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Comparative Biochemistry and Physiol

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

aDepartamento de Biodiversidad y Biologıa Experimental, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellon II,

Ciudad Universitaria (C1428EHA) Buenos Aires, ArgentinabCONICET (Consejo Nacional de Investigaciones Cientıficas y Tecnicas) 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; Pequeux, 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

1095-6433/$ - see front matter D 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.cbpb.2004.07.001

* 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).

(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

ogy, Part A 139 (2004) 103–109

J. Halperin et al. / Comparative Biochemistry and Physiology, Part A 139 (2004) 103–109104

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 intra-

cellular second messenger likely involved in the response

to these neurohormones is 3V–5-Vcyclic adenosine mono-

phosphate (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 semi-

terrestrial 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 osmoregula-

tory 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 long-

term 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 Tuyu,

Buenos Aires, Argentina. Animals were kept in plastic

containers with aerated artificial brackish water of 10xsalinity and fed twice a week with pellets of rabbit food.

Temperature was kept a 20F1 8C with a 12L:12D photo-

period. 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 approx-

imately 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 meas-

ured 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 ACiml�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

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.

J. Halperin et al. / Comparative Biochemistry and Physiology, Part A 139 (2004) 103–109 105

scintillation counter. Unidirectional sodium influx was

calculated according to the formula reported by Lucu and

Siebers (1986):

J ¼ 22Na� S� �

= SRA�Wð 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 pre-

incubated 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

11,000�g. 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 ıl 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 heptamo-

lybdate 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).

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

analytical grade. Dopamine was a gift from Dr. J. Calvete,

Laboratorios Fabra, Argentina. Ouabain, 8-(4-chlorophe-

nylthio)-cAMP (cp-cAMP), 3-isobutyl-1methyl-xanthine

(IBMX), and forskolin were obtained from Sigma. 22Na

was purchased from Amersham Pharmacia Biotech, Argen-

tina. 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 two-

way 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 pro-

duced significant hyperpolarization of gill Vte. Perfusion

with increasing concentrations of the cAMP analogue, cp-

cAMP, 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

J. Halperin et al. / Comparative Biochemistry and Physiology, Part A 139 (2004) 103–109106

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. 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. 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 hyper-

polarization 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. 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).

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.

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 KT5720-

treated 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. Pre-

incubation 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).

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.

J. Halperin et al. / Comparative Biochemistry and Physiology, Part A 139 (2004) 103–109108

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 pre-

incubation 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 trans-

epithelial 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 monitor-

ing 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 phosphor-

ylation 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 ion-

transporting 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. This complex response could also be related

to the existence of different DA receptors in the gills of this

species. Trausch et al. (1989) have prevented the effects of

DA on the Vte of isolated gills of E. sinensis by perfusing

with the D2 antagonist, chlorpromazine. More recently, Mo

et al. (2002) have characterized D1 dopamine receptors in

the gills of the same species by binding of the D1

antagonist, SCH23390. The temporal pattern of the response

to dopamine as well as the presence and interaction of D1

and D2 receptors in ion-regulating crab gills still require

extensive investigation.

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