Camp. Biochem. Physiol. Vol. 103C, No. 3, pp. 521-526, Printed in Great Britain
0306~4492/92 $5.00 + 0.00 0 1992Pergamon Press Ltd
DOPAMINE AS A MODULATOR OF IONIC TRANSPORT AND GLYCOLYTIC FLUXES IN THE GILLS OF THE CHINESE CRAB, ERIOCHEIR SINENSIS D.
Department of Biology, Facultes Universitaires Notre-Dame de la Paix. B-5000 Namur, Belgium (Tel. 08 l-72-4286; Fax 08 l-72-4420) (Received 8 January 1992; accepted for publicalion 17 May 1992) Abstract-l. Dopamine and dibutyryl CAMP induce a significant hyperpolarization when the so-called anterior and posterior gills isolated from the crab Eriocheir sinensis acclimated to freshwater are perfused with the same saline on both sides. 2. When compared to the anterior ones, the posterior gills show higher concentrations of fructose 2,6-biphosphate (Fru 2,6-bP) and a lower ATP/ADP ratio. 3. Perfusion with a freshwater or seawater saline decreases both the level of Fru 2,6-bP and the ATP/ADP ratio whereas dopamine and dibutyryl CAMP significantly increase the Fru 2,6-bP content for the posterior and the anterior gills.
Recent studies on the hormonal control of osmotic regulation in decapod crustaceans show that neurohormones such as dopamine, serotonin and octopamine may activate the transport of ions into isolated gill tissue. The perfusion of gills with dopamine induces an increased influx of Na+ in the tissue of Callinectes supidus (Kamemoto and Oyama, 1985). Dopamine and CAMP increase also sodium uptake and Na, K-ATPase activity in the gills of Curcinus maenus acclimated to fresh water (Sommer and Mantel, 1988). This supports the results presented by Lohrmann and Kamemoto (1987) showing that perfusion with dibutyryl CAMP increases Na + uptake by isolated gills of C. sapidus. A physiological situation in which the action of dopamine might be central for
the acclimation to a dilute medium is suggested by Zatta (1987) who found that the concentration of biogenic amine in the hemolymph of C. maenas increased within a short time when it was placed in a dilute medium. Sommer and Mantel (1988) recently found that the injection of a pericardial organ extract led to an increase in the Na, K-ATPase activity and the influx of Na+ in gills of the green crab. In addition, incubation of isolated gills with dibutyryl CAMP increased the enzyme activity. The same authors demonstrated that the injection of dopamine could indeed increase the concentration of CAMP in the gills of C. maenas (Sommer and Mantel, 1991). Moreover, a short-time exposure but not a long-term acclimation of crabs to 40% seawater (SW) also increased the CAMP content of the posterior and anterior gills. These results point to a possible role of neuroendocrine factors in osmoregulation among crustaceans. They also indicate that the physiological effects of dopamine on gills are important in the early stages of
acclimation and that they are related to an increase in the concentration of CAMP. The relationship existing between CAMP and the actual transport of Na+ and Cl- is strengthened by a recent study on isolated gills of Eriocheir sinensis (Bianchini and Gilles, 1990) which indicates that increased concentrations of CAMP brought about by internal perfusion result in an increased transepithelial potential difference and an increase influx of both Na+ and Cl-. Such effect on the active ionic transport must require renewable energy in order to allow a continuous working of ionic pumps and channels when crabs are acclimated to a dilute medium. In this connection, the ATP/ADP ratio and the concentration of fructose 2,6-biphosphate are two important glycolytic signals. In eucaryotic cells, fructose 2,6-biphosphate has been found to act as a key control metabolite for the coordination of glycolysis and gluconeogenesis. The function of this phosphate ester appears to be to concentrate signals from various metabolic processes at one point of control, PFK, the limiting reaction of glycolysis (Hue, 1987). In mammalian tissues the concentrations of fructose 2,6-biphosphate are modulated by extracellular signals such as hormones or fuel supply (starvation-diabetes-anoxia), the relative availability of gluconeogenic substrates and muscular work (Hers and Van Schaftingen, 1982; Kuwajima and Uyeda, 1982). Hue and Rider (1987) showed a rather close parallelism between the rate of glycolysis and the concentration of fructose 2,6-biphosphate in all studied tissues except cells (like cultured astrocytes, sperm cells and red blood cells) that depend exclusively on glycolysis for their energy requirements. In invertebrate animals, fructose 2,6-biphosphate activates the PFK from insect flight muscle and from foot muscle of the marine whelk (Storey, 1985). It is interesting to note that an ability to survive long periods of anoxia is a major requirement for many species of marine invertebrates which inhabit the intertidal zone. Storey (1988) suggests that during 521
