DOPAMINE AS A MODULATOR OF IONIC TRANSPORT AND NA+/K+-ATPASE ACTIVITY IN THE GILLS OF THE CHINESE CRAB ERIOCHEIR SINENSIS Ji Ling Mo, Pierre Devos, a n d G e r a r d Trausch A B S T R A C T Dopamine and dibutyryl cAMP induced a significant increase of 22Na+ influx in salt-transporting posterior gills isolated from the Chinese crab Eriocheir sinensis acclimated to fresh water. When isolated gills were incubated and perfused in Cl--free saline (Cl replaced by gluconate), dopamine or dibutyryl c A M P still produced a significant increase of 22Na+ influx. After 1-h perfusion, dopamine increased intracellular c A M P content and Na+/K+-ATPase activity as compared with control perfusions without dopamine. Chinese crabs, Eriocheir sinensis (H. Milne Edwards), freely migrate from the sea-water environment into fresh-water steams, where they maintain an osmotic gradient by active uptake of ions such as Na+ and Cl- across the posterior gills (Pequeux and Gilles, 1988). Active electrogenic N a ' absorption proceeds mainly via apical Na+ channels and basolateral Na4/K'-pumps, whereas electrogenic Cl uptake proceeds via apical Cl-/HCO,- antiport and basolateral Cl- channels (Onken and Zeiske, 1991) and is driven by an apical Vtype H ' pump (Putzenlechner et al., 1992). There is now increasing evidence suggesting that neuroendocrine factors, such as dopamine, affect osmoregulation especially by activating the transport of ions through the gills. It has been reported that the pericardial organ (PO), a neurohemal organ, contains dopamine that, perfused through the gill of Callinectes sapidus Rathbun, induces an increase of the uptake of sodium by the gill (Kamemoto and Oyama, 1985). Moreover, injection of a PO extract or dopamine increases the influx of sodium in gills of Carcinus maenas (Linnaeus) acclimated to brackish water (Sommer and Mantel, 1988). The nucleotide c A M P appears to be involved in the neurohormone activity. Isolated gills of Callinectes sapidus perfused with dopamine show an increase in the c A M P level after a 10-min perfusion period (Kamemoto, 1991). On the other hand, when membrane-permeable derivatives of c A M P were used in perfusing isolated posterior gills of Eriocheir sinensis, the influxes of Na+ and Clwere stimulated (Bianchini and Gilles, 1989). Previous data from our laboratory have shown that dopamine and dibutyryl cAMP (db-cAMP) induce an increase in the concentration of fructose 2,6-biphosphate when the posterior gills isolated from E. sinensis acclimated to fresh water are perfused with the same saline on both sides (Detaille et al., 1992). Such effects of bioamines on ion exchange systems seem to result from action on the activity of the Na�/K�-ATPase. This enzyme is well known as an adaptive enzyme for hyperregulatory crabs. Increased Na*/K*-ATPase activity has been reported in the posterior gills of crabs with acute exposure or with acclimation to dilute external media, such as Callinectes sapidus (see Towle et al., 1976), Eriocheir sinensis (see P6queux et al., 1984), Uca pugilator (Bosc) (see D'Orazio and Holliday, 1985), (7ca pugnax (Smith) (see Holliday, 1985), or Carcinus maenas (see Siebers et al., 1985). Adjustments of enzyme activity by neurohormones may be the result of induction or/and activation of existing enzyme for a short-term acclimation and synthesis of new enzyme for a long-term acclimation (Henry and Wheatly, 1988; Sommer and Mantel, 1988; Pequeux, 1995). The idea was proposed by Trausch et al. (1989) that stimulation of protein phosphorylation through bioamine receptors and the influence of phosphorylation of the Na+/K+-ATPase could be assessed as a mechanism defining the role of such an enzyme in crustacean osmoregulating processes. Such study indicates that effects of bioamines are unlikely to be direct, but instead appear related at least to a second messenger, cAMP. Present investigation thus focuses on the effect of dopamine on Na+/K+-ATPase activ- ity a n d Na+ t r a n s p o r t b y using a perfused preparation o f posterior gills isolated f r o m E r i o c h e i r sinensis acclimated to fresh water. O u r findings support the idea that dopam i n e effects o n Na+ m o v e m e n t s i n v o l v e in s o m e w a y the activation of Nal/K+-ATPase activity via a second m e s s e n g e r that m a y be cAMP. MATERIALS