Modulation of ion uptake across posterior gills of the crab by dopamine and cAMP

Comparative Biochemistry and Physiology, Part A 139 (2004) 103 – 109 www.elsevier.com/locate/cbpa Modulation of ion uptake across posterior gills of the crab Chasmagnathus granulatus by dopamine and cAMP J. Halperina,b,*, G. Genovesea,b, M. Tresguerresa, C.M. Luqueta,b a Departamento de Biodiversidad y Biologı́a Experimental, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón II, Ciudad Universitaria (C1428EHA) Buenos Aires, Argentina b CONICET (Consejo Nacional de Investigaciones Cientı́ficas y Técnicas) Rivadavia 1917 (C1033AAJ) Buenos Aires, Argentina Received 12 December 2003; received in revised form 27 May 2004; accepted 20 July 2004 Abstract Cyclic AMP (cAMP) and dopamine modulate ion uptake across isolated and perfused posterior gills of Chasmagnathus granulatus acclimated to 10x salinity. Addition of cAMP agonists, such as cp-cAMP, forskolin, and IBMX, produced a significant increase in the transepithelial potential difference (Vte), which reflects ion transport activity. Dopamine (DA) also had a stimulatory effect on ion uptake, increasing Vte and Na+ influx, although this effect was transient, since both variables remained elevated for less than 30 min. In addition, the dose–response curve for DA concentration-Vte was biphasic, and the maximum stimulation was obtained with 10 Amol l
1. When the effects of forskolin and DA on the Na+/K+-ATPase activity were tested, they correlated well with the Vte and Na+ influx experiments; the enzyme activity increased significantly after preincubation of gill fragments for 10 min with forskolin or DA (51 and 64%, respectively), but there was no effect after pre-incubation with DA for 20 min. Finally, KT5720, a specific inhibitor of cAMP-dependent protein kinase (PKA), completely abolished the stimulatory effect of DA on Vte, suggesting the involvement of PKA in this mechanism. D 2004 Elsevier Inc. All rights reserved. Keywords: cAMP; Chasmagnathus granulatus; Crab; Dopamine; Gills; Ion transport; Na+/K+-ATPase; PKA; Transepithelial potential differences 1. Introduction Hyper-regulating crabs compensate for diffusive salt loss by actively absorbing NaCl across the posterior gills (Mantel, 1985). These organs are lined with a specialized epithelium composed of thick cells with great numbers of mitochondria that supply the energy for the active transport of ions against their diffusional gradients (Taylor and Taylor, 1992; Péqueux, 1995). Located at the basolateral membrane of these cells, the enzyme Na+/K+-ATPase plays a principal role in ion uptake from dilute media, driving sodium uptake (and, in some cases, also chloride) across the gill epithelium of crabs experiencing hypo-osmotic stress * Corresponding author. Department of Physiology and Biophysics, University of Illinois at Chicago, 835 S. Wolcott St., M/C 901, Chicago, IL 60612, USA. Tel.: +1 312 996 7688; fax: +1 312 996 1414. E-mail address: halperj@uic.edu (J. Halperin). 1095-6433/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2004.07.001 (Siebers et al., 1985; Towle and Kays, 1986; Onken and Riestenpatt, 1998; Onken et al., 2003). Na+/K+-ATPase shows both a long-term and a rapid regulation in hypo-osmotic conditions. The former typically takes 1–2 weeks after transferring crabs from full-strength to dilute seawater, and it implies an increase in the enzyme activity measured in posterior gill homogenates (see Lucu and Towle, 2003, for a review). Recently, this response was linked to increased expression of Na+/K+-ATPase a-subunit mRNA in the blue crab Callinectes sapidus (Lucu and Towle, 2003), suggesting that the long-term response depends on the synthesis of more units of this enzyme. On the other hand, rapid activation of Na+/K+-ATPase can be achieved in several different ways, the most studied being neurohormonal action. Injections of hemolymph from crabs acclimated to low-salinity (Savage and