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anerobiosis the degradation of fructose 2,6-biphosphate in gills of mussels may be involved in limiting the use of carbohydrate for purposes other than the energy production. In this work, we study the behaviour of two metabolic indicators (fructose 2,6-biphosphate and ATP/ADP ratio) when posterior and anterior gills of E. sinensis are perfused in the presence of different solutions (FW or SW) with or without dopamine and dibutyryl CAMP. Moreover, the effects of these molecules are tested on the physiological activity of perfused gills with the purpose to establish a relation between ionic transport and glycolytic activity. MATERIALS
Experiments were performed on isolated and perfused gills of the euryhaline Chinese crab Eriocheir sinensis (H. Milne-Edwards) acclimated to freshwater. Specimens caught in freshwater lakes near Emden. northern Germany, were transferred and kept at the laboratory in tanks filled with circulating and oxygenated freshwater (FIS”C). The anterior and posterior gills correspond respectively to Arthrobranchiae III, IV, V and to the 3 most posteriorly located sets of gills, Arthrobranchia VI and Pleurobranchiae I and II. Gills are handled, incubated in and perfused with the same seawater or with the same freshwater saline (FW saline), the composition of which corresponds to the ionic composition of the blood (hemolymph) of a freshwater crab. It contains (in mM): NaCl, 240; KCI, 5; MgCI,, 5; CaCl,, 12.5 and H,BO,, 8.8. The seawater saline (SW saline) contains (in mM): NaCI, 480; KCI, 10; MgCI,, 10; CaCI,, 25 and H,BO,, 8.8. The pH of both solutions is adjusted to 7.6 with Tris-Base. Gill perfusion is achieved following a procedure already described (Pequeux and Gilles. 1978). The transepithelial potential difference (PD) is measured with calomel eiectrodes between the incubation medium and the collecting beakers (Gilles ef a!., 1988). Effecters of ionic transport (dopamine, dibutyryl CAMP) are added to the perfusion saline. These perfused gills are quickly frozen and kept at - 2o’C up to homogenization. The homogenate is assayed for fructose 2.6-biphosphate by a modified procedure described by Bruni ef al. (1989). The extract, obtained after homogenization in 0.25 M HCIO, and centrifugation for 3 min at 3000g (IEC Centra4B), is assayed for the ATP/ADP ratio by a bioluminescent method using a lyophilized kit (ATP ASSAY MIX) obtained from Sigma Chemical Co. The determination of ADP concentrations requires in addition pyruvate kinase and phosphoenolpyruvate as described by Hampp. 1985. ANOVA 1 test is used to compare means for statistical significance and a P i 0.05 is interpreted as being significant. Data are expressed, in graphs, as means i: S.E.M. (for t2 > 2).
posterior gills (PG). The PD returned to control values upon washing out with control saline. Subsequent addition of dibutyryl CAMP (150 PM) in the perfusion saline produced a rapid hyperpolarization (Fig. 1) which was of the same intensity as the one observed when perfusion was made with dopamine. The bioamine had no significant effect when added to the incubation saline (data not shown). We observed the same results when perfusing in identical experimenta conditions the anterior gills with dopamine or with dibutyryl CAMP; these compounds modified the PD which became more electropositive inside (Fig. 1). When the gills of the crab Eriocheir sinensis are perfused with a seawater saline, a condition where ionic transport is drastically reduced (see Pequeux and Gilles, 1981), for the posterior gills, PD values are near OmV and for the anterior ones, they remain slightly positive. In such conditions, additions of 1OOpM dopamine or 150 FM dibutyryl CAMP to the perfusion saline have no significant effect on the PD (data not shown). PDfmV1
-30 I -40: -5Oi -60 , _,O i -0
Time of Derfusion (min.)
PO fmV) 70 SOL
50 40 / 30,t
Effect of dopamine and dibutyryl CAMP on the transepithelial potential difference (PD)
When bathed with the same control FW saline on both sides, anterior and posterior gills show polarities of opposite signs, positive inside for the anterior gills and negative inside for the posterior ones. When added to the perfusion saline (serosal side), dopamine ( 100 p M) immediately increases the inside electronegativity of the epithelium of the
40 Time of perfusion
Fig. 1. Effect of 100 MM dopamine and 150 PM dibutyryl CAMP on the transepithelial potential difference (PD) of perfused posterior or anterior gills isolated from the freshwater-acclimated crab Eriocheirsinensis.Dopamine was first
added to the perfusion medium (IN). After 60 min. the perfusion solution containing the bioamine was washed out with freshwater (FW) saline. Then, the cyclic nucleotide was added to the same bath (IN).