AND M E T H O D S A n i m a l s . - E x p e r i m e n t s were performed on intact gills isolated from Chinese crabs, E r i o c h e i r sinensis, acclimated to fresh water (FW). Animals captured in F W lakes near Emden, Germany, were transferred to the laboratory and kept in tanks filled with circulating and oxygenated tap water (±15°C). The experiments were conducted between December 1996 and March 1997. Salines a n d C h e m i c a l s . - I s o l a t e d gills were bathed on both sides with the same "FW saline" to measure potential difference (PD). FW saline contains (in mmol/1): NaCI, 240; KCI, 5; MgCl,, 5; CaCl2, 12.5, and H , B O " 8.8. The pH was adjusted to 7.6 with Tris-Base. In substitution experiments, gluconate was used to replace all the CI- o f the perfusion and incubation salines. N a ' influxes were measured with an incubation medium prepared by diluting 250 times the "FW saline" or gluconate saline while keeping its pH and buffer concentration constant. According to the experimental scheme, 10 mmol/I NaCl or 10 mmol/I Na-gluconate were added to the medium bathing the apical face of the gills (out) and undiluted "FW saline" or gluconate saline was used for the perfusion medium (in). Drugs, Na-gluconate, dopamine hydrochloride (in the dark), dibutyryl cyclic AMP, phosphoenol-pyruvate, NADH, ATP, and LDH/PK enzymes were obtained from Sigma Chemical Company (St. Louis, Missouri, U.S.A.). C a C l " H�BO" K-gluconate, Mg-gluconate, and Ca-gluconate, were obtained from Fluka Chemical Company, Buchs, Switzerland. All other chemical compounds were obtained from Janssen Chemical Company, Beerse, Belgium. '-=Na' (sodium chloride in water) was purchased from Amersham Company, Slough, England. P e r f ' u s i o n . - T h e posterior gills were cut off at their base and prepared for perfusion. Polyethylene catheters were introduced in the afferent and efferent blood vessels and gently fastened by means of a neoprene-plexiglass clamp. The afferent catheters were then connected to the perfusion reservoir and the efferent catheters to the collecting beaker. The gill was dipped in the incubation medium. The perfused saline ran from the afferent vessel across the lamellae to the efferent vessel, mimicking normal hemolymph circulation, and was collected for analysis. The height between the perfusion reservoir and the collecting beaker was kept at 25 cm. This gives a pressure inside the gills similar to the pressure that drives blood in the open circulatory system of crustaceans, which is in agreement with data given by Belman (1976). Air was continuously bubbled through the incubation medium. This procedure has been already described by Pequeux and Gilles (1978). The transepithelial PD was measured by means of calomel electrodes dipped into the incubation medium and the collecting beakers connected to the perfusion system. In this case, both sides of the epithelium were bathed with the same Ringer saline (Gilles et al., 1988). Preliminary experiments have shown that after a 30-min period of stabilization, the transepithelial PD remains stable for at least 6 h. The inward movements of N a ' were estimated by use of the radioactive tracer 22 Na* (9.25 kBq/ml). It was added to incubation saline and its appearance was measured on the other side. Samples were collected each 10 min for 120-140 min. Dopamine 0.2 mmol/I (a high concentration was used, because dopamine is known to become rapidly auto-oxidized at physiological pH) or db-cAMP 0.15 mmol/I were added to perfusion salines. The radioactivity of samples was counted using a B E C K M A N LS 6 0 0 0 IC counter. At the end of the perfusion period, the gills were blotted on filter paper and weighed. ==Na' influx measurements were expressed as pmol/(g tissue/h). Fluxes rates are calculated by using the method described by Pequeux and Gilles (1981).). After 1-h perfusion with or without dopamine, gills were quickly frozen and kept at - 2 0 ° C until homogenization. When assayed, half a gill was used for N a v y * ATPase activity measurement and the rest for c A M P determination. It was determined that the results were not dependent on the way gills were cut. Mea.surement o f N a ' K ' - A T P a s e A c t i v i t y . - T h e gills were weighed and homogenized in a cold imidazole buffer (50 mmol/I imidazole, 250 mmol/I