Robinson, 1983), dopamine (DA), and pericardial organ extract (Sommer and Mantel, 1988) have all been reported to 104 J. Halperin et al. / Comparative Biochemistry and Physiology, Part A 139 (2004) 103–109 produce a short-term activation of the Na+/K+-ATPase in aquatic crabs. In addition, Trausch et al. (1989) have shown an increase of protein phosphorylation through DA and serotonin (5-HT) receptors in the gills of the Chinese mitten crab Eriocheir sinensis and a positive influence of this phosphorylation on Na+/K+-ATPase activity. The intracellular second messenger likely involved in the response to these neurohormones is 3V–5-Vcyclic adenosine monophosphate (cAMP), since DA, 5-HT, and octopamine elevate its intracellular content in the gills of C. sapidus and E. sinensis (Kamemoto and Oyama, 1985; Lohrmann and Kamemoto, 1987; Bianchini and Gilles, 1990; Mo et al., 1998). However, in E. sinensis, there are contradictory reports about the relationship between Na+/K+-ATPase and cAMP. While incubation of isolated gills with dibutyryl (db)-cAMP produced an activation of the enzyme (Mo et al., 1998), Riestenpatt et al. (1994) working on split gill lamellae mounted in an Ussing chamber found that the principal effect of db-cAMP on Na+ transport was to increase the current and conductivity of this ion at constant electromotive force. These authors concluded that db-cAMP increased the transcellular Na+ conductivity through apical Na+ channels without direct stimulation of the Na+/K+ATPase or that, at least, this stimulation was unimportant. Despite these conflicting results on the Chinese crab, the association between hypo-osmotic stress–DA–cAMP–Na+/ K+–ATPase and a fast activation of ion uptake in aquatic hyper-regulating crabs is well documented and generally accepted (see Morris, 2001 for a review). In contrast, the role of dopamine in the regulation of ion uptake in terrestrial and semi-terrestrial crabs might be different, due to the rapidly changing conditions that these crabs must face regularly. The animals must cope with sudden changes in the osmotic conditions due to tide action, exposure to air with the consequent evaporation of gill chamber water, or entrance into rain pools. In the semiterrestrial purple shore crab Leptograpsus variegatus, DA or cAMP increases the branchial Na+/K+-ATPase activity when injected into intact animals (Morris and Edwards, 1995). It was concluded that DA enhanced Na+/K+-ATPase activity via cAMP, however, direct evidence for this relationship was not provided. Chasmagnathus granulatus (Dana, 1851) is a grapsoid crab that inhabits estuaries in Brazil, Uruguay and Argentina (Boschi, 1964). Adults of this species actively leave water and are able to regulate acid–base balance after several hours of air exposure (Luquet et al., 1998). C. granulatus is well adapted to cope with extreme tidal and seasonal fluctuations in salinity (D’Incao et al., 1992; Anger et al., 1994; Spivak et al., 1994), thanks to its great osmoregulatory capacity both in oligohaline and hyperhaline media (Charmantier et al., 2002). This capacity is based on active absorption and excretion of ions through the epithelium of the posterior gills, using the Na+/K+-ATPase as the main driving force (Luquet et al., 2002; Onken et al., 2003). The aim of this work was to investigate the role of dopamine and cAMP in the regulation of ion uptake and Na+/K+-ATPase activity in a species that experiences both rapid and longterm salinity variations. 2. Materials and methods 2.1. Animals Adult male crabs in intermoult stage C (Drach and Tchernigovtzeff, 1967) of C. granulatus species were collected at Punta Rasa Beach, San Clemente del Tuyú, Buenos Aires, Argentina. Animals were kept in plastic containers with aerated artificial brackish water of 10x salinity and fed twice a week with pellets of rabbit food. Temperature was kept a 20F1 8C with a 12L:12D photoperiod. Animals with an average carapace width of 29.7F0.4 mm were chosen for the study. 