Dopamine modulates ionic transport
experimental situations. After a 30 min perfusion with FW saline, addition of 100 PM dopamine to the perfusion medium induced a significant decrease of this ratio in the anterior gills but not in the posterior gills where it was already low. We observed the same results when the gills were perfused with 150 PM dibutyryl CAMP (Fig. 4). Perfusion with SW saline in the presence of dopamine induced also a reduction of the ATP/ADP ratio in both types of gills (Fig. 5). DISCUSSION
Fig. 2. Fru 2,6-bP concentrations in posterior and anterior gills, perfused with FW saline (n = 6), with dopamine 100pM (n = 4) or with dibutyryl CAMP 150pM (n = 2). Each column represents the mean of n experiments f S.D.
Evidence is now accumulating which points to a neuroendocrine control of the active branchial ion uptake (Kamemoto, 1982; Mantel, 1985). A mechanism controlling the activity of ionic pumps must exist. The probable bioamine/cAMP regulatory system found in the euryhaline crab E. sinensis (Trausch et al., 1989) may be similar to that discovered in mussels (Dietz and Scheide, 1982; Scheide and Dietz, 1986) and in Aplysia californica gills (Weiss and Drummond, 1981). Bioamines, as dopamine or serotonin, increase the CAMP levels and stimulate adenylate cyclase, the enzyme responsible for the production of CAMP, in Apiysia gill tissue. Previous studies in our laboratory have demonstrated the presence of a CAMP dependent protein kinase activity in the posterior and anterior gills of the freshwater acclimated crab E. sinensis (Trausch, data not published). It is therefore reasonable to consider that, in our system, dopamine can act by activating adenylate cyclase which increases the intracellular level of CAMP (perfusion with dibutyryl CAMP leads also to an increase in this 2d messenger). This compound acts by activating a protein kinase A, which in turn phosphorylates various proteins allowing an increase in the activity of the Na+/K+ exchanger. In addition, unpublished results have shown that dibutyryl CAMP increases the ionic transport. These results are consistent with those of Bianchini and Gilles (1989) who have shown, via PD measurement,
Effects of dopamine and dibutyryl CAMP on the levels of fructose 2,6-biphosphate Vru 2,6-bP)
The posterior gills show definitively higher concentrations of fru 2,6-bP than do the anterior gills in both control and test situations. Following a 30 min perfusion with FW saline, a further perfusion of 60 mm in the presence of 100 p M dopamine increased significantly the concentration of fru 2,6-bP in both types of gills (Fig. 2). The perfusion with 150 ,uM dibutyryl CAMP induced also a statistically significant increase of the level of fru 2,6-bP in the gills of the Chinese crab (Fig. 2). When the posterior and anterior gills were perfused with SW saline, we observed the same results as with a FW medium: dopamine increased significantly the concentration of fru 2,6-bP (Fig. 3). Effects of dopamine and dibutyryl CAMP on ATPI ADP ratio in the gills of the crab
The anterior gills show a greater ATP/ADP ratio than do the posterior gills in both control and
0.0 Control SW
Fig. 3. Fru 2,6-bP concentrations in posterior and anterior gills, perfused with SW saline (n = 2) or with dopamine (n = 5). Each column represents the mean of n experiments k S.D.
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Fig. 4. ATP/ADP ratio in posterior and anterior gills, perfused with FW saline (n = 4), with dopamine 100 PM (n = 4) or with dibutyryl CAMP 150 PM (n = 2). Each column represents the mean of n experiments + S.D.