sucrose, and 5 mmol/I EDTA at pH 7.4 with HCI). The Na+/K*-ATPase activity was determined by coupled assay as described by Norby (1988). The cuvette contained 2 ml reaction mixture with and without 5 mmol/I ouabain. The reaction mixture includes (in mmol/1) Tris-CI, 25; MgCI;, 5; EGTA 0.25 at pH 7.4; NaCl, 100; KCI, 25; phosphoenol-pyruvate, 1.5; NADH, O.15; ATP, 5; LDH/PK enzymes (6U/test). After incubation at room temperature for 10 min, the N a ' / K ' - A T P a s e reaction was initiated by addition of homogenate. The Na7K*-ATPase activity was expressed as the activity in the presence of ouabain (specific inhibitor of Na7K*-ATPase activity) subtracted from the activity obtained in the absence of ouabain. Results were given as units of Na'/K'ATPase activity per mg protein. One unit (U) is defined as 1 micromole N A D H oxidized per min. The protein concentration was assayed by a modified procedure described by Lowry et al. ( 1 9 5 1). Measurement o f cAMP C o n c e n t r a t i o n . - T h e gills were homogenized in 9 vol of ice-cold 3% perchloric acid and centrifuged for 5 min at 5,000 g (IEC Centra-4B) at 0°C. The supernatants were adjusted to pH 6 - 7 with I mol/1 potassium bicarbonate. c A M P concentration was measured by using a '=5I-labeled radioimmunoassay (RIA) kit obtained from NEN-Dupont Nemours Co., Boston, Massachusetts, U.S.A. Statistical A n a / y s i s . - D a t a were expressed as mean ± SD. Student's t-test or paired t-test were utilized to compare means. Statistical significance was taken as P � 0.05. RESULTS Effect of Dopamine and db-cAMP on 22Na+ I n f l u x e s Earlier studies p r e s e n t e d b y G i l l e s e t al. ( 1 9 8 8 ) h a v e d e m o n s t r a t e d that Na+ efflux w a s Fig. 1. Effect of dopamine 0.2 mmola and db-cAMP 0.155 mmol/1 added to the perfusion medium (in) on 22nay influxes of posterior gills isolated from fresh-water-acclimated Chinese crabs, Eriocheir sinen.si.s. Data represent means ± SD of 5 experiments (control and test). Perfusion medium: 240 mmol/I NaCl. Incubation medium: 10 mmot/I NaCl. undetectable in the posterior gills of Eriocheir sinensis. We therefore restricted our approach to measuring Na+ influxes through the posterior gills o f E. sinensis acclimated to fresh water. On the other hand, Pequeux and Gilles (1981) described that, for such gills, the influx of Na+ is dependent on the external concentration of the ions, showing saturation kinetics with values lower than 50 mmol/1 external NaCl concentration. In first experiments, isolated posterior gills from Eriocheir sinensis acclimated to fresh water were perfused with a FW saline and incubated in 10 mmol/1 NaCl as described in Materials and Methods. After an initial 30-min period of stabilization, we added 22N a+ outside and measured radioactivity of samples for up to 90 min. In such experimental conditions, influxes values of 16.35 ± 5.23 gmol/(g tissue/h) ( N = 10) were observed. When doing parallel experiments with dopamine 0.2 mmol/1 or db-cAMP 0.15 mmol/1 to the perfusion saline (inside), we induced a significant increase in the Na+ influxes (Fig. 1). A further minimal 30-min period of washing with the fresh-water saline without dopamine or db-cAMP reduced the Na+ influxes close to the control values. When isolated posterior gills were incubated and perfused in Cl--free conditions (Cl- Fig. 2. Effect of dopamine 0.2 mmol/I (N = 6) and dbcAMP 0.15 mmol/I (N = 7) added to the perfusion medium (in) on 22Na* influxes of posterior gills isolated from fresh-water-acclimated Chinese crabs, Eriocheir sinen.si.s. Data represent means ± SD of N experiments. Perfusion medium: 240 mmol/I Na-gluconate. Incubation medium: 10 mmol/1 Na-gluconate. replaced by gluconate), there was a depolarization of PD values going from - 1 0 ± 5mV to +44 ± 30mV (P �0.001 with N = 11) (personal data). Such PD values indicate that we blocked Cl- inward movements through the gills as described by Pequeux and Gilles (1988). Dopamine 0.2 mmol/1 or db-cAMP 0.15 mmol/1 still produced a significant increase of ZZNa* inward movement from Nagluconate 10 mmol/1 outside and 240 mmol/1 inside (Fig. 2). Effect of Dopamine on cAMP Content and Na*K*-ATPase Activity Cyclic nucleotides may function as second messengers for crustacean neurohormones such as dopamine. We investigated for the same