of the exchange of radiosodium in isolated muscle. Nature 160, 262. Ussing, H.H., 1978. Interpretation of tracer fluxes. In: Giebisch, G., Andersen, O.S. (Eds.), Membrane Transport in Biology, Concepts and Models, Vol. 1. Springer Verlag, Berlin, Heidelberg, New York, pp. 115–140.