2.2. Gill perfusion Crabs were sacrificed by destroying the nervous system with a spike and scissors. After removing the dorsal carapace, gills number 6 (the largest among posterior gills) were gently excised, and prepared according to Siebers et al., 1985. The afferent and efferent vessels were connected by fine polyethylene tubing (a=0.4 mm) to a peristaltic pump (afferent) and to a collecting tube (efferent). The tubing was held in position by an acrylic clamp covered with smooth neoprene to minimize gill damage. The preparation was bathed in a beaker containing approximately 25 ml of aerated saline and was perfused at a rate of 0.1 ml min
1. The perfusate was collected in a second beaker. Under these conditions, the preparation remains viable for several hours; gills not showing stable Vte after the usual stabilization period were discarded. 2.3. Transepithelial potential difference (Vte) Ag/AgCl electrodes were connected via agar bridges to the external bath and to the beaker collecting the perfusate (internal side). Electrical potential differences were measured with reference to the external medium and recorded with a chart recorder. 2.4. Sodium flux Unidirectional sodium influx was measured in perfused gills by applying 22Na in the bathing solution according to Luquet et al. (2002) at a final concentration of 0.25 ACi ml
1. After Vte stabilization, at least three samples were collected from the perfusate leaving the gills at 10-min intervals. Finally, dopamine was added to the perfusate at a final concentration of 30 Amol l
1 and additional six samples were collected in the same way. Radioactivity was measured with a Canberra Series 35 plus gamma J. Halperin et al. / Comparative Biochemistry and Physiology, Part A 139 (2004) 103–109 scintillation counter. Unidirectional sodium influx was calculated according to the formula reported by Lucu and Siebers (1986):
J ¼ 22 Na  S =ðSRA  W Þ where J is the calculated unidirectional flux of sodium in Aeq h
1 g-1, 22Na is the radioactivity (cpm) collected at each interval, S is the number of samples collected during 1 h, SRA is the specific radioactivity (cpm Aeq
1), and W is the fresh mass of the gill in grams. 2.5. Na+/K+-ATPase Gills were gently removed, cut into small pieces and preincubated at room temperature in normal saline with and without 10 Amol l
1 dopamine. After that, gills were homogenized with a Teflon-glass homogenizer, 1:20 (w/v) in cold buffer (12.5 mmol l
1 NaCl; 1 mmol l
1 HEPES; 0.5 mmol l
1 EDTA; 0.5 mmol l
1 PMSF adjusted to pH 7.6 with 5% NaOH) and centrifuged for 20 min at 4 8C at 11,000g. Pellets were resuspended in 350 Al of the same buffer and stored on ice. This fraction was used since previous reports on the same species showed that it contains most of the Na+/K+-ATPase activity (Rodriguez Moreno et al., 1998; Genovese et al., 2004). Na+/K+-ATPase activity was determined as described by Lucu and Flik (1999) in 500 ı̀l of (A) buffer solution containing (all in mmol l
1) 100 NaCl, 15 imidazole, 3 Na2ATP, 5 MgCl2, 0.1 EDTA and 12.5 KCl, and in (B) the same buffer without KCl and containing 1 mmol l
1 ouabain. Both solutions were adjusted to pH 7.5 with 0.5 mol l
1 histidine-imidazole. Aliquots of 10 Al of each gill sample were added to the assay buffer (A and B) and incubated in a thermostatic bath at 37 8C for 15 min. The reaction was stopped by the addition of 1 ml of 8.6% cold TCA. Liberated phosphate was quantified colorimetrically by adding 1 ml of a solution containing 9.2% Fe2SO4 and 1.14% ammonium heptamolybdate in 3.63% H2SO4. Absorbance was measured at 700 nm with a Jasco7850 UV/VIS spectrophotometer. The difference between the determinations (A and B) was attributed to Na+/K+-ATPase activity. Protein concentration was determined in 30-Al samples according to Lowry’ et al. (1951) and specific Na+/K+-ATPase activity was expressed in Amol Pi h
1 mg protein