gills the intracellular cAMP concentration and Na+/K+-ATPase activity in mediating the dopamine effect. After a 30-min perfusion of isolated posterior gills of Eriocheir sinensis with the "FW saline" on both sides, dopamine 0.1 mmol/1 was added to the perfusion medium; at the end of a 1-h perfusion period, the gills were quickly placed in liquid nitrogen and cAMP content as well as Na+/K+-ATPase activity were measured on the same gill as described in Materials and Methods. Compared with control perfusions, we Fig. 3. Effect of dopamine 0.1 mmol/I on cAMP concentration after 1-h perfusion of posterior gills isolated from fresh-water-acclimated Chinese crabs, Eriocheir sinensis. Data represent means ± SD of 6 experiments (control and test). found (Fig. 3) a significant increase in c A M P content expressed in pmol/g tissue from 575 ± 77 to 1,179 ± 219 (for both N = 6). Moreover, independently of the perfusion time itself, dopamine 0.1 m m o l / 1 significantly stimulated Na+/K+-ATPase activity expressed in U/mg protein from 0.258 ± 0.021 (N = 12) to 0.387 ± 0.054 (N = 19) (Fig. 4). DiscussION The present study demonstrates that, when added to the perfusion saline, dopamine 0.2 mmol/1 or db-cAMP 0.15 mmol/1 induced a significant increase of 22Na++ influxes through the isolated posterior gills of Eriocheir sinensis acclimated to fresh water and perfused inside with 240 mmol/1 NaCl plus incubated outside with 10 mmol/1 NaCI. Endocrine control of ion transport has been demonstrated for various epithelial tissues, including crustacean gills with dopamine as a major catecholamine controlling the active branchial ion uptake (Sommer and Mantel, 1988 and 1991; Kamemoto and Oyama, 1985; Detaille et al., 1992; Morris and Edwards, 1995). Transduction of dopamine effects into intracellular signals seems to be often mediated through an interaction implicating G-pro- Fig. 4. Effect of dopamine 0.1 mmol/I on Na*/K*-ATPase activity after I -h perfusion of posterior gills isolated from fresh-water-acclimated Chinese crabs, Eriocheir .sinensis. Data represent means ± SD of N experiments (control N = 12 and test N = 19). tein coupled receptors (Dohlman et al., 1987). Since the posterior gills of euryhaline crabs are a major site for osmoregulation (Gilles et al., 1988; Pequeux, 1995), they are likely to be a major target organ for neuroendocrine factors controlling osmoregulatory processes. Lohrman and Kamemoto (1987) briefly described the effect of db-cAMP on Na+ uptake in the posterior gills of Callinectes sapidus. Such an effect seems to be mediated through activation of the serosal side of the Na+/K'pump. Previous studies have shown that a membrane-permeable cyclic AMP derivative, such as dibutyryl cAMP, increases the influxes of Na+ and Cl- across the posterior gills of Eriocheir sinensis (see Bianchini and Gilles, 1989). Studies from our laboratory have reported that dopamine induces a hyperpolarization and not a depolarization when PD were measured (Detaille et al., 1992). Clearly, cAMP is activated by dopamine with a direct effect upon N a ' uptake and Na+/K+-ATPase activity in the gills of crustaceans; dopamine, after 1-h perfusion, increased the intracellular cAMP concentration in the posterior gills. Such results may be due to an activating effect of cAMP on the CI- channels located at the serosal side, which short-circuits the K+ diffusion potential (Pequeux and Gilles, 1988). On the other hand, it is demonstrated that, for Eriocheir sinensis, Na+ and Cl can be absorbed independently. The characteristics of a positive Na+-dependent short-circuit current with external Cl--free saline indicate that active Na� uptake proceeds via apical Na+ channels and basolateral N a ' / K ' - p u m p s (Zeiske et al., 1992). Moreover, an apical side H+-ATPase is believed to drive active, electrogenic, and Na+-independent Cl- absorption. This happens by maintaining a high transapical HCO , gradient that drives Cl- uptake via C1-/HCO , antiport and by hyperpolarizing the cellular potential, so that Cl- ions are conducted through channels across the basolateral membrane (Gilles et al., 1988; Onken and Graszynski, 1989; Onken and Putzenlechner, 1995). Riestenpatt et al. (1994) indicated that stimulation of N a+ uptake across the posterior gills of the Eriocheir sinensis by intracellular cAMP