that CAMP is implicated in the control of transepithelial movements of NaCl in E. sinensis gills by activation of both the Na + /K + exchanger and the Cl channels located on the serosal side of the epithelium. Dopamine, added to the perfusion saline, induced also an increase of the Na+ influx at least in the posterior gills (data not shown). Such results indicate that dopamine could be in some way implicated in the modulation of the transepithelial ionic transport in posterior and anterior gills of the Chinese crab acclimated to fresh water. The CAMP and dopamine effects observed in the present study can be interpreted in terms of changes in the activity of various components which have recently been reported as taking part in the ion movements and in the establishment of the potential difference between the inner and outer side of the gills of E. sinensis (Gilles et al., 1988). Lohrmann and Kamemoto (1987) briefly described the effect of dibutyryl CAMP on Na + uptake in the posterior gills of C. sapidus; such a modulator can be thought to activate the serosal Na+/K+ pumping system. The activation of this pump should result in an increased
turnover of K+ , the result of which being the depolarization of the epithelium by hyperpolarization of its serosal side. The fact that a hyperpolarization and not a depolarization does occur may be due to an activating effect of CAMP and/or dopamine on the Cl- channels located at the serosal side; indeed the Cl- outward movement at that side short-circuits the K+ diffusion potential (Pequeux and Gilles, 1988). Such explanation is only valuable for the posterior gills because in the anterior ones, Cl- fluxes are barely significant (Gocha et al., 1987). The hyperpolarization observed in these gills is explained principally by assuming the presence of a Na+ /K + pump located on the serosal side, and the presence of a system which relates the PD to the K+ diffusion potential generating the activity of that pump (Pequeux and Gilles, 1988). On the other hand, our results show that dopamine and dibutyryl CAMP change the metabolic activity of perfused gills. The level of fru 2,6-bP in the posterior and anterior gills increases largely in the presence of dopamine or CAMP in the perfusion medium (FW or SW). However, the relationship between ionic transport and glycolysis is not obvious. We found indeed the same physiological and metabolic events independently of the transport activity of the gills: perfusion with a seawater saline induced a diminution of ionic transport especially for the posterior gills whereas fru 2,6-bP concentrations remained rather above normal values. These results are in agreement with those of Gilles and Pequeux (1985) who have shown that the acclimation to seawater leads to a disappearance of the pumping sites and to a progressive loss of the ability to transport actively. It is logical that, in such conditions, the dopamine or dibutyryl CAMP present in the perfusion saline have no more effect on the PD of perfused gills. Whatever the transport activity of perfused gills, the effect of dopamine and CAMP on the glycolytic flux is the same. In order to sustain its metabolic activity during the perfusion, the gill draws from its own energetic reserves (glycogen). Considering the ATP/ADP ratio, we recorded its decrease during perfusion with dopamine or dibutyryl CAMP independently of the “carrier” ability of the gills. But 3.0
00 Control SW
Fig. 5. ATP/ADP ratio in posterior and anterior gills, perfused with SW saline (n = 2) or with dopamine (n = 3). Each column represents the mean of n experiments & S.D.
Dopamine modulates ionic transport there was a difference between the posterior and anterior gills that reflected a higher energetic metabolism for the posterior gills. in fact, Wanson et al. (1983) suggested that, due to an increased energy demand, the ATP production is unable to balance the ATP utilization precisely in the epithelial structures known to actively take up Na+ from the environment. These data are thus in good agreement with the idea that acclimation of hyperosmore~lating crabs like E. sinensis to dilute media leads to an increase in pumping activity that can be related to a decrease in ATP/ADP ratio, this decrease inducing in turn the well known increase in oxidative metabolism and oxygen consumption. The acclimation to seawater results on the contrary in an adenylate metabolism running in a steady state because of a fairly low energy demand; indeed the ion-pumping processes are extremely reduced. However, the reduction in the rate of ionic transport induced by perfusion with seawater saline did not modify the effects of dopamine or dibutyryl CAMP on the ATP/ADP ratio and fructose 2,6-biphosphate concentrations. Therefore, even though a close corretation exists between the energetic metabolism and the ionic transport, our experimental conditions of perfusion do not allow us to underline the contribution of glycolysis to this transport. In conclusion, we think that our results support the idea of the mobilization of systems of phosphorylationidephosphorylation inducing the phosphorylation of proteins which in turn produce either an increase of both fru 2,6-bP concentration and glycolytic flux or an increase of activity of pumping systems. Trausch et al. (1989) recently showed that bioamines by stimulating adenylate cyclase, increase the CAMP concentration and induce the phosphorylation of proteins via a CAMP dependent protein kinase. The stimulation of protein phosphorylation through bioamine receptors and its effect on the metabolic activity can be assessed as the mechanism involved in crustacean osmoregulating process. In addition, we observed that fru 2,6-bP is a reliable glycoiytic indicator for crustaceans. As in all mammalian tissues, it is tempting to assume that fru 2,6-bP plays the major role in the control of glycolysis in the gills of E. sinensis and that its action is modulable by different effecters like dopamine and dibutyryl CAMP. ~cknow~~dgeme~is-This study was supported by the Fonds de la Recherche Fondamentale Collective (grant No. 2.4516.86). REFERENCES Bianchini A. and Gilles R. (1990) Cyclic AMP as a modulator of NaCl transport in gills of the euryhaline Chinese crab Eriocheir sinensis. Mar. Biol. 104, 191-195. Bruni P.. Vasta V. and Farnaro P. (1989) An endpoint enzymatic assay for fructose 2,6-biphosphate performed in 96-well mates. Anal. Biochem. 178, 324-326. Dietz T. H. a’nd Seheide J. I. (1982) Monoamine transmitters and CAMP stjmulation of Na transport in freshwater mussels. Can J. Zoo/. 60, 1408-1411. CBPC)
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