1. Time constancy and linearity with protein content under the same conditions of this study were tested in a previous work of our laboratory (Genovese et al., 2004). 105 analytical grade. Dopamine was a gift from Dr. J. Calvete, Laboratorios Fabra, Argentina. Ouabain, 8-(4-chlorophenylthio)-cAMP (cp-cAMP), 3-isobutyl-1methyl-xanthine (IBMX), and forskolin were obtained from Sigma. 22Na was purchased from Amersham Pharmacia Biotech, Argentina. KT5720 was obtained from Alomone Labs, Jerusalem, Israel. Forskolin and KT5720 were prepared from 20 and 1 mmol l
1 stock solutions in DMSO, respectively. Before perfusing the gills with these two drugs, DMSO was added to the control perfusate and Vte was recorded. No significant effect of DMSO was observed. DMSO was also added to the control solutions in the Na+/K+-ATPase activity assays, when the effect of forskolin was studied. 2.7. Statistics Data were analyzed by repeated measures one or twoway analysis of variance or paired Student’s t-test when appropriate (Sokal and Rohlf, 1981). All data were presented as meanFstandard error of mean (S.E.M.). Differences were considered significant with pb0.05. 3. Results 3.1. Effect of cAMP on transepithelial potential differences (Vte) All treatments leading to an increase in intracellular cAMP concentration or to mimicking cAMP action produced significant hyperpolarization of gill Vte. Perfusion with increasing concentrations of the cAMP analogue, cpcAMP, elevated the outside-positive Vte, reaching the maximum effect at a concentration of 10 Amol l
1 (from 2.4F0.3 to 20.4F1.6 mV; pb0.001; n=8; Fig. 1). Forskolin (100 Amol l
1), an activator of adenylate cyclase (Seamon et 2.6. Solutions and chemicals Perfusate and bath solutions contained (in mmol l
1): 468 NaCl, 9.46 KCl, 7.5 MgCl2, 12.53 CaCl2, 5 HEPES, 2.5 NaHCO3. Perfusates also contained 2 mmol l
1 glucose (Luquet et al., 2002). All salts and reagents used were of Fig. 1. Effect of increasing cp-cAMP concentrations on the transepithelial potential difference (Vte) across isolated and perfused posterior gills of C. granulatus acclimated to 10x salinity. MeansFS.E.M. of eight values are given in each column. Asterisks (*) indicate significant differences with pb0.001. 106 J. Halperin et al. / Comparative Biochemistry and Physiology, Part A 139 (2004) 103–109 al., 1981), also augmented Vte dramatically (from 2.5F0.7 to 15.0F1.5 mV; pb0.001; n=5). Phosphodiesterase is the enzyme that catalyzes the degradation of cAMP. Inhibition of this enzyme with 500 Amol l
1 IBMX (Chasin and Harris, 1976) caused a much less pronounced effect on Vte than cp-cAMP but a significant stimulation was still observed (from 2F0.2 to 2.7F0.1 mV; pb0.05; n=6). All Fig. 3. Representative time courses of the effect of dopamine on the transepithelial potential difference (Vte) across isolated and perfused posterior gills. The arrow indicates the addition of different concentrations of dopamine (DA) to the perfusate. 10 Amol l
1 of DA produced the maximum stimulation, producing a more than twofold increase in the control Vte ( pb0.05, n=7). these effects were sustained for at least 1 h. Representative time courses of these experiments are shown in Fig. 2. 3.2. Effect of dopamine (DA) on Vte Representative time courses of Vte stimulation by different concentrations of DA are shown in Fig. 3. The minimum concentration that produced significant hyperpolarization was 5 Amol l
1, while the highest response was obtained with 10 Amol l
1 (from 3.2F0.4 to 8.3F0.5 mV; pb0.05, N=7). At higher concentrations (50 and 500 Amol l
1), the effect was still significant but less pronounced. Hence, the dose–response curve of Vte of gills vs. DA concentration showed a biphasic profile (Fig. 4). All the DA Fig. 2. Representative time courses of transepithelial potential differences (Vte) across isolated and perfused posterior gills of C. granulatus acclimated to 10x salinity. Arrows indicate addition of cp-cAMP 10 Amol mol l