is mainly achieved by an increased amount of apical N a ' channels, whereas the cAMP-induced increase of Cl uptake seems to be caused by a stimulation of apical H+ATPase, with an eventual activation of basolateral Cl- channels. Furthermore, substitution of Cl- by gluconate on both sides of the epithelium gives a depolarization effect of cAMP on the serosal side, explained as an activation of the Na+/K+-pump and/or a leak system (Bianchini and Gilles, 1989). In order to focus our attention on an effect of dopamine only on Na+ transport, we substituted gluconate for Cl- on both sides. Our results demonstrated a significant increase in Na� inward movement for Cl--free saline when dopamine 0.2 mmol/1 or db-cAMP 0.15 mmol/1 were added in the perfusion salines; 2�Na+ influxes increased from 44 ± 25 to 81 ± 40 �mol/(g tissue/h) and from 35 ± 27 to 72 ± 40 Ilmol/(g tissue/h), respectively (as shown in Fig. 2). Such data are in agreement with the idea of Riestenpatt et al. (1994) who showed that analysis of amiloride (known to block Na+ apical transport) induced current-noise revealing a marked increase of apical Na+ channels, whereas stimulation of the basolateral Na'/K+-pump cannot be excluded. However, initial experimental evidence showed that when isolated gills were perfused with Cl -free salines on both sides and ami- loride 1 mmol/1 was added in the external saline, Na+ influxes were largely reduced. If db-cAMP 0.15 mmol/1 was added in perfusion saline, we observed a small ascent in the Na+ influx abolished with ouabain. Since the passive paracellular characteristics of the gill seem not to be modified by db-cAMP, as shown by Riestenpatt et al. (1994), we cannot exclude the possibility that cAMP increases N a ' influxes by stimulating basolateral Na�/K�-ATPase activity. The gill Na'/K+-ATPase is an adaptive enzyme in osmoregulating crabs. The enzyme activity is increased when crabs are exposed to dilute media (Sommer and Mantel, 1988; Corotto and Holliday, 1996), inducing in turn an increase in sodium uptake by the gills. With the hyperregulating blue crab Callinectes sapidus, Savage and Robinson (1983) demonstrated that a factor present in the hemolymph of 30% sea-water-acclimated crabs could increase Na+/K+ -ATPase activity in 100% sea-water crabs. More recently, sinus gland extracts from Pachygrapsus marmoratus (Fabricius) stimulated the influx of Na+ ions through the gills and increased Na+/K+ATPase activity by 54% in incubated posterior gills (Eckhardt et al., 1995). Injection of dopamine or membrane-permeable cyclic A M P (db-cAMP) into intact Leptograpsus variegatus (Fabricius) increases branchial Na+/K+-ATPase up to 67% and 63%, respectively (Morris and Edwards, 1995). The proposal of a direct activation of the Na+/K+ pump is substantiated first by the finding of Sommer and Mantel (1988) that dopamine or db-cAMP when injected into intact Carcinus maenas causes an increase of Na+/ K*-ATPase activity. In mammals, it has been debated whether dopamine inhibits or activates Na7K+-ATPase activity. Satoh et al. (1993) and Bertorello and Katz (1993) proved that dopamine modulates its intracellular messengers to inhibit Na+/K+-ATPase activity. On the contrary, Breton et al. (1994) and Giesen et al. (1984) observed a stimulation of Na*/K*-ATPase activity by increasing cytoplasmic cAMP level. This elicited a major question as to whether direct or indirect phosphorylation of the Na+/K+ pump stimulates or inhibits Na+/K+ATPase activity, and/or alters its subcellular localization or availability at the cell surface (Ewart and Klip, 1992; Rodrigo and Novoa, 1995). On the other hand, Ibarra et al. (1993) proposed that the effect of dopamine may involve a prior increase in intracellular Na+ and that the a,-subunit of Na'/K'-ATPase exists predominantly in a dephosphorylated active state at high N a ' concentration and in a phosphorylated inhibited state at low N a ' level. Our present data showed that, in experimental conditions stimulating Na' influxes and increasing intracellular cAMP concentration in the posterior gills of Eriocheir sinensis acclimated to fresh water, dopamine activated Na+/ K+-ATPase with specific activities increasing (expressed in U/mg protein) from 0.258 ± 0.021 (N = 12) to 0.387 ± 0.054 (N = 19). 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