1 (a), forskolin 100 Amol l
1 (b) and IBMX 500 Amol l
1 (c). Fig. 4. Effect of different dopamine concentrations on the transepithelial potential difference (Vte) across isolated and perfused posterior gills of C. granulatus acclimated to 10x salinity. (a) Time course and (b) DVte=gain in Vte (treated
control). DVte increased significantly from the corresponding controls (*pb0.05; n=4–10) with all concentrations tested except for 1 Amol l
1 ( pN0.05, n=6). J. Halperin et al. / Comparative Biochemistry and Physiology, Part A 139 (2004) 103–109 107 concentrations tested produced transient effects; Vte remained stimulated for less than 30 min. 3.3. Effect of DA on sodium influx DA at 10 Amol l
1 did not produce consistent effects in this experiment. A higher concentration, 30 Amol l
1, produced a significant but transient stimulation of sodium influx, similar to the effect of DA on Vte. Maximum augmentation of 34% (from 398F73 to 534F119 Aeq h
1 g
1; pb0.05; n=5) was recorded 30 min after the addition of dopamine. Sodium influx declined to control values 30 min later (60 min after the addition of DA, see Fig. 5). 3.4. Linkage between DA action and cAMP-dependent protein kinase In order to assess whether dopamine stimulates ion transport through a pathway involving the cAMP-dependent protein kinase (PKA), KT5720, a specific cell permeable inhibitor of this enzyme (Kase et al., 1987) was added to the perfusate before and during perfusion with dopamine. As shown in Fig. 6a, 250 nmol l
1 KT5720 almost completely abolished the stimulation of Vte caused by dopamine. Fig. 6b depicts the effects of dopamine on control and KT5720treated gills. KT5720 alone had no significant effect. 3.5. Na+/K+-ATPase The stimulatory effects of cAMP and DA are likely due to an activation of one or several ion-transporting proteins, thus producing an increase in ion uptake that is reflected in the Vte. Since the Na+/K+-ATPase generates the main driving force for ion uptake in this epithelium (Luquet et al., 2002; Onken et al., 2003), we tested the effect of forskolin and DA on the activity of this enzyme. Preincubation of gill fragments with 100 Amol l
1 forskolin for Fig. 5. Representative time course of the effect of dopamine on sodium influx across an isolated and perfused posterior gill of C. granulatus acclimated to 10x salinity. Arrow indicates the addition of 30 Amol l
1 dopamine (DA). Fig. 6. Effect of KT5720 on the transepithelial potential differences (Vte) across isolated and perfused posterior gills treated with dopamine. (a) Representative time course (filled circles: dopamine alone; empty circles: dopamine plus KT5720). (b) MeanFS.E.M of five values for each treatment. Asterisks indicate statistical differences after dopamine treatment ( pb0.05). Dopamine (DA) was added at a concentration of 10 Amol l
1. KT5720 (a specific PKA inhibitor) at 250 nmol l
1. 10 min produced a significant increase of Na+/K+-ATPase activity (from 20.5F2.5 to 30.9F2.8 Amol Pi h
1 mg
1; n=5). After 10 min of pre-incubation with 10 Amol l
1 dopamine, a significant increase in Na+/K+-ATPase activity, from 24F3.1 to 39.3F2.8 Amol Pi h
1 mg
1, was also recorded (N=13). When the gills were incubated with Fig. 7. Na+/K+-ATPase activity in posterior gills pre-incubated with forskolin (100 Amol l
1, 10 min, n=6) or dopamine (10 Amol l
1, 10 or 20 min, n=13 for each time). The data are given as percentages (FS.E.M.) of control value (Na+/K+-ATPase activity of the paired, untreated gill). Asterisks indicate statistical differences between treated and control gills, with pb0.05. 108 J. Halperin et al. / Comparative Biochemistry and Physiology, Part A 139 (2004) 103–109 dopamine for 20 min, no significant increase from control values was observed. These results correlate well with the Vte and sodium influx experiments. Fig. 7 illustrates the percentage increase in Na+/K+-ATPase activity after preincubation with forskolin and dopamine. 4. Discussion There is a general consensus within the literature about the role of dopamine as a neuroendocrine factor involved in the response of euryhaline aquatic crabs to dilution of the ambient salinity. The prevailing concept is that DA enhances ion uptake by activating the Na+/K+-ATPase, and that the second messenger involved in this regulation seems to be cAMP. Activation of this signaling pathway has frequently been observed to result in increased sodium influxes that compensate for salt lost during hypo-osmotic stress (see Kamemoto, 1991; Morris, 2001 for reviews). In this study, we showed that DA enhances transepithelial potential difference (Vte), sodium uptake and Na+/K+-ATPase activity in posterior gills of C. granulatus. Activators of the PKA transduction pathway, cp-cAMP, forskolin and IBMX, produced comparable stimulation of Vte and Na+/K+-ATPase activity. The connection between DA and the cAMP-PKA transductional pathway was studied by adding a specific inhibitor of PKA (KT5720) to perfused gills before the addition of DA, while monitoring the Vte. The complete abolition of the DA effect by KT5720 is direct evidence showing that the dopamine stimulation of ion transport across C. granulatus gills depends on PKA. Trausch et al. (1989) have demonstrated dopamine and serotonin-dependant phosphorylation of proteins in a microsomal fraction containing Na+/K+ATPase of E. sinensis gill homogenates. Moreover, they have been able to inhibit the serotonin-induced phosphorylation with ouabain, a specific inhibitor of Na+/K+-ATPase. Recently, Towle et al. (2001) have shown a putative site for phosphorylation by PKA in the sequence of C. sapidus gill Na+/K+-ATPase. All these findings together lead to the conclusion that dopamine binds to specific receptors at the gill ionocyte basolateral membrane and increases cAMP levels, leading to PKA-mediated phosphorylation of Na+/ K+-ATPase. So far, the action of DA in the gills of C. granulatus follows the model proposed for aquatic crabs. Novel results are obtained when the time course of the response is taken into account. In all of our experiments, the effect of DA is transient, lasting no more than 30 min, in contrast to the sustained stimulation caused by all the agonists of the PKA pathway tested. Vte and sodium influx show the same response after the addition of DA: an instant but transient increase followed by a return to control levels. Modulation of the Na+/K+-ATPase activity by DA follows the same temporal pattern, indicating that it is one of the iontransporting proteins involved in this mechanism. Perfusion experiments performed on gills of aquatic hyper-regulating crabs have shown sustained effects of dopamine, positively linked to cAMP (Kamemoto and Oyama, 1985; Mo et al., 1998). The transient effects of DA in C. granulatus may indicate a more complex regulatory system, probably related to the amphibious lifestyle of this species, which experiences sudden changes from full to diluted or even to concentrated seawater. In another amphibious crab, L. variegatus, DA has been reported to increase Na+/K+-ATPase activity, presumably by increasing cAMP levels (Morris and Edwards, 1995). However, nothing is known about the time course of this response, and, interestingly, it was only obtained in studies of intact animals; no effect was seen on isolated gills. The few fully terrestrial crabs studied show very different regulatory patterns, including a response to serotonin but not to DA or cAMP in Gecarcoidea natalis and inhibiting effects of dopamine and cAMP in Birgus latro (see Morris, 2001 for a review). Although the activation of the PKA pathway by DA is evident from our results, the transient effect of this bioamine in C. granulatus suggests the presence of either a second signaling pathway leading to the total reversion of the initial changes or a rapid deactivation of the receptors or signaling intermediates. 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